Research On Methanogens In Anaerobic Granular Sludge By Clon Library Based On Functional Genes

Methanogens are considered to be important functional populations in anaerobicgranular sludge, and mcrA gene encoding α subunit of Methyl coenzyme-M reductase(MCR) is unique for methanogens. Methanogenic community in anaerobic granularsludge samples from full-scale UASB bioreactors treating VB12and avermactinwastewater were investigated by PCR-DGGE and clone library based on mcrA gene. Thediversity of methanogenic community and the phylogenetic relationship of dominantmethanogeinc populations were analyzed and compared between different samples. Theeffects of genetically engineered microorganisms on methane-producing capacity ofsludge samples were also discussed. The results were shown as follows.The difference in DGGE band pattern between two sludge samples indicated that thestructure of methanogens and dominant species varied in different treatment environment.Shannon diversity index, Margalef richness index and Berger-Parker dominance indexwere calculated from DGGE band patterns of two different sludge samples, whichshowed higher diversity and more richness of methanogens in sludge sample treatingVB12wastewater compared to sludge sample treating avermactin wastewater, due todifferent wastewater treatment environment.DGGE bands of both samples were sequenced to determined the dominant speciesof methanogens in anaerobic granular sludge and analyze the phylogenetic relationship ofmethanogens. In sludge sample treating VB12wastewater, methanosaeta belonging tomethanosarcinales was dominant and methanobacteriales was also detected. In sludgesample treating avermactin wastewater, the dominant species was also determined to bemethanosaeta. In addition, methanosarcina belonging to methanosarcinales andethanobacteriales was also detected.The clone library analysis of the methanogents in both samples based on mcrA geneindicated that methanosarcinales, methanobacteriales and methanomicrobiales weredetected in sludge sample treating VB12wastewater, and methanosarcinales wasdominant. In addition, only methanosarcinales and methanobacteriales were detected insludge sample treating avermactin wastewater, and methanosaeta was dominant.There was obvious difference in methanogenic community analysis between clonelibrary and PCR-DGGE. For sludge sample treating VB12wastewater, methanosarcinales was determined to be dominant using both clone library and PCR-DGGE, butmethanomicrobiales was detected only in clone library analysis. It seemed that clonelibrary could provide more information of microbial populations.The effect of genetically engineered microorganisms obtained during construction offunctional gene clone library on methane-producing capacity of anaerobic granularsludge was investigated. The results indicated that inoculation of genetically engineeredmicroorganisms reduced the total produced methane amount but increased methaneproducing rate. At the same time, inoculation of genetically engineered microorganismsresulted in rapid system pH decreasing. So the participation of genetically engineeredmicroorganisms might accelerate acidification process, which promoted the production ofmethane. However, accumulation of organic acid led to pH decreasing and as a result, theactivity of methanogens was inhibited and the total produced methane amount wasreduced.