摘要
应用基于16S rDNA的PCR-DGGE对超声波-好氧/缺氧污泥消化过程中微生物种群的多样性进行研究.通过SDS细胞裂解法提取不同时期污泥中的基因组DNA,采用通用引物进行V3区域PCR扩增,长约190 bp的PCR产物经DGGE分离后,获得污泥微生物群落的DNA特征指纹图谱,对条带进行切胶测序,使用序列数据进行同源性分析并建立系统发育树.DGGE图谱表明,在反应器运行的不同时期,微生物群落结构发生动态演替.5、10、15、20、25 d微生物相似性与0 d相比分别为61.2%、48.2%、46.4%、42.6%、41.7%,总细菌Shannon指数经历了一个从逐渐减少到趋于稳定的过程,这表明超声波改变污泥内部性质,导致微生物多样性的降低.UPGMA聚类分析将DGGE图谱区分为三大族群并对应于不同运行时期.测序结果表明,超声波-好氧/缺氧污泥消化中微生物群落主要为Firmicute、Genuscitrobacter、Bacilli、α-Proteobacteria、β-Proteobacteria.
The microbial community structure of sludge aerobic/anoxic digestion after ultrasonic pretreatment was studied by PCR amplification and DGGE based on 16S rDNA.The genomic DNA of sludge at different stages was extracted with SDS cell lysate method.After purification of DNA,the 16S rDNA genes（V3 region） were amplified by using the universal primers（F357GC and R518）.The results of agarose gel（1.5%）electrophoresis showed that the PCR products were about 190 bp in length.The amplified DNA fragments were separated by paralleled DGGE with the denaturant（urea and acrylamide） from 30% to 60%.The sequences were used for homology analysis and phylogenetic trees were constructed.The DGGE profiles showed that the change of microbial diversity was in correspondence to different periods.Compared with 0 d,the diversities of microorganisms were 61.2%,48.2%,46.4%,42.6% and 41.7%,respectively after 5 d,10 d,15 d,20 d,25 d.Shannon density index of bacteria experienced a process from a gradual reduction to stable state.This suggested that ultrasonic pretreatment had a significant impact on bacterial community structures.Cluster analysis of DGGE by UPGMA（unweighted air group method,arithmetic mean） divided all lanes into three clusters,which corresponded to different periods during the whole experiment.The sequences indicated that Firmicute,Genuscitrobacter,Bacilli,α-Proteobacteria,β-Proteobacteria were the predominant microbial populations in the process of sludge aerobic/anoxic digestion after ultrasonic pretreatment.
出处
《环境科学》
EI
CAS
CSCD
北大核心
2012年第2期618-624,共7页
Environmental Science
基金
广东省教育部产学研合作专项(2008B0905200253)
