反硝化基因工程菌株YP4S的构建及对污水除氮特性研究

从养殖场污泥中筛选出菌株YP4,经16S rDNA分子发育树的同源序列比对,确定为克雷伯什菌属(Klebsiella sp.)。由NCBI数据库查编码亚硝酸还原酶(Nir)的基因nirS序列,设计引物,以铜绿假单胞菌PAOI基因组DNA为模板,应用PCR技术扩增目的片段nirS,经过双酶切、克隆和转化,得到重组质粒pYP4S,然后转化野生菌株YP4,构建反硝化基因工程菌YP4S。菌株生长曲线测定表明,工程菌株YP4S与YP4的生长特性基本一致。工程菌株YP4S对模拟污水COD、TN、NH_4^+-N和NO_3^--N具有较高的去除率,YP4S与YP4相比,对NO_2^--N积累的减少量为(32.44±3.96)%,明显减少了NO_2^--N的积累。通过正交试验获得工程菌株YP4S在C/N=10、T=30℃、r=200 r/min和pH=7.0的最佳组合条件下,对模拟污水TN去除率较高。应用工程菌株YP4S处理猪场沉淀池的实际污水,COD、TN、TP、NH_4^+-N和NO_3^--N去除率分别为(95.87±0.82)%、(76.38±3.84)%、(97.13±0.54)%和(75.35±2.57)%,NO_2^--N积累量为(3.31±1.24) mg/L,表明工程菌株YP4S具有较好反硝化作用,对含氮量高的实际污水修复具有潜在的应用前景。

Construction of Denitrifying Genetically Engineered Strain YP4S and Study on Its Nitrogen Removal Characteristics of Wastewater

The strain YP4 was screened from the farm sludge. It was identified as Klebsiella sp. through the homologous sequence alignment with a 16 S r DNA phylogenetic tree. The gene sequence of nirS encoding nitrite reductase(Nir) was obtained by searching NCBI database, and the primers were designed. Taking the genomic DNA of Pseudomonas aeruginosa PAOI as template, the target fragment nirS was amplified by PCR, and the recombinant plasmid pYP4 S was obtained by double enzyme digestion, cloning and transformation. Finally, wild strain YP4 was transformed to construct denitrifying genetic engineering strain YP4 S. The identification result of strain growth curve showed that the growth characteristics of engineering strain YP4 S and YP4 were basically the same. The engineering strain YP4 S had a high removal rate of COD, TN, NH4^+-N, NO3^--N in simulated wastewater. Compared with strain YP4, stain YP4 S could significantly reduce NO2^--N accumulation by(32.44±3.96)%, which significantly reduced the accumulation of NO2^--N. Stain YP4 S, which was obtained by means of orthogonal test, had a higher removal rate of TN in simulated wastewater under the optimum conditions with C/N=10, T=30℃, r=200 r/min and pH =7. Applying the engineering strain YP4 S to treat the actual wastewater in pig farm sedimentation tank, the removal rates of COD, TN, TP, NH4^+-N, NO3^--N of strain YP4 S were(95.87±0.82)%,(76.38±3.84)%,(97.13±0.54)%,(75.35±2.57)% respectively. The accumulation of NO2^--N was(3.31±1.24) mg/L, indicating that the engineering strain YP4 S had a high-efficient denitrification and it had potential application prospects for the actual sewage remediation with high nitrogen content.