一株耐酸性反硝化细菌的分离鉴定及反硝化特性研究

发布时间：2018-03-12 23:05

  本文选题：耐酸反硝化细菌　切入点：反硝化特性　出处：《东华理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：酸法地浸采铀过程中,常采用硝酸铵为淋洗剂,致使硝酸盐进入到含矿含水层,造成地浸采矿退役后地下水环境硝酸盐的超标。微生物反硝化脱氮法因其效率高、成本低常被广泛应用于处理含氮废水。但反硝化细菌的作用条件一般为中性或偏碱性,酸性或偏酸性条件下的较为少见。本论文针对酸性矿山地下水中pH低、硝酸盐含量高等问题,采用选择性培养基,从新疆某铀矿山含矿含水层地下水样品中富集、分离和驯化耐酸性硝酸盐还原菌(Denitrifying Bacteria,DNB);采用稀释涂布和平板划线法纯化菌种;采用Biolog生理生化试验和16S rDNA基因序列分析鉴定所获得菌株(G16X-D),采用兼性厌氧液体培养法研究不同初始pH值、温度、碳源、C/N等因素对G16X-D生长和反硝化特性的影响,优化其反硝化培养条件;分析不同初始NO_3~--N浓度与G16X-D反硝化速率的动力学关系、考察重金属离子(Zn~(2+)、Cd~(2+)、U~(6+))对其去除NO_3~--N的影响。此外,在优化的环境条件下研究了该G16X-D对微生物浸铀浸出液中NO_3~--N去除的效果,为今后的应用提供理论依据。本文主要获得以下研究结果:通过富集分离与驯化,得到一株耐酸性反硝化细菌G16X-D,形态学观察、Biolog生理生化试验以及16S rDNA基因序列分析鉴定结果表明,菌株G16X-D属于铜绿假单胞菌(Pseudomonas aeruginosa)。影响因素试验结果表明,pH4.50的条件下,菌株G16X-D具有较高的反硝化能力,反硝化作用主要发生在对数生长期。菌株G16X-D优化的反硝化培养条件为:最适碳源为葡萄糖,C/N=3.0,温度为35℃,此条件下72 h内初始NO_3~--N浓度为160mg·L~(-1)的还原率达到92.74%,比生长速率μ=0.0472 h~(-1),最短倍增时间t_d=14.68 h。菌株G16X-D的最适pH值为5.77~7.70。动力学研究表明,NO_3~--N浓度S=350.84 mg·L~(-1)时,其最大反硝化速率V_(max)=2.99mg·L~(-1)·h~(-1),半饱和常数K_m=162.50 mg·L~(-1),抑制常数K_i=757.47 mg·L~(-1)。Cd~(2+)对菌株的反硝化作用具有抑制作用,浓度越高抑制性越强;U~(6+)浓度在7.50mg·L~(-1)以下时,促进反硝化作用的进行,大于7.50 mg·L~(-1)时,具有抑制作用;少量的锌离子有助于反硝化作用的进行,50 mg·L~(-1)以上则会有抑制作用;3种重金属离子对G16X-D的毒性大小顺序为Cd~(2+)U~(6+)Zn~(2+)。在优化的反硝化培养条件下,以铀矿石微生物浸出液为试验原水样,未稀释的试验原水样对G16X-D的反硝化作用具有强烈的抑制性;G16X-D以1%接种于在初始NO_3~--N浓度分别为86.52 mg·L~(-1)和163.52 mg·L~(-1)的试验水样中,培养8 d后,NO_3~--N去除率分别为64.4%和41.4%;试验废水中最佳接种量为7.5%时,初始浓度为160mg·L~(-1)的NO_3~--N经96 h去除率可达91.2%。G16X-D对pH4.50的偏酸性矿山废水中NO_3~--N的去除取得了良好的效果。
[Abstract]:In the process of in-situ leaching of uranium by acid method, ammonium nitrate is often used as leaching agent, which causes nitrate to enter the ore-bearing aquifer, which results in the excess of nitrate in groundwater environment after the decommissioning of in-situ leaching and mining. The microbial denitrification and denitrification process has high efficiency. Low cost is widely used in the treatment of nitrogen-containing wastewater. However, denitrifying bacteria are generally neutral or alkaline, and rare under acidic or acidic conditions. In order to solve the problem of high nitrate content, selective culture medium was used to enrich and domesticate the acid tolerant nitrate reducing bacteria (Denitrifying Bacteriae DNBBN) from the groundwater samples of a uranium ore bearing aquifer in Xinjiang, and the bacteria were purified by dilution coating and plate marking. The strain G16X-DX was identified by Biolog physiological and biochemical test and 16s rDNA gene sequence analysis. The effects of different initial pH, temperature and carbon source C / N on the growth and denitrification of G16X-D were studied by facultative anaerobic liquid culture. The conditions of denitrification culture were optimized, the kinetic relationship between different initial NO_3~--N concentrations and the denitrification rate of G16X-D was analyzed, and the effect of heavy metal ions on NO_3~--N removal was investigated. The effect of G16X-D on the removal of NO_3~--N from microorganism leaching uranium leachate was studied under the optimized environmental conditions, which provided theoretical basis for the future application. The following results are obtained in this paper: enrichment, separation and acclimation. A strain of acid tolerant denitrifying bacteria G16X-Dwas obtained. The physiological and biochemical tests of Biolog and the sequence analysis of 16s rDNA gene showed that the strain G16X-D belonged to Pseudomonas aeruginosa. Strain G16X-D had high denitrification ability, and denitrification mainly occurred in logarithmic growth period. The optimum denitrification conditions of strain G16X-D were as follows: glucose C / N = 3.0 and temperature 35 鈩，

本文编号：1603701