盐湖环境中苯酚降解菌的分离与鉴定及其降解特性

发布时间：2018-09-02 07:30

【摘要】：自然界中存在广泛的微生物资源,其生存环境各异,且有丰富的基因多样性。从自然界中广泛分离污染物分解菌,研究其污染物降解活性和基因特征之间的关系是从分子水平揭示微生物的污染物降解活性及其影响因素,从而研制出高效污染物降解菌的最有效方法。本文旨在揭示自然高盐环境中耐盐苯酚降解菌的生物多样性、代谢多样性以及对苯酚的降解活性,为今后基因序列和苯酚降解活性之间的关系研究提供参考和依据。本文以内蒙古地区额济淖尔盐湖、吉兰泰盐湖以及查干淖尔盐湖为研究对象,驯化分离了耐盐苯酚降解菌。研究发现,从额济淖尔湖驯化培养出的菌群能够显著降低培养液中的苯酚浓度。应用以苯酚为唯一碳源的固体培养基分离出了该菌群中的微生物,并挑选其中两株进行了16SrDNA法鉴定。结果表明挑选出的两株苯酚降解菌分别属于丙酸杆菌属和盐单胞菌属。利用苯酚单加氧酶基因、邻苯二酚1,2双加氧酶基因以及邻苯二酚2,3双加氧酶基因的简并引物检测了苯酚降解过程中的关键酶的编码基因。在属于丙酸杆菌属的苯酚降解菌中检测到了苯酚单加氧酶基因和邻苯二酚1,2双加氧酶基因,说明该菌可能利用苯酚单加氧酶将苯酚转化为邻苯二酚,并将邻苯二酚进一步分解为粘康酸。虽然对PCR条件进行了多次优化,在属于盐单胞菌的苯酚降解菌中未能检测到任何功能基因,说明该菌降解基因序列与目前发现的降解基因序列有较大的差异。应用含200 mg／L苯酚,10%盐度的培养液初步试验了分离出的菌株对苯酚的降解活性。经一轮的驯化培养后,丙酸杆菌能够在12天之内降解99.5%的苯酚,盐单胞菌在12天之内降解99.0%的苯酚。研究发现,50mg／L的酵母提取物对丙酸杆菌的苯酚降解活性没有影响,而不存在酵母提取物时盐单胞菌对苯酚的降解活性显著降低。本文对于自然环境中耐盐苯酚降解菌多样性和降解基因多样性了解以及污染物处理工程菌的开发利用具有重要的科研价值。
[Abstract]:There are a wide range of microbial resources in nature, their living environment is different, and there is abundant genetic diversity. Pollutant decomposing bacteria were widely isolated from nature. The relationship between their pollutant degradation activity and genetic characteristics was revealed at molecular level. Therefore, the most effective method for the degradation of pollutants was developed. The purpose of this paper is to reveal the biological diversity, metabolic diversity and degradation activity of phenol-tolerant bacteria in natural high-salt environment, and to provide a reference and basis for the study of the relationship between gene sequence and phenol degradation activity in the future. In this paper, salt-tolerant phenol degrading bacteria were domesticated and isolated from Ejinaoer Salt Lake, Jilantai Salt Lake and Chagannur Salt Lake in Inner Mongolia. It was found that the microbial communities domesticated from Eziannur Lake could significantly reduce the concentration of phenol in the culture medium. The microbes in the microflora were isolated in solid medium with phenol as the sole carbon source and two of them were selected for identification by 16SrDNA. The results showed that the two phenol-degrading bacteria belonged to Propionibacterium and Salmonella respectively. The degenerate primers of phenol monooxygenase gene, catechol 1 + 2 dioxygenase gene and catechol 2 + 3 double oxygenase gene were used to detect the key enzyme coding genes in the process of phenol degradation. Phenol monooxygenase gene and catechol 1 / 2 dioxygenase gene were detected in phenol-degrading bacteria belonging to Propionibacterium, indicating that phenol could be transformed into catechol by phenol monooxygenase. The catechol was further decomposed into mucoronic acid. Although the PCR conditions were optimized many times, no functional gene was detected in phenol degrading bacteria belonging to Salmonella, which indicated that the degradation gene sequence of this strain was different from that of the current one. The degradation activity of phenol was preliminarily tested in the culture medium containing 10% salt of 200 mg/L phenol. After one round of acclimation, propionic acid bacillus could degrade 99.5% phenol within 12 days, and Salmonella could degrade 99.0% phenol within 12 days. It was found that the yeast extract of 50 mg / L had no effect on phenol degradation activity of Propionibacterium propioniae, but the phenol degradation activity of Salmonella Salmonella was significantly decreased when there was no yeast extract. This paper has important scientific research value for understanding the diversity of phenol-tolerant bacteria and degrading gene diversity in natural environment and for the development and utilization of pollutant treatment engineering bacteria.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X172;X703

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