一株耐低温异养硝化-好氧反硝化菌的分离鉴定及其脱氮特性  

Isolation and Identification of a Psychrotolerant Heterotrophic Nitrification-aerobic Denitrification Bacterium and Its Nitrogenremoving Characteristics

作　　者：董怡华[1,2] 王凌潇 任涵雪 陈峰 DONG Yi-hua;WANG Ling-xiao;REN Han-xue;CHEN Feng(Key Laboratory of the Ministry of Education for Eco-restoration of Regional Contaminated Environment,Shenyang University,Shenyang 110044;College of Environment,Shenyang University,Shenyang 110044)

机构地区：[1]沈阳大学区域污染环境生态修复教育部重点实验室,沈阳110044 [2]沈阳大学环境学院,沈阳110044

出　　处：《生物技术通报》2023年第12期237-249,共13页Biotechnology Bulletin

基　　金：沈阳市科技计划项目(21-108-9-25);“十三五”国家水体污染控制与治理科技重大专项(2018ZX07601-002)。

摘　　要：为了提高低温废水的生物脱氮效率,从寒冷地区冬季土壤和底泥中分离筛选耐低温异养硝化-好氧反硝化细菌,研究其脱氮特性及途径。通过菌落和细胞形态特征观察、16S rRNA基因序列分析鉴定菌种。分别以NH_(4)^(+)-N、NO_(3)^(-)-N、NO_(2)^(-)-N为唯一氮源,以NH_(4)^(+)-N和NO_(3)^(-)-N为混合氮源,考察菌株在低温条件(10℃)的硝化、反硝化以及同步硝化反硝化性能。采用聚合酶链式反应(polymerase chain reaction,PCR)对菌株的脱氮功能酶基因扩增,推测低温脱氮途径。结果表明,从河水底泥中筛选出一株异养硝化-好氧反硝化菌,经鉴定为Pseudomonas veronii,命名为P.veronii DH-3。该菌分别以相同初始含氮量(105 mg/L)的NH_(4)^(+)-N、NO_(3)^(-)-N和NO_(2)^(-)-N为唯一氮源,在10℃好氧培养48 h时,氮的去除率分别为99.07%、96.89%和90.29%,且在脱氮过程中几乎无亚硝酸盐的累积。以NH_(4)^(+)-N和NO_(3)^(-)-N为混合氮源时,NH_(4)^(+)-N在48 h内被完全去除,NO_(3)^(-)-N的去除率为87.09%;氮平衡分析结果表明,以NO_(3)^(-)-N和NO_(2)^(-)-N为唯一氮源时含氮气体和细胞内生物氮的转化率均低于NH_(4)^(+)-N,表明该菌株的异养硝化能力强于好氧反硝化能力。脱氮功能基因hao、napA、nirS、nirK、cnorB和nosZ的成功表达,进一步证实该菌株具有硝化反硝化能力。根据上述研究结果,推测该菌株低温脱氮的主要途径为异养硝化-好氧反硝化作用和同化作用。菌株P.veronii DH-3具有良好的异养硝化-好氧反硝化性能,为低温含氮废水的生物净化提供了理论支持。In order to improve the biological nitrogen removal efficiency of low-temperature wastewater,a psychrotolerant heterotrophic nitrification and aerobic denitrification bacterium was isolated from soil and river sediment in cold regions,and its nitrogen removal characteristics and pathway were investigated.The isolated bacterium was identified based on its colony and cell morphological observation as well as 16S rRNA gene sequence analysis.The nitrification,denitrification,and simultaneous nitrification and denitrification performance of the strain were investigated with NH_(4)^(+)-N,NO_(3)^(-)-N,and NO_(2)^(-)-N as the sole nitrogen source and NH_(4)^(+)-N and NO_(3)^(-)-N as mixed nitrogen sources.The nitrogen-removing functional enzyme genes of the strain were amplified by polymerase chain reaction(PCR)to speculate the nitrogen-removing pathway at low-temperature.A heterotrophic nitrification and aerobic denitrification bacterium was screened from the river sediment and identified as Pseudomonas veronii,named P.veronii DH-3.When strain P.veronii DH-3 was cultured aerobically for 48 h at 10℃with NH_(4)^(+)-N,NO_(3)^(-)-N,and NO_(2)^(-)-N as the sole nitrogen sources(N 105 mg/L),the corresponding nitrogen removal efficiencies were 99.07%,96.89%,and 90.29%,respectively,without accumulation of nitrite.When NH_(4)^(+)-N and NO_(3)^(-)-N were used as mixed nitrogen sources,NH_(4)^(+)-N was completely removed in 48 h and the NO_(3)^(-)-N removal rate was 87.09%,indicating that strain P.veronii DH-3 had excellent simultaneous nitrification and denitrification performance at 10℃.The nitrogen balance analysis results showed that when NO_(3)^(-)-N and NO_(2)^(-)-N were the sole nitrogen sources respectively,the conversion rates of nitrogenous gas and intracellular nitrogen were lower than those of NH_(4)^(+)-N,indicating that the heterotrophic nitrification ability of the strain DH-3 was stronger than that of aerobic denitrification.Meanwhile,the successful expressions of denitrification functional genes hao,napA,nirS,

关 键 词：耐低温菌 生物脱氮 异养硝化 好氧反硝化 分离鉴定 脱氮功能基因 假单胞菌 

分 类 号：X172[环境科学与工程—环境科学] X703