张多 高环多环芳烃的降解

多环芳烃降解的影响因素及机理的研究论文关键词：多环芳烃降解的影响因素降解机理论文摘要：多环芳烃是一类普遍存在的环境污染物，微生物的降解是PAHs去除的主要途径。

本文介绍了多环芳烃性质及目前国内外研究状况，以及降解多环芳烃的微生物，阐述了三大因素：基质、微生物活性和环境因子等对微生物降解多环芳烃的影响，微生物降解多环芳烃的机理。

并对今后的几个研究发展方向进行了展望。

PAHs are a class of widespread pollutants in the environmental, microbial degradation is the main way of removing PAH. This article presented the nature of the polycyclic aromatic hydrocarbon and research situation at home and abroad, and the microbial of degradating polycyclic aromatichydrocarbon, analysis the three factors, such as matrix, microbial activity and environmental factors, on which the effects of microbial degradatation of polycyclic aromatic hydrocarbon, and the mechanism of microbial degradation of polycyclic aromatic hydrocarbon. Finally, forecasted a few research directions of future development.Key words: polycyclic aromatic hydrocarbon, degradation factors, degradation mechanism多环芳烃(Polycyclic aromatic hydrocarbons,PAHs)是环境中普遍存在的一类有机污染物，是指两个或两个以上苯环以线状、角状或簇状排列的稠环化合物，是有机物不完全燃烧或高温裂解的副产物[1]。

多环芳烃降解菌的分离鉴定及其降解条件的优化的开题报
告
题目：多环芳烃降解菌的分离鉴定及其降解条件的优化
研究背景和意义：
多环芳烃（PAHs）是一类广泛存在于自然环境中的有机污染物，具有很强的毒
性和致癌性，长期大量积累会对环境和人体健康造成危害。

PAHs的去除是环境污染修复的重要内容之一。

目前，生物降解被认为是一种高效、经济、环保的PAHs去除技术。

在这种情况下，多环芳烃降解菌的筛选和应用是非常重要的。

研究内容和方法：
本研究的主要内容包括以下两个方面：
1.多环芳烃降解菌的分离和鉴定
选取不同环境样品（如石油污染地和化工厂附近土壤等）并分离筛选含有降解PAHs能力的菌株。

利用微生物学方法（如形态学特征、生理生化特性等）和分子生物学方法（如16S rDNA）对所筛选到的降解菌株进行鉴定，并推测其降解PAHs的作用
机制。

2.菌株降解条件的优化
选取不同种类的PAHs作为底物，研究不同生长条件对菌株降解能力的影响。

例
如调整菌株生长适宜温度、pH值、底物初始浓度等因素，优化其降解PAHs的效率。

此外，还将考虑利用联合培养和基因修饰等方法提高菌株的降解效率。

研究预期结果：
本研究旨在分离筛选出一批高效的PAHs降解菌株，并优化其降解条件，从而为
生物修复技术在PAHs污染处理中的实际应用提供了有用的参考。

预期可以发现一些高效降解PAHs的菌株，并对其降解机制进行探究。

此外，通过菌株生长条件的优化，也有望在菌株降解过程中获得更好的降解效果。

多环芳烃降解菌的筛选、降解机理及降解性能研究共3篇多环芳烃降解菌的筛选、降解机理及降解性能研究1多环芳烃（PAHs）是一类具有广泛应用的化学物质，由于在生产、运输等环节中不当处理而形成的污染物使得PAHs在环境中广泛存在。

而PAHs在自然环境中的生物降解速度缓慢，引发环境问题和生态危害，因此，在环境治理和污染修复方面，PAHs的降解成为一项重要的研究方向。

多环芳烃降解菌因其在PAHs分解中发挥重要作用而备受关注。

多环芳烃降解菌的筛选是研究PAHs降解的关键步骤。

目前，已经成功分离得到了许多对PAHs具有高水平降解能力的菌株，例如Sphingomonas、Pseudomonas和Mycobacterium等。

这些降解菌在土壤、水源等环境中都能有效地分解PAHs污染物，具有很强的应用价值。

多环芳烃降解菌的降解机理和降解能力是研究重点之一。

多环芳烃具有复杂性和多样性，降解机制也各异不同。

常见的PAHs降解途径包括：氧化、脱氢、脱环等反应，这些反应的发生都需要通过特定酶类的催化作用才能实现。

例如，多环芳烃阵列氢氧化酶（PAH-OH）可以将PAHs转化为相应的二元酸或酮类物质。

据研究表明，多环芳烃降解菌的降解能力与菌株自身的代谢活性、酶类酶学特性等密切相关。

多环芳烃降解菌的降解性能研究将对其应用于实际环境治理具有指导作用。

因为PAHs的化学结构复杂，降解过程中需要较高反应能量和完整的降解途径。

由于不同的菌株在PAHs降解稳定性、耐受性、适应性等方面存在差异，所以选择适合的菌株在实际应用中具有很高的重要性。

因此，深入研究PAHs降解菌株的降解性能，探究其在不同养分、温度、pH等环境变化下的生存、刺激响应和降解速率等特性，有助于更好地了解多环芳烃降解菌的整体性能和应用潜力，并为之后的环境修复工作提供更有针对性的建议和指导。

综上所述，多环芳烃降解菌的筛选、降解机理和降解性能研究对PAHs污染治理具有重要意义。

今后，研究人员将在这个领域展开更深入的研究，努力为保护环境、构建共享绿色家园做出贡献综合研究表明，多环芳烃降解菌的筛选、降解机理和降解性能研究是解决PAHs污染治理问题的重要途径。

第31卷第5期2003年10月浙江工业大学学报JOU RNAL O F ZH EJ I AN G UN I V ER S IT Y O F T ECHNOLO GY V o l .31N o.5O ct .2003文章编号:100624303(2003)0520528206收稿日期:2003202215;修订日期:2003206215作者简介:包贞(1979—),女,浙江金华人,硕士,主要从事固体废弃物处置研究。

环境中多环芳烃的分布及降解包　贞,潘志彦,杨　晔,俞尚清(浙江工业大学生物与环境工程学院,浙江杭州310032)摘要:多环芳烃(PA H s )在环境中大量存在,由于其具有致癌性和致突变性而受到国内外学者的广泛关注。

介绍了多环芳烃(PA H s )的来源以及在环境中的分布,综述了其在大气、水体、土壤和植被中的迁移转化规律,重点概括了环境中多环芳烃(PA H s )的几种降解方法,包括微生物降解、生物修复及其它物理化学方法,并简单提出了目前针对环境中的多环芳烃(PA H s )污染国内外采取的降解技术以及相应的控制措施。

关键词:多环芳烃;来源;迁移转化;降解中图分类号:X 503;X 132 文献标识码:AThe d istr ibution and decom position of PAHs i n the env ironm en tBAO Zhen ,PAN Zh i 2yan ,YAN G Ye ,YU Shang 2qing(Co llege of B i o logical and Environm ental Engineering ,Zhejiang U niversity of T echno logy ,H angzhou 310032,Ch ina )Abstract :Po lycyclic arom atic hydrocarbon s (PA H s )are o rgan ic po llu tan ts w h ich ex ist in the environm en t ,and have attracted popu lar atten ti on due to their carcinogen ic o r m u ta 2gen ic natu re .T he sou rces and the distribu ti on of PA H s in the environm en t are in troduced .Fu rther m o re ,it exp lain s the pattern s of m igrati on ,the tran sfo r m ati on of PA H s in the air ,w ater ,so il and vegetati on ,and in p articu lar generalizes the decom po siti on of PA H s in the environm en t ,including m icrobe degradati on ,the b i o logic treatm en t fo r rem ediati on and som e p hysical chem ical rem oval m ethods .It review s som e decom po sab le techno logy especially app lied to treat PA H s po llu tan ts and its con tro lling m ethods .Key words :po lycyclic arom atic hydrocarbon s (PA H s );sou rces ;m igrati on and tran sfo r 2m ati on ;decom po siti on0　引　言多环芳烃(Po lycyclic arom atic hydrocarbon s ,简称PA H s ),是指两个或两个以上苯环以线状、角状或簇状排列的稠环化合物[1]。

多环芳烃的微生物降解魏花朵河南大学环境与规划学院摘要:环境污染已成为当今世界所面临的一个重要问题。

应用生物降解能力使有害废物无害化或低毒害化,是当今环境治理的主要研究方向。

微生物作为生物界的主要降解类群,在水体污染、固体废弃物污染、重金属污染、化合物污染、石油及大气污染等治理过程中,均取得显著效果。

纯培养微生物的单一菌株及混合菌株的多环芳烃降解的研究已有很多年了。

为了更好地应用生物修复技术治理被多环芳烃污染的环境, 有必要对降解微生物、降解机制、环境影响因子等因素进行进一步的研究,从而选择出最优化的方案来治理污染环境。

关键词:多环芳烃微生物生物降解1环境污染治理的微生物学原理:微生物是肉眼不易看见、必须在电子显微镜或光学显微镜下才能看见的单细胞或简单多细胞或无细胞结构的微小生物的总称。

自然界中存在着丰富的微生物种群,在生物圈中着重充当分解者的角色。

微生物对物质的降解与转化,保证了自然界中正常的物质循环。

微生物对污染物的降解与转化是环境污染治理的基础。

由于微生物自身特点和代谢活动表现出在环境中的化学作用,决定了它对污染物具有强大的降解与转化能力。

1.1 微生物适合环境污染治理的特点微生物对污染物具有强大降解与转化能力,主要是因为微生物具有以下特点:1.1.1微生物个体微小,比表面积大,代谢速率快微生物的这个特点,使之具有惊人的代谢活性,有利于营养物的吸收和废物的排泄,有利于污染物的快速降解与转化。

1.1.2微生物种类多,分布广,代谢类型多样环境的多样性决定了微生物类型的多样性。

微生物种类多,代谢类型多样,为当今日益复杂的环境污染治理提供了更多的功能菌,对环境中形形色色的物质的降解转化,起着至关重要的作用。

1.1.3微生物繁殖快,易变异,适应性强微生物巨大的比表面积使之对生成条件下的变化具有极强的敏感性,加之微生物繁殖快、数量多,可在短时间内产生大量变异的后代,对进入环境中的“新”污染物,微生物可通过基因突变,改变原来的代谢类型而适应、降解之。

河北科技师范学院本科毕业论文（设计）多环芳烃—芘的降解研究院（系、部）名称：专业名称：学生姓名：学生学号：指导教师：2009年11月5日河北科技师范学院教务处制摘要多环芳烃是指分子中含有两个或两个以上苯环的碳氢化合物，多环芳烃是一类具有很强致癌性，致突变性和致畸性的环境污染物，具有低水溶性、高辛醇-水分配系数、高沉积物-水分配系数和较低的蒸汽压等特点。

它可以通过大气沉降、城市污水排放以及雨水冲刷作用进入水体，对整个生态系统的健康造成威胁，水体中多环芳烃呈3种状态：吸附于颗粒物、溶解态、悬浮态，是环境污染中最重要的检测项目之一，其已越来越受到人们的重视。

降解多环芳烃的方法有很多，常规方法有物理方法，化学方法等，物理法和普通的化学方法不仅降解的效果差，效率低，而且降解产物不彻底，光氧化法的降解效率高，产物稳定，具有很好的实际应用价值。

在光氧化过程中，水中的多环芳烃是在光诱发所产生的单线态氧、臭氧或羟基游离基的作用下发生氧化降解的。

本实验旨在观察在实验室中水萃效率最高时的实验条件以及在实验室控制的条件下多环芳烃降解的半衰期。

关键词：多环芳烃、光氧化、降解目录摘要 ....................................错误！未定义书签。

第1章文献综述 ...........................错误！未定义书签。

1.1 研究意义 .........................错误！未定义书签。

1.1.1 多环芳烃概述................错误！未定义书签。

1.1.2 多环芳香烃的来源与危害 (1)1.1.3 环境光化学基础 (2)1.2 多环芳烃的光降解 (3)1.3 研究进展 (4)1.3.1 PA HS光降解影响因素 (4)1.3.2多环芳烃分析测定 (5)1.4 主要研究内容 (6)第二章、实验部分 (6)2.1实验材料与方法 (6)2.2实验仪器与试剂 (7)2.2.1实验仪器设备 (7)2.2.2实验试剂 (7)2.3反应溶液介质和溶液的配制 (8)2.4实验步骤 (8)2.5质量控制与质量保证 (10)2.6色谱条件 (11)第三章、实验结果与讨论 ..................错误！未定义书签。

多环芳烃污染物在生态系统中的生物降解机制研究多环芳烃（Polycyclic Aromatic Hydrocarbons，PAHs）是一类含有多个苯环结构的化学物质，广泛存在于自然界和工业领域。

这些化合物因其高寿命、低生物降解性和致癌性等特点而引起了人们的关注。

研究多环芳烃污染物在生态系统中的生物降解机制对环境保护和生物安全具有重要意义。

多环芳烃能够通过微生物降解而被去除，这一过程通常包括两个步骤：吸附和酶解。

吸附是指多环芳烃能够被微生物表面的细胞壁或胞外多糖所吸附，并通过吸附将污染物转移到细胞内部。

而酶解则是指微生物通过酶的作用将多环芳烃降解成较小的低分子化合物，最终得到CO2、H2O等无害的物质。

目前，研究者从多个角度对多环芳烃污染物在生态系统中的生物降解机制进行了深入研究。

其中，菌株筛选、基因重组和基因组学等技术的应用得到了广泛关注。

在菌株筛选方面，研究者通常采用微生物分类学和生态学两个方法来筛选高降解能力的微生物。

微生物分类学方法是指根据微生物的形态、生理生化特征以及分子遗传学特征等来对微生物进行分类。

而生态学方法则是指根据多环芳烃的来源、污染地点和环境条件等因素，寻找适应该环境的微生物种群。

在菌株筛选方面的研究表明，面包酵母、肠球菌、铜绿假单胞菌等能够有效去除多环芳烃。

在基因重组方面，研究者通常将微生物的基因组和降解基因进行分析、比较和重组。

通过这种方法，研究者可以将低降解能力的微生物降解基因进行转移，从而提高微生物的降解能力。

在基因重组方面的研究表明，转化了naphthalene dioxygenase基因的异养杆菌能够有效去除多环芳烃。

在基因组学方面，研究者可以快速鉴定新的降解基因和降解途径，从而加速微生物降解多环芳烃的速率。

通过测序微生物基因组，研究者可以发现这些微生物所拥有的各种降解途径和蛋白质。

在基因组学方面的研究表明，依靠微生物基因组的分析，可以快速鉴定降解基因和降解途径，例如海洋细菌Marinobacter sp.和Mycolicibacterium strain CU5能够降解多环芳烃。

Biodegradation of polycyclic aromatic hydrocarbons by a halotolerant bacterial strain Ochrobactrum sp.VA1P.Arulazhagan a ,⇑,N.Vasudevan ba Department of Civil and Environmental Engineering,Sung Kyun Kwan University,300CheonCheon-Dong,Jangan-Gu,Suwon,Gyeonggi-Do 440-746,Republic of Korea bCentre for Environmental Studies,Anna University,Chennai 600025,Indiaa r t i c l e i n f o Keywords:BiodegradationPolycyclic aromatic hydrocarbons Halotolerant Ochrobactruma b s t r a c tPolycyclic aromatic hydrocarbons (PAHs)are ubiquitous pollutants in the environment and are derived from both man-made and natural resources.The present study is focused on the degradation of PAHs by a halotolerant bacterial strain under saline conditions.The bacterial strain VA1was isolated from a PAH-degrading consortium that was enriched from marine water samples that were collected from dif-ferent sites at Chennai,India.In the present study,a clearing zone formed on PAH-amended mineral salt agar media confirmed the utilization of PAH by the bacterial strain VA1.The results show that the strain VA1was able to degrade anthracene (88%),phenanthrene (98%),naphthalene (90%),fluorene (97%),pyr-ene (84%),benzo(k)fluoranthene (57%)and benzo(e)pyrene (50%)at a 30g/L NaCl concentration.The present study reveals that the VA1strain was able to degrade PAHs in petroleum wastewater under saline conditions.The promising PAH-degrading halotolerant bacterial strain,VA1,was identified as Ochrobac-trum ing biochemical and molecular techniques.Ó2010Elsevier Ltd.All rights reserved.1.IntroductionThe biodegradation of polycyclic aromatic hydrocarbons (PAHs)plays a vital role,considering the ubiquitous distribution and dele-terious effects of PAHs on human health.The hydrophobic nature of polycyclic aromatic hydrocarbons makes their clean up extremely difficult and allows them to persist for longer periods.Polycyclic aromatic hydrocarbons are released into the environment through the incomplete combustion of solid and liquid fuels (Ramdahl and Bjorseth,1985).They are suspected to possess toxic,mutagenic and carcinogenic properties (Heitkamp and Cerniglia,1988;Keith and Telliard,1979;Yuan et al.,2000).The genotoxicity of PAHs also increases with their size,up to at least four or five fused benzene rings (Cerniglia,1992).Oil spillage leads to the contamination of seawater by hydrocarbons.PAHs require effective remediation strategies due to their growing production from anthropogenic sources (Wilson and Jones,1993).Several bacterial species,such as Bacillus napthovorans ,Halomonas eurihalina,Sphingomonas sp.,Cycloclasticus sp.and Pseudoalteromonas sp.(Zhuang et al.,2002;Martínez-Checa et al.,2002;Ye et al.,1996;Geiselbrecht et al.,1998;Hedlund and Staley,2006),were isolated from areas with dif-ferent salt concentrations and their biodegradation effects were analyzed.Sohn et al.(2004)isolated a high-molecular-mass PAH-degrading bacterium (Novosphingobium pentaromativorans sp.nov.)from estuarine sediment.The strain was able to degrade a mixture of PAHs (pyrene,chrysene,benzo(a)pyrene,benzo(a)anthracene and benzo(b)fluoranthene)in the presence of 10%(w/v)2-hydroxypropyl b -cyclodextrin in a buffered medium with 2.5%salinity.The microbial degradation of petroleum hydrocarbon pollutants is limited by an utilizable source of nitrogen and phos-phorus (Rosenberg et al.,1998).Because petroleum contains only traces of nitrogen,the required nitrogen must come from the sur-rounding environment (Oren et al.,1992).The present study deals with the degradation of different PAHs by a halotolerant bacterial strain (Ochrobactrum sp.VA1)that was isolated from a PAH-degrading bacterial consortium (Arulazhagan and Vasudevan,2009).The strain was able to degrade both low molecular weight (two to three benzene rings)and high molecu-lar weight (four to six benzene rings)PAHs under saline condi-tions.The study also details the role of additional nutrients in PAH degradation at high saline conditions (NaCl:60g/L).The deg-radation of PAHs and the importance of nitrogen and phosphate were detailed in the treatment of petroleum wastewater by Ochrobactrum sp.VA1.2.Materials and methods 2.1.Media compositionThe carbon-free mineral salt medium contained 2.5g NH 4Cl,5.46g KH 2PO 4,4.76g Na 2HPO 4,0.20g MgSO 4and 30.0g NaCl in0025-326X/$-see front matter Ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.marpolbul.2010.09.020Corresponding author.Tel.:+82312996699.E-mail address:arulazhagan_p@yahoo.co.in (P.Arulazhagan).1L distilled water,pH7.4±0.2.Thefinal pH of the medium was adjusted to7.4with0.1N NaOH,and the medium was sterilized by autoclaving(121°C for15min)prior to the addition of the PAHs substrates.Stock solutions of each PAH were prepared in ethyl ace-tate and stored at4°C.2.2.PAHsThe PAHs were purchased from Sigma–Aldrich(98–99%purity), and all other chemicals(Analar grade)were purchased from Merck, India.The PAHs used in the study were selected based on the num-ber of rings(two to six benzene rings)and their molecular weights. The molecular weights of the PAHs used in the study were naph-thalene(128),fluorene(166),phenanthrene(178),anthracene (178),pyrene,benzo(e)pyrene and benzo(k)fluoranthene.The con-centrations of PAHs used were3ppm anthracene,3ppm phenan-threne,3ppm naphthalene,3ppmfluorene,3ppm pyrene, 1ppm benzo(e)pyrene and1ppm benzo(k)fluoranthene.Anthra-cene and pyrene were selected as representatives of LMW and HMW PAHs,respectively,for studies at different concentrations (25,50,75and100ppm).2.3.Bacterial strainThe PAH-degrading bacterial consortium was enriched from water samples collected from seven different petroleum-or coal-contaminated sites from the port of Chennai,India(Arulazhagan and Vasudevan,2009).The bacterial strain Ochrobactrum sp.VA1 was isolated from the PAHs-degrading bacterial consortium.The bacterial strain(VA1)was grown on phenanthrene,and the cell count was checked daily and during every transfer to fresh med-ium.The cell morphology and motility of exponentially growing li-quid cultures were examined on freshly prepared wet mounts by light microscopy.Plate counting was done in nutrient agar medium.2.4.Enrichment of bacterial strainThe PAH phenanthrene dissolved in ethyl acetate was added to 250mL conicalflasks.After the ethyl acetate had evaporated,the mineral medium(100mL)was added.The bacterial cells(5mL) were added to the mineral medium containing the PAH(phenan-threne,3ppm)as the sole carbon source.The conicalflasks were shaken at150rpm at37°C for48h.After the growth was visual-ized,5mL of enrichment cultures were then transferred to a fresh medium and incubated under the same conditions.Subsequently, three to four identical transfers were performed in the respective PAH-containing medium.2.5.PAH clearing zone-spray-plate techniquePAH degradation by the bacterial strain was analyzed by the spray-plate technique using the PAH as the sole carbon and energy source.Agar was added to the mineral salt medium for plating in Petri dishes.After solidification,acetone-dissolved anthracene was sprayed on top of the medium.The bacterial strain was inoc-ulated on the PAH amended medium and incubated at37°C for 48h.After incubation,the clearing zones formed around the colo-nies indicated the degradation of the PAH sprayed on the medium (Kiyohara et al.,1982).2.6.Analysis of PAH degradationFor the degradation study,the bacterial strain was inoculated in mineral medium containing PAH.The different compositions used in the degradation of PAH were(i)medium+PAH+bacterial strain;(ii)medium+PAH and(iii)medium+bacterial strain, where(ii)and(iii)served as controls.The bacterial strain was added to the medium at a concentration of104–105cfu/mL.The cultures,prepared in duplicate,were incubated at37°C in a shaker at150rpm and were extracted at every24h time interval for 5days.The samples were extracted twice with ethyl acetate(v/v) after acidification to pH2.5with1N HCl.The extracts werefiltered through anhydrous sodium sulfate and condensed to1mL for the chromatographical analysis of PAH degradation using a condensa-tion unit(Buchi,Germany).The condensed sample wasfiltered through a0.2-mm syringe filter and was analyzed using high performance liquid chromatog-raphy(HPLC).HPLC analysis was performed with a KNAUER(K501, Knauer,Germany)unit equipped with a PAH-specific column(Ultr-asep ES,B590/02,250Â4mm,Knauer,Germany)with a UV–VIS detector connected to the WINCHROME software,which was used to process the data.The mobile phase was acetonitrile.Standard solutions of different PAHs were used as a reference.Theflow rate of the mobile phase was maintained at1mL/min.The samples were injected individually,and the utilization rate of PAHs was cal-culated based on the peak area percent and the retention time.2.7.Metabolite formation-TLC and GCMSDuring the degradation study,different kinds of metabolites were formed;these metabolites were identified using thin layer chromatography(TLC).The condensed samples were loaded on a TLC plate with the help of capillary tubes(10l L).The chromato-gram was run with solvents in ratios as follows:50:50benzene/ hexane,50:50benzene/acetone and80:10:10benzene/acetone/ acetic acid.After removing the plates from the solvents,2%Gibbs reagent was sprayed on the plate,and the plate was observed un-der ultraviolet light at265nm to identify the PAH metabolites.A Hewlett–Packard6890gas chromatograph,equipped with a 5973mass spectrometer with an HP-5MS(30mÂ0.25mm I.D.Â0.25l m)fused-silica capillary column,was used for the analysis.The column temperature program was set at100°C held for1min,15°C/min to160°C and5°C/min to300°C held for 7min.The GC injector was held isothermally at280°C with a split-less period of3min.Helium was used as the carrier gas at aflow rate of1ml/min using electronic pressure control.The GC/MS interface temperature was maintained at280°C.The MS was oper-ated in electron impact(EI)ionization mode with an electron en-ergy of70eV,and the scan to determine the appropriate masses for the selected ion monitoring ranged from50to500amu(atom to mass unit).Standards from Sigma–Aldrich were used for the PAH(Fluorene)and their metabolites.A GC–MS library search was used to confirm the metabolites without standards.2.8.CO2evolution testIn this study,PAH+MSM and MSM+VA1bacterial strain served as the controls and PAH+MSM+VA1bacterial strain was used as the test sample.Mineral salt medium,in combination with both PAH and the VA1bacterial strain,was used as the sample. PAH(3ppm phenanthrene)dissolved in ethyl acetate was added to sterile saline bottles(100mL).After the evaporation of the sol-vent,25mL of mineral salts medium was added to the bottle. The bottle was completely sealed(airtight)with an aluminum stopper.The medium was kept in an orbital shaker at150rpm. Samples were collected at24h time intervals and analyzed for CO2evolution in a gas chromatograph.The carbon dioxide content was measured in a Porapak Q column(80/100mesh,2m)with a thermal conductivity detector using an external standard.The carrier gas was helium,and the column temperature was50°C. The temperature of the injector and of the detector was100°C.P.Arulazhagan,N.Vasudevan/Marine Pollution Bulletin62(2011)388–394389Samples(250l L)of the headspace gas of the cultureflask were withdrawn with a gas-tight syringe and injected into the gas chro-matograph for CO2determination.The samples in the saline bottles were extracted and analyzed on a PAH-specific column using HPLC to measure the degradation of PAH.b-scale study on the treatment of petroleum wastewaterA lab-scale study using petroleum wastewater(produced water)from Cairn Energy Private Limited,India,with2.9%salinity was conducted in a500mLflask.The nitrogen and phosphate lev-els in the wastewater were5.6ppm and3.9ppm,respectively.Due to the low concentration of nutrients,the bacterial strain VA1was unable to grow on PAHs present in the wastewater.To support and enhance the growth of VA1,ammonium chloride(0.03g/L)and potassium dihydrogen phosphate(0.015g/L)were added to the wastewater.2.10.Phenotypic and phylogenetic analysisThe bacterial strain was analyzed for phenotypic characters using KB003:Hi24‘‘Enterobacteriaceae Identification Kit”, (Himedia,India).The phenotypic characteristics of the strain were analyzed using the Enterobacteriaceae Identification Kit to confirm the utilization of different carbon sources and also to identify the genus of the strain.2.11.Extraction and amplification of bacterial DNADNA from the bacterial cells was extracted using a Qiagen(QIA-ampÒDNA stool Mini kit Cat.No.51504)DNA isolation ing the protocol from the manufacturer,DNA was eluted in200l L of AE buffer and stored at4°C for further use.The concentrated DNA samples were amplified by polymerase chain reaction(PCR) using a thermal cycler(MastercyclerÒpersonal,Eppendorf AG,Ger-many).Amplification was performed using a forward primer(27F) and a reverse primer(1520R)that are complementary to highly conserved regions of bacterial16S rRNA genes.The sequence of the forward primer(25pmol)was50-AGAGTTTGATCCTGGCTCAG-30(hybridizing at positions8–27,according to the Escherichia coli numbering system),and the sequence of the reverse primer (25pmol)was50-AAGGAGGTGATCCAGCCGCA-30(hybridizing at positions1541–1522),for a combined concentration of50pmol. The PCR supermix(Invitrogen Cat.No.10572-014,USA)consisted of22mM Tris–HCl(pH8.4),55mM KCl,1.65mM MgCl2,220l M dGTP,220l M dATP,220l M dTTP,220l M dCTP and22U recom-binant Taq DNA polymerase/mL.The PCR supermix(40l L)was mixed with the primers(5l L)and the DNA(5l L)for a total vol-ume of50l L in0.2mL PCR tubes and was then loaded in the ther-mal cycler.The PCR was performed with an initial denaturation at 94°C for3min,followed by30cycles of denaturation at94°C for 1min,primer annealing at60°C for0.45min,and primer exten-sion at72°C for2min.For thefinal step,the samples were incu-bated at72°C for10min.PCR amplification was verified by electrophoresis,performed in a horizontal submarine apparatus with1%agarose gel.TAE buffer was used as the tank buffer.The electrophoresis was performed for2h at50V.The gel was visual-ized in an UV illuminator.3.16S rRNA sequential analysis of the PAH-degrading bacterial strainThe cyclic sequencing reaction was performed using the BigDye TerminatorV3.1cycle sequencing kit containing Ampli Taq DNA polymerase(Applied Biosystems,P/N:4337457).The sequencing reaction mix was prepared by combining1l L of BigDyeV3.1, 2l L of5Âsequencing buffer and1l L of50%DMSO.To4l L of sequencing reaction mix,4pmol of primer(2l L)and a sufficient amount of purified PCR product was added.The resulting solution was denatured at95°C for5min.Cycling began by denaturing at 95°C for30s,annealing at52°C for30s and extension for4min at60°C;the cycle was repeated a total of30times in an MWG thermocycler.The reaction was then purified on a Sephadex plate (Edge Biosystems),and centrifugation was used to remove un-bound labeled and unlabeled nucleotides and salts.The purified reaction was loaded onto the96capillary ABI3700automated DNA analyzer,and electrophoresis was carried out for4h.The nucleotide sequences were registered in the computer that is at-tached to the ABI3700DNA analyzer.The nucleotide sequences obtained from the ABI DNA analyzer were studied using the BLAST software available on the NCBI web-site().After editing the sequence,the BLAST software was used to identify the specific type of bacterium corre-sponding to the nucleotide sequence.4.ResultsThis study was conducted to understand the degradation of dif-ferent PAHs at different concentrations by the halotolerant bacte-rial strain(Ochrobactrum sp.VA1)under saline conditions.The study also details the importance of additional substrates for PAH degradation at higher salinity.4.1.Isolation of the PAH-degrading bacterial strainThe bacterial consortium that was enriched from the marine environment contained three bacterial strains(VA1,VA2and VA3).Among the three bacterial strains,strain VA1grew better on different LMW PAHs than did VA2and VA3.When the strain VA1was grown on PAH-coated(anthracene)mineral agar plates, clearing zones were visualized,indicating the PAH-utilizing ability of the strain.Clearing zones on mineral salts medium coated with PAH(anthracene)were analyzed for the VA1strain using Gordona sp.BP9as a positive control.A clearing zone of1.8mm diameter was observed for the positive control,whereas for the VA1strain, it was2.1mm on anthracene.Thin layer chromatographic analysis revealed that VA1was able to degrade PAHs.Different colored spots indicated the metabolite formation.The amount of CO2pro-duced in the controlflask with the VA1strain and medium was 152ppmv.The ratio between the amount of carbon dioxide pro-duced and the residual hydrocarbons gives a complete picture of the hydrocarbon degradation(Penet et al.,2004).The stoichiome-tric equation used to calculate the amount of CO2produced during phenanthrene(C14H10)degradation was as follows:2C14H10þ33O2!28CO2þ10H2OThe study confirms the mineralization potential of the bacterial strain to degrade phenanthrene as a sole carbon source in30g/L NaCl.The bacterial strain VA1degraded phenanthrene(92%)and released1274ppmv CO2(Fig1).Duringfluorene(3ppm)degradation,the different metabolites formed were analyzed using thin layer chromatography(TLC).The samples collected on the fourth day showed different colored spots on the TLC plate,which indicated the metabolite formation.The formation of metabolites during the mineralization offluorene was further confirmed by GC–Mass Spectral analysis(Table1). The analysis showed six peaks,out of which two peaks were con-firmed as monohydroxyfluorene(m/z,254),fluorenone(m/z,310) and phthalic acid(m/z,310).390P.Arulazhagan,N.Vasudevan/Marine Pollution Bulletin62(2011)388–394Fig.3.Degradation of naphthalene by Ochrobactrum sp.VA1(NaCl:30g/L).Fig.4.Degradation offluorene by Ochrobactrum sp.VA1(NaCl:30g/L). Fig.2.Degradation of anthracene by Ochrobactrum sp.VA1(NaCl:30g/L).7.Degradation of benzo(k)fluoranthene by Ochrobactrum sp.VA1(NaCl:30g/L).Fig.9.Treatment of petroleum wastewater using Ochrobactrum sp.VA1.oxidase positive.The strain grows at37°C and at pH7.4.When the strain was analyzed for physiological characteristics,the strain showed negative results for ONPG,phenylalanine deamination, methyl red,Voges-Proskauer and indole reactions.The strain uti-lized9out of13carbon sources.Arabinose,adonitol,trehalose and lactose are the carbon sources that were not utilized by the strain(Table4).Thus,based on the phylogenetic analysis and phe-notypic characterization,the bacterial strain VA1has been identi-fied as Ochrobactrum sp.5.DiscussionThe halotolerant,PAHs-degrading bacterial strain,Ochrobac-trum sp.VA1,was isolated from a PAH-degrading bacterial consor-tium.Among the three bacterial strains present in the consortium, strain VA1showed more potential growth and degradation of PAHs than strains VA2(Enterobacter cloacae)and VA3(Stenotrophomonas maltophilia).The strain VA1also potentially degraded different concentrations of LMW and HMW PAHs and the PAHs present in petroleum wastewater under saline conditions.Initially,degradation of PAH by the VA1strain was confirmed using clearing zones formed on anthracene-coated mineral salt agar medium.Further studies on the mineralization of PAH,based on CO2evolution by the bacterial strain,proved the degradation. During the mineralization of PAHs,the amount of CO2evolved indicates the extent of degradation(Solano-Serena et al.,1999). Bouchez et al.(1996)reported that the complete degradation of PAH results in the evolution of CO2,the formation of biomass and water-soluble metabolites.The bacterial strain used in the present study utilized phenanthrene(92%)as a sole carbon source and released1274ppmv of CO2.The stoichiometrically calculated amount of CO2that evolved during phenanthrene degradation was almost equal to90%of phenanthrene converted into CO2. The degradation of PAH by the bacterial strain was enhanced by the addition of substrate to the medium.The complete mineralization of PAH by the bacterial strain was further confirmed by analyzing the metabolites formed duringflu-orene degradation.Metabolites,such as monohydroxyfluorene,fluorenone and phthalic acid,forfluorene-utilizing bacteria,were also reported for Pseudomonas sp.F274(Grifoll et al.,1994).Phtha-lic acid that is formed duringfluorene degradation may be further degraded to CO2and water(Yamazoe et al.,2004).Thus,in the present study,the formation of such metabolites may be due to enzymatic action on the PAHs.LMW and HMW PAHs were used as sole carbon sources for the growth of the VA1bacterial strain.VA1degraded more than85%of LMW PAHs in4days at30g/L NaCl,except for anthracene(5days), which showed that LMW PAHs are easily degraded by VA1.The re-sults depicted in Figs.3–5show the degradation of naphthalene (3ppm),fluorene(3ppm)and phenanthrene(3ppm)by the VA1 strain at89%,97%and92%,respectively.Moody et al.(2001)re-ported that the Mycobacterium sp.strain PYR-1was dosed with anthracene or phenanthrene and,after14days of incubation,had degraded92%and90%of the added anthracene and phenanthrene.The strain Ochrobactrum sp.VA1was also found to be effective in degrading the HMW PAHs,such as pyrene(84%),benzo(k)fluo-ranthene(57%)and benzo(e)pyrene(51%).An increase in the molecular weight decreased the percent degradation of PAHs by the bacterial strain.Martínez-Checa et al.(2002)reported that the Halomonas eurihalina strain H-28was able to grow and produce surfactant on different carbon sources(n-hexadecane,petrol, n-tetradecane and crude oil)in the presence of glucose,yeast extract,malt extract and proteose-peptone at51.3g/L NaCl.The strain degraded naphthalene(95%),phenanthrene(50%),fluo-ranthene(50%)and pyrene(58%).Daane et al.(2001)reported that the bacterial strain Paenibacillus group-PR-P3,isolated from the rhizosphere of salt marsh plants,degraded the naphthalene and phenanthrene that was present in the sediments completely.They also reported that71%of thefluorene was degraded by the Paeni-bacillus group-PR-P1strain.Pseudomonas cepacia F297is able to grow withfluorene as the sole source of carbon and energy(Grifoll et al.(1995).Hedlund et al.(1999)reported that the Neptunomonas naphthovorans(NAG-2N-113and NAG-2N-126)strains,isolated from creosote-contaminated sediments,were capable of degrading 5ppm naphthalene completely in7days.When compared to the above reports,the halotolerant strain VA1used in the present study degraded both LMW and HMW PAHs under saline conditions without any additional substrate.Salinity plays an important role in the treatment of petroleum wastewater.When the salinity of the medium was increased to 60g/L NaCl,the percent degradation of PAHs decreased,and the need for additional substrates occurred(Kargi and Dincer,1996). To support the growth of the bacterial strain,yeast extract(0.2g/L) was added to the medium.The addition of yeast extract enhanced the percent degradation of the PAHs by Ochrobactrum sp.at60g/L NaCl.Thus,the need for additional substrate for PAH degradation at high salinity is reported from this study.The need for additional substrate has been emphasized previously(Kargi and Dincer,1996).The strain VA1was also studied at different concentrations of PAHs(anthracene and pyrene),and the results showed above 60%degradation up to75ppm.When the concentration of PAH in-creased to100ppm,the percent degradation decreased.Zhuang et al.(2002)reported that Bacillus naphthovorans MN-003,isolated from marine fuel oil-contaminated tropical marine sediments (optimum salinity1.75–3.50%),was used in a bioreactor to treat naphthalene-contaminated seawater at a removal rate of0.8g/L/ day.Kumar et al.(2007)isolated a halotolerant and thermotolerant Bacillus sp.,which degraded a mixture of hydrocarbons(0.02%) with diesel,gas oil,alkanes,crude oil and kerosene(0.2%)The strain also produced a surface-active emulsifying pared to the previous reports,the strain Ochrobactrum sp.VA1used in the present study degraded PAHs without any additional substrate at 30g/L NaCl.In the lab scale petroleum wastewater study,due to the low concentration of nitrogen and phosphate,the strain was unableTable4Phenotypic analysis of PAHs degrading Ochrobactrum sp.VA1strain. of the biochemical test VA11.Oxidase+2.ONPGÀ3.Lysine decarboxylase+4.Ornithine+5.Urease+6.Phenylalanine deaminationÀ7.Nitrate reductionÀ8.H2S production+9.Citrate utilization+10.Methyl redÀ11.Voges ProskauresÀ12.IndoleÀ13.Malonate+14.Esculin+15.ArabinoseÀ16.Xylose+17.AdonitolÀ18.Rhamnose+19.Cellobiose+20.Melibiose+21.Saccharose+22.Raffinose+23.TrehaloseÀ24.Glucose+ctoseÀONPG–ortho nitro phenylene b-galactopyranoside.P.Arulazhagan,N.Vasudevan/Marine Pollution Bulletin62(2011)388–394393to degrade PAHs.Therefore,the addition of nutrients(nitrogen and phosphate)for the degradation of PAHs under saline condi-tions was detailed.With this addition of nutrients,Ochrobactrum sp.VA1was also found to be potent in the degradation of PAHs in petroleum wastewater.The strain was capable of reducing66% of COD and effectively degraded PAHs(fluorene and benzo(e)pyr-ene)in the petroleum wastewater.Based on the phylogenetic analysis and phenotypic characterization,it was confirmed that the PAH degrading isolate VA1belongs to the Ochrobactrum sp. According to the earlier reports by Holmes et al.(1988),El-Sayed et al.(2003),Lebuhn et al.(2000),and Trujillo et al.(2005),the phenotypic results in the present study confirmed that the stain belongs to Ochrobactrum sp.The strain VA2was identified as Enterobacter cloacae,based on the phenotypic characteristics re-ported by Saadoun(2002),who isolated Enterobacter cloacae from soil capable of growing on diesel.The bacterial strain VA3was identified as Stenotrophomonas maltophilia,based on the pheno-typic characteristics reported by Juhasz et al.(2000),who iso-lated the Stenotrophomonas maltophilia strain VUN10,003from soil near port Melbourne,Australia for its potential in the degra-dation of HMW PAHs.The Ochrobactrum strain isolated by El-Sayed et al.(2003)was capable of degrading phenol(100ppm). The gene sequences of the strains were submitted to GenBank, and the respective accession numbers are EU722312(Ochrobac-trum sp.VA1),EU722313(Enterobacter cloacae VA2)and EU722314(Stenotrophomonas maltophilia VA3)(Arulazhagan and Vasudevan,2009).From the present study,it may be concluded that the Ochrobac-trum sp.strain VA1(EU722312)is halotolerant and capable of degrading both LMW and HMW PAHs under saline conditions. 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中国科学院研究生院硕士学位论文高分子量多环芳烃降解菌群驯化过程中的演变及降解特性姓名：***申请学位级别：硕士专业：微生物学指导教师：***2012-05摘要摘要将焦化厂污染土壤样品，在以芘、荧葸作为底物的矿物盐（ＭＳＭ）培养基中富集驯化，得到两个能够降解高分子量多环芳烃的混合菌群（芘驯化菌群Ｐ，荧葸驯化菌群Ｆ）。

通过培养和非培养方法进行了菌群多样性调查。

通过构建１６ＳｒＲＮＡ基因文库的方法，分析焦化厂原始土壤中菌群组成和混合菌群转接３次（Ｐ．３，Ｆ．３）、６次（Ｐ．６，Ｆ．６）和９次（Ｐ．９，Ｆ．９）后的组成变化。

系统进化分析表明（１）变形菌纲是土壤原样及Ｐ．３，Ｆ．３混合菌群中的主要类群，分别占１００％，８３％和７１％：（２）与原土壤样品相似，Ｐ．３中ｙ－Ｐｒｏｔｅｏｂａｃｔｅｒｉａ（占变形菌门的７７％）中假单胞菌属的菌依然占主导地位，但由于菌群的多样性增加，假单胞菌属所占比例减少。

传代过程中，菌群组成发生改变，随着传代次数的增加，菌群的多样性进一步增加，），－Ｐｒｏｔｅｏｂａｃｔｅｒｉａ的比例在混合菌群中的比例下降（Ｐ．６中占变形菌门的３３％，Ｐ．９中占变形菌门的１８％），而１３－Ｐｒｏｔｅｏｂａｃｔｅｒｉａ在混合菌群中的比例上升（Ｐ．６中占变形菌门的３６％，Ｐ．９中占变形菌门的５５％）；Ｆ菌群中ＩＢ－Ｐｒｏｔｅｏｂａｃｔｅｒｉａ的比例也逐渐上升（Ｆ．３，３０％；Ｆ．６，６３％；Ｆ．９，７６％），而０‘．Ｐｒｏｔｅｏｂａｃｔｅｒｉａ和ｙ－Ｐｒｏｔｅｏｂａｃｔｅｒｉａ的组成比例逐渐下降（Ｆ．３，４１％，２７％；Ｆ．６，２０％，１５％：Ｆ．９，１３％，１０％。

）。

通过培养，从混合菌群中分离纯化得到１７株菌，系统进化分析表明分别属于Ａｃｈｒｏｍｏｂａｃｔｅｒ，Ｂａｃｉｌｌｕｓ，Ｍｉｃｒｏｂａｃｔｅｒｉｕｍ，Ａｒｔｈｒｏｂａｃｔｅｒ，Ｅｘｉｇｕｏｂａｃｔｅｒｉｕｍ，Ａｌｃａｌｉｇｅｎｅｓ和Ｐａｒａｐｅｄｏｂａｃｔｅｒ属。