A strain of Bacillus amyloliquefaciens that inhibits bacterial growth and reduces ammonia nitrogen and its application

Abstract

This invention belongs to the field of biotechnology and discloses a strain of Bacillus amyloliquefaciens ZJ02 that inhibits bacterial growth and reduces ammonia nitrogen. Its accession number is CCTCC NO: M20252399, the accession date is October 30, 2025, and the depositary institution is the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. ZJ02 exhibits significant antibacterial effects against Escherichia coli and Salmonella, with an inhibition zone size of 18.11 mm against Salmonella (SA) after 24 hours. Compared to other strains, it demonstrates superior ammonia nitrogen degradation under the same conditions, achieving a degradation rate of 76.58%, reaching as high as 87.57% at an initial ammonia nitrogen concentration of 500 mg / L. Furthermore, it possesses excellent ability to reduce ammonia release from animal feces. In ammonia removal tests conducted in pig farm environments, compared to the control group, the NH3 concentration decreased by 60.27% on day 6, significantly outperforming Bacillus subtilis and Bacillus licheniformis. This invention also provides the application of this Bacillus amyloliquefaciens.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a strain of Bacillus amyloliquefaciens that inhibits bacterial growth and reduces ammonia nitrogen, and its applications. Background Technology

[0002] Modern livestock and poultry farming is gradually shifting from traditional feeding methods to modern intensive farming. However, air pollution caused by livestock and poultry manure has become a serious problem restricting the sustainable development of the industry. Livestock and poultry produce large amounts of manure during production, and manure piles release toxic and harmful gases, affecting livestock and poultry production, the living environment of surrounding residents, and their health. These harmful gases include many components, with NH3 being the main toxic gas emitted during livestock and poultry production. High concentrations of NH3 in the farming environment can reduce livestock and poultry production performance, induce respiratory diseases, and even lead to death. Therefore, solving the problem of NH3 emissions from livestock and poultry under intensive farming has become one of the most crucial issues in the livestock industry.

[0003] The treatment of malodorous gases in livestock and poultry manure relies on various intensive technological approaches, primarily categorized into three types: physical, chemical, and biological methods. Compared to traditional physical and chemical methods, biological methods, which primarily utilize probiotics, are widely favored due to their low cost, high safety, high efficiency, and ability to inhibit ammonia production at its source. Commonly used strains include Bacillus subtilis, yeast, and Bacillus subtilis. In addition to these, other types of bacteria may exist in nature, potentially offering even more effective reductions in ammonia release from animal manure compared to known strains.

[0004] Therefore, the technical problem to be solved in this case is: how to discover a strain of Bacillus amyloliquefaciens that has high antibacterial properties, degrades ammonia nitrogen, and reduces ammonia release from animal feces. Summary of the Invention

[0005] The purpose of this invention is to provide a Bacillus amyloliquefaciens ZJ02 that inhibits bacteria and reduces ammonia nitrogen, with accession number CCTCC NO: M20252399, accession date October 30, 2025, deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0006] In addition, the present invention also provides the application of this Bacillus amyloliquefaciens.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A strain of Bacillus amyloliquefaciens ZJ02, which inhibits bacterial growth and reduces ammonia nitrogen, has been deposited on October 30, 2025, with accession number CCTCC NO: M20252399. The depositary institution is the China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, China.

[0009] Furthermore, this invention discloses the application of Bacillus amyloliquefaciens as described above in reducing ammonia emissions from animal feces.

[0010] Preferably, the animal is one of a dog, a pig, or a cow.

[0011] Furthermore, this invention discloses the application of Bacillus amyloliquefaciens as described above in degrading ammonia nitrogen levels in polluted water bodies.

[0012] Furthermore, this invention discloses the application of Bacillus amyloliquefaciens as described above in the preparation of antibacterial agents.

[0013] Preferably, the bacteria is at least one of Escherichia coli and Salmonella.

[0014] Furthermore, the present invention discloses a deodorizing agent containing Bacillus amyloliquefaciens as described above.

[0015] Furthermore, the present invention discloses a wastewater treatment agent, characterized in that the wastewater treatment agent contains Bacillus amyloliquefaciens as described above.

[0016] Finally, the present invention also discloses an antibacterial agent, characterized in that the antibacterial agent contains Bacillus amyloliquefaciens as described above.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] The Bacillus amyloliquefaciens ZJ02 provided by this invention has a significant inhibitory effect on Escherichia coli and Salmonella. Specifically, the inhibition zone size against Salmonella (SA) is 18.11 mm after 24 hours. Furthermore, compared to other strains such as Lactobacillus plantarum and Lactobacillus acidophilus, it exhibits better ammonia nitrogen degradation under the same conditions, with an ammonia nitrogen degradation rate of 76.58%, reaching as high as 87.57% at an initial ammonia nitrogen concentration of 500 mg / L. Most importantly, it has an excellent ability to reduce ammonia release from animal feces. In ammonia removal tests in pig farm environments, compared to the control group, the NH3 concentration decreased by 60.27% on day 6, significantly better than Bacillus subtilis and Bacillus licheniformis. Attached Figure Description

[0019] Figure 1 The ammonia nitrogen standard curve used in Example 1;

[0020] Figure 2 This describes the colony morphology of Bacillus amyloliquefaciens ZJ02 on a solid culture medium in an embodiment of the present invention.

[0021] Figure 3 This is a Gram morphology diagram of Bacillus amyloliquefaciens ZJ02 observed under a microscope according to an embodiment of the present invention.

[0022] Figure 4 Phylogenetic tree diagram of the 16S rRNA gene of strain ZJ02;

[0023] Figure 5 The growth curve of strain ZJ02;

[0024] Figure 6 The ammonia nitrogen standard curve used in Example 4. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the described reagents are all commercially available reagents.

[0026] Strain information:

[0027] Bacillus amyloliquefaciens ZJ02, accession number CCTCC NO: M20252399, accession date October 30, 2025, deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0028] Lactobacillus acidophilus, Bacillus coagulans, Lactobacillus plantarum, and Enterococcus faecalis: purchased from Jiangxi Haoshiwo Biotechnology Co., Ltd.

[0029] Bacillus licheniformis and Bacillus subtilis: purchased from Changsha Green Leaf Biotechnology Co., Ltd.

[0030] Example 1: Isolation and Screening of Strains

[0031] 1.1 Culture medium and reagents:

[0032] Nutrient Broth (NB): 10 g / L peptone, 3.0 g / L beef extract, 5.0 g / L NaCl, pH 7.2±0.2, autoclaved at 121℃ for 15 min.

[0033] Nutrient agar (NA): peptone 10 g / L, beef extract 3.0 g / L, NaCl 5.0 g / L, agar 15.0 g / L, pH 7.3 ± 0.1, autoclaved at 121℃ for 15 min.

[0034] Ammonia nitrogen liquid culture medium (g / L): glucose 5.0 g, ammonium sulfate 0.2 g, sodium chloride 2 g, magnesium sulfate heptahydrate 0.5 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.5 g.

[0035] LB Broth (LB): Tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, pH 7.0 ± 0.1, autoclaved at 121°C for 15 min.

[0036] Mueller-Hinton Agar (MHA): 6.0 g / L beef extract, 1.5 g / L soluble starch, 17.5 g / L acid-hydrolyzed casein, 17.0 g / L agar, pH 7.3±0.1, autoclaved at 121℃ for 15 min.

[0037] 1.2 Initial screening and isolation of strains

[0038] Weigh 1g of Tibetan pig feces sample and place it in 9mL of sterile physiological saline. Mix the liquid in a vortex mixer for 30s to obtain a bacterial suspension. Place the bacterial suspension in an 80℃ water bath for 5min, remove and cool rapidly. Dilute the treated bacterial suspension 10-fold to 10 ... 5 ~10 7 Take 100 μl of the culture medium and spread it evenly on the nutrient agar medium. Incubate at 37°C for 20 h. Pick different single colonies that have been cultured on the nutrient agar plate and streak them 3 to 4 times. A total of 58 Bacillus strains were isolated and named ZJ01-ZJ58.

[0039] 2. Antibacterial test for rescreening

[0040] The common pathogenic bacterium Escherichia coli E2 was selected, and the inhibitory ability of the isolated Bacillus against the pathogenic bacterium was evaluated by the size of the inhibition zone diameter (mm).

[0041] 2.1 Preparation of test bacterial solutions

[0042] The isolated Bacillus strains ZJ01-ZJ58 were first cultured on plates, and then single colonies were picked using an inoculation loop and inoculated into nutrient broth medium. The culture was then incubated at 37°C and 200 r / min for 20 h to prepare the test bacterial suspension, with a bacterial count of 10⁻⁶. 8 ~10 9 CFU / ml, for later use.

[0043] 2.2 Preparation of Pathogens

[0044] Glycerol tubes containing pathogenic Escherichia coli E2 were inoculated into nutrient broth medium and cultured at 37°C and 200 rpm for 16-18 hours before use.

[0045] 2.3 Escherichia coli inhibition test

[0046] The antibacterial test was conducted using the double-layer agar diffusion method. First, approximately 10 mL of MHA medium was poured into a petri dish and allowed to solidify. Then, an Oxford cup was evenly placed on top of the MHA medium. Next, an appropriate amount of the cultured indicator bacteria was added to the MHA medium at approximately 50°C to achieve a bacterial concentration of approximately 1 x 10⁻⁶. 8 The concentration of cfu / mL was measured, and then 15 mL of MHA medium containing indicator bacteria was poured into the petri dish. After the MHA medium solidified, all Oxford cups were removed, and 100 μL of cultured Bacillus subtilis was added to each well. A negative control of physiological saline and a positive control of 5 μg / mL gentamicin sulfate were included. The mixture was incubated at 37℃ for 20 h, and the presence of inhibition zones was observed. If an inhibition zone was present, its diameter was measured, taking measurements from three directions in each well, and the average value was calculated as the size of the inhibition zone. The results are shown in Table 1 below.

[0047] Table 1 Results of antibacterial test

[0048]

[0049] 3. Secondary screening of ammonia nitrogen degradation capacity

[0050] 3.1 Preparation of the ammonia nitrogen standard curve

[0051] Preparation of the standard curve: Add 0.00, 0.50, 100, 2.00, 4.00, 6.00, 8.00, and 10.00 ml of ammonia nitrogen standard working solution to 50 mL colorimetric tubes, respectively. Add distilled water to the mark and mix well. Add 1 ml each of potassium sodium tartrate solution and Nessler's reagent to each tube. Incubate at room temperature for 10 min, and measure the absorbance at 420 nm. The standard curve is shown below. Figure 1 As shown.

[0052] 3.2. Strains with significant antibacterial activity were further screened using Nessler's reagent method.

[0053] Selected Bacillus strains with antibacterial effects were inoculated into 25 ml of LB liquid medium at a 1 vol% inoculum. After incubation at 37°C with shaking for 16 h, the cultured bacterial solution was added at a 0.2 vol% ratio to 100 ml of ammonia nitrogen medium with an initial ammonia nitrogen concentration of 200 mg / L. The CTRL group used ammonia nitrogen medium without bacterial inoculum. After 24 h, the ammonia nitrogen content of the water sample was measured: accurately pipette 10 ml of the water sample to be tested into a 50 mL stoppered colorimetric tube, perform color development according to the above steps, measure the absorbance value, and calculate the ammonia nitrogen content in the water sample according to the standard curve. If the water sample has high turbidity, it should be filtered first. If the ammonia nitrogen content of the water sample exceeds the range of the standard curve, it should be appropriately diluted before measurement. Ammonia nitrogen degradation rate = (initial ammonia nitrogen content - final ammonia nitrogen content) / initial ammonia nitrogen content * 100%.

[0054] The results are shown in Table 2. Five strains with degradation capabilities were screened out.

[0055] Table 2 Ammonia Reduction Rate Data Table

[0056] Group 24-hour ammonia reduction rate (%) CTRL group 11.93% Group ZJ01 9.12% Group ZJ02 76.67% ZJ14 group 37.02% ZJ15 group 55.70% ZJ17 Group 56.50%

[0057] As shown in Tables 1 and 2, ZJ02 was used for subsequent experiments based on the determination of antibacterial ability and ammonia nitrogen reduction ability.

[0058] Example 2: Identification and biological characteristics of ZJ02

[0059] 1. Colony morphology identification

[0060] The colony morphology of ZJ02 is as follows: Figure 2 As shown, strain ZJ02 exhibited significant growth dominance. Colonies of strain ZJ02 were round, pale yellow, opaque, with a rough surface, irregular edges, and raised features. Single colonies were picked for Gram staining and microscopic examination (refer to the Gram staining kit instructions for specific procedures). Under an optical microscope, the bacteria appeared as short rods and showed Gram-positive staining. Figure 3 As shown.

[0061] 2. 16S rRNA molecular identification

[0062] Genomic DNA was extracted from strain ZJ02 using a bacterial genomic DNA extraction kit manufactured by Beijing Solarbio Science & Technology Co., Ltd. After dilution of the extracted DNA sample, it was used as a template for PCR reaction. Universal primers were used to amplify the 16S rDNA template of strain ZJ02 using the Qingke 1 xTSE101 Gold Mix.

[0063] The 27F primer sequence was 5'-AGAGTTTGATCMTGGCTCAG-3'; the 1492R primer sequence was 5'-GGTTACCTTGTTACGACTT-3'. After PCR amplification, the PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with sequences in the NCBI GenBank database using BLAST. Sequences associated with the type strain with high homology were selected, and a phylogenetic tree of the 16S rRNA gene of strain ZJ02 was constructed using the neighbor-joining method with MEGA 6.0 software. Figure 4 As shown, strain ZJ02 clusters within the genus *Bacillus amyloliquefaciens*, with *Bacillus amyloliquefaciens* (PP897336.1) forming a branch. Therefore, strain ZJ02 is preliminarily identified as *Bacillus amyloliquefaciens*.

[0064] 3. Growth curve of ZJ02

[0065] After activation, strain ZJ02 was inoculated into 25 mL of LB medium at a 1% (v / v) inoculation rate and cultured at 37°C and 180 rpm in a shaker. The culture was performed every 2 hours, and the absorbance was measured at 600 nm. The OD value was plotted with the culture time on the x-axis. 600 Plot a growth curve with the vertical axis as the ordinate, such as... Figure 5 As shown.

[0066] Example 3: Antibacterial test of strain ZJ02

[0067] 1. Preparation of test bacterial suspension

[0068] The isolated Bacillus amyloliquefaciens was first cultured on plates, and then single colonies were picked and inoculated into nutrient broth medium. The culture was then incubated at 37°C and 200 rpm for 16-24 hours to prepare the test bacterial suspension, with a bacterial count of 10⁻⁶. 9 CFU / ml, for later use.

[0069] 2. Preparation of pathogens

[0070] Pathogenic Escherichia coli E2, Escherichia coli K88, Salmonella, and pathogenic bacteria were inoculated into nutrient broth medium and cultured at 37°C and 180 rpm for 18 hours before use.

[0071] Several common pathogens causing livestock and poultry diseases were selected: *Escherichia coli* K88, *Salmonella*, and *Escherichia coli* E2. The double-layer Oxford cup method described in Example 1 was used for the antibacterial experiment. The inhibitory ability of strain ZJ02 against these pathogens was evaluated by the diameter (mm) of the inhibition zone. A positive control of 5 μg / mL gentamicin sulfate was also included. The results are shown in Table 3. The inhibitory effects of strain ZJ02 against *Escherichia coli* K88, *Salmonella*, and *Escherichia coli* E2 were significantly better than the positive control treatment of 5 μg / mL gentamicin sulfate.

[0072] Table 3 Antibacterial effect of strain ZJ02

[0073]

[0074] Example 4: Study on the degradation characteristics of ammonia nitrogen by strain ZJ02

[0075] The ammonia nitrogen standard curve used in Example 4 is as follows: Figure 6 As shown.

[0076] 1. Experimental culture medium

[0077] Ammonia nitrogen liquid culture medium (g / L): glucose 5.0 g, ammonium sulfate 0.2 g, sodium chloride 2 g, magnesium sulfate heptahydrate 0.5 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.5 g.

[0078] 2. Effects of different time periods on the degradation of ammonia nitrogen by strain ZJ02

[0079] Bacillus amyloliquefaciens ZJ02 was inoculated at a rate of 0.2 vol% into 100 ml of liquid culture medium with an initial ammonia nitrogen concentration of 200 mg / L. The inoculation concentration was controlled at 10. 9 The concentration of ammonia nitrogen in the culture medium was adjusted to CFU / mL to pH 7, with three replicates per group. The medium was incubated at 37°C with shaking at 160 r / min. Every 12 hours, the concentration of ammonia nitrogen in the culture medium was measured according to the method in Example 1, and the degradation rate of ammonia nitrogen in each culture medium was calculated.

[0080] The results of ammonia nitrogen degradation at different time points are shown in Table 4. When the initial ammonia nitrogen concentration was 200 mg / L, the ammonia nitrogen degradation rate of ZJ02 reached a maximum of 78.74% after 24 hours.

[0081] Table 4. Results of ammonia nitrogen degradation by strain ZJ02 at different time periods

[0082]

[0083] 3. Effects of different initial concentrations on the degradation of ammonia nitrogen by strain ZJ02

[0084] The initial ammonia nitrogen concentration in the culture medium was adjusted to 50 mg / L, 100 mg / L, and 500 mg / L, and the pH was adjusted to 7. *Bacillus amyloliquefaciens* ZJ02 was inoculated at a rate of 0.2 vol% into 100 ml of liquid culture medium at each of the above different ammonia nitrogen concentrations. The inoculum concentration was controlled at 10... 9 CFU / mL, with three replicates per group. The culture was carried out at 37°C with shaking at 160 r / min for 24 h. After 24 h, the concentration of ammonia nitrogen in the culture medium was measured according to the method in Example 1, and the degradation rate of ammonia nitrogen in each culture medium was calculated.

[0085] The effects of initial ammonia nitrogen concentration on the degradation of ammonia nitrogen in ZJ02 are shown in Table 5. When the initial ammonia nitrogen concentration is 500 mg / L, the degradation rate of ZJ02 can reach 87.57%.

[0086] Table 5. Effects of different initial ammonia nitrogen concentrations on ammonia nitrogen degradation by strain ZJ02

[0087]

[0088] 4. Effects of different temperatures on the degradation of ammonia nitrogen by strain ZJ02

[0089] The temperature of the constant temperature shaking incubator was changed to 10°C, 15°C, 20°C, 25°C, 30°C, and 40°C. The pH was adjusted to 7. *Bacillus amyloliquefaciens* ZJ02 was inoculated at a rate of 0.2 vol% into 100 ml of liquid culture medium with an initial ammonia nitrogen concentration of 200 mg / L. The inoculum concentration was controlled at 10... 9 CFU / mL, with three replicates per group. Different temperatures were set according to the above requirements, and the culture was incubated with shaking at 160 r / min. After 24 h, the concentration of ammonia nitrogen in the culture medium was measured according to the method in Example 1, and the degradation rate of ammonia nitrogen in each culture medium was calculated.

[0090] The effects of different culture temperatures on the degradation rate of ammonia nitrogen in ZJ02 are shown in Table 6. The degradation rate of ammonia nitrogen was highest at a culture temperature of 30°C.

[0091] Table 6. Effects of different culture temperatures on the degradation rate of ZJ02 ammonia nitrogen.

[0092]

[0093] 5. Effects of different pH values ​​on the degradation of ammonia nitrogen by strain ZJ02

[0094] The pH of the ammonia nitrogen medium was adjusted to 5.0, 6.0, 7.0, 8.0, and 10.0 respectively, using ammonia nitrogen concentration of 200 mg / L. Bacillus amyloliquefaciens ZJ02 was then inoculated into the ammonia nitrogen medium at different pH values ​​at an inoculum concentration of 0.2 vol%. The inoculum concentration was controlled at 10. 9 CFU / mL, with three replicates per group. The culture was carried out at 37°C with shaking at 160 r / min. After 24 h, the concentration of ammonia nitrogen in the culture medium was measured according to the method in Example 1, and the degradation rate of ammonia nitrogen in each culture medium was calculated.

[0095] The effects of different pH values ​​on the ammonia nitrogen degradation rate of strain ZJ02 are shown in Table 7. The ammonia nitrogen degradation rate was highest at pH 6, reaching 85.56%.

[0096] Table 7. Effects of different pH values ​​on the ammonia nitrogen degradation rate of strain ZJ02

[0097]

[0098] 6. Comparison of ZJ02 strain with other ammonia nitrogen degrading strains

[0099] 0.2 vol% of ZJ02 bacterial suspension and other ammonia-degrading strains (Lactobacillus acidophilus, Bacillus coagulans, Lactobacillus plantarum, and Enterococcus faecalis) were added to 100 ml of ammonia nitrogen culture medium with an ammonia nitrogen concentration of 200 mg / L for testing. The inoculum concentration was controlled at 10%. 9 CFU / mL, with three replicates per group. Cultured at 37℃ and 160 r / min with shaking for 24 h. After 24 h, the concentration of ammonia nitrogen in the culture medium was determined according to the method in Example 1, and the degradation rate of ammonia nitrogen in each culture medium was calculated.

[0100] The results are shown in Table 8. It can be seen that strain ZJ02 has a better ammonia nitrogen degradation rate compared with other strains.

[0101] Table 8 Comparison of ammonia nitrogen degradation rates between strain ZJ02 and other strains

[0102] Group Ammonia nitrogen degradation rate / % CTRL group 24.56% Lactobacillus acidophilus group 69.86% Bacillus coagulans group 75.04% Group ZJ02 76.58% Lactobacillus plantarum group 56.36% Enterococcus faecalis group 74.39%

[0103] Example 5: Study on the ability of strain ZJ02 to remove ammonia from livestock and poultry manure

[0104] 1. Preparation of ZJ02 bacterial suspension

[0105] The selected strain ZJ02 was activated by culturing in liquid Lb medium and cultured at 37℃ for 14 h to obtain seed culture. The supernatant was discarded after centrifugation at 8000 r / min for five minutes, and physiological saline was added. The concentration of the bacterial suspension was adjusted by McFarland turbidimeter for subsequent experiments.

[0106] 2. Statistical methods

[0107] All experiments were repeated three times using SPSS 26.0 software. Multiple comparisons were performed using one-way ANOVA and Duncan's test in one-way ANOVA. Data are expressed as uniform mean. P < 0.05 was considered statistically significant, and data with no identical letters on the top label were considered statistically significant.

[0108] 3. Measurement of ammonia release from livestock and poultry manure

[0109] Inoculate 50g of fresh manure with 10% of the manure mass, placing the inoculation solution in a 1L covered plastic container. Place a 50mL sterile beaker containing 20mL of 2vol% boric acid solution (used to absorb NH3) inside the container. Seal the container opening quickly with plastic wrap, then replace the lid. Place the sample in a constant-temperature fermentation room. Add an equal volume of sterile physiological saline to the control group. Each treatment was repeated in triplicate. NH3 release was measured in all groups using the Kjeldahl method after 24 hours.

[0110] 3.1 Study on the ability of strain ZJ02 to remove ammonia from cattle dung

[0111] Bacillus amyloliquefaciens ZJ02 was first cultured on agar plates, then a single colony was picked with an inoculation loop and inoculated into nutrient broth medium. The culture was then incubated at 37°C and 180 rpm for 14 h to prepare the test bacterial solution. Subsequently, a ZJ02 bacterial suspension was prepared, and the bacterial count was adjusted to 5 × 10⁻⁶. 8 CFU / ml. 50g of cow dung was inoculated with 5ml of ZJ02 bacterial suspension in a plastic bucket, and ammonia release was calculated using the Kjeldahl method with boric acid absorption.

[0112] The results are shown in Table 9. Compared with the control group, the experimental group with added ZJ02 bacterial suspension significantly reduced the release of ammonia from cow dung in the range of 24-72 h.

[0113] Table 9 Results of the ZJ02 strain's ability to remove ammonia from cow dung.

[0114] 24-hour ammonia release (mg / L) Ammonia release over 48 hours (mg / L) Ammonia release over 72 hours (mg / L) ZJ02 <![CDATA[30.37 bc ]]> <![CDATA[52.13 b ]]> <![CDATA[69.70 b ]]> CTRL <![CDATA[40.69 a ]]> <![CDATA[80.69 a ]]> <![CDATA[127.50 a ]]> p-value ＜0.01 ＜0.01 ＜0.01

[0115] 3.2 Study on the ability of strain ZJ02 to remove ammonia from pig manure

[0116] Bacillus amyloliquefaciens ZJ02 was first cultured on agar plates, then a single colony was picked and inoculated into nutrient broth medium and cultured at 37°C and 180 rpm for 14 h to prepare the test bacterial solution. Subsequently, a ZJ02 bacterial suspension was prepared and the bacterial count was adjusted to 10⁻⁶. 8CFU / ml. 50g of pig manure was inoculated with 5ml of ZJ02 bacterial suspension in a plastic bucket, and ammonia release was calculated using the Kjeldahl method with boric acid absorption.

[0117] The results are shown in Table 10. Compared with the control group, the experimental group with added ZJ02 bacterial suspension significantly reduced the release of ammonia gas from pig feces from 48 to 96 hours.

[0118] Table 10 Results of the ammonia removal capacity of strain ZJ02 from pig manure

[0119] Ammonia release over 48 hours (mg / L) Ammonia release over 72 hours (mg / L) Ammonia release rate (mg / L) over 96 hours ZJ02 <![CDATA[23.12 c ]]> <![CDATA[177.25 b ]]> <![CDATA[332.52 c ]]> CTRL <![CDATA[88.85 a ]]> <![CDATA[267.92 a ]]> <![CDATA[496.85 a ]]> p-value ＜0.01 ＜0.01 ＜0.01

[0120] 3.3 Study on the ability of bacterial strains to remove ammonia from dog feces

[0121] Bacillus amyloliquefaciens ZJ02 was first cultured on agar plates, then a single colony was picked and inoculated into nutrient broth medium and cultured at 37°C and 180 rpm for 14 h to prepare the test bacterial solution. Subsequently, a ZJ02 bacterial suspension was prepared and the bacterial count was adjusted to 10⁻⁶. 8 CFU / ml. 50g of dog feces was inoculated with 5ml of ZJ02 bacterial suspension in a plastic bucket, and ammonia release was calculated using the Kjeldahl method with boric acid absorption.

[0122] The results are shown in Table 11. Compared with the control group, the experimental group with added ZJ02 bacterial suspension significantly reduced the release of ammonia gas from dog feces at 72h.

[0123] Table 11 Results of the ammonia removal capacity of strain ZJ02 from dog feces

[0124] Ammonia release over 72 hours (mg / L) ZJ02 52.13 CTRL <![CDATA[135.66 a ]]> p-value 0.049

[0125] 3.4. Test on the ammonia removal capacity of strain ZJ02 in a simulated pig farm environment and comparison with other strains

[0126] Bacillus amyloliquefaciens ZJ02, Bacillus subtilis, and Bacillus licheniformis were cultured separately on plates. Single colonies were then picked and inoculated into nutrient broth medium and cultured at 37°C and 180 rpm to prepare the test bacterial suspensions. The bacterial count for each strain was 10⁻⁶. 11 CFU / ml, the above different test bacterial solutions were added to 100g of pig farm manure at a ratio of 10wt%, and the ammonia release was calculated at different time periods by boric acid absorption Kjeldahl nitrogen determination method.

[0127] The results are shown in Table 12. On day 6 of treatment, the NH3 concentration of Bacillus amyloliquefaciens ZJ02 was only 54.04 mol / L, which was significantly lower than that of other strain treatment groups.

[0128] Table 12 Results of ammonia removal capacity test of strain ZJ02 in simulated pig farm environment and comparison with other strains

[0129] Processing group Fecal matter mass (g) Absorption liquid volume (mL) <![CDATA[NH3 concentration in 1 day (mol / L)]]> <![CDATA[4-day NH3 concentration (mol / L)]]> <![CDATA[6-day NH3 concentration (mol / L)]]> CTRL 100 20 443.44 396.86 136.01 Bacillus subtilis 100 20 354.40 341.34 108.07 Bacillus licheniformis 100 20 339.10 316.74 98.75 ZJ02 bacteria 100 20 313.39 253.36 54.04

[0130] In summary, the Bacillus amyloliquefaciens ZJ02 provided by this invention has a significant inhibitory effect on Escherichia coli and Salmonella, with an inhibition zone size of 18.11 mm against Salmonella (SA) after 24 hours. Furthermore, compared to other strains such as Lactobacillus plantarum and Lactobacillus acidophilus, it exhibits better ammonia nitrogen degradation under the same conditions, with an ammonia nitrogen degradation rate of 76.58%, reaching as high as 87.57% at an initial ammonia nitrogen concentration of 500 mg / L. Most importantly, it has an excellent ability to reduce ammonia release from animal feces. In ammonia removal tests in pig farm environments, compared to the control group, the NH3 concentration was reduced by 60.27% on day 6, significantly superior to Bacillus subtilis and Bacillus licheniformis.

[0131] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens ZJ02 that inhibits bacterial growth and reduces ammonia nitrogen, characterized in that, The accession number is CCTCC NO: M20252399, the accession date is October 30, 2025, the depositary institution is China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, China.

2. The application of Bacillus amyloliquefaciens as described in claim 1 in reducing ammonia emissions from animal feces.

3. The application according to claim 2, characterized in that, The animal in question is one of the following: dog, pig, or cow.

4. The application of Bacillus amyloliquefaciens as described in claim 1 in degrading ammonia nitrogen content in polluted water bodies.

5. The application of Bacillus amyloliquefaciens as described in claim 1 in the preparation of antibacterial agents.

6. The application according to claim 2, characterized in that, The bacteria mentioned are at least one of Escherichia coli and Salmonella.

7. A deodorizing agent, characterized in that, The deodorizing agent contains Bacillus amyloliquefaciens as described in claim 1.

8. A wastewater treatment agent, characterized in that, The wastewater treatment agent contains Bacillus amyloliquefaciens as described in claim 1.

9. An antibacterial agent, characterized in that, The antibacterial agent contains Bacillus amyloliquefaciens as described in claim 1.

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