| With the development of livestock and poultry farming,antibiotics are used in large quantities for the control of animal diseases.Antibiotics,drug-resistant bacteria and drug-resistant genes in livestock and poultry manure enter the environment,posing a threat to human public health security.Aerobic composting is a common way to realize the harmlessness of livestock and poultry manure,which has the advantages of short composting cycle and high maturity.In this paper,florfenicol,a widely used antimicrobial drug in farms,was used as the research object,and aerobic composting was carried out with waste rod-livestock manure as raw material to explore the reduction effect of florfenicol and its drug resistance genes during aerobic composting,and to discuss the correlation between the change of drug resistance gene abundance and the change of bacterial population.The main research results are as follows:(1)A high performance liquid chromatography-mass spectrometry method was developed for the determination of florfenicol in chicken manure.After freeze-drying,chicken manure was extracted by ammonia-ethyl acetate and purified by MCX solid phase extraction column.The results showed that there was a good linear relationship between the peak area and the concentration in the range of 0 ng/m L～200 ng/m L,and the correlation coefficient R~2=0.99933.When the concentration of florfenicol in chicken manure was 1μg/kg,10μg/kg and 100μg/kg,the average recovery rate was73.75%～81.78%,the intra-day relative standard deviation was 2.14%～3.75%,and the inter-day relative standard deviation was 6.49%～9.08%.The detection limit of this method is 0.01μg/kg,and the quantitative limit is 0.03μg/kg.The reduction effect of aerobic composting on florfenicol was explored by detecting the content of florfenicol in composting samples at different periods.The results showed that the content of florfenicol decreased significantly after 30 days of aerobic composting,and the degradation rate reached 94.45%.The degradation mainly occurred in the heating period and high temperature period of composting,indicating that the high temperature during composting could effectively reduce the content of florfenicol.(2)Using chicken manure and edible mushroom bran as raw materials,composting experiments were designed and composting indexes were tested.The results showed that the compost entered the high temperature period on the day 3 of composting,and the high temperature stage lasted for 10 days with the highest temperature of 53.6℃.The temperature of the pile was basically consistent with the ambient temperature after 23 days,when the compost had entered a relatively stable state.During the composting process,the contents of humus,huminic acid and fulvic acid in the pile showed a trend of first decreasing and then increasing.At the end of composting,the humus content was 7.36 g/kg,which increased by 7.88%compared with the beginning of composting.As the composting progressed,the lignocellulose content gradually decreased.The degradation rates of cellulose,lignin,and hemicellulose were 40.26%,20.52%,and 32.19%,respectively,and their degradation mainly occurred at the high and cool-down stages.The seed germination index(GI)integrally reflected the phytotoxicity of the compost.At the beginning of composting,the GI was 67.47%.When the composting was finished,the GI was 229.58%,indicating that this composting had fully reached the decomposition conditions.(3)Drug-resistant Escherichia coli and Staphylococcus were screened from chicken manure,and 10 strains of Staphylococcus and 9 strains of Escherichia coli were obtained and tested for florfenicol resistance genes.The results showed that five Escherichia coli strains were detected with the flo R gene,10 strains of Staphylococcus were detected with the optr A,cfr and fex B genes,and seven strains of Staphylococcus were detected with the fex A gene.The 16S r RNA gene and florfenicol resistance gene(flo R,optr A,cfr,fex A,fex B)in compost samples were quantitatively analyzed by real-time fluorescence quantitative PCR,and their relative abundance was calculated.The experimental results showed that flo R,optr A,cfr,fex A and fex B could not be detected at the high temperature stage of composting,indicating that aerobic composting had a significant effect on the reduction of florfenicolresistance gene.(4)The changes of microbial community structure during composting were analyzed by 16S r RNA and ITS high-throughput sequencing techniques.The results showed that the Chao1 index and Shannon index of bacteria were significantly higher at the end of composting than at the beginning of composting,indicating that aerobic composting of chicken manure could improve the diversity of bacterial communities.The Chao1 index of fungi increased,but the difference between the Shannon index and the beginning of composting was small,indicating that this composting changed the diversity of fungal communities less.The results of bacterial community composition at the phylum level showed that Protebacteria,Bacteroidota and Firmicutes were the dominant phyla during the warming period.Protebacteria,Bacteroidota,Actinobacteriota,Patescibacteria and Gemmationadota were the dominant bacteria in the maturity stage,and the bacterial community structure changed significantly during composting.Planctomycetota and Gemmationadota were not detected in chicken manure,while the relative abundance of Planctomycetota and Gemmationadota increased and the relative abundance of Firmicutes decreased during the heating and high temperature periods.Combined with the trend of ablation of florfenicol and its resistance genes,it suggests that the ablation of florfenicol resistance genes is related to Firmicutes and that the florfenicol degrading bacteria may belong to Planctomycetota and Gemmationadota.The results of fungal community composition at the phylum level showed that Ascomycota,Basidiomycota and Basidiomycota were dominant in the early stage of composting.At the maturity stage,the relative abundance of Ascomycota,Basidiomycota and Basidiomycota did not change much compared with the initial stage,indicating that compost had no significant effect on fungal community structure. |
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