Screening Of Bacillus Subtilis Strain J530 With Urease Inhibitory Activity And Its Effect On Reducing Ammonia Remission In Poultry Manure

The ammonia produced by poultry manure not only endangers the health of poultry and breeders,but also pollutes the surrounding environment,causing increasingly serious harm to the atmosphere,soil,and water.Controlling ammonia emissions from poultry manure is of great significance to the sustainable development of the poultry breeding industry and the improvement of air quality.In this study,we screened Bacillus that efficiently utilizes ammonia nitrogen and produces urease inhibitors,which can be used for ammonia reduction in poultry manure and provide a new source of bacteria for the development of composting bacteria or microbial feed additives.Take fresh chicken manure as a sample,use ammonium sulfate as the sole nitrogen source for continuous subculture enrichment and isolation of Bacillus,inoculate the isolated Bacillus in a medium with known ammonium nitrogen content for cultivation,and select highly efficient ammonium nitrogen from it Use strains;then inoculate urea phenol red medium plates and urea shake flask medium with strains that efficiently utilize ammonium nitrogen,and select strains that do not produce urease;finally use the crude soybean urease extract as a reference to screen for soybean urease activity.The strains with the highest inhibition rate,and use physiological,biochemical and molecular biology methods to identify their strains.After ammonium nitrogen enrichment culture,562 strains that can grow well with ammonium nitrogen were isolated and obtained,of which 218 strains with ammonium nitrogen utilization rate above 85%;23 strains were screened for non-urease production,and then 6 strains with an inhibition rate of urease activity above 50%.Among them,the ammonium nitrogen utilization rate of strain J530 was 89.6%,and its fermentation supernatant had the highest inhibition rate on soybean urease activity,reaching 99.3%.The J530 strain was identified as Bacillus subtilis.Taking spore yield as an indicator,single factor experiment and orthogonal experiment were used to optimize the fermentation conditions of strain J530 in shake flask.After optimization,the optimal fermentation conditions of J530 are corn flour 1.5%,soybean meal 1.5%,MgSO4·7H2O 0.3%,Na2HPO4 0.2%,KH2PO4 0.2%,fermentation time 36 h,filling volume 50 mL/250 mL,initial pH 8.0,inoculation volume 4.0%,culture temperature 37 ?,shaker speed 150 r/min.After optimization,the spore concentration(7.3×109cfu/mL)of the shake flask fermentation broth of strain J530 was increased by 21.32 times(P<0.01).compared with that before optimization(3.27×108 cfu/mL).In order to explore the influence of J530 inoculum on the related indicators of chicken manure ammonia reduction,the prepared J530 inoculum was inoculated with fresh broiler manure,incubated at 30? for 5 days,and the pH value of chicken manure,the number of bacillus,urease activity,and ammonia release were measured.And the content of ammonia nitrogen,urea,uric acid,total nitrogen and so on.The test showed that after 5 days of inoculation of broiler feces in vitro with J530,the pH value of feces in the test group was reduced by 1.35(P<0.05),the number of Bacillus was increased by 60.29 times(P<0.01),and the urease activity was reduced by 80.78%(P<0.01),ammonia release decreased by 67.78%(P<0.01),ammonia nitrogen content decreased by 48.57%(P<0.01),urea,uric acid and total nitrogen content increased by 24.79%(P<0.05),0.91%(P>0.05)and 8.53%(P>0.05).In order to explore the application effect of J530 inoculum as a feed additive,a total of 90 Hong y u d380 broilers about 20 days old were selected and divided into 3 groups with 30 in each group.The control group was fed with basic diet,test group 1 and test group 2 add 0.1%and 1.0%J530 inoculants to the basal diet,and the feeding test was 25 days in total.On 0,2,4,6,8,10,15,20,25 days,measure the pH of each group of broiler feces,the number of Bacillus,urease activity,ammonia nitrogen content,ammonia release,urea nitrogen,uric acid content and other indicators;At the end of the test period,slaughter was carried out,and blood ammonia,serum urea nitrogen,and uric acid content were measured after intravenous blood sampling before slaughter.The results showed that during the 0-6d feeding period,the difference between the parameters in the feces of the two test groups and the control group gradually increased,and reached the maximum on the 8th day,after which the difference stabilized.Compared with the control group,the average pH of the feces of broiler chickens in the 8-25d test group 1 decreased by 0.58(P<0.05),the average number of Bacillus increased by 101.72%(P<0.01),urease activity,ammonia nitrogen content and ammonia the average values of air release amount decreased by 60.14%(P<0.01),27.04%(P<0.01)and 38.42%(P<0.01),respectively,and the average content of urea nitrogen and uric acid increased by 39.13%(P<0.01)and 2.56%(P>0.05);compared with the control group,the average pH of the feces of broiler chickens in the 8-25d test group 2 decreased by 0.61(P<0.05),and the average number of Bacillus increased by 99.12%(P<0.01).The average values of urease activity,ammonia nitrogen content and ammonia release amount were reduced by 64.01%(P<0.01),30.94%(P<0.01)and 38.08%(P<0.01),respectively.The average contents of urea nitrogen and uric acid were reduced respectively increased by 41.00%(P<0.01),and 1.61%(P>0.05).At the end of the experiment,the number of Bacillus and Lactobacillus in the feces of the experimental group 1 increased by 87.69%(P<0.01)and 12.27%(P<0.05),respectively,and the number of Escherichia coli and Salmonella decreased by 9.65%(P<0.05)and 13.95%(P<0.05);the number of Bacillus and Lactobacillus in the feces of the test group 2 increased by 89.63%(P<0.01)and 11.62%(P<0.05)respectively compared with the control group.Escherichia coli and Salmonella were respectively it was reduced by 10.03%(P<0.05)and 15.06%(P<0.05).Poultry breeding has a good application prospect.It is beneficial to environmental protection in terms of fecal ammonia reduction,and it can also significantly reduce intestinal ammonia and blood ammonia,which is beneficial to poultry.Healthy and improve the growth performance of poultry.This research provides experimental basis for further industrial application of J530.Compared with the control group,the average levels of serum urea nitrogen,uric acid and blood ammonia in the test group 1 were reduced by 9.76%(P<0.05),1.10%(P>0.05)and 41.98%(P<0.01),respectively;the test group 2 Compared with the control group,the average content of urea nitrogen and blood ammonia decreased by 11.38%(P<0.05)and 47.79%(P<0.01),and the average serum uric acid content increased by 0.50%(P>0.05).However,there is no significant difference between the test group 1 and the test group 2 in the above parameters,suggesting that the addition amount of bacterial agents only needs to be 0.1%.In summary,J530 microbial agent used as a feed additive in the poultry breeding industry has the effect of reducing ammonia emissions,while also significantly reducing serum urea nitrogen and blood ammonia,which is beneficial to the health of poultry and improves the growth performance of poultry.This research provides experimental basis for the further application of J530.