A composite microbial agent, its preparation method and application
By leveraging the synergistic effect of compound microbial agents, the problems of environmental pollution and health threats in cow manure treatment have been solved, achieving rapid composting and harmless treatment, and improving compost quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HUBEI MAOSHENG BIOLOGY CO LTD
- Filing Date
- 2025-11-27
- Publication Date
- 2026-07-31
AI Technical Summary
Existing technologies are ineffective in treating cow manure, leading to environmental pollution and health threats. Furthermore, there are risks of ammonia nitrogen volatilization loss and the spread of pathogenic microorganisms during the composting process.
The use of compound microbial agents, including Bacillus thermophilus, Bacillus brucellosis and Aspergillus chevallis, accelerates the composting process through synergistic effects, kills pathogens and insect eggs, reduces the risk of transmission, and achieves resource utilization.
It achieves rapid composting of cow manure, effectively kills pathogens and insect eggs, reduces the risk of transmission, improves compost quality, and achieves the goal of harmless and resource-based treatment.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a compound microbial agent, its preparation method, and its application. Background Technology
[0002] Agricultural waste is a general term for waste generated from agricultural production, agricultural product processing, livestock and poultry farming, and rural residential waste. It includes four main categories: plant waste, animal waste, processing waste, and rural and urban domestic waste. With the rapid development of industrial and agricultural production and the dramatic increase in population, these wastes are increasing year by year. In particular, with the rapid development of livestock and poultry farming, especially the shift from scattered household farming to large-scale, intensive farms, the carrying capacity per unit of land is increasing, thus placing a greater pollution load on the local environment. The relocation of livestock and poultry farms from rural and pastoral areas to urban suburbs has led to a disconnect between agriculture and animal husbandry, resulting in large-scale local concentrations of livestock waste that are not properly treated. For example, large amounts of untreated manure are directly applied to the soil, exceeding the soil's self-purification capacity, leading to incomplete degradation and anaerobic decomposition, producing malodorous substances and harmful substances such as nitrites. This alters the composition and properties of the soil, destroying its original basic functions and reducing soil quality, causing enormous damage to the ecological environment and posing a serious threat to the health of animals and humans. Furthermore, if feces are not treated in a timely manner and are left to accumulate, they will breed a large number of mosquitoes and flies, which will cause pathogens and parasite eggs to multiply rapidly and easily spread, worsening the surrounding environmental sanitation and even causing the spread of infectious diseases in humans and animals.
[0003] Composting is a relatively traditional biological fermentation technology. Initially, it involved piling and fermenting the compost, which took 4-6 months to mature under natural temperatures. To accelerate maturation and eliminate the cost of turning the compost pile, ventilation pipes could be installed at the bottom of the pile for forced or static ventilation, shortening the maturation time. This method is low-cost but requires a large area, has a long processing time, and is easily affected by weather. By utilizing the fermentation action of microorganisms, the microbial process of converting degradable organic matter into stable humus is artificially promoted, reducing the composting time from over 100 days of natural composting to 30-50 days, or even less, achieving rapid maturation.
[0004] Currently, there is limited research on the treatment of cow manure. Furthermore, there are many problems with the composting of cattle farm manure, such as the high water content and foul odor of the manure. In addition, a large amount of ammonia nitrogen is easily lost through volatilization during the treatment process. The manure also contains a large number of pathogenic microorganisms and weed seeds, all of which pose a threat to the environment. Therefore, there is an urgent need to develop a microbial agent for the composting of cow manure to promote its decomposition and achieve the goal of harmless treatment. Summary of the Invention
[0005] The purpose of this invention is to provide a compound microbial agent that effectively decomposes livestock and poultry manure, with good decomposition effect, high seed germination index, effectively kills pathogens and insect eggs in the manure, reduces toxicity, significantly reduces the risk of pathogen transmission, and achieves the purpose of harmless and resource-based treatment and utilization.
[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A compound microbial agent comprising *Bacillus thermophilus*, *Thermophyton floccosum*, and *Aspergillus chevallaris*; the *Bacillus thermophilus* was purchased from the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC1.3474, original accession date September 11, 2003; the *Thermophyton floccosum* was purchased from the CGMCC, accession number CGMCC1.8658, original accession date September 17, 2008; and the *Aspergillus chevallaris* was purchased from the CGMCC, accession number CGMCC3.12662, original accession date July 10, 2008. All strains used in this invention can be purchased openly through the culture catalogs of the culture centers, eliminating the need for repeated biological preservation.
[0007] Furthermore, the compound microbial agent is in powder form.
[0008] This invention provides a method for preparing a compound microbial agent, comprising the following steps: (1) After thawing, *Bacillus thermophilus* and *Thermophilus brevicornu* were activated in LB solid medium, and then single colonies were picked and inoculated into LB liquid medium and cultured until OD. 600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10%. When the effective viable count reached 1×10 9 When the concentration of CFU / mL was reached, the culture was stopped, and two bacterial cultures were obtained. (2) After thawing Aspergillus chevalerate, activate and culture it in PDA solid medium for 5-7 days. Spores are scraped from the surface of the medium and inoculated into PDA liquid medium. Culture for 36-48 hours to prepare a spore suspension. Inoculate the fermenter with 10% of the spore suspension. When the effective viable count reaches 1×10⁻⁶, the spore suspension is inoculated into the fermenter. 9 When the concentration of CFU / mL is reached, stop the culture to obtain Aspergillus cheivae culture. (3) The bacterial suspensions of Bacillus thermophilus, Bacillus brucellus, and Aspergillus serrata prepared in step (1) and the bacterial suspensions of Aspergillus serrata in step (2) are mixed in a volume ratio of 1:1:1 and then freeze-dried into freeze-dried powder to obtain the final product, composite microbial agent.
[0009] The present invention also provides the application of a compound microbial agent, which is used to accelerate the decomposition of livestock and poultry manure and improve the quality of compost.
[0010] Furthermore, the livestock and poultry manure is cow manure.
[0011] In a specific implementation plan, the weight ratio of the compound microbial agent to the feces is (1-3):1000.
[0012] Beneficial effects The composite microbial agent prepared in this invention includes Bacillus thermophilus, Thermophyton floccosum, and Aspergillus chevallis. Bacillus thermophilus secretes cellulase, hemicellulase, and protease; Thermophyton floccosum can secrete lipase and cellulase; and Aspergillus chevallis can secrete cellulase and hemicellulase. The combination of these three microorganisms can secrete a variety of biological enzymes, which work synergistically to significantly promote the composting process.
[0013] This invention contains *Bacillus thermophilus*, *Thermophyton floccosum*, and *Aspergillus chevallaris*. These three selected strains exhibit a temperature gradient distribution between 25-70°C, enabling normal physiological metabolism. This ensures that the strains continue to compost the feces even as the temperature of the compost pile changes, thus accelerating the composting process. *Bacillus thermophilus* and *Thermophyton floccosum* are both thermophilic strains, capable of rapidly dehydrating and composting the feces at high temperatures of 60-70°C. This ensures that composting can continue even when most microorganisms cannot perform normal physiological metabolism at temperatures exceeding 50°C. Furthermore, the sustained high temperature helps kill pathogens, insect eggs, and weed seeds in the feces, reducing toxicity and significantly lowering the risk of pathogen transmission, achieving the goal of harmless and resource-based treatment and utilization. The synergistic effect of these three strains achieves full-cycle coverage of the composting process, with efficiency far exceeding that of a single microbial agent or an improperly formulated combination.
[0014] The compound microbial agent prepared by this invention can rapidly decompose various organic matter. The decomposed manure has a high degree of decomposition, low toxicity, and a seed germination index of over 95%. Detailed Implementation
[0015] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.
[0016] Example 1 A compound microbial agent comprising *Bacillus thermophilus*, *Thermophyton floccosum*, and *Aspergillus chevallis*; the *Bacillus thermophilus* has the accession number CGMCC1.3474; the *Thermophyton floccosum* has the accession number CGMCC1.8658; and the *Aspergillus chevallis* has the accession number CGMCC3.12662.
[0017] The compound microbial agent is in powder form.
[0018] A method for preparing a compound microbial agent includes the following steps: (1) After thawing, *Bacillus thermophilus* and *Thermophilus brevicornu* were activated in LB solid medium, and then single colonies were picked and inoculated into LB liquid medium and cultured until OD. 600 =0.6 to obtain seed culture, and the seed culture was inoculated into the fermenter at a rate of 10%. When the effective viable count reached 1×10 9 When the concentration of CFU / mL was reached, the culture was stopped, and two bacterial cultures were obtained. (2) After thawing Aspergillus chevalerate, activate and culture it in PDA solid medium for 5 days. Spores are scraped from the surface of the medium and inoculated into PDA liquid medium. After culturing for 48 hours, a spore suspension is prepared. The spore suspension is then inoculated into the fermenter at an inoculation rate of 10%. When the effective viable count reaches 1×10⁻⁶, the spore suspension is further inoculated into the fermenter. 9 When the concentration of CFU / mL is reached, stop the culture to obtain Aspergillus cheivae culture. (3) The bacterial suspensions of Bacillus thermophilus, Bacillus brucellus, and Aspergillus serrata prepared in step (1) and the bacterial suspensions of Aspergillus serrata in step (2) are mixed in a volume ratio of 1:1:1 and then freeze-dried into freeze-dried powder to obtain the final product, composite microbial agent.
[0019] Comparative Example 1 Compared with Example 1, this comparative example changed the volume ratio of Bacillus thermophilus, Thermophyton floccosum, and Aspergillus chevallaris to 2:1:1.
[0020] Comparative Example 2 Compared with Example 1, this comparative example changed the volume ratio of Bacillus thermophilus, Thermophyton floccosum, and Aspergillus chevallaris to 1:2:1.
[0021] Comparative Example 3 Compared with Example 1, this comparative example changed the volume ratio of Bacillus thermophilus, Thermophyton floccosum, and Aspergillus chevallaris to 1:1:2.
[0022] Comparative Example 4 Compared with Example 1, this comparative example uses Bacillus thermophilus and Thermophyton floccosum in a 1:1 volume ratio in the compound microbial agent.
[0023] Comparative Example 5 Compared with Example 1, this comparative example uses Bacillus thermophilus and Aspergillus serrata in a 1:1 volume ratio for the compound microbial agent.
[0024] Comparative Example 6 Compared with Example 1, this comparative example uses a compound microbial agent containing *Thermophyton floccosum* and *Aspergillus chevallaris* in a volume ratio of 1:1.
[0025] Comparative Example 7 Compared with Example 1, this comparative example uses only Bacillus thermophilus in the compound microbial agent.
[0026] Comparative Example 8 Compared with Example 1, this comparative example uses only Thermophyton floccosum in the compound microbial agent.
[0027] Comparative Example 9 Compared with Example 1, this comparative example uses only Aspergillus chevalerate in the compound microbial agent.
[0028] Performance testing Determination of cellulose degradation ability of each strain: Three strains were prepared into seed suspensions and inoculated into Congo red medium at an inoculum of 10%. The suspensions were cultured at 50°C with shaking. Cellulase activity was measured at 3, 4, 5, 6 and 7 days, with 3 replicates.
[0029] Standard curve preparation: Anhydrous glucose was dried at 80℃ to constant weight to prepare a 1 mg / mL standard solution. 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution were taken, and distilled water was added to bring the volume to 2.0 mL. 1.5 mL of DNS was added, and the mixture was boiled in a water bath for 5 min. After cooling, the volume was adjusted to 25 mL, and the OD value was measured at 540 nm to prepare the standard curve.
[0030] Cellulase activity assay: After fermentation, the Congo red medium was centrifuged at 10000 r / min for 10 min to obtain crude enzyme solution. 0.1 mL of this solution was added to 1.9 mL of 1% CMC-Na solution and hydrolyzed at 50℃ for 20 min. Then, 1.5 mL of DNS colorimetric solution was added and the solution was boiled in a water bath for 5 min. The volume was adjusted to 25 mL, and the OD value was measured at 540 nm. The result was compared with the standard curve to calculate the glucose content (m1). Separately, 0.1 mL of the supernatant was added to 1.9 mL of water, followed by 1.5 mL of DNS. The mixture was boiled in a water bath for 5 min and the volume was adjusted to 25 mL. The glucose content (m2) of the crude enzyme solution was measured at 540 nm.
[0031] Enzyme activity calculation formula: Cellulase activity (U / mL) = (m1 - m2) × 10 × 1000 / t Protein degradation ability determination of each strain: Standard curve preparation: Prepare L-tyrosine standard solutions of different concentrations and perform measurements immediately after dilution. Take 1 mL of each standard solution, add 5 mL of sodium carbonate solution and 1 mL of Folin-phenol reagent to each, shake well, and develop the color in a 40 ℃ water bath for 20 min. Measure the absorbance (OD) value at 680 nm and plot the standard curve. Use the regression equation to calculate the amount of tyrosine (μg) when the absorbance is 1, which is the absorbance constant K value. The K value should be between 95 and 100.
[0032] Preparation of crude enzyme solution: The strain was inoculated into seed culture medium and cultured at 50°C with shaking for 12 h. 1% of the strain was then inoculated into fermentation medium and cultured at 50°C with shaking for 48 h. The supernatant was then collected by centrifugation.
[0033] Protease activity assay: Preheat 2% casein solution to 40℃ for 5 min; add 1 mL of crude enzyme solution to 2 mL of casein solution, mix well, and incubate at 40℃ for 10 min. Add 1 mL of 10% trichloroacetic acid to terminate the reaction. Take 1 mL of supernatant, add 5 mL of 0.55 mol / L Na2CO3, add 1 mL of Folin-phenol reagent, and incubate at 40℃ for 20 min for color development. Measure the absorbance at 680 nm. Use the reaction system with added water as a blank.
[0034] Enzyme activity calculation formula: Protease activity (U / mL) = A × K × V / t; where A is the OD value of the fermentation broth, K is the absorbance constant, V is the total volume of the reaction reagents, and t is the reaction time.
[0035] Determination of lipid degradation capacity of each strain: Take several 100 mL conical flasks, one as a control flask and the others as test flasks, as shown in Table 1 below. Titrate with 0.05 M standard sodium hydroxide solution until a faint pink color appears, and record the volume of alkaline solution used in the titration.
[0036] Table 1. Methods for Lipase Assay The formula for calculating enzyme activity is: Lipase activity (U / mL) = (AB) × N × f / t; where A is the alkali solution consumed by the sample (mL), B is the alkali solution consumed by the control group (mL), N is the concentration of the alkali solution (0.05μmol), the dilution factor of the crude enzyme solution, and t is the reaction time (min).
[0037] Hemicellulase activity assay of each strain: Hemicellulase activity assay: 1.0 mL of crude enzyme solution was mixed with 1.5 mL of 1% xylan solution at pH 4.8 (for the control, 1.0 mL of diluted enzyme solution was mixed with 3.0 mL of acetate buffer at pH 4.8, without adding xylan solution), and the mixture was incubated at 50 °C for 60 min. After incubation, 4.0 mL of DNS reagent was added and shaken well. The mixture was then immediately incubated in a boiling water bath for 10 min. After cooling, water was added to bring the volume to 25 mL, and the OD value was measured at 550 nm.
[0038] One unit of hemicellulase activity is defined as the amount of enzyme that, when in 1.0 mL of enzyme solution at 50°C and pH 4.8, produces one microgram of reducing sugar (xylose) per minute from a 1.0% xylan solution. The enzyme activity calculation formula is: Hemicellulase activity (U / mL) = (A × N × 1000) / 60; where A is the OD value. 550nm The xylose concentration corresponding to the absorption value is given below, where N is the enzyme dilution factor.
[0039] Table 2. Results of enzyme activity assays for each strain Specific applications of microbial agents The cow manure used in the experiment was fresh cow manure from a livestock farm. The basic properties of the cow manure were: moisture content 83.7%, dry matter 17.5%, and total organic matter 81.2%. Due to the high moisture content of the cow manure, it was combined with corn stalks for composting, with a corn stalk to fresh cow manure mass ratio of 3:10. The mixed materials were placed in a foam box with dimensions of 100cm long, 50cm wide, and 80cm high for composting. Ten treatment groups were set up: microbial agents prepared in Example 1 and Comparative Examples 1-9 were applied, respectively. The microbial agents were added at a rate of 1‰ of the mixed materials, and the compost piles were turned and mixed thoroughly on days 1, 3, 5, 7, 10, 13, 17, 21, 25, and 30 of composting. Samples were taken every morning, collecting samples from five directions and three depths in each pile and mixing them thoroughly. 0.1kg was collected from each pile, with three replicates.
[0040] Testing methods for each indicator: Temperature: The temperature of the pile is measured at 5 directions and 3 depths at the same time every day; Moisture content: The moisture content of compost should be determined according to the vacuum oven method in GB / T 8576-2010; Seed germination index: The germination index of compost seeds was determined according to the standard NY / T 525-2021. Water-soluble organic carbon: Refer to the potassium dichromate titration method in NY / T 525-2021; the lower the value, the better the degradation. Total nitrogen: Refer to the potassium dichromate titration method in NY / T 525-2021; Fecal coliform count: Determination method as specified in GB / T 19524.1-2004; Ascaris egg mortality rate: Refer to the determination method in GB / T 19524.2-2004; Moisture content, seed germination index, water-soluble organic carbon and total nitrogen, as well as the mortality rates of fecal coliform bacteria and Ascaris eggs were all measured after the composting process was completed. The above test data are shown in Table 3.
[0041] Table 3. Composting test results for each treatment group As shown in Table 3, after continuous high-temperature treatment, the microbial agent prepared in Example 1 of this invention almost completely killed Escherichia coli in the compost, and all Ascaris eggs also died. This indicates that the microbial agent prepared in this invention achieves the harmless treatment requirements after composting cow manure. Furthermore, the seed germination rate reached over 95%, indicating a high degree of manure composting. In addition, the compost after composting with the microbial agent of this invention had a low content of water-soluble organic matter, indicating that the organic matter was effectively decomposed. In contrast, in the comparative examples 1-9, where the composition of the agent was changed, the synergistic balance of the three bacteria—Bacillus thermophilus, Bacillus brucellosis, and Aspergillus chevallaris—was disrupted, leading to varying degrees of decreased composting efficiency. Therefore, all three functional strains selected in the microbial composting agent of this invention are indispensable.
[0042] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.
Claims
1. A compound microbial agent, characterized in that, The compound microbial agent includes *Bacillus thermophilus*, *Thermophyton floccosum*, and *Aspergillus chevallaris*; the strain number of *Bacillus thermophilus* is CGMCC1.3474; the strain number of *Thermophyton floccosum* is CGMCC1.8658; and the strain number of *Aspergillus chevallaris* is CGMCC3.12662. The preparation method of the compound microbial agent includes the following steps: (1) Thermoanaerobacterium thermosaccharolyticum and Thermus brokians were thawed and activated in LB solid medium, respectively, and then single colonies were inoculated into LB liquid medium to culture to OD 600 =0.6 to obtain seed liquid, which was inoculated into a fermenter at an amount of 10%, and when the effective viable cell count reached 1×10 9 CFU / mL, the culture was stopped to obtain two kinds of bacterial liquid; (2) After thawing Aspergillus chevalerate, activate and culture it in PDA solid medium for 5-7 days. Spores are scraped from the surface of the medium and inoculated into PDA liquid medium. Culture for 36-48 hours to prepare a spore suspension. Inoculate the fermenter with 10% of the spore suspension. When the effective viable count reaches 1×10⁻⁶, the spore suspension is inoculated into the fermenter. 9 When the concentration of CFU / mL is reached, stop the culture to obtain Aspergillus cheivae culture. (3) The bacterial suspensions of Bacillus thermophilus, Bacillus brucellus, and Aspergillus serrata prepared in step (1) and the bacterial suspensions of Aspergillus serrata in step (2) are mixed in a volume ratio of 1:1:1 and then freeze-dried into freeze-dried powder to obtain the final product, composite microbial agent.
2. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent is in powder form.
3. The application of the compound microbial agent according to claim 1, characterized in that, The compound microbial agent is used to accelerate the decomposition of livestock and poultry manure and improve the quality of compost.
4. The application of the compound microbial agent according to claim 3, characterized in that, The livestock and poultry manure mentioned is cow manure.
5. The application of the compound microbial agent according to claim 3, characterized in that, The mass ratio of the compound microbial agent to the feces is (1-3):1000.