【作者】 曲媛媛；

【导师】 周集体；

【作者基本信息】 大连理工大学 ， 环境工程， 2005， 博士

【摘要】 本论文的研究目的是考察1株新分离的鞘氨醇单胞菌（Sphingomonas xenophaga QYY）对蒽醌染料中间体溴氨酸的降解特性,并对菌株进行生理生化及16S rDNA分子鉴定,考察其生长及降解溴氨酸的特性,推测出该菌株降解溴氨酸的途径;同时,利用该菌株作为生物强化制剂对溴氨酸进行强化降解研究,通过现代分子生态技术揭示强化系统中微生物群落动态及结构变化。 菌株QYY是高效的溴氨酸降解菌,结合中国科学院微生物所的生理生化鉴定及本实验室对菌株QYY的形态观察、16S rDNA序列分析,菌株QYY归属于变形细菌α亚类,Sphingomonas xenophaga菌种。菌株QYY已作为专利菌种保存于中国普通微生物菌种保藏中心,注册编号为:CGMCC No.1172。菌株QYY与S. xenophaga的模式菌株DSM6383^T的16S rDNA序列同源性高达99%,其16S rDNA序列登录GenBank,注册号为:AY611716。采用细菌中大质粒提取方法,从菌株QYY中分离得到1个非功能野生型质粒pSx-QYY（5683bp）。该质粒包括4个开放阅读框架,其中包含编码大肠杆菌复制子蛋白的基因,质粒序列登录GenBank,注册号为AY787146。 考察了菌株QYY生长及降解溴氨酸的基本特性,为生物强化实验奠定基础。菌株QYY对链霉素具有抗性,对其它所试抗生素均无抗性。菌株QYY最适的生长与降解条件为:温度30℃,pH=7.0,摇床转速为150r/min,接种量8%。菌株在以溴氨酸为唯一碳氮源培养基中可耐受的溴氨酸浓度为1000mg/L。优化了菌株QYY的休眠细胞降解溴氨酸的条件:温度30℃,pH=8.5,摇床转速为150r/min,并考察了休眠细胞对不同浓度溴氨酸的降解情况。确定了用海藻酸钙包埋菌株QYY的固定化方法,考察了固定化细胞对溴氨酸的最佳降解条件:摇床转速150r/min,温度30℃,pH 4.0-8.0。 利用紫外-可见光谱、离子色谱、液相色谱-质谱、核磁共振等先进的分析手段对菌株QYY降解溴氨酸的机理进行了研究,鉴定了降解的主要终产物,推测了可能的代谢途径。溴氨酸在菌株QYY作用下,总有机碳、COD_Cr的去除率分别为52.58%及56.79%,蒽醌环结构断裂。推测该降解过程是在环羟化双加氧酶与环断裂双加氧酶作用下发生的,主要的降解终产物为2-氨基-3-羟基-5-溴苯磺酸和2-氨基-4-羟基-5-溴苯磺酸。更多还原

【Abstract】 The purpose of this dissertation is to investigate the characteristics of a newly isolated Sphingomonas xenophaga QYY with the ability to degrade bromoamine acid (BAA). The physio-biochemical characterization and 16S rDNA molecular identification were carried out. The characteristics of strain QYY growth and BAA degradation were also investigated and the possible metabolic pathway of BAA degradation by strain QYY was presumed. Meanwhile, bioaugmentation of BAA degradation with strain QYY as augmented inoculums was studied, and the dynamic and structural changes of microbial community in augmented system were revealved by modem molecular ecological techniques.Strain QYY is an efficient BAA-degrading strain. It was identified as Sphingomonas xenophaga belonging to Proteobacteria a subclass based on morphological and physio-biochemical characteristics and 16S rDNA sequence analysis. Strain QYY is deposited as a patent strain in China General Microorganism Culture Center with the accession number CGMCC 1172. The 16S rDNA sequence of strain QYY shared high similarity (99%) with that of type strain DSM6383. The 16S rDNA sequence of strain QYY was submitted to GenBank with the number AY611716. A novel non-function wide plasmid (pSx-QYY) about 5683bp was isolated from strain QYY by the mega plasmid extraction strategy. The plasmid contains four opening reading frames (ORFs), of which contains gene encoding replication protein of E. coli. The sequence of pSx-QYY was submitted to GenBank with the accession number AY787146.The characteristics of strain QYY growth and BAA degradation were investigated with the purpose of building a foundation for bioaugmentation experiments. Strain QYY is resistant to streptomycin and sensitive to other antibiotics. The optimal conditions for both growth and degradation process are as follows: pH=7.0, temperature 30℃, shaking speed 150r/min and inoculation amount 8%. Strain QYY can tolerate up to 1000mg/L BAA. The optimal degradation conditions of resting cells were also obtained as follows: pH=8.5, temperature 30℃, shaking speed 150r/min. The entrapped immobilization method of alginate sodium was adopted. The optimal degradation conditions for immobilized cells are obtained as follows: shaking speed 150r/min, temperature 30℃, pH=4.0-8.0.The metabolic pathway of BAA degradation by strain QYY was investigated by advanced analytical instruments such as UV-Vis, Ion Chromatogram, HPLC-MASS, H-NMR, etc. The final products were identified and the possible metabolic pathway was presumed. During the更多还原