| In recent years,the fast-paced progress in animal husbandry has resulted in the production of substantial amounts of livestock and poultry manure.Unfortunately,this byproduct is frequently neglected,leading to considerable environmental contamination and wastage of resources.By utilizing the fermentation process of microorganisms,composting technology can convert the unstable organic matter present in livestock and poultry manure into a more stable organic matter,thereby achieving harmless treatment and fertilizer utilization of the manure.Fertilization can effectively and quickly achieve harmless treatment of livestock and poultry manure,and transform it into a usable organic fertilizer resource,making it the preferred option for of livestock and poultry manure.In this experiment,beneficial bacteria screened from fresh pig manure were used as exogenous inoculants added to the livestock manure heap,and explored the impact of screened exogenous bacteria on microbial diversity and metabolites in the manure heap through conventional fermentation and high-throughput sequencing technology.The main research results are as follows:(1)Screening and characterization of beneficial bacteria in fresh pig manure.Two strains of bacteria were screened from fresh Dianlu pig manure samples,and identified through colony morphology,Gram staining,16 S r DNA homology analysis,and construction of a phylogenetic tree.The results showed that two strains were screened in the experiment,which were Gram-positive bacteria with gray-white,circular,smooth and neat edges on LB agar after 24-hour cultivation in a constant temperature incubator at 30℃.Further identification by biological technology revealed that both strains belonged to the Bacillus,and were named Bacillus YF-13 and Bacillus YF-15.After sequencing,the accession numbers of YF-13 and YF-15 in Gen Bank were obtained as OQ629339 and OP349652,respectively.The study of bacterial growth characteristics indicated that both strains could grow normally in LB culture medium.The antibiotic sensitivity test showed that YF-13 was insensitive to compound sulfamethoxazole,moderately sensitive to spectinomycin and polymyxomycin,and highly sensitive to 11 other antibiotics.Phylogenetic and homology analysis revealed that the similarity between the two strains and the strain Bacillus-aryabhattai(MT525305.1)was as high as 98%,making it impossible to determine the genus relationship of Bacillus.YF-13 strain was further selected for genome sequencing analysis,and the results showed that the average GC content of effective data was 38.19%,and more than 96.75% of the base sequencing accuracy was 99%,and more than 91.23% of the base sequencing accuracy was 99.9%.The K-mer distribution was generally in a Poisson distribution,and there are two obvious peaks due to the uneven curve caused by frequency fluctuations.A total of 2984 genes were annotated in the GO database for biological process functions,2169 genes for cellular components,and 583 genes for molecular functions.YF-13 strain was identified as a Bacillus megaterium using NR database annotation.(2)The effect of Bacillus YF-13 and YF-15 on the fermentation of a mixture of duck and horse manure.The screened Bacillus strains were added to the horse and duck manure mixture for fermentation.The results showed that the YF-13-added group had a faster heating rate than the control group during the fermentation process,reaching over 50°C on the second day of the composting,and the high-temperature period(>55°C)lasted for 10 days with a maximum temperature of 61.16°C,and the high-temperature period(>60°C)lasted for 5 days,while the temperature of both YF-15 group and control group lasted for 3 days with > 50°C.During the fermentation process,covering the manure using black film affected the evaporation of water,but the overall moisture content showed a downward trend.The p H of the manure showed a trend of rising first and then falling.Among the three groups,the YF-13 group had a significantly higher p H change than the control group,while the difference between the YF-15 group and the control group was not significant.In the whole composting process,the temperature,duration,and other physical and chemical parameters of Bacillus YF-13 were superior than those of YF-15 and control groups.(3)The effects of YF-13 and YF-15 on the microbial community structure and metabolites of a mixture of duck and horse manure.The microbial diversity and non-targeted metabolomics analysis based on the OUT level with 97% similarity were across 27 samples,yielding 39 phyla,110 classes,296 orders,549 families,1176 genera,and 2181 species.One-way analysis of variance showed no significant difference in α diversity(p>0.05).At the phylum level,the dominant phyla were sequentially Proteobacteria,Bacteroidetes,Chloroflexi,Actinobacteria,and Firmicutes.At the genus level,the dominant genera with relative abundance greater than 1 were SBR1031,Genus Rumininum,Phyllostachys,Rhizobiaceae,Pseudomonas,and Actinomyces..(4)The non-targeted metabolomics identified metabolic differential compounds.after data preprocessing,there were 572 positive ions accounting for 17.39% and 277 negative ions accounting for 8.93%,suggesting qualified data.A total of 465 metabolites were annotated by KEGG classification,accounting for 54.77%,including 4.6% antibiotics,9.2% hormones and transmitters,10.77% steroids,and43.08% lipids.Among the 224 metabolites annotated by KEGG pathway,the ABC transporters pathway had the largest number of participating metabolites among the top 20 pathways,while the number of metabolites involved in the Lysine degradation pathway is fewest.Comparing the control group,differential metabolite analysis revealed YF13 had 52 differential metabolites,including 22 down-regulated metabolites and 13 up-regulated metabolites,while YD15 had 123 differential metabolites,including 54 down-regulated metabolites and 25 up-regulated metabolites.Correlation analysis suggested that the genus Photinia and the phylum Actinobacteria played important roles in the microbial metabolism during the composting process.In summary,we screened two strains of Bacillus YF-13 and YF-15 from fresh pig manure,which had good biological characteristics and could grow in livestock manure containing antibiotics.Especially,strain YF-13 had the advantages of fast growth,quickly rising temperature and improved composting maturation,and can be wildey used as a beneficial bacteria to improve the degradation efficiency and quality of compost. |
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