| China is one of the countries with the most abundant straw resources in the world.The agricultural straw output has reached 1.04 billion tons,and the straw output is showing a growing trend.Due to the complex structure of crop straws,the anti-degradation barrier formed by the interweaving of various components is the key bottleneck for the effective utilization of crop straws.Taking advantage of the multi-enzyme collaboration of microbial consortium to break through the straw degradation resistance barrier and promoting the synthesis of methane in anaerobic fermentation of straw raw materials has become a research hotspot.In this study,an anaerobic lignocellulose-degrading consortium TC-5 that can degrade untreated wheat straw was selected from samples from different sources by adding biochemical resuscitation factors.The efficiency of TC-5 to degrade wheat straw and the effect of different pretreatment techniques on straw degradation were explored,and the initial oxygen content of the medium was the key factor affecting the stability of TC-5.The microbial consortium TC-5 was inoculated as a biological fortification agent in the process of pig manure-rice straw semi-continuous combined anaerobic fermentation,and the gas generation process and microorganisms of anaerobic fermentation process were examined by exogenous inoculation of biological fortification agent TC-5 The dynamic impact of diversity.At the same time,this study also isolated a new bacterial strain HXT-9 from contaminated river sediments,and conducted a number of classification studies.The research content of this paper is mainly divided into the following aspects:1.Screening of anaerobic lignocellulose degrading microflora based on compound biochemical resuscitation factorsThrough the recombinant strain constructed,the desired proteins M-Rpf and CH-Rpf were produced.Compound biochemical resuscitation factors were formed by combining two biological resuscitation factors and sodium pyruvate,and applied to the enrichment of difficult-to-cultivate microorganisms in rumen fluid.The results of microbial diversity sequencing showed that the addition of composite biochemical resuscitation factors significantly increased the unknown in rumen fluid.The proportion of microorganisms.The biochemical resuscitation factor was further used to successfully enrich five anaerobic bacterial groups capable of degrading lignocellulosic substrates from samples from different sources,of which TC-1 and TC-5 derived from high-temperature compost straw-degrading bacterial groups could simultaneously degrade Xylan and cellulose components have the ability to directly degrade straw,and the degradation rate of TC-5 is better than that of TC-1.Based on metagenomic sequencing analysis,the dominant species of the TC-5 were identified,and the relative abundance of the difficult-to-cultivate microorganism Clostridium cellulosi was found to be as high as 51.56%.Genomic analysis of splicing and assembly confirmed that Anaerobic Clostridium is the main contributor to the efficient degradation of wheat straw by TC-5.2.Study on the degradation characteristics of anaerobic lignocellulose degrading consortium TC-5Based on the anaerobic lignocellulose-degrading consortium TC-5 was enriched from the medium containing wheat straw,so the effect of TC-5 on the anaerobic degradation of wheat straw was explored.According to the degradation rate of various components of wheat straw,the content change of organic acid products during the degradation process and the analysis of the enzyme activity results,it can be seen that TC-5 is beneficial to the degradation of wheat straw and can increase the degradation rate to a certain extent.Wheat straw was pretreated by lye soaking and extruding,the results showed that both pretreatment methods can significantly improve the degradation rate of wheat straw,respectively increased by 55.53%and 46.21%,accompanied by higher CMCase and hemicellulose enzyme activity.Since oxygen concentration can affect the growth of anaerobic bacteria,the effect of different initial oxygen concentrations on the ability of TC-5 to decompose wheat straw was explored.From the analysis of straw degradation rate and bacterial group structure,it is found that the initial concentration of oxygen in the culture system has a significant effect on the degradation rate of straw.The lower the initial oxygen concentration,the greater the activity of anaerobic bacteria and the higher the degradation rate of straw.3.The effect of adding anaerobic lignocellulose-degrading consortium TC-5 on pig manure-rice straw semi-continuous combined anaerobic fermentationIn the pig manure-rice straw semi-continuous combined anaerobic fermentation experiment,three experiment stages A,B and C were run.Phase A is the fermentation start-up phase,that is,a batch-type batch test with a complete feed,totaling 20 days;Phase B is the semi-continuous fermentation of the input and output materials with an SRT of 20 days;Stage C is the semi-continuous fermentation with an SRT of 20 days after adding anaerobic lignocellulose-degrading consortium TC-5.From all the gas production results,it can be seen that,compared with the A and B stages,the anaerobic consortium TC-5 stage C has a significantly higher gas production rate,indicating that the addition of the consortium can help biogas and methane production increased gas rate.The high-throughput sequencing technology was used to measure the dynamic changes of microbial colonies during fermentation,and it was found that the species richness of bacteria Christensenellaceae_R-7_group,Clostridium sensu stricto 1,Flavobacterium and vadinBC27 wastewater sludge group were higher than those without inoculation,and the archaea Methanosaeta degrees are also increasing.This shows that the addition of bacteria TC-5 contributes to the degradation of straw lignocellulose,which in turn is conducive to the increase of biogas and methane production.4.Screening,isolation and identification of strain HXT-9Gram-negative aerobic bacteria isolated from contaminated river sediments,named HXT-9.The strain cells look like rods,without flagella and cannot move.The pH range of the strain growth is 4.0-8.0(optimum 7.0);the strain can grow in the range of 10-42?(optimum 30?);the salt tolerance range is 0-2.5%(w/v)(optimum 0.5%).Phylogenetic tree based on 16S rRNA gene sequence shows that strain HXT-9 belongs to the genus Parvibaculum,and has the highest affinity with P.lavamentivorans DS-1(96.1%).The average nucleotide identity(ANI)and DNA-DNA hybridization(DDH)values between strains HXT-9 and DS-1 were 75.2%and 20.8%,respectively.The G+C content of the strain HXT-9 genome was 62.1mol%.The main fatty acids(>10%)are C16:0 3-OH(12.5%),C19:0 cyclo ?8c(13.8%)and summed feature 8(C18:1 ?7c and/or C18:1 ?6c)(54.3%).The main respiratory quinone is Q-11.The main polar lipids are DPG(diphosphatidylglycerol),PG(phosphatidylglycerol),PME(phosphatidylmonom ethylethanolamine),PE(phosphatidylethanolamine),PC(phosphatidylcholine)and AL(not identified amino lipids).According to phylogenetic analysis and physiological and biochemical data,the strain HXT-9 represents a new species in the genus Parvibaculum,whose name is Parvibaculum sedimenti sp.nov.HXT-9T(=CCTCC AB 2019273T=KCTC 72547T). |
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