PCR-DGGE技术在农村户用沼气发酵微生物研究中的初步应用

通过PCR-DGGE技术,对北京郊区一运行良好的农村户用沼气池的细菌多样性分析,分别在1、3、4、5月份采集沼气池的厌氧活性污泥样品,提取厌氧活性污泥微生物总DNA,对其16S rDNA基因的V3可变区进行扩增,结合DGGE(变性梯度凝胶电泳)技术,跟踪检测微生物的动态变化,并利用软件对DGGE图像进行聚类分析.测定了厌氧活性污泥DGGE胶上优势和差异条带16S rDNAV3区片段序列,通过与NCBI数据库中的序列基因库比对,初步确定细菌的种属.结果表明:农村户用沼气池中发酵微生物种类较为丰富,但有明显的优势种群,随着气温变化,微生物多样性基本稳定,但种群丰度有明显变化,DGGE图谱中的优势条带16S rDNA基因序列经比对后都属于未培养的细菌.

A PRELIMINARY APPLICATION OF PCR- DGGE IN MICROBIAL COMMUNITY STRUCTURE ANALYSIS OF RURAL HOUSEHOLD BIOGAS DIGESTERS

Based on PCR-DGGE technology,the bacterial community of rural household biogas digesters was observed and monitored.Genomic DNA was extracted directly from the activated sludges which were sampled in January,March,April and May of 2006.The 16S rDNA genes V3 region were amplified by using the bacterial specific primers,and separated by DGGE.Then DGGE patterns were analyzed with associated softwares.The sequences of 16S rDNA DGGE predominant bands fragments were determined by comparison with NCBI database.The results showed that the bacterial diversity was abundant and relatively stable in rural household biogas digesters,but the bacterial community structure was changed variously in different months.And most of the predominant bacteria were uncultured bacteria.