遗传差异及环境因子对芽孢杆菌反硝化性能与产气模式的影响

The denitrifying Bacillus showed a broad application prospect for the nitrogen bioremediation of surface water, while its theoretical research was relatively weak，some phenomenon or problems appeared in the process of application could not be explained and resolved, and the efficient genetic engineering strains were also difficult to be constructed. This application was mainly aimed to research the denitrification mechanism, the distribution law of the denitrification-related genes and the role of N2O and N2 production in the Bacillus group by use a variety of modern biological engineering methods. The expected results would explain the aerobic denitrification mechanism of Bacillus from the molecular perspective, and find out the relationship between environmental factors and genetic differences and gaseous product of denitrification. In addition, we would also clone the complete coding sequences of several key denitrification-genes, and inspect the variation of denitrification features by high expressing or deleting these genes in Bacillus. This work would provide the meaningful theoretical supports for the rational use of the denitrifying Bacillus probiotics and the efficient genetic engineered strains construction.

反硝化芽孢杆菌在地表水氮素污染控制中展示了广阔的应用前景，但其机理研究相对薄弱，对在应用过程中出现的现象及问题无从解释和解决，也制约了高性能菌株的遗传改造。本申请以反硝化芽孢杆菌群体为研究对象，运用现代生物工程手段，针对芽孢杆菌的反硝化机理、反硝化酶系的分布特点及N2O及N2的释放规律等开展深入研究，试图从分子水平阐明有氧条件下芽孢杆菌的反硝化作用机制、解析环境因子及基因组成差异与芽孢杆菌反硝化性能及产物模式之间的关系。此外，对几个关键的反硝化酶编码基因进行全序列克隆，并研究这些基因丰度表达或缺失对芽孢杆菌反硝化特征的影响。预期成果有望为合理利用反硝化芽孢杆菌微生态制剂以及构建高效基因工程菌株提供坚实的理论依据。

项目执行期间，建立了包括442株反硝化芽孢杆菌的菌群，获得47株总氮脱除能力大于20%的芽孢杆菌，其中Bacillus firmus和Bacillus megaterium是自然界最主要的反硝化芽孢杆菌，同时还发现多株芽孢杆菌能够实现高浓度污水的同步脱除。实验首次克隆及异源表达了芽孢杆菌的nir K基因，并对其酶学性质进行了深入分析。在B. subtitlis GY-48中丰度表达了nirK基因，使得重组菌的亚硝酸盐最大积累量从100.4 mg/L降低到2.1 mg/L。筛选了1株能够在8%盐浓度下实现高效好氧脱氮的海杆菌NY-4，并考察了氧气梯度对其反硝化能力及反硝化相关基因的影响，随着氧气浓度从0%至100%，NY-4的总氮脱除率从66.19%下降至 36.36%；narG, nirS 及nosZ的基因表达量也分别下降了12.19、72.08 及 40.74倍。在海杆菌NY-4中丰度表达了nir S基因，使菌株的亚硝酸耐受能力从0.92 g/L提高到1.65 g/L，亚硝酸盐能力提高了1.8倍。本研究获得的多株细菌及构建的基因工程菌均具有潜在的应用前景，建立的酶活分析方法及遗传操作平台也为类似的研究工作提供了参考。