Isolation Of Ammonium Nitrogen Degrading Microoganism Strains And Determine Its Functional Genes

Among the pollution emissions from livestock and poultry farming,ammonia emission is the most concerned issue so far.In recent years,there has been a lot of research on ammonia reduction at home and abroad.Microorganisms have been the focus of attention due to their high efficiency,low cost and no pollution.There were a lot of basic researches on the screening of ammonium nitrogen degrading microoganism strains,but the key functional gene expression and mechanism of ammonium nitrogen degrading microoganism strains are still unclear.In this study,we screen strains from cecal contents and feces of laying hens that can efficiently degrade ammonia nitrogen to explore the mechanism of ammonia reduction and find key genes that degrade ammonia nitrogen.Provide a basis for seeking efficient ammonia reduction measures for laying hens.This study is divided into three parts.In the first experiment,10 strains of ammonianitrogen-tolerant bacteria were screened by enrichment culture and gradient acclimation.The strain with strong ammonia nitrogen degradation ability was further screened by ammonia nitrogen degradation test.In the second experiment,two strains of ammonia-nitrogendegrading bacteria were selected for the in vitro fermentation test.We set three treatments which included control group(CK),the Bacillus coagulans group(B1)and the Enterococcus faecium group(C2)and each treatment had three repetitions.We determined the ammonia yield and analyzed the physical and chemical indicators,the total number of bacteria and the transcriptional expression of ammonia assimilation genes to explore the ability of strains to reduce ammonia gas and its mechanism.In the third experiment,the expression of ammonium assimilation genes of strains B1 and C2 under ammonia-nitrogen stress was determined by real-time fluorescent PCR,and the key genes for the degradation of ammonia nitrogen in the strain were determined.The E.coli overexpression vector were constructed and that were added in the vitro fermentation test.We set five treatments which included control group(CK),the empty vector group(PET),the GDH gene overexpression group(GDH),the GS gene overexpression group(GS),and the GMPS gene overexpression group(GMPS)and each treatment had three repetitions.We determined the ammonia yield and analyzed the physical and chemical indicators,the total number of bacteria and the transcriptional expression of ammonia assimilation genes.The effects of GDH gene,GS gene and GMPS gene on ammonia emission of laying hens were verified,and the mechanism of ammonia reduction of the strains was further explained from the genetic point of view.The main results are as follows:(1)10 of ammonia-tolerant bacteria were selected,which belonged to Pediococcus acidilactici strain,Lactobacillus johnsonii strain,Pediococcus acidilactici strain,Lactobacillus rhamnosus strain LS8,Bacillus coagu Lans strain,Bacillus coagu Lans strain,Bacillus coagu Lans strain,Enterococcus faecium strain,Enterococcus faecium strain,Bacillus subtilis strain.Bacillus coagulans(B1)and Enterococcus faecium(C2)have good degradation ability to ammonia nitrogen and have good tolerance to high ammonia nitrogen environment.The degradation rates were 99.78% and 97.00%,respectively,which is significantly higher than other strains(P<0.05).(2)Adding Enterococcus faecium(C2)and Bacillus coagulans(B1)significantly reduced ammonia emissions from the fermentation broth,with a reduction ratio of 53.60% and 31.38%,respectively(P<0.05).Compared with the control group,the addition of Enterococcus faecium(C2)and Bacillus coagulans(B1)significantly reduced urease activity(P<0.05),and increased the total number of bacteria(P<0.05).The expression levels of GDH,GS,GMPS,and GOGAT genes in the B1 and C2 groups were up-regulated relative to the control group(P<0.05).(3)Under the stress of ammonia nitrogen,the GMPS gene and GS gene of B1 strain were expressed in large amount(P<0.05),and the GDH gene of C2 strain was expressed in large amount(P<0.05).Combined with in vitro fermentation test,GDH gene may be the key gene for the reduction of ammonia gas in the chicken body by Enterococcus faecium,and the gene of GS,GMPS may be the key genes for the reduction of ammonia by Bacillus coagulans.Three overexpressing strains expressing GDH,GS and GMPS genes were constructed separately.(4)The GDH,GMPS and GS groups significantly reduced ammonia production(P<0.05),which decreased by 55.5%,43.6%,and 54.8%,respectively.The ammonia production in GDH and GS groups was significantly lower than that in PET(P<0.05).The p H of GS group was significantly lower than that of PET group(P<0.05).The urease activity and total nitrogen content were significantly higher than that of PET group(P<0.05).The nitrate content of GMPS group was significantly higher than that of PET(P<0.05).The urease activity of GDH group was significantly lower than that of the CK group(P<0.05).In summary,the Enterococcus faecium(C2)and Bacillus coagulans(B1)screened in this experiment can significantly reduce the production of cecal ammonia in laying hens,and the emission reduction efficiency can reach 53.60% and 31.38%.Enterococcus faecium(C2)and Bacillus coagulans(B1)promote the proliferation of microorganisms,inhibit the urease activity of microorganisms,and reduce ammonia emissions by regulating the expression of GDH gene and GS gene,respectively.