DG-DGGE分析产氢发酵系统微生物群落动态及种群多样性

Application of DG-DGGE to analyze microbial community diversity and population dynamics in fermentative hydrogen-producing system

应用双梯度-变性梯度凝胶电泳(DG-DGGE)对生物制氢反应器微生物种群的动态变化及多样性进行监测。间隔7d从反应器取厌氧活性污泥,以细菌16SrDNA通用引物进行V2～V3区域PCR扩增,长约450bp的PCR产物经DGGE分离后,获得污泥微生物群落的16SrDNA指纹图谱。污泥接种到反应器后微生物群落中既有原始种群的消亡和增长,也有次级种群的强化和演变。反应器在运行初期群落演替迅速,15d时微生物群落结构变化最大。群落结构的相似性随着演替时间的增加而逐渐升高,种群动态变化后形成稳定的群落结构。29d时微生物多样性基本保持不变,微生物优势种属达到19个OTU。在细菌竞争和协同作用制约下,种群多样性降低后趋于稳定,形成顶级群落。有些种群在群落结构中一直存在,是群落建成的原始种群,原始种群与次级种群在代谢过程中具有协同作用,表现出群落的综合生态特征。

Anaerobic microflora enriched from sewage sludge produces hydrogen by using molasses wastewater as its substrate. Use has been made of the double gradient-denaturing gradient gel eleetrophoresis （DG-DGGE） to monitor the genetic diversity and dynamics of microbial communities in a bio-hydrogen producing reactor. Samples of the anaerobic sludge were analyzed every 7 days, and genomic DNA of the microbial community was extracted. After purification of the DNA by using the DNA gel recovery kit, the 16S rRNA genes （V2 to V3 region） were amplified by using the universal primers （F63GC and R518）.The results of agarose gel （2%） electrophoresis show that the PCR products are about 450bp in length. These amplified DNA fragments were separated by parallel DGGE with the denaturant （urea and formamide） from 30% to 60%. The profile of DGGE changes when sludge is inoculated in the reactor, both the primary community dies out or increases, and a second micriobial community increases quickly. The change of the community structure is the greatest on the 15th day. As community succession increases with time, the community structure declines in complexity and a stable community structure forms. On the 29th day, the community diversity stops changing and the amount of the microbial dominant populations reach 19 OTUs. The community diversity tends to be stable after the decreasing under the competition and the cooperation of bacteria, and a climax community forms. Some kinds of populations have been existing in the community structure, they are primary communities. There are some great differences in composition and quantity of the dominant population between the primary community and the climax community. The primary community and the second community have a cooperation effect in the metabolizing process; show the complex ecological characteristics of the microbial community. This is because different niche conditions lead to different enrichment of bacteria, and forms different microbial community structures. Only by enriching an^d selecting the needed dominant populations fast can produce hydrogen effectively in the reactor.