High-throughput Sequencing Of Compost Flora In Organic Fertilizer Plants And Study On Biological Characteristics Of Some Flora

In recent years,with the continuous development of large-scale and intensive farming in China's cattle industry,more and more pollutants such as cow manure have been produced.If not handled properly,it will cause pollution in air,water,soil and pathogenic microorganisms.Cow manure contains a large amount of cellulose and nutrients such as nitrogen,phosphorus,and potassium,and is a kind of biomass resource with good development prospects.At present,the main technology for harmless treatment of cow manure is composting technology,which can achieve the harmless,resourceful and reduction of cow manure pollutants.Composting technology refers to a technology that promotes the degradation of degradable organic substances in livestock and poultry manure through the metabolism of microorganisms,and the development of high-efficiency microbial agents for degrading cattle manure is conducive to improving the composting efficiency of livestock and poultry manure.In this study,16 S rRNA gene high-throughput sequencing was used to analyze the changes in bacterial composition during the composting process of cow manure and to understand the changes in the predominant flora during the composting process;Isolation,screening and identification of bacteria and determination of enzyme activity have screened out the predominant strains that have the effect of efficiently degrading cow manure,and provide the predominant strains for the research and development of microbial agents that efficiently degrade cow manure.The research results are as follows:1.High-throughput sequencing of 16 S rRNA genes was performed on 10 samples at different stages of cow manure composting,and a total of 1042384 original sequences were obtained.After optimization filtering,521192 high-quality sequences were obtained.All high-quality sequences were classified into 22 gates and 43 Class,96 orders,202 families,366 genera,515 species and 777 OTU.An analysis of the Alpha diversity of the bacterial community at a similar level of 97% found that during the composting process,the bacterial community diversity decreased first,then increased,and finally stabilized.The results of the composition of the sample flora showed that at the phylum classification level,the relativeabundance of the Firmicutes,Proteobacteria,Chloroflexi and Actinobacteria in the early stage of composting was relatively high,which was the main dominant phylum;with the composting The main dominant bacteria in the middle and late stages of composting were Firmicutes,Bacteroidetes,Actinobacteria,Proteobacteria and Chloroflexi.At the family classification level,Planococcaceae,Bacillaceae,Flavobacteriaceae,Anaerolineaceae and Trueperaceae are the main dominant families in the composting process.At the genus classification level,the predominant genus of bacteria in the early stage of composting is Sporosarcina,Truepera,Bacillus,Pseudogracilibacillus,Luteibacter,Actinomadura,Pseudomonas,Thermobifida.2.Through cellulose Congo red medium and casein medium,57 strains were isolated and purified from cow manure compost samples.Colony morphology observation,staining microscopy,biochemical identification and PCR amplification identification of 16 S rRNA gene,57 Among the strains,56 are bacteria of the genus Bacillus sp.and one is bacteria of the genus Lysinibacillus sp.The sequence similarity of the 16 S rRNA gene sequences of 57 strains and their genus bacteria is above 98%.Further identification using multiplex PCR revealed that strain X10 was Bacillus amyloliquefaciens,strains S15 and FD2 were Bacillus subtilis,and 18 strains were Bacillus pumilus.3.The 57 strains obtained by separation and purification were subjected to cellulase and protease enzyme activity screening tests,and 10 strains of cellulase and 8 strains with higher protease enzyme activity were screened;CMC enzyme saccharification force method and Folin were selected respectively Reagent colorimetric method was used to conduct further enzymatic activity re-screening on the strains obtained from the initial screening.The strain with the highest cellulase activity was X10,and its enzyme activity was 31.9 U/mL;the strain with the highest protease activity was S3,which The enzyme activity was 40.83 U/mL.