Study On Microbial Diversity And Antibiotic Resistance Gene Changes During Degradation Of Cow Manure In Ectopic Fermentation Bed

With the rapid development of intensive large-scale dairy farming,the amount of fecal sewage in the breeding process has also increased significantly.The direct discharge or untreated recycling of fecal sewage will not only pollute the ecological environment,but seriously restrict the sustainable and healthy development of the dairy farming industry.Therefore,the harmless treatment and resource utilization of fecal sewage have become an urgent problem to be solved in the dairy farming industry.In this study,the Ectopic fermentation bed(EFB)was used as the research object,and divided into two groups: A and B(group A: 1d composting and group B:2d composting).The changes of microbial diversity and Antibiotic resistance gene(ARGs)during the dynamic degradation of cow feces were studied.The specific research results are as follows:1.Changes of microbial diversity during fecal and sewage degradation in ectopic fermentation bed16s rRNA high-throughput sequencing technology was used to analyze the changes of microbial community structure in the process of manure degradation.It was found that the main phylum in the fermentation process were Firmicutes,Proteobacteria,Bacteroidetes,Chloroflexi and Actinobacteria.Among them,in the early stage(0-12 d),Firmicutes reached more than 60%,in the middle stage(19-40d),Chloroflexi,Proteobacteria and Actinobacteria accounted for more than 50%,and in the later stage(47-54 d),Bacteroidetes and Firmicutes accounted for about40%.Succiniclasticum,Butyrivibrio,Prevotella,Clostridium,Turicibacter,Steroidobacter and Ruminofilibacter are the genera with high abundance in the fermentation process.The results of Alpha diversity analysis showed that Chao1,Observed-species and Shannon index did not change significantly from 0 to 40 d during the early stage of fermentation,but increased significantly after 40 d,indicating that the species richness in the late stage(40-61 d)was higher than that in the middle stage(0-40 d)before fermentation.The Simpson index remained stable during the whole fermentation process,indicating that the microbial uniformity of the fermentation bed changed little during the whole fermentation process.Based on the comprehensive analysis,it was deduced that the microbial diversity was increased during the whole fermentation process.In the late stage of fermentation(47–61 d),more microorganisms with inhibited activity in the early and middle stages appeared,but their relative abundance was low,thus exerting little influence on the uniformity of the overall microbial community.Through PCo A analysis,it was concluded that fermentation time was the main factor affecting the difference in microbial composition of the fermentation bed,and the change of fermentation time was mainly reflected in the temperature change of the fermentation bed.Therefore,it was speculated that temperature was the main factor affecting the microbial diversity in the fermentation process,and it was found that the turnover frequency had no significant effect on the microbial composition of the fermentation bed.2.Removal effect of ARGs in ectopic fermentation bed degraded by manureThe qualitative detection of ARGs was carried out on the degradation products of fecal sewage and fecal sewage in dairy farms by PCR.Twenty-four common ARGs and four mobile genetic elements including tetracyclines,sulfonamides,aminoglycosides,beta-lactams,macrolides,lincosamides and type b sterptogramin(MLSB)and fluoroquinolone-chloramphenicol resistance gene(FCA),PCR detection was performed.A total of 11 ARGs and 2 transposon genes(Tn916and ISCR1)from tet W,tet G,sul2,sul1,aac(6’)-ib-cr,fex A,bla TEM-1,erm X,erm Q,optr A,and isa(E)were detected in cattle manure samples.These results indicated that there existed a variety of ARGs and Mobile genetic elements(MGEs)in the feces of dairy farms,which would pose a potential threat to the environment if the feces were directly discharged or treated improperly.The above genes were also detected in the detection of two groups of fermentation bed samples during the fermentation process,and the presence of intⅠ1 was also detected,indicating that fecal sewage was the main source of ARGs in the fermentation bed.However,the types of ARGs detected in the samples on days 33 and 40 were decreased.Only tet G,tet W,sul1,sul2 and aac(6’)-ib-cr were detected in the samples of groups A and B,while MGEs was not detected,suggesting that the ectopic fermentation bed had certain effects on the removal of ARGs and MGEs in feces.Based on the situation that sulfadiazine,penicillin,ampicillin,oxytetracycline and lincomycin were commonly used for disease treatment in our dairy farm,the q PCR technology was used to detect six ARGs(sul1,sul2,tet G,tet W,bla TEM-1 and erm Q)and intⅠ1.The removal rates of bla TEM-1,intⅠ1 and erm Q in Group A were > 99%(the relative content of Args was below the detection limit)at the end of the fermentation compared with that on day 0.The removal rates of tet W,tet G and sul1 were 87.88%,16.69% and 54.59% respectively,but the removal rate of sul2 increased more than 4 times.The removal rates of bla TEM-1,intⅠ1,erm Q,tet W,sul1 and sul2 in Group B were 97.89%,99.92%,41.90%,98.43%,67.53% and57.14%,respectively.tet G increased by 13.11%.These results indicated that the ectopic fermentation bed had a good removal effect of common ARGs in fecal sewage,and different stacking times had no significant effect on the removal effect of ARGs.In this study,the microbial diversity in the ectopic fermentation process was analyzed,and the dynamic change laws of dominant phylum and genus in different stages were discussed.This study laid a foundation for further clarifying the role of microorganisms in the fecal sewage degradation process of the ectopic fermentation bed,and at the same time helped to identify the functional microbial strains that can improve the fermentation effect,optimize the combination of fermentation agents,and improve the effect of EFB on the degradation of fecal sewage.By studying the changes in the relative abundance of ARGs during the degradation of fecal sewage,it was found that the application of EFB to degrade dairy cow fecal sewage could remove most of ARGs in the fecal sewage,and it was speculated that temperature might be the key factor affecting the reduction of ARGs,but its action mechanism required further study.