Nitrogen-preserving and deodorizing compound microbial agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses a nitrogen-preserving and deodorizing microbial agent as well as a preparation method and application thereof in livestock and poultry manure compost, and the nitrogen-preserving and deodorizing microbial agent is particularly suitable for efficiently removing ammonia gas and hydrogen sulfide. The microbial agent is composed of two kinds of functional deodorization strains, and the two kinds of functional deodorization strains are the bacillus subtilis for composting and the bacillus sojae for soybean, and the two kinds of functional deodorization strains are the bacillus subtilis for composting and the bacillus sojae for soybean. Experiments prove that the two strains have no antagonism, and show good synergistic nitrogen conservation and deodorization capability after being compounded. In practical application, the complex microbial inoculant not only enables the total nitrogen to be increased by 24.95%-31.58% compared with a control group, but also can remarkably reduce the release concentration of NH3 and H2S in the livestock and poultry manure composting process, and the removal efficiency reaches 73.75%-82.32% and 79.73%-96.11% respectively. In addition, the microbial inoculum is simple and convenient in preparation process, convenient to operate in the using process and low in cost, effectively solves the problems of weak nitrogen conservation and deodorization effects, slow effect and the like of the existing microbial inoculum, and has good engineering application value and popularization prospect.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a nitrogen-retaining and deodorizing composite microbial agent, a preparation method and an application thereof. Background Art

[0002] With the increasing intensification of animal husbandry, the generation of large quantities of livestock and poultry manure has become a major issue in agricultural environmental governance. This creates immense pressure for resource utilization. If improperly handled, it not only causes significant nitrogen volatilization losses, leading to eutrophication of soil and water bodies, but also releases malodorous gases, negatively impacting both the ecological and human habitats. Therefore, how to efficiently utilize livestock and poultry manure as a resource while simultaneously addressing the nitrogen losses and odor pollution generated during its treatment has become a pressing technical challenge and research hotspot.

[0003] Aerobic composting is an important technological approach for stabilizing and repurposing livestock and poultry manure. It effectively converts organic pollutants into stable organic fertilizer, recycles nutrients such as nitrogen, phosphorus, and potassium, and reduces pathogenic microorganisms and toxic and hazardous substances to a certain extent. However, in actual composting processes, nitrogen loss rates generally exceed 30%, and the release of large amounts of odor is an unavoidable problem, seriously affecting the quality of the compost product and the surrounding ecological environment. Ammonia (NH3) and hydrogen sulfide (H2S) are the main odorous components. Typically, NH3 release from the compost pile is concentrated during the high-temperature period, likely due to the inhibition of nitrifying bacteria, the reduced conversion efficiency of ammonium nitrogen to nitrate nitrogen, and the direct volatilization of free ammonia. H2S release, on the other hand, occurs more frequently during the warming period and the initial high-temperature period. This is likely due to the rapid decomposition of easily degradable organic matter in the early stages of composting, the high oxygen consumption, the formation of a localized anaerobic microenvironment, and the promotion of the reduction metabolism of sulfur-containing organic matter. The release of NH3 and H2S not only leads to nitrogen loss and reduces composting efficiency, but also poses potential risks to the environment and health.

[0004] Studies have shown that adding microbial agents with specific functions to compost piles can help regulate nitrogen and sulfur metabolism and significantly inhibit odor release. Therefore, screening microorganisms with efficient NH3 and H2S degradation capabilities and developing composite agent products for livestock and poultry manure composting is an important technical means to achieve synergistic nitrogen conservation, deodorization, and resource utilization. Summary of the Invention

[0005] In view of the problems of poor adaptability of the existing composting nitrogen-retaining and deodorizing microbial agents, significant antagonism, and low degradation efficiency, the present invention aims to provide a high-efficiency nitrogen-retaining and deodorizing composite microbial agent, its preparation method, and its application in livestock and poultry manure composting. The agent is in the form of a liquid preparation with a simple preparation process. It not only increases the total nitrogen content by 24.95-31.58% in pig manure and chicken manure composting systems compared to the control group, but also shows excellent removal effects on NH3 and H2S, with NH3 removal rates reaching 73.75% (pig manure) and 82.32% (chicken manure), respectively, and H2S removal rates reaching 96.11% (pig manure) and 79.73% (chicken manure), respectively.

[0006] The two core strains involved in the present invention are: compost Bacillus (Bacillus stercoris), strain CTH-1, deposited with China General Microbiological Culture Collection Center, deposit number CGMCC NO.33898, and deposit date May 26, 2025; soybean marine Bacillus (Oceanobacillus sojae), strain CTH-2, deposited with China General Microbiological Culture Collection Center, deposit number CGMCC NO.33899, and deposit date May 26, 2025.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions:

[0008] The invention provides a nitrogen-retaining and deodorizing composite microbial agent, which comprises the following two strains: a composting Bacillus (Bacillus stercoris), which is a strain CTH-1 and has a preservation number of CGMCC No. 33898; and a soybean marine Bacillus (Oceanobacillus sojae), which is a strain CTH-2 and has a preservation number of CGMCC No. 33899.

[0009] Furthermore, the mixing ratio of the composting Bacillus CTH-1 and the soybean marine Bacillus CTH-2 in terms of the number of viable bacteria is 1:1.

[0010] Furthermore, the bacterial agent is a liquid preparation.

[0011] Furthermore, the effective viable bacterial concentrations of the composting Bacillus CTH-1 and the soybean marine Bacillus CTH-2 are ≥10 8 cfu / mL.

[0012] The present invention also provides a preparation method of the above-mentioned nitrogen-retaining and deodorizing composite microbial agent, which comprises: inoculating composting Bacillus CTH-1 and soybean marine Bacillus CTH-2 into a liquid culture medium respectively, culturing them for 24 hours under shaking conditions of 25-30° C. and 180-200 r / min to obtain a composting Bacillus active bacteria seed liquid and a soybean marine Bacillus active bacteria seed liquid; detecting the live bacteria concentration of the composting Bacillus active bacteria seed liquid and the soybean marine Bacillus active bacteria seed liquid by a plate count method, adjusting the effective live bacteria concentration with sterile water, and mixing the composting Bacillus active bacteria seed liquid and the soybean marine Bacillus active bacteria seed liquid in a clean bench to obtain the nitrogen-retaining and deodorizing composite microbial agent.

[0013] 1. Strain Screening Method

[0014] The present invention provides a method for screening composting Bacillus and soybean marine Bacillus, comprising the following steps: taking 1 g of sample from a co-composting system of pig manure and corn straw, adding 10 mL of sterile water, shaking on a rotary shaker (150 rpm, 30° C.) for 7 days to obtain a compost dilution solution, taking the supernatant after standing, and gradiently diluting to 10 -8 , take 0.1 mL of the dilution solution and inoculate it into solid culture medium. After incubation at 37°C, select colonies with different morphologies and dominant growth, purify them by streaking on plates, and prepare slant-type strains for preservation.

[0015] 2. Antagonism Test

[0016] The two target strains were cultured to the logarithmic growth phase and tested for mutual growth inhibition using the stand-off culture method or plate diffusion method. The results showed that there was no significant antagonism between Bacillus composting and Bacillus sojae, and they could coexist synergistically.

[0017] 3. Identification of NH3 degradation ability

[0018] Prepare an NH3-selective culture medium and sterilize it by autoclaving at 121°C for 15 minutes before use. Add 10 mL of culture medium and 10 mL of 25% ammonia solution to each bottle, inoculate 500 μL of the bacterial solution in each bottle, seal the bottle, and incubate in a shaker at 37°C and 200 rpm for 5 days. Degradation ability was assessed by observing the turbidity and pH changes of the bacterial solution. Both strains demonstrated excellent NH3 degradation capabilities.

[0019] 4. Identification of H2S degradation ability

[0020] An H2S-selective culture medium was prepared and sterilized at 120°C with high-pressure steam for 15 minutes. Afterwards, the medium was placed in a beaker and inoculated with 500 μL of the activated bacterial solution. A centrifuge tube was placed inside the beaker, and 12 mL of 25% H2SO4 was added, followed by 4 g of FeS as an H2S release source. The beaker was sealed and incubated at 37°C and 200 rpm for 5 days. The degradation capacity of the culture medium was assessed by observing the turbidity and pH changes. Both strains demonstrated excellent H2S degradation.

[0021] 5. Preparation of liquid composite bacterial agent

[0022] The two strains were inoculated into their appropriate liquid culture medium and cultured at 25°C in a shaker (180 rpm) for 24 hours until they entered the logarithmic phase. The viable bacterial concentration was determined by plate count and diluted with sterile water to 1.0 × 10 8 ~2.0×10 8 cfu / mL, and mixed in a volume ratio of 1:1 to obtain a liquid composite bacterial agent.

[0023] The present invention also provides an application of the above-mentioned nitrogen-retaining and deodorizing composite microbial agent in livestock and poultry manure composting.

[0024] Furthermore, a method for using the nitrogen-retaining and deodorizing composite microbial agent for nitrogen retention and deodorization in livestock and poultry manure compost comprises spraying the nitrogen-retaining and deodorizing composite microbial agent on the surface of the livestock and poultry manure compost raw material at a rate of 5% to 15% of the mass of the odor source material, and mixing the mixture evenly.

[0025] Furthermore, the components of the odor mainly include ammonia and hydrogen sulfide; and the livestock and poultry manure includes pig manure and / or chicken manure.

[0026] Furthermore, the livestock and poultry manure composting specifically includes: using corn stalks as auxiliary material, mixing it with pig manure and / or chicken manure according to a set carbon-nitrogen ratio.

[0027] Furthermore, the particle size of the corn straw auxiliary material is less than 0.5 cm and the moisture content is lower than 10%.

[0028] The specific application method for compost deodorization includes spraying the prepared liquid composite microbial agent at a rate of 5% to 15% of the total compost mass, and then stirring evenly after spraying. The compost material is pig manure or chicken manure supplemented with corn straw to adjust the carbon-nitrogen ratio to 25-30 and the original moisture content to approximately 60%.

[0029] Among them, corn straw is crushed to a particle size of less than 0.5 cm and a moisture content of less than 10%, which is used to improve the material structure and regulate moisture to create a composting environment that is more conducive to the growth of microorganisms.

[0030] Finally, compared with the prior art, the present invention has at least the following beneficial effects:

[0031] 1. The strains used are derived from the actual livestock and poultry manure composting system. They are dominant indigenous bacteria with strong environmental adaptability and high metabolic activity.

[0032] 2. The two strains have no antagonistic effect and have outstanding ability to synergistically degrade NH3 and H2S;

[0033] 3. The bacterial agent is a liquid preparation, and the number of viable bacteria is stable at 10 8 cfu / mL or more, easy to use, and can achieve rapid on-site inoculation;

[0034] 4. Experimental data show that in pig manure and chicken manure composting systems, not only can the total nitrogen content be increased by 24.95-31.58% compared to the control group, but the release of NH3 and H2S can also be reduced to 73.75%-82.32% and 79.73%-96.11% respectively;

[0035] 5. It helps to improve the fertilizer efficiency of compost products, reduce nitrogen loss, reduce the risk of odor pollution, and improve the resource utilization efficiency of livestock and poultry manure;

[0036] 6. It has good application prospects and can be promoted in the manure emission reduction and harmless treatment systems of large-scale livestock and poultry farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the appearance of the plate culture of compost Bacillus and soybean marine Bacillus;

[0038] Figure 2 This is the experimental result of the degradation ability of NH3 and H2S by composting Bacillus and soybean marine Bacillus;

[0039] Figure 3 This is a comparison of NH3 release concentrations during composting under different treatments;

[0040] Figure 4 Comparison of NH3 release concentration during composting under different treatments. DETAILED DESCRIPTION

[0041] To more fully illustrate the technical solution of the present invention, the present invention is described in detail below with reference to diagrams and typical embodiments.

[0042] Example 1: Screening of strains and antagonism experiments

[0043] (1) Strain isolation and purification

[0044] 1g sample was collected from the pig manure and corn straw compost pile, 10mL sterile water was added, and the sample was placed in a rotary shaker (150r / min, 30℃) for 7 days to fully release the microorganisms in the pile. After standing, the supernatant was taken and diluted in 10-fold series (10 -2 to 10 -8 ), spread 0.1 mL of each dilution onto the surface of LB solid medium and incubate upside down in a 37°C incubator for 24 hours. Select dominant strains based on colony morphology, purify them by plate streak, and transfer them to LB slant medium after purification. Figure 1 The culture appearance of typical target strains is shown: compost Bacillus is white to beige, with neat edges and a slightly raised morphology; soybean marine Bacillus is light yellow, with neat edges and a flat morphology.

[0045] The LB culture medium was prepared as follows: 3.0 g / L beef extract; 10.0 g / L peptone; 5.0 g / L NaCl; and 18 g / L agar. The mixture was dissolved in deionized water, pH 7.4-7.6, and sterilized by high-pressure steam at 121° C. for 30 min.

[0046] (2) Strain antagonism test

[0047] After culturing the two target bacterial strains in LB liquid medium at 30°C and 180 rpm for 24 hours, 5 μL of each culture was inoculated onto nutrient broth agar plates for confrontation. The colonies were observed for the presence of inhibition zones in the contact area. If the strains grew normally with no distinct boundary between them, it was considered non-antagonistic. The results showed no antagonism between Bacillus composting and Bacillus sojae marineus, demonstrating a good foundation for their combination.

[0048] Example 2: Identification of NH3 and H2S degradation capabilities

[0049] (1) NH3 degradation ability

[0050] Prepare NH3 selective medium (see recipe below) and autoclave (121°C, 15 min) for later use. Add 10 mL of medium and 10 mL of 25% ammonia to each bottle, inoculate 500 μL of activated bacterial solution, seal, and culture in a shaker at 37°C, 200 rpm, for 5 days. Observe the turbidity and pH of the culture.

[0051] The formula of NH3 selective culture medium is as follows: sucrose 50.0 g, ammonia water 10.0 mL, KH2PO4 2.0 g, MgSO4·7H2O 0.5 g, FeSO4 0.1 g, 1% ZnSO4 solution 5.0 mL, NaCl 2.0 g, make up to 1000 mL, sterilize at 121°C for 30 min, pH natural.

[0052] (2) H2S degradation ability

[0053] Prepare H2S-selective culture medium (see recipe below), sterilize by autoclaving (120°C, 15 minutes), and transfer to a beaker. Inoculate 500 μL of activated bacterial solution. Place a 50 mL sterile centrifuge tube in the center of the beaker, add 12 mL of 25% H2SO4 and 4 g of FeS, seal the tube, and incubate the culture in a shaker at 37°C, 200 rpm, for 5 days. Monitor the turbidity and pH of the culture medium to confirm H2S degradation capacity.

[0054] The formula of H2S selective culture medium is as follows: glucose 5.0 g, K2HPO4 0.5 g, KNO3 1.0 g, MgCl2 0.5 g, NaCl0.5 g, NH4Cl 0.5 g, Na2CO3 1.0 g, FeCl2 0.01 g, make up to 1000 mL, sterilize at 121°C for 30 min, pH natural.

[0055] Figure 2 The figure shows the comparison of the strains' ability to degrade NH3 and H2S. Both strains caused the NH3 selective culture medium and the H2S selective culture medium to change from initially clear to turbid, indicating that the two strains have the ability to degrade NH3 and H2S. In addition, both strains caused the pH of the NH3 selective culture medium to decrease by 1.0 to 1.2 and caused the pH of the H2S selective culture medium to increase by 0.4 to 0.6, further confirming that the two strains have the ability to degrade NH3 and H2S.

[0056] Example 3: Preparation of liquid deodorizing composite bacterial agent

[0057] (1) Expansion culture of strain seed liquid

[0058] The composting Bacillus and soybean marine Bacillus obtained by screening in Example 1 were inoculated into LB liquid culture medium respectively, and cultured at 25° C. and 180 r / min shaking conditions for 24 hours to obtain active bacterial seed liquid.

[0059] (2) Compounding of microbial agents

[0060] The viable bacterial concentration was determined by plate counting method and adjusted to 1.5 × 10 8 cfu / mL, and mixed the bacterial liquids at a ratio of 1:1 according to the number of viable bacteria in an ultra-clean workbench to prepare a liquid composite microbial agent.

[0061] Example 4: Application effect of liquid bacterial agent in composting

[0062] (1) Test materials and equipment

[0063] Raw materials: Pig manure / chicken manure and corn straw, sourced from farms in Qinhuangdao area.

[0064] Testing equipment: electronic thermometer (Shenzhen Xindeli Electronics), Kjeldahl nitrogen analyzer (Shanghai Yihong Instrument), fluorescence spectrometer (Hitachi, Japan), pump-suction gas detector (Changzhou Baiboli Electronics).

[0065] (2) Composting test design and operation: The raw materials were crushed to about 1.0 cm and homogenized. Four treatment groups were set up: T1, pig manure; T2, pig manure + compound microbial agent; T3, chicken manure; T4, chicken manure + compound microbial agent. Corn straw was used to adjust the C / N ratio to 25, and the initial moisture content was uniformly adjusted to 60%. The compost was turned every 3 days, and the fermentation cycle was 15 days. The amount of microbial agent applied was 10% of the total mass of the raw materials. An electronic thermometer was installed at the bottom to monitor the reaction and ambient temperature in real time. A gas detector was used at a fixed point 2 to 3 cm deep on the surface of the compost to continuously measure for 5 minutes and record the changes in NH3 and H2S concentrations. Compost samples were collected using the five-point sampling method before and after composting and mixed evenly for humification index and total nitrogen analysis.

[0066] (3) Application results

[0067] The results of the humification test are shown in Table 1.

[0068] Table 1 Comparative data of humification index and total nitrogen after composting under different treatments

[0069]

[0070] As can be seen from Table 1, the humification index of group T1 was 1.48, and the humification index of group T2 was 2.01, an increase of 35.81%; the humification index of group T3 was 1.87, and the humification index of group T4 was 3.44, an increase of 83.96%.

[0071] The total nitrogen test results are shown in Table 1. The total nitrogen concentration of the T1 group was 15.03 g / kg, and the total nitrogen concentration of the T2 group was 18.78 g / kg, an increase of 24.95%; the total nitrogen concentration of the T3 group was 17.89 g / kg, and the total nitrogen concentration of the T4 group was 23.54 g / kg, an increase of 31.58%.

[0072] The test results of NH3 application are as follows Figure 3 As shown, the NH3 concentration in the T1 group decreased by 29.91%, in the T2 group decreased by 73.75%, and increased by 43.84%; in the T3 group decreased by 33.62%, and in the T4 group decreased by 82.32%, and increased by 48.70%.

[0073] Application H2S test results such as Figure 4 As shown, the H2S concentration in the T1 group decreased by 40.93%, in the T2 group decreased by 96.11%, and increased by 55.18%; in the T3 group decreased by 28.25%, and in the T4 group decreased by 79.73%, and increased by 51.48%.

[0074] The experimental results confirmed that the prepared composite bacterial agent can, on the one hand, promote the humification degree of compost, on the other hand, increase the total nitrogen concentration of compost, and finally significantly promote the synergistic removal of NH3 and H2S during the composting process, and has good application potential for nitrogen retention and deodorization.

[0075] Those skilled in the art will appreciate that the above-described embodiments are specific examples of the present invention, and that in actual applications, various changes in form and detail may be made thereto without departing from the spirit and scope of the present invention. Any person skilled in the art may make changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope defined in the claims.

Claims

1. A nitrogen-retaining and deodorizing composite microbial agent, characterized in that: These include the following two strains: Composting Bacillus (Bacillus stercoris), strain CTH-1, its deposit number is CGMCC NO.33898; The strain of Oceanobacillus sojae is CTH-2, and its deposit number is CGMCC NO.33899.

2. The nitrogen-retaining and deodorizing composite microbial agent according to claim 1, wherein The mixing ratio of the composting Bacillus CTH-1 and the soybean marine Bacillus CTH-2 is 1:1 in terms of the number of viable bacteria.

3. The nitrogen-retaining and deodorizing composite microbial agent according to claim 1, wherein The bacterial agent is a liquid preparation.

4. The nitrogen-retaining and deodorizing composite microbial agent according to claim 3, wherein The effective live bacterial concentrations of composting Bacillus CTH-1 and soybean marine Bacillus CTH-2 are ≥10 8 cfu / mL.

5. A method for preparing a nitrogen-retaining and deodorizing composite microbial agent according to any one of claims 1 to 4, characterized in that: The preparation method comprises: The composting Bacillus CTH-1 and the soybean marine Bacillus CTH-2 were inoculated into liquid culture medium respectively, and cultured at 25-30°C and 180-200 r / min shaking conditions for 24 hours to obtain the composting Bacillus active bacterial seed liquid and the soybean marine Bacillus active bacterial seed liquid; The live bacteria concentrations of the compost Bacillus subtilis active bacteria seed liquid and the soybean marine Bacillus subtilis active bacteria seed liquid are detected by a plate count method, and the effective live bacteria concentrations are adjusted with sterile water. The compost Bacillus subtilis active bacteria seed liquid and the soybean marine Bacillus subtilis active bacteria seed liquid are mixed in a clean bench to obtain the nitrogen-retaining and deodorizing composite microbial agent.

6. Use of the nitrogen-retaining and deodorizing composite microbial agent according to any one of claims 1 to 4 in composting livestock and poultry manure.

7. The use according to claim 6, characterized in that The method of using the nitrogen-retaining and deodorizing composite microbial agent for nitrogen retention and deodorization in livestock and poultry manure compost comprises: spraying the nitrogen-retaining and deodorizing composite microbial agent on the surface of livestock and poultry manure compost raw materials at a mass ratio of 5% to 15% of the odor source material and mixing the mixture evenly.

8. The use according to claim 7, characterized in that The components of the odor mainly include ammonia and hydrogen sulfide; the livestock and poultry manure includes pig manure and / or chicken manure.

9. The use according to claim 7, characterized in that The livestock and poultry manure composting specifically includes: using corn stalks as auxiliary material, mixing with pig manure and / or chicken manure according to a set carbon-nitrogen ratio to form a compost.

10. The use according to claim 9, characterized in that The corn stalk auxiliary material has a particle size of less than 0.5 cm and a moisture content of less than 10%.

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