A compound microbial agent, its preparation method and space application

Abstract

This invention relates to a compound microbial agent, its preparation method, and its spatial application, belonging to the field of microbial technology. The compound microbial agent provided by this invention includes Bacillus cladei, Bacillus stolonifera, Bacillus sturdierus, Bacillus horikoshi, Bacillus megaterium, Bacillus licheniformis, Bacillus cereus, and Bacillus spheroidis. The compound microbial agent prepared by this invention does not require additional auxiliary materials, processing techniques, or production lines, simplifying the production process and equipment. It can be placed in an automatic integrated spraying device to achieve timed and quantitative automatic spraying. When applied on a large scale in livestock and poultry breeding spaces, it can achieve direct, efficient, and rapid odor removal, realizing rapid air purification.

Description

Technical Field

[0001] This invention relates to a compound microbial agent, its preparation method, and its space application, belonging to the field of microbial technology. Background Technology

[0002] Traditional livestock farming generates enormous quantities of manure and its strong, pungent odor, causing serious air, water, and soil pollution. This hinders rural non-point source pollution control efforts, restricts the long-term development of animal husbandry, and impacts people's quality of life. Among the malodorous gases, ammonia and hydrogen sulfide are the most harmful to human and animal health.

[0003] Chinese patent document CN101412975A (application number 200710163670.5) discloses an organic waste treatment microbial agent, its cultivation method, inoculation method, and application in toilets. The agent includes *Bacillus stolonifer*, *Bacillus cereus*, *Bacillus subtilis*, *Bacillus licheniformis*, and *Bacillus spheroidosa*. This agent is used to treat organic waste, and the final decomposition products are carbon dioxide and water vapor, meeting national emission standards. The application of this agent requires continuous reproduction and decomposition of organic waste within a specific temperature- and humidity-controlled mixing chamber.

[0004] Chinese patent document CN108441447A (application number 201810271418.4) discloses a method for preparing a novel biological deodorizing agent. The preparation method is as follows: a) Selection of microbial strains: select the following microbial strains according to the weight ratio: yeast, Bacillus subtilis, Bacillus licheniformis, Lactobacillus plantarum, and Bacillus megaterium; b) Primary culture; c) Secondary culture; d) Mix evenly; e) Vacuum freeze-drying; f) Select 300-320 parts of activated carbon, 250-280 parts of diatomaceous earth, and 230-270 parts of zeolite according to the weight ratio, pulverize them to a particle size of 26-35 mesh, and mix them evenly with the solid microbial powder to prepare a novel biological deodorizing agent. This invention uses microbial strains to ferment and decompose odorous organic matter, transforming the odorous substances into odorless substances.

[0005] Chinese patent document CN111821847A (application number 202010734658.0) discloses a composite biological deodorizer and its preparation method. The composite biological deodorizer includes tea seed cake extract, tea seed shell extract, Enterococcus faecalis, Bacillus megaterium, Bacillus cereus, methyltrophic Bacillus, and Rhodopseudomonas palustris. The preparation method is to mix and dilute the extracts of tea seed cake and tea seed shell, and then mix them with a microbial community composed of fermented Enterococcus faecalis, Bacillus megaterium, Bacillus cereus, methyltrophic Bacillus, and Rhodopseudomonas palustris.

[0006] Chinese patent document CN102872475A (application number 201210365005.3) discloses a deodorizing and chromium-reducing biological agent and its preparation method, comprising the following components: (1) a compound microbial agent accounting for 10% of the total weight, wherein the compound microbial agent is composed of *Saccharomyces cerevisiae*, *Bacillus spheroidae*, *Lactobacillus plantarum* and *Rhodotorula gravidarum*; (2) a microbial carrier accounting for 90% of the total weight, comprising 20-50 parts of wheat bran or rice bran; 160-300 parts of organic bentonite; and 40-80 parts of water.

[0007] Biological deodorization methods primarily involve fermentation and composting with the pollution source, but reports on direct spraying into the air for deodorization are scarce. Furthermore, products containing carriers such as bran, rice bran, activated carbon, diatomaceous earth, and zeolite, as well as requiring reaction containers like reaction chambers, are unsuitable for spatial applications. Existing reports on microbial deodorizers include those that utilize the prepared microbial deodorizer to ferment and compost odor-emitting organic matter for odor removal; others combine microbial deodorizers with other substances for odor removal.

[0008] There are no reports in the existing technology on the use of microbial inoculants in combination for space deodorization. Nor have any patent documents been found regarding the use of Bacillus clausti or Bacillus sphaeroides for biological deodorization. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a compound microbial agent, its preparation method, and its space applications.

[0010] This invention mentions that compound microbial agents can be directly used for air spray deodorization.

[0011] The technical solution of the present invention is as follows:

[0012] A compound microbial agent comprising Bacillus clausii, Solibacillus silvestris, Bacillus firmus, Sutcliffiella horikoshii, Bacillus megaterium, Bacillus licheniformis, Bacillus cereus, and Bacillus sphaerieus.

[0013] According to a preferred embodiment of the present invention, the compound microbial agent contains the following strains: Bacillus clausii (strain number: BORWO-1, preservation number No. 202191), Solibacillus silvestris (strain number: BORWO-2, preservation number No. 202192), Bacillus firmus (strain number: BORWO-3, preservation number No. 202193), Sutcliffiella horikoshii (strain number: BORWO-4, preservation number No. 202194), Bacillus megaterium (strain number: BORWO-5, preservation number No. 202195), Bacillus licheniformis (strain number: BORWO-6, preservation number No. 202196), and Bacillus cereus (strain number: BORWO-6, preservation number No. 202196). Bacillus cereus (strain number: BORWO-7, accession number No. 202197) and Bacillus sphaerieus (strain number: BORWO-8, accession number No. 202198) are both deposited in the Microbial Culture Collection of the Institute of Ecology, Shandong Academy of Sciences.

[0014] According to a preferred embodiment of the present invention, the total viable count in the compound microbial agent is 10. 7 ~10 10 CFU / mL.

[0015] The preparation method of the above-mentioned compound microbial agent includes the following steps:

[0016] (1) Activation of strains: Each of the above 8 strains was inoculated into a sterile liquid culture medium for culture and activation to obtain bacterial solution;

[0017] (2) The bacterial culture obtained in step (1) is inoculated into liquid culture medium at an inoculation amount of 0.1% to 10% by volume to obtain seed culture.

[0018] (3) Bacterial culture: The seed cultures of the eight strains prepared in step (2) are inoculated into a sterile fermentation medium at an inoculation rate of 1.0-10% by mass. The inoculation is prepared by inoculating the seed cultures of Bacillus clausti, Bacillus agronomycetes, Bacillus sturdius, Bacillus horikoshi, Bacillus megaterium, Bacillus licheniformis, Bacillus cereus and Bacillus spheroidum in a mass ratio of (0.5-3.5):(0.5-3.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5). The culture is prepared by inoculating the seed cultures of the eight strains prepared in step (2) in a mass ratio of (0.5-3.5):( ...1.5):(0.5-1.5):(0.5-1.5). The culture is prepared by inoculating the seed cultures of the eight strains prepared in step (2)

[0019] (4) The compound bacterial solution prepared in step (3) is used directly as a compound bacterial agent;

[0020] Alternatively, the composite bacterial solution prepared in step (3) can be dried at room temperature or low temperature to form a wettable powder, thereby obtaining a composite bacterial agent.

[0021] According to a preferred embodiment of the present invention, in step (1), the liquid culture medium is LB culture medium.

[0022] According to a preferred embodiment of the present invention, in step (1), the culture conditions are 28-37℃, 100-200 rpm, and the culture time is 12-48h.

[0023] According to a preferred embodiment of the present invention, in step (2), the liquid culture medium is LB culture medium.

[0024] According to a preferred embodiment of the present invention, in step (2), the culture conditions are 28-37℃, 100-200 rpm, and the culture time is 12-48h.

[0025] According to a preferred embodiment of the present invention, in step (2), the viable bacterial concentration of the seed liquid is 10 for Bacillus clausti. 9 ~10 10 / mL; the viable bacterial concentration of soil Bacillus was 10 9 ~10 10 / mL; the viable bacterial concentration of Bacillus thuringiensis was 10. 7 ~10 9 / mL; the viable bacterial concentration of Bacillus Horikoshi was 10. 7 ~10 9 / mL; the viable bacterial concentration of Bacillus megaterium was 10. 7 ~10 9 / mL; the viable bacterial concentration of Bacillus licheniformis was 10 7 ~10 9 / mL; the viable bacterial concentration of Bacillus cereus was 10. 7 ~10 9 / mL; the viable bacterial concentration of Bacillus spheroidis is 10 7 ~10 9 / mL.

[0026] According to a preferred embodiment of the present invention, in step (3), the components per liter of the fermentation medium are as follows:

[0027] Yeast powder 3.5–15 g / L, peptone 7.0–15 g / L, sodium chloride 3.5–10 g / L, dipotassium hydrogen phosphate 0.5–1.5 g / L, potassium dihydrogen phosphate 0.5–1.5 g / L, magnesium sulfate 0.1–0.5 g / L, ammonium sulfate 0.5–1.0 g / L, calcium chloride 0.002–0.05 g / L, balance water.

[0028] The application of the above-mentioned compound microbial agent in eliminating odors.

[0029] Application of the aforementioned compound microbial agent in the space field.

[0030] According to a preferred embodiment of the present invention, the above-mentioned compound microbial agent is used in the elimination of odors in the spatial domain.

[0031] According to a preferred embodiment of the present invention, the application method is as follows: dilute the above-mentioned compound microbial agent with water at a volume ratio of 10 to 1000 times, place it in a spraying device, spray for 1 to 30 minutes, and spray at a rate of (0.5 to 1.5) L / min / m³. 3 Spray it in spaces polluted by odors to remove them.

[0032] According to a preferred embodiment of the present invention, the odor-polluting space is an odor-polluting space in the aquaculture industry.

[0033] More preferably, the odor pollution space in the aquaculture industry includes aquaculture sheds, large-scale aquaculture farms, and aquaculture wastewater storage areas.

[0034] Beneficial effects

[0035] 1. This invention addresses the problem of strong, pungent odors and spatial odor pollution generated during traditional aquaculture processes. The pure microbial compound agent prepared is suitable for use in spatial environments. When sprayed in odor-polluted spaces in aquaculture sheds or large-scale farms, it can significantly and quickly eliminate odors and solve the spatial odor problem in aquaculture farms. The ammonia degradation rate can reach 95%, and the hydrogen sulfide degradation efficiency can reach 90%. After deodorization, it can maintain its effect for 6 to 120 hours under conditions where no new pollution sources are generated.

[0036] 2. The compound microbial agent prepared by this invention does not require additional auxiliary materials and the production process and equipment are more simplified; it can be placed in an automatic integrated spraying device to achieve timed and quantitative automatic spraying; when applied on a large scale in livestock and poultry breeding spaces, it can achieve the purpose of direct, efficient and rapid odor removal and rapid air purification. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0038] Unless otherwise specified, the drugs and reagents used in the examples are common market products. Any content not described in detail in the examples is based on the prior art.

[0039] Microbial source

[0040] Bacillus clausii (strain number: BORWO-1, accession number No. 202191), Solibacillus silvestris (strain number: BORWO-2, accession number No. 202192), Bacillus firmus (strain number: BORWO-3, accession number No. 202193), Sutcliffiella horikoshii (strain number: BORWO-4, accession number No. 202194), Bacillus megaterium (strain number: BORWO-1, accession number No. 202194), Bacillus clausii (strain number: BORWO-2, accession number No. 202194), Bacillus clausii (strain number: BORWO-3, accession number No. 202193), Sutcliffiella horikoshii (strain number: BORWO-4, accession number No. 202194), Bacillus megaterium (strain number: BORWO-1, accession number No. 202191), Solibacillus silvestris (strain number: BORWO-2, accession number No. 202192), Bacillus firmus (strain number: BORWO-3, accession number No. 202193), Bacillus horikoshii (strain number: BORWO-4, accession number No. 202194), Bacillus megaterium (strain number: BORWO-4, accession number No. 202195), Bacillus clausii (strain number: BORWO-1, accession number No. 202191), Solibacillus silvestris (strain number: BORWO-2, accession number No. 202195), Bacillus clausii (strain number: BORWO-3, accession number No. The following strains were isolated in-house and are deposited in the Microbial Culture Collection of the Institute of Ecology, Shandong Academy of Sciences. They are: B. megaterium (strain number: BORWO-5, accession number No. 202195), B. licheniformis (strain number: BORWO-6, accession number No. 202196), B. cereus (strain number: BORWO-7, accession number No. 202197), and B. sphaerieus (strain number: BORWO-8, accession number No. 202198). These strains are available for purchase from the collection.

[0041] The bacterial species involved in the embodiments are referred to as "Bacillus clausti BORWO-1, Bacillus stropharia BORWO-2, Bacillus sturdier BORWO-3, Bacillus horikoshi BORWO-4, Bacillus megaterium BORWO-5, Bacillus licheniformis BORWO-6, Bacillus cereus BORWO-7, and Bacillus spheroidae BORWO-8".

[0042] Experimental Example

[0043] The specific source of the microbial strains used in this invention is obtained through the following steps:

[0044] (1) Take 1-10g of compost sample that has been stored for 1 year and add it to 50-200mL of MRS liquid medium and NB liquid medium respectively. Place it on a shaker and shake it at 28-37℃ and 100-200rpm for 12-72h.

[0045] (2) Take 0.1–10 mL of the above-mentioned cultured liquid sample and dilute it serially to 10 mL. -1 ~10 -7 Spread 0.05-0.5 mL onto MRS solid medium and NB solid medium respectively, and incubate upside down at 28-37℃ and 100-200 rpm for 12-72 h;

[0046] (3) Select colonies that grow normally on the solid plates above for 16S identification and obtain 8 Bacillus species. The strain numbers are Bacillus clausii BORWO-1, Solibacillus silvestris BORWO-2, Bacillus firmus BORWO-3, Sutcliffiella horikoshii BORWO-4, Bacillus megaterium BORWO-5, Bacillus licheniformis BORWO-6, Bacillus cereus BORWO-7, and Bacillus sphaerieus BORWO-8. It is deposited at the Institute of Ecology, Shandong Academy of Sciences, at No. 28789, Jingshi East Road, Lixia District, Jinan City.

[0047] Example 1

[0048] A method for preparing a compound microbial agent includes the following steps:

[0049] (1) Activation of strains: Eight strains of Bacillus clausii BORWO-1, Bacillus agronomycetes BORWO-2, Bacillus sturdier BORWO-3, Bacillus Horikoshi BORWO-4, Bacillus megaterium BORWO-5, Bacillus licheniformis BORWO-6, Bacillus cereus BORWO-7, and Bacillus spheroidis BORWO-8, which were stored at -80℃, were inoculated into sterile LB liquid medium at an inoculation rate of 0.1% by volume and cultured at 30℃ and 150 rpm for 48 h to fully activate the strains.

[0050] (2) Seed culture: The above 8 kinds of bacterial solutions that have been fully activated were inoculated into sterile LB liquid medium at an inoculation amount of 0.1% by volume and cultured at 30℃ and 150rpm for 24h to obtain seed culture.

[0051] In step (2), the viable bacterial concentration of the seed solution was 10 for Bacillus clausti. 10 / mL; the viable bacterial concentration of soil Bacillus was 10 10 / mL; the viable bacterial concentration of Bacillus thuringiensis was 10. 7 / mL; the viable bacterial concentration of Bacillus Horikoshi was 10. 7 / mL; the viable bacterial concentration of Bacillus megaterium was 10. 7 / mL; the viable bacterial concentration of Bacillus licheniformis was 10 7 / mL; the viable bacterial concentration of Bacillus cereus was 10. 7 / mL; the viable bacterial concentration of Bacillus spheroidis is 10 7 / mL.

[0052] (3) Bacterial culture: The seed cultures of the eight strains prepared in step (2) were cultured in the following mass ratio: Bacillus clausti, Bacillus agronomycetes, Bacillus sturdierus, Bacillus horikosa, Bacillus megaterium, Bacillus licheniformis, Bacillus cereus, and Bacillus spheroidis. The seed cultures were inoculated into a sterile fermentation medium at a volume fraction of 1.0% and cultured at 30°C and 150 rpm for 24 hours to obtain a composite bacterial culture. The viable cell concentration of the composite bacterial culture was 10. 7 The CFU / mL compound bacterial solution was used as a compound bacterial agent.

[0053] The components per liter of the fermentation medium are as follows: yeast extract 5.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, dipotassium hydrogen phosphate 1.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 0.8 g / L, calcium chloride 0.02 g / L, and the balance being water.

[0054] The compound microbial agent prepared above is used to remove odors in breeding sheds. The specific method is as follows:

[0055] Dilute the above-mentioned compound bacterial solution 10 times by volume, place it in a spraying device, and spray it in the breeding shed for 10 minutes at a spraying rate of 1.5 L / min / m². 3 It can eliminate odors, with an ammonia degradation rate of 91% and a hydrogen sulfide degradation efficiency of 90%. After deodorization, it can maintain its effect for 120 hours without the generation of new pollution sources.

[0056] The detection method for ammonia is based on GB / T18204.25-2000 "Method for Determination of Ammonia in Air in Public Places", and the detection method for hydrogen sulfide is based on GB / T14678-93 "Determination of Hydrogen Sulfide, Methanethiol, Dimethyl Sulfide and Dimethyl Disulfide in Air - Gas Chromatography".

[0057] Example 2

[0058] A method for preparing a compound microbial agent includes the following steps:

[0059] (1) Activation of strains: Eight strains of Bacillus clausii BORWO-1, Bacillus agronomycetes BORWO-2, Bacillus sturdier BORWO-3, Bacillus Horikoshi BORWO-4, Bacillus megaterium BORWO-5, Bacillus licheniformis BORWO-6, Bacillus cereus BORWO-7, and Bacillus spheroidis BORWO-8, which were stored at -80℃, were inoculated into sterile LB liquid medium at a volume fraction of 10% and cultured at 30℃ and 150rpm for 24 hours to fully activate the strains.

[0060] (2) Seed culture: The above 8 kinds of bacterial solutions that have been fully activated are inoculated into sterile LB liquid medium at an inoculation amount of 10% by volume and cultured at 30℃ and 150rpm for 12h to obtain seed culture.

[0061] In step (2), the viable bacterial concentration of the seed solution was 10 for Bacillus clausti. 9 / mL; the viable bacterial concentration of soil Bacillus was 10 10 / mL; the viable bacterial concentration of Bacillus thuringiensis was 10. 8 / mL; the viable bacterial concentration of Bacillus Horikoshi was 10. 8 / mL; the viable bacterial concentration of Bacillus megaterium was 10. 8 / mL; the viable bacterial concentration of Bacillus licheniformis was 10 8 / mL; the viable bacterial concentration of Bacillus cereus was 10. 7 / mL; the viable bacterial concentration of Bacillus spheroidis is 10 7 / mL.

[0062] (3) Bacterial culture: The seed cultures of the eight strains prepared in step (2) were cultured in the following mass ratio: Bacillus cladei, Bacillus agronomycetes, Bacillus sturdierus, Bacillus horikosa, Bacillus megaterium, Bacillus licheniformis, Bacillus cereus, and Bacillus spheroidis. The seed cultures were inoculated into a sterile fermentation medium at a volume fraction of 10% and cultured at 30°C and 150 rpm for 12 hours to obtain a composite bacterial culture. The viable cell concentration of the composite bacterial culture was 10%. 10 The compound bacterial solution was dried at low temperature to obtain a wettable powder, with a concentration of CFU / mL, thus obtaining the compound bacterial agent.

[0063] The components per liter of the fermentation medium are as follows: yeast extract 3.5 g / L, peptone 7.0 g / L, sodium chloride 3.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.1 g / L, ammonium sulfate 0.5 g / L, calcium chloride 0.002 g / L, and the balance being water.

[0064] The compound microbial agent prepared above is used to remove odors from odor-polluted spaces in large-scale farms. The specific method is as follows:

[0065] Dissolve the prepared compound microbial agent (wettable powder) in water, dilute it 1000 times by volume, place it in a spraying device, and spray it for 30 minutes in the odor-polluted space of the large-scale farm. The spraying rate is 1.0 L / min / m². 3 It can eliminate odors, with an ammonia degradation rate of up to 95% and a hydrogen sulfide degradation efficiency of up to 89%. After deodorization, it can maintain its effect for 84 hours under conditions where no new pollution sources are generated. The detection methods for ammonia and hydrogen sulfide are the same as in Example 1.

[0066] Example 3

[0067] A method for preparing a compound microbial agent includes the following steps:

[0068] (1) Activation of strains: Eight strains of Bacillus clausii BORWO-1, Bacillus agronomycetes BORWO-2, Bacillus sturdier BORWO-3, Bacillus Horikoshi BORWO-4, Bacillus megaterium BORWO-5, Bacillus licheniformis BORWO-6, Bacillus cereus BORWO-7, and Bacillus spheroidis BORWO-8, which were stored at -80℃, were inoculated into sterile LB liquid medium at a volume fraction of 1% and cultured at 30℃ and 150rpm for 24h to fully activate the strains.

[0069] (2) Seed culture: The above 8 kinds of bacterial solutions that have been fully activated were inoculated into sterile LB liquid medium at an inoculation amount of 1% by volume and cultured at 30℃ and 150rpm for 15h to obtain seed culture.

[0070] In step (2), the viable bacterial concentration of the seed solution was 10 for Bacillus clausti. 9 / mL; the viable bacterial concentration of soil Bacillus was 10 9 / mL; the viable bacterial concentration of Bacillus thuringiensis was 10. 9 / mL; the viable bacterial concentration of Bacillus Horikoshi was 10. 9 / mL; the viable bacterial concentration of Bacillus megaterium was 10. 9 / mL; the viable bacterial concentration of Bacillus licheniformis was 10 9 / mL; the viable bacterial concentration of Bacillus cereus was 10. 9 / mL; the viable bacterial concentration of Bacillus spheroidis is 10 9 / mL.

[0071] (3) Bacterial culture: The seed cultures of the eight strains prepared in step (2) were cultured in the following mass ratio: Bacillus cladei, Bacillus agronomycetes, Bacillus sturdierus, Bacillus horikosa, Bacillus megaterium, Bacillus licheniformis, Bacillus cereus, and Bacillus spheroidis. The seed cultures were inoculated into a sterile fermentation medium at a volume fraction of 5.0% and cultured at 30°C and 150 rpm for 15 h to obtain a composite bacterial culture. The viable cell concentration of the composite bacterial culture was 10. 9 The compound bacterial solution was dried at low temperature to obtain a wettable powder, with a concentration of CFU / mL, thus obtaining the compound bacterial agent.

[0072] The components per liter of the fermentation medium are as follows: yeast extract 15 g / L, peptone 15 g / L, sodium chloride 10 g / L, dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.5 g / L, ammonium sulfate 1.0 g / L, calcium chloride 0.05 g / L, and the remainder is water.

[0073] The compound microbial agent prepared above is used to remove odors from odor-polluted spaces in large-scale farms. The specific method is as follows:

[0074] Dissolve the prepared compound microbial agent (wettable powder) in water, dilute it 500 times by volume, place it in a spraying device, and spray it for 15 minutes in the odor-polluted space of the large-scale farm. The spraying rate is 0.5 L / min / m². 3It can eliminate odors, with an ammonia degradation rate of 94% and a hydrogen sulfide degradation efficiency of 87%. After deodorization, it can maintain its effect for 96 hours under conditions where no new pollution sources are generated. The detection methods for ammonia and hydrogen sulfide are the same as in Example 1.

[0075] Comparative Example 1

[0076] A single bacterial strain was used to prepare the corresponding bacterial agent, resulting in an agent containing only Bacillus megaterium, with a viable bacterial concentration of 10⁻⁶. 9 / mL. Dilute the above bacterial solution 10 times by volume, place it in a spraying device, and spray it in the breeding shed for 10 minutes at a spraying rate of 1.5L / min / m. 3 The odor was not significantly eliminated; the ammonia degradation rate was only 21%, and the hydrogen sulfide degradation rate was only 13%.

[0077] Comparative Example 2

[0078] The difference from Example 1 is that Bacillus subtilis (TTL1, Z206D4) is used instead of Bacillus spp. in the compound microbial agent, while all other aspects are the same in preparing the compound microbial agent.

[0079] Dilute the above-mentioned compound bacterial solution 10 times by volume, place it in a spraying device, and spray it in the breeding shed for 10 minutes at a spraying rate of 1.5 L / min / m². 3 The ammonia degradation rate reaches 35%, and the hydrogen sulfide degradation efficiency reaches 19%. After deodorization, it can be maintained for 1 hour under the condition that no new pollution source is generated. The detection methods for ammonia and hydrogen sulfide are the same as in Example 1.

[0080] Comparative Example 3

[0081] The difference from Example 1 is that Bacillus clausti was not added when preparing the compound microbial agent, and Bacillus licheniformis was used instead of Bacillus clausti. All other aspects are the same when preparing the compound microbial agent.

[0082] The above-mentioned compound bacterial solution was diluted 10 times by volume and placed in a spraying device. Spraying it in the breeding shed for 10 minutes resulted in a 51% degradation rate of ammonia and a 43% degradation rate of hydrogen sulfide. After deodorization, the effect could be maintained for 5 hours without the generation of new pollution sources. The detection methods for ammonia and hydrogen sulfide were the same as in Example 1.

[0083] Comparative Example 4

[0084] A single bacterial strain was used to prepare the corresponding bacterial agent, resulting in an agent containing only Bacillus clausti, with a viable cell concentration of 10⁻⁶. 9 / mL. Dilute the above bacterial solution 10 times by volume, place it in a spraying device, and spray it in the breeding shed for 10 minutes at a spraying rate of 1.5L / min / m.3 The odor was not significantly eliminated; the ammonia degradation rate was only 26%, and the hydrogen sulfide degradation rate was only 15%. The detection methods for ammonia and hydrogen sulfide were the same as in Example 1.

[0085] Comparative Example 5

[0086] A single bacterial strain was used to prepare the corresponding inoculum, resulting in an inoculum containing only *Bacillus stolonifer*, with a viable bacterial concentration of 10⁻⁶. 9 / mL. Dilute the above bacterial solution 10 times by volume, place it in a spraying device, and spray it in the breeding shed for 10 minutes at a spraying rate of 1.5L / min / m. 3 The odor was not significantly eliminated; the ammonia degradation rate was only 30%, and the hydrogen sulfide degradation rate was only 22%. The detection methods for ammonia and hydrogen sulfide were the same as in Example 1.

[0087] The compound microbial agent prepared by this invention requires no additional auxiliary materials, simplifying the production process and equipment. It can be placed in an automated integrated spraying device for timed and quantitative automatic spraying. When applied on a large scale in livestock and poultry farming spaces, it can directly, efficiently, and rapidly remove odors, achieving rapid air purification. The pure microbial compound microbial agent prepared by this invention is suitable for use in open spaces. Spraying it in odor-polluted spaces in livestock sheds or large-scale farms can significantly and quickly eliminate odors, solving the problem of odor in livestock farms.

Claims

1. A complex microbial agent, characterized in that, Bacillus clausii (ATCC® 35837®), Bacillus clausii Bacillus agri (ATCC® 6636®), Solibacillus silvestris Bacillus firmus (ATCC® 19538®), Bacillus firmus Bacillus horikoshii (ATCC® 33678®), Sutcliffiella horikoshii Bacillus megaterium (ATCC® 14945®), Bacillus megaterium Bacillus licheniformis (ATCC® 14580®), Bacillus licheniformis Bacillus cereus (ATCC® 11778®), and Bacillus cereus Bacillus sphaericus (ATCC® 14573®). Bacillus sphaerieus Bacillus clausii (ATCC® 35837®), Bacillus clausii Bacillus agri (ATCC® 6636®), Solibacillus silvestris Bacillus firmus (ATCC® 19538®), Bacillus firmus Bacillus horikoshii (ATCC® 33678®), Sutcliffiella horikoshii Bacillus megaterium (ATCC® 14945®), Bacillus megaterium Bacillus licheniformis (ATCC® 14580®), Bacillus licheniformis Bacillus cereus (ATCC® 117 Bacillus clausii (Bacillus clausii) Bacillus clausii Strain No. BORWO-1, Accession No. 202191, Bacillus agri (Bacillus agri) Solibacillus silvestris Strain No. BORWO-2, Accession No. 202192, Bacillus firmus (Bacillus firmus) Bacillus firmus Strain No. BORWO-3, Accession No. 202193, Bacillus horikoshii (Bacillus horikoshii) Sutcliffiella horikoshii Strain No. BORWO-4, Accession No. 202194, Bacillus megaterium (Bacillus megaterium) Bacillus megaterium Strain No. BORWO-5, Accession No. 202195, Bacillus licheniformis (Bacillus licheniformis) Bacillus licheniformis Strain No. BORWO-6, Accession No. 202196, Bacillus cereus (Bacillus cereus) Bacillus cereus Strain No. BORWO-7, Accession No. 202197, Bacillus sphaericus (Bacillus sphaericus) Bacillus sphaerieus Strain No. BORWO-8, Accession No. 202198; all preserved in the Microbial Culture Collection of Shandong Provincial Institute of Ecology.

2. The complex microbial agent according to claim 1, wherein The total number of viable bacteria in the complex microbial agent is 10 7 ~10 10 CFU / mL.

3. The method for preparing the complex microbial agent according to any one of claims 1-2, characterized in that, comprising the following steps: (1) strain activation: inoculate each of the eight strains of claim 1 into a sterile liquid culture medium to activate, and prepare a bacterial liquid; (2) inoculate the bacterial liquid prepared in step (1) into a liquid culture medium at a volume fraction of 0.1% to 10% respectively, and prepare a seed liquid; (3) bacterial liquid culture: inoculate the seed liquid of the eight strains prepared in step (2) into a sterile fermentation medium at a volume fraction of 1.0-10%, and culture at 28-37°C, 100-200 rpm for 12-72 h to prepare a compound bacterial liquid, wherein the mass ratio of Bacillus clausii, Solibacillus silvestris, Bacillus firmus, Sutcliffiella horikoshii, Bacillus megaterium, Bacillus licheniformis, Bacillus cereus and Bacillus sphaerieus is (0.5-3.5):(0.5-3.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5); (4) the compound bacterial liquid prepared in step (3) is directly used as a compound microbial agent; or, the compound bacterial liquid prepared in step (3) is dried at room temperature to prepare a wettable powder, and obtain a compound microbial agent.

4. The preparation method of the complex microbial agent according to claim 3, characterized in that, In step (1), the liquid culture medium is LB medium; In step (1), the culture conditions are 28-37°C, 100-200 rpm, and the culture time is 12-48 h.

5. The preparation method of the complex microbial agent according to claim 3, characterized in that, In step (2), the liquid culture medium is LB medium; In step (2), the culture conditions are 28-37°C, 100-200 rpm, and the culture time is 12-48 h.

6. The preparation method of the complex microbial agent according to claim 3, characterized in that, In step (2), the viable cell concentration of the seed solution was 10 9 ~10 10 / mL for Bacillus clausii, 10 9 ~10 10 / mL for Bacillus agri, 10 7 ~10 9 / mL for Bacillus firmus, 10 7 ~10 9 / mL for Bacillus horikoshii, 10 7 ~10 9 / mL for Bacillus megaterium, 10 7 ~10 9 / mL for Bacillus licheniformis, 10 7 ~10 9 / mL for Bacillus cereus, and 10 7 ~10 9 / mL for Bacillus sphaericus, respectively.

7. The preparation method of the complex microbial agent according to claim 3, characterized in that, In step (3), the components of the fermentation medium per liter are as follows: yeast powder 3.5-15 g / L, peptone 7.0-15 g / L, sodium chloride 3.5-10 g / L, dipotassium hydrogen phosphate 0.5-1.5 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, magnesium sulfate 0.1-0.5 g / L, ammonium sulfate 0.5-1.0 g / L, calcium chloride 0.002-0.05 g / L, and the balance is water.

8. The use of the composite microbial agent according to any one of claims 1-2 in eliminating odor. The odor is generated by ammonia and / or hydrogen sulfide.

9. The use according to claim 8, wherein the compound is ###00007### or a pharmaceutically acceptable salt thereof. The use of the composite microbial agent in eliminating odor in the field of space.

10. Use as claimed in claim 9, characterised in that, The application method is: diluting the complex microbial agent with water, diluting 10-1000 times in volume ratio, placing in a spraying device, spraying time 1-30 minutes, spraying amount (0.5-1.5) L / min / m 3 Spraying to remove odor in the odor pollution space.

11. The use according to claim 9, wherein the compound is ###00010### or a pharmaceutically acceptable salt thereof. The odor-polluted space is an odor-polluted space in the breeding industry.

12. The use according to claim 11, characterized in that, The odor-polluted space in the breeding industry is a breeding shed, a large-scale breeding farm, or a breeding wastewater storage area.