Domestication And Screening Of High-Temperature Lignin Degradtion Bacteria And Its Application In Sheep Manure-Mushroom Residue Composting

With the rapid development of livestock and poultry breeding in China,the pollution of livestock and poultry manure is becoming more and more serious.Mixed composting of livestock and poultry manure with lignocellulosic raw materials,such as crop straw,bacterial residue and forestry waste,is an important technical way to realize the harmless,resource and recycling of livestock and poultry manure.However,in the process of composting,lignocellulosic components in composting materials are difficult to degrade,resulting in long fermentation cycle,incomplete use of materials,and low degree of decay.Therefore,the use of high efficiency lignin degradation bacteria to promote the degradation of lignocellulose,improve the compost maturity and utilization rate of feces material,so as to effectively solve the pollution problem of livestock and poultry feces.In this paper,we obtained a group of strains with high temperature tolerance and high lignin degradation efficiency from a high temperature habitat rich in lignin through acclimation,combination and screening,and studied the effects of these strains on the fermentation of sheep dung and fungal residue composting and the composting of cellulose and lignin.The results are as follows:(1)Lignin was the only carbon source and was acclimated and screened at 50℃.A group of high efficiency lignin degrading bacteria was obtained and named as SD.The degradation rate of lignin reached 41.60%at high temperature(50℃).(2)Isolation by pure culture,a total of 7 cultivable strains were obtained,named SD-1,SD-4,SD-6,SD-9.SD-14,SD-18 and SD-22,their lignin degradation rates under condition of 50℃ are 8.50%,7.54%,7.92%,4.29%,13.95%,16.72% and 33.75%,respectively.After 16 S r DNA sequence homology comparison,these seven strains were initially identified as Aneurinibacillus humi of the Aneurinibacillus,Paenibacillus cookii of the Paenibacillus,Bacillus ginsengihumi;Bacillus tequilensis and Bacillus sp.of the Bacillus,and Brevibacterium sp.of the Brevibacterium.(3)The lignin-degrading microbial agent Z made by SD strain was added to the mixed compost of sheep dung and fungus residue.The results showed that the pile entered the high temperature period for 48 hours,the highest temperature reached 55.47℃,and the high temperature period lasted for 13 d.Compared with the blank control group,the compost temperature entered the high temperature period 24 h earlier,the duration was longer by 8 days,and the maximum temperature increased by 2.80℃.By the end of composting,the degradation rates of cellulose and lignin in the pile were 56.77% and 27.17%,respectively,which were significantly higher than the blank control group;the C/N ratio of the pile was 14.46,and total nitrogen,available phosphorus and available potassium were also significant higher than the blank control group.The number of effective viable bacteria,the number of fecal coliforms,and the mortality of roundworm eggs all meet the standards of the agricultural industry.Therefore,the addition of microbial agent Z during the composting process can increase the temperature of the pile,effectively degrade cellulose and lignin,speed up the composting process,and improve the quality of the compost.(4)Using high-throughput sequencing technology to study the composition of microbial communities at different stages of composting,the results are as follows: Alpha diversity results show that the addition of microbial inoculants increases the abundance and diversity of bacterial and fungal flora in the compost.The Beta diversity results showed that with the progress of composting,the bacterial and fungal communities showed obvious temporal succession patterns during the composting process.The analysis of the flora structure of each sample showed that,at the level of phyla classification,Proteobacteria(13.34%-57.38%),Firmicutes(6.50%-61.11%),Actinobacteriota(7.44%-48.68%),Chloroflexi(0.44%-29.75%)and Bacteroidota(1.73%-28.58%)are the dominant bacterial groups;Ascomycota(71.30%-99.80%),Basidiomycota(0.16%-21.50%)are The dominant group of fungi.At the level of genus classification,in the initial stage of composting(0 d),the dominant genera of bacterial groups mainly include Pseudomonas,Corynebacterium,Escherichia-Shigella,and Glutabacterium.The dominant genera of the fungal group include Aspergillus,Cephaliophora,Trichoderma,Penicillium,Microascus and Chrysosporium;the dominant genera of the bacterial group includes Pseudomonas in the early stage of the high temperature stage(3rd d),Corynebacterium,Rhodococcus,Paenibacillus,Methylocaldum,Escherichia-Shigella,Bacillus,Romboutsia,and Solibacillus.The dominant genera of the fungal group include Aspergillus,Ascobolus and Trichoderma;at the end of the high temperature period(9th d),the dominant genera of the bacterial group are Corynebacterium,Romboutsia,Hydrogenispora,and Rummeliibacillus,Bacillus,Pseudomonas.The dominant genera of the fungal group included Aspergillus,Scedosporium,Volvariella,and Talaromyces;the dominant genera of each test group(CK,Z,R,RZ)changed after the composting was completed(on the 32 nd day).The dominant genera of the bacterial group mainly include Actinocorallia,Ruminofilibacter,Rhodococcus,Streptomyces,Truepera and Nocardia,and Candidatus＿Chloroploca.The dominant genera of the fungal group include Cephaliophora,Ascomycota unclassified genera,Ascobolus unclassified genera,Pseudallescheria,Zopfiella,Iodophanus,Leucothecium;Aspergillus and Kernia.(5)Using redundant correlation analysis and correlation Pheatmap to analyze the correlation between environmental factors and microbial communities,the results show that organic carbon(TOC)is the main factor affecting the composition of microbial communities in the composting process.Other environmental factors such as reactor p H,temperature,total nitrogen,total phosphorus,total potassium and C/N ratio also have a certain impact on community composition.Environmental factors such as temperature,p H and C/N ratio are compatible with Aestuariicella,Actinomadura,Escherichia-Shigella,Glutamicibacter,Corynebacterium,Dietzia,Brevibacterium,Brachybacterium,Facklamia,Enterococcus,Solibacillus and other bacterial groups;Aspergillus,Thermomyces,Pseudallescheria,Penicillium,Microascus,Acaulium,Pithoascus,Trichosporon,Acremonium,etc.have significant correlations.The enzyme activity of the lignocellulose degrading enzyme system has a significant correlation with the microbial community composition at the end of the composting high temperature stage(9th day).It is related to bacterial groups such as Hyphomicrobiaceae,Peptostreptococcaceae,Alcaligenaceae,Methylococcaceae,Rhodothermaceae,Hungateiclostridiaceae,Gracilibacteraceae,Hydrogenispora,Bacillus,Lutispora,Actinomadura,Sneathiella and Aestuariicella;Talaromyces,Mortierella,Trichoderma,Wallemia,Aspergillus,Thermomyces,Beauveria and Acrostalagmus have significant correlations.