High-temperature deodorization and greenhouse gas emission reduction biological agent and application

Abstract

The present application relates to high-temperature deodorization and greenhouse gas emission reduction biological bacterial agent in the field of microorganisms and application, and mainly solves the problem of how to remove various harmful gases under high-temperature conditions. The present application provides a composite bacterial agent, the active ingredients of which are composed of Pediococcus acidilactici and Bacillus licheniformis, wherein Pediococcus acidilactici ( Pediococcus acidilactici ) JLL113 has a preservation number of CGMCC No: 32316 in the China General Microbiological Culture Collection Center; and Bacillus licheniformis ( Bacillus licheniformis ) CW has a preservation number of CGMCC No: 32182 in the China General Microbiological Culture Collection Center. The composite bacterial agent of the present application can be used for deodorization and emission reduction in the process of livestock and poultry manure (normal state, composting) treatment, and has good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to high-temperature deodorization and greenhouse gas emission reduction biological bacteria agents in the field of microorganisms and applications. BACKGROUND

[0002] In the process of industrialization and urbanization, waste disposal and agricultural waste management have become an important aspect of environmental challenges, especially the emission of malodorous gases and greenhouse gases (such as ammonia, methane and nitrous oxide) has become increasingly prominent. Traditional physical and chemical treatment methods are not efficient in treating malodorous gases and greenhouse gases, and there is a risk of secondary pollution. Biological deodorization and greenhouse gas emission reduction technology has become a key development direction for malodorous and greenhouse gas treatment due to its high efficiency, no secondary pollution, simple equipment, convenient operation, low cost, and easy management and maintenance. However, the strains currently used have several limitations. First, most functional strains are not resistant to high temperatures, limiting their application in high-temperature composting processes; second, single strains can usually only target specific types of malodorous gases or greenhouse gases, and have weak environmental resistance. Since malodorous gases and greenhouse gases are usually a mixture of multiple gases, single strains have limited effect in actual high-temperature deodorization and greenhouse gas emission reduction processes, so it is particularly urgent to develop composite bacterial agents. SUMMARY

[0003] The main problem to be solved by the present application is how to remove multiple harmful gases under high-temperature conditions.

[0004] To solve the above problems, the present application provides a microbial composite bacterial agent.

[0005] The active ingredients of the composite bacterial agent provided by the present application are composed of Pediococcus acidilactici and Bacillus licheniformis.

[0006] In the above composite bacterial agent, the Pediococcus acidilactici is Pediococcus acidilactici (JLL113) with a preservation number of CGMCC No. 32316 in the General Microbiological Center of the Chinese Microbial Strain Preservation Management Committee; and the Bacillus licheniformis is Bacillus licheniformis (CW) with a preservation number of CGMCC No. 32182 in the General Microbiological Center of the Chinese Microbial Strain Preservation Management Committee. Pediococcus acidilactici Bacillus licheniformis

[0007] The active ingredients of the above bacterial agent can be the above-mentioned Pediococcus acidilactici or / and Bacillus licheniformis and / or metabolites of the above-mentioned Pediococcus acidilactici or / and Bacillus licheniformis, and the active ingredients of the above bacterial agent can also contain other biological components or non-biological components. Other active ingredients of the above bacterial agent can be determined by those skilled in the art according to the removal effect of malodorous gases in waste.

[0008] ​​In the above, the metabolite can be obtained from the fermentation broth of the Pediococcus acidilactici or / and Bacillus licheniformis. The metabolite can be a sterile metabolite of the Pediococcus acidilactici or / and Bacillus licheniformis or a metabolite containing bacteria of the Pediococcus acidilactici or / and Bacillus licheniformis. The sterile metabolite of the Pediococcus acidilactici or / and Bacillus licheniformis (sterile fermentation filtrate) can be prepared by culturing the Pediococcus acidilactici or / and Bacillus licheniformis in a liquid culture medium, and filtering the Pediococcus acidilactici or / and Bacillus licheniformis in the liquid culture (fermentation broth) to obtain the sterile metabolite of the Pediococcus acidilactici or / and Bacillus licheniformis. The metabolite containing bacteria of the Pediococcus acidilactici or / and Bacillus licheniformis can be prepared by culturing the Pediococcus acidilactici or / and Bacillus licheniformis in a liquid fermentation medium, and collecting the fermentation broth, which is the metabolite containing bacteria of the Pediococcus acidilactici or / and Bacillus licheniformis.

[0009] In the above-mentioned microbial agent, in addition to the active ingredient, a carrier is also contained. The carrier can be a carrier commonly used in the field of environmental governance and biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a high molecular compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomite; the plant material can be at least one of corn meal, soybean meal and starch; the high molecular compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent, a vegetable oil, a mineral oil or water; the organic solvent can be decane and / or dodecane.

[0010] The above-mentioned microbial agent can be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule. If necessary, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH regulator, etc. can also be added to the above-mentioned microbial agent. The complex microbial agent can contain a carrier.

[0011] Specifically, the liquid carrier is xanthan gum, sodium alginate and potassium dihydrogen phosphate. The mass / volume percentage of the xanthan gum in the microbial liquid agent is 0.01%, the mass / volume percentage of the sodium alginate in the microbial liquid agent is 0.03%, and the mass / volume percentage of the potassium dihydrogen phosphate in the microbial liquid agent is 0.05%.

[0012] The present application also provides a method for culturing the above-mentioned Pediococcus acidilactici, which comprises the step of culturing the Pediococcus acidilactici in a culture medium for culturing Pediococcus acidilactici.

[0013] The present application also provides a method for culturing the above-mentioned Bacillus licheniformis, which comprises the step of culturing the Bacillus licheniformis in a culture medium for culturing Bacillus licheniformis.

[0014] Furthermore, in the compound microbial agent, the CFU content ratio of the *Pediococcus lactis* and the *Bacillus licheniformis* is 0.9 × 10⁻⁶. 8 CFU / ml: 1.7×10 9 CFU / ml.

[0015] The present invention also provides a *Pediococcus lactis* strain, wherein the *Pediococcus lactis* is *Pediococcus lactis* (… Pediococcus acidilactici JLL113, its accession number at the China General Microbiological Culture Collection Center is CGMCC No.32316.

[0016] In one specific embodiment, the content of the *Pediococcus lactis* inoculum prepared from the *Pediococcus lactis* is 1.7 × 10⁻⁶. 8 CFU / ml.

[0017] The present invention also provides a Bacillus licheniformis, wherein the Bacillus licheniformis is a Bacillus licheniformis (B. licheniformis) Bacillus licheniformis CW, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 32182.

[0018] In one specific embodiment, the content of the Bacillus licheniformis inoculant prepared from the Bacillus licheniformis is 3.4 × 10⁻⁶. 9 CFU / ml.

[0019] The present invention also provides a microbial agent for preparing compost, the microbial agent containing the aforementioned Pediococcus lactis and / or the aforementioned Bacillus licheniformis.

[0020] This invention also provides a method for preparing a compound microbial agent for composting. In a specific embodiment, the method includes mixing the above-mentioned compound microbial agent with a carrier to obtain the compound microbial agent.

[0021] Specifically, the method includes the following steps:

[0022] (1) Mix equal volumes of the fermentation broth of *Pediococcus lactis* and the fermentation broth of *Bacillus licheniformis* to obtain a mixed bacterial solution;

[0023] (2) Centrifuge the fermentation broth of the single strain or the mixed bacterial broth and collect the precipitate; dissolve the precipitate and add xanthan gum, sodium alginate and potassium dihydrogen phosphate to obtain the single bacterial agent or the compound microbial agent.

[0024] In the above method, the Pediococcus acidilactici fermentation broth is obtained by inoculating Pediococcus acidilactici into liquid MRS (de Man, Rogosa, Sharpe) medium for fermentation culture; the MRS medium is composed of solvent and solute, the solvent is water, and the concentration of each solute in the medium is as follows: tryptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, potassium dihydrogen phosphate 2.0 g / L, sodium acetate 5.0 g / L, triammonium citrate 2.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, and Tween 80 1.0 g / L. The fermentation culture conditions are as follows: 50°C, 180-200 rpm / min for 36-48 h.

[0025] The Bacillus licheniformis fermentation broth is obtained by inoculating Bacillus licheniformis into beef extract-tryptone medium for fermentation culture; the beef extract-tryptone medium is composed of solvent and solute, the solvent is water, and the concentration of each solute in the medium is as follows: beef extract 5.0 g / L, tryptone 10.0 g / L, NaCl 5 g / L, and pH 7.2-7.4. The fermentation culture conditions are as follows: 55°C, 180-200 rpm / min for 36-48 h.

[0026] In the above method, step (2) comprises the following steps: centrifuging the single bacterial liquid or mixed bacterial liquid at 5000 rpm / min for 20 min; collecting the precipitate; dissolving the precipitate with deionized water and mixing with xanthan gum, sodium alginate and potassium dihydrogen phosphate to obtain a compound microbial liquid inoculant. The mass fraction of xanthan gum, sodium alginate and potassium dihydrogen phosphate in the compound microbial liquid inoculant is 0.01%, 0.03% and 0.05%, respectively; and the amount of deionized water is 1 / 10 of the amount before centrifugation, thereby obtaining a single bacterial inoculant or a compound bacterial inoculant.

[0027] The application further provides the use of the compound bacterial inoculant described above in any one of the following:

[0028] A1) deodorization or odor removal or greenhouse gas emission reduction;

[0029] A2) preparation of a product for deodorization or odor removal or greenhouse gas emission reduction;

[0030] A3) removal of odor gas and greenhouse gas from waste;

[0031] A4) preparation of a product for removing odor gas or greenhouse gas from waste;

[0032] A5) removal of odor gas and greenhouse gas generated in the fermentation process of organic materials or organic waste;

[0033] A6) Prepare products that remove odorous gases or greenhouse gases produced during the fermentation of organic materials or organic waste.

[0034] The present invention also provides the use of the aforementioned *Pediococcus lactis* and / or *Bacillus licheniformis* in any of the following:

[0035] A1) Deodorization or deodorization, or greenhouse gas emission reduction;

[0036] A2) Prepare products for deodorization or deodorization or greenhouse gas emission reduction;

[0037] A3) Remove odorous and greenhouse gases from waste;

[0038] A4) Prepare products that remove odorous gases or greenhouse gases from waste;

[0039] A5) Removes odorous gases and greenhouse gases produced during the fermentation of organic materials or organic waste;

[0040] A6) Prepare products that remove odorous gases or greenhouse gases produced during the fermentation of organic materials or organic waste.

[0041] In the above applications, the odorous gas is ammonia, and the greenhouse gas is carbon dioxide / methane / nitrous oxide.

[0042] In the above applications, the organic materials or organic waste include cellulose substances and / or livestock and poultry manure.

[0043] In one specific embodiment, the compound microbial agent can be added to a high-temperature composting bed composed of chicken manure and sawdust to deodorize and reduce greenhouse gas emissions during the composting process.

[0044] The advantage of this invention is that it utilizes the thermophilic heterotrophic nitrifying bacteria Bacillus licheniformis (B. licheniformis) Bacillus licheniformis CW and thermostable Pediococcus lactis ( Pediococcus acidilactici JLL113 works synergistically to enhance the deodorization and greenhouse gas emission reduction effects of different microorganisms, thereby improving the stability and efficiency of microbial deodorization and greenhouse gas emission reduction.

[0045] The deodorizing and emission-reducing compound microbial agent provided by this invention can be used for deodorization and emission reduction in the treatment of livestock and poultry manure (both routine and composting processes), effectively reducing emissions of ammonia, nitrous oxide, methane, and carbon dioxide. The microbial compound agent of this invention reduces carbon dioxide emissions by more than 50%, methane by more than 30%, nitrous oxide by more than 40%, and ammonia by more than 70%. This microbial agent has significant emission reduction effects, is safe and harmless, and will play an important role in the deodorization of solid waste such as livestock and poultry manure and the reduction of greenhouse gas emissions, showing promising application prospects.

[0046] Strain name: Pediococcus acidilactici

[0047] Latin name: Pediococcus acidilactici

[0048] Strain number: JLL113

[0049] Preservation agency: China General Microbiological Culture Collection Center

[0050] Abbreviation of preservation agency: CGMCC

[0051] Address: No. 3, Beichen West Road, Haidian District, Beijing

[0052] Preservation date: October 23, 2024

[0053] Preservation center registration number: CGMCC No. 32316.

[0054] Strain name: Bacillus licheniformis

[0055] Latin name: Bacillus licheniformis

[0056] Strain number: CW

[0057] Preservation agency: China General Microbiological Culture Collection Center

[0058] Abbreviation of preservation agency: CGMCC

[0059] Address: No. 3, Beichen West Road, Haidian District, Beijing

[0060] Preservation date: October 11, 2024

[0061] Preservation center registration number: CGMCC No. 32182. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 Temperature change of different treatments in composting process. DETAILED DESCRIPTION

[0063] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.

[0064] The experimental methods in the following examples are all routine methods, unless otherwise specified, according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0065] Quantitative experiments in the following examples were set up in triplicate, and the results were averaged.

[0066] The culture medium in the following examples was configured as follows:

[0067] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, solvent: distilled water, pH 7.0.

[0068] MRS medium: tryptone 10 g / L, beef extract powder: 5 g / L, yeast extract powder, 4 g / L, glucose 20 g / L, Tween 80: 1 ml / L, potassium phosphate dibasic 2 g / L, sodium acetate 5 g / L, triammonium citrate 2 g / L, magnesium sulfate (MgSO4·7H2O) 0.2 g / L, manganese sulfate (MnSO4·4H2O) 0.05 g / L, solvent: distilled water, pH 7.0.

[0069] Beef extract tryptone medium: beef extract 5.0 g / L, tryptone 10.0 g / L, NaCl 5 g / L, pH 7.2-7.4.

[0070] Ammonia selective medium: sucrose 50 g, ammonia water 10 ml, KH2PO4 2 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.1 g, 1% ZnSO4 5 ml, NaCl 2 g, deionized water 1000 ml, pH: natural.

[0071] Sulfur bacteria medium: Na2S2O3·5H2O 10 g, K2HPO4 3 g, CaCl2·6H2O 0.2 g, NH4Cl 2 g, MgCl2 0.5 g, deionized water 1000 ml, pH: 6.0-6.2.

[0072] After the above culture medium was configured, it was sterilized by high temperature 121℃ for 30 min.

[0073] The following examples use SPSS 16.0 statistical software to process data, and the experimental results are expressed as mean ± standard deviation, One-way ANOVA test is used, P<0.05 (*) indicates significant difference, P<0.01 (**) indicates extremely significant difference, P<0.001 (*** ) indicates extremely significant difference.

[0074] Example 1, Lactococcus lactis (Lactococcus lactis) Bacillus pallidus Example 2, Bacillus licheniformis (Bacillus licheniformis) Bacillus licheniformis Isolation, purification, identification and preservation of JLL113 and CW

[0075] I. Isolation, purification, identification and preservation of Pediococcus acidilactici strain JLL113 Bacillus pallidus

[0076] 1. Sample collection information

[0077] Sample source: collected from animal (chicken) feces; collector: Zhang Dongyan; collector's organization: Beijing Academy of Agricultural and Forestry Sciences; collector's contact information: zhdy203@126.com

[0078] 2. Isolation and screening of strains

[0079] Strains were isolated from chicken feces samples, and conventional gradient dilution coating separation was used. The bacteria with large morphological differences and pure culture were picked on MRS medium at 55°C, and then purified and stored on MRS medium. Finally, the strains with deamination and sulfur odor were screened on ammonia selective medium and sulfur bacteria medium. Finally, a bacterial strain was obtained, which was named strain JLL113.

[0080] 3. Identification of strains

[0081] The morphological, physiological and biochemical characteristics and partial conserved sequences of strain JLL113 were analyzed. The physiological and biochemical characteristics and environmental tolerance characteristics were determined according to the methods described in "Principles and Methods of Soil Microbial Research" (Lin Xianggui, Principles and Methods of Soil Microbial Research [M], Higher Education Press, 2010).

[0082] (1) Morphological characteristics of strain JLL113: The bacterial cells of strain JLL113 were spherical or ellipsoidal under 400 times optical microscope, with uniform size, usually arranged singly or in pairs. The bacterial surface was smooth, and no spores were formed. The strain had certain motility. The strain did not produce pigment on MRS medium, and the colony was milky white, opaque, round or nearly round, smooth and slightly glossy on the surface. With the extension of culture time, the colony gradually increased and became milky white, with a relatively neat edge and moist texture.

[0083] (2) Physiological and biochemical characteristics of strain JLL113: Gram staining positive, no capsule formation, methyl red, acetyl methyl methanol, nitrate reduction, indole reaction, contact enzyme, urease positive, can hydrolyze gelatin, starch and esculin, oxidase reaction, tyrosine decomposition negative, do not produce hydrogen sulfide, utilize citrate, do not utilize malonate, arginine, lysine, ornithine decarboxylase reaction positive, phenylalanine deaminase reaction negative, glucose, sucrose produce acid and gas, can utilize glucose, maltose, sorbitol, rhamnose, inulin, galactose, inositol, xylose, mannose, mannitol, ribose, etc., do not utilize arabinose and sorbose.

[0084] ​The environmental tolerance of Pediococcus acidilactici JLL113 is that it can grow normally in the medium containing 1%-6% (mass content) sodium chloride, cannot grow in the medium containing 8% (mass content) sodium chloride, can grow normally in the medium with pH 4.5-8.5, grows weakly at pH 3.0, cannot grow at pH 9.0, grows weakly at the environmental temperature of 15°C, grows normally at the temperature of 25°C-55°C, and the optimum growth temperature is 50°C.

[0085] (3) Molecular biology analysis

[0086] The genomic DNA of the strain JLL113 is extracted by using a bacterial genomic DNA extraction kit (Solea bacterial DNA kit, D1600), and the strain JLL113 is specifically amplified by using 16S rDNA, and the strain JLL113 has the 16S rDNA with the nucleotide sequence of sequence 1 in the sequence table (Table 1).

[0087]

[0088] Based on the above morphological characteristics, physiological and biochemical characteristics, and 16S rDNA gene sequence analysis results, strain JLL113 was identified as *Pediococcus lactis*. Pediococcus acidilactici ).

[0089] 4. Preservation of strain JLL113

[0090] Pediococcus lactis JLL113 was deposited on October 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 32316. It will be referred to as Pediococcus lactis CGMCC No. 32316 or strain JLL113.

[0091] II. Bacillus licheniformis ( Bacillus licheniformis Isolation, purification, identification and preservation of CW

[0092] 1. Sample collection information

[0093] The samples were obtained from high-temperature composting samples of livestock and poultry manure from the Beijing Academy of Agricultural and Forestry Sciences.

[0094] 2. Isolation and screening of strains

[0095] Strains were isolated from high-temperature composted sludge samples using conventional serial dilution plating. They were cultured on three media: LB broth, beef extract peptone medium, and ammonia-selective medium at 55°C. Bacteria with significant morphological differences and suitable for pure culture were selected and purified on LB broth. Finally, they were selectively cultured on ammonia-selective medium and sulfur-containing bacteria medium to screen for microorganisms with deammonia- and sulfur-odor-removing effects. A single bacterial strain was obtained and named strain CW.

[0096] 3. Identification of strains

[0097] Morphological, physiological and biochemical characteristics, and some conserved sequences of strain CW were analyzed. Physiological, biochemical, and environmental tolerance characteristics were determined according to the methods described in "Principles and Methods of Soil Microbiology Research" (Lin Xiangui, Principles and Methods of Soil Microbiology Research [M], Higher Education Press, 2010).

[0098] (1) Morphological characteristics of strain CW:

[0099] Under a 400x optical microscope, the cells of strain CW appear rod-shaped, often arranged singly or in chains, and can form spores. The spores are oval-shaped and located in the center or off-center of the cell. The cells do not swell. On LB medium, they do not produce pigment, are opaque, flat or round, with regular edges, and a smooth or slightly rough surface. As the incubation time increases, the colonies thicken and may dry out.

[0100] (2) Physiological and biochemical characteristics of the strain CW: Gram staining positive, capable of forming a bacterial film. Methyl red test negative, acetyl methyl methanol test (VP) positive, nitrate reduction test positive, indole reaction negative, contact enzyme positive, oxidase negative, urease positive, can hydrolyze starch, gelatin, cellulose, decompose tyrosine negative, can utilize citrate, not utilize malonate. Arginine, lysine, ornithine decarboxylase positive, phenylalanine decarboxylase negative. Can ferment glucose, sucrose, maltose, etc. to produce acid, not produce gas, can utilize glucose, maltose, sucrose, xylose, mannose, ribose, mannitol, but not sorbitol, arabinose.

[0101] The strain CW has strong tolerance to the environment. It can grow in a culture medium containing 1%-10% (mass content) sodium chloride, grow normally in the range of 1%-7%, grow slowly or be inhibited in 10% sodium chloride. It can grow normally in the range of pH 5.5-9.5, grow weakly at pH 4.5, and cannot grow above pH 10. It can grow normally at the environmental temperature of 40℃-65℃, the optimum growth temperature is 55℃, and it can still grow normally after being treated at 80℃ for 15 min.

[0102] (3) Molecular biology analysis

[0103] The genomic DNA of the strain CW was extracted by using a bacterial genomic DNA extraction kit (Solei Biological Bacterial DNA Kit, D1600), and 16S rDNA was selected for specific amplification of the strain CW. The nucleotide sequence of the strain CW is 16S rDNA of sequence 2 in the sequence table (Table 1).

[0104]

[0105] Based on the above morphological characteristics analysis, physiological and biochemical characteristics and 16S rDNA gene sequence analysis results, strain CW was identified as Bacillus licheniformis (Bacillus licheniformis) Bacillus licheniformis )。

[0106] 4. Preservation of Bacillus licheniformis CW

[0107] Bacillus licheniformis CW has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 11, 2024, with the preservation number CGMCC No. 32182. Hereinafter referred to as Bacillus licheniformis CGMCC No. 32182 or strain CW.

[0108] Example 2, preparation of microbial liquid inoculant

[0109] 1. Preparation of Pediococcus acidilactici JLL113 inoculant

[0110] A single colony of Pediococcus acidilactici JLL113 was inoculated in MRS medium and incubated at 50°C for 24h. Then 7.5ml was aseptically inoculated in a blue cap bottle containing 250 mL medium and incubated at 50°C for 36-48h. The concentration was 5.2x10 7 CFU / ml, and Pediococcus acidilactici JLL113 strain culture solution was obtained.

[0111] 2. Preparation of Bacillus licheniformis CW inoculant

[0112] A single colony of Bacillus licheniformis CW was inoculated in beef extract peptone medium and incubated at 55°C with 180-200 rpm / min shaking. After 24h, 7.5ml was aseptically inoculated in a 250ml flask containing 150 mL medium and incubated at 55°C with 180-200 rpm / min shaking for 36-48h. The concentration was 9.1x10 8 CFU / ml, and Bacillus licheniformis CW strain culture solution was obtained.

[0113] JLL113 strain culture solution and CW strain culture solution were prepared into single inoculant and compound inoculant. The content of JLL113 single inoculant was 1.7x10 8 CFU / ml; the content of CW single inoculant was 3.4x10 9 CFU / ml; the contents of Pediococcus acidilactici and Bacillus licheniformis in the mixed inoculant were 0.9x10 8 CFU / ml and 1.7x10 9 CFU / ml, respectively.

[0114] Example 3, Evaluation of deodorization effect of high-temperature composting process

[0115] The composting test site was located in the greenhouse of the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences (longitude 116.29°, latitude 39.95°). The composting device was a 56L cylindrical metal intelligent fermentation tank, which consisted of a tank body, a sealing cover, a sponge insulation layer, a continuous temperature monitoring probe, and a filter screen. The inner diameter of the fermentation tank was 36cm, the outer diameter was 46cm, the inner height was 55cm, the outer height was 83cm, and there was a 5cm thick insulation material to reduce heat loss. The temperature sensor connected through the system control cabinet was inserted into the center of the pile to monitor the pile temperature. A stainless steel screen was placed at the bottom of the composting tank to support the composting material, evenly diffuse air, and provide space for leachate discharge.

[0116] Wood chips and fresh chicken manure (volume ratio of wood chips to chicken manure about 1:1, weight ratio about 1:2) were thoroughly mixed to obtain a mixture, and the C / N ratio of the mixture was about 25, and the moisture content was 60%.

[0117] The microbial inoculant prepared in Example 2 was used as a deodorizing agent, and four treatments were set up. 0.1% by volume of water was added as a control treatment (referred to as CK), 0.1% by volume of Pediococcus acidilactici JLL113 inoculant was added (referred to as JLL113, the concentration was 1.7×10 5 CFU / mL), 0.1% by volume of Bacillus licheniformis CW inoculant was added (referred to as CW, the concentration was 3.4×10 6 CFU / mL), and 0.1% by volume of compound inoculant was added (referred to as compound, JLL113 at a concentration of 0.9×10 5 CFU / mL, and CW at a concentration of 1.7×10 6 CFU / mL).

[0118] The corresponding proportion of water and the above-mentioned inoculants were added to the mixture to prepare compost by fermentation. Each treatment was repeated three times, and forced aeration + manual turning was used to provide oxygen during composting. The aeration rate was 0.2L·(kg·min) -1 The ventilation rate was intermittent from the bottom of the composting tank (30min ventilation, 30min stop).

[0119] The emission fluxes of CO2, CH4 and N2O during composting were collected by static boxes, and the collection time was from 9:00 to 11:00 am. The static box was made of opaque PVC board, with an inner diameter of 320mm and a height of 200mm. The gas in the box was collected with a sealed gas cylinder after 0, 5, 15 and 30 minutes, respectively. The concentrations of CO2, CH4 and N2O were determined using a gas chromatograph (HP6890N, Agilent).

[0120] NH3 emission was measured by titration method, using a borate acid absorption bottle containing 2% concentration of boric acid to directly absorb the gas, and using a standard concentration of acid for titration. The measurement time was from 9:00 to 11:00 am. The composting lasted for 28 days, and turning and sampling were performed at 1, 3, 5, 7, 10, 14, 21, and 28 d during the composting process.

[0121] During the entire composting process, the temperature of the three experimental groups treated with bacterial inoculants was higher than that of the blank control treatment, and the temperature of the experimental group treated with the compound bacterial inoculant was higher than that of the other two single bacterial inoculant treatments. Figure 1 Compared with the control treatment, the treatment with bacterial inoculants could reduce the emission of carbon dioxide, and the treatment with compound bacterial inoculants could significantly reduce the emission of carbon dioxide (Table 1). On the 28th day of the experiment, the detection value of carbon dioxide (1.05 ± 0.16 mg / (m 2 ·h) -1 ) of the compound bacterial inoculant treatment was only 6.65% of that of the CK treatment (15.80 ± 2.47 mg / (m 2 ·h) -1 ).

[0122]

[0123]

[0124]

[0125]

[0126] The results of the gas detection experiment during the composting process are shown in Tables 1-4. During the composting fermentation process, microbial inoculants can reduce the amount of methane, carbon dioxide, nitrous oxide, and ammonia in the air, and the emission reduction effect of compound bacterial inoculants is significantly better than that of single bacterial strains, which can significantly reduce the emission of the above four kinds of greenhouse gases and odor. The specific results are as follows:

[0127] 1) During the 28-day composting process, the compound bacterial inoculant significantly reduced the emission of carbon dioxide (Table 1). From the 7th day of composting, the carbon dioxide emission of the compound bacterial inoculant was reduced by more than 50%, to more than 70% on the 10th day, and to more than 90% on the 28th day (compared with the control treatment).

[0128] 2) The compound bacterial inoculant also significantly reduced the emission of methane (except for the 3rd day detection result) (Table 2). During the entire composting process, the methane emission was reduced by more than 30% compared with the control (except for 22.5% on the 3rd day).

[0129] 3) The composite microbial agent can significantly reduce the emission of nitrous oxide (Table 3), and the emission of nitrous oxide is reduced by more than 40% compared with the control during the whole composting process, and the emission of nitrous oxide is reduced by 54.4% compared with the blank control on the 14th day.

[0130] 4) During the whole composting process, the composite microbial agent significantly reduces the emission of ammonia, which is reduced by more than 70.0 compared with the control group, and the emission of ammonia is not detected after 21 days of composting (Table 4).

[0131] In summary, the single microbial agent and the composite microbial agent treatment of the present application can effectively reduce the production of the above four gases during composting, and the composite microbial agent has the best effect, which can significantly reduce the production of the four gases (except for the non-significant reduction of methane on the 3rd day), and compared with the control, the composite microbial agent prepared by mixing Pediococcus acidilactici and Bacillus licheniformis can reduce the emission of the four gases by more than 50% (carbon dioxide), 30% (methane), 40% (nitrous oxide) and 70% (ammonia) on average.

[0132] The present application has been described in detail above. For those skilled in the art, the present application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the purpose and scope of the present application, and without unnecessary experiments. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application intends to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are out of the scope disclosed in the present application.

Claims

1. A compound microbial agent, characterized in that, The active ingredients of the compound microbial agent consist of Pediococcus lactis and Bacillus licheniformis; The lactic acid cocci are *Pediococcus lactis* ( Pediococcus acidilactici JLL113, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 32316; the Bacillus licheniformis is Bacillus licheniformis ( Bacillus licheniformis CW, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 32182.

2. The compound microbial agent according to claim 1, characterized in that: In the compound microbial agent, the CFU content ratio of *Pediococcus lactis* and *Bacillus licheniformis* is 0.9 × 10⁻⁶. 8 CFU / ml: 1.7×10 9 CFU / ml.

3. The use of the compound microbial agent according to claim 1 or 2 in any of the following: A1) Deodorization or deodorization, or greenhouse gas emission reduction; A2) Prepare products for deodorization or deodorization or greenhouse gas emission reduction; A3) Remove odorous and greenhouse gases from waste; A4) Prepare products that remove odorous gases or greenhouse gases from waste; A5) Removes odorous gases and greenhouse gases produced during the fermentation of organic waste; A6) Prepare products that remove odorous gases or greenhouse gases produced during the fermentation of organic waste.

4. The application according to claim 3, characterized in that, The odorous gas is ammonia, and the greenhouse gas is carbon dioxide / methane / nitrous oxide.

5. The application according to claim 3 or 4, characterized in that, The organic waste includes cellulose materials and / or livestock and poultry manure.

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