摘要

      基于厌氧氨氧化反应的新型生物脱氮技术，具有能耗和物耗低、污泥产量少等优点，在废水处理领域具有广阔的应用前景。然而，厌氧氨氧化细菌存在的细胞产率低、代时长、生存条件苛刻等缺点使其在工程方面的应用受到了极大的限制。本实验旨在通过分子生物学手段构建含有厌氧氨氧化关键酶——亚硝酸盐还原酶（NIR）和肼合成酶（HZS）的基因工程菌并考察其降解氨氮和亚硝酸盐的能力。主要结果如下：

     （1）目的基因nirS和hzsB片段是通过提取厌氧氨氧化颗粒污泥中微生物的总DNA，并进行PCR特异性扩增获得。将目的基因片段与克隆载体pEASY-Blunt相连，转化至克隆受体菌Trans1-T1中，并进行PCR筛选测序鉴定。结果显示成功构建重组质粒pEASY-Blunt- nirS和pEASY-Blunt-hzsB；

     （2）提取重组质粒，对其进行PCR特异性扩增nirS和hzsB基因片段。将扩增产物与表达载体pEASY-Blunt E1相连，并在Trans1-T1细胞内转化；提取Trans1-T1细胞内的表达载体，导入表达受体菌E.coli BL21(DE3)的细胞中，并进行PCR鉴定。结果表明，成功构建基因工程菌E.coli BL21 (DE3) (pEASY-Blunt E1-nirS)和E.coli BL21 (DE3) (pEASY-Blunt E1-hzsB)；

     （3）将构建的基因工程菌置于含有一定浓度氨氮和亚硝酸盐的培养基中培养28h，定时检测培养基中两种物质浓度。结果显示，培养基中NH4+-N最高去除率大于95%，NO2—-N最高去除率达到25.51%。因此，基因工程菌E.coli BL21 (DE3) (pEASY-Blunt E1-nirS)的nirS基因和E.coli BL21(DE3)(pEASY-Blunt E1-hzsB)的hzsB基因表达成功。

关键词：厌氧氨氧化; 基因工程菌; nirS; hzsB

                                               Abstract

    Anaerobic ammonia oxidation (ANAMMOX), as a new biological nitrogen removal technology, has attracted more and more attention in the field of wastewater treatment because of its various advantages, such as low energy and material consumption and low sludge production. However, it has been restricted in application to wastewater treatment due to the slow growth of ANAMMOX bacteria. This study aimed at constructing ANAMMOX genetic engineering bacteria containing nitrite reductase (NIR) or hydrazine synthase (HZS) genes, by using molecular biological method. The results were showed as follows:

    (1) The genetic recombination of plasmid DNA——pEASY-Blunt-nirS and pEASY-Blunt-hzsB were successfully constructed. The targeting gene fragments（nirS and hzsB）were obtained through extracting genomic DNA from ANAMMOX bacteria and amplifying them with PCR technology. The recombinant plasmids, which connected targeting gene fragments with cloning vector pEASY-Blunt, transformed into the cloning receptor bacteria Escherichia coli cells Trans1-T1 for screening and identification.

    (2) The genetic engineering bacteria —— E.coli BL21(DE3)(pEASY-Blunt E1-nirS) and E.coli BL21 (DE3)(pEASY-Blunt E1-hzsB) were successfully constructed. The genetic recombination of plasmid DNA was extracted, connected with the expression vector pEASY-Blunt E1, and amplified in Trans1-T1. The expression vectors in Trans1-T1 were extracted and transformed into the expressional receptor bacteria E.coli BL21 (DE3) and identified by PCR amplification.
    (3) The genes including nirS and hzsB in the constructed genetic engineering bacterial cells were successfully expressed through the gene expression experiments. The constructed genetic engineering bacteria were cultivated in culture media with ammonia and nitrite in special concentrations for 28h. The concentrations of ammonium and nitrite were monitored in particular time. The removal rate of ammonia was over 95% and the removal rate of nitrite reached 25.51%.
Key Words: ANAMMOX; gene engineering bacteria; nirS; hzsB

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