Preparation and fermentation method and application of compound microbial soil

Abstract

The invention relates to a preparation method of compound microorganism bacterium soil, microorganisms in the compound microorganism bacterium soil comprise bacillus subtilis YCBA0319 with the preservation number of CGMCC No.33006 and trichoderma longibrachiatum YCFA0522 with the preservation number of CGMCC No.41655. The preparation method comprises the following steps: selecting relatively clean clay or loam, and removing stones and plant residues; mixing the fine soil, the soybean meal and the cottonseed cake, stirring by using a double-shaft paddle type mixer, and spraying sterile water to adjust the water content; sub-packaging into polypropylene woven bags, sterilizing, and cooling for later use; preparing a seed solution of the bacillus subtilis YCBA0319; preparing a spore suspension of the trichoderma longibrachiatum YCFA0522; the bacillus subtilis is 2.5 * 10 < 9 > CFU / g, and the trichoderma longibrachiatum is 5.0 * 10 < 9 > CFU / g. According to the technical scheme, a granular structure is formed with soil in the fermentation process, so that an appropriate living microenvironment is provided for microorganisms, effective microbial communities are protected from being influenced by external adverse conditions, and the activity and function exertion of the effective microbial communities after field application are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a preparation method of a composite microbial soil, a fermentation method of a composite microbial soil, and an application of a composite microbial soil. Background Art

[0002] In recent years, problems such as soil quality decline, structural degradation, and reduction of organic matter content have become important factors restricting the sustainable development of agriculture. Long-term unreasonable fertilization, excessive use of chemical pesticides, and single tillage methods have led to the destruction of soil aggregate structure and the decline of microbial diversity, thereby reducing the water and fertilizer retention capacity of the soil, and further affecting the growth and yield of crops. To improve this situation, microbial inoculants, as a green and sustainable soil improvement means, have received extensive attention due to their multiple functions such as optimizing soil structure, promoting nutrient cycling, and enhancing the healthy growth of crops.

[0003] Bacillus subtilis and Trichoderma longibrachiatum are two types of functional microorganisms that have been relatively deeply studied in the field of agricultural microorganisms. Bacillus subtilis can improve the degradation efficiency of organic matter in the soil, enhance the availability of nutrients, and inhibit a variety of soil-borne pathogens through the competitive exclusion mechanism by secreting extracellular enzymes and secondary metabolites. Trichoderma longibrachiatum can promote the growth of crop roots, enhance plant stress resistance, and induce plant systemic resistance. Its metabolites, such as extracellular polysaccharides and organic acids, can promote the formation of soil aggregates, thereby improving the physical and chemical properties of the soil. In addition, these two strains have good compatibility in the soil ecosystem, and their combined action helps to significantly improve the soil health level.

[0004] Most traditional microbial inoculants adopt liquid fermentation technology, and due to the weak stress resistance of microorganisms, they often have low survival rates due to insufficient adaptability or weak competitiveness in complex soil environments, thus affecting the stability of soil improvement effects. Therefore, improving the adaptability of microbial inoculants has become the key to their application effects. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art or related technologies.

[0006] For this reason, the purpose of the present invention is to provide a preparation and fermentation method of a composite microbial soil and its application. By forming aggregate structures with the soil during the fermentation process, a suitable survival microenvironment is provided for microorganisms, protecting beneficial microbial communities from the influence of external adverse conditions, and ensuring their activity and function play after field application.

[0007] To achieve the above object, the technical solution of the first aspect of the present invention provides a method for preparing a composite microbial soil, wherein the microorganisms in the composite microbial soil include Bacillus subtilis YCBA0319 with a preservation number of CGMCC No. 33006 and Trichoderma longibrachiatum YCFA0522 with a preservation number of CGMCC No. 41655. The preparation method includes the following steps:

[0008] Select relatively clean clay or loam, crush it and pass it through a 2-mm sieve to obtain fine soil, and remove stones and plant residues;

[0009] Mix the fine soil, soybean meal, and cotton cake in a mass ratio of 90:6:4 with an error of ±0.5%, stir with a double-shaft paddle mixer at a rotation speed of 25 rpm for 15 minutes, and spray sterile water to adjust the water content to 40%-45%;

[0010] Pack it into polypropylene woven bags with an air permeability ≥ 200 L / m 2 ·s, 20 kg per bag, perform high-temperature sterilization on the sterilized bags at 121°C for 30 minutes, and cool to below 40°C for standby;

[0011] Preparation of Bacillus subtilis YCBA0319 seed liquid: Streak the preserved strain on an LB agar plate and culture it at 37°C for 24 h;

[0012] Pick a single colony and inoculate it into an LB liquid medium containing 0.5% soybean meal leachate, culture it at 37°C and 180 rpm for 18 h until the bacterial density reaches 2.8×10 9 CFU / mL;

[0013] Preparation of Trichoderma longibrachiatum YCFA0522 spore suspension: Scrape the spores on the PDA slant and dilute them with sterile water containing 0.05% Tween-80 to 1×10 8 spores / mL;

[0014] Inoculate the spore liquid into sterilized cotton cake powder with a water content of 50%, and culture it at 28°C for 72 h until the mycelium coverage rate ≥ 90%;

[0015] Mix them according to a viable bacteria ratio of 1:2, wherein, Bacillus subtilis is 2.5×10 9 CFU / g, and Trichoderma longibrachiatum is 5.0×10 9 CFU / g.

[0016] The technical solution of the second aspect of the present invention provides a fermentation method for the composite microbial soil prepared by the technical solution of the first aspect of the present invention, including the following steps:

[0017] The composite microbial soil is mixed with the sterilized raw materials in a mixer according to a total inoculation amount of 5% to form an inoculated material, and the inoculated material is stacked into a strip stack with a bottom width of 1.2 m and a height of 0.8 m;

[0018] On the first day, a transparent polyethylene film with a thickness of 0.1 mm and a light transmittance of ≥90% is covered on the strip stack to promote temperature rise;

[0019] From the second day: the transparent polyethylene film is replaced with a waterproof non-woven fabric with an air permeability of 150 L / m 2 ·s, and 5 layers of weed-proof cloth are covered on the outer layer;

[0020] Low-temperature regulation: when the ambient temperature < 20°C, a heat-insulating cotton quilt (thermal conductivity ≤ 0.035 W / m·K) is covered;

[0021] Turning pile management: turning the pile once every 4 days;

[0022] Monitor the fermentation state in real time. When the inoculated material meets the following determination indexes, the inoculated material is stored after fermentation:

[0023] Physical and chemical indexes:

[0024] The temperature naturally drops to the ambient temperature ±2°C;

[0025] The proportion of water-stable aggregates (>0.25 mm) ≥ 35% (determined by wet sieving method);

[0026] Biological indexes:

[0027] The viable bacteria count ≥ 1×10 8 CFU / g (plate counting method);

[0028] Apparent characteristics:

[0029] The surface of the pile is covered with white hyphae (the coverage rate of Trichoderma hyphae ≥ 80%);

[0030] The material forms lumps and becomes loose and granular after being gently tapped.

[0031] In the above technical solution, preferably, storing the inoculated material includes:

[0032] Performing a drying treatment on the inoculated material, spreading the thickness ≤ 20 cm, drying in the shade at 25-30°C until the water content ≤ 15% and the relative humidity ≤ 40% to prevent spore germination;

[0033] Using an aluminum foil composite bag with an oxygen permeability ≤ 0.5 cm 3 / m 2 ·day for vacuum packaging and storage, storage conditions: storing in the dark at 4-10°C, shelf life ≥ 6 months.

[0034] The technical solution of the third aspect of the present invention provides an application of the composite microbial bacterial soil prepared by the technical solution of the first aspect of the present invention in improving soil organic matter or promoting the formation of soil aggregates.

[0035] In the above technical solution, preferably, the form of the application is to apply it into the soil by broadcasting and plowing.

[0036] Compared with the prior art, the advantages of the preparation and fermentation method of the composite microbial bacterial soil provided by the present invention and its application are as follows: In view of the problems of low content of soil organic matter, poor aggregate structure of a large number of cultivated lands in China at present, and poor field application effect of microbial inoculants, the present invention provides a solid fermentation method of composite microbial bacterial soil based on Bacillus subtilis YCBA0319 and Trichoderma longibrachiatum YCFA0522. By optimizing the strain ratio and culture conditions, the survival rate and function of the inoculant in the soil are enhanced. The application of this inoculant can significantly increase the content of soil organic matter, promote the formation of soil aggregates, enhance the survival ability of microorganisms in the field, thereby improving soil fertility and crop yield. This technology provides a green and efficient soil improvement and crop yield increase solution for modern agriculture.

[0037] Biological preservation description:

[0038] Bacillus subtilis YCBA0319 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 9, 2024, with the deposit number CGMCC No. 33006. The address of the depositary institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0039] Trichoderma longibrachiatum YCFA0522 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 9, 2024, with the deposit number CGMCC No. 41655. The address of the depositary institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Description of the drawings

[0040] The above and / or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, where:

[0041] Figure 1 Shows the completed fermentation diagram of the composite microbial bacterial soil. Detailed implementation manners

[0042] In order to more clearly understand the above objects, features, and advantages of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other.

[0043] In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the limitations of the specific embodiments disclosed below.

[0044] Embodiment 1 of the present invention provides a fermentation method.

[0045] Embodiment 2 of the present invention provides an application method.

[0046] Embodiment 1: Fermentation method

[0047] Raw material pretreatment

[0048] Select relatively clean clay or loam, crush it and pass through a 2 mm sieve to sieve it into fine soil, removing stones and plant residues;

[0049] Soybean meal: produced in Shandong, crude protein ≥ 46%, moisture ≤ 10%, crushed to 0.6 - 0.8 mm by a hammer mill;

[0050] Cotton cake: treated with phenol removal in Xinjiang (free gossypol content 0.015%), crushed to 1 mm, dried at 60 °C to moisture ≤ 8%.

[0051] Mixed sterilization

[0052] Raw material ratio

[0053] Weigh according to the mass ratio of fine soil: soybean meal: cotton cake = 90:6:4 (error ± 0.5%).

[0054] Mixed moisture adjustment

[0055] Stir with a double-shaft paddle mixer (rotation speed 25 rpm) for 15 minutes;

[0056] Spray sterile water to adjust the water content to 40% - 45% (measured value 42.3 ± 0.8%).

[0057] Sterilization treatment

[0058] Pack into polypropylene woven bags (air permeability ≥ 200 L / m 2 ·s), 20 kg per bag;

[0059] Use an autoclave with the temperature set to 121°C (i.e., the temperature of the autoclave is set to 121°C, and the pressure of the autoclave is not adjusted, ranging from 0.1 MPa to 0.15 MPa higher than the atmospheric pressure) to perform high-temperature sterilization on the sterilization bag for 30 minutes, and cool it to below 40°C for standby.

[0060] Alternative: Cover with a PE film and expose to the sun for 5 days, with a cumulative effective accumulated temperature of ≥50°C for 120 hours.

[0061] Strain activation and compounding

[0062] Bacillus subtilis YCBA0319:

[0063] Seed liquid preparation: Streak the preserved strain on an LB agar plate and culture at 37°C for 24 h;

[0064] Liquid expansion culture: Pick a single colony and inoculate it into an LB liquid medium containing 0.5% soybean meal leaching solution, and culture it at 37°C and 180 rpm for 18 h until the bacterial density reaches 2.8×10 9 CFU / mL;

[0065] Trichoderma longibrachiatum YCFA0522:

[0066] Sporulation suspension preparation: Scrape the spores on the PDA slant and dilute them with sterile water containing 0.05% Tween-80 to 1×10 8 spores / mL;

[0067] Solid pre-culture: Inoculate the spore suspension into sterilized cotton cake powder (water content 50%) and culture at 28°C for 72 h until the mycelium coverage rate ≥90%;

[0068] Strain compounding: Mix in a live bacteria ratio of 1:2 (Bacillus subtilis 2.5×10 9 CFU / g, Trichoderma longibrachiatum 5.0×10 9 CFU / g).

[0069] Solid-state fermentation control

[0070] The strain is mixed with the sterilized raw materials in a blender at a total inoculation amount of 5%.

[0071] Pile body construction: Stack the inoculated materials into a strip stack with a bottom width of 1.2 m and a height of 0.8 m.

[0072] Covering material:

[0073] On the first day, use a transparent polyethylene film (thickness 0.1 mm, light transmittance ≥90%) to promote temperature rise;

[0074] From the second day: Replace it with a breathable and waterproof non-woven fabric (breathability rate 150 L / m 2 ·s), and cover the outer layer with 5 layers of weed-proof cloth;

[0075] Low-temperature regulation: When the environmental temperature is < 20°C, cover with a heat-insulating cotton quilt (thermal conductivity ≤ 0.035 W / m·K).

[0076] Process monitoring:

[0077] Temperature: Record the temperatures of the upper, middle, and lower layers of the stack every hour, and control the central temperature at 38 - 42°C.

[0078] Oxygen: Use a portable oxygen analyzer (accuracy ±0.5%) to detect the pore oxygen concentration in the stack and maintain it at 8 - 12%.

[0079] Turning management: Turn the stack once every 4 days, and appropriately supplement water during turning.

[0080] Judgment of fermentation end point:

[0081] Physical and chemical indicators:

[0082] The temperature naturally drops to the environmental temperature ±2°C;

[0083] The proportion of water-stable aggregates (> 0.25 mm) ≥ 35% (determined by wet sieving method);

[0084] Biological indicators:

[0085] The number of viable bacteria ≥ 1×10 8 CFU / g (plate counting method);

[0086] Apparent characteristics:

[0087] The surface of the stack is covered with white mycelia (the coverage rate of Trichoderma mycelia ≥ 80%);

[0088] The material forms into lumps and becomes loose and granular after gently tapping.

[0089] Preservation of fungus soil

[0090] Drying treatment:

[0091] The paving thickness ≤ 20 cm, and air-dry at 25 - 30°C until the water content ≤ 15%;

[0092] Under light-proof conditions, the relative humidity ≤ 40% to prevent spore germination.

[0093] Encapsulation and storage:

[0094] Use an aluminum foil composite bag (oxygen transmission rate ≤ 0.5 cm 3 / m 2 ·day) for vacuum packaging;

[0095] Storage conditions: Store in the dark at 4 - 10°C, and the shelf life ≥ 6 months.

[0096] The completed fermentation diagram of the compound microbial bacterial soil is as Figure 1 shown below.

[0097] Example 2: Application of the fermented microbial bacterial soil

[0098] A field experiment was conducted using a compound microbial inoculant in the corn planting area of the Shuitou Farm in Xia County, Yuncheng City. The previous crop was wheat, and the straw was returned to the field in full amount. The compound microbial bacterial soil and 15-15-15 compound fertilizer were applied into the soil by broadcasting and plowing. Four groups were set for the application amount of the bacterial soil, namely CK: 0 kg / mu, T1: 100 kg / mu, T2: 150 kg / mu, and T3: 200 kg / mu; the application amount of compound fertilizer in each group was 50 kg / mu. After the corn was harvested, the organic matter content and water-stable aggregates of each group were measured.

[0099] Table 1 Effects of microbial bacterial soil on the number of soil aggregates and organic matter content

[0100] Treatment Proportion of aggregates ≥ 0.25 mm (%) Organic matter content (g / kg) CK 12.51 2.35 T1 23.54 2.86 T2 25.78 2.78 T3 26.47 2.80

[0101] From the change of soil water-stable aggregates, in the CK group: the proportion of aggregates ≥ 0.25 mm was only 12.51%, the soil structure was loose, and the anti-erosion ability was weak. In the T1-T3 groups: with the increase of the application amount of the bacterial soil, the proportion of aggregates increased significantly (T1: 23.54% → T3: 26.47%), and the T3 group increased by 111.6% compared with CK, indicating that microbial metabolites (such as exopolysaccharides) effectively bonded soil particles to form a stable aggregate structure. The difference between T2 (150 kg / mu) and T3 (200 kg / mu) was small (25.78% vs 26.47%), suggesting that the aggregate formation efficiency tended to be saturated after the application amount of the bacterial soil exceeded 150 kg / mu.

[0102] From the change of soil organic matter, in the CK group: the organic matter content was 2.35%, and the straw returning to the field did not significantly improve the humification efficiency. In the T1 group: the organic matter content reached 2.86% (21.7% higher than CK), and the microbial community accelerated the decomposition of straw and synthesized humic acid. In the T2-T3 groups: the organic matter content was slightly lower than that of T1 (2.78 - 2.80%), which might be due to the excessive consumption of some carbon sources by microbial respiration caused by the excessive bacterial soil.

[0103] The application amount of the microbial bacterial soil was positively correlated with the improvement of soil aggregates, and the recommended field application amount was 150 kg / mu (T2 group).

[0104] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a composite microbial soil, characterized in that: The microorganisms in the composite microbial soil include Bacillus subtilis YCBA0319, with a preservation number of CGMCC No.33006, and Trichoderma longifolia YCFA0522, with a preservation number of CGMCC No.41655. The preparation method includes the following steps: Choose relatively clean clay or loam, crush it and pass it through a 2mm sieve to make fine soil, remove stones and plant residues; Fine soil, soybean meal and cotton cake were mixed in a mass ratio of 90:6:4 with an error of ±0.5%, stirred in a double-shaft paddle mixer at a speed of 25 rpm for 15 minutes, and sprayed with sterile water to adjust the moisture content to 40%-45%; Packed into polypropylene woven bags, air permeability ≥ 200L / m 2 ·s, 20kg per bag, sterilize the sterilization bag at 121℃ for 30 minutes, cool to below 40℃ for later use; Preparation of Bacillus subtilis YCBA0319 seed solution: streak the preserved strain on LB agar plate and culture at 37°C for 24 h; A single colony was selected and inoculated into LB liquid medium containing 0.5% soybean meal extract, and cultured at 37°C and 180 rpm for 18 h until the bacterial density reached 2.8×10 8 CFU / mL; Preparation of spore suspension of Trichoderma longifolia YCFA0522: Scrape spores from PDA slant and dilute to 1 × 10 with sterile water containing 0.05% Tween-80. 8 Spores / mL; Inoculate the spore liquid to a moisture content of 50% to form sterilized cotton cake powder, and culture at 28°C for 72h until the mycelium coverage is ≥90%; The live bacteria ratio was 1:2, of which 2.5×10 9 CFU / g, Trichoderma longifolia 5.0×10 9 CFU / g.

2. A fermentation method of the composite microbial soil prepared by the preparation method according to claim 1, characterized in that: The following steps are involved: The composite microbial soil is mixed with the sterilized raw materials in a mixer according to a total inoculation amount of 5% to form an inoculation material, and the inoculation material is piled into a strip with a bottom width of 1.2m and a height of 0.8m; On the first day, cover the windrow with a transparent polyethylene film with a thickness of 0.1 mm and a light transmittance of ≥ 90% to promote temperature rise; From the second day: the transparent polyethylene film was replaced with a film with an air permeability of 150L / m 2 ·s waterproof non-woven fabric, with 5 layers of anti-weed fabric on the outside; Low temperature control: When the ambient temperature is less than 20℃, cover with a thermal quilt (thermal conductivity ≤ 0.035W / m·K); Compost turning management: Turn the compost once every 4 days; Monitor the fermentation status in real time. When the inoculated material meets the following criteria, stop fermentation and save the inoculated material: Physical and chemical indicators: The temperature naturally drops to the ambient temperature ±2°C; The proportion of water-stable aggregates (>0.25mm) is ≥35% (determined by wet sieving method); Biological indicators: Viable count ≥1×10 8 CFU / g (plate count method); Appearance characteristics: The surface of the pile is covered with white hyphae (Trichoderma hyphae coverage ≥ 80%); The material is in lumps and becomes loose and granular after tapping.

3. The fermentation method according to claim 2, characterized in that Preserving the inoculated material comprises: The inoculated material is dried, with a paving thickness of ≤20 cm, and is dried in the shade at 25-30° C. to a moisture content of ≤15% and a relative humidity of ≤40% to prevent spore germination; Use oxygen permeability ≤ 0.5cm 3 / m 2 ·Day aluminum foil composite bag is vacuum packed and stored at 4-10℃ away from light. The shelf life is ≥ 6 months.

4. Use of the composite microbial soil as claimed in claim 1 in increasing soil organic matter or promoting soil aggregate formation.

5. The use according to claim 2, characterized in that: The application form is by broadcasting and tilling into the soil.

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