脱氮除磷假单胞菌的筛选及定量检测

Screening and detecting of denitrifying phosphorus-removing Pseudomonas sp.

摘要:

【目的】筛选具有较强脱氮除磷能力的细菌，建立结合S1酶保护分析的分子探针技术，以分析该菌在发酵过程中的数量变化情况。【方法】采用缺磷培养基厌氧培养、富磷培养基好氧培养和硝酸盐还原产气实验进行脱氮除磷菌筛选。通过16S rRNA基因序列分析及同源性比对，结合菌株的生理生化鉴定试验，鉴定筛选株。设计相应的16S rRNA探针组，建立结合S1酶保护分析的分子探针技术。【结果】筛选的菌株被鉴定为假单胞菌Pseudomonas sp.，命名为LY10。菌株LY10在富磷培养基中好氧培养24 h，总磷去除率达90.01%。在反硝化聚磷培养基中培养48 h，总氮和总磷去除率分别为84.71%和89.37%。针对假单胞菌16S rRNA基因序列设计了一组用于结合S1酶保护分析的分子探针Probe-P.sp，该探针具有很高的甄别灵敏度，能够将LY10与丛毛单胞属(Commonas)等5种细菌区分开；分子探针定量分析假单胞菌LY10，其细胞量与吸光值呈线性关系，检测的线性范围为103-106 cells/mL，线性方程为：y=?0.967 87+ 0.372 99x (R2=0.996 7，n=5)。【结论】新筛的假单胞菌LY10的脱氮除磷能力较强，具有生物脱氮除磷的工业化应用潜质。所建立的结合S1酶保护分析的分子探针技术的特异性和灵敏度良好，有望应用于混菌体系中的假单胞菌的定性定量分析。

Abstract:

[Objective] We selected a strain with high capability of denitrifying and phosphorus removal. Besides we established an assay of sandwich hybridization with S1 nuclease protection to analyze the microbial community of this strain in fermentation process. [Methods] We used anaerobic culture in a phosphorus deficient medium, aerobic culture in a phosphorus-rich medium and nitrate-reducing experiment for bacterial isolation. Physiological and biochemical tests and 16S rRNA gene probing technique were used to identify the selected strain. A set of DNA probe was synthesized and used in quantification. [Results] The selected strain was identified as Pseudomonas sp. and named as LY10. The phosphorus removal rate of LY10 was 90.31% under aerobic condition in 24 hours, the denitrifying and phosphorus removal rate were 84.71% and 89.37%, respectively, under anoxic condition in 48 hours. Bacterial detection with the reported technique showed LY10 could be well identified from other strains such as Commonas with the probe Probe-P. sp. which was complementary to the conservative region of 16S rRNA of Pseudomonas genus. The cell density range was from 103 to 106 cells per milliliter and the equation was y=?0.967 87+0.372 99x (R2=0.996 7, n=5). [Conclusion] The selected strain showed strong activity on removing compounds with N and P, thus it can have potential application in industrial bio-denitrifying and phosphorus removal.