Preparation method of agricultural liquid microbial agent and microbial agent

Abstract

The invention belongs to the technical field of agricultural microbial agents, and relates to an agricultural liquid microbial agent preparation method and a microbial agent, the method comprises the following steps: mixing bacillus amyloliquefaciens and trichoderma reesei, and carrying out primary aerobic fermentation; mixing a product subjected to primary aerobic fermentation with lactobacillus acidophilus subjected to expanding culture, and carrying out secondary micro-aerobic fermentation; and carrying out membrane separation treatment on the product subjected to secondary microaerobic fermentation, and concentrating the bacterial liquid. According to the microbial agent, through the synergistic effect of multiple strains, the inhibition rate of soil-borne pathogenic bacteria is increased, the occurrence rate of crop rhizosphere diseases is reduced, and then the yield and quality of crops are synergistically optimized. High-activity flora is reserved through gradient fermentation and membrane separation technologies, the continuous colonization capacity of thalli in deep soil is maintained in combination with biochar, a long-term stable field protection barrier can still be formed through flora interaction even if mechanical turning is not needed, and therefore disease prevention and control are achieved under the no-tillage or minimal-tillage condition.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microbial agents, and in particular to a preparation method of a liquid agricultural microbial agent and the microbial agent. Background Art

[0002] In modern agricultural production, the "bio-organic" synergistic model, combining microbial inoculants with organic fertilizers, is gaining widespread attention to achieve sustainable agricultural development and improve soil fertility and crop health. This model utilizes beneficial microorganisms in microbial inoculants, such as rhizobia, nitrogen-fixing bacteria, and Bacillus, in combination with organic fertilizers. This model not only provides abundant nutrients for crops, but also improves soil structure and inhibits harmful pathogens through microbial activity, thereby increasing crop yield and quality.

[0003] However, in actual application, this model faces many technical difficulties. Most of the microorganisms in existing microbial agents are aerobic microorganisms, and their growth and metabolism require an adequate supply of oxygen. They rely on aerobic metabolism to inhibit pathogens, but they cannot spread to deep soil. Because the oxygen concentration in deep soil is lower than 0.1% and there are problems such as salinization and heavy metal pollution, the activity of aerobic bacteria drops sharply or even becomes inactivated. At present, existing technologies often rely on mechanical turning to force the dispersion of microbial agents, such as using a trough-type turning machine to turn the land. It not only destroys the soil structure, resulting in decreased air permeability and imbalance of the microbial community. It also increases the labor links of farmers, consumes a lot of manpower and material resources, increases production costs, reduces production efficiency, and makes it difficult for many farmers to bear. Summary of the Invention

[0004] Given that aerobic bacteria in existing microbial agents become ineffective in deep soil due to lack of oxygen, the conventional reliance on mechanical compost turning increases the labor time of farmers.

[0005] The first aspect of the present application provides a method for preparing a liquid microbial agent for agricultural use, comprising: Step 1: inoculating Bacillus amyloliquefaciens, Trichoderma reesei, and Lactobacillus acidophilus into their respective culture media for cultivation, and obtaining corresponding revived bacterial liquids; Step 2: performing gradient expansion culture on the revived Bacillus amyloliquefaciens solution obtained in step 1; performing solid-state pre-culture on the revived Trichoderma reesei solution; and performing anaerobically expanding culture on the revived Lactobacillus acidophilus solution; Step 3: Bacillus amyloliquefaciens obtained in step 2 and Trichoderma reesei are mixed in a ratio of (8-10):1 and subjected to primary aerobic fermentation, with glucose and magnesium sulfate added during the fermentation; Step 4: The product of the primary aerobic fermentation is mixed with the expanded Lactobacillus acidophilus for secondary microaerobic fermentation, during which catalase and glutathione are added; Step 5: The product of the secondary microaerobic fermentation is subjected to membrane separation treatment to desalinate and concentrate the bacterial liquid; Step 6: Add biochar and citric acid to the concentrated bacterial solution and homogenize.

[0006] Furthermore, in step 1, Trichoderma reesei spores are inoculated into a liquid culture medium, the temperature is controlled at 25-30° C., shaking culture is carried out for 30-48 hours, and the mycelial suspension is collected to obtain a Trichoderma reesei revived bacterial liquid.

[0007] Furthermore, in step 2, the solid-state pre-culture includes inoculating the revived Trichoderma reesei liquid obtained in step 1 into a rice husk substrate, transferring the pre-cultured mycelium to a potato glucose liquid culture medium after the mycelium is dispersed, and adding maltose during the fermentation process.

[0008] Furthermore, in step 2, the gradient expansion culture comprises transferring the revived Bacillus amyloliquefaciens liquid obtained in step 1 to a liquid culture medium containing soybean meal hydrolyzate and sucrose at an inoculum rate of 1%-2%, controlling the temperature at 25-30° C., and culturing for 10-14 hours to obtain a first-level seed liquid; Transfer the first-level seed liquid to the fermentation tank culture medium at an inoculation rate of 0.3%-0.5%, culture for 12-16 hours, and obtain the second-level seed liquid.

[0009] Furthermore, in step 2, the anaerobic expansion includes transferring the revived Lactobacillus acidophilus liquid obtained in step 1 into the culture medium at an inoculation rate of 1-2% and placing it in an anaerobic tank, injecting nitrogen to an oxygen concentration of ≤0.1%, and culturing it at 35-40°C for 18-24 hours.

[0010] Further, membrane separation treatment is performed, wherein molecules with a molecular weight cutoff greater than 100 kDa are retained by an ultrafiltration membrane, and molecules with a molecular weight cutoff greater than 500 Da are retained by a nanofiltration membrane, and desalination treatment is performed.

[0011] Furthermore, after adding the biochar, citric acid with a mass fraction of 0.2%-0.3% was added in multiple times.

[0012] Furthermore, in step 2, a feed treatment is performed during the gradient expansion process; During the first-level seed liquid culture stage of Bacillus amyloliquefaciens, add 0.1%-0.3% glucose and 0.01%-0.05% magnesium sulfate by mass when the fermentation is longer than 12 hours, and maintain the dissolved oxygen ≥5%; During the secondary seed liquid culture stage, when the fermentation time was greater than 24 h, 0.1% soybean meal hydrolyzate was added to induce the secretion of secondary metabolites.

[0013] Furthermore, in step 6, the biochar and citric acid are added by pre-treating the biochar, activating the rice husk charcoal at 600°C-630°C for 2-3 hours, grinding it to 100-200 mesh, and adding 1%-2% by mass; Add 0.2%-0.3% citric acid in 3-5 times, with at least 10 minutes between each addition, and control the pH to 6.2±0.1.

[0014] The second aspect of the present application mentions a liquid microorganism for agricultural use, which is prepared using the method for preparing a liquid microbial agent for agricultural use in the first aspect.

[0015] The beneficial effects of this application are:

[0016] The present application discloses a method for preparing a liquid microbial agent for agricultural use and a microbial agent, wherein the method comprises: inoculating Bacillus amyloliquefaciens, Trichoderma reesei, and Lactobacillus acidophilus into respective culture media for cultivation, and obtaining corresponding revived bacterial liquids; subjecting the revived Bacillus amyloliquefaciens bacterial liquid to gradient expansion culture; subjecting the revived Trichoderma reesei bacterial liquid to solid-state pre-culture; subjecting the revived Lactobacillus acidophilus bacterial liquid to anaerobically expansion culture; mixing Bacillus amyloliquefaciens and Trichoderma reesei for primary aerobic fermentation, and adding glucose and magnesium sulfate during fermentation; mixing the product of the primary aerobic fermentation with the expanded Lactobacillus acidophilus for secondary microaerobic fermentation, and adding catalase and glutathione during fermentation; subjecting the product of the secondary microaerobic fermentation to membrane separation treatment, desalting and concentrating the bacterial liquid; adding biochar and citric acid to the concentrated bacterial liquid, and homogenizing it.

[0017] This application systematically solves technical bottlenecks through the synergistic effect of phased gradient oxygen regulation and functional enhancement strategies: To address the problem of aerobic bacteria failing in deep hypoxia, the bacterial community is rapidly multiplied through primary aerobic fermentation, combined with secondary microaerobic fermentation to establish a bacterial community oxygen demand gradient, allowing Bacillus amyloliquefaciens to adapt to low-oxygen environments through facultative metabolism and achieve functional survival in deep soils; at the same time, a dual slow-release system is formed by membrane separation and concentration of bacterial agents and biochar addition, combined with citric acid to regulate the soil microenvironment pH, significantly extending the colonization period of bacterial agents in deep soils, replacing traditional compost turning operations and fundamentally solving the dependence on mechanical compost turning. Through two-stage oxygen gradient fermentation to construct a low-oxygen-tolerant bacterial community, supplemented by an antioxidant enzyme system to maintain cell membrane stability, ultimately achieving the sustained metabolic function and survival ability of aerobic bacteria in deep anoxic soils. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0019] Figure 1This is a process flow chart of a method for preparing a liquid microbial agent for agricultural use mentioned in the present invention; Figure 2 This is a schematic diagram of the dynamic changes in the survival rate of Bacillus in the field in the method for preparing a liquid microbial agent for agricultural use mentioned in the present invention. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0021] For this purpose, refer to Figure 1 The first aspect of the present application relates to a method for preparing a liquid microbial agent for agricultural use, comprising: Step 1: Bacillus amyloliquefaciens, Trichoderma reesei, and Lactobacillus acidophilus are inoculated into their respective culture media for cultivation, and corresponding revived bacterial liquids are obtained respectively.

[0022] Exemplarily, Bacillus amyloliquefaciens, Trichoderma reesei, and Lactobacillus acidophilus are inoculated into LB medium, PDA liquid medium, and MRS medium, respectively.

[0023] The method involves inoculating Trichoderma reesei spores into a liquid culture medium, controlling the temperature at 25-30°C and shaking the culture for 30-48 hours, collecting the mycelial suspension, and obtaining a Trichoderma reesei revived culture medium. Bacillus amyloliquefaciens is then inoculated, supplemented with 0.1% glucose, and shaken at 200 rpm to accelerate the recovery of the cells from a dormant state.

[0024] It's important to note that LB medium (Luria-Bertani) contains tryptone, yeast extract, and NaCl, providing nitrogen and carbon sources to support the rapid resurgence of Bacillus amyloliquefaciens and the secretion of secondary metabolites. PDA liquid medium (potato dextrose) contains potato extract (providing cellulose to induce enzyme secretion) and glucose (a carbon source) to promote the dispersion of Trichoderma reesei mycelium and chitinase synthesis. MRS medium (Man, Rogosa, Sharpe) contains beef extract, yeast extract, and Tween 80, specifically for the cultivation of lactic acid bacteria. It maintains a low pH environment (4.5-5.0), inhibiting contaminants and promoting lactic acid secretion.

[0025] Bacillus amyloliquefaciens quickly revived in LB medium, providing highly active seed liquid for subsequent fermentation; the mycelium dispersion of Trichoderma in PDA medium was improved (viscosity reduced by 40%), enhancing subsequent enzyme production capacity; Lactobacillus produced lactic acid in MRS medium, inhibiting miscellaneous bacteria and creating an acidic environment for mixed fermentation.

[0026] For example, Bacillus amyloliquefaciens can be supplemented with 0.1% glucose to accelerate metabolism under 200 rpm shaking conditions, increase the number of viable bacteria, and provide highly active seeds for subsequent fermentation.

[0027] The tryptone in LB medium contains tryptophan, which induces Bacillus amyloliquefaciens to synthesize lipopeptide antibiotics and enhances its antibacterial ability.

[0028] After Trichoderma reesei is cultured in PDA liquid, the viscosity of the Trichoderma reesei fermentation broth is reduced, the degree of mycelial fragmentation is improved, and the contact area during subsequent co-culture with Bacillus is increased, thereby improving the synergistic antibacterial efficiency.

[0029] The lactic acid produced by Lactobacillus acidophilus effectively inhibits miscellaneous bacteria and provides a clean microenvironment for subsequent mixed fermentation.

[0030] Step 2: performing gradient expansion culture on the revived Bacillus amyloliquefaciens solution obtained in step 1; performing solid-state pre-culture on the revived Trichoderma reesei solution; and performing anaerobic expansion culture on the revived Lactobacillus acidophilus solution.

[0031] In the present technical solution, in step 2, the gradient expansion culture comprises transferring the revived Bacillus amyloliquefaciens solution obtained in step 1 to a liquid culture medium containing soybean meal hydrolyzate and sucrose at an inoculum rate of 1%-2%, controlling the temperature at 25-30°C, and culturing for 10-14 hours to obtain a first-level seed solution; Transfer the first-level seed liquid to the fermentation tank culture medium at an inoculation rate of 0.3%-0.5%, culture for 12-16 hours, and obtain the second-level seed liquid.

[0032] In this technical solution, in step 2, a feed treatment is performed during the gradient expansion process; During the primary seed liquid culture stage of Bacillus amyloliquefaciens, 0.1%-0.3% glucose and 0.01%-0.05% magnesium sulfate were added with a mass fraction of 0.1%-0.3% when the fermentation was longer than 12 hours to maintain the dissolved oxygen ≥5%.

[0033] It should be noted that Bacillus amyloliquefaciens has a high demand for carbon sources during the exponential growth phase. Supplementing glucose can alleviate the carbon source limitation and prolong the logarithmic growth phase. The high sugar environment promotes Bacillus to secrete surfactant, thereby improving the antibacterial rate.

[0034] It should be noted that magnesium ions It is a cofactor of ATPase and protease. A concentration of 0.01%-0.05% can increase the metabolic efficiency of bacteria by 20%. Magnesium ions stabilize the cell membrane structure and reduce the interference of dissolved oxygen fluctuations on the respiratory chain.

[0035] It should be noted that when dissolved oxygen is ≥5%, Bacillus mainly metabolizes aerobically, avoiding the pH drop caused by acetic acid accumulation under anaerobic conditions and maintaining the pH at 6.5-6.8.

[0036] Adding glucose and magnesium sulfate in stages can extend the logarithmic growth phase and increase the number of live bacteria.

[0037] In the secondary seed liquid culture stage, when the fermentation is more than 24 hours, adding 0.1% soybean meal hydrolyzate can induce the secretion of chitinase and β-1,3-glucanase.

[0038] It should be noted that soybean meal hydrolyzate provides plant-derived amino acids, such as glutamine and aspartic acid, which relieve nitrogen source limitations and trigger secondary metabolic pathways; while the oligopeptides in the hydrolyzate act as signal molecules to activate the chitinase gene expression of Trichoderma and further enhance enzyme activity.

[0039] It should be noted that the chitinase secreted by Trichoderma reesei degrades chitin in soybean meal hydrolysate to produce reducing sugars such as glucose and mannose, which promote the production of surfactant by Bacillus.

[0040] It should be noted that secondary metabolites, such as glucanase, can enhance the bacteria's ability to degrade soil fiber, improve field colonization efficiency, and enhance stress resistance.

[0041] It should be noted that the addition of glucose must be completed before the dissolved oxygen begins to decrease (<5%) to avoid metabolic imbalance; soybean meal hydrolyzate must be added after the pH of the fermentation liquid stabilizes (6.0-6.5) to prevent acid inhibition.

[0042] Glucose can be added in batches, such as 0.2% after 12 hours and 0.1% after 18 hours, to avoid excess carbon source leading to acetic acid accumulation; The concentration of magnesium sulfate must be strictly controlled below 0.05% to prevent excessive inhibition of the respiratory chain by magnesium ions.

[0043] For example, a dissolved oxygen electrode can be used for real-time feedback, and a stirring or aeration system can be linked to maintain dissolved oxygen ≥5%.

[0044] This application achieves precise regulation of metabolic pathways by adding glucose / magnesium sulfate and soybean meal hydrolyzate in stages, prolongs the logarithmic growth period, and increases the number of viable bacteria by 30%; strengthens secondary metabolites, and synergistically improves enzyme activity and antibiotic production.

[0045] This application provides a standardized solution for the preparation of high-density, highly functional microbial agents through the interaction of dynamic feeding and metabolites. Soybean meal hydrolysate provides small peptides and amino acids to Bacillus amyloliquefaciens, inducing the synthesis of secondary metabolites. Sucrose acts as a slow-release carbon source to mitigate the glucose effect. Furthermore, soybean meal hydrolysate provides a plant-derived nitrogen source, promoting the secretion of lipopeptide antibiotics by Bacillus.

[0046] In the present technical solution, in step 2, the solid-state pre-culture includes inoculating the revived Trichoderma reesei liquid obtained in step 1 into the rice husk matrix, transferring the pre-cultured mycelium to a potato glucose liquid culture medium after the mycelium is dispersed, and adding maltose during the fermentation process.

[0047] In this technical solution, Trichoderma reesei is inoculated into a rice husk matrix. For example, the rice husk matrix is 80% rice husk and 20% bran, with a particle size of 100-200 mesh and a controlled humidity of 70%. The rough surface of the rice husk matrix stimulates branching of Trichoderma reesei hyphae through physical friction, increasing biomass. The bran provides lignocellulose, a supplemental nitrogen source, promotes chitinase secretion, pre-activates the cellulase system, and enhances enzyme activity during subsequent liquid fermentation.

[0048] Solid-state pre-cultivation provides physical protection. Furthermore, the rice hull matrix induces the dispersion of Trichoderma mycelium, reduces its aggregation rate, and enhances its ability to physically encapsulate pathogens. Inoculating the revived Trichoderma reesei culture medium with the rice hull matrix for solid-state pre-cultivation improves mycelial dispersion. After transfer to liquid culture, mycelial biomass increases, enhancing physical inhibition of pathogens. Adding maltose during fermentation prolongs the logarithmic growth phase and increases chitinase production.

[0049] In the present technical solution, in step 2, the anaerobic expansion includes transferring the revived Lactobacillus acidophilus liquid obtained in step 1 into the culture medium at an inoculation rate of 1-2% and placing it in an anaerobic tank, injecting nitrogen to an oxygen concentration of ≤0.1%, and culturing it at 35-40°C for 18-24 hours.

[0050] It should be noted that Lactobacillus acidophilus produces lactic acid (concentration ≥ 1.5%) under hypoxic conditions, which synergizes with the antibiotics of Bacillus to inhibit bacteria; Lactobacillus metabolites chelate heavy metals , reducing soil toxicity.

[0051] It should be noted that nitrogen oxygen control (≤0.1%) simulates a strictly anaerobic environment, promotes the production of extracellular polysaccharides (EPS) by Lactobacillus acidophilus, and enhances the protective effect of bacterial cell wrapping during subsequent co-culture with Bacillus.

[0052] For example, nitrogen was injected at a rate of 0.5 L / min to reduce the oxygen concentration in the anaerobic jar to ≤ 0.1%, simulating anoxic conditions deep in the soil. Under hypoxic conditions, Lactobacillus acidophilus switches from aerobic respiration to fermentative metabolism, prioritizing carbon sources for EPS synthesis over ATP production. Under anaerobic conditions, Lactobacillus acidophilus secretes EPS (e.g., rhamnosyl lactic acid glycans). Simultaneously, the EPS forms a slime layer around the spores, mitigating damage to the spores from salt stress, high temperatures, and ultraviolet radiation.

[0053] At the same time, the colonization rate of EPS-wrapped Bacillus in the soil is improved and its stress resistance is enhanced; further, the two form a metabolic mutualism: the sugars in EPS are utilized by Bacillus, promoting its production of surfactant.

[0054] Step 3: The Bacillus amyloliquefaciens obtained in step 2 is mixed with Trichoderma reesei for primary aerobic fermentation, and glucose and magnesium sulfate are added during the fermentation.

[0055] The ratio of Bacillus amyloliquefaciens to Trichoderma reesei mycelium is (8-10):1. The mycelial network of Trichoderma reesei envelops Bacillus amyloliquefaciens, enhancing the bacterial community's ability to survive in high-salt environments and significantly increasing its survival rate. Bacillus amyloliquefaciens secretes surfactant, a bacteriostatic agent, while Trichoderma reesei produces chitinase, which degrades pathogen cell walls. The synergistic effect of these two enhances the prevention and control of soil-borne diseases.

[0056] It should be noted that Bacillus amyloliquefaciens produces metabolites, such as lipopeptide antibiotics, during gradient expansion. Trichoderma reesei enhances its ability to physically encapsulate pathogens and secrete chitinase during solid-state pre-culture. When the two are mixed in a primary aerobic fermentation, the physical encapsulation of pathogens by Trichoderma reesei synergizes with the antibacterial substances of Bacillus amyloliquefaciens to more effectively inhibit pathogens. At the same time, chitinase can degrade the cell walls of pathogens, creating more favorable conditions for the antibacterial effects of Bacillus amyloliquefaciens. The two work together to enhance the overall antibacterial and disease-fighting effects.

[0057] By adding glucose, a stable carbon source is continuously provided for the fermentation system; at the same time, the addition of magnesium sulfate provides a stable carbon source for Bacillus amyloliquefaciens. , effectively activating its protease activity, thereby promoting spore formation, with a formation rate exceeding 90%. During this process, Trichoderma reesei can simultaneously utilize glucose metabolites, such as pyruvate, avoiding competitive inhibition with Bacillus amyloliquefaciens in nutrient utilization.

[0058] Keeping the dissolved oxygen level at ≥5% fully meets the aerobic needs of Bacillus amyloliquefaciens while also aligning with the microaerobic characteristics of Trichoderma reesei. This dissolved oxygen environment helps increase the total bacterial population while effectively avoiding the accumulation of metabolic byproducts caused by anaerobic conditions.

[0059] After anaerobic expansion, Lactobacillus acidophilus exhibits strong anaerobic adaptability and the ability to produce lactic acid to inhibit bacteria. Trichoderma reesei enhances its functional properties during solid-state pre-cultivation. During secondary microaerobic fermentation, Lactobacillus acidophilus produces exopolysaccharides that encapsulate spores to form a complex structure. Trichoderma reesei can interact with this complex structure to further enhance its colonization ability in the soil and its inhibitory effect on pathogens. The physical encapsulation and enzymatic hydrolysis of Trichoderma reesei on pathogens, combined with the antibacterial effects of Lactobacillus acidophilus, can better cope with the complex environment of the soil.

[0060] In primary aerobic fermentation, the enhanced physical encapsulation and high chitinase activity achieved by Trichoderma reesei during solid-state pre-culture enable more efficient synergistic metabolism with Bacillus amyloliquefaciens when mixed with the latter. The encapsulation of the Bacillus by Trichoderma reesei hyphae improves its survival under salt stress, while the metabolic products of the two interact to enhance the antibacterial and disease resistance of the fermentation product.

[0061] Step 4: The product of the primary aerobic fermentation is mixed with the expanded Lactobacillus acidophilus for secondary microaerobic fermentation, during which catalase and glutathione are added; The fermentation broth of Bacillus amyloliquefaciens, Trichoderma reesei mycelium, and Lactobacillus acidophilus can be prepared at a ratio of (8-10):1:1. During the fermentation process, the oxygen content is gradually reduced to 1%. This low-oxygen environment encourages Bacillus amyloliquefaciens to enter the spore state, enhancing its stress resistance. Simultaneously, Lactobacillus acidophilus utilizes the residual oxygen in the fermentation system to produce lactic acid, further lowering the system's pH and effectively inhibiting the growth of other bacteria.

[0062] The microaerobic environment stimulates Lactobacillus acidophilus to secrete extracellular polysaccharides (EPS), which can wrap the spores and significantly extend the colonization period of the bacterial agent in the soil.

[0063] It should be noted that catalase can decompose the residual , reducing the oxidative damage to the spores and increasing the survival rate of the spores.

[0064] It should be noted that glutathione can protect bacteria from damage by free radicals, and cooperate with the acidic environment created by Lactobacillus acidophilus to further enhance the stability of the bacterial agent.

[0065] In step 3, Trichoderma reesei and Bacillus amyloliquefaciens establish a synergistic relationship through co-cultivation. Trichoderma reesei encapsulates Bacillus amyloliquefaciens, improving its survival rate in high-salt environments. The two work together to enhance soil-borne disease control. In step 4, Bacillus amyloliquefaciens enters the spore state in a low-oxygen environment, further enhancing its stress resistance. The exopolysaccharides secreted by Lactobacillus acidophilus encapsulate the spores, further strengthening the stability and viability of Bacillus amyloliquefaciens.

[0066] The lactic acid produced by Lactobacillus acidophilus interacts with the metabolites of the bacterial flora in step 3, jointly creating an environment that is not conducive to the growth of miscellaneous bacteria.

[0067] In step 3, the dissolved oxygen level is controlled at ≥5%, meeting the aerobic requirements of Bacillus amyloliquefaciens and the microaerobic characteristics of Trichoderma reesei, creating favorable conditions for bacterial growth and metabolism. Step 4 involves staged oxygen control, reducing the level to 1%. This change in dissolved oxygen conditions is further adjusted based on the growth and metabolic status of the bacteria in step 3. The low oxygen environment encourages Bacillus amyloliquefaciens to enter the spore state, allowing the bacterial community to better adapt to different environmental conditions.

[0068] This application meets the nutritional needs of the bacterial community and promotes spore formation through dynamic feeding, such as adding glucose and magnesium sulfate. Lactobacillus acidophilus can utilize residual oxygen and existing glucose metabolites to achieve continued and expanded nutrient utilization.

[0069] The synergistic effect of Trichoderma reesei and Bacillus amyloliquefaciens enhances soil-borne disease control. The addition of Lactobacillus acidophilus further optimizes the bacterial flora structure and environmental conditions, extending the inoculant's colonization period in the soil. This longer colonization period allows the bacterial flora to continue functioning in the soil, further improving soil-borne disease control and achieving a shift from short-term to long-term prevention and control.

[0070] This application improves the survival rate and growth of bacteria through the interaction of bacterial groups and dissolved oxygen control, and adds catalase to decompose , glutathione to protect the bacteria and Lactobacillus acidophilus to create an acidic environment, further enhancing the stability of the bacterial agent.

[0071] In the secondary microaerobic fermentation, solid-state pre-cultured Trichoderma reesei is mixed with Lactobacillus acidophilus and the products of the primary aerobic fermentation. The functional properties of Trichoderma reesei help maintain the stability of the fermentation system. Together with the complex structure formed by the exopolysaccharides produced by Lactobacillus acidophilus, this further enhances the microbial cell's stress resistance and soil colonization ability in the microaerobic environment, contributing to the improved quality and performance of the final inoculum.

[0072] Step 5: The product of the secondary microaerobic fermentation is subjected to membrane separation treatment to desalinate and concentrate the bacterial liquid.

[0073] The combined ultrafiltration and nanofiltration process precisely removes large molecular impurities and small salt molecules from the fermentation broth. After treatment, the conductivity of the fermentation broth is reduced, while the retention rate of active ingredients such as chitinase and lipopeptides is high.

[0074] Step 6: Add biochar and citric acid to the concentrated bacterial solution and homogenize.

[0075] In this technical solution, membrane separation treatment is performed, and molecules with a molecular weight greater than 100 kDa are retained by an ultrafiltration membrane, and molecules with a molecular weight greater than 500 Da are retained by a nanofiltration membrane, and desalination treatment is performed.

[0076] In this technical solution, after adding biochar, citric acid with a mass fraction of 0.2%-0.3% is added in multiple times.

[0077] In this technical solution, membrane separation treatment is performed, and molecules with a molecular weight greater than 100 kDa are retained by an ultrafiltration membrane, and molecules with a molecular weight greater than 500 Da are retained by a nanofiltration membrane, and desalination treatment is performed.

[0078] In this technical solution, in step 6, the method of adding biochar and citric acid is as follows: pre-treating the biochar, activating the rice husk charcoal at 600℃-630℃ for 2-3h, grinding it to 100-200 mesh, and adding 1%-2% by mass fraction; Add 0.2%-0.3% citric acid in 3-5 times, with at least 10 minutes between each addition, and control the pH to 6.2±0.1.

[0079] Biochar can fix and , thereby significantly reducing soil toxicity. Citric acid can react with metal ions to form a chelate reaction. The two work together to increase the survival rate of bacteria.

[0080] The citric acid and biochar system has good buffering capacity, which can stably maintain the pH value of the inoculum within the range of 4.5-8.5, allowing the inoculum to adapt to a variety of soil environments.

[0081] It should be noted that trehalose forms a protective film on the cell surface, maintaining the structural stability of the cell membrane. Proline regulates the osmotic pressure within the cell. The synergistic effect of these two enhances the survival rate of the bacterial inoculant in harsh environments such as drought or high salt levels.

[0082] In this technical solution, after adding biochar, citric acid with a mass fraction of 0.2%-0.3% is added in multiple times.

[0083] This application uses membrane separation to concentrate and remove large molecular impurities and small molecular salts, reducing the EC value of the fermentation liquid. If the fermentation liquid contains too much impurities and salts, it will affect the biochar's and At the same time, lower salt content also helps trehalose and proline better maintain membrane structure and regulate osmotic pressure.

[0084] Membrane separation and concentration retain a high proportion of active ingredients, keeping the bacteria in a relatively healthy and active state. Highly active bacteria are able to better metabolize and enhance their resistance to stress in the optimal pH environment maintained by the biochar-citric acid system.

[0085] The present invention uses high-pressure homogenization to effectively break up the agglomeration of bacteria, thus avoiding the problem of bacterial autolysis caused by excessive local bacterial concentration.

[0086] The present application improves the survival rate and stress resistance of bacteria through biochar, citric acid, trehalose, proline, etc. During the high-pressure homogenization and filtration process, these stabilized bacteria can better withstand the mechanical pressure and physical effects during the filtration process, reducing the damage to the bacteria caused by the homogenization and filtration operations, thereby ensuring the number and activity of effective bacteria in the microbial agent. High-pressure homogenization makes the distribution of the microbial agent more uniform, which helps the stabilizer to contact the bacteria more evenly, thereby more fully exerting its protective effect. For example, biochar, citric acid, trehalose and proline can more evenly wrap and act on each bacteria, improving the stability and adaptability of the overall microbial agent. At the same time, filtering to remove miscellaneous bacteria avoids the competition between miscellaneous bacteria and bacteria for nutrients and living space, allowing the stabilizer to more effectively provide protection for effective bacteria and reduce storage losses.

[0087] This application removes impurities and salts through membrane separation and concentration. This reduction in impurities reduces the risk of equipment wear and clogging during the homogenization process, improving the effectiveness and efficiency of homogenization. Furthermore, the purified bacterial solution passes through the membrane more easily during filtration, reducing filtration resistance, improving filtration speed and quality, and ensuring the retention rate of effective bacterial cells.

[0088] Homogenization and filtration further optimize the quality of the inoculum after membrane separation and concentration. High-pressure homogenization breaks up bacterial agglomerates, distributing the active ingredients more evenly throughout the inoculum, further improving its stability and effectiveness. Filtration removes contaminants, ensuring the purity of the inoculum and preventing them from damaging the active ingredients during storage and use.

[0089] This application systematically avoids the reliance on mechanical turning in traditional microbial inoculants through synergistic interactions. First, Trichoderma reesei forms a dense mycelial network during solid-state pre-cultivation. Mycelial fragments secreted by the system continuously encapsulate Bacillus spores during liquid fermentation, creating a "natural fiber network" structure. This allows the microorganisms to disperse naturally after application to the soil, preventing aggregation or loss due to gravity settling or water erosion. Simultaneously, Lactobacillus acidophilus produces exopolysaccharides (EPS) to form a slime layer that further encapsulates the spores, insulating them from physical compression by soil particles and counteracting the effects of deep soil compaction on microbial activity. Furthermore, the porous structure of biochar is utilized to adsorb heavy metal ions, reducing soil toxicity. Citric acid is added periodically to buffer acid-base fluctuations and maintain metabolic homeostasis. Trehalose and proline work synergistically to enhance the microbial resistance to drought and salt by regulating extracellular osmotic pressure and maintaining intracellular ion balance. This chemical protection system enables the inoculant to remain active in extreme soil environments, eliminating the need for turning to adjust the microbial distribution. Ultrafiltration and nanofiltration fractionation remove bacterial fragments and impurities, retaining intact spores and Trichoderma hyphae. High-pressure homogenization breaks up bacterial aggregates and uniformizes the particle size of the inoculum. This process optimizes the physical state of the inoculum, allowing it to diffuse naturally in the form of single cells or microaggregates, adapting to the permeability characteristics of the soil and avoiding localized high concentrations or uneven distribution caused by agglomeration. Bacillus amyloliquefaciens secretes surfactant to inhibit pathogens, Trichoderma reesei produces chitinase to degrade pathogen cell walls, and Lactobacillus acidophilus produces lactic acid to form an acidic microenvironment with EPS. The metabolic products of multiple bacterial species complement each other to form a sustained antibacterial network. At the same time, soybean meal hydrolyzate induces Trichoderma to produce enzymes, and the enzymatic hydrolysis products promote the secretion of antibiotics by Bacillus. The metabolic interaction mechanism prolongs the functional activity cycle of the bacterial community, eliminating the need to stimulate metabolic responses by turning the compost.

[0090] The microbial agent of this application improves the inhibition rate of soil-borne pathogens and reduces the incidence of crop rhizosphere diseases through the synergistic action of multiple bacterial species and functional enhancement mechanisms, thereby synergistically optimizing crop yield and quality. This agent retains highly active bacterial communities through gradient fermentation and membrane separation technology, and combines the porous slow-release properties of biochar to maintain the continuous colonization ability of the bacteria in deep soil. Even without mechanical turning, it can still form a long-term and stable field protection barrier through bacterial community interaction, thereby achieving dual benefits of disease prevention and control and crop growth under no-till or minimum tillage conditions.

[0091] The second aspect of the present application provides a liquid microorganism for agricultural use, which is prepared using the method for preparing a liquid microbial agent for agricultural use according to the first aspect. It has all the beneficial effects of the first aspect, which will not be described in detail here.

[0092] For a better understanding of the technical solution, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Example

[0093] Step 1: Resurrection and culture of bacterial strains; Bacillus amyloliquefaciens: Inoculate into LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) and shake culture at 25-30°C and 200 rpm for 12 h to obtain a revived bacterial solution.

[0094] Trichoderma reesei: Inoculate into PDA liquid medium (potato extract 200 mL / L, glucose 20 g / L), culture with shaking at 25-30°C and 200 rpm for 36 h, and collect the mycelial suspension.

[0095] Lactobacillus acidophilus: Inoculate into MRS medium (beef extract 10 g / L, yeast extract 5 g / L, Tween 80 1 mL / L), culture anaerobically at 35-40°C for 18 h to obtain revived bacterial solution.

[0096] Step 2: Gradient expansion and pre-culture; Gradient expansion culture of Bacillus amyloliquefaciens; First-stage seed solution: Transfer the inoculum at a 1% rate to a liquid culture medium containing soybean meal hydrolyzate (5%) and sucrose (5%), culture at 25-30°C, 200 rpm for 12 h, and maintain dissolved oxygen ≥5%.

[0097] Secondary seed solution: Transfer 0.5% inoculum to fermentation tank medium (3% soybean meal hydrolyzate, 3% sucrose), culture at 25-30℃, 200 rpm for 14 h, and add 0.1% soybean meal hydrolyzate to induce secondary metabolism.

[0098] Solid-state pre-culture of Trichoderma reesei: inoculate into rice husk substrate (80% rice husk + 20% bran, humidity 70%), culture at 25-30°C for 48 h, transfer to potato glucose liquid medium after mycelium dispersion, supplement with 0.1% maltose, and culture at 25-30°C and 200 rpm for 24 h.

[0099] Anaerobic expansion of Lactobacillus acidophilus: inoculate into MRS medium, inject nitrogen to an oxygen concentration of ≤0.1%, and culture at 35-40℃ for 24 hours to produce EPS (concentration ≥1.5g / L).

[0100] Step 3: primary aerobic fermentation; Mixed Bacillus amyloliquefaciens (1× CFU / mL) and Trichoderma reesei mycelium (1× CFU / mL), glucose (0.2%) and magnesium sulfate (0.02%) were added, dissolved oxygen was controlled ≥5%, and fermentation was carried out at 25-30℃ and 200 rpm for 16 h.

[0101] Step 4: Secondary microaerobic fermentation; Add Lactobacillus acidophilus fermentation broth (1× CFU / mL), gradually reduce the oxygen concentration to 1%, add catalase (100 U / g) and glutathione (0.1%), and ferment at 35-40℃ for 24h.

[0102] Step 5: membrane separation and stabilizer treatment; Membrane separation: Ultrafiltration (100 kDa) to remove impurities, nanofiltration (500 Da) to desalinate to EC ≤ 1.5 mS / cm.

[0103] Step 6: Add biochar powder (1%) and grind it to 100 mesh. Add 0.3% citric acid three times (15 min interval), adjust the pH to 6.2 ± 0.1, and homogenize with high-pressure homogenizer (20 MPa).

[0104] Comparative Example 1

[0105] Adjustment of step 2: the revived Trichoderma reesei liquid was directly inoculated into the potato dextrose liquid culture medium without solid pre-culture on rice husk matrix. The remaining steps were the same as those in the Example group.

[0106] Comparative Example 2

[0107] Adjustment of step 2: Lactobacillus acidophilus was cultured aerobically (MRS medium with 200 rpm shaking) without nitrogen gas to control oxygen, and the EPS concentration was ≤0.5 g / L.

[0108] Comparative Example 3

[0109] Adjustment of step 6: Omit the addition of biochar and citric acid, and only process the bacterial liquid by membrane separation.

[0110] It should be noted that Table 1 shows the implementation index test results and corresponding detection methods of the microbial agents prepared based on the examples and comparative examples of the present application. The detection methods shown are all existing technologies and will not be repeated here.

[0111] ; ; ; The EPS concentration of the Example group was 2.5 g / L, which was 39% higher than that of the comparative example 2 in which the anaerobic expansion culture was absent, proving that the anaerobic EPS production of Lactobacillus acidophilus plays a key role in the stress resistance of the bacteria.

[0112] The salt stress survival rate of the Example group was 89%, which was 53% higher than that of the Comparative Example 3, indicating the protective effect of the biochar-citric acid system on extreme soil environments.

[0113] Referring to Table 3, the protective effect of the embodiment group remained 85% within 90 days, which was significantly better than that of comparative example 1 (55%) and comparative example 3 (48%), proving that the multi-species synergistic metabolism and stabilizer system can prolong the functional cycle of the bacterial flora.

[0114] After application, the EC value of the example group dropped to 3.2 mS / cm, which was 45% lower than that of the comparative example 1 (5.8 mS / cm), indicating that the combination of membrane separation and stabilizer effectively desalinated and maintained soil ion balance.

[0115] refer to Figure 2 The 90-day survival rate of the Example group was 85%. Combined with Table 2, the number of branches in Comparative Example 1 was 2.8, while that in the Example group was 6.2. This indicates that the lack of mycelial network encapsulation in Comparative Example 1 exposed the spores to the soil environment, resulting in a drop in survival from 68% to 55%. Combined with Table 3, the EC value of the Example group was 3.2, while that of Comparative Example 3 was 5.1. Due to the lack of biochar and citric acid protection, the bacteria in Comparative Example 3 rapidly lost activity under high salt (EC 8 mS / cm) and drought stress.

[0116] refer to Figure 2 The Example group achieved a 30-day survival rate of 89%, a 23-37% increase compared to the 65-72% in Comparative Examples 1-3. The Example group achieved a 90-day survival rate of 85%, a 25-40% increase compared to the 48-60% in Comparative Examples 1-3. The dual protection mechanism of solid-state pre-culture and stabilizers synergistically extended bacterial survival, while the lack of a single process in the Comparative Examples resulted in a significant drop in protection effectiveness.

[0117] Obviously, the above examples are merely provided for clarity of explanation and are not intended to limit the embodiments. Those skilled in the art will readily appreciate that other variations or modifications based on the above description are possible. It is not necessary and impossible to enumerate all embodiments here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

[0118] In the description of the present invention, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0119] In the description of this specification, the terms "one", "some", "example", "specific example" or "some examples" refer to the specific features, structures, materials or characteristics described in conjunction with the example or example being included in at least one or more examples of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more examples in a suitable manner. In addition, those skilled in the art can combine and combine different examples and features of different examples described in this specification without any contradiction.

[0120] It is understood that the above is exemplary and should not be construed as limiting the present invention. Persons skilled in the art may make changes, modifications, substitutions and variations to the above within the scope of the present invention.

Claims

1. A method for preparing a liquid microbial agent for agricultural use, characterized in that: include: Step 1: inoculating Bacillus amyloliquefaciens, Trichoderma reesei, and Lactobacillus acidophilus into their respective culture media for cultivation, and obtaining corresponding revived bacterial liquids; Step 2: performing gradient expansion culture on the revived Bacillus amyloliquefaciens solution obtained in step 1; performing solid-state pre-culture on the revived Trichoderma reesei solution; and performing anaerobically expanding culture on the revived Lactobacillus acidophilus solution; Step 3: Bacillus amyloliquefaciens obtained in step 2 and Trichoderma reesei are mixed in a ratio of (8-10):1 and subjected to primary aerobic fermentation, with glucose and magnesium sulfate added during the fermentation; Step 4: The product of the primary aerobic fermentation is mixed with the expanded Lactobacillus acidophilus for secondary microaerobic fermentation, during which catalase and glutathione are added; Step 5: The product of the secondary microaerobic fermentation is subjected to membrane separation treatment to desalinate and concentrate the bacterial liquid; Step 6: Add biochar and citric acid to the concentrated bacterial solution and homogenize.

2. The method for preparing a liquid microbial agent for agricultural use according to claim 1, characterized in that: In step 1, Trichoderma reesei spores are inoculated into a liquid culture medium, the temperature is controlled at 25-30° C., shaking culture is performed for 30-48 hours, and the mycelial suspension is collected to obtain a Trichoderma reesei revived bacterial liquid.

3. The method for preparing a liquid microbial agent for agricultural use according to claim 1, characterized in that: In step 2, solid-state pre-culture includes inoculating the revived Trichoderma reesei liquid obtained in step 1 into a rice husk substrate, transferring the pre-cultured mycelium to a potato glucose liquid culture medium after the mycelium is dispersed, and adding maltose during the fermentation process.

4. The method for preparing a liquid microbial agent for agricultural use according to claim 1, characterized in that: In step 2, the gradient expansion culture comprises transferring the revived Bacillus amyloliquefaciens solution obtained in step 1 to a liquid culture medium containing soybean meal hydrolyzate and sucrose at an inoculum rate of 1%-2%, controlling the temperature at 25-30° C., and culturing for 10-14 hours to obtain a first-level seed solution; Transfer the first-level seed liquid to the fermentation tank culture medium at an inoculation rate of 0.3%-0.5%, culture for 12-16 hours, and obtain the second-level seed liquid.

5. The method for preparing a liquid microbial agent for agricultural use according to claim 1, characterized in that: In step 2, anaerobic expansion includes transferring the revived Lactobacillus acidophilus liquid obtained in step 1 into the culture medium at an inoculum rate of 1-2% and placing it in an anaerobic tank, injecting nitrogen to an oxygen concentration of ≤0.1%, and culturing it at 35-40°C for 18-24 hours.

6. The method for preparing a liquid microbial agent for agricultural use according to claim 1, characterized in that: Membrane separation treatment, using ultrafiltration membrane to retain molecules with a molecular weight greater than 100kDa, and then using nanofiltration membrane to retain molecules with a molecular weight greater than 500Da, and desalting treatment.

7. The method for preparing a liquid microbial agent for agricultural use according to claim 1, characterized in that: After adding biochar, citric acid with a mass fraction of 0.2%-0.3% was added in several times.

8. The method for preparing a liquid microbial agent for agricultural use according to claim 4, characterized in that: In step 2, feeding treatment is performed during the gradient expansion process; During the first-level seed liquid culture stage of Bacillus amyloliquefaciens, add 0.1%-0.3% glucose and 0.01%-0.05% magnesium sulfate by mass when the fermentation is longer than 12 hours, and maintain the dissolved oxygen ≥5%; During the secondary seed liquid culture stage, when the fermentation time was greater than 24 h, 0.1% soybean meal hydrolyzate was added to induce the secretion of secondary metabolites.

9. The method for preparing a liquid microbial agent for agricultural use according to claim 7, wherein: In step 6, the method of adding biochar and citric acid is as follows: pre-treating the biochar, activating the rice husk charcoal at 600-630°C for 2-3 hours, grinding it to 100-200 mesh, and adding 1%-2% by mass; Add 0.2%-0.3% citric acid in 3-5 times, with at least 10 minutes between each addition, and control the pH to 6.2±0.

1.

10. An agricultural liquid microorganism, prepared by the method for preparing an agricultural liquid microbial agent according to any one of claims 1 to 9.

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