降酚微生物分子检测技术优化及在含酚工业废水治理中的应用

发布时间：2018-02-13 13:22

  本文关键词： 降酚微生物 引物优化 微生物群落结构 降解特性　出处：《湖南农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：酚类化合物作为重要的化工原料与副产物,广泛存在于工业废水之中,造成的环境污染负荷日益剧增,并逐渐成为当前水体的主要污染物之一。在含酚废水的众多处理方法中,由于成本低、效率高、操作简单、无二次污染等特点,生物法逐渐成为治理含酚工业废水的主要方法。为了进一步提高生物法的效率和速度,本研究在优化降酚微生物分子检测技术的基础上,研究含酚工业废水对微生物群落结构的影响,并筛选出高浓度高效降酚菌,深入研究其降解特性、影响因素,从而为通过定向调控或添加外源降酚微生物,提高含酚工业废水治理效率提供理论依据。主要结论如下：(1)根据已知的降酚微生物16S rDNA基因和苯酚羟化酶基因,通过生物信息学方法设计出4对降酚微生物16S rDNA引物和3对苯酚羟化酶基因的引物,并以江西景德镇某焦化厂活性污泥为对象,分析了各对引物的有效检出率,从而筛选出最优引物对。研究发现,苯酚羟化酶基因的最优引物对是M2; 16S rDNA的最优引物是M7。(2)通过高通量测序,研究了江西景德镇某焦化厂厌氧活性污泥和好氧活性污泥的微生物群落结构及季节对微生物群落结构的影响。研究发现焦化厂活性污泥中微生物菌群丰度相对较低,但多样性较高。活性污泥中的微生物可分16个分类单元,其中变形菌门为优势菌落,占48%,且随着气温升高而降低。从活性污泥中微生物的种属来看,Diaphorobacter sp.和Ottowia sp.为优势菌,分别占20.81%和13.48%。从不同季节来看,同季节的好氧活性污泥与厌氧活性污泥在结构上基本一致,不同季节优势菌群不同。其中2月和8月的优势菌群为Diaphorobacter sp.,占29.38%,而5月和11月的优势菌群为Ottowia sp.,占17.06%。(3)通过富集培养分离纯化到5株高浓度高效降酚菌,分别命名为pd-A、pd-B、 pd-C、pd-D、pd-E,耐酚能力均在2000 mg/L以上,其中pd-D降酚效果最佳。当苯酚初始浓度为1000 mg/L时,48 h内pd-D菌株的苯酚降解率达72.11%。研究发现,pd-D菌株的最佳降解条件为：初始酚浓度1000mg/L、30℃、pH7.0。在该条件下,96h内pd-D菌株能将苯酚完全降解,且COD去除率大于80%。(4)研究了以pd-D菌株为阳极催化剂,利用微生物燃料电池处理苯酚废水的新模式。研究表明,当在苯酚浓度为1000 mg/L, pd-D菌株的产电周期约7 d,产电电压达0.5 V,COD去除明显升高,苯酚降解时间缩短了24 h。(5)经形态学观察、生理生化实验及16S rDNA基因序列比对分析,初步确定pd-D为梭状芽孢杆菌属(Clostridium sp.)成员。
[Abstract]:Phenolic compounds, as important chemical raw materials and by-products, widely exist in industrial wastewater, resulting in an increasing environmental pollution load, and gradually become one of the main pollutants in water. Because of the characteristics of low cost, high efficiency, simple operation and no secondary pollution, biological method has gradually become the main method for the treatment of industrial wastewater containing phenol. On the basis of optimizing the molecular detection technology of phenol reducing microorganism, the effect of phenol industrial wastewater on microbial community structure was studied, and the high concentration and high efficiency phenol reducing bacteria were screened out, and its degradation characteristics and influencing factors were studied deeply. It provides a theoretical basis for improving the treatment efficiency of phenol-containing industrial wastewater by regulating or adding exogenous phenol-reducing microorganisms. The main conclusions are as follows: (1) based on the known 16s rDNA gene and phenol-hydroxylase gene of phenol-reducing microorganism, Four pairs of 16s rDNA primers and three pairs of primers for phenol hydroxylase gene were designed by bioinformatics. The effective detection rate of each pair of primers was analyzed by using activated sludge of a coking plant in Jingdezhen, Jiangxi Province. The results showed that the optimal primer pair of phenol hydroxylase gene was M2, and the best primer of 16s rDNA was M7.2) by high-throughput sequencing. The microbial community structure of anaerobic activated sludge and aerobic activated sludge in a coking plant in Jingdezhen, Jiangxi Province, and the effect of season on the microbial community structure were studied. It was found that the abundance of microbial flora in activated sludge of coking plant was relatively low. But the diversity is high. The microorganism in activated sludge can be divided into 16 taxonomic units, among which Proteus is the dominant colony, accounting for 48, and decreases with the increase of temperature. From the species of microorganisms in activated sludge, Diaphorobacter sp. and Ottowia sp. are the dominant bacteria. From different seasons, the structure of aerobic activated sludge and anaerobic activated sludge in the same season was basically the same. In February and August, the dominant flora was Diaphorobacter sp., accounting for 29.38%, while in May and May, the dominant flora was Ottowia sp., which accounted for 17.06. 3) five strains of high concentration high concentration phenol reducing bacteria were isolated and purified by enrichment culture. They were named pd-An pd-B, pd-Ca-pd-Dd-Pd-E, respectively, and the phenolic tolerance was more than 2 000 mg/L. When the initial concentration of phenol was 1000mg / L, the phenol degradation rate of pd-D strain reached 72.11. The optimum degradation conditions of pd-D strain were found as follows: initial phenol concentration 1000mg / L ~ 30 鈩，

本文编号：1508260