Analyse On Microbial Community Structure Of Rural Household Biogas Digesters By The Molecular Methods

With the development of society, the questions of lacking energy and environmental pollution are getting prominent, already seriously threatening humanity's survival and development. The methane technology is an effective approach to resolve energy crisis and environmental pollution in the countryside and actualize sustainable agricultural development. Because of the limitations of traditional researching method, we know little about the methane fermentation microbial community structure at present, hindering the project and the craft improvement, leading to the difficulty to resolve the problems that the low efficiency and poor controllable of function. The successful application of modern molecular ecological techniques in the research of environmental microorganism provides a new approach to break the technical bottlenecks. This article analyzed the microbial community structure in the rural household biogas digesters with molecular ecology techniques on the basis of 16S rDNA clone libary and DGGE.By improving DNA extracted method for actived sludge, an effective method for anaerobic actived sludge total DNA extraction and purification was established. And DNA yield, the integrity and the PCR purity can meet the need of molecular technology.Because its composition had never been studied on the rural household biogas digesters, it was necessary to optimize 16S rDNA V3 PCR-DGGE condition of the rural household biogas digesters: 1ng as the template, the Touchdown PCR-DGGE, 35%～56% denatured gel density, 600ng PCR product and 150V 270min in the 25μl PCR reacting system can display more accurate information of microbial diversity.The bacteria and archaea libraries were constructed separately. Researches were carried out to the diversity of the fermention application from rural household biogas digesters by analyzing these libraries. The results showed: 72 and 9 different clones had been separated, which were obtained from Bacterial and Archaea 16S rDNA library. Through the public databases (GenBank, RDP) blasted; To bacteria, the sequenced OTUs, five clusters: Firmicutes, Protecobacteria, dBacteroidetes, Chloroflexi and Genera_incertae_sedis_OP11. Among them, the Firmicutes including Clostridia and Bacillisubgroup,was predominant, and 54% of all the clones, and the second cluster was the Bacteroidetes accounting for 36%; To arhaea, all the sequenced OTUs belonged to Euryarchaeota, including 9 populations: Methanosaeta, Methanocorpusculum, Methanosarcina, Methanoplanus, Methanoculleus, Uncultured euryarchaeote and Methanocorpusculum parvum.The microbial diversity can be tracked by using PCR-denaturing grandient gel electrophoresis, in normal and acid pits, simultaneously their dynamic changes. The results showed: the microorganisms were rich in two pits, the profile of the bacteria microbial community structures displayed more complex than the archaea. The richness of different population existenced obviously different, has the minority richness population, this result as same as 16S rDNA clones library. The bacteria community structure of the acid pit was different from the normal one. The amount of Clostridia in the acid pit is more than the normal pit, and Bacteroidetes to be less; but the archaea community structures were similar in two different pits. So the primary conclusion was that the bacteria was the possible main factor correlative with fermention state, not archara. On different phase, the diversity of archaea community structures were stable on the whole, the number of some microorganisms were changing with the fermentation temperature changing. There was positive relationship between the quantity of the Clostridia and thefermentation temperature, nevertheless the Bacteroidetes in reverse.The paper analyzed the microbial community structure of the rural household biogas digesters, provided scientific basis to optimize the microbial community structure and adjusted the function for further. It has laid the foundation for realized the effective and controllable methane fermentation in our countryside.