Compound microbial agent as well as preparation method and application thereof

Abstract

The invention discloses a compound microbial agent as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. The compound microbial agent comprises geobacillus thermophilus, Thermus buchneri and Aspergillus shermangii, and the three screened strains ensure that manure is thoroughly decomposed by the strains in the change process of the temperature of a pile body, so that the thoroughly decomposing process is accelerated. Wherein the geobacillus thermophilus and the Thermus buchneri are thermophilic strains, so that the composting effect can still be performed when most microorganisms cannot perform normal physiological metabolism when the temperature of a pile body exceeds 50 DEG C. The three strains have a synergistic effect, complete-cycle coverage of the decomposition process is realized, various organic matters can be quickly decomposed, the manure decomposition degree is high, the seed germination index is as high as 95% or above, in addition, continuous high temperature is also beneficial to killing pathogenic bacteria, worm eggs and weed seeds in the manure, toxicity is reduced, and the transmission risk of pathogens is greatly reduced; and the purposes of harmless and resourceful treatment and utilization are achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a compound microbial agent and a preparation method and application thereof. BACKGROUND

[0002] Agricultural waste is a general term for waste discharged by agricultural production, agricultural product processing, livestock and poultry breeding industry and rural residents, including four categories of plant waste, animal waste, processing waste and rural and urban household garbage. With the rapid development of industrial and agricultural production and the rapid growth of population, these wastes are increasing year by year. In particular, with the rapid development of livestock and poultry breeding industry, especially with the change of breeding mode from scattered feeding of farmers to large-scale and intensive breeding farms, the unit land load of livestock is getting higher and higher, so that the pollution load of the local environment is getting larger and larger; livestock and poultry breeding farms are transferred from rural and pastoral areas to urban suburbs, resulting in the disconnection between farming and grazing, leading to the local large-scale concentration of breeding waste, which cannot be properly treated, such as a large amount of manure directly applied to the soil without harmless treatment, which exceeds the self-purification capacity of the soil, resulting in incomplete degradation and anaerobic decomposition, producing harmful substances such as malodorous substances and nitrite, causing changes in the composition and properties of the soil, destroying its original basic functions and reducing the soil quality, which not only causes great damage to the ecological environment, but also poses a serious threat to the health of animals and humans. In addition, if the manure is not treated in time and is randomly stacked, a large number of mosquitoes and flies will breed, and the pathogenic microorganisms and parasitic eggs in the manure will multiply rapidly and easily spread, worsening the surrounding environmental sanitation and even causing the spread of human and animal infectious diseases.

[0003] Composting is a relatively traditional biological fermentation technology. Initially, it uses the method of composting, and can be composted at natural temperature for 4-6 months. In order to accelerate composting and save the cost of turning, a ventilation pipe can be set at the bottom of the pile for forced or static ventilation, which can shorten the composting time. This method has low cost, but occupies a large area, takes a long time to process, and is easily affected by weather. By using the fermentation of microorganisms, the process of artificially promoting the transformation of degradable organic matter into stable humus can shorten the composting time from more than 100 days of natural composting to 30-50 days or even shorter time to achieve rapid composting.

[0004] At present, there are few studies on the treatment of cow dung, and there are many problems in the composting treatment of cow dung, such as high water content of manure, foul smell, and easy loss of ammonia nitrogen during the treatment process, and a large amount of pathogenic microorganisms and weed seeds in the manure, which will pose a threat to the environment. Therefore, it is urgent to develop a microbial agent for cow dung composting to promote cow dung composting and achieve the purpose of harmless treatment. SUMMARY

[0005] The present application aims to provide a kind of complex microbial inoculant, effectively composting livestock and poultry manure, with good composting effect, high seed germination index, effectively killing pathogenic bacteria and insect eggs in manure, reducing toxicity, significantly reducing the risk of pathogen transmission, achieving the purpose of harmless, resource utilization.

[0006] To achieve the above technical purposes, the technical scheme adopted by the present application is: A kind of complex microbial inoculant, the complex microbial inoculant includes Bacillus caldotenax, Thermus brockianus and Aspergillus sydowi;The Bacillus caldotenax is purchased from China General Microbiological Culture Collection Center, with preservation number CGMCC1.3474, and original preservation date September 11, 2003;The Thermus brockianus is purchased from China General Microbiological Culture Collection Center, with preservation number CGMCC1.8658, and original preservation date September 17, 2008;The Aspergillus sydowi is purchased from China General Microbiological Culture Collection Center, with preservation number CGMCC3.12662, and original preservation date July 10, 2008.The strains used in the present application can be purchased through the preservation center strain directory query, without repeating biological preservation.

[0007] Further, the complex microbial inoculant is a powder.

[0008] The present application provides a preparation method of complex microbial inoculant, comprising the following steps: (1) after thawing Bacillus caldotenax and Thermus brockianus, activate them in LB solid culture medium respectively, then inoculate single colonies into LB liquid culture medium respectively, culture to OD 600 =0.6 to obtain seed liquid, inoculate the seed liquid into fermentation tank at an amount of 10%, when the effective viable count reaches 1×10 9 CFU / mL, stop the culture, to obtain two kinds of bacterial liquid; (2) after thawing Aspergillus sydowi, activate and culture it in PDA solid culture medium for 5-7 days, inoculate spores on the surface of culture medium into PDA liquid culture medium to culture for 36-48h to prepare spore suspension, inoculate the spore suspension into fermentation tank at an inoculation amount of 10%, when the effective viable count reaches 1×10 9 CFU / mL, stop the culture, to obtain Aspergillus sydowi bacterial liquid; (3) mix the Bacillus caldotenax bacterial liquid, Thermus brockianus bacterial liquid prepared in step (1) and Aspergillus sydowi bacterial liquid in step (2) according to the volume ratio of 1:1:1, then freeze-dry into freeze-dried powder, to obtain the final product complex microbial inoculant.

[0009] The present application also provides the application of complex microbial inoculant, which is used for accelerating the composting of livestock and poultry manure and improving 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% to cultivate. 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 chevallis*; the strain number of *Bacillus thermophilus* is CGMCC1.3474; the strain number of *Thermophyton floccosum* is CGMCC1.8658; and the strain number of *Aspergillus chevallis* is CGMCC3.12662.

2. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent is in powder form.

3. The method for preparing the composite microbial agent according to any one of claims 1-2, characterized in that, 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% to cultivate. 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.

4. 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.

5. The application of the compound microbial agent according to claim 4, characterized in that, The livestock and poultry manure mentioned is cow manure.

6. The application of the compound microbial agent according to claim 4, characterized in that, The mass ratio of the compound microbial agent to the feces is (1-3):1000.

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