Enrichment And Identification Of Methanogens And Preliminary Application In Low Temperature At Yanbian Area

In order to improve the activity of methanogens in the biogas anaerobic fermentation system,and find ways to improve the efficiency of biogas production in the reactor at low temperatures in northern.We use wetland sludge samples from Yanbian Korean Autonomous Prefecture to filter methanogen samples by DGGE and high-throughput sequencing technology.And we use Hungate separation technology to enrich and isolate methanogens at low temperature.Using anaerobic pecked-bed reactor and measuring the daily gas production,methane content,pH,volatile fatty acid content,COD removal rate and microbial diversity to preliminary research the influences of enriched methanogens in reactor operation.The research results are as follows:(1)Methanomicrobiales and Methanosarcinales are dominant bacteria in the microbial community composition of DH,HC,YJ and HL in Yanbian Korean Autonomous Prefecture.The results shows that we give preference to choose the samples DH5,DH40,HC5,HC25,and HC40 to enrichment and isolation.The abundance of Methanomicrobiales and Methanosarcinales are higher in these samples which is easy to isolate methanogens.(2)The enriched methanogens of DH40 had the highest OD600 and strong growth ability at 6 d.A methanogenic strain L2 was isolated from the wetland sludge of Yanbian Korean Autonomous Prefecture.The isolated strain was negative for Gram staining,rod-shaped,and strictly anaerobic.The 16S rRNA gene phylogenetic analysis indicated that L2 belongs to Methanomicrobiaceae,Methaneacinia.(3)Anaerobic pecked-bed reactors A and B were successfully started and stable.The daily gas production are similar,methane content of A and B are balanced at 60%and 50%,the COD removal rate is stable at about 80%,the pH and VFA decomposition are in a stable state,which is suitable for anaerobic fermentation.During the operation phase,the daily gas production and cumulative gas production of reactors A and B showed a significant upward trend.The methane content of reactor B inoculated with DH sludge increased rapidly,and the COD removal rate increased to 70%.Enrichment of methanogens in fixed bed reators is feasible.The microbial diversity on the carbon fiber carrier was higher than that of the sediment.Methermicoccus and Methanobacterium were the dominant bacteria in two reactors,Methanosalsum,Methanomassicicoccus and Methanosaeta were only present in reactor B.(4)Adding enriched methanogens to the Anaerobic pecked-bed reactor under low temperature,the daily gas production increased rapidly,reaching the highest peak of 730 mL on the 30 d,and the methane content increased from 61%to 65.8%,there is no fluctuation in pH and VFA,the COD removal rate increased 15%,and the OD600 of the liquid increased 0.213.Methanolacinia and Methcmohalophilus were the unique bacteria after adding enriched methanogens.The addition of enriched methanogens has a positive effect on the various indexes of the reactor,and the effect remains after the addition of enriched methanogens is stopped.Enriched methanogens can be initially applied to low temperature anaerobic reactors.