PCR-DGGE技术解析生物制氢反应器微生物多样性

为了揭示发酵法生物制氢反应器厌氧活性污泥的微生物种群多样性 ,从运行不同时期取厌氧活性污泥 ,通过细胞裂解直接提取活性污泥的基因组DNA .以细菌 16SrRNA基因通用引物F338GC/R5 34进行V3高变异区域PCR扩增 ,长约 200bp的PCR产物经变性梯度凝胶电泳 (DGGE)分离后 ,获得微生物群落的特征DNA指纹图谱 .研究表明 ,不同时期的厌氧活性污泥中存在共同种属和各自的特异种属 ,群落结构和优势种群数量具有时序动态性 ,微生物多样性呈现出协同变化的特征 .微生物多样性由强化到减弱 ,群落结构之间的相似性逐渐升高 ,演替速度由快速到缓慢 .优势种群经历了动态演替过程 ,最终形成特定种群构成的顶级群落 .

Application of PCR-DGGE to Resolve Microbial Diversity in Bio-Hydrogen Producing Reactor

To reveal microbial diversity of anaerobic activated sludge in bio-hydrogen producing reactor, sludge in different period were sampled and their genomic DNA of microbial community was extracted directly. After purification of the genomic DNA using DNA gel recovery kit, the 16S rRNA genes (V3 region) were amplified by using the universal primers (F338GC and R534). The result of agarose gel (2%) electrophoresis show that the PCR products were about 200bp. These amplified DNA fragments were separated by denaturing gradient gel electrophoresis (DGGE) with the denaturant (urea and formamide) from 30% to 60%. The profile of DGGE show that different sludge had the different bands' patterns. In the profiles of DGGE, there exist some common bands in all sludge samples, which indicate that some kinds of microorganisms exist in each period. On the other hand, the specific bands in some sludge show that different period had their own specific microorganisms. Dynamics of community structure and amount of dominant populations were responsible to different period. Shift of microbial diversity correspond to period of running reactor, microbial diversity increased before it decreased. Similarity between communities gradually increased, speed of shift gradually decreased in succession. Finally, microbial climax community made up of specific populations was formed.