【作者】 王春明；

【导师】 刘世贵；

【作者基本信息】 四川大学 ， 遗传学， 2007， 博士

【摘要】 多环芳烃具有致癌、致畸和致突变的特性，对生物体产生遗传毒性，而且能够通过食物链在生物体内富集，对人体具有潜在危害性。同时该类化合物还具有高度的稳定性，在环境中难以降解。随着石油开采和使用量的增加，大量的石油及其加工品进入环境，原油的采集、精炼、运输、燃烧等过程中伴随产生大量的多环芳烃，不可避免地对环境造成污染，多环芳烃的积累已经越来越严重地威胁着人类的健康，已成为世界各国共同关注的有机污染物。微生物在生物修复中具有不可忽视的作用，筛选高效功能菌株是生物修复的基础。作者以多环芳烃萘、菲、蒽、芘为筛选培养基，从石油污染土壤和油田外排水中筛选出18株多环芳烃降解菌，通过降解过程中细菌的生长现象和降解能力等复筛出4株多环芳烃高效降解茵。为确定这些菌株的分类地位，对这四株细菌进行了形态学观察、生理生化指标测定及分子生物学鉴定。16S rDNA序列分析显示，这四株细菌分别与Microbacterium、Cellulosimicrobium、Chelatococcus和Sphingopyxis四个属有较高的序列同源性，同源性分别为100％，97．8％、98．2％和99．0％。结合表型特征和16S rDNA序列分析，用DNAMAN构建系统净化树，并用Bootstraping法对其评价，初步将这四株多环芳烃降解菌分别归为属，命名为Microbacterium sp．3-28、Cellulosimicrobium sp．NF、Chelatococcus sp．EW和Sphingopyxis sp．EY。本文系统研究了单菌株3-28、NF、EW和EY对PAHs的降解能力和环境因子pH值、盐度、温度及振荡条件对这些多环芳烃降解菌的生长活性及其对PAHs降解的影响以及其对不同浓度PAHs的降解能力。结果表明，这些多环芳烃菌株能很好地利用不同分子量的PAHs-萘、菲、蒽、芘作为其生长的碳源和能源，经过一段时间的培养，3-28可分别将培养液中的萘、菲、蒽、芘降解100％、100％、97.54％和90.20％，NF、EW、EY可分别将培养液中的萘、菲、蒽、芘降解100％、100％、96.22％和87.00％，100％、100％、88.08％和69.20％以及100％、100％、94.34％和84.20％：单菌株作用于萘、菲、蒽、芘混合液中物质，3-28对混合液中萘、菲、蒽、芘的降解率分别是100.0％、100.0％、94.38％和86.40％，NF、EW、EY对混合液中萘、菲、的降解率分别是100.0％、100.0％、91.52％和77.40％，100.0％、100.0％、79.02％和61.20％，以及100.0％、100.0％、84.74％和72.60％。相同的培养时间多底物培养液中的菌群浓度高于单底物系。这些多环芳烃降解菌生长的最适pH值为7.0-8.0，在pH6.0-9.0的范围内可以良好的生长，培养液中pH值低于6.0和超过9.0都会抑制微生物的生长；在盐度为10-30的培养液中可以良好地生长，在盐度为40-50的培养液中生长明显受到抑制。浓度较低的PAHs培养中，降解菌生长快，培养液中的PAHs能在短时间内被迅速降解，碳源很快消耗，微生物的生长水平不高(D_600nm=0.60左右)；浓度为250-500mg／L的萘、菲和200～400mg／L蒽、芘是细菌降解的最适浓度，萘、菲浓度为1000mg／L和蒽、芘800mg／L以上会抑制微生物的生长，生物降解需要更长的时间。这4株细菌在静置缺氧条件下对PAHs仍具有较高的降解性能。为了进一步研究多环芳烃降解菌的降解途径，作者对降解过程中产生的酶和代谢产物进行了分析。研究表明，在多环芳烃降解过程中能够检测到脱氢酶和多酚氧化酶，这2种酶体系在多环芳烃降解过程中具有一定的作用，但对于多环芳烃的降解而言，不是主要的酶体系。双加氧酶在多环芳烃的开环过程中起主要作用，也就是说，本研究中菲、蒽、芘的代谢都是双加氧酶体系作用的结果，代谢产物中均检测到二羟基-PAHs的产生。微生物采油技术（MEOR）的主要机理之一就是利用微生物降解原油中的胶质、沥青质等重质组分，降低原油粘度和凝固点，以达到改善原油物化性质的目的。从多环芳烃降解菌中选育出一株能有效降解稠油中重质组分的菌种3-28。使用该菌株对渤海和新疆等油田的稠油进行微生物降解实验，分析了细菌降解对原油粘度等物理特征的改变以及饱和烃、芳烃以及胶质、沥青质各组分在原油中相对含量和内部组成的影响。结果表明，细菌作用引起原油性质发生明显变化，粘度降低20％以上，凝固点降低近10℃，原油中沥青质含量降低30％以上，其组成也发生了一定的变化，原油饱和烃轻重组分比值变大，大大改善了原油的物化性质。更多还原

【Abstract】 Polycyclic aromatic hydrocarbons (PAHs) compose a large and heterogeneousgroup of organic contaminants. It contains compounds with two to six rings, fused ina linear, angular, or cluster arrangement. PAHs are formed and emitted as a result ofthe incomplete combustion of organic material. They may be also released into theenvironment through the disposal of coal tars and other coal processing wastes,petroleum sludges, asphalts, creosote and other wood preservative wastes, chemicalwastes, and soots. The fate of these compounds and their degradation products in theenvironment is of concern due to their toxic, mutagenic, and carcinogenic properties.Sixteen PAHs are listed as priority pollutants by the US Environmental ProtectionAgency.Microbes play an important role in bio-remediation technologies, so that it is abasic study to find out efficiently functional strains for environmentalbio-remediation. In this paper four PAHs-biodegrading strains were been screenedwith a selective culture medium of PAHs from oily wastewater andoil-contamination soil. The four strains were been identified through morphological,physiological and biochemical characteristics investigation and gene analysis. The16S rDNA of the strains were amplified by PCR and was sequenced 581, 582, 1209,1230 bp respectively, and then were compared with those available in the GenBank databases. The results show that 3-28 16S rDNA sequence is most similar to that ofMicrobacterium esteraromaticum, NF similar to Cellulosimicrobium cellulans, EWsimiar to Chelatococcus asacharovorans, EY similar to Sphingopyxis terrae, and thesequence similarities are 100%, 97.8%, 98.2% and 99.0%, respectively. The fourstrains belong to Microbacterium, Cellulosimicrobium, Chelatococcus andSphingopyxis sp. The phylogenetic trees including the four strains were derivedwith DNAMAN and analyzed with Bootstraping. The results demonstrate that 3-28,NF, EW and EY strains were close consanguinity to Microbacterium sp.,Cellulosimicrobium sp., Chelatococcus sp. and Sphingopyxis sp.Enzymes and their metabolic products in PAHs bidegradation process havebeen analyzed to determine the possible reduction trajectories of PAH-biodegradingstrains. The results show that the activities of dehydrogenase and polyphenoloxidase can be examined during the reduction process of PAHs, and the reductionprocess is influenced somehow by the two enzymatic systems. However,dehydrogenase and polyphenol oxidase seems not to be the principal enzymaticsystems in the PAHs biodegradation. In fact, the results indicate that bioxygenasesystem plays an important role in the ring cleavage process, i.e., all of metabolismsof phenanthrene, anthracene a pyrene result from bioxygenase system mostly, anddihydroxy-PAHs can also be detected in all of their metabolite.One of the mechanisms of microbial enhanced oil recovery (MEOR) is toutilize microbes to biodegrade the heavy fractions of crude oil such as the resins andasphaltene and reduce its viscidity and freezing point in order to improve thephysicochemical characteristics of the thicked oil. A strain of Microbacterium sp.was screened to biodegrade the asphaltene fraction of the crude oil from Bohai oilfield and Xinjiang oil field effectively and the change of the components among thesaturated fraction, aromatic fraction, resins and asphaltene fraction and theircomposition were analyzed. The results show that the physicochemicalcharacteristics of the crude oil have been changed obviously under the effect ofMicrobacterium that the contents of the resin and asphaltene fraction decrease above 9% and the saturated and aromatic fraction increase to a certain extent and theviscidity and the freezing point reduce 15% and 20% respectively.更多还原