Deodorizing microecological bacterial agent, preparation method and application thereof

Abstract

The application discloses a kind of deodorization microecological bacterial agent and its preparation method and application, belong to environmental microbiological technology field.The application provides a kind of deodorization microbial composition, the deodorization microbial composition includes enterococcus faecium GXSCU1, compost bacillus T-32, lactobacillus casei T-369 and Pichia pastoris T-371.The microecological bacterial agent prepared by specific proportion and process in the application, the synergistic effect between each strain, improve the treatment effect and stability.The method preparation process of the microecological bacterial agent in the application is simple, low in cost, without secondary pollution, meet the green breeding concept, with wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, and particularly relates to a deodorizing microecological agent, its preparation method and application. Background Technology

[0002] With the rapid development of livestock and poultry farming, the problem of livestock and poultry manure treatment has become increasingly prominent. Livestock and poultry manure contains large amounts of organic matter, nitrogen, phosphorus, and other nutrients, as well as large amounts of malodorous gases such as ammonia, hydrogen sulfide, and methane. These malodorous gases not only pollute the surrounding environment but also adversely affect the health of livestock, poultry, and humans. Therefore, how to effectively treat livestock and poultry manure and reduce the emission of malodorous gases has become a crucial issue facing the livestock and poultry farming industry.

[0003] Traditional methods for treating livestock and poultry manure mainly include physical, chemical, and biological methods. Physical methods primarily remove odorous gases through adsorption and filtration, but their effectiveness is limited and costs are high. Chemical methods mainly remove odorous gases through neutralization and oxidation reactions using chemical agents, but the use of these agents may cause secondary pollution to the environment. Biological methods primarily utilize the metabolic processes of microorganisms to degrade organic matter, reducing the generation of odorous gases. They offer advantages such as environmental friendliness, economy, and sustainability, and have therefore become a current research hotspot.

[0004] In biological treatments, the application of probiotics is gaining increasing attention. Probiotics are a class of live microorganisms beneficial to the host, capable of improving the health and productivity of livestock and poultry by regulating intestinal flora balance, inhibiting the growth of harmful bacteria, and promoting nutrient absorption. In recent years, research has also found that probiotics have significant effects in the treatment of livestock and poultry manure. Adding probiotics can accelerate the degradation of organic matter in manure, reduce the production of foul odors, and improve the fertilizer efficiency of manure, thus achieving resource recycling.

[0005] Currently, there are some research reports on the application of probiotics in livestock and poultry manure treatment. For example, studies have shown that probiotics such as lactic acid bacteria and Bacillus can effectively degrade organic matter in manure and reduce the emission of ammonia, hydrogen sulfide, and odor concentrations. However, the treatment effect of a single strain is often limited and cannot meet the needs of practical applications. Therefore, developing a microecological agent for deodorizing livestock and poultry manure, improving deodorization effect, reducing cost, and ensuring its stability and safety under different environmental conditions, has important practical significance and application value. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a deodorizing microbial composition that can effectively reduce the concentration of ammonia, hydrogen sulfide and odor, improve the deodorizing effect, and ensure its stability and safety under different environmental conditions.

[0007] Another object of the present invention is to provide a fermentation broth for a deodorizing microbial composition.

[0008] Another object of the present invention is to provide a method for preparing the fermentation broth.

[0009] Another objective of this invention is to provide a microecological agent.

[0010] Another object of the present invention is to provide a method for preparing the aforementioned microbial agent.

[0011] Another object of the present invention is to provide the application of the deodorizing microbial composition, the fermentation broth, or the microecological agent in the preparation of deodorizing products.

[0012] Another object of the present invention is to provide an application of the deodorizing microbial composition, the fermentation broth, or the microecological agent in the treatment of malodorous gases.

[0013] Another object of the present invention is to provide a deodorization method.

[0014] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0015] This invention provides a deodorizing microbial composition comprising Enterococcus faecalis (… Enterococcus faecium GXSCU1, Bacillus compostii ( Dung bacillus T-32, Lactobacillus casei ( Lactobacillus casei T-369 and Pichia pastoris (ethanol) Pichia ethanolica The following are listed: T-371; the accession number of Enterococcus faecalis GXSCU1 is GDMCC No: 62041; the accession number of Bacillus compostii T-32 is GDMCC No: 66237; the accession number of Lactobacillus casei T-369 is GDMCC No: 63763; and the accession number of Pichia pastoris T-371 is GDMCC No: 66239.

[0016] The present invention also provides a fermentation broth of a deodorizing microbial composition, which is the fermentation broth of the deodorizing microbial composition.

[0017] The present invention also provides a method for preparing the fermentation broth, the method comprising: inoculating the deodorizing microbial composition into a culture medium and culturing it to obtain the fermentation broth.

[0018] The present invention also provides a microecological agent, which comprises the deodorizing microbial composition or the fermentation broth.

[0019] The present invention also provides a method for preparing the microecological agent, the method comprising: progressively scale-up cultivation of the deodorizing microbial composition or the fermentation broth to obtain the microecological agent.

[0020] The present invention also provides the application of the deodorizing microbial composition, the fermentation broth, or the microecological agent in the preparation of deodorizing products.

[0021] The present invention also provides the application of the deodorizing microbial composition, the fermentation broth, or the microecological agent in the treatment of malodorous gases.

[0022] Preferably, the malodorous gas includes one or more of ammonia, trimethylamine, hydrogen sulfide, methanethiol, dimethyl sulfide, dimethyl disulfide, carbon disulfide, and styrene.

[0023] Preferably, the application includes any one of the following:

[0024] (1) Control of odor pollutants from livestock and poultry manure in agricultural environments;

[0025] (2) Purification of odorous gases during the composting process of organic waste;

[0026] (3) Biodegradation of volatile odorous substances in contaminated sites.

[0027] The present invention also provides a deodorization method, the deodorization method comprising: adding the deodorizing microbial composition or the fermentation broth or the microecological agent to the material to be deodorized.

[0028] The beneficial effects of this invention are:

[0029] This invention combines *Enterococcus faecalis* GXSCU1, *Bacillus compostii* T-32, *Lactobacillus casei* T-369, and *Pichia pastoris* T-371. Through optimized strain combination and synergistic effects among the strains, it effectively reduces the concentrations of ammonia, hydrogen sulfide, and odor in livestock and poultry manure, achieving maximum degradation rates of 41.43%, 40.70%, and 16.91%, respectively, thus improving deodorization efficiency while ensuring stability and safety under different environmental conditions. This demonstrates that the deodorizing microbial composition of this invention helps degrade pollutants in manure, improves the odorous environment of livestock and poultry farms, reduces the emission of odorous gases, and possesses highly efficient deodorization and manure degradation capabilities. It also enhances the fertilization efficiency of manure, achieving resource recycling, and is suitable for manure treatment in large-scale livestock and poultry farms.

[0030] This invention provides a microbial agent prepared through a specific ratio and process, in which the various bacterial species work synergistically to improve treatment efficacy and stability. The method for preparing this microbial agent is simple, low-cost, and produces no secondary pollution, aligning with green aquaculture principles and possessing broad application prospects.

[0031] Biological Preservation Information

[0032] This invention relates to Enterococcus faecalis GXSCU1, which is classified and named Enterococcus faecalis ( Enterococcus faecium The sample was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on November 5, 2021. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The accession number is GDMCC No: 62041.

[0033] This invention relates to Bacillus compostingus T-32, which is classified and named Bacillus compostingus (… Dung bacillus The sample was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on April 28, 2025. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The accession number is GDMCC No:66237.

[0034] This invention relates to *Lactobacillus casei* T-369, classified and named *Lactobacillus casei* (… Lactobacillus cheese The sample was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on August 31, 2023. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The accession number is GDMCC No: 63763.

[0035] This invention relates to Pichia pastoris T-371, classified and named Pichia pastoris (…). Pichia ethanolica The sample was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on April 28, 2025. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The accession number is GDMCC No:66239. Attached Figure Description

[0036] Figure 1 The images show colony observations of the four strains in Example 1, where A is a colony observation of Enterococcus faecalis GXSCU1, B is a colony observation of Bacillus compostii T-32, C is a colony observation of Lactobacillus casei T-369, and D is a colony observation of Pichia pastoris T-371.

[0037] Figure 2 The images show the cell morphology of the four strains in Example 1, where A is the cell morphology of Enterococcus faecalis GXSCU1, B is the cell morphology of Bacillus compostii T-32, C is the cell morphology of Lactobacillus casei T-369, and D is the cell morphology of Pichia pastoris T-371.

[0038] Figure 3 The images show Gram staining observations of the four strains in Example 1, where A is a Gram staining observation of Enterococcus faecalis GXSCU1, B is a Gram staining observation of Bacillus compostii T-32, C is a Gram staining observation of Lactobacillus casei T-369, and D is a Gram staining observation of Pichia pastoris T-371.

[0039] Figure 4 The following is a phylogenetic tree diagram of the four strains in Example 1, where A is the phylogenetic tree of Enterococcus faecalis GXSCU1, B is the phylogenetic tree of Bacillus compostii T-32, C is the phylogenetic tree of Lactobacillus casei T-369, and D is the phylogenetic tree of Pichia pastoris T-371.

[0040] Figure 5 The changes in ammonia levels between the candidate strains and the control group in Example 2;

[0041] Figure 6 The changes in hydrogen sulfide between the candidate strains and the control group in Example 2;

[0042] Figure 7 The changes in odor between the candidate strains and the control group in Example 2;

[0043] Figure 8 The graphs show the degradation effects of different deodorizing microbial compositions on the concentrations of ammonia, hydrogen sulfide, and odor in fecal waste in Example 2. In Example 2, A represents the degradation effect on ammonia, B represents the degradation effect on hydrogen sulfide, and C represents the degradation effect on odor.

[0044] Figure 9 This is a flowchart of the activation process of the microecological agent in Example 4. Detailed Implementation

[0045] This invention provides a deodorizing microbial composition comprising Enterococcus faecalis (… Enterococcus faecium GXSCU1, Bacillus compostii ( Dung bacillus T-32, Lactobacillus casei ( Lactobacillus casei T-369 and Pichia pastoris (ethanol) Pichia ethanolica The following are the accession numbers for the bacteria: T-371; Enterococcus faecalis GXSCU1, with accession number GDMCC No: 62041; Bacillus compostii T-32, with accession number GDMCC No: 66237; Lactobacillus casei T-369, with accession number GDMCC No: 63763; and Pichia pastoris T-371, with accession number GDMCC No: 66239.

[0046] In this invention, the 16S rDNA sequence of Enterococcus faecalis GXSCU1 is shown in SEQ ID NO.1; the 16S rDNA sequence of Bacillus compostii T-32 is shown in SEQ ID NO.2; the 16S rDNA sequence of Lactobacillus casei T-369 is shown in SEQ ID NO.3; and the 16S rDNA sequence of Pichia pastoris T-371 is shown in SEQ ID NO.4.

[0047] In this invention, Enterococcus faecalis GXSCU1 and Bacillus compostii T-32 are Gram-positive, VP test negative, methyl red test positive, nitrate utilization test positive, and propionate and citrate utilization tests negative; Lactobacillus casei T-369 and Pichia pastoris T-371 are Gram-positive, VP test negative, methyl red test positive, and nitrate, propionate and citrate utilization tests positive.

[0048] In this invention, *Enterococcus faecalis* GXSCU1, *Bacillus compostii* T-32, and *Lactobacillus casei* T-369 can ferment and utilize various carbon sources such as glucose, sucrose, inulin, maltose, D-raffinose, cellobiose, and inositol, and metabolize them to produce organic acids (such as lactic acid and acetic acid); *Pichia pastoris* T-371 can utilize propionate and citrate as carbon sources for growth and metabolize them to produce organic acids (such as acetic acid), and can also convert nitrates (NO3) into organic acids. - ) is reduced to nitrite (NO2) - It may be further reduced to products such as nitrogen (N2).

[0049] The *Enterococcus faecium* GXSCU1, *Bacillus compostii* T-32, *Lactobacillus casei* T-369, and *Pichia pastoris* T-371 of this invention, when used individually, all exhibit good degradation effects on ammonia and odor, but their degradation effect on hydrogen sulfide is not significant. When these strains are combined, the synergistic effect among them effectively reduces the concentrations of ammonia, hydrogen sulfide, and odor in livestock and poultry manure, with the highest degradation rates reaching 50.93%, 100.00%, and 100.00%, respectively. Compared to single strains, the deodorizing microbial composition of this invention can significantly improve the odor degradation rate, while also shortening the degradation time and prolonging the degradation effect. This indicates that the deodorizing microbial composition of this invention helps to degrade pollutants in manure and improve the malodorous environment of livestock and poultry farms.

[0050] The deodorizing microbial composition of this invention can regulate the microbial community in pig manure, including Treponema, Clostridium, Ruminococcus, Alistipes, and Solobacterium. After treating pig manure with this deodorizing microbial composition, the microbial community structure of the pig manure changes significantly, with the dominant microbial community mainly composed of Clostridium, Treponema, Mobilisporobacter, Ruminococcus, and Tissierella. It can effectively degrade malodorous substances such as indole and skatole, reducing odor generation.

[0051] The present invention also provides a fermentation broth of a deodorizing microbial composition, which is the fermentation broth of the deodorizing microbial composition.

[0052] In this invention, the preferred method for preparing the fermentation broth includes: inoculating the deodorizing microbial composition into a culture medium and culturing it to obtain the fermentation broth.

[0053] In some embodiments of the present invention, the microorganisms in the deodorizing microbial composition are preferably activated and cultured to obtain bacterial suspensions, the bacterial suspensions are mixed to obtain a bacterial suspension of the deodorizing microbial composition, and then inoculated into a culture medium for culture to obtain a fermentation broth.

[0054] The activation method preferably includes activation by streak plating. In some embodiments, the activation method preferably includes: inoculating Enterococcus faecalis GXSCU1, Bacillus compostii T-32, Lactobacillus casei T-369, and Pichia pastoris T-371 into an activation medium and culturing them, respectively.

[0055] The preferred formulation of the activation culture medium for Enterococcus faecalis GXSCU1 and Bacillus compostii T-32 is: 0.5%~4% glucose, 0.5%~3% peptone, 0.1%~2% yeast extract, 0.5%~3% sodium chloride, 0.01%~0.5% potassium dihydrogen phosphate, 0.01%~0.5% dipotassium hydrogen phosphate, and 2%~3% agar powder; more preferably, it is 2% glucose, 1% peptone, 0.5% yeast extract, 1% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, and 2% agar powder; the preferred pH is 5~8, for example, 5, 6, 7 or 8.

[0056] The preferred culture temperature for Enterococcus faecalis GXSCU1 and Bacillus compostii T-32 in the activated medium is 32℃~38℃, for example 32, 33, 34, 35, 36, 37 or 38℃; the preferred culture time is 24~48 h, for example 24, 28, 32, 36, 40, 44 or 48 h.

[0057] The preferred formulation of the activation culture medium for *Lactobacillus casei* T-369 and *Pichia pastoris* T-371 is: 0.5%–2.5% peptone, 0.5%–3% beef extract, 1%–3% glucose, 0.1%–2% yeast extract, 0.1%–1% sodium acetate, 0.05%–1.5% potassium dihydrogen phosphate, 0.001%–1% magnesium sulfate, 0.001%–1.5% manganese sulfate, and 0.01%–1.5%... The ingredients are: 1% triammonium citrate, 0.05%~2.5% Tween 80 and 2%~3% agar powder, more preferably 1% peptone, 0.5% beef extract, 2% glucose, 0.4% yeast extract, 0.5% sodium acetate, 0.2% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 0.005% manganese sulfate, 0.02% triammonium citrate, 0.1% Tween 80 and 2% agar powder; the pH is preferably 5~8, for example 5, 6, 7 or 8.

[0058] The preferred culture temperature for Lactobacillus casei T-369 and Pichia pastoris T-371 in the activation medium is 32 ℃ to 38 ℃, for example, 32, 33, 34, 35, 36, 37 or 38 ℃; the preferred culture time is 24 to 48 h, for example, 24, 28, 32, 36, 40, 44 or 48 h.

[0059] After activation, it is preferable to select a single colony and inoculate it into the corresponding liquid culture medium for culture. The culture solution is then inoculated into the liquid culture medium for further culture to obtain a bacterial suspension.

[0060] The liquid culture medium is preferably an activated culture medium formula without agar powder, and the pH of the liquid culture medium is preferably the natural pH. The culture temperature in the liquid culture medium is preferably 32 ℃~38 ℃, for example 32, 33, 34, 35, 36, 37 or 38 ℃; the culture time for a single colony is preferably 24 h~48 h, for example 24, 28, 32, 36, 40, 44 or 48 h; the inoculation amount of the culture medium is preferably 2%~10% of the volume of the liquid culture medium, for example 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%; the culture time is preferably 24~48 h, for example 24, 28, 32, 36, 40, 44 or 48 h.

[0061] In some embodiments, the method for obtaining the bacterial suspension preferably includes: using a sterile inoculating loop to pick up the seed culture from a glycerol tube, streaking it onto a plate for rejuvenation, culturing it at 32 ℃~38 ℃ for 24 h~48 h, using a sterile inoculating loop to pick up a single colony and inoculate it into a liquid culture medium, culturing it at 32 ℃~38 ℃ for 24 h~48 h, and after the culture is completed, continuing to inoculate into the liquid culture medium at an inoculation rate of 2%~10% (v:V), and culturing it at 32 ℃~38 ℃ for 24 h~48 h.

[0062] The preferred viable bacteria concentration of the bacterial suspension is 1×10⁻⁶. 7 ~1×10 9 CFU / mL, for example 1×10 7 5×10 7 1×10 8 5×10 8 Or 1×10 9 CFU / mL. In the bacterial suspension of the deodorizing microbial composition, the preferred ratio of viable bacteria concentrations of Enterococcus faecalis GXSCU1, Bacillus compostii T-32, Lactobacillus casei T-369, and Pichia pastoris T-371 is (1~5):(1~5):(1~5):(1~5), more preferably (1~3):(1~3):(1~3):(1~3), and even more preferably 1:1:1:1.

[0063] In this invention, the inoculation amount of the deodorizing microbial composition bacterial suspension is preferably 1% to 10% of the culture medium volume, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.

[0064] In this invention, the type of culture medium can be conventionally selected according to actual needs. In some embodiments, the culture medium preferably comprises the following ingredients by weight percentage: 0.5%~4% glucose, 0.5%~3% peptone, 0.1%~2% yeast extract, 0.5%~3% sodium chloride, 0.01%~0.5% potassium dihydrogen phosphate, 0.01%~0.5% dipotassium hydrogen phosphate, with the balance being water; more preferably, it comprises the following ingredients by weight percentage: 2% glucose, 1% peptone, 0.5% yeast extract, 1% sodium chloride, 0.2% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate. The pH of the culture medium is preferably 5~8, for example, 5, 6, 7, or 8.

[0065] In this invention, the culture temperature is preferably 32 ℃~37 ℃, for example 32, 33, 34, 35, 36 or 37 ℃; the culture time is preferably 24 h~48 h, for example 24, 28, 32, 36, 40, 44 or 48 h.

[0066] The present invention also provides a microecological agent, which comprises the deodorizing microbial composition or the fermentation broth.

[0067] In this invention, the preferred method for preparing the microecological agent includes: progressively scaling up the deodorizing microbial composition or the fermentation broth to obtain the microecological agent.

[0068] In some embodiments of the present invention, it is preferable to activate and culture the microorganisms in the deodorizing microbial composition to obtain bacterial suspensions, mix the bacterial suspensions to obtain a bacterial suspension of the deodorizing microbial composition, inoculate it in a culture medium to obtain a fermentation broth, and then conduct a step-by-step scale-up culture to obtain a microecological agent.

[0069] In some embodiments of the present invention, the step of progressive scale-up cultivation preferably includes: mixing the fermentation broth with a primary nutrient pack for fermentation to obtain a primary activated broth; and mixing the primary activated broth with a secondary nutrient pack for fermentation to obtain a secondary activated broth.

[0070] The primary nutrient package preferably comprises the following ingredients in parts by weight: 5-15 parts glucose, 0.1-1 parts sodium chloride, 0.1-0.5 parts peptone, and 0.1-0.5 parts yeast powder; more preferably, 7-12 parts glucose, 0.3-0.7 parts sodium chloride, 0.2-0.3 parts peptone, and 0.2-0.3 parts yeast powder; and even more preferably, 9 parts glucose, 0.5 parts sodium chloride, 0.25 parts peptone, and 0.25 parts yeast powder.

[0071] The preferred mass ratio of the fermentation liquid to the primary nutrient pack is 25:(5~15), for example, 5:1, 25:6, 25:7, 25:8, 25:9, 5:2, 25:11, 25:12, 25:13, 25:14 or 5:3.

[0072] The fermentation broth is preferably added to a container with a volume capacity of 20 times its weight and sealed for fermentation, and water is added to make up the difference.

[0073] The fermentation time for mixing the fermentation broth with the primary nutrient pack is preferably 24 h to 48 h, for example, 24, 28, 32, 36, 40, 44 or 48 h.

[0074] The secondary nutrient package preferably comprises the following ingredients in parts by weight: 30-50 parts glucose, 1-10 parts sodium chloride, 0.5-5 parts peptone, and 0.5-5 parts yeast powder; more preferably, 40-45 parts glucose, 3-7 parts sodium chloride, 0.6-2 parts peptone, and 0.6-2 parts yeast powder; and even more preferably, 43 parts glucose, 5 parts sodium chloride, 1 part peptone, and 1 part yeast powder.

[0075] The preferred mass ratio of the primary activation solution to the secondary nutrient pack is 50:(4~10), for example, 50:4, 10:1, 50:6, 50:7, 50:8, 50:9 or 5:1.

[0076] The primary activation solution is preferably added to a container with a volume capacity 10 times that of the fermentation solution, and water is added to make up the difference.

[0077] The optimal fermentation time for the primary activation solution and the secondary nutrient pack is 24 to 48 hours, for example, 24, 28, 32, 36, 40, 44, or 48 hours. The fermentation time can be determined through sensory evaluation based on site conditions. For example, in summer, when temperatures are higher, activation is better, the pH drops rapidly to below 4.0, and the fermentation liquid becomes cloudy with an acidic, wine-like aroma. In winter, when temperatures are lower, fermentation is slower, thus requiring a longer fermentation time.

[0078] The pH of the secondary activation solution is preferably ≤4.0; the bacterial concentration of the secondary activation solution is preferably ≥5×10⁻⁶. 7 CFU / mL.

[0079] In this invention, the number of times the scale-up culture is carried out is not specifically limited and can be selected conventionally according to actual production needs. The deodorizing microbial composition of this invention is scaled up step by step through activation, shake flask culture (500 mL), scale-up culture (5000 mL), seed fermentation tank (250 L), and large fermentation tank (1.5 T), ultimately achieving a daily production of 1.5 T of microbial agent liquid.

[0080] The present invention also provides the application of the deodorizing microbial composition, the fermentation broth, or the microecological agent in the preparation of deodorizing products.

[0081] In this invention, the type of product is not specifically limited and can be conventionally selected according to actual needs. The product may also include acceptable excipients or additives, and the specific types and amounts of excipients or additives can be conventionally selected according to actual needs.

[0082] The present invention also provides the application of the deodorizing microbial composition, the fermentation broth, or the microecological agent in the treatment of malodorous gases.

[0083] In this invention, the malodorous gas preferably includes one or more of ammonia, trimethylamine, hydrogen sulfide, methanethiol, dimethyl sulfide, dimethyl disulfide, carbon disulfide, and styrene.

[0084] In this invention, the treatment of odorous gases preferably includes fecal deodorization, garbage deodorization, and wastewater deodorization; the locations for the treatment of odorous gases preferably include livestock and poultry farms, manure storage tanks, public toilets, landfills, garbage transfer stations, composting plants, and wastewater treatment plants.

[0085] In this invention, the application preferably includes any one of the following:

[0086] (1) Control of odor pollutants from livestock and poultry manure in agricultural environments;

[0087] (2) Purification of odorous gases during the composting process of organic waste;

[0088] (3) Biodegradation of volatile odorous substances in contaminated sites.

[0089] The present invention also provides a deodorization method, the deodorization method comprising: adding the deodorizing microbial composition or the fermentation broth or the microecological agent to the material to be deodorized.

[0090] In this invention, the type of material to be deodorized is not particularly limited. In some embodiments, the material to be deodorized preferably includes livestock and poultry manure, garbage, or organic waste.

[0091] In this invention, the method of addition is not particularly limited. In some embodiments, the deodorizing microbial composition, the fermentation broth, or the microecological agent is sprayed onto the material to be deodorized for deodorization. The amount sprayed is not particularly limited and can be conventionally selected according to actual needs.

[0092] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0093] Unless otherwise specified, the following embodiments are all conventional methods.

[0094] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0095] Example 1: Physicochemical characteristics analysis and 16S rDNA sequencing identification of four bacterial strains in the deodorizing microbial composition

[0096] 1. Strains Isolation and Purification

[0097] Enterococcus faecalis GXSCU1, Bacillus compostii T-32, Lactobacillus casei T-369, and Pichia pastoris T-371 were isolated and purified from fermented feed and organic fertilizer, respectively.

[0098] 2. Colony morphology identification

[0099] The colony morphology, cell morphology, and Gram staining of the four bacterial strains were observed separately, and the results are as follows: Figure 1-Figure 3 As shown.

[0100] It can be seen that the morphological characteristics of Enterococcus faecalis GXSCU1 are as follows: after the strain is cultured on optimized plates (2% glucose, 1% peptone, 1% sodium chloride, 0.5% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, 2% agar, natural pH) at 35 ℃±1 ℃ for 24 h~48 h, the colonies are round, with neat edges, smooth and glossy surface, and milky white color. Observation under a microscope by Gram staining shows that this strain is a Gram-positive bacterium, with a round or oval shape, and is arranged singly, in pairs or short chains.

[0101] The morphological characteristics of Bacillus compostii T-32 are as follows: After being cultured on optimized plates (2% glucose, 1% peptone, 1% sodium chloride, 0.5% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, 2% agar, natural pH) at 35 ℃ ± 1℃ for 24 h to 48 h, the colonies are flat, with neat edges, slightly rough surface, and pale yellow color. Gram staining and microscopic observation show that this strain is a Gram-positive bacterium, rod-shaped, and produces spores.

[0102] The morphological characteristics of Lactobacillus casei T-369 are as follows: After being cultured on MRS plates at 35 ℃ ± 1 ℃ for 24 h to 48 h, the colonies are round with neat edges, smooth surface, and milky white color. Observation under a microscope after Gram staining shows that this strain is a Gram-positive bacterium, with short or long rods in shape, arranged mostly in short chains or long chains, non-motile, and does not produce spores.

[0103] The morphological characteristics of Pichia pastoris T-371 are as follows: After being cultured on MRS plates at 35 ℃±1 ℃ for 24 h to 48 h, the colonies are round with neat edges, smooth and raised surfaces, and are milky white. Gram staining and microscopic observation show that this strain is a Gram-positive bacterium with morphologies such as spherical, oval, and elliptical.

[0104] 3. Physiological and biochemical characteristics analysis

[0105] Physiological and biochemical identification of four strains was performed in accordance with the common strain identification methods in the Manual of Bacterial Systematic Identification. The identification results are shown in Table 1.

[0106] Table 1. Physiological and biochemical identification results of the four bacterial strains.

[0107]

[0108] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0109] It can be seen that Enterococcus faecalis GXSCU1 and Bacillus compostii T-32 are Gram-positive, VP test negative, methyl red test positive, nitrate utilization test positive, and propionate and citrate utilization tests negative; Lactobacillus casei T-369 and Pichia pastoris T-371 are Gram-positive, VP test negative, methyl red test positive, and nitrate, propionate and citrate utilization tests positive.

[0110] Enterococcus faecalis GXSCU1, Bacillus compostii T-32, and Lactobacillus casei T-369 can ferment and utilize various carbon sources such as glucose, sucrose, inulin, maltose, D-raffinose, cellobiose, and inositol, and metabolize them to produce organic acids (such as lactic acid and acetic acid). Pichia pastoris T-371 can utilize propionate and citrate as carbon sources for growth and metabolize them to produce organic acids (such as acetic acid), and can also convert nitrates (NO3) into organic acids. - ) is reduced to nitrite (NO2) - It may be further reduced to products such as nitrogen (N2).

[0111] 4. 16S rDNA sequencing identification and phylogenetic tree analysis

[0112] Sequencing analysis revealed that the 16S rDNA sequence length of *Enterococcus faecalis* GXSCU1 was 1465 bp, as shown in SEQ ID NO.1; the 16S rDNA sequence length of *Bacillus compostii* T-32 was 1391 bp, as shown in SEQ ID NO.2; the 16S rDNA sequence length of *Lactobacillus casei* T-369 was 1494 bp, as shown in SEQ ID NO.3; and the ITS sequence length of *Pichia pastoris* T-371 was 545 bp, as shown in SEQ ID NO.4. Sequence alignment of the four strains using the NCBI database was performed, and the results are shown in Table 2. The phylogenetic tree analysis results of the four strains are as follows: Figure 4 As shown.

[0113] Table 2. Results of 16S rDNA sequence alignment for the four bacterial strains.

[0114]

[0115] It can be seen that the 16S rDNA sequence of Enterococcus faecalis GXSCU1 is similar to... Enterococcus faecium Completely identical, with 100% sequence similarity; the 16S rDNA sequence of Bacillus composting T-32 is identical to... Dung bacillus Completely identical, with 100% sequence similarity; the 16S rDNA sequence of Lactobacillus casei T-369 is identical to... Lactobacillus casei Completely identical, with 100% sequence similarity; the 16S rDNA sequence of Pichia pastoris T-371 is identical to... Pichia ethanolicaCompletely identical, with a sequence similarity of 100%.

[0116] Four strains were deposited at the Guangdong Provincial Microbial Culture Collection Center: Enterococcus faecalis GXSCU1 (GDMCC No. 62041), Bacillus compostii T-32 (GDMCC No. 66237), Lactobacillus casei T-369 (GDMCC No. 63763), and Pichia pastoris T-371 (GDMCC No. 66239).

[0117] Example 2: Evaluation of the effect of the deodorizing microbial composition on the degradation of ammonia, hydrogen sulfide and odor concentration in fecal waste.

[0118] 1. Screening of strains

[0119] Bacillus subtilis UBIT-1, Enterococcus faecalis GXSCU1, Bacillus licheniformis UBIT-31, Bacillus compostii T-32, Bacillus amyloliquefaciens UBIT-93, Bacillus widmannii UBIT-108, Bacillus bereaves UBIT-167, Lactobacillus casei T-369, and Pichia pastoris T-371 were selected strains. The selected strains were activated, and the concentration of each bacterial suspension was adjusted to 1×10⁻⁶. 8 CFU / mL.

[0120] The control group used a mixture of 900 mL sterile water and 100 mL fecal matter as the baseline, while the experimental group used a mixture of 900 mL bacterial suspension and 100 mL fecal matter as the baseline. Both groups added 100 mL of fecal matter daily for at least 15 days. Ammonia, hydrogen sulfide, and odor concentrations were measured daily. Data was measured and recorded before each addition of fecal matter. The deodorizing effect of the deodorizing bacteria on water-soaked feces was evaluated by comparing daily data changes. The results are as follows: Figure 5-Figure 7 As shown.

[0121] The results showed that with the addition of fecal waste, the odor concentration in the control group reached a stable peak after 7 days, while the experimental group reached a stable peak after a maximum of 19 days, which was 12 days longer than the control group. The hydrogen sulfide concentration in both the control and experimental groups reached a stable peak after 7 days, indicating no significant degradation effect on hydrogen sulfide. Considering the influence of various strains, five strains with strong acid-producing ability, adaptability, and high degradation efficiency (Enterococcus faecalis GXSCU1, Bacillus proteolyticus T-32, Bacillus wedmannii UBIT-108, Lactobacillus casei T-369, and Candida albicans T-371) were preferentially selected for dynamic simulated deodorization experiments using a compound microbial agent.

[0122] 2. Evaluation of the effectiveness of the deodorizing microbial composition in degrading ammonia, hydrogen sulfide, and odor concentration in fecal waste.

[0123] Antagonistic experiments on the above strains revealed that Bacillus wedmannii UBIT-108 had antagonistic effects with the other four strains. Therefore, the above four strains (Enterococcus faecalis GXSCU1, Bacillus compostii T-32, Lactobacillus casei T-369, and Pichia pastoris T-371) were subjected to a dynamic simulation deodorization test using compound microbial agents.

[0124] (1) Experimental grouping

[0125] Experimental group 1: Enterococcus faecalis GXSCU1 + Lactobacillus casei T-369 (strain ratio 1:1);

[0126] Experimental group 2: Enterococcus faecalis GXSCU1 + Lactobacillus casei T-369 + Pichia pastoris T-371 (strain ratio 1:1:1).

[0127] Experimental group 3: Enterococcus faecalis GXSCU1 + Bacillus compostii T-32 + Lactobacillus casei T-369 + Pichia pastoris T-371 (strain ratio 1:1:1:1).

[0128] (2) Preparation of fermentation broth for deodorizing microbial composition

[0129] a. Activate the above-mentioned strains using the streak plate method:

[0130] The activation medium formulation for Enterococcus faecalis GXSCU1 and Bacillus compostii T-32 was as follows: 2% glucose, 1% peptone, 0.5% yeast extract, 1% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, and 2% agar powder, pH 5-8. Seed culture from glycerol tubes was streaked onto the above-mentioned plates using a sterile inoculation loop and incubated at 37 ℃ ± 1 ℃ for 48 h. After incubation, a single colony was picked up using a sterile inoculation loop and inoculated into the liquid optimized medium (activation medium formulation omitting agar powder), and incubated at 37 ℃ ± 1 ℃ for 24 h. Subsequently, inoculation was continued at a rate of 5% (v:v) into the above liquid optimized medium, and incubated at 37 ℃ ± 1 ℃ for 48 h to obtain the bacterial suspension.

[0131] The activation medium formulation for *Lactobacillus casei* T-369 and *Pichia pastoris* T-371 was as follows: 1% peptone, 0.5% beef extract, 2% glucose, 0.4% yeast extract, 0.5% sodium acetate, 0.2% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 0.005% manganese sulfate, 0.02% triammonium citrate, 0.1% Tween 80, and 2% agar powder, pH 5-8. Seed culture from glycerol tubes was streaked onto the above-mentioned plates using a sterile inoculation loop and incubated at 37 ℃ ± 1 ℃ for 48 h. After incubation, a single colony was picked up using a sterile inoculation loop and inoculated into the liquid optimized medium (activation medium formulation omitting agar powder), and incubated at 37 ℃ ± 1 ℃ for 24 h. Subsequently, inoculation was continued at a rate of 5% (v:v) into the above liquid optimized medium, and incubated at 37 ℃ ± 1 ℃ for 48 h to obtain a bacterial suspension.

[0132] b. Combine the above-mentioned strains:

[0133] After the seed culture is completed, adjust the concentration of each bacterial suspension to 1×10⁻⁶. 8 CFU / mL, according to the above experimental groups, the bacterial suspension was mixed and inoculated into the deodorizing bacterial agent medium (2% glucose, 1% peptone, 1% sodium chloride, 0.5% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, natural pH) at an inoculation rate of 5% (v:v). The medium was then incubated at 35 ℃ ± 2 ℃ for 48 h. After incubation, the bacterial suspension concentration was adjusted to 1×10⁻⁶. 8 CFU / mL was used to obtain the fermentation broth of each group of deodorizing microbial compositions.

[0134] The control group used 900 mL of sterile water mixed with 100 mL of fecal matter as the baseline, while the experimental groups used 900 mL of fermentation broth from the deodorizing microbial composition (Experimental Group 1, Experimental Group 2, and Experimental Group 3) mixed with 100 mL of fecal matter as the baseline. Both the control and experimental groups added 100 mL of fecal matter daily for 15 days. Ammonia, hydrogen sulfide, and odor concentrations were measured daily. Data was measured and recorded before each addition of fecal matter. The deodorizing effect of the deodorizing microbial composition on water-soaked feces was evaluated by comparing daily data changes. The results are shown in Table 3. Figure 8 As shown.

[0135] Table 3. Degradation effect of deodorizing microbial composition on fecal odor gases

[0136]

[0137] The results showed that the deodorizing microbial composition in experimental group 3 effectively reduced the concentrations of ammonia, hydrogen sulfide, and odor, with the highest degradation rates reaching 41.43%, 40.70%, and 16.91%, respectively. This indicates that the deodorizing microbial composition of this combination helps to degrade pollutants in manure and improve the malodorous environment of livestock and poultry farms.

[0138] 3. Evaluation of the effect of different proportions of deodorizing microbial compositions on the degradation of pH, ammonia, hydrogen sulfide and odor concentration in fecal waste.

[0139] The fermentation broth of the deodorizing microbial composition from experimental group three above, and a concentration of 1×10 8 Single strains of Enterococcus faecalis GXSCU1, Bacillus compostii T-32, Lactobacillus casei T-369, and Pichia pastoris T-371 (CFU / mL) were inoculated into 250 mL sample bottles containing feces at concentrations of 10% (① 90% feces), 30% (② 70% feces), and 50% (③ 50% feces), respectively. Control groups were prepared by adding 10% (①), 30% (②), and 50% (③) sterile water, while a blank group was prepared without any treatment. All bottles were sealed with breathable sealing film and incubated at room temperature. Ammonia, hydrogen sulfide, odor, and pH were measured daily to evaluate the degradation effect of the strains on fecal odor. The results are shown in Table 4. The degradation rate was calculated using the following formula:

[0140]

[0141] Table 4. Degradation effect of microbial compositions for deodorizing fecal odors without varying proportions.

[0142]

[0143]

[0144]

[0145] The results showed that increasing the amount of deodorizing microbial composition helped control pollutant emissions and reduce peak pollutant emissions. The deodorizing microbial composition in experimental group 3③ achieved maximum degradation rates of 50.93%, 100.00%, and 100.00% for ammonia, hydrogen sulfide, and odor concentration, respectively. Compared to a single strain, the deodorizing microbial composition in experimental group 3 significantly improved the degradation rate. This indicates that the combined deodorizing microbial composition helps degrade pollutants in manure and improves the odorous environment of livestock and poultry farms.

[0146] Example 3: Microbial colony composition in fecal samples treated with the deodorizing microbial composition

[0147] The experimental samples from the control group and experimental group 3 in Example 2, "Evaluation of the effect of deodorizing microbial composition on the degradation of ammonia, hydrogen sulfide and odor concentration in fecal sewage", were sent to Qingdao Ouyi Biotechnology Co., Ltd. for whole genome analysis. Representative sequences from the non-redundant gene set were compared with the NCBI NR database using DIAMOND software, and sequences with e < 1 were selected. -5 Proteins with the highest sequence similarity were selected from the annotations to obtain functional annotation information. Species annotations were obtained from the taxonomic information database corresponding to the NR library. Then, the abundance of the species was calculated using the sum of gene abundance corresponding to the species. The abundance of the species in each sample was statistically analyzed at each taxonomic level: Domain, Kingdom, Phylum, Class, Order, Family, Genus, and Species. This constructed abundance spectra at the corresponding taxonomic levels to explore the composition of the bacterial community. The results are shown in Table 5.

[0148] Table 5. Microbial community composition of the top 15 microorganisms in fecal samples.

[0149]

[0150]

[0151] The results showed that the fecal samples from the control group mainly consisted of *Treponema*, *Clostridium*, *Ruminococcus*, *Alistipes*, and *Solobacterium*. The fecal samples from the experimental group mainly consisted of *Clostridium*, *Treponema*, *Mobilisporobacter*, *Ruminococcus*, and *Tissierella*. The changes in the composition of the two groups directly reflected the adjustment of microbial function during fecal fermentation, which is significant for the efficiency of fecal treatment, product quality, and environmental impact. The newly added genera in the experimental group (such as *Tissierella* and *Anaerobic Columnar Bacteria*) were more inclined to participate in amino acid metabolism and crude fiber degradation, which improved the nitrogen and carbon conversion efficiency in the fecal samples and was more conducive to the accumulation of nutrients in the fermentation products. By comparing the metagenomic composition of the control group and the experimental group after manure fermentation, it was found that the deodorizing microbial composition helps to reshape the microbial community structure and improve the efficiency of manure fermentation, which has positive significance for the actual utilization of manure resources.

[0152] Example 4: Evaluation of the effect of microbial agents on degrading malodorous gases from pig farm manure

[0153] Using 1,500 pigs weighing approximately 60 kg each as the research subjects, about 20 tons of microecological bacteria were injected into a water-filled manure pit as the experimental group to evaluate its degradation effect on malodorous gases in manure. No intervention was performed as the control group.

[0154] The specific activation process of the selected microbial agent is as follows (e.g. Figure 9 (as shown)

[0155] (1) Preparation of deodorizing probiotic stock solution: According to the ratio of experimental group three in Example 2, the fermentation broth of the deodorizing microbial composition was prepared according to the preparation method in Example 2 to obtain the deodorizing probiotic stock solution;

[0156] (2) Primary activation liquid: Add 25 kg of deodorizing probiotic stock solution and the corresponding nutrient pack (9 kg glucose, 0.5 kg sodium chloride, 0.25 kg peptone, 0.25 kg yeast powder) to a 500 L capacity sealable plastic bucket, add water to make up to 500 kg, stir thoroughly, seal and let stand for fermentation for 48 h to make primary activation liquid;

[0157] (3) Secondary activation solution: Add the primary activation solution (500 kg) and the corresponding nutrient pack (43 kg glucose, 5 kg sodium chloride, 1 kg peptone, 1 kg yeast powder) to a 5 m³ solution. 3 A secondary activation container with a volume of [volume value missing] is then filled with clean water to make up to 5 m³. 3 After thorough mixing, seal and allow to ferment for 48 hours. The pH should be ≤4.0 and the bacterial concentration ≥5×10⁻⁶ using precise pH test paper. 7 CFU / mL, which is used to prepare a secondary activation solution.

[0158] Portable gas detectors were used to monitor and record environmental data on the concentrations of ammonia, hydrogen sulfide, and odor in the pigsty. The effectiveness of the implementation was evaluated by comparing the daily environmental data. The results are shown in Table 6.

[0159] Table 6. Degradation effect of microbial agents on malodorous gases from pig farm manure.

[0160]

[0161] The results showed that injecting microbial agents into the water-filled manure pit reduced the daily average concentrations of ammonia, hydrogen sulfide, and odor in the pigsty by 22.56%, 57.14%, and 15.24% at 10 days, and by 22.44%, 50.00%, and 8.13% at 30 days, respectively, effectively improving the air quality in the pigsty and enhancing the health of the pig herd.

[0162] The microbial agent prepared by this invention through a specific ratio and process has significant deodorizing effect and manure degradation ability, and is suitable for manure treatment in large-scale livestock and poultry farms, and has important environmental and economic value.

[0163] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A deodorizing microbial composition, characterized in that, The deodorizing microbial composition consists of Enterococcus faecalis with a live bacteria concentration ratio of (1~5):(1~5):(1~5):(1~5) in the form of (1~5):(1~5):(1~5). Enterococcus faecium GXSCU1, Bacillus compostii ( Bacillus stercoris T-32, Lactobacillus casei ( Lacticaseibacillus casei T-369 and Pichia pastoris (ethanol) Pichia ethanolica The composition is as follows: T-371; the preservation number of Enterococcus faecalis GXSCU1 is GDMCC No:62041; the preservation number of Bacillus compostii T-32 is GDMCC No:66237; the preservation number of Lactobacillus casei T-369 is GDMCC No:63763; and the preservation number of Pichia pastoris T-371 is GDMCC No:66239.

2. A fermentation broth for a deodorizing microbial composition, characterized in that, The fermentation broth is the deodorizing microbial composition according to claim 1.

3. The method for preparing the fermentation broth according to claim 2, characterized in that, The preparation method includes: inoculating the deodorizing microbial composition of claim 1 into a culture medium and culturing it to obtain a fermentation broth.

4. A microecological agent, characterized in that, The microbial agent comprises the deodorizing microbial composition of claim 1 or the fermentation broth of claim 2.

5. The method for preparing the microecological agent according to claim 4, characterized in that, The preparation method includes: culturing the deodorizing microbial composition of claim 1 or the fermentation broth of claim 2 through stepwise scale-up cultivation to obtain a microecological agent.

6. The application of the deodorizing microbial composition of claim 1, the fermentation broth of claim 2, or the microecological agent of claim 4 in the preparation of deodorizing products.

7. The application of the deodorizing microbial composition of claim 1, the fermentation broth of claim 2, or the microecological agent of claim 4 in the treatment of malodorous gases.

8. The application according to claim 7, characterized in that, The malodorous gases include one or more of ammonia, trimethylamine, hydrogen sulfide, methanethiol, dimethyl sulfide, dimethyl disulfide, carbon disulfide, and styrene.

9. The application according to claim 7, characterized in that, The application includes any one of the following: (1) Control of odor pollutants from livestock and poultry manure in agricultural environments; (2) Purification of odorous gases during the composting process of organic waste; (3) Biodegradation of volatile odorous substances in contaminated sites.

10. A deodorization method, characterized in that, The deodorization method includes adding the deodorizing microbial composition of claim 1, the fermentation broth of claim 2, or the microecological agent of claim 4 to the material to be deodorized.

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