【作者】 刘彬彬；

【导师】 赵立平；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 微生物分子生态学发展的一个重要的方面是对尚未得到纯培养的微生物的分类地位与其功能进行关联,从而得到这些微生物的功能信息。随着分子生物学的发展,通过对16S rRNA基因等具有进化标记的基因的克隆和测序,越来越多的不可培养的微生物被发现,同时,大量研究发现微生物区系的组成和功能通常会随着环境的变化而同时发生变化。我们可以根据这一模式深入了解微生物区系的结构与功能动态变化情况,并鉴定主要的功能菌。随着人们环保意识增强,可持续发展观念的理解深入,人们对难降解有机物在环境中的降解、迁移及危害越来越关注并开发出了很多高效的去除这些有机物的方法。然而,在很多生物处理的工艺中,人们对处理过程的生物降解机理并不是很清楚,对处理过程的微生物的区系结构及主要的功能菌也知之甚少。在本研究中,首先对处理含有高浓度喹啉废水的反应器进行了解析,喹啉是一种主要的含氮杂环化合物,是存在于一些水体和某些土壤中的主要的污染有机物之一,在工业废水处理过程中,喹啉是一种常见的难降解的复杂有机物之一。实验中反硝化生物膜反应器和一个平行的厌氧反应器是从同一份种子污泥出发驯化建立的。两个反应器的其它条件完全一致,只是在反硝化反应器中加入了一定量的硝酸根。经过六个星期的驯化,两个反应器均达到了稳定运行状态。经过五天的连续监测,在反硝化反应器中喹啉的平均去除率为90.2%,COD的平均去除率为81.1%,而厌氧反应器中为喹啉平均去除率为53.6%,COD的平均去除率60.4%。为了找出反硝化条件下及厌氧条件下的主要功能菌,我们对种子污泥、反硝化反应器和厌氧反应器中生物膜样品的微生物种群进行了对比分析。首先通过16S rRNA v3区基因的PCR-DGGE分析,对种子污泥和两个反应器中的微生物组成进行了对比。通过对比发现样品的微生物区系组成有很大的差异,表明种子污泥在含喹啉的合成废水中进行驯化时,其微生物组成发生了很大的变化,其中那些能在反硝化和厌氧条件下降解喹啉的微生物被富集。对其中的优势条带进行割胶,克隆测序分析表明在反硝化反应器中的最优势的条带所代表的序列与Thauera和Azoarcus属的微生物具有很近的亲缘关系。而在厌氧反应器中变形菌纲Gamma亚纲与Desulfobacter postgatei种的微生物是优势的条带。同时我们还构建了16S rDNA的全长序列的克隆文库。根据Kemp,P.F.等人的方法对所建文库的有效性进行了验证,结果表明三个文库均已足够大。通过两个统计学参数Shannon Wiener index (H)和reciprocal of Simpson’s index (1/D)分析表明三个文库的多样性顺序为SS>DR>AR。说明在驯化过程中微生物的多样性降低了。通过对克隆文库进行进化地位分析表明,在反硝化反应器中,所有的Phylotype均属于变形菌纲Beta亚纲,而种子污泥中的克隆分属于8个纲。其中反硝化反应器中73.6%的克隆与Thauera和Azoarcus属的微生物具有最高的同源性。而在种子污泥中,与这两个属同源性最高的微生物只有4%,在厌氧反应器的克隆文库中没有发现属于这两个属的微生物。厌氧反应器中变形菌纲Gamma亚纲与Desulfobacter postgatei种的微生物显著增加,这种动态变化表明这些细菌可能是在反硝化和厌氧条件下对喹啉的降解起关键作用的微生物。为了进一步定量的验证所得到的结果,我们应用实时荧光定量PCR(Real-time quantitative PCR, RTQ-PCR)的方法,选取了专一性的针对Thauera和Azoarcus属微生物的引物。对降解喹啉的反硝化反应器的种子污泥样品和稳定运行后的生物膜样品进行了定量分析。结果表明在每微克湿重的污泥中待检测的基因片段的拷贝数在种子污泥中为为3.70±0.16×105,在反硝化反应器中为3.69±0.97×106。这表明在驯化过程中,Thauera和Azoarcus属微生物的细胞数增加了一个数量级。也进一步表明了,这两个属的微生物很有可能是在反硝化条件下的去除复杂有机物的重要的功能菌。用类似的方法我们分析了以焦化废水为底物的两个反应器,系统运行稳定后,GC/MS分析表明,苯酚、甲苯、嘧啶在DR中的去除效率较高,而咔唑和苯胺在AR中具有更高的去除效率。反硝化装置中的平均COD去除率为51.4%,厌氧反应器中为18.3%。PCR-DGGE分析和克隆文库分析都表明,两个反应器中发生了不同的微生物区系变化,在反硝化反应器内,Thauera属的微生物发展成为优势菌群。这与喹啉部分的实验结果吻合。在厌氧反应器中最优势的微生物与一个未鉴定的硫酸盐还原菌具有最高的同源性。在本研究中,通过对系统中的微生物区系结构进行动态监测,与反应器的整体功能变化相关联,在分子水平上鉴定了厌氧和反硝化条件下降解喹啉及焦化废水的优势功能菌类群。更多还原

【Abstract】 An important object of microbial ecology is to linking bacterial phylogenetics with the biological functionality. With the development of the molecular techniques, more and more 16S rRNA genes were sequenced and submitted to the gene sequence databases. These sequences provide enormous information about the uncultured bacteria. But the functions and the environmental roles of most of these bacteria are unknown. At the same time, many studies are finding that the microbial community composition and function often shift together in response to environmental stimuli. Utilizing this paradigm, functional important strains can be identified through functionally monitoring and dissecting the microbial communities.In this study, the biofilm reactors treating quinoline-containing wastewater were set up using the same seed sludge. The microbial communities were analyzed by the molecular techniques. An 18L tank was used as the quinoline reactor and the hydraulic retention time (HRT) was kept at 24h. All conditions were same except the nitrate was added to the denitrifying reactor. After 6 weeks acclimations, the reactor reached the stable stage. The average quinoline removal efficiency was 90.2% and the average COD removal efficiency was 81.1% in the denitrifying reactor. In the anaerobic reactor the average quinoline removal efficiency was 53.6% and the average COD removal efficiency was 60.4% in the denitrifying reactor. The DGGE profiling indicated that dramatic microbial community structure shift was occurred in the two reactors. The dominant bands in the gel were excised and sequenced, the most dominant band in the denitrifying reactor was affiliated with the genera Thauera and Azoarcus. While in the anaerobic reactor Gamma Proteobacteria and Desulfobacter postgatei were the most dominant band sequences. 16S rRNA gene libraries were constructed for the three samples. Both of the two estimators reached an asymptote for the libraries. This indicated that the libraries were large enough to yield stable phylotype richness estimates. Two statistic indices, the Shannon Wiener index (H) and the reciprocal of Simpson’s index (1/D), were calculated for the three libraries and indicated that the order of the phylotype richness is SS>DR>AR. Phylogenetic analyses indicated that all clones from the DR belonged to theβsubclass of Proteobacteria, while eight classes were identified in the SS. Clones affiliated with the genera Thauera and Azoarcus were 74% in DR and only 4% in SS. The Gamma Proteobacteria and Desulfobacter postgatei become dominant during the acclimation period in the anaerobic reactor.In order to verify the population level changes of the microorganisms belong to genus Thauera and Azoarcus. Real-time quantitative PCR (RTQ-PCR) was performed to the seeding sludge sample and the biofilm sample in the quinoline-containing denitrifying reactor. The copy number of a Thauera and Azoarcus specific gene fragment were 3.70±0.16×105 and 3.69±0.97×106 per microgram of wet weight of biofilm samples in SS and DR, respectively. This indicated that the cell number of the genera Thauera and Azoarcus increased by about one order of magnitude during the acclimation. The greater abundance of Thauera and Azoarcus in association with higher efficiency after acclimation confirmed that these phylotypes might play an important role for quinoline and COD removal under denitrifying conditions.Using the similar methods, two reactors treating coking wastewater were analyzed. The COD removal efficiencies in the anaerobic and the denitrifying reactors were 18.3% and 51.4%, respectively. GC/MS analysis showed that phenol, toluene and pyridine were more efficiently removed in denitrifying reactor than in anaerobic reactor, while carbazole and aniline had higher removal efficiency in anaerobic reactor. DGGE and 16S rDNA library analysis indicated distinct microbial communities formed under the anaerobic and denitrifying conditions. Phylogenetic analysis of the 16S rRNA gene libraries indicated that the most dominant population is Thauera in denitrifying reactor and an unclassified sulfate-reducing bacterium in anaerobic reactor.In this study,the microbial community structure changes were monitored by the molecular techniques, through linking the bacterial phylogenetics with the overall functionality of the reactors, the dominant functional members were identified.更多还原