| Although biogas fermentation technology is widely used in China,temperature is the limiting factor of biogas fermentation.Due to the long and cold winter in northern China and Xinjiang,there is a problem of low gas production in biogas fermentation in winter.The potential of biogas fermentation cannot be fully developed.Low temperature is the main factor affecting the promotion of biogas and restricting the fermentation efficiency.It is of great practical significance to study the addition of exogenous microbial agents to the microbial flora changes in low temperature environments.In this study,high-throughput sequencing and metagenomic technology were used to study the changes of microbial community structure in low-temperature biogas fermentation of cow manure supplemented with Enterococcus sp.(L1).Adding different concentrations of L1 in the biogas fermentation reactor bacterium agent(0,1%,5%,10%),placed in 15? constant temperature incubator for fermentation.Recorded the daily gas output,and extracted the total DNA of the sludge at different stages of fermentation.Analyzed the microbial community succession in the biogas fermentation process with high-throughput sequencing and metagenomic technology.Understand the changing rules in different fermentation stages of the selected three strains with Real-time fluorescence quantitative PCR.To lay a theoretical foundation for further optimization of fermentation conditions for low temperature biogas fermentation of cattle manure.Experiment 1:The purpose was to study the physiological and biochemical characteristics and fermentation conditions of a hydrogen-producing strain selected in the previous experiment.A strain of Enterococcus sp.(L1)was successfully isolated from the biogas fermentation in a large amount of low-temperature biogas fermentation of cow and sheep manure with the pre-experiment.Observed the morphological characteristics of the bacteria with optical microscope.And Gram staining,16S rDNA identification,physiological and biochemical characteristics and culture medium optimization were performed.The results of optical microscope showed that L1 bacteria was gram-positive,without spore and capsule.The result of 16S rDNA identification showed that the bacterium was Enterococcus sp..Studies on physiological and biochemical characteristics showed that L1 bacteria could ferment lactose to produce lactic acid.L1 used glucose,sucrose,maltose and lactose produced acid,and used starch produced alkali.Optimum conditions of the optimization results showed that the best carbon source for maltose,the best nitrogen source for mixed nitrogen source,the optimum pH value of 7,the optimum growth temperature 30?,the optimal Na2HPO4 adding amount was 0.2%,the optimal medium combination was maltose 1 g/L,yeast extract 10 g/L,peptone 5 g/L,Na2HPO4 2 g/L.Experiment 2:The purpose of this study was to investigate the effect of adding different concentrations of L1 bacteria on the gas production of methane under low temperature(15?)fermentation conditions.The test results showed that the 10%L1 strain had the largest gas production,the average daily gas production rate reached 0.02 m3/(m3·d).Experiment 3:The purpose of this study is to use high-throughput sequencing technology to study the changes of bacterial and archaea flora during the simulated biogas fermentation of cow manure at low temperature(15?)by adding different concentrations of L1 bacteria.In this chapter,50 mL of fermentation broth was taken on the 10th,30th,60th,and 90th days of fermentation.Extracted the total DNA of the microbial microorganisms.Used HiSeq2500 PE250 for high-throughput sequencing.The results showed that at the level of the phylum:the archaea group mainly consisted of Euryarchaeota and Tenericumtes.The bacterial flora is mainly composed of Proteobacteria,Firmicutes and Bacteroidetes.Euryarchaeota and Bacteroidetes have relatively large changes with the abundance in different fermentation stages,with both increasing and decreasing first.At the genus level:At the 60d of fermentation(the peak gas-producing stage),the dominant archaea in each treatment is mainly Methanosarcina and the dominant bacteria is mainly Ruminococcaceae UGG-010.Experiment 4:The purpose of this study was to analyze the changes of bacterial community during the simulated low-temperature(15 ?)simulated biogas fermentation process in the 10%treatment with large gas production by using metagenomic sequencing technology.The experiment used Illumina HiSeq sequencing platform to obtain 87063.52 Mbp Raw Data and 86754.85 Mbp of Clean Data after quality control.The proportions of the genus and phylum were 55.56%and 76.67%.The results showed that the Firmicutes and Bacteroidetes were the dominant communities of the methanogenic process,and the Spirochaetae,Synergistetes and Chloroflexi were New.The dominant genus at the peak of gas production is Methanosarcina.Metabolic pathway prediction is mainly Carbohydrate Metabolism and Amino Acid Metabolism.Experiment 5:The purpose of the study simulate the variation of different fermentation stages of biogas fermentation used Real-time PCR with the three strains(Methanoculleus,Methanosarcina and Enterococcus)obtained in the previous analysis at low temperature(15 ?).The results showed that Methanosarcina had the largest number of Copies in each treatment group on the 60th day of fermentation.The Copies of CK,1%treatment,5%treatment,and 10%treatment were 30.98×107 Copies/?L,63.77×107 Copies/?L,28.6×107 Copies/?L,and 24.35×107 Copies/?L.And the increase in Copies of Methanosarcina was also the largest in the 60th day of fermentation.The following opinions can be concluded from the current research:(1)The optimal addition of L1 bacteria was 10%treatment.(2)The dominant bacteria that maintain the normal fermentation system at low temperature(15?)is Methanosarcina.(3)The main reason for increasing the gas production of simulated biogas fermentation under the condition of adding 10%L1 bacteria to promote low temperature(15?)is that provide H for biogas fermentation. |
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