Preparation method of agricultural liquid microbial agent and microbial agent

Abstract

The application belongs to the technical field of agricultural microbial agent, and relates to a preparation method of an agricultural liquid microbial agent and the microbial agent.The method comprises the following steps: mixing bacillus amyloliquefaciens and trichoderma reesei to perform primary aerobic fermentation; mixing the product subjected to the primary aerobic fermentation and lactobacillus acidophilus subjected to expansion to perform secondary micro-aerobic fermentation; and performing membrane separation treatment on the product subjected to the secondary micro-aerobic fermentation and concentrating the bacterial liquid.The microbial agent can improve the inhibition rate of soil-borne pathogens and reduce the incidence of crop rhizosphere diseases through the synergistic effect of multiple strains, so as to optimize the yield and quality of crops. Gradient fermentation and membrane separation technology are used to retain high-activity bacterial groups, and biological carbon is used to maintain the continuous colonization ability of bacterial bodies in deep soil. Even without mechanical turning, a long-term stable field protection barrier can be formed through bacterial group interaction, so that the disease prevention and control can be realized under the condition of no-tillage or reduced tillage.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial inoculants, and in particular to a method for preparing a liquid microbial inoculant for agricultural use and the microbial inoculant itself. Background Technology

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

[0003] However, this model faces numerous technical challenges in practical application. Most microorganisms in existing microbial agents are aerobic, requiring a sufficient oxygen supply for growth and metabolism. While they rely on aerobic metabolism to inhibit pathogens, they cannot diffuse into deeper soil layers. Due to the oxygen concentration in deeper soils being below 0.1% and the presence of salinization and heavy metal pollution, the activity of aerobic bacteria decreases sharply or even becomes inactive. Currently, existing technologies often rely on mechanical turning to forcibly disperse the microbial agent, such as using trough-type compost turners. This not only damages soil structure, leading to decreased aeration and microbial community imbalance, but also increases farmers' workload, consuming significant manpower and resources, raising production costs, and reducing efficiency, making it difficult for many farmers to bear the burden. Summary of the Invention

[0004] Given that the aerobic bacteria in existing microbial inoculants become ineffective in oxygen-deficient deep soil, there are technical problems with the conventional reliance on mechanical turning, which increases the labor involved for farmers.

[0005] The first aspect of this application mentions a method for preparing liquid microbial inoculants for agricultural use, comprising:

[0006] Step 1: Inoculate Bacillus amyloliquefaciens, Trichoderma reesei, and Lactobacillus acidophilus into their respective culture media and culture them to obtain the corresponding revived bacterial solutions;

[0007] Step 2: Perform gradient expansion culture of the reactivated Bacillus amyloliquefaciens broth obtained in Step 1; perform solid-state pre-culture of the reactivated Trichoderma reductoid broth; and perform anaerobic expansion culture of the reactivated Lactobacillus acidophilus broth.

[0008] Step 3: Mix the Bacillus amyloliquefaciens obtained in Step 2 with Trichoderma reesei at a ratio of (8-10):1 and carry out primary aerobic fermentation. Add glucose and magnesium sulfate during fermentation.

[0009] Step 4: The product of the first-stage aerobic fermentation is mixed with the expanded Lactobacillus acidophilus for secondary micro-aerobic fermentation, and catalase and glutathione are added during the fermentation process;

[0010] Step 5: The product of the secondary micro-aerobic fermentation is subjected to membrane separation treatment, desalination, and concentration of the bacterial solution;

[0011] Step 6: Biochar and citric acid are added to the concentrated bacterial solution, and homogenization treatment is performed.

[0012] Further, in step 1, Trichoderma reesei spores are inoculated into a liquid culture medium, the temperature is controlled at 25-30°C, and the mycelium suspension is collected after 30-48h of shaking culture to obtain the Trichoderma reesei revival bacterial solution.

[0013] Further, in step 2, the solid-state pre-culture includes inoculating the Trichoderma reesei revival bacterial solution obtained in step 1 into rice husk substrate, and after the mycelium is dispersed, the pre-culture mycelium is transferred to a potato dextrose liquid culture medium, and during the fermentation process, maltose is supplemented.

[0014] Further, in step 2, the gradient expansion includes transferring the Bacillus amyloliquefaciens revival bacterial solution obtained in step 1 to a liquid culture medium containing soybean meal hydrolysate and sucrose at an inoculation amount of 1%-2%, controlling the temperature at 25-30°C, and culturing for 10-14h to obtain a first-stage seed solution.

[0015] The first-stage seed solution is transferred to a fermentation tank culture medium at an inoculation amount of 0.3%-0.5%, and cultured for 12-16h to obtain a second-stage seed solution.

[0016] Further, in step 2, the anaerobic expansion includes transferring the Lactobacillus acidophilus revival bacterial solution obtained in step 1 to the culture medium and placing it in an anaerobic tank, injecting nitrogen gas to an oxygen concentration of ≤0.1%, and incubating at 35-40°C for 18-24h.

[0017] Further, the membrane separation treatment is performed by using an ultrafiltration membrane to remove molecules with a molecular weight greater than 100kDa, and then using a nanofiltration membrane to remove molecules with a molecular weight greater than 500Da, and desalination treatment.

[0018] Further, after adding biochar, citric acid with a mass fraction of 0.2%-0.3% is added in multiple times.

[0019] Further, in step 2, a feed supplement is added during the gradient expansion process.

[0020] During the culture of the Bacillus amyloliquefaciens first-stage seed solution, 0.1%-0.3% glucose and 0.01%-0.05% magnesium sulfate are supplemented after more than 12h of fermentation to maintain the dissolved oxygen at ≥5%.

[0021] In the secondary seed liquid culture stage, 0.1% soybean meal hydrolysate is added to induce the secretion of secondary metabolites when the fermentation time is greater than 24h.

[0022] Further, in step 6, the method of adding biochar and citric acid is as follows: the biochar is pretreated, the rice husk charcoal is activated at 600-630℃ for 2-3h, ground to 100-200 mesh, and added in an amount of 1%-2% by mass fraction;

[0023] 0.2%-0.3% citric acid is added in 3-5 portions, each time with an interval of at least 10min, and the pH is controlled to 6.2±0.1.

[0024] The second aspect of the present application refers to an agricultural liquid microorganism prepared by the method for preparing an agricultural liquid microbial agent of the first aspect.

[0025] The present application has the following beneficial effects:

[0026] The present application refers to a method for preparing an agricultural liquid microbial agent and a microbial agent, wherein the method comprises: inoculating Bacillus amyloliquefaciens, Trichoderma reesei and Lactobacillus acidophilus into respective culture media for culture, and obtaining corresponding revived bacterial liquids, respectively; gradient expanding the Bacillus amyloliquefaciens revived bacterial liquid; solid-state pre-culturing the Trichoderma reesei revived bacterial liquid; anaerobically expanding the Lactobacillus acidophilus revived bacterial liquid; mixing the Bacillus amyloliquefaciens and the Trichoderma reesei for primary aerobic fermentation, and adding glucose and magnesium sulfate during the fermentation; mixing the product after the primary aerobic fermentation and the expanded Lactobacillus acidophilus for secondary micro-aerobic fermentation, and adding catalase and glutathione during the fermentation; performing membrane separation treatment on the product after the secondary micro-aerobic fermentation, desalting and concentrating the bacterial liquid; and adding biochar and citric acid to the concentrated bacterial liquid and homogenizing the same.

[0027] The present application systematically solves the technical bottleneck through the synergistic effect of the phased gradient oxygen regulation and the functional strengthening strategy: for the deep-layer anoxic failure problem of aerobic bacteria, the bacterial population is rapidly proliferated through primary aerobic fermentation, and an oxygen demand gradient of the bacterial population is constructed through secondary micro-aerobic fermentation, so that the Bacillus amyloliquefaciens can adapt to the low-oxygen environment through facultative metabolism, and realize functional survival in deep soil; at the same time, a double slow-release system is formed by membrane separation and concentration of the microbial agent and addition of biochar, and the soil microenvironment pH is regulated by citric acid, which significantly prolongs the colonization period of the microbial agent in deep soil, and replaces the traditional turning operation, thereby fundamentally solving the dependence on mechanical turning. Through two-stage oxygen gradient fermentation, a low-oxygen tolerant bacterial population is constructed, and an antioxidant enzyme system is used to maintain the stability of the cell membrane, so as to ultimately realize the sustained metabolic function and survival ability of aerobic bacteria in deep anoxic soil. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to make the content of the present application more easily understood, the present application is further described in detail below according to the specific embodiments of the present application and in conjunction with the drawings.

[0029] Figure 1 A process flow chart of a preparation method of an agricultural liquid microbial agent mentioned in the present application;

[0030] Figure 2 A dynamic change diagram of survival rate of field bacillus in a preparation method of an agricultural liquid microbial agent mentioned in the present application. DETAILED DESCRIPTION

[0031] The present application is further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the examples are not as a limitation of the present application.

[0032] For this purpose, with reference to Figure 1 , the first aspect of the present application mentions a preparation method of an agricultural liquid microbial agent, comprising:

[0033] Step 1: Bacillus amyloliquefaciens, Trichoderma reesei, Lactobacillus acidophilus are inoculated into respective culture media for cultivation, and respective revival bacterial liquids are obtained.

[0034] Exemplarily, Bacillus amyloliquefaciens, Trichoderma reesei, Lactobacillus acidophilus are inoculated into LB culture medium, PDA liquid culture medium, and MRS culture medium, respectively.

[0035] Among them, Trichoderma reesei spores are inoculated into liquid culture medium, the temperature is controlled at 25-30℃, and the mycelium suspension is collected after 30-48h of shaking cultivation, to obtain Trichoderma reesei revival bacterial liquid. Bacillus amyloliquefaciens is inoculated, 0.1% glucose is supplemented, and 200rpm shaking condition is controlled to accelerate the recovery of bacterial bodies from the dormant state.

[0036] It should be noted that LB culture medium (Luria-Bertani): containing tryptone, yeast extract, NaCl, providing nitrogen source and carbon source, supporting rapid revival of Bacillus amyloliquefaciens and secretion of secondary metabolites. PDA liquid culture medium (potato dextrose): containing potato extract (providing cellulase induction enzyme secretion) and glucose (carbon source), promoting dispersion of Trichoderma reesei mycelium and chitinase synthesis. MRS culture medium (Man, Rogosa, Sharpe): containing beef extract, yeast extract, Tween 80 and other components, dedicated to lactic acid bacteria culture, maintaining a low pH environment (4.5-5.0), inhibiting miscellaneous bacteria and promoting lactic acid secretion.

[0037] Bacillus amyloliquefaciens is quickly revived in LB medium to provide high-activity seed liquid for subsequent fermentation; Trichoderma is improved in mycelium dispersibility (40% reduction in viscosity) in PDA medium to enhance subsequent enzyme production capacity; Lactobacillus produces lactic acid in MRS medium to inhibit miscellaneous bacteria and create an acidic environment for mixed fermentation.

[0038] Exemplarily, Bacillus amyloliquefaciens can be supplemented with 0.1% glucose to accelerate metabolism under the condition of 200 rpm oscillation, thereby increasing the number of viable bacteria and providing high-activity seed for subsequent fermentation.

[0039] The tryptophan contained in the tryptone in the LB medium induces Bacillus amyloliquefaciens to synthesize lipopeptide antibiotics, thereby enhancing the bacteriostatic capacity.

[0040] After liquid culture of Trichoderma reesei in PDA, the viscosity of the Trichoderma reesei fermentation liquid is reduced, the degree of mycelium fragmentation is increased, the contact area during subsequent co-culture with Bacillus is increased, and the synergistic bacteriostatic efficiency is improved.

[0041] Lactic acid produced by Lactobacillus acidophilus effectively inhibits miscellaneous bacteria, thereby providing a clean microenvironment for subsequent mixed fermentation.

[0042] Step 2: Gradient expansion of the revived Bacillus amyloliquefaciens liquid obtained in step 1 is performed; solid-state pre-culture of the revived Trichoderma reesei liquid is performed; anaerobic expansion of the revived Lactobacillus acidophilus liquid is performed.

[0043] In the technical solution, in step 2, the gradient expansion includes transferring the revived Bacillus amyloliquefaciens liquid obtained in step 1 into a liquid culture medium containing soybean meal hydrolysate and sucrose at an inoculation amount of 1%-2%, controlling the temperature to be 25-30°C, and culturing for 10-14 h to obtain a primary seed liquid.

[0044] The primary seed liquid is transferred into a fermentation tank culture medium at an inoculation amount of 0.3%-0.5%, and cultured for 12-16 h to obtain a secondary seed liquid.

[0045] In the technical solution, in step 2, a feeding treatment is performed during the gradient expansion.

[0046] During the culture of the primary seed liquid of Bacillus amyloliquefaciens, 0.1%-0.3% glucose and 0.01%-0.05% magnesium sulfate are supplemented when the fermentation is greater than 12 h, and the dissolved oxygen is maintained to be greater than or equal to 5%.

[0047] It should be noted that Bacillus amyloliquefaciens has a high demand for carbon sources during the exponential growth phase, and the supplementation of glucose can alleviate the carbon source limitation and prolong the logarithmic growth phase; a high-sugar environment promotes the secretion of surfactin by Bacillus amyloliquefaciens, thereby improving the bacteriostatic rate.

[0048] It should be noted that magnesium ions ATP enzyme, cofactor of protease, 0.01%-0.05% concentration can improve the metabolic efficiency of bacteria by 20%; magnesium ions stabilize the cell membrane structure, reduce the interference of dissolved oxygen fluctuation on the respiratory chain.

[0049] It should be noted that when the dissolved oxygen is greater than or equal to 5%, the bacillus metabolizes mainly in an aerobic manner, avoiding the pH drop caused by acetic acid accumulation under anaerobic conditions, and maintaining the pH at 6.5-6.8.

[0050] Supplementing glucose and magnesium sulfate in stages can prolong the logarithmic growth phase and increase the number of viable bacteria.

[0051] During the secondary seed liquid culture stage, under the condition that the fermentation is greater than 24h, supplementing 0.1% soybean meal hydrolysate can induce the secretion of chitinase and β-1,3-glucanase.

[0052] It should be noted that the soybean meal hydrolysate provides plant-derived amino acids such as glutamine and aspartic acid, which can relieve nitrogen limitation and trigger the secondary metabolic pathway; and the oligopeptides in the hydrolysate act as signal molecules to activate the expression of chitinase genes in trichoderma, further improving enzyme activity.

[0053] 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 promotes the production of surfactin by bacillus.

[0054] It should be noted that secondary metabolites such as glucanase can enhance the ability of bacteria to degrade soil fibers, improve field colonization efficiency, and enhance stress resistance.

[0055] It should be noted that the addition of glucose should be completed before the dissolved oxygen begins to decrease (<5%) to avoid metabolic imbalance; the soybean meal hydrolysate should be added after the pH of the fermentation broth is stable (6.0-6.5) to prevent acid inhibition.

[0056] Glucose can be added in stages, such as 0.2% after 12h and 0.1% after 18h, to avoid excessive carbon source accumulation leading to acetic acid accumulation.

[0057] The concentration of magnesium sulfate should be strictly controlled below 0.05% to prevent excessive magnesium ions from inhibiting the respiratory chain.

[0058] Exemplarily, a dissolved oxygen electrode can be used for real-time feedback, and a stirring or aeration system can be linked to maintain the dissolved oxygen at greater than or equal to 5%.

[0059] The present application realizes precise regulation of metabolic pathways by supplementing glucose / magnesium sulfate and soybean meal hydrolysate in stages, prolongs the logarithmic growth phase, and increases the number of viable bacteria by 30%; and strengthens secondary metabolites, enzyme activity, and antibiotic production.

[0060] The application provides a standardized scheme for high-density and high-functioning bacterial agent preparation by dynamic feeding and metabolic product interaction. Soybean meal hydrolysate provides small peptides and amino acids for Bacillus amyloliquefaciens to induce secondary metabolic product synthesis, and sucrose as a slow-release carbon source avoids the glucose effect. Meanwhile, the soybean meal hydrolysate provides plant-derived nitrogen source to promote the secretion of lipopeptide antibiotics by Bacillus.

[0061] In the technical solution, in step 2, the solid pre-culturing comprises inoculating the Trichoderma reesei revival bacterial liquid obtained in step 1 into rice husk substrate, dispersing the mycelium, and then transferring the pre-cultured mycelium into potato dextrose liquid medium, and adding maltose during the fermentation process.

[0062] In the technical solution, the Trichoderma reesei is inoculated into rice husk substrate, and the rice husk substrate is 80% rice husk + 20% bran, the particle size of the rice husk substrate is 100-200 mesh, and the humidity is controlled to be 70%. The rough surface of the rice husk substrate can stimulate the branching of the Trichoderma reesei mycelium through physical friction, so that the biomass is increased; the bran provides lignocellulose and supplements the nitrogen source, promotes the secretion of chitinase, pre-activates the cellulase system, and improves the enzyme activity in the subsequent liquid fermentation.

[0063] The solid pre-culturing can provide physical protection, in addition, the rice husk substrate can induce the dispersion of the Trichoderma reesei mycelium, reduce the agglomeration rate, and enhance the physical wrapping ability to the pathogenic bacteria; the Trichoderma reesei revival bacterial liquid is inoculated into the rice husk substrate for solid pre-culturing, so that the mycelium can be better dispersed, the biomass of the mycelium is increased after being transferred into the liquid medium, and the physical inhibition ability to the pathogenic bacteria is enhanced. Maltose is added during the fermentation process, the logarithmic growth phase is prolonged, and the yield of chitinase is increased.

[0064] In the technical solution, in step 2, the anaerobic expansion comprises transferring the Lactobacillus acidophilus revival bacterial liquid obtained in step 1 into a culture medium at an inoculation amount of 1-2% and placing the culture medium in an anaerobic tank, injecting nitrogen gas to make the oxygen concentration ≤0.1%, and then culturing at 35-40°C for 18-24h.

[0065] It should be noted that the Lactobacillus acidophilus produces lactic acid (concentration ≥1.5%) under low-oxygen conditions, which cooperates with the antibiotics of Bacillus to inhibit bacteria; the metabolic product of the Lactobacillus acidophilus chelates heavy metals , and reduces the toxicity of the soil.

[0066] It should be noted that the nitrogen gas controls the oxygen (≤0.1%) to simulate a strict anaerobic environment, promotes the Lactobacillus acidophilus to produce extracellular polysaccharide (EPS), and enhances the bacterial body wrapping protection effect during the subsequent co-culturing with Bacillus.

[0067] Exemplarily, nitrogen gas is injected at a rate of 0.5 L / min to reduce the oxygen concentration in the anaerobic tank to ≤0.1%, simulating the deep anaerobic environment of soil. Under low-oxygen conditions, L. acidophilus switches from aerobic respiration to fermentation metabolism, and the carbon source is preferentially used for EPS synthesis rather than ATP generation. Under anaerobic conditions, L. acidophilus secretes EPS (such as rhamnose lactosaccharide). At the same time, EPS forms a mucus layer to wrap Bacillus, reducing the damage of salt stress, high temperature and ultraviolet radiation to Bacillus spores.

[0068] At the same time, EPS-wrapped Bacillus spores have improved colonization rate in soil and enhanced stress resistance; further, the two form metabolic mutualism: the sugars in EPS are utilized by Bacillus spores, promoting the production of surface active agents.

[0069] Step 3: Mix the B. amyloliquefaciens obtained in Step 2 with T. reesei to perform primary aerobic fermentation, and add glucose and magnesium sulfate during fermentation.

[0070] The ratio of B. amyloliquefaciens to T. reesei mycelium is (8-10):1, and the T. reesei mycelial network wraps the B. amyloliquefaciens, enhancing the survival ability of the microbial community in a high-salt environment and greatly improving its survival rate. B. amyloliquefaciens can secrete surfactin with antibacterial effects, while T. reesei can produce chitinase for degrading the cell walls of pathogenic bacteria. The two work together to improve the control effect on soil-borne diseases.

[0071] It should be noted that B. amyloliquefaciens produces metabolic products such as lipopeptide antibiotics during gradient expansion. T. reesei enhances the physical wrapping ability of pathogenic bacteria and the secretion of chitinase during solid-state pre-culture. When the two are mixed for primary aerobic fermentation, the physical wrapping of pathogenic bacteria by T. reesei can work synergistically with the antibacterial substances of B. amyloliquefaciens to more effectively inhibit pathogenic bacteria. At the same time, chitinase can degrade the cell walls of pathogenic bacteria, creating more favorable conditions for the antibacterial effects of B. amyloliquefaciens. The two work together to improve the overall antibacterial and disease resistance effects.

[0072] By adding glucose, a stable carbon source is continuously provided for the fermentation system; at the same time, the addition of magnesium sulfate provides for B. amyloliquefaciens, effectively activating the activity of its protease, and then promoting the formation of spores, with a formation rate of over 90%. In this process, T. reesei can simultaneously utilize metabolic products of glucose, such as pyruvic acid, avoiding competition inhibition in nutrient utilization with B. amyloliquefaciens.

[0073] Controlling the dissolved oxygen level at ≥5% can fully meet the aerobic needs of B. amyloliquefaciens and also meet the micro-aerobic characteristics of T. reesei. Under this dissolved oxygen environment, it is helpful to increase the total amount of bacterial cells, while effectively avoiding the accumulation of metabolic byproducts due to anaerobic limitations.

[0074] Lactobacillus acidophilus has strong anaerobic adaptability and the ability to produce lactic acid to inhibit the growth of other bacteria after anaerobic expansion. Trichoderma reesei enhances its functional properties during solid-state pre-culture. During secondary micro-aerobic fermentation, Lactobacillus acidophilus produces extracellular polysaccharides to encapsulate spores to form a complex structure, and Trichoderma reesei can interact with this complex structure to further improve its colonization ability in soil and its inhibitory effect on pathogenic bacteria. The physical encapsulation and enzymatic degradation of Trichoderma reesei combined with the antibacterial effect of Lactobacillus acidophilus can better cope with the complex environment in soil.

[0075] During primary aerobic fermentation, Trichoderma reesei has enhanced physical encapsulation ability and higher chitinase activity during solid-state pre-culture, which enables it to more efficiently synergize with Bacillus amyloliquefaciens when they are mixed for fermentation. The mycelium of Trichoderma reesei encapsulates Bacillus amyloliquefaciens, improving its survival rate under salt stress, and the metabolic products of the two organisms complement each other, enhancing the antibacterial and disease-resistant ability of the fermentation products.

[0076] Step 4: Mix the product of the primary aerobic fermentation with the expanded Lactobacillus acidophilus for secondary micro-aerobic fermentation, and add catalase and glutathione during the fermentation process.

[0077] Among them, the ratio of Bacillus amyloliquefaciens, Trichoderma reesei mycelium, and Lactobacillus acidophilus fermentation broth can be (8-10):1:1. During the fermentation process, the oxygen content is gradually reduced to 1%. Low oxygen environment stimulates Bacillus amyloliquefaciens to enter spore state, enhancing its stress resistance. At the same time, Lactobacillus acidophilus produces lactic acid using the residual oxygen in the fermentation system, further reducing the pH value of the system, thereby effectively inhibiting the growth of other bacteria.

[0078] Micro-aerobic environment stimulates Lactobacillus acidophilus to secrete extracellular polysaccharides (EPS), which can encapsulate spores, significantly prolonging the colonization period of the microbial agent in soil.

[0079] It should be noted that catalase can decompose the residual hydrogen peroxide during the fermentation process, reducing its oxidative damage to spores and increasing the survival rate of spores.

[0080] It should be noted that glutathione can protect the bacterial cell from free radical damage, and in combination with the acidic environment created by Lactobacillus acidophilus, it further enhances the stability of the microbial agent.

[0081] ​In step 3, Trichoderma reesei and Bacillus amyloliquefaciens establish a synergistic relationship through co-culture, Trichoderma reesei encapsulates Bacillus amyloliquefaciens, improves its survival rate in high salt environment, and the two work together to improve the control effect of soil-borne diseases. In step 4, Bacillus amyloliquefaciens enters spore state under low oxygen environment, its stress resistance is further enhanced, and the extracellular polysaccharide secreted by Lactobacillus acidophilus encapsulates spores, further consolidating the stability and survival ability of Bacillus amyloliquefaciens.

[0082] Lactobacillus acidophilus produces lactic acid which interacts with the metabolic products of the microbial community in step 3, together creating an environment that is not conducive to the growth of miscellaneous bacteria.

[0083] In step 3, by controlling the dissolved oxygen ≥ 5% to meet the aerobic demand of Bacillus amyloliquefaciens and the micro-aerobic characteristics of Trichoderma reesei, good conditions are created for cell growth and metabolism. Step 4 carries out segmented oxygen control, reducing to 1%, the change of dissolved oxygen conditions is based on the growth state and metabolism of the cells in step 3 for further adjustment. Low oxygen environment promotes Bacillus amyloliquefaciens to enter spore state, so that the microbial community can better adapt to different environmental conditions.

[0084] The present application meets the nutritional needs of the microbial 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, etc., to achieve the continuation and expansion of nutrient utilization.

[0085] The synergistic effect of Trichoderma reesei and Bacillus amyloliquefaciens improves the control effect of soil-borne diseases, and the addition of Lactobacillus acidophilus further optimizes the microbial community structure and environmental conditions, prolonging the colonization period of the microbial inoculant in the soil. Longer colonization period enables the microbial community to continuously exert its effect in the soil, thereby further improving the control effect of soil-borne diseases, and realizing the transformation from short-term prevention to long-term prevention and control.

[0086] The present application improves the survival rate and growth of the microbial cells through the interaction of the microbial community and the control of dissolved oxygen, and further enhances the stability of the microbial inoculant through the decomposition of hydrogen peroxide by the added catalase, the protection of the microbial cells by glutathione, and the creation of an acidic environment by Lactobacillus acidophilus.

[0087] In the secondary micro-aerobic fermentation, Trichoderma reesei pre-cultured in solid state is mixed with Lactobacillus acidophilus and the product of the first aerobic fermentation. The functional characteristics of Trichoderma reesei help maintain the stability of the fermentation system, and together with the complex structure formed by the extracellular polysaccharide produced by Lactobacillus acidophilus, further improve the stress resistance and soil colonization ability of the microbial cells in the micro-aerobic environment, contributing to the improvement of the quality and performance of the final microbial inoculant.

[0088] Step 5: The product of the secondary micro-aerobic fermentation is treated by membrane separation to remove salt and concentrate the microbial liquid. ​

[0089] The combined process of ultrafiltration and nanofiltration can accurately remove macromolecular impurities and small molecular salts in the fermentation broth. After treatment, the conductivity of the fermentation broth is reduced, and the retention rate of active ingredients such as chitinase and lipopeptide is high.

[0090] Step 6: Add biochar and citric acid to the concentrated broth and homogenize.

[0091] In the technical solution, the membrane separation treatment retains molecules with a molecular weight greater than 100 kDa through an ultrafiltration membrane, and then retains molecules with a molecular weight greater than 500 Da through a nanofiltration membrane, and desalination treatment.

[0092] In the technical solution, after adding biochar, 0.2%-0.3% citric acid is added in multiple times.

[0093] In the technical solution, the membrane separation treatment retains molecules with a molecular weight greater than 100 kDa through an ultrafiltration membrane, and then retains molecules with a molecular weight greater than 500 Da through a nanofiltration membrane, and desalination treatment.

[0094] In the technical solution, in step 6, the method of adding biochar and citric acid is to pretreat the biochar, activate the rice husk charcoal at 600-630°C for 2-3h, grind it to 100-200 mesh, and add it according to the mass fraction of 1%-2%;

[0095] Add 0.2%-0.3% citric acid in 3-5 times, each time at least interval greater than 10 min, and control pH to 6.2±0.1.

[0096] Biochar can fix and in the soil, thereby significantly reducing the toxicity of the soil. Citric acid can undergo chelation with metal ions. The two work together to improve the survival rate of the bacterial cells.

[0097] The citric acid and biochar system has good buffering capacity and can maintain the pH value of the bacterial agent in the range of 4.5-8.5, so that the bacterial agent can adapt to a variety of soil environments.

[0098] It should be noted that trehalose can form a protective film on the cell surface to maintain the structural stability of the cell membrane. Proline can regulate the osmotic pressure in the cell. The two work together to improve the survival rate of the bacterial agent in harsh environments such as drought or high salt.

[0099] In the technical solution, after adding biochar, 0.2%-0.3% citric acid is added in multiple times.

[0100] The present application removes macromolecular impurities and small molecular salts through membrane separation and concentration, reducing the EC value of the fermentation broth. If the fermentation broth contains excessive impurities and salts, it will affect the fixation effect of biochar on and , as well as the chelation of citric acid on metal ions. At the same time, lower salt content is also conducive to the better function of trehalose and proline in maintaining membrane structure and regulating osmotic pressure.

[0101] Membrane separation and concentration retain a high proportion of active ingredients, keeping the bacteria in a relatively healthy and active state. Higher activity of the bacteria can better perform metabolic activities in the suitable pH environment maintained by the biochar-citric acid system, enhancing their stress resistance.

[0102] The present application can effectively break the agglomeration of bacteria through high-pressure homogenization, avoiding the problem of bacterial autolysis caused by excessive local bacterial concentration.

[0103] The present application improves the survival rate and stress resistance of bacteria through biochar, citric acid, trehalose, proline, etc. During high-pressure homogenization and filtration, these stabilized bacteria can better withstand mechanical pressure and physical action during filtration, reducing damage to bacteria caused by homogenization and filtration, thereby ensuring the number and activity of effective bacteria in the inoculant. High-pressure homogenization makes the inoculant more evenly distributed, 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 bacterium, improving the stability and adaptability of the overall inoculant. At the same time, removing unwanted bacteria avoids competition for nutrients and living space, allowing the stabilizer to more effectively protect the effective bacteria and reduce storage loss.

[0104] The present application removes impurities and salts through membrane separation and concentration, reducing the risk of equipment wear and blockage during homogenization, improving the effectiveness and efficiency of homogenization. At the same time, pure bacterial solution is easier to pass through the filter membrane during filtration, reducing filtration resistance and improving filtration speed and quality, ensuring the retention rate of effective bacteria.

[0105] Homogenization and filtration further optimize the quality of the inoculant after membrane separation and concentration. High-pressure homogenization breaks the agglomeration of bacteria, making active ingredients more evenly distributed in the inoculant, further improving the stability and effectiveness of the inoculant. Filtration removes unwanted bacteria, ensuring the purity of the inoculant and avoiding damage to active ingredients during storage and use.

[0106] The application avoids the dependence on mechanical turning in the application of traditional microbial inoculants through synergistic effect. Firstly, Trichoderma reesei forms dense mycelial network through solid-state pre-culture, and the mycelial fragments secreted by Trichoderma reesei continuously wrap Bacillus in liquid fermentation, thereby constructing a “natural fiber network” structure. The bacteria can naturally disperse after being applied to the soil, thereby avoiding aggregation or loss caused by gravity sedimentation or water flow scouring. At the same time, Lactobacillus acidophilus produces extracellular polysaccharide (EPS) to form a mucilage layer, which further wraps Bacillus, isolates the physical extrusion of soil particles, and resists the influence of deep soil compaction on the activity of bacteria. Furthermore, the porous structure of biochar adsorbs heavy metal ions, thereby reducing the toxicity of the soil; citric acid is added in batches to buffer the acid-base fluctuation, thereby maintaining the metabolic homeostasis of the bacteria. The synergistic effect of trehalose and proline enhances the drought and salt tolerance of the bacteria through extracellular osmotic pressure regulation and intracellular ion balance. The chemical protection system enables the microbial inoculant to maintain activity in extreme soil environments without the need to adjust the distribution of the microbial flora through turning. The bacteria fragments and impurities are removed through ultrafiltration and nanofiltration classification treatment, and the intact Bacillus and Trichoderma mycelium are retained; high-pressure homogenization breaks the bacterial aggregates, thereby homogenizing the particle size of the microbial inoculant. The process optimizes the physical state of the microbial inoculant, so that the microbial inoculant naturally disperses in the form of single cells or micro-aggregates, thereby adapting to the soil permeation characteristics and avoiding the problem of excessively high local concentration or uneven distribution caused by aggregation. Bacillus amyloliquefaciens secretes surfactin to inhibit pathogenic bacteria, Trichoderma reesei produces chitinase to degrade the cell wall of pathogenic bacteria, and Lactobacillus acidophilus produces lactic acid and EPS to form an acidic microenvironment. The metabolic products of multiple strains complement each other to form a sustained antibacterial network, and the soybean meal hydrolysate induces Trichoderma to produce enzymes, and the enzyme hydrolysate promotes the secretion of antibiotics by Bacillus. The metabolic interaction mechanism prolongs the functional activity period of the microbial flora, and there is no need to stimulate metabolic response through turning.

[0107] The microbial inoculant of the application improves the inhibition rate of soil-borne pathogens and reduces the incidence of crop rhizosphere diseases through the synergistic effect of multiple strains and functional enhancement mechanism, and further optimizes the yield and quality of crops. The microbial inoculant retains high-activity microbial flora through gradient fermentation and membrane separation technology, and maintains the sustained colonization ability of the bacteria in deep soil in combination with the porous slow-release characteristics of biochar. Even without mechanical turning, a long-term stable field protection barrier can be formed through microbial flora interaction, thereby realizing the dual gain of disease control and crop growth under no-tillage or reduced-tillage conditions.

[0108] The second aspect of the application refers to a liquid microorganism for agriculture, which is prepared by the preparation method of the liquid microbial inoculant for agriculture of the first aspect. The liquid microorganism for agriculture has all the beneficial effects of the first aspect, which will not be repeated here.

[0109] In order to better understand the technical solutions, the exemplary embodiments of the application will be described in more detail below with reference to the accompanying drawings. Embodiment

[0110] Step 1: Revive culture of strains;

[0111] Bacillus amyloliquefaciens: inoculate into LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl), 25-30℃, 200 rpm shaking culture for 12 h, and obtain the resuscitation bacterial solution.

[0112] Trichoderma reesei: inoculate into PDA liquid medium (200 mL / L potato extract, 20 g / L glucose), 25-30℃, 200 rpm shaking culture for 36 h, and collect the mycelium suspension.

[0113] Lactobacillus acidophilus: inoculate into MRS medium (10 g / L beef extract, 5 g / L yeast extract, 1 mL / L Tween 80), 35-40℃ anaerobic culture for 18 h, and obtain the resuscitation bacterial solution.

[0114] Step 2: Gradient expansion and pre-culture;

[0115] Bacillus amyloliquefaciens gradient expansion;

[0116] Primary seed solution: inoculate into liquid medium containing soybean hydrolysate (5%) and sucrose (5%) at a 1% inoculation amount, 25-30℃, 200 rpm culture for 12 h, and the dissolved oxygen is ≥5%.

[0117] Secondary seed solution: inoculate into fermentation tank medium (soybean hydrolysate 3%, sucrose 3%) at a 0.5% inoculation amount, 25-30℃, 200 rpm culture for 14 h, and add 0.1% soybean hydrolysate to induce secondary metabolism.

[0118] Trichoderma reesei solid-state pre-culture: inoculate into rice hull substrate (80% rice hull + 20% bran, humidity 70%), 25-30℃ culture for 48 h, and transfer to potato glucose liquid medium after the mycelium is dispersed, add 0.1% maltose, 25-30℃, 200 rpm culture for 24 h.

[0119] Lactobacillus acidophilus anaerobic expansion: inoculate into MRS medium, inject nitrogen gas to an oxygen concentration of ≤0.1%, 35-40℃ static culture for 24 h, and produce EPS (concentration ≥1.5 g / L).

[0120] Step 3: Primary aerobic fermentation;

[0121] Mix Bacillus amyloliquefaciens (1× CFU / mL) and Trichoderma reesei mycelium (1× CFU / mL), add glucose (0.2%) and magnesium sulfate (0.02%), control the dissolved oxygen to be ≥5%, 25-30℃, 200 rpm fermentation for 16 h.

[0122] Step 4: Secondary micro-aerobic fermentation;

[0123] Lactobacillus acidophilus fermentation broth (1x CFU / mL) was added, the oxygen concentration was gradually reduced to 1%, hydrogen peroxide enzyme (100 U / g) and glutathione (0.1%) were added, and the fermentation was carried out at 35-40°C for 24h.

[0124] Step 5: Membrane separation and stabilizer treatment;

[0125] Membrane separation: ultrafiltration (100 kDa) to remove impurities, nanofiltration (500 Da) to desalt to EC≤1.5 mS / cm.

[0126] Step 6: Add biochar powder (1%) to 100 mesh, add 0.3% citric acid in 3 times (interval 15 min), adjust pH to 6.2±0.1, homogenize by high pressure homogenization (20 MPa).

[0127] Comparative Example 1

[0128] Step 2 adjustment: Trichoderma reesei revival broth was directly inoculated into potato dextrose liquid medium without rice husk substrate solid pre-culture, and the rest of the steps were the same as the example group.

[0129] Comparative Example 2

[0130] Step 2 adjustment: Lactobacillus acidophilus was expanded in aerobic conditions (MRS medium + 200 rpm shaking), without nitrogen control oxygen, and the EPS concentration was ≤0.5g / L.

[0131] Comparative Example 3

[0132] Step 6 adjustment: omit the addition of biochar and citric acid, only process the broth by membrane separation.

[0133] It should be noted that Table 1 shows the detection of the implementation index results of the microbial inoculant prepared based on the examples and comparative examples of the present application and the corresponding detection methods. The detection methods shown are prior art and will not be described here.

[0134] ;

[0135] ;

[0136] ;

[0137] The EPS concentration of the example group was 2.5 g / L, which was 39% higher than that in Comparative Example 2 without anaerobic expansion, proving the key role of Lactobacillus acidophilus anaerobic EPS production in stress resistance of the bacteria.

[0138] The salt stress survival rate of the example group was 89%, which was 53% higher than that of Comparative Example 3, indicating the protective effect of the biochar-citric acid system on extreme soil environment.

[0139] Referring to Table 3, the survival rate of the example group is 85% in 90 days, which is significantly better than that of Comparative Example 1 (55%) and Comparative Example 3 (48%), proving that the multi-strain synergistic metabolism and stabilizer system can prolong the functional cycle of the microbial community.

[0140] After the application of the example group, the EC value is reduced to 3.2 mS / cm, which is reduced by 45% compared with Comparative Example 1 (5.8 mS / cm), indicating that the combination of membrane separation and stabilizers effectively desalts and maintains the ion balance of the soil.

[0141] Referring to Table 2, the survival rate of the example group is 85% in 90 days, which is significantly better than that of Comparative Example 1 (55%) and Comparative Example 3 (48%), proving that the multi-strain synergistic metabolism and stabilizer system can prolong the functional cycle of the microbial community. Figure 2 , the survival rate of the example group is 85% in 90 days, combined with Table 2, the branch number of Comparative Example 1 is 2.8, and the branch number of the example group is 6.2, so it can be known that the survival rate of Comparative Example 1 is reduced from 68% to 55% because the mycelium network is missing and the spores are exposed to the soil environment. Combined with Table 3, the EC value of the example is 3.2, and that of Comparative Example 3 is 5.1. The microorganisms in Comparative Example 3 rapidly inactivate under high salt (EC 8 mS / cm) and drought stress because of the absence of protection by biochar and citric acid.

[0142] Referring to Table 2, the survival rate of the example group is 85% in 90 days, which is significantly better than that of Comparative Example 1 (55%) and Comparative Example 3 (48%), proving that the multi-strain synergistic metabolism and stabilizer system can prolong the functional cycle of the microbial community. Figure 2 In the 30-day control effect, the survival rate of the example group is 89%, which is increased by 23-37% compared with Comparative Examples 1-3 (65-72%); in the 90-day control effect, the survival rate of the example group is 85%, which is increased by 25-40% compared with Comparative Examples 1-3 (48-60%). Based on the dual protection mechanism of solid-state pre-culture and stabilizers, the survival cycle of the microorganisms is prolonged, and the protection effect of the single process of the comparative examples drops sharply.

[0143] Obviously, the above is only an example for the sake of clarity, and is not a limitation on the embodiments. Based on the above description, those skilled in the art can make other different forms of changes or variations. Here, it is not necessary or possible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

[0144] In the description of the present application, it should be understood that the terms "first", "second" are only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features limited by "first", "second" can explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of "multiple" is two or more, unless otherwise specifically limited.

[0145] In the description of the specification, the description of the terms "one", "some", "example", "specific example" or "some examples" or the like means that the specific feature, structure, material or characteristic described in connection with the or examples is included in at least one or examples of the present application. In the specification, the illustrative representation of the above terms does not necessarily refer to the same example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in one or more or examples. Furthermore, the person skilled in the art can combine and combine the different examples described in the specification and the features of the different examples, without mutual contradiction.

[0146] It can be understood that the above is exemplary and cannot be understood as a limitation of the present application. Those skilled in the art can modify, modify, replace and modify the above within the scope of the present application.

Claims

1. A method for preparing liquid microbial inoculant for agricultural use, characterized in that, include: Step 1: Inoculate Bacillus amyloliquefaciens, Trichoderma reesei, and Lactobacillus acidophilus into their respective culture media and culture them to obtain the corresponding revived bacterial solutions; Step 2: Perform gradient expansion culture of the reactivated Bacillus amyloliquefaciens broth obtained in Step 1; perform solid-state pre-culture of the reactivated Trichoderma reductoid broth; and perform anaerobic expansion culture of the reactivated Lactobacillus acidophilus broth. Step 3: Mix the Bacillus amyloliquefaciens obtained in Step 2 with Trichoderma reesei at a ratio of (8-10):1 and carry out primary aerobic fermentation. Add glucose and magnesium sulfate during fermentation. Step 4: Mix the product from the primary aerobic fermentation with the expanded Lactobacillus acidophilus and carry out secondary microaerobic fermentation. Add catalase and glutathione during the fermentation process. Step 5: The product from the secondary microaerobic fermentation is subjected to membrane separation to desalinate and concentrate the bacterial solution; Step 6: Add biochar and citric acid to the concentrated bacterial solution and homogenize it.

2. The method for preparing an agricultural liquid microbial inoculant according to claim 1, characterized in that, In step 1, Trichoderma reesei spores are inoculated into liquid culture medium, the temperature is controlled at 25-30℃, and the culture is shaken for 30-48 hours. The mycelial suspension is collected to obtain Trichoderma resurrected bacterial solution.

3. The method for preparing an agricultural liquid microbial inoculant according to claim 1, characterized in that, In step 2, the solid-state pre-culture includes inoculating the Trichoderma resurrection culture obtained in step 1 into a rice husk substrate, dispersing the mycelium, transferring the pre-cultured mycelium to potato dextrose liquid medium, and adding maltose during fermentation.

4. The method for preparing an agricultural liquid microbial inoculant according to claim 1, characterized in that, In step 2, the gradient expansion culture includes transferring the revived Bacillus amyloliquefaciens broth obtained in step 1 to a liquid culture medium containing soybean meal hydrolysate and sucrose at an inoculation rate of 1%-2%, controlling the temperature at 25-30℃, and culturing for 10-14 hours to obtain the primary seed culture; The primary seed culture was transferred to the fermenter medium at an inoculation rate of 0.3%-0.5% and cultured for 12-16 hours to obtain the secondary seed culture.

5. The method for preparing an agricultural liquid microbial inoculant according to claim 1, characterized in that, In step 2, the anaerobic expansion culture includes transferring the revived Lactobacillus acidophilus culture obtained in step 1 to the culture medium at an inoculation rate of 1-2% and placing it in an anaerobic tank, injecting nitrogen gas until the oxygen concentration is ≤0.1%, and incubating it statically at 35-40℃ for 18-24 hours.

6. The method for preparing an agricultural liquid microbial inoculant according to claim 1, characterized in that, Membrane separation treatment involves using an ultrafiltration membrane to retain molecules with a molecular weight greater than 100 kDa, followed by a nanofiltration membrane to retain molecules with a molecular weight greater than 500 kDa, and then desalination treatment.

7. The method for preparing an agricultural liquid microbial inoculant according to claim 1, characterized in that, After adding biochar, add citric acid at a mass fraction of 0.2%-0.3% in multiple batches.

8. The method for preparing an agricultural liquid microbial inoculant according to claim 4, characterized in that, In step 2, feeding is performed during the gradient amplification process; During the primary seed culture stage of Bacillus amyloliquefaciens, if fermentation exceeds 12 hours, supplement with 0.1%-0.3% glucose and 0.01%-0.05% magnesium sulfate to maintain dissolved oxygen ≥5%. During the secondary seed culture stage, if fermentation lasts longer than 24 hours, 0.1% soybean meal hydrolysate is added to induce the secretion of secondary metabolites.

9. The method for preparing an agricultural liquid microbial inoculant according to claim 7, characterized in that: In step 6, the biochar and citric acid are added by pretreating the biochar, activating the rice husk char at 600℃-630℃ for 2-3 hours, grinding it to 100-200 mesh, and adding it at a mass fraction of 1%-2%. Add 0.2%-0.3% citric acid in 3-5 portions, with an interval of 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 as described in any one of claims 1-9.

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