High-temperature-resistant efficient nitrosation complex microbial inoculant and application of high-temperature-resistant efficient nitrosation complex microbial inoculant in aerobic composting of livestock and poultry manure

Abstract

The invention belongs to the field of environmental microorganisms, and discloses a high-temperature-resistant efficient nitrosation complex microbial inoculant and an application of the high-temperature-resistant efficient nitrosation complex microbial inoculant in aerobic composting of livestock and poultry manure, and the complex microbial inoculant comprises bacillus firmus CCTCC M 20241091, vinca bacillus CCTCC M 20241092, and achromobacter xylosus CCTCC M 2024620. The compound strain can accelerate conversion of ammonium nitrogen into nitrite nitrogen during aerobic composting of livestock and poultry manure, so that the amount of NH4 < + >-N converted into NH3 in the composting process is reduced, and nitrogen loss caused by volatilization of ammonia gas is reduced. The nitrosobacteria agent has the advantages of high conversion efficiency, simple operation and no secondary pollution, and is an effective method for reducing nitrogen loss and ammonia volatilization in the aerobic composting process.

Description

Technical Field

[0001] The invention belongs to the field of environmental microorganisms, and in particular relates to a high-temperature resistant and efficient nitrite-forming composite bacterial agent and its application in aerobic composting of livestock and poultry manure. Background Art

[0002] The rapid development of animal husbandry meets people's demand for livestock products to a certain extent, and is also an important aspect of environmental issues, among which livestock and poultry manure is one of the main influencing factors. According to data released by China's livestock and poultry waste treatment industry, the amount of livestock and poultry manure produced nationwide has continued to grow in the past seven years, bringing huge pressure to environmental governance. The resulting environmental problems such as gas, odor, pathogens, dust and microorganisms are often reported. As a resource that is misplaced, the utilization value of a large amount of manure is reflected in more and more studies on the resource utilization of manure. Reasonable use of manure can not only generate energy, but also prevent environmental pollution. It can promote the green and healthy development of livestock and poultry farming. As a major breeding country, my country is in urgent need of resource utilization of livestock and poultry manure in farms. In order to achieve green, healthy and sustainable development of livestock and poultry farming, how to realize the resource utilization of livestock and poultry manure is the key.

[0003] Composting is widely used as an easy-to-use and economical way to manage organic waste. Composting technology is an aerobic, thermophilic, solid-state fermentation process mediated by microorganisms. Aerobic composting is an aerobic biotransformation process that converts organic matter into stable, relatively non-toxic compounds under the action of microorganisms and the corresponding enzymes they secrete, thereby reducing the volume and mass of feces and killing pathogenic microorganisms by high temperatures during the composting process. However, the large-scale loss of nitrogen has long been a major problem that has not been solved in aerobic composting. It not only causes the emission of nitrogen-containing gases such as ammonia (NH3), but also reduces the quality of the final compost product, hindering the development of composting technology. It is reported that the amount of nitrogen lost through NH3 emissions during the composting process of different materials can reach 21%-77% of the initial composting level.

[0004] Microorganisms play an important role in aerobic composting. Bacteria and actinomycetes as well as fungi and protozoa are the main microorganisms in aerobic composting. Nitrogen conversion in the composting process can be divided into ammoniation, nitrification, assimilation, denitrification, DNRA, anaerobic ammonium oxidation, nitrogen fixation and other processes. Ammoniation is the first step in the nitrogen cycle in composting, which mainly occurs during the composting warming period and high temperature period. It is the process by which ammonifying bacteria mineralize organic nitrogen in livestock and poultry manure into NH4 +-N process. Part of the ammonium nitrogen produced during the ammoniation process will be converted into ammonia gas, and the other part will enter the nitrification process. Nitrification is a key step to accelerate the conversion of ammonium nitrogen into oxidized nitrogen (nitrite and nitrate) and reduce ammonia volatilization. This process is carried out in two steps: the first step is to oxidize ammonium nitrogen into nitrite nitrogen, and then nitrite nitrogen is oxidized into nitrate nitrogen. Nitrate nitrogen allows nitrogen to be retained in organic fertilizers more stably, thereby achieving the effect of improving quality and retaining nitrogen.

[0005] The Bacillus firmus YA10 provided by the present invention is a Bacillus strain separated from an aerobic composting process and capable of converting ammonium nitrogen into nitrite nitrogen. It can convert ammonium nitrogen into nitrite nitrogen during the aerobic composting process, has a certain high temperature resistance, and has broad application prospects.

[0006] Changchun Barga YS5, Achromobacter xylosylating H-2 and Bacillus firmus YA10 were all isolated from aerobic composting. This nitrosating composite bacterial agent was first reported to be used in aerobic composting of livestock and poultry. Since it comes from aerobic composting of livestock and poultry manure, it has the advantages of high efficiency, high temperature resistance, no pollution to the environment, and reduced nitrogen loss and ammonia emission during aerobic composting, and has broad application prospects. Summary of the invention

[0007] The invention aims to provide a high-efficiency high-temperature resistant nitrosating composite bacterial agent. The nitrosating composite bacterial agent comprises Bacillus firmus YA10, Bhargavaea changchunensis YS5 and Achromobacter xylosoxldans H-2, and the preservation numbers are CCTCC NO: M20241091, CCTCC NO: M 20241092 and CCTCC NO: M 2024620, respectively.

[0008] Another object of the present invention is to provide the use of the above-mentioned nitrite-containing bacteria agent in aerobic composting of livestock and poultry manure.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A high-temperature resistant and efficient nitrosating composite bacterial agent, wherein the main effect bacterial agent of the composite bacterial agent is obtained:

[0011] Acquisition of Bacillus firmus YA10:

[0012] The applicant isolated a Bacillus strain from the aerobic composting sample, which was identified as Bacillus firmus. It was sent to the China Center for Type Culture Collection for preservation on May 29, 2024, and the classification name was: Bacillus firmus YA10; the preservation number was CCTCC NO: M 20241091; location: Wuhan University, Wuhan, China.

[0013] Bacillus firmus YA10 colonies grew well in LB medium.

[0014] Acquisition of Bhargavaea changchunensis YS5:

[0015] The applicant isolated a strain from an aerobic composting sample, which belonged to Bhargavaea changchunensis after 16SrDNA sequencing combined with physiological and biochemical identification. It was sent to the China Center for Type Culture Collection for preservation on May 29, 2024, and was classified and named: Bhargavaea changchunensis YS5; the preservation number is CCTCC NO: M 20241092; address: Wuhan University, Wuhan, China.

[0016] Obtaining Achromobacter xylosoxldans H-2:

[0017] The applicant isolated a Bacillus strain from an aerobic composting sample, which was found to belong to Achromobacter xylosoxldans after 16SrDNA sequencing combined with physiological and biochemical identification. It was sent to the China Center for Type Culture Collection for preservation on April 3, 2024, and was classified and named: Achromobacter xylosoxldans H-2; the preservation number is CCTCC NO: M 2024620; address: Wuhan University, Wuhan, China.

[0018] The protection scope of the present invention includes:

[0019] A high-efficiency high-temperature resistant nitrosating composite bacterial agent comprises Bacillus firmus YA10, Bhargavaea changchunensis YS5 and Achromobacter xylosoxldans H-2, and the preservation numbers are CCTCC NO: M 20241091, CCTCC NO: M20241092 and CCTCC NO: M 2024620, respectively.

[0020] In the composite bacterial agent described above, preferably, the effective bacterial concentration of Bacillus firmus YA10, Bargainella changchunii YS5 and Achromobacter xylosylating H-2 is: 2-3:1~2:1~2.

[0021] The best strain ratio of the composite bacterial agent described above, Bacillus firmus YA10, Bacillus var. changchunba YS5 and Achromobacter xylosylating H-2, is 2:1:1.

[0022] The protection scope of the present invention also includes:

[0023] The application of the above-mentioned composite bacterial agent in aerobic composting of livestock and poultry manure.

[0024] The application process described above includes mixing the composite bacterial agent provided by the present invention into the fermentation process during the high temperature fermentation period.

[0025] The application of the above-mentioned composite bacterial agent in the preparation of high temperature resistant composting microbial agent.

[0026] The application of the above-mentioned composite bacterial agent in promoting the conversion of ammonium nitrogen into nitrite nitrogen.

[0027] The above-mentioned composite bacterial agent is used to promote nitrogen conversion in aerobic composting.

[0028] The application of the above-mentioned composite bacterial agent in reducing ammonia emissions in aerobic composting.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] The invention provides a high-efficiency high-temperature resistant nitrosating composite bacterial agent, which can adapt to a high-temperature environment above 50°C, withstand a high-temperature fermentation period in composting, prolong the duration of high temperature, and shorten the composting fermentation cycle.

[0031] Compared with the simple use of existing EM bacterial agents, the present invention effectively reduces the emission of ammonia in aerobic composting by utilizing composite bacterial agents and EM bacterial agents, and at the same time can accelerate the conversion of ammonium nitrogen into nitrite nitrogen and further into nitrate nitrogen, which is retained in the compost sample, thereby reducing nitrogen loss during the composting process. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the colony morphology of Bacillus firmus YA10 strain.

[0033] Figure 2 This is the colony morphology of the YS5 strain of Bargaea changchunensis.

[0034] Figure 3 This is the colony morphology of Achromobacter xylosylating H-2 strain.

[0035] Figure 4 Figure 3 shows the trend graph of NH3 emission rate change (a) and cumulative amount (b) of composting treatment. DETAILED DESCRIPTION

[0036] In order to better explain the present invention, the main contents of the present invention are further illustrated below in conjunction with specific examples, but the contents of the present invention are not limited to the following examples. The technical solutions of the present invention, if not otherwise specified, are conventional techniques in the art, and the reagents or materials, if not otherwise specified, are all derived from commercial channels.

[0037] Embodiment 1:

[0038] Obtaining Bacillus firmus YA10, Bargainella changchunensis YS5 and Achromobacter xylosylating H-2:

[0039] (1) Subculture screening of screening medium with ammonium nitrogen as the only nitrogen source:

[0040] The applicant isolated three strains of bacteria from aerobic composting samples, named YA10, YS5 and H-2. After preliminary identification, these three strains can convert ammonium nitrogen into nitrite nitrogen. The applicant also subcultured these three strains and identified their ammonium nitrogen conversion ability. It was found that with the increase of generations, their ammonium nitrogen conversion ability gradually increased. The method for determining the ability to convert ammonium nitrogen into nitrite nitrogen is as follows:

[0041] The fermentation broth of the strain to be tested was inoculated into the screening medium with ammonium nitrogen as the only nitrogen source, and cultured in a 100mL / 250mL system, 37°C, and 180r / min shaking table. After 48h, the NO2 - -N production, each group was repeated 3 times. The ammonium sulfate concentration was gradually increased by 0.5g / L until the bacteria stopped growing, enhancing the strain's adaptability to ammonium nitrogen. After 1 week of culture, 15% of the inoculum was transferred to a new culture medium for growth, and the ammonium sulfate concentration was gradually increased from 0.5g / L to 4g / L. In this way, it was kept in an isolated culture medium with ammonium nitrogen as the only nitrogen source and continued to be subcultured to adapt to growth until the NO2 produced - -N amount no longer increases,

[0042] The formula of the screening culture medium with ammonium nitrogen as the sole nitrogen source is: 0.5g ammonium sulfate, 0.2g potassium dihydrogen phosphate, 0.04g calcium chloride dihydrate, 0.04g magnesium sulfate heptahydrate, 0.5mg phenol red, 0.5mg Fe-EDTA, 1000mL distilled water, and 5% Na2CO3 to adjust the pH to 7.8-8.0.

[0043] Table 1-3 shows the NO2 of strains at different generations - -N Generate amount:

[0044] Table 1 NO2 of different generations of YA10 strains - -N Generation

[0045]

[0046] Table 2 NO2 of YS5 strain in different generations - -N Generation

[0047]

[0048] Table 3 NO2 of H-2 strains in different generations - -N Generation

[0049]

[0050] As can be seen from Tables 1 to 3, the transformation abilities of strains YA10, YS5 and H-2 were stable and no longer increased at the 70th generation. Therefore, these three strains were subjected to high temperature adaptation mutation.

[0051] (2) Evolution of heat-resistance adaptability of YA10, YS5 and H-2 strain composite inoculants

[0052] The bacterial suspensions of YA10, YS5 and H-2 (the concentration of the bacterial suspensions was 1.0×10 8 cfu / ml) were mixed in a volume ratio of 2:1:1 to prepare a composite bacterial agent, and temperature adaptability evolution was performed:

[0053] The composite bacterial agent was cultured in batches in a 100 mL Erlenmeyer flask containing 60 mL of nitrite medium. The OD value of the inoculated population in each Erlenmeyer flask was approximately 0.2. Batch screening was first performed at 42°C, with continuous shaking culture at 180 rpm. Every week, a small volume (the volume required for inoculation at an OD of 0.2) of the expanded culture was transferred to 60 mL of fresh culture medium to allow the culture to grow through a normal growth curve. Afterwards, adaptive evolution was performed at 47°C to allow the culture to grow through a normal growth curve. Finally, adaptive growth was performed at 50°C. Table 4 shows the NO2 growth of the composite bacterial agent at different temperatures and at different generations. - -N Generate amount.

[0054] Nitrite medium: ammonium sulfate 0.5 g, potassium dihydrogen phosphate 0.2 g, calcium chloride dihydrate 0.04 g, magnesium sulfate heptahydrate 0.04 g, phenol red 0.5 mg, Fe-EDTA 0.5 mg, distilled water 1000 mL, 5% Na2CO3 to adjust the pH to 7.8-8.0.

[0055] Table 4 NO2 of composite bacterial agent at different generations and different temperatures - -N Generation

[0056]

[0057] From Table 4, we can see that by the 200th generation, the composite bacterial agent had a significant effect on NO2 - -N production has stabilized and no longer increases. Therefore, the composite bacterial agent was re-streaked and cultured, and single clones were picked and sequenced by 16SrDNA combined with physiological and biochemical identification. The three mutant strains obtained are as follows:

[0058] YA10 is Bacillus firmus YA10. The strain was sent to the China Center for Type Culture Collection for preservation on May 29, 2024, and the classification name is: Bacillus firmus YA10; the preservation number is CCTCC NO: M20241091; location: Wuhan University, Wuhan, China.

[0059] Bacillus firmus YA10 grows well in LB medium ( Figure 1 , Table 5).

[0060] In the present invention, Bacillus firmus is referred to as YA10.

[0061] Morphological characteristics: irregular oval, white, rough and dull colony surface, with irregular edges.

[0062] Physiological and biochemical characteristics of strains: as shown in Table 5

[0063] Table 5: Physiological and biochemical characteristics of Bacillus firmus YA10

[0064]

[0065]

[0066] +: positive reaction; -: negative reaction;

[0067] YS5 strain is Bhargavaea changchunensis ( Figure 2, Table 6), was sent to the China Center for Type Culture Collection for preservation on May 29, 2024, and the classification name is: Bhargavaeachangchunensis YS5; the preservation number is CCTCC NO: M20241092; address: Wuhan University, Wuhan, China.

[0068] Morphological characteristics: small yellow round colonies with neat edges, shiny and opaque surface.

[0069] Physiological and biochemical characteristics of strains: as shown in Table 6

[0070] Table 6 Physiological and biochemical characteristics of Bargaea changchunensis YS5

[0071]

[0072] +: positive reaction; -: negative reaction;

[0073] The H-2 strain is Achromobacter xylosoxldans, which was sent to the China Center for Type Culture Collection for preservation on April 3, 2024. The classification name is: Achromobacterxylosoxldans H-2; the preservation number is CCTCC NO: M 2024620; address: Wuhan University, Wuhan, China.

[0074] Morphological characteristics: small yellow round colonies with neat edges, shiny and opaque surface.

[0075] Physiological and biochemical characteristics of strains: as shown in Table 7, Figure 3 .

[0076] Table 7 Physiological and biochemical characteristics of Achromobacter xylosylating H-2

[0077]

[0078] +: positive reaction; -: negative reaction;

[0079] Embodiment 2:

[0080] The preparation method of a high-efficiency high-temperature resistant nitrosating composite bacterial agent comprises:

[0081] Fermentation of Bacillus firmus YA10 (the percentages in the following culture medium formula are all mass fractions):

[0082] Bacillus firmus YA10 was revived, and 100 μL of YA10 bacterial solution was inoculated into 5 mL of LB liquid medium. The culture was shaken at 37°C and 180 r / min for 12 h until the stable phase. The effective bacterial concentration was 1.8×108 cfu / ml, adjust the concentration to 1.0×10 8 cfu / ml.

[0083] Fermentation of Bargaea changchunensis YS5 (the percentages in the following medium formula are all mass fractions):

[0084] The Changchun Barga strain YS5 was revived, and 100 μL of YS5 bacterial solution was inoculated into 5 mL of LB liquid medium. The culture was shaken at 37°C and 180 r / min for 12 h until the stable phase. The effective bacterial concentration was 2.5×10 8 cfu / ml, adjust the concentration to 1.0×10 8 cfu / ml.

[0085] Fermentation of Achromobacter xylosylating H-2 (the percentages in the following culture medium formula are all mass fractions):

[0086] The xylosylating Achromobacter H-2 was revived, and 100 μL of H-2 bacterial solution was inoculated into 5 mL of LB liquid medium. The culture was shaken at 37°C and 180 r / min for 12 h until the stable phase. The effective bacterial concentration was 2.8×10 8 cfu / ml, adjust the concentration to 1.0×10 8 cfu / ml.

[0087] The above bacterial liquids were mixed at a volume ratio of 2:1:1 to obtain a high-temperature resistant and efficient nitrite-forming composite bacterial agent, which was used in the following examples.

[0088] Embodiment 3:

[0089] Application of high-efficiency high-temperature resistant nitrite-forming compound bacteria in aerobic composting of livestock and poultry manure:

[0090] The composting test was conducted at the Huazhong Agricultural University Premium Pig Farm in January 2024. The raw materials of the test, chicken manure, came from the Huazhong Agricultural University Premium Chicken Farm, and sawdust was purchased from the Huazhong Agricultural University Veterinary Hospital. Some characteristics of the raw materials are shown in Table 8.

[0091] The composting experiment was carried out in a cylindrical composting reactor with a volume of 50L, in which one reactor was inoculated with only EM bacteria as control group 1 (CK1), the one without adding microbial bacteria as control group 2 (CK2), and the one with adding high-temperature resistant high-efficiency nitrite compound bacteria and EM bacteria as treatment group (TM). At the beginning of composting, 15kg of fresh chicken manure and 5kg of sawdust were mixed, and the carbon-nitrogen ratio of the composting raw materials was adjusted to about 25. The moisture content was set to about 60% by adding sterile water, and the composting raw materials were fully mixed. The temperature was monitored in real time during composting. Fresh air was provided to the two composting reactors for aeration through an air supply pump at an air supply rate of 0.3L / min. Aeration frequency: aeration for 10min, intermittent for 1min. About 100g of samples were taken from each treatment group at the 0th, 1st, 3rd, 5th, 7th, 10th, 13th, 16th and 20th day, and a part of them was used to determine NH4 + -N、NO3 - -N, NO2 - -N content, and the other part was stored at 20℃.

[0092] Table 8 Determination of C, N and moisture content in initial materials sawdust and pig manure

[0093]

[0094] Divided into two groups: control group and experimental group, each with 3 replicates.

[0095] Test group: composting started after the compost raw materials were mixed, and the day was recorded as composting day 0. About one day (composting day 1), the high temperature period (temperature 50°C and above) was reached, and the highest temperature in the high temperature period was 59.5°C. When entering the high temperature period (temperature 50°C), 1% (v / w) of the high temperature resistant and efficient nitrite-forming composite bacterial agent prepared in Example 2 was added and mixed evenly. The high temperature period lasted for about 5 days; then it entered the cooling period. On the 8th day of composting, when the pile temperature dropped to about 37°C, 1% (v / w) of EM bacterial agent was added and mixed evenly. On the 11th day of composting, the pile temperature was close to the natural environment temperature, and the pile was basically decomposed.

[0096] Control group 1: Composting started after the composting materials were mixed. The day was recorded as the 0th day of composting. It would reach the high temperature period (temperature above 50°C) in about one day (the first day of composting). The highest temperature in the high temperature period was 55.5°C. When entering the high temperature period (temperature 50°C), sterile water equal to the above bacterial solution was added and mixed evenly. The high temperature period lasted for about 4 days, and then entered the cooling period. On the 7th day of composting, the temperature of the pile dropped to about 37°C, 1% (v / w) EM bacterial agent was added and mixed evenly. When the composting was carried out for 15 days, the temperature of the pile was close to the natural environment temperature, and the pile was basically mature.

[0097] Control group 2: Composting started after the composting materials were mixed. The day was recorded as the 0th day of composting. It would reach the high temperature period (temperature above 50°C) in about one day (the 1st day of composting). The highest temperature in the high temperature period was 55.5°C. When entering the high temperature period (temperature 50°C), sterile water equal to the above bacterial solution was added and mixed evenly. The high temperature period lasted for about 4 days, and then entered the cooling period. On the 7th day of composting, the temperature of the pile dropped to about 37°C. Sterile water equal to the above bacterial solution was added and mixed evenly. When the composting was carried out for 17 days, the temperature of the pile was close to the natural environment temperature, and the pile was basically mature.

[0098] NH4 in compost samples + -N, NO2 - -N、NO3 - Determination of -N

[0099] Weigh 5 g of fresh sample and put it into a conical flask, add 50 ml of sodium chloride solution (1 mol / L), shake in a constant temperature shaker at 180 r / min for 30 min, take out and filter for subsequent determination.

[0100] a.NH4 + -N determination

[0101] Take a certain volume of water sample in a 50mL colorimetric tube, dilute it to the mark with water, add 1mL potassium sodium tartrate and 1mL Nessler's reagent. After leaving it for 10 minutes, compare the color at 420nm.

[0102] Preparation of standard solution: Weigh 3.819 + 0.004g ammonium chloride, dissolve in water, transfer to a 1000ml volumetric flask, dilute to scale, obtain a 1000μg / ml solution, pipette 10ml into a 1000ml volumetric flask, dilute to scale, and prepare before use. (Standard curve: 10μg / ml ammonium chloride standard solution. Add 0, 0.5, 1, 2, 3, 5, 7, 10ml. Add water to scale)

[0103] b.NO2 - -N determination

[0104] Take a certain volume of water sample in a 50mL colorimetric tube, dilute with water to the mark, add 1mL of color developer, and compare the color at 540nm within 20 minutes or 2 hours.

[0105] Color developer: Put 250ml water and 50ml phosphoric acid in a 500ml beaker, add 20.0g 4-aminobenzenesulfonamide (NH-2C6H4SO2NH-2), and then dissolve 1g N-(1-teayl)ethylenediamine dihydrochloride in the above solution, transfer to a 500ml volumetric flask, dilute with water to the mark, and shake well. This solution is stored in a brown reagent bottle at 2-5℃ and can be stable for at least one month.

[0106] Preparation of standard solution: Weigh 1.232g sodium nitrite (NaNO2), dissolve in 150ml water, make up to 1000ml volumetric flask, dilute with water to the mark, shake well, store in a brown reagent bottle, add 1ml chloroform, it can be stable for at least 1 month, and get 250μg / ml. Take 50ml and make up to 250ml to get 50μg / ml. The brown bottle can be stored for one week. Take 10ml and make up to 500ml to get 1μg / ml nitrite nitrogen working solution, prepare it now for use. (Standard curve: 1.00mg / L nitrite nitrogen working solution, add 0, 1, 3, 5, 7, 10ml. Dilute with water to the mark)

[0107] c.NO3 - -N determination

[0108] Take a certain volume of water sample in a 50mL colorimetric tube, dilute with water to the mark, add 1mL hydrochloric acid solution, 0.1mL aminosulfonic acid solution, and compare at 220nm and 275nm. Here is 1mol / L hydrochloric acid. 0.8% aminosulfonic acid: weigh 8g of amino acid and dissolve it in water, dilute to 1L.

[0109] Preparation of standard solution: Weigh 0.7221g of high-grade pure potassium nitrate dried at 105-110℃ for 2h and dissolve it in water, dilute to 1L, i.e. 100μg / ml storage solution. Use a pipette to take 25ml of storage solution and dilute to 100ml to prepare 25μg / ml standard solution (standard curve: 20μg / ml potassium nitrate standard solution, add 0, 0.5, 1, 2, 3, 4ml. Then add water to the scale)

[0110] The experimental results show that the treatment group with high-efficiency nitrite-reducing compound bacteria added has a higher NH4 + -N content was lower than that of the control group (Table 9). + -N reached the maximum value, which was 3.315g / kg for control group 1, 3.361g / kg for control group 2 and 3.009g / kg for the experimental group.

[0111] Table 9 NH4 in compost + -N Variations

[0112]

[0113] The test results show that the test group with the addition of high-efficiency high-temperature resistant nitrite-reducing compound bacteria has a significant effect on NO2 - -N content was higher than that in the control group (Table 10).

[0114] Table 10 NO2 in compost - -N Variations

[0115]

[0116] The test results show that the test group with the addition of high-efficiency high-temperature resistant nitrite-reducing compound bacteria has a higher NO3 - -N content was higher than that in the control group (Table 11).

[0117] Table 11 NO3 in compost - -N Variations

[0118]

[0119]

[0120] In summary, the addition of nitrite-forming bacteria can accelerate the conversion of ammonium nitrogen into nitrite nitrogen and further into nitrate nitrogen, which is retained in the compost sample and reduces nitrogen loss during the composting process.

[0121] Embodiment 12:

[0122] The ability of high-efficiency high-temperature resistant nitrite-reducing compound bacteria to reduce ammonia emissions during aerobic composting:

[0123] Ammonia determination

[0124] The composting test was conducted at the Huazhong Agricultural University Premium Pig Farm in January 2024. The raw materials for the test, chicken manure, came from the Huazhong Agricultural University Premium Chicken Farm, and sawdust was purchased from the Huazhong Agricultural University Veterinary Hospital. Some characteristics of the raw materials are shown in Table 12.

[0125] The composting experiment was carried out in a cylindrical composting reactor with a volume of 50L, in which one reactor was inoculated with only EM bacteria as control group 1 (CK1), the one without adding microbial bacteria as control group 2 (CK2), and the one with adding high-efficiency nitrite-reducing compound bacteria and EM bacteria as treatment group (TM). At the beginning of composting, 15kg of fresh chicken manure and 5kg of sawdust were mixed, the carbon-nitrogen ratio of the composting raw materials was adjusted to about 25, and the moisture content was set to about 60% by adding sterile water, and the composting raw materials were fully mixed. The temperature was monitored in real time during composting. Fresh air was provided to the two composting reactors for aeration through an air supply pump at a supply speed of 0.3L / min. Aeration frequency: aeration for 10min, intermittent for 1min.

[0126] Composting was carried out for 20 days. Composting started on the morning of day 0, and the next day was counted as the first day of composting. From the first day, gas collection began at 9 am every morning. After the greenhouse gas was collected, the wooden plug connected to the hose was plugged at the vent of the fermenter, and a glass bottle containing 20ml of 2% boric acid was placed on the atmospheric sampler, and the bottle mouth was connected to the other side of the hose to collect ammonia. This experiment used the boric acid absorption method to determine ammonia. The absorbed boric acid solution was titrated with 0.05mol / L sulfuric acid, and the titration amount was recorded. The recorded data was processed and analyzed to calculate the ammonia emission.

[0127] Table 12 Determination of C, N and moisture content in initial materials sawdust and pig manure

[0128]

[0129] Divided into two groups: control group and experimental group, each with 3 replicates.

[0130] Test group: composting started after the compost raw materials were mixed, and the day was recorded as the 0th day of composting. It would reach the high temperature period (temperature above 50°C) in about one day (the 1st day of composting), and the highest temperature in the high temperature period was 59.5°C. When entering the high temperature period (temperature 50°C), 1% (v / w) of the high temperature resistant and efficient nitrite-forming composite bacterial agent prepared in Example 2 was added and mixed evenly. The high temperature period lasted for about 5 days; then it entered the cooling period. On the 8th day of composting, the temperature of the pile dropped to about 37°C, 1% (v / w) of EM bacterial agent was added and mixed evenly. On the 11th day of composting, the temperature of the pile was close to the natural environment temperature, and the pile was basically decomposed.

[0131] Control group 1: Composting started after the composting materials were mixed. The day was recorded as the 0th day of composting. It would reach the high temperature period (temperature above 50°C) in about one day (the 1st day of composting). The highest temperature in the high temperature period was 55.5°C. When entering the high temperature period (temperature 50°C), sterile water equal to the above bacterial solution was added and mixed evenly. The high temperature period lasted for about 4 days; then it entered the cooling period. On the 7th day of composting, when the temperature of the pile dropped to about 37°C, 1% (v / w) EM bacterial agent was added and mixed evenly. When the composting was carried out for 15 days, the temperature of the pile was close to the natural environment temperature, and the pile was basically mature.

[0132] Control group 2: Composting started after the composting materials were mixed. The day was recorded as the 0th day of composting. It would reach the high temperature period (temperature above 50°C) in about one day (the 1st day of composting). The highest temperature in the high temperature period was 55.5°C. When entering the high temperature period (temperature 50°C), sterile water equal to the above bacterial solution was added and mixed evenly. The high temperature period lasted for about 4 days; then it entered the cooling period. On the 7th day of composting, the temperature of the pile dropped to about 37°C. Sterile water equal to the above bacterial solution was added and mixed evenly. When the composting was carried out for 17 days, the temperature of the pile was close to the natural environment temperature, and the pile was basically mature.

[0133] The test results show the change of NH3 emission over time. During the high temperature period, NH3 emission increased rapidly and reached a peak on the first day, with control group 1 at 1.906g / d, control group 2 at 1.867g / d, and treatment group at 1.749g / d. It is worth noting that during the high temperature stage, the total cumulative NH3 emission (5.581g) of the treatment group with the addition of high temperature resistant and efficient nitrite compound bacteria was lower than that of control group 1 (6.699g) and control group 2 (6.783g) ( Figure 4 ).

Claims

1. A high-efficiency high-temperature resistant nitrosating composite bacterial agent, the nitrosating composite bacterial agent comprising Bacillus firmus Bacillus firmus YA10, Bargaea changchunensis Bhargavaea changchunensis YS5 and Achromobacter xylosoxldans H-2, the deposit numbers are CCTCC NO: M 20241091, CCTCC NO: M20241092 and CCTCC NO: M 2024620, respectively.

2. According to the composite bacterial agent of claim 1, the effective bacterial concentrations of Bacillus firmus YA10, Bargainella changchunii YS5 and Achromobacter xylosylating H-2 are: 2-3:1~2:1~2.

3. The composite bacterial agent according to claim 2, wherein the optimal strain ratio of Bacillus firmus YA10, Bacillus var. changchunyi YS5 and Achromobacter xylosus H-2 is 2:1:

1.

4. Application of the above-mentioned composite bacterial agent in aerobic composting of livestock and poultry manure.

5. The use according to claim 4, wherein the use process comprises mixing the composite bacterial agent according to claim 1 into the high temperature fermentation period of aerobic composting of livestock and poultry manure for fermentation.

6. Use of the composite bacterial agent according to claim 1 in the preparation of a high temperature resistant composting microbial agent.

7. Use of the composite bacterial agent according to claim 1 in promoting nitrogen conversion in aerobic composting.

8. Use of the composite bacterial agent according to claim 1 in promoting the conversion of ammonium nitrogen into nitrite nitrogen.

9. Use of the composite bacterial agent according to claim 1 in reducing ammonia emission in aerobic composting.

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