利用流式细胞术研究鞘氨醇单胞菌GY2B降解菲过程中细菌表面特性的变化

微生物与污染物接触是生物降解的第一步,为进一步了解降解过程中有机污染物对降解菌的影响,通过对不同条件下鞘氨醇单胞菌GY2B(Sphingomonas sp.GY2B)降解菲的研究,并结合Propidium Iodide(PI)染料和流式细胞仪分析不同底物及污染物浓度对GY2B菌体细胞表面性质的影响.结果表明,随着菲的降解,PI染色的GY2B菌株细胞增多,说明细菌膜结构受到一定的破坏,通透性不断增强.污染物浓度越高,降解菌的膜完整性的破坏越为严重.60 h时,300 mg·L-1浓度条件下,染色细胞/未被染色细胞的比值可达12.44,而在100 mg·L-1和1.2 mg·L-1浓度条件下,比值分别为1.95和1.11.同时降解过程中细菌细胞的傅里叶红外光谱检测,死亡、受损和完整细胞的区分以及Zeta电位分析也进一步验证了细菌细胞表面性质的改变.利用流式细胞术与染料结合分析降解过程中细菌细胞膜完整性的变化,从单细胞水平上了解细菌降解污染物过程中其细胞表面性质的改变,有助于更好地研究GY2B对菲的降解机制.

Using Flow Cytometry to Explore the Changes of Sphingomonas sp. GY2B Bacterial Surface Characteristics in the Process of Degrading Phenanthrene

The first step of biodegradation is the contact of microorganism and pollutants,in order to examine the influence of phenanthrene on Sphingomonas sp.GY2B's surface properties during its degrading process, the bacteria was cultivated at different conditions,and detected by flow cytometry combined with fluorescent dyes for its surface changes. The results indicated that, the membrane structure had been certainly damaged during the degrading process, leading to an increased membrane permeability. Moreover, the destruction of bacteria membrane integrity became more serious with a higher pollutant concentration. At the concentration of 300 mg·L^-1, the ratio of stained bacterial cells/unstained cells was 12.44 after cultured for 60 h, while at 100 mg·L^-1 and 1.2 mg·L^-1, the ratios were 1.95 and 1.11, respectively. The results of fourier transform infrared(FT-IR) absorbance spectroscopy detection, the discrimination of death, injured and intact cells, and Zeta potential detection further verified the bacterial cell surface permeability changes. Flow cytometry combined with fluorescent dye propidium iodide was used to monitor the changes of bacterial membrane integrity on single-cell level which exhibited a good potential for exploring the changes of bacterial surface properties during the degrading progress and more deeply for investigating the degradation mechanism.