Synergistic organic-inorganic complex microbial inoculant for promoting yield increase of rice and preparation method of synergistic organic-inorganic complex microbial inoculant

Abstract

The invention relates to the technical field of organic-inorganic complex microbial inoculant manufacturing, and discloses a synergistic interaction type organic-inorganic complex microbial inoculant for promoting rice yield increase and a preparation method thereof.The preparation method comprises the steps that an organic carrier is subjected to pH adjustment to form an active interface with negative charges, then two multivalent metal cations with different coordination capacities are adopted to construct a gradient stable structure, and the gradient stable structure is formed; microorganisms and inorganic nutrients are jointly anchored on an interface in situ in an ion bridge form, and finally, low-temperature solidification is performed, and through a chemical structure pre-constructed in a manufacturing stage, each functional component forms an inseparable synergistic unit, so that functional dissociation and action opportunity mismatch caused by physical scouring of the functional components in soil are avoided, and the soil remediation efficiency is improved. And an internal response relationship between nutrient supply and microorganism demand is established.

Description

TECHNICAL FIELD

[0001] The present application relates to a synergistic organic-inorganic compound microbial agent for promoting rice yield increase and a preparation method thereof, and belongs to the technical field of organic-inorganic compound microbial agent manufacturing. BACKGROUND

[0002] In the field of organic-inorganic compound microbial agent manufacturing, combining organic carriers, inorganic nutrients and beneficial microorganisms in the same particle is a recognized technical strategy to achieve synergistic effects. Therefore, the industry generally uses physical methods such as mechanical mixing and spray granulation to aggregate the independently pretreated components. The main goal is to obtain a particle product that is uniform in composition and has sufficient physical strength to meet the requirements of storage, transportation and mechanized application.

[0003] However, when such products made by physical methods are applied to the dynamic water environment of rice fields, the mismatch between the manufacturing method and the application conditions leads to functional deficiencies. The manufacturing method only achieves physical aggregation of components in product form and does not establish a functional stable connection between components. When the particle encounters water, water-soluble inorganic nutrients will quickly dissolve and migrate with the water flow, spatially separating from the microorganisms still attached to the organic carriers. At the same time, microorganisms need a certain start-up time from dormancy to activity, which causes a time lag, missing the window period of using the available nutrients, and making the pre-set synergistic goal difficult to achieve due to the functional dissociation of components in the application environment.

[0004] To delay nutrient loss, the industry also uses methods such as coating or enhancing adhesion to improve the physical integrity of the particles. However, these improvements are still limited to the macro-physical structure, i.e., slowing down the overall disintegration rate of the particles. The core issue that this approach cannot solve is that once the components are dissolved from the particles, they are still independent units in the soil microenvironment, following their inherent physical and chemical migration rules. The functional fragmentation between them has not changed. Specifically, the existing technology mainly has the following deficiencies: 1. Separation of functional components, i.e., water-soluble nutrients and microbial carriers are spatially separated under the action of soil water flow, hindering the synergistic effect; 2. Mismatch of action timing, i.e., the rapid loss of available nutrients and the slow activation of microorganisms do not match in time, reducing the effectiveness of nutrient utilization; 3. Lack of functional connection, i.e., the manufacturing process fails to give the internal stable connection of the components to interact in the soil microenvironment. Therefore, how to provide a manufacturing method that can pre-construct a micro-unit with stable structure and integrated function during the preparation of fertilizer particles, in which inorganic nutrients, microorganisms and organic carriers are inseparably combined, so that the release of nutrients is directly related to the life activities of microorganisms, has become a technical problem to be solved by the present application. SUMMARY

[0005] The present application provides a synergistic organic-inorganic compound microbial agent for promoting rice yield increase and a preparation method thereof, and aims to solve the problem of the lack of internal functional connection of the components of the compound microbial agent manufactured by physical mixing in the prior art.

[0006] To achieve the above-mentioned purpose, the present application provides a preparation method of a synergistic organic-inorganic compound microbial agent for promoting rice yield increase, comprising the following steps executed in the following order: Step a, placing an organic carrier containing an organic acid functional group in a weak alkaline chemical environment with a pH value of 7.5 to 8.5 to form a negatively charged active colloid interface on the surface of the organic carrier; Step b, mixing an aqueous suspension containing microorganisms and inorganic nutrients with a solution containing a first type of multivalent metal cation and a second type of multivalent metal cation to obtain a mixture, wherein the coordination ability of the second type of multivalent metal cation to the organic acid functional group on the surface of the organic carrier is stronger than that of the first type of multivalent metal cation; Step c, applying the mixture obtained in step b to the negatively charged active colloid interface in step a, constructing a basic ion cross-linking network resistant to physical washing on the surface of the organic carrier by interfacial complexation of the first type of multivalent metal cation with the active colloid interface, and forming coordination lock points in the basic ion cross-linking network that have specific responses to chelators in plant root exudates by using the second type of multivalent metal cation, thereby anchoring the microorganisms and inorganic nutrients together on the surface of the organic carrier to form a composite structure; Step d, drying the composite structure at a low temperature below 50 to solidify the composite structure formed by interfacial complexation.

[0007] Preferably, the organic carrier is a mixture of rapeseed oil cake and humic acid; the first type of multivalent metal cation is calcium ion; and the second type of multivalent metal cation is zinc ion or manganese ion.

[0008] Preferably, in step a, a chemical reducing agent is added at the same time as the organic carrier is placed in the weak alkaline chemical environment to pre-convert the quinone functional group on the surface of the organic carrier into a reduced state of hydroquinone group, thereby forming an antioxidant microenvironment on the interface.

[0009] Preferably, in step b, the first type of multivalent metal cation and the second type of multivalent metal cation are provided in the form of a pre-prepared water-soluble heteronuclear metal complex precursor containing both metal ions.

[0010] Preferably, in step c, the molar amount of the first type of multivalent metal cation is The ratio of the molar amount of the organic acid functional groups on the surface of the organic carrier to the molar amount of the first type of polyvalent metal cations is limited to a sub-saturation range which satisfies: wherein, represents the total molar amount of the first type of polyvalent metal cations, represents the total molar amount of the organic acid functional groups provided by the organic carrier and involved in the complexation.

[0011] Preferably, the microorganism in step b comprises at least one of Bacillus subtilis and Bacillus mycoides; the microorganism anchored in step c is in a dormant state; the inorganic nutrient in step b comprises phosphate or sulfate; in step c, the phosphate or sulfate anions in the inorganic nutrient form a slightly soluble precipitate with the first type of polyvalent metal cations and the second type of polyvalent metal cations and are locked in situ in the complex structure being formed.

[0012] Preferably, the chemical reducing agent is sodium sulfite or ascorbic acid.

[0013] Preferably, the water-soluble heteronuclear metal complex precursor is a calcium-zinc-citrate complex or a calcium-manganese-citrate complex prepared by reacting an alkali containing calcium ions with an oxide containing zinc ions or manganese ions and citric acid in an aqueous solution.

[0014] Preferably, in step c, the mixture is sprayed in atomized form onto the surface of the organic carrier in a state of dynamic stirring; in step d, the low-temperature drying is continued until the final water content of the composite structure is less than 10%.

[0015] Compared with the prior art, the present application has the following advantages: 1. By adjusting the pH of the organic carrier containing organic acid functional groups to form a negatively charged active colloidal interface, and then applying a suspension containing microbial inorganic nutrients and polyvalent metal cations thereon, a manufacturing method different from traditional physical mixing is established; in this method, the organic carrier is no longer just an inert filler, but becomes a reaction skeleton, and the surface-activated functional groups of the organic carrier are bridged by polyvalent metal cations to fix the microbial cells and inorganic nutrient anions in situ, forming a composite structure in which the organic carrier is the skeleton and the three components are tightly combined and inseparable by chemical forces, which makes the components form stable synergistic units during the manufacturing stage before being applied to the soil, avoiding the problem of spatial separation of functional components and mismatch of action timing in traditional fertilizers due to physical washing in the soil.

[0016] 2、Based on the formed composite structure, the inorganic nutrients inside are not in a simple physical form, but are locked in the form of slightly soluble precipitates or complexes on the colloidal interface inhabited by microorganisms. This structure makes the release rate of nutrients no longer mainly dependent on its own solubility, but directly related to the life activities of microorganisms. The organic acids produced by microbial metabolism locally change the pH of the microenvironment at the interface, thereby affecting the balance of ion complexation, thus establishing an internal response relationship between nutrient supply and microbial demand, so that nutrients can be gradually released with the activity of microorganisms, realizing the synchronization of nutrient supply timing and microbial demand in the rhizosphere of crops.

[0017] 3、When adjusting the pH in step a, the chemical reducing agent is added to the organic carrier. The method also utilizes the quinone structure of humic acid in the organic carrier during the construction of the composite structure. The introduced reducing agent converts the quinone group at the carrier interface into a reduced hydroquinone group in advance, which makes the organic skeleton of the finally formed composite structure itself have long-term antioxidant ability. In the subsequent storage and transportation process, the oxygen invading the particles will preferentially react with the more active hydroquinone group, thereby providing a continuous and spontaneous biochemical protection microenvironment for the dormant microbial cells fixed on the interface, avoiding the attenuation of the number of active bacteria due to oxidative stress damage.

[0018] 4、When the multivalent metal cations in step c contain at least two metal cations with significantly different coordination abilities for organic acid functional groups, such as calcium ions and zinc ions, the composite structure constructed by the method forms a locking system with gradient stability inside. The calcium ions with weaker coordination ability form a basic ion crosslinking network resistant to physical erosion, while the zinc ions with strong coordination ability form a few but stable coordination locking points. This structure shows high resistance to non-specific acidic signals such as hydrogen ions commonly present in the soil environment, avoiding premature disintegration of the particles, and shows specific response to specific chelating agents such as citric acid secreted by plant roots. Because the affinity of these chelating agents for zinc ions is sufficient to destroy the key coordination locking points, triggering the accelerated disintegration of the entire structure, realizing the targeted release of functional components in the rhizosphere microdomain, the product still maintains its on-demand release characteristics in complex soil environments such as acidic or alkaline environments. BRIEF DESCRIPTION OF DRAWINGS

[0019] Fig. 1 Schematic diagram of the preparation and functional release process of the composite microbial agent of the present application; Fig. 2 Test data graph showing the influence of key stoichiometric ratios on product performance of the present application; Fig. 3 Schematic diagram of the four-stage intelligent release mechanism of the composite microbial agent of the present application. DETAILED DESCRIPTION

[0020] In order to make the technical solutions and advantages of the present application clearer, the present application will be further described in detail below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0021] The present application discloses a synergistic organic-inorganic compound microbial agent for promoting rice yield increase and a preparation method thereof. The purpose is to form a synergistic organic-inorganic compound microbial agent with integrated structure and function in the manufacturing stage by chemical construction. The overall process mainly includes the preparation and application of the chemical activation functional component suspension on the surface of the organic carrier, the interface complex anchoring based on the multivalent metal cation, and the final low-temperature solidification forming. Through the regulation of the chemical conditions of each stage, the granular microbial agent formed finally has an inseparable unit between the internal functional components, so that in soil application, the separation of each component due to physical washing and the action time sequence mismatching are avoided.

[0022] In one specific embodiment, the starting step of the preparation method, i.e. step a, involves the pretreatment of the organic carrier as the basic skeleton, in order to change the condition that the organic carrier only serves as an inert physical filler, the method places the organic carrier containing organic acid functional groups in a weakly alkaline chemical environment with a pH value of 7.5 to 8.5, the organic carrier is preferably a mixture of rapeseed oil cake and humic acid, in which the rapeseed oil cake provides the basic organic matter, and the humic acid is rich in a large number of carboxyl and phenolic hydroxyl functional groups; under this pH condition, the deprotonation reaction of these organic acid functional groups occurs, thereby forming an active colloidal interface with stable negative charge on the solid-liquid interface of the organic carrier particles, which changes the function of the organic carrier as only a physical filler and makes it a pre-set reaction skeleton ready for subsequent chemical reactions; in order to improve the shelf life stability of the product, while adjusting the pH of the organic carrier in step a, a chemical reducing agent can also be added to the system, which is preferably sodium sulfite or ascorbic acid, the mechanism of which is that the surface of the humic acid component in the organic carrier contains quinone functional groups, and the introduced chemical reducing agent can convert these quinone functional groups into more active reduced hydroquinone groups in a weakly alkaline environment, which pre-stores the reducing capacity on the carrier interface, so that in the subsequent storage and transportation process, the oxygen invading the interior of the particles will be preferentially reacted with the hydroquinone groups and consumed, thereby providing a sustained anti-oxidation microenvironment for the dormant microbial cells anchored on the interface to avoid the decline in the number of live bacteria due to oxidative stress damage; the next step b is the preparation of a functional component suspension, which aims to integrate the microorganisms that need to be anchored later and inorganic nutrients into a unified liquid system, in this step, the aqueous suspension containing microorganisms and inorganic nutrients is mixed with a solution containing the first and second types of multivalent metal cations to obtain a mixture, wherein the microorganisms can include at least one of Bacillus subtilis and Bacillus mycoides, which exist in the form of dormant state, and the inorganic nutrients can include phosphates or sulfates commonly used in agricultural production, the coordination ability of the second type of multivalent metal cation with the organic acid functional groups on the surface of the organic carrier is stronger than that of the first type of multivalent metal cation, for example, the first type of multivalent metal cation can be selected as calcium ion, and the second type of multivalent metal cation can be selected as zinc ion or manganese ion with stronger coordination ability, this gradient selection of coordination ability of different cations is the basis for constructing the subsequent responsive release structure.

[0023] To cope with the problem of micro-distribution inhomogeneity caused by random competitive reactions when two independent metal salt solutions are co-sprayed in large-scale industrial production, in a preferred embodiment, the first type of polyvalent metal cation and the second type of polyvalent metal cation in step b are provided in the form of a pre-prepared water-soluble heteronuclear metal complex precursor containing both metal ions. For example, a stable calcium-zinc-citrate complex or a calcium-manganese-citrate complex can be prepared by reacting calcium hydroxide containing calcium ions with zinc oxide or manganese oxide containing zinc ions or manganese ions, respectively, with citric acid in an aqueous solution. The use of such a single precursor solution ensures that the two metal ions are delivered to the reaction interface in a precise stoichiometric ratio inherent in the molecule, thereby ensuring the functional homogeneity and reliability of the final product. Step c is the key step in forming the composite structure of the present application. In this step, the mixture obtained in step b is continuously sprayed in atomized form onto the surface of the organic carrier treated in step a under dynamic stirring. In this process, the polyvalent metal cations in the mixture form an interfacial complexation with the negatively charged active colloidal interface on the surface of the organic carrier, thereby anchoring the microbial cells and inorganic nutrients in the suspension to the surface of the organic carrier to form a composite structure. Specifically, the first type of polyvalent metal cation, i.e. calcium ions, which have relatively weak coordination ability, will form ionic cross-linking with a large number of organic acid functional groups to build a basic ionic cross-linking network on the surface of the organic carrier that resists physical washing by conventional soil water flow. The second type of polyvalent metal cation, i.e. zinc ions or manganese ions, which have stronger coordination ability, will form a small number of chemically very stable coordination bonds with some organic acid functional groups in this basic network to form coordination lock points that are specifically responsive to specific chelating agents in plant root exudates. This gradient stable structure allows the bacterial agent particles to remain structurally intact in a conventional soil environment, but when they come into contact with the multi-dentate chelating agents such as citric acid excreted by rice roots, the affinity of these chelating agents for zinc ions or manganese ions is sufficient to disrupt the key coordination lock points, thereby triggering the accelerated disintegration of the entire structure and achieving targeted release of functional components in the rhizosphere microdomain.

[0024] To achieve the above-mentioned targeted disintegration property, the stoichiometric ratio of the key components in step c is limited, i.e. the molar ratio of the first type of polyvalent metal cation to the molar amount of organic acid functional groups on the surface of the organic carrier is limited to a subsaturation range, which satisfies: wherein, represents the total molar amount of the first type of polyvalent metal cation, represents the total mole amount of organic acid functional groups provided by the organic carrier and involved in the complexation, and the setting of the range is based on the fact that if the ratio is lower than 0.33, the ion cross-linked network formed is not dense enough, and the composite structure is physically unstable in the soil water environment and prone to premature disintegration, and if the ratio is higher than 0.5, the cross-linked network is too dense and stable, and even when contacted with the plant root exudates as strong metal ion chelators, the response of the structure disintegration becomes sluggish and cannot timely release nutrients and microorganisms, therefore, limiting the ratio to the subsaturation range is a necessary engineering implementation means for the composite structure to obtain the properties of maintaining structural stability in the conventional soil water environment and targeted structural disintegration in the rhizosphere microdomain; in addition, during the complexation process of step c, the inorganic nutrients in step b are also fixed in situ by chemical action, specifically, the phosphate or sulfate anions in the inorganic nutrients will react with the first type of multivalent metal cations and the second type of multivalent metal cations in the system to form slightly soluble precipitates such as calcium phosphate or zinc phosphate, and are locked in situ in the complexing structure being formed, which makes the release rate of inorganic nutrients no longer mainly dependent on its own solubility, but directly related to the stability of the entire complexing structure and the life activities of microorganisms, for example, the organic acids produced by microbial metabolism can locally change the pH of the interface microenvironment, thereby affecting the balance of ion complexation, and thus a response relationship is established between nutrient supply and microbial demand; the last step d is to dry the formed composite structure at low temperature to finally solidify the complex structure formed by the interface complexation, in order to protect the biological activity of the anchored microbial bodies, the drying process is carried out at a temperature lower than 50 °C, and continues until the final moisture content of the composite structure is less than 10%, this step gently removes the water in the system, so that the composite structure of organic carrier-metal ion-microorganism-inorganic nutrient self-assembled in the liquid phase is stably solidified to form the final granular product; the synergistic organic-inorganic composite microbial agent directly obtained by the above preparation method is a granular composite structure, which microscopically contains an organic carrier as the core, and an interface complexing layer completely covering the surface of the organic carrier core, the interface complexing layer contains microorganisms, inorganic nutrients, first type of multivalent metal cations and second type of multivalent metal cations, wherein the microorganisms and inorganic nutrients are anchored together on the surface of the organic carrier core through the first type and second type of multivalent metal cations to form a basic ion cross-linked network and a gradient stable structure with specific response to chelators in plant root exudates.

[0025] In one specific embodiment, to obtain a composite microbial agent possessing both structural stability and targeted responsiveness, a preparation scheme comprising the following components and process parameters is provided: an organic carrier core, composed of 500g rapeseed oil cake and 500g humic acid (dry basis), wherein the total molar amount of organic acid functional groups available from 1000g of the organic carrier at pH 8.5 is determined by potentiometric titration. The functional suspension consists of 2.0 mol of deionized water, 100 g of potassium dihydrogen phosphate (as an inorganic nutrient), 50 g of dormant Bacillus subtilis powder, and a metal cation precursor solution. The metal cation precursor solution contains 0.4 mol of calcium hydroxide. With 0.1 mol zinc oxide The reaction was carried out in a sufficient amount of aqueous citric acid solution to obtain a clear calcium-zinc-citrate complex precursor solution, which provides a first-class multivalent metal cation for the subsequent interfacial complexation step. Its total molar amount It is 0.8 mol; and provides a second type of multivalent metal cation. Its total molar amount is 0.1 mol. Therefore, the ratio of the molar amount of the first type of polyvalent metal cations to the molar amount of the organic acid functional groups on the surface of the organic support is... The value was set to 0.4, falling within the range of 0.33 to 0.5. In this technical solution, the roles and interrelationships of each core component are defined as follows: the organic carrier (a mixture of rapeseed oil cake and humic acid) serves as the reaction framework; its surface carboxyl and phenolic hydroxyl functional groups are deprotonated in an environment of pH 7.5 to 8.5, forming a negatively charged reaction interface, providing anchoring points for subsequent ion complexation; the first type of multivalent metal cation... As a basic structural building agent, it undergoes interfacial complexation with the organic acid functional groups on the surface of the organic carrier, forming a basic ionic cross-linking network covering the entire carrier surface. The main function of this network is to provide the composite structure with macroscopic stability against the physical erosion of soil water flow; second type of multivalent metal cations Its coordination ability with organic acid functional groups is stronger than In the basic ionic cross-linked network, a few chemically stable coordination locking sites are formed. These locking sites are crucial for coordination in conventional soil environments. It is not sensitive, but it has a specific response to multidentate chelating agents (such as citric acid) in plant root exudates.

[0026] In a typical southern acidic red soil rice planting area, the area encounters sustained heavy rainfall at the early tillering stage after rice transplanting, for the organic and inorganic compound microbial agent prepared by using the conventional physical mixing method, after application, the available nitrogen and potassium nutrients in the particles are rapidly dissolved and lost with surface runoff, and the microorganisms such as Bacillus subtilis attached to the organic carrier need a certain start-up time from the dormant state to the active period, when they begin to proliferate and play a role in the new environment, the available nutrients required for their growth have been largely lost, resulting in slow growth of rice seedlings and yellowing of leaves, at the same time, the lost nutrients enter the surrounding water, causing eutrophication of the water body; under the same soil and climate conditions, the application of the synergistic organic and inorganic compound microbial agent prepared by the method of the application, the granular composite structure, because each functional component has been anchored on the surface of the organic carrier core by interfacial complexation, forms a basic ion crosslinking network constructed by the first type of multivalent metal cation calcium ion, which maintains the integrity of the structure under the physical washing of heavy rain, and stably maintains the microorganisms and inorganic nutrients such as phosphate fixed in situ in the rhizosphere soil of rice, avoiding the spatial separation of functional components and the immediate loss of nutrients.

[0027] With the growth of rice roots, the root tips begin to secrete organic acids such as citric acid and malic acid, when these plant root secretions as strong metal ion chelators contact the surface of the composite structure, they will react with a few but stable coordination lock points formed by the second type of multivalent metal cation zinc ion in the complex structure, due to the affinity of these chelators for zinc ions, which is enough to destroy the key coordination lock points, and then trigger the accelerated disintegration of the entire gradient stable structure, this process makes the fixed microorganisms and inorganic nutrients concentratedly released at the most active absorption interface of rice roots, so that the nutrient supply and crop demand tend to be synchronized in time and space; finally, in this application scenario, the rice tillering period grows healthily, the root system is developed, the leaf color is dark green, and the soil nutrient monitoring data in the field shows that the effective phosphorus concentration in the rhizosphere area is maintained at a high level, while the nitrogen and phosphorus contents at the field drainage outlet are significantly lower than those in the control area applying conventional fertilizers, the application of the composite structure not only meets the nutrient demand of crops, but also reduces the impact on the surrounding environment caused by nutrient loss.

[0028] Example 2: To verify the effect of the composite structure prepared by the method of the present invention on nutrient retention and plant growth, the following pot simulation experiment was set up. Acidic red soil was used as the cultivation substrate and filled into polyethylene experimental flower pots of uniform size. The test crop was rice. The entire experiment was conducted in a greenhouse to maintain consistent environmental conditions such as temperature, light, and humidity to eliminate interference from non-experimental factors. This experiment set up five treatment groups, namely: the experimental group, which used the synergistic organic-inorganic composite microbial agent prepared by the complete method of the present invention, wherein the molar amount of the first type of polyvalent metal cation was... Molar amount of organic acid functional groups on the surface of organic carrier The ratio was set to 0.4; Control group 1 consisted of the same material components as the experimental group, which were physically and mechanically mixed without pH adjustment and chemical complexation steps; Control group 2 used the preparation method of this invention, but in step b, only the first type of polyvalent metal cation calcium ions were used, without adding the second type of polyvalent metal cation zinc ions; Control group 3 used the complete preparation method of this invention, but... and The ratio was set to 0.2; control group 4 used the complete preparation method of this invention, but with... and The ratio is set to 0.7.

[0029] In each treatment group, fertilizer was evenly applied to the potting substrate according to the equivalent nitrogen application rate. On the 7th day after fertilization, a simulated heavy rainfall leaching test was conducted on all potted plants. That is, an equal amount of deionized water was evenly applied to the surface of each potting substrate, and all leachate was collected. The concentration of phosphorus in the leachate was determined by inductively coupled plasma atomic emission spectrometry. Afterward, the rice was cultivated for 90 days, and the agronomic traits of the plants were recorded regularly. At the end of the experiment, the aboveground parts were harvested, dried, and weighed to obtain biomass data. The leaching test and crop growth data are summarized in Table 1.

[0030] Table 1: Comparison of nutrient leaching and crop growth data in different treatment groups.

[0031] Referring to Table 1, the phosphorus concentration data of the leachate show that the phosphorus loss in the experimental group was 1.8 mg / L, while that in control group 1 was 15.6 mg / L, indicating that the physical mixture without chemical complexation cannot effectively retain water-soluble nutrients. The phosphorus loss in control group 2 was 5.3 mg / L, higher than that in the experimental group, showing that the lack of coordination locking sites for the formation of second-type polyvalent metal cations reduces the stability of the overall structure. Control group 3, due to... and The ratio was 0.2, lower than the optimal range, indicating insufficient ionic cross-linking network and a phosphorus loss of 12.8 mg / L. The phosphorus loss in control group 4 was 2.1 mg / L, close to the experimental group, but its crop growth data, especially the number of effective tillers and aboveground dry weight, were lower than the experimental group, indicating excessive phosphorus loss. and While the ratio enhances structural stability, it also reduces the structure's responsiveness to plant root exudates, affecting the timely release of nutrients. Considering growth indicators such as plant height, effective tiller number, and aboveground dry weight, all values ​​in the experimental group were higher than those in all control groups. This result corresponds to the lowest nutrient loss rate and the release characteristics regulated by the gradient-stabilized structure. The experimental data show that the preparation method of this invention, by constructing a specific chemical structure and controlling the stoichiometry of key components within a defined subsaturated range, can maintain nutrient stability while responding to crop rhizosphere signals for release, thereby improving nutrient utilization efficiency and crop biomass.

[0032] To verify the protective effect of adding a chemical reducing agent in step a on the microbial activity of the composite structure, a comparative experiment was set up. Experimental sample group A used the complete preparation method of the experimental group in Example 2, i.e., ascorbic acid was added as a chemical reducing agent in step a. Comparative sample group B used the exact same preparation method as experimental sample group A, but without adding any chemical reducing agent in step a. Both groups of samples were heated at 30°C. Accelerated storage tests were conducted under conditions of 75% relative humidity. After 6 months of storage, the number of viable bacteria in the two groups of samples was determined by plate counting. The results showed that the decay rate of viable bacteria in experimental group A was lower than that in control group B. This data indicates that the antioxidant microenvironment pre-formed on the interface of the organic carrier by adding a chemical reducing agent effectively protects the fixed dormant microorganisms from oxidative stress damage during long-term storage.

[0033] Example 3: This example combines Figs. 1 to 3 This paper describes a synergistic organic-inorganic compound microbial agent for promoting rice yield and its preparation method. Fig. 1 As shown, the process begins in the raw material supply stage, where organic carriers and chemical reagents are fed into the activated organic carrier unit to form intermediate D1 activated organic carrier. Simultaneously, microorganisms, inorganic nutrients, and polyvalent metal cations are fed into the functional suspension preparation unit to form intermediate D2 functional suspension. Subsequently, intermediates D1 and D2 enter the interfacial complexation stage to generate wet composite structure D3. This wet structure undergoes low-temperature curing and molding treatment to obtain the final product D4 granular bacterial agent. After the final product enters the application environment of rice fields, its internal targeted release functional components are triggered by root exudate chelating agents, thereby releasing nutrients and microorganisms into the application environment.

[0034] like Fig. 2 As shown, the horizontal axis of the graph represents the ratio of the molar amount of type I polyvalent metal cations to the molar amount of organic acid functional groups on the surface of the organic support, i.e. / The ratio, with the ordinate representing the phosphorus leaching rate (%), is plotted on two curves. The solid line marked with circles represents the structural stability test results under deionized water leaching conditions, showing that the phosphorus leaching rate increases with... / The ratio increases and then decreases sharply before leveling off, while the dashed line marked with a triangle represents the target responsiveness test results under citric acid leaching conditions, showing that the phosphorus leaching rate is... / The ratio begins to decrease significantly after it exceeds 0.5; both curves together reveal that when... / When the ratio is in the range of 0.33 to 0.5, the composite structure can simultaneously maintain high structural stability in conventional aquatic environments and high targeting responsiveness in chelating agent environments.

[0035] like Fig. 3 As shown, the mechanism revolves around a core synergistic unit that cycles through the process. Step 1, interface activation, involves adjusting the pH to create a charged reaction interface on the organic carrier. Subsequently, in step 2, chemical construction, the interface is activated by... and The ion bridges and coordination locking sites formed by the multivalent metal cations represented by the cation anchor the functional components, thus entering step 3 to stabilize and wait, forming a structurally stable particulate product to resist physical erosion. Finally, in step 4, the intelligent release stage, the root system signal triggers the structural disintegration and releases nutrients in a coordinated manner. The released components then affect the pH of the rhizosphere microenvironment, providing feedback to the interface state in step 1, thereby forming a functional closed loop.

[0036] Example 4: This example provides a standardized engineering calibration procedure for determining the preferred subsaturation range of the preparation method of the present invention when applied to a novel organic support containing organic acid functional groups. This procedure is used to determine the molar amount of the first type of polyvalent metal cation. Molar amount of organic acid functional groups on the surface of the new organic carrier The proportional relationship between them; first, the new organic carrier is characterized to determine its The value of the organic acid functional group content of the organic carrier is determined as follows: a certain mass of dry organic carrier sample is placed in deionized water to form a suspension, and the pH of the solution is monitored using an acidimeter with a resolution of 0.01 pH unit. The suspension is potentiometrically titrated with a standard sodium hydroxide solution of known concentration under constant stirring, and the titration end point is set at pH 8.5. The total molar amount of organic acid functional groups per unit mass of the organic carrier that can participate in the reaction at this pH is calculated based on the volume and concentration of the sodium hydroxide standard solution consumed ; then, based on the determined value, a series of test samples are prepared in which the ratio of and is systematically changed while all other components and process parameters remain consistent with the above, and the ratio is set as a series of gradients starting from 0.1, increasing by 0.1 to 0.8, thereby obtaining eight groups of composite structure particle samples with the ratio of and being 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8, respectively.

[0037] Next, the eight groups of samples are subjected to two performance tests respectively. The first is a structure stability test in which a fixed mass of sample particles is placed in a chromatographic column filled with quartz sand, and eluted with deionized water at a constant flow rate. All the eluate collected within a specified time is measured for the concentration of phosphorus as a tracer. The second is a target responsiveness test using the same device and operation process, but replacing the eluent with a solution containing a specific concentration of citric acid, and measuring the concentration of phosphorus in the eluate within the same time. Finally, the data obtained from the two tests are analyzed. When the ratio of and is less than 0.3, the phosphorus elution rate in the structure stability test shows a jump; when the ratio is higher than 0.6, the phosphorus elution rate in the target responsiveness test shows a sharp drop. Through the inflection point of the change in the two test data, an optimal subsaturation range that takes into account the structure stability and target responsiveness of the new organic carrier can be determined.

[0038] Example 5: To verify the structural stability and response specificity of the composite structure of the present application in different soil pH environments, the composite structure particles containing calcium ions and zinc ions prepared by the above method were placed in two kinds of buffer solutions simulating soil chemical environments for testing. The first solution had a pH value of 4.5 to simulate an acidic soil environment. After the particles were immersed in the acidic solution, it was observed that the particles swelled but the core structure remained intact, accompanied by a very low level of phosphorus element release. The reason for this was that the hydrogen ions in the solution could act on the low-intensity basic ion crosslinking network formed by the first type of multivalent metal cation, calcium ions, but were not enough to destroy the more chemically stable coordination lock point formed by the second type of multivalent metal cation, zinc ions, and organic acid functional groups, so that the core of the composite structure was maintained, avoiding premature non-targeted release of functional components.

[0039] The second solution had a pH value of 8.5 to simulate an alkaline soil environment. The composite structure particles were pre-wetted in the alkaline solution, and then a solution containing citric acid was added to simulate contact with plant roots. After the addition of citric acid, it was observed that the particles changed from a stable state to structural disintegration, accompanied by a large release of phosphorus elements. The reason for this phenomenon was that the targeted disintegration mechanism of the composite structure was triggered not mainly by the acidity brought by the root exudates, but by the strong coordination capture ability of multidentate chelating agents such as citric acid on the second type of multivalent metal cation, zinc ions. This coordination capture action was still effective in an alkaline environment, thereby destroying the key coordination lock point and triggering the coordinated disintegration of the structure. Thus, the gradient stable structure of the present application enables the composite microbial agent to maintain its structural stability and targeted release function in different chemical conditions of the soil.

[0040] Example 6: In the large-scale production of synergistic organic-inorganic composite microbial agents, to ensure that the stoichiometric ratio of the gradient stable structure composed of two different multivalent metal cations is constant on each particle, and to avoid micro-distribution heterogeneity caused by independent ion source co-spraying, this embodiment provides a standardized engineering procedure for preparing a water-soluble heteronuclear metal complex precursor, which is in the form of a single component to provide ion sources for subsequent interfacial complexation. The procedure is carried out in a stainless steel reactor with a heating jacket and a mechanical stirring device. First, a calculated amount of citric acid hydrate is added to deionized water, heated to 60 and stirred until completely dissolved to form a clear citric acid solution. Then, under the conditions of maintaining the solution temperature and continuous stirring, pre-accurately weighed zinc oxide powder and calcium hydroxide powder meeting the industrial first-class product standards are slowly and synchronously added to the citric acid solution according to the preset calcium-zinc molar ratio. After the addition is completed, the temperature of the reaction system is maintained at 70 And the reaction is continuously stirred for 2 hours until there is no visible solid particles in the reactor and the system presents a uniform clear solution state, which is the reaction end point.

[0041] After the reaction is completed, the heating is stopped and the solution is cooled to room temperature. The calcium ion and zinc ion concentrations in the solution are detected by inductively coupled plasma atomic emission spectrometer to verify the consistency of the molar ratio and the feeding ratio, and the final concentration of the precursor solution is calibrated. The calcium-zinc-citrate complex solution prepared by this procedure is stable in nature and can be directly used as a raw material for the spraying process in step c. By using this single-component precursor, the first type of multivalent metal cation and the second type of multivalent metal cation are uniformly delivered to the active colloid interface of the organic carrier at a constant stoichiometric ratio, thereby ensuring the batch-to-batch stability of the final product function.

[0042] To further verify the necessity and non-obviousness of the synergistic effect of the functional components achieved by the specific chemical structure of the present application, the following comparative examples are set up.

[0043] Comparative Example 1: In this comparative example, an organic carrier (500 g of a mixture of rapeseed oil cake and 500 g of humic acid), inorganic nutrients (100 g of potassium dihydrogen phosphate), microorganisms (50 g of dormant Bacillus subtilis powder) and metal salts (equivalent to 0.8 mol of calcium ions and 0.1 mol of zinc ions) are used, but steps a, b, c and d of the present application are not performed. Instead, the conventional mechanical mixing and physical granulation method mentioned in the background art is used, which is as follows: all the above-mentioned dry powdered materials are placed in a V-type mixer for sufficient mechanical mixing until the appearance color is uniform. Then, water is used as a wetting agent to prepare granular products with a particle size comparable to the test group in Example 2 by a disc granulator. Finally, the granules are dried at a temperature lower than 50

[0044] Table 2: Comparison of nutrient leaching and crop growth data between Comparative Example 1 and the test group.

[0045] ​Conclusion analysis: the test results show that the leachate phosphorus concentration of Comparative Example 1 is as high as 16.5 mg / L, higher than 1.8 mg / L of the test group, which confirms that the granules prepared by conventional physical mixing, the water-soluble inorganic nutrients in the interior will be quickly dissolved and lost in large quantities when encountering water erosion, accordingly, the plant height, effective tillering number and aboveground dry weight and other crop growth indicators after 90 days are significantly lower than the test group, even slightly lower than the control group 1 in Example 2.

[0046] It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and the present application can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application.

[0047] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for preparing a synergistic organic-inorganic compound microbial agent for promoting rice yield increase, characterized in that, This includes the steps performed in the following order: Step a: The organic carrier containing organic acid functional groups is placed in a weakly alkaline chemical environment with a pH of 7.5 to 8.5 to form a negatively charged active colloidal interface on the surface of the organic carrier. Step b: Mix an aqueous suspension containing microorganisms and inorganic nutrients with a solution containing first-type polyvalent metal cations and second-type polyvalent metal cations to obtain a mixture, wherein the second-type polyvalent metal cations have a stronger coordination ability with the organic acid functional groups on the surface of the organic carrier than the first-type polyvalent metal cations. Step c: The mixture obtained in step b is applied to the negatively charged active colloidal interface in step a. Through interfacial complexation between the first type of multivalent metal cations and the active colloidal interface, a basic ionic cross-linking network resistant to physical erosion is constructed on the surface of the organic carrier. The second type of multivalent metal cations are used to form coordination locking sites in the basic ionic cross-linking network that specifically respond to chelating agents in plant root exudates. In this way, microorganisms and inorganic nutrients are anchored together on the surface of the organic carrier to form a composite structure. Step d: The composite structure is subjected to a temperature below 50°C. Low-temperature drying is used to solidify the composite structure formed by interfacial complexation.

2. The method for preparing a synergistic organic-inorganic compound microbial agent for promoting rice yield increase according to claim 1, characterized in that, The organic carrier is a mixture of rapeseed oil cake and humic acid; the first type of polyvalent metal cation is calcium ion; the second type of polyvalent metal cation is zinc ion or manganese ion.

3. The method for preparing a synergistic organic-inorganic compound microbial agent for promoting rice yield increase according to claim 1, characterized in that, In step a, while the organic support is placed in a weakly alkaline chemical environment, a chemical reducing agent is also added to pre-convert the quinone functional groups on the surface of the organic support into reduced hydroquinone groups.

4. The method for preparing a synergistic organic-inorganic compound microbial agent for promoting rice yield increase according to claim 1, characterized in that, In step b, the first type of multivalent metal cation and the second type of multivalent metal cation are provided together in the form of a pre-prepared water-soluble heteronuclear metal complex precursor containing the two metal ions.

5. The method for preparing a synergistic organic-inorganic compound microbial agent for promoting rice yield increase according to claim 1, characterized in that, In step c, the molar amount of the first type of multivalent metal cations Molar amount of organic acid functional groups on the surface of organic carrier The ratio is limited to a subsaturated range, which satisfies: ,in, Represents the total molar amount of type I multivalent metal cations. It represents the total molar amount of organic acid functional groups provided by the organic carrier and participating in the complexation.

6. The method for preparing a synergistic organic-inorganic compound microbial agent for promoting rice yield increase according to claim 1, characterized in that, In step b, the microorganisms include at least one of Bacillus subtilis and Bacillus mucilaginosus; in step c, the anchored microorganisms are in a dormant state; in step b, the inorganic nutrients include phosphate or sulfate; in step c, the phosphate or sulfate anions in the inorganic nutrients form slightly soluble precipitates with the first type of polyvalent metal cations and the second type of polyvalent metal cations, and are locked in situ in the forming complex structure.

7. The method for preparing a synergistic organic-inorganic compound microbial agent for promoting rice yield increase according to claim 3, characterized in that, The chemical reducing agent is sodium sulfite or ascorbic acid.

8. The method for preparing a synergistic organic-inorganic compound microbial agent for promoting rice yield increase according to claim 4, characterized in that, Water-soluble heteronuclear metal complex precursors are calcium-zinc-citrate complexes or calcium-manganese-citrate complexes prepared by reacting a base containing calcium ions with an oxide containing zinc or manganese ions in an aqueous solution of citric acid.

9. A method for preparing a synergistic organic-inorganic compound microbial agent for promoting rice yield increase according to claim 1, characterized in that, In step c, the mixture is sprayed onto the surface of the organic carrier in a dynamically stirred state in the form of atomization; in step d, low-temperature drying continues until the final moisture content of the composite structure is less than 10%.

10. A synergistic organic-inorganic composite microbial agent obtained by the preparation method according to any one of claims 1 to 9, characterized in that, The microbial agent is a granular composite structure, which includes: an organic carrier core; an interfacial complexing layer completely covering the surface of the organic carrier core; the interfacial complexing layer contains microorganisms, inorganic nutrients, first-class polyvalent metal cations, and second-class polyvalent metal cations. The microorganisms and inorganic nutrients are anchored to the surface of the organic carrier core through the first-class and second-class polyvalent metal cations to form a basic ionic cross-linking network and a gradient stable structure with coordination locking points that specifically respond to chelating agents in plant root exudates.

Images