1株2,4-D降解菌的gfp标记及其在废水生物处理系统中的检测方法

Study on gfp Labeling of a 2,4-D Degrading Strain and Its Detection in a Wastewater Biotreatment System

通过mini-Tn7转座子系统将绿色荧光蛋白基因(gfp)插入到2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid,2,4-D)降解菌Achromobacter sp.的染色体上,考察了标记前后该菌株的生长、发光及降解污染物特性,并探讨了将其投加到不同废水生物处理系统(活性污泥和颗粒污泥系统)后的定量检测方法.结果表明,Achromobacter sp.标记前后生长和降解2,4-D特性基本不变,在103～112 h内可将初始浓度约为100 mg/L的2,4-D完全降解.标记后菌株在生长和降解2,4-D过程中都能够稳定表达绿色荧光,降解过程荧光强度/D600稳定在4 500左右.向活性污泥系统投加该标记菌,可通过直接测定混合液荧光强度对该标记菌进行定量检测,在标记菌质量分数为0～75%的范围内,绿色荧光蛋白的表达水平与该标记菌的质量分数线性相关(R2=0.995 2).向颗粒污泥系统投加该标记菌,需要对混合液破碎均质化处理后测定荧光强度,在标记菌质量分数为0～42%的范围内,绿色荧光蛋白的表达水平与该标记菌的质量分数线性相关(R2=0.980 1).基于Tn7插入gfp的标记方法可以用来跟踪检测生物处理系统中的特异微生物.

A 2,4-dichlorophenoxyacetic acid（2,4-D） degrading special bacteria Achromobacter sp.was chromosomally labeled with a green fluorescent protein gene（gfp） using a mini-Tn7 transposon delivery system.The growth status,fluorescence expression and degradation ability of the strain before and after labeling were compared.Methods to quantify the strain in different biotreatment systems（activated sludge or granular sludge system） after inoculation were also investigated.Results showed that the labeled Achromobacter sp.and its control strain demonstrated a similar growth pattern and 2,4-D degradation ability： both of them could completely remove 2,4-D of about 100 mg/L within 103-112 h.The labeled strain could express fluorescence stably during the course of growth and degradation with fluorescence intensity/D600 stabilized at about 4 500.For an activated sludge system bioaugmented with this labeled strain,its abundance could determined through direct measuring fluorescence emitted by the sludge mixture,for it was linearly associated to the percentage of the strain in the range of 0-75%（R2 = 0.995 2）.For a granular sludge system bioaugmented with this strain,fluorescence of the sludge mixture could be measured after homogenous pretreatment,and the percentage of the strain in the range of 0-42% was also linearly related to the fluorescence intensity emitted by the sludge mixture（R2 = 0.980 1）.Overall,this gfp labeling method based on Tn7 delivery system can be used to monitor specific bacteria in a biotreatment system.