A strain with high-temperature ammoniation and ammonia oxidation capacity and application thereof

Abstract

The application provides a strain with high-temperature ammoniation and ammoxidation capacity and application thereof. Anoxybacillus kestanbolensis The strain is preserved in the China General Microbiological Culture Collection Center on November 26, 2025, and the preservation number is CGMCC No. 36771. The strain has high efficient ammoniation capacity and ammoxidation capacity under high-temperature conditions, can be used for reducing ammonia emission in the process of organic waste treatment, reducing nitrogen loss and improving the quality of compost products.

Description

Technical Field

[0001] This invention belongs to the field of microbial resources and the resource utilization technology of organic solid waste, and relates to a strain with both high-temperature ammoniation and ammonia oxidation capabilities and its application. Specifically, it relates to a thermophilic bacterium, *Bacillus kestenbergii*, obtained through domestication and screening from high-temperature composting samples and possessing both high-temperature ammoniation and ammonia oxidation capabilities. Anoxybacillus kestanbolensis And its applications. Background Technology

[0002] my country's annual livestock and poultry manure production is approximately 3.05 billion tons. When used as a nitrogen-rich raw material in composting, it easily leads to the volatilization of large amounts of NH3 (30%–40%), which not only reduces the fertilizer efficiency of the product but also causes air pollution. Nitrogen conversion during composting mainly involves two key stages: ammonification (organic nitrogen → NH4). + ) and ammonia oxidation (NH4) + →NO2 - The balance between NH3 emissions and nitrogen retention during the composting process directly affects the balance between these two factors.

[0003] The use of additives is one of the important ways to reduce nitrogen loss in composting. Physical and chemical additives are fast-acting, but their application in actual industry is limited due to the risks of high salt ion concentration and soil accumulation, as well as their non-reusability which increases composting costs. Conversely, microbial inoculants have significant advantages in terms of cost and environmental friendliness. However, current research and applications are mostly geared towards promoting compost temperature rise, accelerating organic matter degradation, and promoting compost maturation. While some studies focus on NH3 emission reduction and nitrogen loss reduction, they primarily utilize traditional room-temperature nitrifying bacteria, which are difficult to adapt to the high-temperature environment of composting. Therefore, the effectiveness of these nitrifying bacteria in composting varies. Consequently, bacteria suitable for the high-temperature environment of composting and aimed at reducing NH3 emissions and nitrogen loss still need to be discovered and developed.

[0004] Kestanbor anaerobic spores Anoxybacillus kestanbolensis It is a type of anaerobic Bacillus ( Anoxybacillus Thermophilic facultative anaerobic bacteria are often used as extreme environment microbial resources to develop heat-resistant industrial enzymes, such as amylase and other carbon-degrading enzymes. However, there are currently no reports on the development and application of this bacterium in the composting process, and its effects on nitrogen conversion and NH3 emission reduction still need to be explored. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. This invention provides a thermophilic bacterial strain, *Bacillus kestenbergii*, possessing both high-temperature ammoniation and ammonia oxidation capabilities. Anoxybacillus kestanbolensisIt possesses both ammonification and ammonia oxidation capabilities under high-temperature conditions, and can promote the conversion of organic nitrogen to NH4. + →NO2 - →NO3 - This bacterial strain, when applied to composting, can promote nitrogen conversion, reduce NH3 emissions and nitrogen loss in the compost pile. Simultaneously, it can supplement the existing library of nitrogen-converting bacteria, providing more options, and can also be combined with other functional strains to work synergistically.

[0006] The first aspect of the present invention provides a Kestambor anaerobic spore-forming bacterium. Anoxybacillus kestanbolensis It was deposited on November 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36771.

[0007] According to an embodiment of the present invention, its 16S rDNA sequence is shown in SEQ ID NO:1.

[0008] A second aspect of the present invention provides a microbial inoculant, comprising the *Bacillus kestenbergii* anaerobic spores described in the first aspect.

[0009] According to an embodiment of the present invention, the microbial agent is a dry powder or a bacterial solution, and the effective viable count of the *Bacillus kestenbergii* anaerobic bacteria in the dry powder or bacterial solution is at least 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL.

[0010] According to an embodiment of the present invention, the microbial agent is obtained by inoculating the *Bacillus kestenbergii* anaerobic spores. Anoxybacillus kestanbolensis It was obtained by activation culture and fermentation in LB medium.

[0011] According to an embodiment of the present invention, the LB culture medium is formulated with Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, deionized water 1000 mL, and pH 7.2~7.4.

[0012] The fermentation culture conditions are as follows: The *Bacillus kestenbergii* anaerobic culture is cultured under the following conditions: Anoxybacillus kestanbolensis After being inoculated into the LB medium, the culture was fermented at 60°C and 180 r / min for 24–48 h.

[0013] According to a third aspect of the present invention, a culture or fermentation broth of the aforementioned *Bacillus kestenbergii* or the aforementioned microbial agent is provided.

[0014] According to a fourth aspect of the present invention, the use of the *Bacillus kestanborella* described in the first aspect or the microbial agent described in the second aspect in the preparation of formulations for reducing ammonia emissions, reducing nitrogen loss, and improving compost quality during the treatment of organic waste is provided.

[0015] According to a fifth aspect of the present invention, a method for reducing ammonia emissions and nitrogen loss and improving compost quality during the treatment of organic waste is provided, comprising: mixing the above-described anaerobic Bacillus kestenbergii or the above-described microbial agent with organic waste and culturing it; The mixed culture is carried out in an in vitro organic waste sample or directly in the pile of organic waste treatment facilities. Through this treatment, organic nitrogen and ammonium nitrogen in the sample or facility can be converted, thereby reducing NH3 emissions and nitrogen loss.

[0016] According to an embodiment of the present invention, the *Bacillus kestenbergii* or the microbial agent is inoculated when the temperature of the isolated organic waste sample or the pile of organic waste in the treatment site reaches 50-60°C.

[0017] According to an embodiment of the present invention, when the organic waste is compost, it is subjected to aerobic composting treatment, and the fermentation cycle is 15 to 25 days.

[0018] According to an embodiment of the present invention, the inoculation amount of the anaerobic Bacillus kestenbergii or the microbial agent is 0.5% to 2% of the fresh weight of the organic waste.

[0019] According to an embodiment of the present invention, the organic waste sample is selected from at least one of livestock and poultry manure, crop straw, and kitchen waste. The organic waste treatment site is selected from at least one of the following: composting, fermentation, and other nitrogen-rich or nitrogen-containing organic waste treatment sites.

[0020] The beneficial effects achieved by this invention are at least as follows: This invention provides a novel high-temperature strain, namely Bacillus kestenbergii anaerobicus, which can maintain vigorous metabolic activity at high temperatures of 45~65℃, overcoming the deficiency of insufficient activity of room-temperature strains during the high-temperature period of composting.

[0021] The Kessenberg anaerobic spores provided by this invention Anoxybacillus kestanbolensis Its application in composting and fermentation can effectively reduce NH3 emissions during the treatment of organic waste and promote the transformation of organic nitrogen into NH4 in the compost pile. + →NO2 - →NO3 - The conversion increases the NO3 content in compost products. -The strain's unique facultative anaerobic and thermophilic growth characteristics ensure stable ammonification and ammonia oxidation functions even under high temperature and localized anaerobic conditions, enabling rapid nitrogen conversion in samples within specific environments, reducing ammonia emissions, minimizing nitrogen loss, and promoting composting.

[0022] Preservation Certificate Classified as *Kestenberg anaerobic spores* Anoxybacillus kestanbolensis The accession number is CGMCC No. 36771, the depository is China General Microbiological Culture Collection Center, the deposit date is November 26, 2025, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0023] Figure 1 is a colony morphology diagram of the WYN-30 strain provided according to an embodiment of the present invention.

[0024] Figure 2 is a phylogenetic tree of strain WYN-30 based on the 16S rDNA sequence provided in an embodiment of the present invention.

[0025] Figure 3 shows the qualitative determination of the ammonification capacity (a) and ammonia oxidation capacity (b) of strain WYN-30 provided according to the embodiments of the present invention (where the test tube on the left in each figure is strain WYN-30 and the control group is on the right).

[0026] Figure 4~ Figure 6 The figures represent the changes in ammonium nitrogen, nitrite nitrogen, and nitrate nitrogen content of strain WYN-30 provided in the embodiments of the present invention.

[0027] Figure 7~ Figure 8 These are the daily and cumulative ammonia emissions of the control group and the group with added WYN-30 strain during the 21-day composting process provided by the embodiments of the present invention.

[0028] Figure 9 shows the change in seed germination index during composting according to an embodiment of the present invention. Detailed Implementation

[0029] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention. Furthermore, some terms used in the present invention are explained and described; these explanations and descriptions are for convenience of understanding only and should not be considered as limiting the scope of protection of the present invention.

[0030] This invention provides a Kestenbor anaerobic spore-forming bacterium. Anoxybacillus kestanbolensisThis strain was deposited on November 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36771. This strain possesses highly efficient ammoniation and ammonia oxidation capabilities under high-temperature conditions, and can be used to reduce NH3 emissions during organic waste composting and promote nitrogen conversion and maturation within the compost pile.

[0031] The 16S rDNA sequence of the provided strain is shown in SEQ ID NO:1.

[0032] The aforementioned Kestanbor anaerobic spores Anoxybacillus kestanbolensis The samples were obtained by acclimatizing pig manure high-temperature compost samples at 60℃ under nitrogen-rich conditions, and then qualitatively screening and quantitatively screening by stepwise dilution in two types of selective culture media, DSM88 and MD, streak separation, and colorimetric tests using Nessler's reagent and Gliese's reagent.

[0033] This invention also provides a microbial inoculant comprising the aforementioned *Bacillus kestenbergii* anaerobic strain as a core functional component. This microbial inoculant can be in the form of dry powder, granules, lyophilized powder, or liquid bacterial solution. When the inoculant is in the form of dry powder, granules, or lyophilized powder, the effective viable count of *Bacillus kestenbergii* is at least 1 × 10⁻⁶. 8 CFU / g, preferably 1×10⁻⁶ 9 ~1×10 10 CFU / g; when the bacterial agent is in liquid form, the effective viable count is at least 1×10⁻⁶. 8 CFU / mL, preferably 1×10⁻⁶ 9 ~1×10 10 CFU / mL.

[0034] The preparation method of the microbial agent includes: using preserved Bacillus kestenbergii anaerobic spores. Anoxybacillus kestanbolensis After activation, the culture medium is inoculated into LB medium for enrichment fermentation culture. The fermentation temperature is 50~60℃, the stirring speed is 150~300 r / min, and the fermentation time is 24~48 h. After fermentation, liquid bacterial agent can be obtained directly.

[0035] According to an embodiment of the present invention, the LB culture medium is formulated with Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, deionized water 1000 mL, and pH 7.2~7.4.

[0036] The aforementioned Bacillus kestenbergii anaerobic spores can be added during the composting process of organic waste. Anoxybacillus kestanbolensis Alternatively, the aforementioned microbial agents can be used to reduce NH3 emissions during the composting process of organic waste, promote nitrogen conversion and decomposition within the compost pile, and reduce nitrogen loss. The organic waste mentioned can be at least one of livestock and poultry manure, crop straw, and kitchen waste.

[0037] This invention also provides a method for reducing ammonia emissions and nitrogen loss and improving compost quality during organic waste treatment, comprising: using the aforementioned *Bacillus kestenbergii* anaerobic bacteria. Anoxybacillus kestanbolensisAlternatively, the aforementioned microbial agents can be co-cultured with organic waste; wherein the co-culture is carried out in an in vitro organic waste sample or directly in the compost pile of an organic waste treatment site. This treatment can rapidly convert nitrogenous substances in the sample or site, reducing NH3 emissions and minimizing nitrogen loss. The mentioned sites include, but are not limited to, at least one of the following: composting, fermentation, or other nitrogen-rich or nitrogen-containing organic waste treatment sites.

[0038] The “ex vivo organic waste sample” mentioned in this article refers to a representative organic waste sample taken from the original organic waste treatment site (such as a composting plant or a manure storage pond in a livestock farm treatment area), and then transferred away from the original environment and placed in a controlled external environment such as a laboratory, a small reactor, an incubator, or a specific container for mixed fermentation.

[0039] The above-mentioned Kestanbor anaerobic spores Anoxybacillus kestanbolensis Alternatively, microbial agents can be applied in the treatment of organic waste, such as in the aerobic composting of livestock and poultry manure. When the compost temperature reaches about 55°C, adding microbial agents can reduce NH3 emissions by 26.32% and increase ammonification (organic nitrogen → NH4) during the high-temperature period. + ) and ammonia oxidation (NH4) + →NO2 - The ability to reduce nitrate nitrogen (NO3) after composting - The content of -N increased by 23.95%, and the seed germination index increased by 15.41%.

[0040] The technical solution of the present invention will be described below through specific embodiments. It should be noted that these embodiments are only used to facilitate understanding by those skilled in the art and should not be considered as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0041] Example 1 Example 1: A strain of *Bacillus kestenbergii* anaerobic was obtained by screening using the following method, specifically including: Five g of pig manure compost samples were collected during the high-temperature period (≥60℃) and placed in a 250 ml Erlenmeyer flask. 100 ml of 1 mM PBS buffer and 5 ml of 1 mM (NH4)2SO4 were added. The mixture was acclimatized at 60℃ and 150 r / min for 28 days. The mixture was used as the mother liquor. The target strains in the mother liquor were initially screened and streaked to separate using DSM88 and organic nitrogen source MD selective medium to construct a high-temperature ammonification and ammonia oxidation functional bacterial library.

[0042] The functional strains obtained from the initial screening were subjected to qualitative and quantitative screening of ammoniation and ammonia oxidation capabilities in organic-inorganic nitrogen source MD liquid medium.

[0043] (1) Qualitative screening of ammoniation ability: The test strain was inoculated into organic nitrogen source type MD liquid medium and cultured at 60℃ and 120r / min for 24 h. 2 mL of culture medium was added to Nessler's reagent, mixed well and allowed to stand for 10 min to observe the color. If the culture medium turned obviously yellow (compared to the blank control), it was determined to be ammoniation positive.

[0044] (2) Qualitative screening of ammonia oxidation capacity: The test strain was inoculated into inorganic nitrogen source type MD medium and cultured at 60℃ and 120r / min for 24 h. 2 mL of culture medium was added to Griess reagent A and B in sequence, mixed well and allowed to stand for 15-20 min. If the culture medium turned red or purplish-red, it was determined to be positive for ammonia oxidation.

[0045] Strains that simultaneously meet the criteria of "ammoniation positive" and "ammonia oxidation positive" are designated as double-positive strains and proceed to the rescreening stage. This double-positive screening strategy ensures that the target strains entering the rescreening stage possess both organic nitrogen degradation and inorganic nitrogen transformation capabilities, which is a prerequisite for subsequent quantitative evaluation and practical application.

[0046] (3) Quantitative rescreening of ammonification capacity: It is necessary to quantitatively determine the ammonification activity of the target strain, that is, to use the sodium salicylate-hypochlorite spectrophotometric method (HJ 536-2009 "Determination of Ammonia Nitrogen in Water - Salicylic Acid Spectrophotometric Method") to determine the NH4 in the culture medium. + -N concentration, calculate ammoniation rate: (Formula 1) (4) Quantitative rescreening of ammonia oxidation capacity: It is necessary to quantitatively determine the ammonia oxidation activity of the target strain, that is, to use the diazo-coupled spectrophotometric method (GB 7493-87 "Determination of Nitrite Nitrogen in Water" spectrophotometric method) to determine NO2 in the culture medium. - NO3- concentration was determined using the hydrazine sulfate reduction-spectrophotometric method. - -N concentration, calculate the ammonia oxidation rate: (Formula 2) In the formula, T represents the culture time (h), and C represents the corresponding nitrogenous form concentration (mg / L). The average rate at two time points, 24 h and 48 h, is taken, and the ammonification rate and ammonia oxidation rate are added together to obtain a comprehensive performance index. The strains are then ranked in descending order according to this index.

[0047] The DSM88 culture medium used included the following: (NH4)2SO4 0.50 g / L, KH2PO4 0.28 g / L, MgSO4·7H2O 0.25 g / L, CaCl2 0.01 g / L, Tryptone 2.00 g / L, Yeast extract 2.00 g / L, Na2MoO4·2H2O 10.3 µL / L, FeCl3 17.3 µL / L, CuSO4 10 µL / L, MnSO4·H2O 13 µL / L, H3BO3 80.9 µL / L, ZnSO4·7H2O 17.4 µL / L, CoCl2·6H2O 19.3 µL / L, deionized water 1000 mL, pH 8.0.

[0048] The organic nitrogen source MD medium formula used includes: 100 mg / L triacetic acid, 8 mg / L NaCl, 100 mg / L MgSO4·7H2O, 111 mg / L KH2PO4, 2 g / L Tryptone, 2 g / L Yeast Extract, 10.3 µL / L Na2MoO4·2H2O, 17.3 µL / L FeCl3, 10 µL / L CuSO4, 13 µL / L MnSO4·H2O, 80.9 µL / L H3BO3, 17.4 µL / L ZnSO4·7H2O, 19.3 µL / L CoCl2·6H2O, 1000 mL deionized water, pH adjusted to 8.0.

[0049] The inorganic nitrogen source MD medium formulation used includes: based on the organic nitrogen source MD medium, replacing Tryptone and Yeast Extract with an equal amount of NH4Cl (in NH4Cl form). + (Using the only nitrogen source), the rest of the formula remains unchanged, pH 8.0.

[0050] The strain obtained through this screening method was named WYN-30. The obtained strain was then characterized as follows: (1) Colony characteristics like Figure 1 As shown, strain WYN-30 is a Gram-positive, facultatively anaerobic bacillus that produces endospores. When cultured on LB solid medium at 60°C for 24 h, it forms round, smooth-surfaced, regularly edged, milky-white to pale yellow colonies, approximately 2–3 mm in diameter.

[0051] (2) Strain identification Genomic DNA was extracted using a DNA extraction kit (manufacturer: Beijing Boyoushun Biotechnology Co., Ltd.). The extracted DNA was used as a PCR template, and amplification was performed using a PCR instrument (Biometra Tgradient) with primers (synthesized by BGI Genomics) to obtain PCR amplification products. All reagents used for amplification, including Taq DNA polymerase, were purchased from TaKaRa.

[0052] The universal primers used include: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2) 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 3) The PCR amplification system consisted of: 2 μL of treated sample (20 ng / μL) template, 2.5 μL of dNTP mixture (2.5 mM), 1.5 μL each of primers (20 μM), and 10×ExTaq buffer (Mg2+). 2+ Add 5 μL of ExTaq enzyme (5 U / μL) and 0.2 μL of ddH2O to a final volume of 50 μL.

[0053] The PCR amplification program was as follows: 94 °C pre-denaturation for 4 min; 94 °C denaturation for 30 s, 56 °C annealing for 30 s, 72 °C extension for 90 s, 35 cycles, 72 °C extension for 10 min.

[0054] After PCR amplification products were detected by 1.5% agarose gel, the target band was recovered and purified using a PCR purification kit (promega). Bidirectional sequencing was then performed using amplification primers (ABI sequencer 3730xl). The sequencing results were assembled, entered into GenBank, and compared with NCBI using BLASTN to construct a phylogenetic tree, as shown below. Figure 2 As shown. This strain was ultimately identified as *Bacillus kestenbergii*, an anaerobic spore-forming bacterium. Anoxybacillus kestanbolensis The 16S rDNA sequence is shown in SEQ ID NO:1. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36771.

[0055] Example 2 Example 2 identified the high-temperature ammoniation and ammonia oxidation capabilities of the strain prepared in Example 1.

[0056] (1) High temperature ammoniation ability To verify the ammoniation (organic nitrogen → NH4) of this strain under high temperature conditions. +The high-temperature ammonification ability of this strain was tested using organic nitrogen-source MD medium (the formulation of which is the same as in Example 1 above), and cultured at 60℃ and 120 r / min for 24 h. 2 mL of culture medium was added to Nessler's reagent, mixed well, and allowed to stand for 10 min. The color was observed, and the absorbance was measured using a spectrophotometer. It was ultimately found that after 24 h of culture, the addition of Nessler's reagent resulted in a distinctly deep yellow culture medium (e.g., ...). Figure 3 As shown in Figure a), the ammoniation rate was determined to be 1.98 mg·L⁻¹. -1 ·h -1 The ammoniation rate mentioned therein was calculated according to Formula 1 in Example 1.

[0057] (2) High-temperature ammonia oxidation capacity To verify the ammonia oxidation (NH4+) activity of this strain under high temperature conditions. + →NO2 - The high-temperature ammonia oxidation capacity of this strain was tested using inorganic nitrogen-source MD medium (the medium formulation is the same as in Example 1 above), and cultured at 60℃ and 120 r / min for 24 h. 2 mL of culture medium was taken and Griess reagent A and B were added sequentially, mixed well, and allowed to stand for 15-20 min. The color was observed, and the absorbance was measured using a spectrophotometer. It was ultimately found that after 24 h of culture, the addition of Griess reagent resulted in a red or purplish-red culture medium (e.g., ...). Figure 3 As shown in Figure b), the ammonia oxidation rate was determined to be 0.06 mg·L⁻¹. -1 ·h -1 The ammonia oxidation rate mentioned therein was calculated according to Formula 2 in Example 1.

[0058] Example 3 Example 3 verified the ammonia emission reduction and nitrogen conversion capabilities of the provided strain during the composting process.

[0059] A 725 ml composting pilot-scale simulated fermenter was selected and placed in a programmable temperature-controlled insulated box. Six temperature plateaus were set to simulate the actual fermentation process. Stage 1: 30℃ for 1 day; Stage 2: 40℃ for 1 day; Stage 3: 50℃ for 1 day; Stage 4: 60℃ for 7 days; Stage 5: 45℃ for 4 days; Stage 6: 30℃ for 7 days.

[0060] Two experimental groups were set up: an experimental group and a control group (CK group), with two replicates in each group. The composting raw materials were pig manure and wheat bran (pig manure:wheat bran = 3:2 mass ratio), totaling 200 g. The initial carbon-to-nitrogen ratio of the materials was approximately 20:1, the moisture content was approximately 65%, the fermentation period was 21 days, the aeration rate was 0.2 L / (min·kg dry weight), and the aeration method was intermittent aeration, i.e., aeration for 30 minutes followed by a 30-minute pause. In the experimental group, *Bacillus kestenbergii* anaerobic spores were inoculated at a 1% inoculum (v / w) when the compost temperature reached 55℃. Anoxybacillus kestanbolensis The inoculum was added to the heap, with an actual inoculation volume of 2 mL. The viable count of the bacteria measured before inoculation was 1 × 10⁻⁶. 8 CFU / mL; the control group consisted of an equal volume of sterile water. Sampling was performed on days 0, 3, 7, 15, and 21.

[0061] By measuring the levels of NH3, ammonium nitrogen, nitrite nitrogen, nitrate nitrogen, and seed germination index within the pile, the anaerobic Bacillus kelsinbohr was investigated. Anoxybacillus kestanbolensis The high-temperature ammoniation and ammonia oxidation capabilities, as well as the effect of reducing NH3 emissions and nitrogen loss, were verified.

[0062] The result is as follows Figure 4 to Figure 9 As shown. After 21 days of fermentation, compared with the control group, inoculation reduced NH3 emissions by 26.32%, nitrogen loss by 26.32%, increased nitrate nitrogen content by 23.95%, and increased seed germination index by 15.41% during the composting process.

[0063] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A type of Kestambor anaerobic spore-forming bacillus Anoxybacillus kestanbolensis Its characteristics are, It was deposited on November 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36771.

2. The *Bacillus kestenbergii* anaerobic spores according to claim 1 Anoxybacillus kestanbolensis Its characteristics are, Its 16S rDNA sequence is shown in SEQ ID NO:

1.

3. A microbial inoculant, characterized in that, Including the *Bacillus kestenbergii* anaerobic spores as described in claim 1 or 2 Anoxybacillus kestanbolensis .

4. The microbial agent according to claim 1, characterized in that, The microbial agent is a dry powder or a bacterial liquid, and the dry powder or bacterial liquid contains the *Bacillus kestenbergii* anaerobic spores. Anoxybacillus kestanbolensis The effective viable count is at least 1×10⁻⁶. 8 CFU / g or 1×10 8 CFU / ml; Optionally, the microbial agent is prepared by passing the *Bacillus kestenbergii* anaerobic spores. Anoxybacillus kestanbolensis It was obtained by activation and fermentation culture on LB medium; Optionally, the LB medium is formulated with Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, deionized water 1000 mL, and pH 7.2-7.

4. Optionally, the fermentation culture conditions are as follows: The *Bacillus kestenbergii* anaerobic spores are fermented under the following conditions: Anoxybacillus kestanbolensis After being inoculated into the LB medium, the culture was fermented at 60°C and 180 r / min for 24–48 h.

5. The culture or fermentation broth of the anaerobic Bacillus kestenbergii as described in claim 1 or 2, or the microbial agent as described in claim 3 or 4.

6. The *Bacillus kestenbergii* anaerobic spores as described in claim 1 or 2 Anoxybacillus kestanbolensis Alternatively, the use of the microbial agent as described in claim 3 or 4 in the preparation of formulations for reducing ammonia emissions, nitrogen loss, and improving compost quality during organic waste treatment.

7. A method for reducing ammonia emissions, nitrogen loss, and improving compost quality during organic waste treatment, characterized in that, include: The *Bacillus kestanborella* as described in claim 1 or 2 Anoxybacillus kestanbolensis Alternatively, the microbial inoculant as described in claim 3 or 4 can be mixed with organic waste for cultivation; The mixed culture is carried out in an in vitro organic waste sample or directly in the pile of organic waste at the organic waste treatment site.

8. The method according to claim 7, characterized in that, The aforementioned Bacillus kestanborella anaerobicans or the aforementioned microbial agent is inoculated when the temperature of the isolated organic waste sample or the pile of organic waste at the treatment site reaches 50-60 degrees Celsius. Optionally, when the organic waste is compost, it shall be subjected to aerobic composting treatment, with a fermentation cycle of 15-25 days.

9. The method according to claim 7, characterized in that, The anaerobic Bacillus kestenberg Anoxybacillus kestanbolensis Alternatively, the inoculation amount of the microbial agent may be 0.5%-2% of the fresh weight of the organic waste.

10. The method according to claim 7, characterized in that, The organic waste samples were selected from at least one of livestock and poultry manure, crop straw, and kitchen waste. The organic waste treatment site is selected from at least one of composting and fermentation.

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