Study On Molecular Characterization Of Microbial Populations In Anaerobic PCP-degrading Bioreactor

Chlorinated organic compounds are among the most significant pollutants in the world and attract great environmental concerns due to their toxicity, persistence and bioaccumulation. Considerable effort has been devoted to find economical remediation technologies, the microbial degradation is known as a major and effective process for reducing pollution caused by such compounds.In order to improve the efficiency and stability of biological treatment system and bioremediation process, it was important to investigate the relationship between community structure, functional property and spatial/temporal variability of the microbial population. Development and application of advanced molecular microbiological techniques such as Polymerase chain reaction(PCR), Denaturing gradient gel electrophoresis (DGGE), Fluorescent in situ hybridization(FISH) would provide new insights into microbial population at the genetic level.The purpose of this present work was to evaluate the molecular characterization of microbial populations in anaerobic PCP-degrading bioreactor using a molecular approach. A laboratory scale EGSB reactor was constinuously operated over 180 days when PCP load was increased stepwise, during anaerobic degradation of PCP and sludge granulation process, spatial and temporal dynamics of the microbial populations were investigated by PCR-DGGE. Following achievements were obtained:1. DGGE analysis of PCR-amplified V3 region of the 16S rDNA gene fragments indicated that both eubacteria and arechaea populations differed obviously during the anaerobic degradation of PCP and sludge granulation process. Therefore, PCP degradation process changed the diversity of the microbial community, it was most probably related to the development of PCP-dechlorinating consortium by species' adaptation to the presence of PCP.2. On the basis of Dice coefficient (Cs), Similarity analysis of the DGGE band patterns indicated that the composition of microbial community at different layers of the reactor exhibited a moderate shift during the stable phase, Cs between the top andbottom layers were 71.4~77.6 for eubacteria and 70.4~76.7 for archaea.3. In the case of PCP shock loading, significant spatial variations of the microbial populations in the reactor occurred, Cs between the top and bottom layers of the reactor was sharply decreased to 43.3 and 35.2 for eubacterial and archaeal communities respectively.In addition, the present results also showed that microbial community structure changed starting in the bottom layer, which indicated that microorganisms especially for methanogens at the bottom layer were relatively more sensitive to PCP shock loading or toxicity than the top layer.4. In order to further understand the composition of the microbial community, major DGGE bands were purified, sequenced and their phylogenetic affiliations were determined. The partial 16S rDNA nucleotide sequences of retrieved DGGE fragments were compared with sequences available from public databases (Genbank) through a blast search.Sequence analysis of successfully retrieved eubacterial DGGE fragments showed a band closely related to an uncultured eubacteria which was found during the dechlorination of 1,2-dichloropropane (DCP). Nucleotide sequences showed up to 98% of the similarity, which indicate the existence of an uncultured PCP-dechlorinating eubacteria in the ractor during the anaerobic degradation of PCP.5. For the Archaea domain, Phylogenetic analysis of successfully retrieved archaeal DGGE sequences demonstrated the presence of Me(hanobacteraceae, Methanosarcinaceae and Methanocorpusulaceae, the predominance of methanogens were closely related to Methanosaeta concilii, Methanobacterium sp. , Methanobrevibacter sp. and Methanothrix soehngenii. Which was in good agreement with the conclusion obtained by other researchers.