【作者】 王春艳；

【导师】 丁永生； 党宏月；

【作者基本信息】 大连海事大学 ， 环境科学， 2008， 博士

【摘要】 为了解富营养、氮素污染严重的胶州湾海域反硝化细菌群落的结构与组成,应用PCR技术为基础的基因文库方法对胶州湾沉积物反硝化细菌nirS基因的分子多样性进行研究。5个站位共筛选了331个克隆,分别属于132个不同的分类操作单元(Operational Taxonomic Units,OTUs)。各个站位OTUs个数30～44不等,显示了较高的多样性。nirS基因序列与GenBank数据库中最相似序列的相似度在62～100%。系统进化分析表明,包括波罗的海、墨西哥湾等12大海域和河口的沉积物或水体的nisS基因序列可被划分为7个簇(Marine ClusterⅠ-Ⅶ),其中MarineClusterⅦ在序列50%相似度水平上又可划分为7个亚簇。其中Marine ClusterⅦg是本研究中特有的亚簇,体现了胶州湾沉积坏境具有不同于其他海域的反硝化细菌基因序列。基于OTUs在各站位的分配和nirS基因序列系统进化关系,对各站位反硝化细菌群落结构进行Cluster Environments、Jackknife Cluster和PCA聚类,结果表明A5、Y1、D1站位反硝化细菌群落关系接近,B2、C4站位关系较远。结合环境因子聚类分析结果,揭示胶州湾沉积物反硝化细菌群落结构与沉积物粒度等多种环境因子相关。陆源污染、河流输入以及水动力条件是影响样地反硝化基因多样性的重要因素。大量抗生素药物的滥用及不合理排污,使得水环境中耐药细菌危害日益严重,为了了解胶州湾耐药细菌污染情况及耐药基因的传播途径,以胶州湾土霉素抗性菌为背景,共筛选得到具有氨苄青霉素抗性细菌66株。采用多重PCR方法,对超β-内酰胺酶(ESBLs)和质粒介导的AmpC酶进行检测,结果表明TEM,SHV是其优势基因型。16S rRNA基因序列分析显示,携带β-内酰胺酶基因的多重耐药菌中93.8%都属肠杆菌群。同时以筛选得到的32株产β-内酰胺酶多重耐药菌为对象研究其传播途径,发现68.8%的多重耐药菌携带整合子,其中Ⅰ类整合酶占有优势。整合子已成为胶州湾多重耐药细菌的主要传播机制。分析表明β-内酰胺抗性细菌空间分布差异较大。胶州湾独特的地理特点、陆源污染和人为活动是影响其耐药细菌分布的主要因素。耐药细菌及其基因分布信息可作为一种生物指示,对揭示海岸环境质量或污染源有潜在作用。为解决渤海海域石油污染日益严重的情况,从大连石化隔油池污水中分离、纯化得到一株以柴油为唯一碳源的石油降解菌DW-1。生理生化性状和Biolog分析结果表明该菌株为假单胞菌(Pseudomonas sp.)。正交实验结果表明,pH 8.5,盐度30‰,氮磷质量比10:1为该菌株最适生长条件。在油培养基浓度3g/L,菌龄为48h的细菌进行接种,平均除油率在70%左右,最高可达80.32%,降解过程中将柴油乳化为小油滴吸附在其表面。海水培养基中絮状物薄而透且形状较大,表明该菌株具有应用于海洋石油污染治理的潜质。更多还原

【Abstract】 To understand the composition and structure of denitrifying communities in the Jiaozhou Bay, the diversity of denitrifying bacterial nirS gene from the sediments of Jiaozhou Bay was investigated by using a PCR-based cloning approach. A total of 331 clones were obtained from 5 stations. Restriction analysis of cloned nirS showed a total of 132 Operational Taxonomic Units （OTUs） were obtained from all samples. The number of OTUs of each station ranged from 30 to 44. All OTUs were sequenced. The similarity of the nirS sequences to their best-match known genes retrieved from the GenBank database varied from 62 to 100. Molecular phylogenetic analysis of nirS sequences retrieved from the sediments or water column of 12 sea areas assigned the nirS sequences into 7 major clusters （Marine ClusterⅠ-Ⅶ）. Marine ClusterⅦwas divided into 7 sub-clusters with 50% similarity and Marine ClusterⅦg was specific for Jiaozhou Bay. Cluster Environment, Jackknife Cluster and Principal Coordinates Analysis （PCA） indicated that the denitrifying communities of A5, Y1 and D1 were more similar, however, B2 and C4 were not. The correlation analysis between geographic locations and its biogeochemical data indicated that multi-environment factors controlled the community structure of denitrifying bacteria. Terrestrial pollution, hydrodynamic factor and grain size of sediments should be key factors to affect the diversity of denitrifying bacteria.Environmental microbiology investigation was performed to determine the molecular diversity ofβ-lactamase genes among ampicillin-resistant bacteria from Jiaozhou Bay. With multiplex PCR,β-lactamase genes detected in 93.8% of the bacterial isolates were identified as Enterobacteriaceae. The most frequently detected gene was bla_TEM, followed by bla_SHV, Most of the isolates （68.8%） were positive for the intI1 integrase gene. Anthropogenic contamination from onshore sewage processing plants might contribute predominantly to theβ-lactamase gene reservoir in the studied coastal waters. Environmental antibiotic-resistant bacteria and resistance genes may serve as bioindicators of coastal environmental quality or biotracers of the potential contamination sources. Integron-mediated horizontal transference plays a particularly important role in the spread of multidrug resistance in aquatic environments.A crude oil degrading strain DW-1, diesel oil as sole carbon, was isolated from oil-polluted wastewater. The strain was identified as Pseudomonas sp. with physiological experiments and analysis of Biolog. Orthogonal factorial experiment showed pH 8.5, salinity 30‰and ratio of nitrogen to phosphorus 10:1 are the optimal growth conditions. Bacteria strain DW-1 cultivated for 48h were inoculated in inorganic culture medium oil with 3g/L oil. The oil degradation rate was about 70% and the highest was 80.32%. The characteristics of flocks in seawater medium indicated the strain had feasibility to disposal marine oil contamination.更多还原