【作者】 杨莉；

【导师】 杨宇；

【作者基本信息】 中南大学 ， 生物学， 2013， 硕士

【摘要】 随着现在工业的迅速发展,环境污染的问题也越来越严重,特别是含砷金矿的开采,由于砷对人及其他生物的毒害,砷污染已经是严重阻碍国民身体健康的关键,寻找一种快速,绿色的方法解决砷污染的问题是当务之急。鉴于上述背景,本研究主要致力于研究如何更好的运用生物学处理含砷废水的问题。湖南石门雄黄矿是一个具有1500年历史的老矿,具有悠久的开采历史。从该矿区尾矿堆积处积水、积水底层污泥表层及其地下400米处的矿坑水中取样,分别命名为SY,NY和XY。对三个样品利用ICP-MS进行全元素分析后,利用变性梯度凝胶电泳(DGGE)和限制性片段长度多态性(RFLP)技术,对三个样品的群落结构及其多样性进行分析。为了获得氧化As3+的混合菌群,利用含砷的培养基对样品中的菌群进行富集,多次传代使菌群稳定,并对三种混合菌群进行砷耐受能力及氧化能力的测定。同时为了获得氧化As3+的单菌,分别将混合菌种利用含砷的培养基富集,平板分离后,利用五价砷和硝酸银的颜色反应,对单菌落的五价砷的氧化能力进行检测,获得可高效氧化As3+的菌株。最后利用获得的可氧化As3+的混合菌群处理从华西集团车河选矿厂取来的含砷废水,检测其处理效果。经过DGGE与RFLP分析可知,NY的群落多样性远远的大于SY和XY。将三个样品中的典型克隆子进行测序分析,所得序列经NCBI比对后,发现与测序结果相似程度较高序列所属菌株,也大都来自于相似的环境条件。对序列进行鉴定后发现,在三个样品中检测出α-变形菌、β-变形菌、γ-变形菌及许多类型的杆菌,而且NY存在一定的假单胞菌,根据相关的研究结果,可能为具有氧化As3+能力的菌株。由于三个样品中都还有较高浓度的硫,因此也检测出了硫杆菌。与样品所处环境相关,XY处于地下400米的位置,所以出现较多类型的嗜氢菌属,而NY中检测出相对较多的硅藻门类。经过对样品中所筛选出的可氧化As3+的菌株16S rDNA序列鉴定,主要为一些变形菌和杆菌等。利用获得的单一菌株处理含砷废水,分别检测每种单菌氧化As3+的能力,都起到不错的效果。同时通过对获得的目的菌株进行氧化As3+的蛋白酶基因的克隆,发现具有氧化能力的菌株,并不一定具有三价砷氧化酶基因。将取来的样品在高于来源砷浓度的富集培养基中培养,获得了可氧化As3+的混合菌群,经检测主要为不动杆菌属、假单胞菌属及各种变形菌。在一定梯度砷浓度的培养基中分别检测这三种菌群的耐受能力,发现这三种菌群在砷浓度高达0.5g/L的条件下,虽生长较慢,但也可以达到108-109。利用这三种菌群处理一定浓度的含砷废水,废水中的As3+几乎完全被氧化,总砷的浓度在一定的时间内也降低了许多。再利用该具有氧化As3+能力的菌群,对含砷废水包括从车河选矿厂获得的废水进行处理,在10h内,废水中的总砷已基本被处理完全。更多还原

【Abstract】 Now with the rapid development of industry, the problem of environmental pollution has become serious increasingly, especially arsenic containing gold mining, because the toxicity of arsenic on human and other organisms, arsenic pollution has been seriously affect our health. Looking for a fast, green method to solve the arsenic pollution is a pressing matter at the moment. In view of the above background, this research mainly focuses on the study of the biological treatment and how to better solve the problem of waste water containing arsenic.Hunan Shimen realgar mine is an old mine with a history of1500, has a long history of mining. Three samples from the pit water of the mine tailings and bottom sludge, the last site of underground for400meters, which were named SY, NY and XY respectively. On three samples by ICP-MS elemental analysis, using denaturing gradient gel electrophoresis (DGGE) and restriction fragment length polymorphism (RFLP) technique, analysis of three samples of community structure and diversity. In order to obtain the oxidation of trivalent arsenic mixed bacteria, the bacteria in the samples were enriched using medium containing arsenic, subculture that bacteria make then stability, and then determine arsenic tolerance and oxidation ability of three kinds of mixed bacteria. At the same time in order to obtain single bacteria oxidation of trivalent arsenic, respectively, using mixed cultures medium containing arsenic enrichment, plate separation, according to the result of the As5+and silver nitrate color reaction, oxidation of single colonies were detected to obtain efficient oxidation strains. Finally by the oxidation of trivalent arsenic mixed bacteria from arsenic containing wastewater treatment of Huaxi Group Chehe concentrator took, to detect the effect of treatment.Through DGGE and RFLP analysis, the NY community diversity is far greater than SY and XY. The typical clones of three samples were sequenced, the sequence obtained by NCBI comparison, discovery and sequencing results higher similarity sequences of strains, mostly from similar environmental conditions. Identification of sequences found, detect α-Proteobacteria,β-Proteobacteria, y-Proteobacteria and many types of bacteria in three samples, and the presence of NY Pseudomonas certain, according to the research results related, probably with oxidation of trivalent arsenic strains. Because there is a higher concentration of sulfur in three samples, so we also detected thiobacillus. XY in the ground, and which related to the sample environment, so many types of Hydrogenophilus appeared.After strain16S rDNA sequence identification of samples screened the oxidation of trivalent arsenic, mainly belong to Pseudomonas, Acinetobacter. Using the obtained pure bacteria were detected in treatment of wastewater containing arsenic, ability of each single bacteria oxidation of trivalent arsenic, can play a good effect. At the same time by cloning protease gene was oxidized trivalent arsenic on objective strain obtained by oxidation, found to have ability of strain, not necessarily with arsenite oxidase gene.Using samples in the above arsenic concentration enrichment medium, the oxidation of trivalent arsenic mixed bacteria obtained, after testing were major for Acinetobacter, Pseudomonas and so on. These three kinds of samples were detected in the culture medium for the tolerance of certain gradient arsenic concentrations, found that three kinds of bacteria in the arsenic concentration of up to0.5g/L under the conditions of growth, although some is slowly, but also can reach108-109. Using the three kinds of bacteria from wastewater containing arsenic treatment of certain concentration, trivalent arsenic in waste water was almost completely oxidized, concentrations of total arsenic in a certain period of time also decreased the number of. The flora with the oxidation of trivalent arsenic capacity of arsenic-containing wastewater, wastewater from Chehe concentrator processing, in the range of10h, the total arsenic in waste water is handled completely.更多还原