沼气池污泥微生物总DNA提取方法的比较

Comparison of Three Protocols for Total DNA Extraction of Microorganism in Biogas Digesters

沼气发酵系统是一个复杂的生态系统,其污泥微生物超过99%是不可培养的。为了优化沼气池纤维素的转化效率、沼气的产率和开展污泥微生物多样性研究,本研究采用化学裂解法、溶菌酶裂解法和QIAampDNA Stool Mini Kit提取了沼气池污泥样品中微生物的总DNA,对三种方法的DNA得率、纯度、大片段提取效果以及是否含有PCR反应抑制剂进行了研究,最后对16S rRNA基因V3区的扩增产物进行了PCR变性梯度凝胶电泳(PCR-DGGE)分析。与化学裂解法和QIAamp DNA Stool Mini Kit法相比,溶菌酶裂解法得到的DNA量大、片段长、片段分布广、PCR扩增效率高;同时PCR-DGGE图谱显示,溶菌酶裂解法可更好地展示沼气池污泥中微生物的多样性。该结果为进一步提高沼气池中纤维素的转化效率和沼气生产优势菌种的质和量打下了一定的前期基础。

Fermentation system of biogas production is a complicated ecology system,and more than 99%of the microorganisms in biogas digesters are unculturable.To optimize conditions for cellulose conversion and biogas production in biogas digesters,and also to investigate the microbial diversity in biogas digesters,Chemical lysis method,lysozvme lysis method and QIAamp DNA Stool Mini Kit were used to extract total microbe DNA of biogas digesters,and then the DNA quality,the DNA yield,the size of DNA fragments,and existence of PCR inhibitor were examined,finally PCR-DGGE profiles were carried out to investigate the diversity of microbe in biogas digesters.The results of pulsed-field gel electrophoresis （PFGE） showed that the DNA obtained from lysozvme lysis method had larger size fragments,better total DNA yield,more widely distributed range of fragments,and more effective PCR amplification efficiency than that of chemical cleavage method and the QIAamp DNA Stool Mini Kit method.The result of PCR and PCR-denaturant gradient gel electrophoresis （PCR-DGGE） indicated that the DNA templates obtained from lysozyme lysis method had better efficiency of PCR amplification,and revealed an more abundant microbe diversity than that of chemical lysis method and QIAamp DNA Stool Mini Kit.This studies lay a basis to improve the conditions of biogas fermentation and to investigate the quality and quantity of dominant microorganisms.