Aerobacillus pallidus and application thereof in reducing ammonia volatilization in aerobic composting

Abstract

The invention discloses an aeribacillus pallidus strain and application thereof in reduction of ammonia volatilization in aerobic composting, and belongs to the technical field of microorganisms, the aeribacillus pallidus strain is named as aeribacillus pallidus GW-C and preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.34226. The invention further discloses a preparation method of the aeribacillus pallidus strain. The aerobacillus pallidus GW-C provided by the invention has better high-temperature resistance and nitrification capacity, and can be applied to aerobic composting to reduce the emission of ammonia gas, effectively reduce the loss of nitrogen in a compost product and relieve the environmental pollution caused by the generation of gas in livestock and poultry aerobic composting.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of *Bacillus aeruginosa* and its application in reducing ammonia volatilization during aerobic composting. Background Technology

[0002] With the rapid development of livestock and poultry farming in my country, livestock and poultry farming has become a major source of pollution. Poultry manure contains a large amount of organic matter, nitrogen, phosphorus, potassium, and trace elements. Improper treatment of livestock and poultry manure can further deteriorate the environment, waste resources, and disrupt the balance of the agricultural ecosystem. Therefore, the rational treatment and utilization of livestock and poultry manure has become one of the main problems to be solved. Aerobic composting technology is one of the most important means of resource utilization and harmless disposal of livestock and poultry manure. However, aerobic composting produces greenhouse gases such as carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O). The emission of these gases seriously affects the quality of compost products, causes secondary pollution, and exacerbates the global greenhouse effect. Some studies have shown that adding microbial agents during aerobic composting can accelerate the composting process, improve the quality of compost products, and change the microbial community structure during composting, enriching functional bacteria, promoting the growth of ammonia-oxidizing bacteria and nitrification, increasing nitrogen fixation, and reducing ammonia production. Microbial additives can also reduce gas emissions during composting. This may be because the addition of exogenous compound microbial agents inhibits the growth of nitrifying microorganisms and the activity of related enzymes, thereby inhibiting the nitrification of nitrous oxide and nitrogen. However, the growth of many mesophilic nitrifying microorganisms is inhibited in the high-temperature environment of composting, leading to a decline in their nitrification capacity and an inability to sustain nitrogen conversion and retain nitrogen. The addition of thermophilic heterotrophic nitrifying bacteria in composting offers numerous advantages, including improved composting efficiency, reduced ammonia emissions, reduced nitrogen loss, enhanced fertilizer efficiency of compost products, and reduced odor pollution. Therefore, using thermophilic heterotrophic nitrifying bacteria to address nitrogen loss in compost is a scientifically effective measure, and the screening and application of thermophilic heterotrophic nitrifying bacteria are of great significance.

[0003] However, the current technology has at least the following problems: (1) Screening of high-temperature strains and optimization of culture conditions: Although a variety of high-temperature strains have been isolated, the adaptability and stability of these strains in actual composting still need to be further verified. For example, high-temperature strains from different isolation sources cannot show good nitrification under different composting raw materials and environmental conditions to effectively accelerate the composting process and solve the problem of ammonia volatilization. The cost of the microbial agent and the preparation time are longer than those of mesophilic nitrifying bacteria. The culture conditions are limited by temperature and the preparation cost is high, which limits its large-scale application. (2) Technical difficulties in the composting process: Although high-temperature strains can significantly increase the composting temperature, how to maintain a stable temperature in the high-temperature stage and how to avoid the decline in microbial activity caused by excessive temperature are still problems that need to be solved. Although high-temperature composting technology can shorten the fermentation cycle, in practical applications, there are still problems of insufficient composting and low humification efficiency. During high-temperature composting, the emissions of ammonia and greenhouse gases (such as N2O) are still high. Therefore, the use of high-temperature microbial agents to deal with the above problems are the focus of current research. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of *Bacillus pallida* to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A strain of Aeribacillus pallidus, named Aeribacillus pallidus GW-C, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34226. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is April 16, 2025.

[0007] Another object of the present invention is to provide a nitrifying agent comprising the above-mentioned *Bacillus pallida*.

[0008] Another object of the present invention is to provide an application of the above-mentioned *Bacillus aeruginosa* or the above-mentioned microbial agent in reducing ammonia volatilization during aerobic composting.

[0009] Furthermore, the *Bacillus aeruginosa* or the bacterial agent converts ammonia nitrogen into nitrate nitrogen and nitrite nitrogen through nitrification, thereby reducing ammonia volatilization.

[0010] Furthermore, the temperature conditions for the nitration process are 55℃-60℃.

[0011] Furthermore, the acid-base environment for the nitration reaction is pH=7-9.

[0012] Furthermore, the carbon source for the nitration is sodium succinate.

[0013] Furthermore, the carbon-to-nitrogen ratio in the nitration process is 10-20.

[0014] The present invention provides a strain of *Bacillus aeruginosa* with a simple cultivation method and a short cultivation cycle. It has good high-temperature resistance and nitrification ability. When applied to aerobic composting, it can reduce ammonia emissions, effectively reduce nitrogen loss in compost products, and reduce environmental pollution caused by gas generation in aerobic composting of livestock and poultry. Attached Figure Description

[0015] Figure 1 This is a morphological diagram of strain GW-C in heterotrophic nitrification medium;

[0016] Figure 2 This is a phylogenetic tree diagram of strain GW-C;

[0017] Figure 3 The growth curve and ammonia nitrogen utilization efficiency of strain GW-C are shown.

[0018] Figure 4 This is a graph showing the utilization of different carbon sources by strain GW-C.

[0019] Figure 5 The utilization of strain GW-C at different temperatures;

[0020] Figure 6 The utilization of strain GW-C at different pH levels;

[0021] Figure 7 The utilization of different C / N ratios by strain GW-C;

[0022] Figure 8 The graph shows the temperature monitoring results of the compost pile during the aerobic composting process for the experimental and control groups.

[0023] Figure 9 The graph shows the ammonia detection results during the aerobic composting process for the experimental and control groups.

[0024] Figure 10 The graph shows the cumulative ammonia emissions during the aerobic composting process for the experimental and control groups.

[0025] Figure 11 The graph shows the nitrate nitrogen detection results during aerobic composting in the experimental and control groups.

[0026] Figure 12 The graph shows the detection results of total nitrogen during aerobic composting in the experimental and control groups. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0028] 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.

[0029] In one embodiment of the present invention, a heat-resistant, heterotrophic nitrification-resistant strain of *Aeribacillus pallidus* sp. GW-C was screened out and named. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34226; the deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is April 16, 2025.

[0030] The *Bacillus globus* GW-C, which has a maximum temperature tolerance of 65°C, is a thermotolerant heterotrophic nitrifying bacterium that reduces ammonia emissions during aerobic composting. It can be used to reduce ammonia emissions during sludge composting. Its properties are as follows:

[0031] like Figure 1 As shown, under culture medium, it appears as a round, white, opaque organism with neat edges, a moist surface, and slightly raised surfaces. Under scanning electron microscopy, it appears as a single, short rod with blunt, rounded edges, and is Gram-negative. Physiological and biochemical tests show that *Bacillus globus gravidarum* GW-C is positive in starch hydrolysis, glucose fermentation, fructose fermentation, sucrose fermentation, gelatin liquefaction, and citrate utilization tests; and negative in indole, hydrogen peroxide, acetylmethane (VP), methyl red, and hydrogen sulfide tests. *Bacillus globus gravidarum* GW-C tolerates a temperature of 65℃; growth is inhibited at 65℃, and the optimal culture temperature is 55℃.

[0032] In another embodiment of the present invention, a nitrifying agent is also provided, which comprises the above-mentioned Bacillus pallida.

[0033] This invention optimizes the culture conditions of Bacillus aeruginosa using single-factor experiments. The microbial agent prepared using Bacillus aeruginosa can solve the problems of large amounts of ammonia and foul odor emissions in existing aerobic composting of livestock and poultry manure.

[0034] Specifically, *Bacillus cereus* GW-C or its inoculum converts ammonia nitrogen into nitrate and nitrite nitrogen through nitrification, thereby reducing ammonia volatilization. When sodium succinate is used as the carbon source, the nitrification temperature is 55℃-60℃, the pH is 7-9, and the carbon-to-nitrogen ratio (C / N) is 10-20.

[0035] When the pale airborne bacillus GW-C is added to compost materials for composting experiments, it can accelerate the rise in the temperature of the compost pile, which is conducive to the continued survival of the strain during the high-temperature period and maintain good nitrification. It also plays an important role in killing pests in the compost materials.

[0036] The following embodiments are implementation examples of the technical solution of the present invention in practical applications, but are not limited thereto. The reagents and experimental equipment involved are all commercially available products.

[0037] Example 1: This example provides a method for isolating strains resistant to high-temperature heterotrophic nitrification, specifically including the following steps:

[0038] S1. High-temperature samples of aerobic compost were collected using the five-point sampling method, placed in sterile sampling bags and stored in ice boxes, and brought back to the laboratory for enrichment, separation and screening.

[0039] S2. Cultivation, isolation, and purification of bacterial strains:

[0040] (1) Preparation of culture medium

[0041] LB medium (g / L): tryptone 10.00, yeast extract 5.00, NaCl 10.00, solid medium supplemented with 18.00 agar powder;

[0042] Enrichment and acclimatization medium (g / L): (NH4)2SO4 2.00, C4H4Na2O4 14.31, Vickers salt solution 50 mL, pH 7.0; Separation medium (g / L): agar powder 18.00, other conditions are the same as enrichment and acclimatization medium.

[0043] Heterotrophic nitrification medium (g / L): (NH4)2SO4 1.00, C4H4Na2O4 7.16, other components same as acclimatization medium;

[0044] Vickers salt solution (g / L): K2HPO4 5.00, MgSO4·7H2O 2.50, NaCl 2.50, FeSO4·7H2O 0.05, MnSO4 0.05.

[0045] Liquid culture medium can be prepared into solid culture medium by adding 1.8% agar. All culture media are sterilized by autoclaving at 121℃ for 20 min before use.

[0046] (2) Take 2 g of the collected compost sample and add it to a 250 mL conical flask containing 200 mL of heterotrophic nitrification medium. Incubate at 180 r / min and 55℃ in a shaker for 3–4 days. Then, take 10% of the enriched solution and add it to a new heterotrophic nitrification medium. Place the new medium in a shaker and incubate at 180 r / min and 55℃. Repeat this process 4–5 times.

[0047] (3) Configure 10 -2 10 -4 10 -6 10 -8 Dilute the bacterial suspension and add 100 μL of the suspension to the surface of the culture medium. Spread the bacterial suspensions of different dilutions onto the isolation medium and incubate at 55°C for 5 days. Then, streak colonies of different colors and sizes until purified bacteria are obtained.

[0048] (4) Preliminary identification of isolated strains: 1 mL of bacterial culture was centrifuged in a pre-prepared sterilized 1.5 mL centrifuge tube, and the supernatant was discarded. Genomic DNA was extracted using a bacterial DNA extraction kit. Universal forward primer 27F and universal reverse primer 1492R were used for polymerase chain reaction (PCR) amplification of 16S rRNA. Sequencing results were analyzed for BLAST similarity in the NCBI database (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the sequences were submitted to the NCBI GenBank database to determine the relevant genera and species of closely related strains. Finally, the Neighbor-Joining method was used to construct a phylogenetic tree. Figure 2 The image shown is an image of the isolation, screening, and phylogenetic tree of strain GW-C. Genomic DNA was extracted from strain GW-C and amplified using 16S rDNA sequencing. The results showed that the strain had a similarity of over 99% to Aeribacillus pallidus sp. PZ-1 (KC441060.1) in the GenBank database, thus confirming the strain as Aeribacillus pallidus sp. The sequence was uploaded to the GenBank database, obtaining its accession number PQ578296.

[0049] (5) Secondary screening of the primary screening strains: The strains that were initially screened were inoculated into liquid heterotrophic nitrification medium and cultured at 55℃ and 160r / min for 96h. The growth OD was measured every 12h. 600 and NH4 +The -N concentration was measured, and then the solution was placed in a shaker at 180 r / min and incubated at 55℃ for 96 h. The supernatant was then used to measure NH4+. + -N concentration was selected by comparison with NH4+. + Strains with high N utilization were subjected to a second screening. Based on the first screening, nitrogen balance was constructed for the selected strains with ammonia nitrogen as the sole nitrogen source. The strain with the highest ammonia assimilation rate, GW-C, was selected for in-depth study and stored in a glycerol solution at -80°C.

[0050] Example 2: Study on nitrogen transformation characteristics of strain GW-C (i.e., Bacillus pallida GW-C) using ammonia nitrogen as the sole nitrogen source, as detailed below:

[0051] Strain GW-C was cultured in a medium with ammonia nitrogen as the sole nitrogen source (nitrogen concentration 400 mg / L, C / N ratio 10), and sodium succinate as the carbon source. Before the experiment, the strain was pre-cultured in an enrichment medium until the OD (oxidative stress) of the strain was reached. 600 ≈1. Take an appropriate amount of bacterial suspension and centrifuge at 10000 r for 5 min at 4℃, then resuspend in PBS at least three times. Then, add 2 mL of the resuspended solution (OD200). 600 ≈1) Inoculate into the three culture media mentioned above. Measure OD every 24 hours. 600 NH4 + -N, NO3 - -N and NO2 - -N, the test methods were ultraviolet spectrophotometry, N-(1-naphthyl)-ethylenediamine spectrophotometry, and Nathaniel's reagent spectrophotometry, respectively. All tests were performed under aseptic conditions, and each test was repeated three times, with uninoculated culture medium as a blank control. The culture medium composition was as follows: (NH4)2SO4 1.8g, C4H4Na2O4 20.1g, Vickers salt solution 50 mL, pH 7.0; Vickers salt solution (g / L): K2HPO4 5.00, MgSO4·7H2O 2.50, NaCl 2.50, FeSO4·7H2O 0.05, MnSO4 0.05.

[0052] The above test results are as follows Figure 3 As shown, it reflects the use of NH4 + The growth curve and nitrogen concentration changes of strain GW-C in a 55℃ medium with -N as the nitrogen source were observed. The results showed that 0-48 h was the rapid growth period of the strain, the growth rate slowed down from 48-72 h, and the OD of the strain peaked at 72 h. 600 The value reached a peak of 1.2, and the decrease after 72 hours is presumably due to the lack of nutrients in the culture medium, leading to bacterial death. NH4+ was present from 0-24 hours. + The -N concentration decreased sharply, and the ammonia nitrogen utilization rate was 5.79 mg / L·h, while NO3- concentration decreased during this process. --N concentration rose to 114.15 mg / L, NO2 - The -N concentration increased to 9.76 mg / L, presumably due to NH4+. + -N is converted into a large amount of NO3 through nitration. - -N and NO2 - -N. NH4 between 24-48h + The NO-N concentration rose to 285.88 mg / L, indicating that some NO2... - -N and NO3 - -N is then reduced to NH4 through assimilation or dissimilation of nitrate. + -N. In summary, with NH4 + In a culture medium where -N is the nitrogen source, strain GW-C can convert NH4+ into nitrogen through nitrification and denitrification. + -N is converted to NO3 - -N and NO2 - -N, suggesting that strain GW-C has good nitrification ability.

[0053] Example 3: Optimization of culture conditions for strain GW-C, as detailed below:

[0054] To evaluate the effects of different environmental conditions on the nitrification performance of the strain, carbon source, carbon-nitrogen ratio, pH, and temperature were used as variables under a nitrogen content of 400 mg / L. Glucose, sodium citrate, sodium succinate, sucrose, and sodium acetate were used as carbon sources to investigate their effects on the strain. The C / N ratio was adjusted to 5-25 to examine its effect on the strain's nitrification performance. The pH was adjusted to 5, 6, 7, 8, 9, and 10 to observe the effect of pH on the strain's nitrification performance. All experiments were conducted at 55℃ and 160 rpm for 96 h. To investigate the effect of temperature on the strain's nitrification performance, the experimental temperatures were adjusted to 45℃, 50℃, 55℃, 60℃, and 65℃, and the strain was cultured at 160 rpm for 96 h. Samples were taken every 12 h to measure the OD (oxidative stress). 600 NH4 + -N, NO3 - -N, NO2 - -N, All experiments were performed under sterile conditions, and each experiment was repeated three times, with uninoculated culture medium as a control.

[0055] To determine the optimal initial carbon source for strain GW-C, five common carbon sources were used as substrates, such as... Figure 4 As shown, when the carbon source is glucose, sucrose, and sodium succinate, the bacterial growth OD... 600At 96 h, the values ​​were 1.12±0.02, 0.93±0.02, and 0.99±0.04, respectively, higher than those of sodium acetate (0.43±0.02) and sodium citrate (0.64±0.01). In terms of ammonia nitrogen utilization, the highest utilization rates (91.63% and 91.52%, respectively) were observed when sodium butyrate and sodium acetate were used as carbon sources. Glucose, sucrose, and sodium citrate showed lower utilization rates of ammonia nitrogen, at 66.00%, 20.49%, and 37.80%, respectively. In conclusion, this strain exhibits the best utilization of sodium succinate, and sodium succinate was selected as the optimal carbon source.

[0056] High temperatures denature proteins, while low temperatures inhibit enzyme activity, thereby reducing metabolic rate or even completely suppressing metabolism. For example... Figure 5 As shown, the bacterial growth OD under different temperature conditions... 600 The corresponding changes in nitrogen concentrations over 0–96 h were observed. At the end of the culture at 60 °C, the OD of strain GW-C was [data missing]. 600 The value is 1.10 ± 0.02, corresponding to the highest NH4 content. + -N utilization was 95.85%, compared to Anoxybacillus contaminans HA under the same temperature conditions. NH4 + -N removal rate was only 71%. Most HN-AD reactions were carried out under mesophilic conditions (20-40℃), under which the strain's NH4+... + -N removal was significantly inhibited. These results indicate that temperature significantly affects the growth of strain GW-E and NH4+ removal. + The ability to remove -N is crucial, and the optimal culture temperature for strain GW-C was ultimately determined to be 50~60℃.

[0057] pH is a crucial factor affecting microbial growth. Most HNADMs adapt to neutral or slightly alkaline environments (pH = 6.0-9.0), and free ammonia is more favorable for heterotrophic nitrification under slightly alkaline conditions. Figure 6 As shown, strain GW-C exhibited growth and NH4+ within a pH range of 5-10. + -N utilization capacity. OD at pH 5-10 after 96 hours of incubation. 600 The values ​​were 0.24±0.01, 0.09±0.02, 1.16±0.03, 0.95±0.02, 1.04±0.07, and 0.68±0.03, respectively, for NH4. + The N-N utilization rates were 51.44%, 64.17%, 85.84%, 90.95%, 75.61%, and 73.70%, respectively. Strain GW-C exhibited the highest NH4+ utilization at pH 8. + -N utilization rate, NH4 at pH 5 and pH 10+ -N removal rate was affected, but after 96 hours of cultivation, it could adapt to extremely acidic and alkaline environments, achieving NH4+ removal under extreme environmental conditions. + The utilization of -N. In summary, pH 7-9 was ultimately chosen for subsequent experiments.

[0058] The ratio of electron donors to electron acceptors in microbial nitrification and denitrification can be reflected by the C / N ratio, which plays a crucial role in denitrification. The results are as follows... Figure 7 The results showed that the OD values ​​of the strains were [data missing] when the C / N ratio was 10, 15, 20, and 25. 600 The values ​​were 1.24±0.02, 1.23±0.02, 1.21±0.01, and 1.07±0.04, significantly higher than the growth OD at a C / N ratio of 5. 600 0.60±0.01. When the C / N ratio is 5, NH4 + The ammonia nitrogen utilization rate was 71.65%. When the C / N ratio was 10–25, the ammonia nitrogen utilization rates of strain GW-C were 98.95%, 99.32%, 99.25%, and 99.18%, respectively. Therefore, when the C / N ratio was 10, the ammonia nitrogen utilization rate of the strain was [missing information]. + -N is the most effective, and the suitable C / N range is 10~25.

[0059] Example 4: An aerobic composting experiment was conducted on strain GW-C, as detailed below:

[0060] The cow manure samples for the compost were sourced from the Xin Gaoyuan Dairy Farm in Lanzhou New Area, and the corn stalks were sourced from Yuzhong County, Lanzhou City, Gansu Province. Before aerobic composting, the stalks were cut to less than 2 cm to ensure good mixing, and the moisture content was adjusted to approximately 65% ​​using deionized water. The main characteristics of the raw materials are detailed in Table 1.

[0061] Table 1 Physicochemical properties of compost raw materials

[0062]

[0063] This experiment was conducted in a 140-liter laboratory-scale reactor for 30 days. Fresh cow manure and straw were mixed at a fresh weight ratio of 12:1, resulting in a C / N ratio of 25. To investigate the effect of strain GW-C inoculant on the aerobic composting process, three treatment groups were set up: control group (CG): cow manure + straw + volume of sterile water; treatment group (TG): cow manure + straw + 1% strain GW-C inoculant. The preparation method of strain GW-C inoculant is as follows: Based on the experimental results of Example 2, after adjusting the environmental factors such as pH, temperature, and C / N ratio of strain GW-C, it was transferred to 500 mL Erlenmeyer flasks for subculture and enrichment culture to prepare a heat-resistant strain GW-C inoculant. The viable count was higher than 10 throughout the entire process.8 CFU. During composting, a timer socket was used to control the blower's operating cycle, starting it every 30 minutes for 5 minutes each time. The blower's power was 200W, and the air volume was 2.4-2.4m³. 3 The ventilation rate is set at 100°C / min to ensure adequate aeration of the compost material and promote aerobic metabolic activity of microorganisms. Furthermore, to prevent localized anaerobic reactions within the compost pile, manual turning is performed once daily before the high-temperature period, and then every two days during the cooling phase, to promote organic matter decomposition and accelerate the composting process.

[0064] Temperature is an important physicochemical property in the composting process, reflecting the internal conditions of the process. Furthermore, temperature is a crucial determinant of the superiority of certain microbial communities over others. For example... Figure 8 As shown, the entire composting process followed the typical composting temperature change trend of heating-high temperature-cooling-maturation. The temperature of the microbial agent treatment group (TG) rose rapidly in the early stage of composting (0-6 days) and then entered the high-temperature stage (50℃). This may be because the unstable organic matter in the early stage of composting is utilized by microorganisms, releasing a large amount of heat in the process. The highest temperatures of the control group (CG) and the microbial agent group (TG) were 52.96℃ and 56.72℃, respectively. Compared with the crude control (CG), the highest temperature of the microbial agent group (TG) was significantly higher (P<0.05), and the high-temperature period lasted longer. This phenomenon can be attributed to the fact that, under the condition of inoculating the same number of viable microbial agents, using the strain GW-C provided in the embodiments of this invention is more conducive to the composting reaction and improves efficiency.

[0065] like Figure 9 and Figure 10 As shown, ammonia emission is the main pathway for nitrogen loss during aerobic composting. In this embodiment of the invention, the two groups of samples exhibited similar ammonia emission patterns during composting, with an initial increase followed by a gradual decrease. NH3 emissions were mainly concentrated between days 6 and 12, accounting for more than 50% of the cumulative emissions throughout the composting process. CG and TG reached their emission peaks on day 8, with peak emissions of 188.3 mg / d and 110.6 mg / d, respectively. The ammonia emission of the inoculant group (TG) was significantly lower than that of CG (P<0.05). The experimental results indicate that adding strain GW-C inoculant to aerobic composting significantly reduced ammonia emissions. In the initial stage of composting, the content of readily degradable organic matter is high, and microorganisms multiply rapidly in the organic-rich environment, leading to a rapid increase in the number of AOBs (acid-free organic matter), accelerating the decomposition of organic nitrogen compounds during composting, and resulting in the release of large amounts of NH3. The total NH3 emissions in the control group and the microbial agent group during the entire composting process were 1386.22 mg and 884.35 mg, respectively. The ammonia emissions in the microbial agent group (TG) were significantly lower than those in the control group (CG) (P<0.05). Compared with the control group, the microbial agent group reduced ammonia volatilization by 36.2%.

[0066] from Figure 11 It can be seen that as nitrification proceeds during composting, the NO3 levels in both the control and treatment groups decrease. - The nitrogen (NO3) content continues to increase. This phenomenon is mainly due to two reasons: firstly, lower ammonia nitrogen levels and lower temperatures are conducive to the growth and activity of nitrogen (NB); secondly, continuous changes in the properties of compost materials, such as increased temperature, pH fluctuations, and continuous oxygen supply, also promote NO3- production. - The generation of -N. Throughout the composting process, the NO3- of the microbial agent group (TG)... - The NO3- content was significantly higher in the microbial agent group than in the other two treatment groups. At the end of composting, the NO3- content in the microbial agent group (TG) was significantly higher. - The -N content (1.56 g / kg) was 38.1% higher than that of the control group (CG) (1.13 g / kg) (P < 0.05). These results indicate that inoculation with strain GW-C effectively promoted the AOB's conversion of NH4+. + -N is converted to NO3 - The process of -N fixation is the biochemical fixation of nitrogen. Simultaneously, from... Figure 12 The total nitrogen results showed that at the end of composting, the total nitrogen in the treatment group (CG) decreased by 11.89%, while that in the microbial agent group (TG) decreased by only 1.79%, indicating that the addition of microbial agents played a significant role in nitrogen retention during composting.

[0067] It should be noted that the measurement indicators involved in the above embodiments are as follows:

[0068] (1) Temperature: The temperature is measured at fixed times (morning, noon and evening) every day. The ground thermometer (-50~200℃) is used to measure the compost temperature from the top of the pile to a depth of 25cm. The average value of 5 points at the four corners and the center of the pile is taken, and the ambient temperature is measured at the same time.

[0069] (2) Ammonia (NH3): NH3 was determined by boric acid absorption-Nessler colorimetric method.

[0070] (3) Nitrate nitrogen: Weigh 20g of fresh sample into a 250mL conical flask, add 2mol / L KCl solution for extraction and filtration, and determine the nitrate nitrogen content in the sample by ultraviolet spectrophotometry.

[0071] (4) Total nitrogen

[0072] In summary, the *Bacillus globosum* GW-C provided in this embodiment of the invention can undergo nitrification under high temperature conditions, converting ammonium ions into nitrate nitrogen. Adding it to the aerobic compost pile can reduce ammonia emissions by 36.2%. The emission peaks were reached on day 8 in the CG and TG groups, with peak emissions of 188.3 mg / d and 110.6 mg / d, respectively. The ammonia emissions in the TG group were lower than those in the CG group, which can effectively reduce the loss of nitrogen in the compost product.

[0073] When using the Bacillus GW-C provided in this embodiment of the invention during composting, the highest temperature of the compost pile can reach 56.72℃, which is 3.76℃ higher than the highest temperature without bacteria, and the high-temperature period during composting can be extended by 3 days.

[0074] Using the *Bacillus globosum* GW-C provided in this invention during composting can significantly reduce ammonia emissions, with a cumulative ammonia emission of 884.35 mg, compared to 1386.22 mg in the control group without the addition of the bacterial agent.

[0075] The composting process uses the *Bacillus globosum* GW-C, NO3 provided in this embodiment of the invention. - -N content can be increased by 38.10%, and total nitrogen loss can be reduced by 10.10%.

[0076] The *Bacillus globosum* GW-C provided in this invention can reduce ammonia emissions and mitigate environmental pollution caused by gas generation during aerobic composting of livestock and poultry.

[0077] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A strain of *Bacillus pallida*, characterized in that, It was named Aeribacillus spallidus GW-C and is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC NO.34226.

2. A microbial agent with nitrification function, characterized in that, It includes the Pale Air Bacillus as described in claim 1.

3. The application of the *Bacillus aeruginosa* as described in claim 1 or the microbial agent as described in claim 2 in reducing ammonia volatilization during aerobic composting.

4. The application according to claim 3, characterized in that, The pale airborne Bacillus or the bacterial agent converts ammonia nitrogen into nitrate nitrogen through nitrification, and finally into total nitrogen, thereby reducing ammonia volatilization.

5. The application according to claim 4, characterized in that, The temperature conditions for the nitration process are 55℃-60℃.

6. The application according to claim 4, characterized in that, The acid-base environment for the nitration process is pH 7-9.

7. The application according to claim 4, characterized in that, The carbon source for the nitration is sodium succinate.

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