Microbial agent for improving saline-alkali soil

Abstract

The invention discloses a microbial agent for improving saline-alkali soil, and belongs to the technical field of compound microbial fertilizer.The microbial agent comprises an acidic flora, an alkaline flora, a double-network gel system and a spent bleaching clay coating layer, the alkaline flora is an alkaline buffer core of streptomyces rochei and CaCO3 microspheres, and the double-network gel system is a double-network gel system. The acidic flora comprises a complex of bacillus subtilis and a humic acid buffer solution and an inner core consisting of trichoderma harzianum and citric acid microcrystals; the double-network gel system disclosed by the invention is composite gel which is formed by assembling CMC-beta-CD network gel of beta-cyclodextrin modified carboxymethyl cellulose for embedding a middle-layer flora and CMCHT network gel of calcium cross-linked carboxymethyl chitosan for embedding an inner-layer flora and an outer-layer flora through a hydrogen bond, wherein the CMC-beta-CD network gel is prepared from CMC-beta-CD network gel of beta-cyclodextrin modified carboxymethyl cellulose for embedding the middle-layer flora and the CMCHT network gel of calcium cross-linked carboxymethyl chitosan for embedding the inner-layer flora and the outer-layer flora; according to the method, the problem of flora survival is solved through the intelligent response and slow release characteristics of the double-network gel, deep soil remediation and waste white clay resource utilization are achieved through acid / alkali flora synergistic metabolism, and the core bottlenecks that flora colonization is difficult, the improvement time is short and the application cost is high in saline-alkali soil treatment are overcome.

Description

Technical Field

[0001] This invention relates to the field of compound microbial fertilizer technology, and in particular to a microbial agent for improving saline-alkali land. Background Technology

[0002] Saline-alkali soil is one of the core problems of global arable land degradation. The causes of salinization are complex, including the phenomenon of "salt coming with water and going with water" caused by groundwater uplift and strong evaporation, as well as the accumulation of alkaline Na2CO3 in soda-type saline-alkali land. Traditional treatments rely on physical desalination and chemical amendments, but these measures are not only costly and time-consuming, but may also disrupt the soil's micro-ecological balance.

[0003] The use of microbial agents for saline-alkali land improvement has become a new direction for saline-alkali land management due to its green, environmentally friendly, and low-cost nature; however, its practical application still has some shortcomings. The most significant of these is the low survival rate of microbial strains due to the unique physicochemical properties of saline-alkali land. Ordinary microbial agents have a survival rate of less than 20% in severely saline-alkali environments with pH > 10 and salinity > 5‰. For example, Bacillus subtilis exhibits a 70% decrease in intracellular enzyme activity under alkaline conditions, often leading to root colonization failure. To improve the survival rate of microbial strains, the technology of loading and encapsulating microorganisms with appropriate carriers has become a research focus. However, existing encapsulation materials, such as commonly used sodium alginate, are prone to disintegration under extreme pH conditions, failing to achieve intelligent response.

[0004] Developing embedding materials with intelligent response mechanisms to improve the survival rate and colonization success rate of microorganisms in saline-alkali land, and even severely saline-alkali land, has become an urgent technical problem to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide a microbial agent for improving saline-alkali land, so as to solve the problems existing in the prior art. This invention proposes a three-in-one microbial agent and preparation method integrating acid-alkali bacteria synergy, intelligent carrier response and solid waste resource utilization, so as to solve the problems of low survival rate of bacteria, insufficient colonization capacity and single function of bacteria in saline-alkali soil.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a microbial agent for improving saline-alkali land. The key feature is that the microbial agent comprises acidic bacteria, alkaline bacteria, a dual-network gel system, and a waste clay coating layer. The aforementioned alkaline bacteria form an alkaline buffer core of *Streptomyces loucheretia* and CaCO3 microspheres, with a viable count of 5 × 10⁻⁶ *Streptomyces loucheretia*. 9 CFU / g ~ 8×10 9CFU / g; the above-mentioned acidic bacterial community includes a complex of Bacillus subtilis and humic acid buffer and a core composed of Trichoderma harzianum and citrate microcrystals, with a viable count of Bacillus subtilis of 2 × 10⁻⁶. 10 CFU / g ~ 3×10 10 CFU / g, viable count of Trichoderma harzianum is 3×10 9 CFU / g ~5×10 9 CFU / g; The above-mentioned dual-network gel system is a composite gel assembled by hydrogen bonds from a CMC-β-CD network gel containing β-cyclodextrin-modified carboxymethyl cellulose that embeds the middle layer of bacteria and a CMCHT network gel containing calcium-crosslinked carboxymethyl chitosan that embeds the inner and outer layers of bacteria; CaCO3 microspheres are CaCO3 particles with a particle size of 33μm to 38μm, and citric acid microcrystals are citric acid crystals with a particle size of 30μm to 50μm.

[0008] Furthermore, the preparation method of the above-mentioned microbial inoculant includes:

[0009] S1 strain activation: Streptomyces loucheri, Bacillus subtilis and Trichoderma harzianum were activated separately to prepare Streptomyces seed liquid, Bacillus subtilis seed liquid and Trichoderma harzianum seed liquid;

[0010] Preparation of the S2 dual-network gel system:

[0011] Preparation of S21 CMC-β-CD network gel: Carboxymethyl cellulose and β-cyclodextrin were dissolved in water, 1,4-butanediol diglycidyl ether was added, the pH was adjusted to 10-11, and the reaction was carried out at 30℃-32℃ for 1-2 hours. After the reaction was completed, the gel was precipitated with anhydrous ethanol and dried to obtain CMC-β-CD network gel.

[0012] Preparation of S22 CMCHT network gel: Carboxymethyl chitosan was dissolved in acetic acid solution, CaCl2 solution A was added dropwise to produce a precipitate, the precipitate was collected by centrifugation, washed with deionized water until the washing solution was neutral, and dried to obtain CMCHT network gel;

[0013] S3 microbial community embedding:

[0014] Preparation of S31 alkaline bacterial colony gel: Streptomyces seed liquid was mixed with CaCO3 microspheres, and 0.1% glutaraldehyde solution was added dropwise. The reaction was carried out for 30 min to 40 min. After the reaction was completed, the mixture was filtered and freeze-dried to obtain CaCO3 microspheres loaded with Streptomyces. CMCHT network gel was added to a solid content of 12% to 18% to obtain alkaline bacterial colony gel.

[0015] Preparation of S32 acidic bacteria:

[0016] Bacillus subtilis seed culture was mixed with humic acid buffer to obtain a bacterial humic acid mixture;

[0017] Trichoderma harzianum seed liquid was centrifuged to obtain Trichoderma harzianum spore mud, and Trichoderma harzianum spore mud was mixed with citric acid microcrystals to obtain Trichoderma citric acid mixture;

[0018] Acidic bacterial colony gel was prepared by mixing bacterial humic acid mixture, Trichoderma citric acid mixture and CMCHT network gel at a mass ratio of 1:1:6-8, adding CaCl2 solution B dropwise, and reacting at a temperature of 22℃-28℃ for 10-20 minutes.

[0019] Composite of S33 dual-network gel system: Alkaline bacterial gel and acidic bacterial gel are mixed, water is added, heated to 55℃~60℃ and shaken at 180r / min~200r / min for 1h~2h. After the reaction is completed, the composite microbial sclerotia are obtained by filtration and freeze drying.

[0020] S4 waste clay coating layer:

[0021] Waste bleaching clay and composite microbial sclerotia are mixed at a mass ratio of 3 to 4:1 and granulated. The mixture is then subjected to drum granulation at a speed of 28 to 32 r / min, steam curing at 95 to 98℃ for 3 to 4 hours, and drying to obtain the microbial agent.

[0022] Preferably, the viable Streptomyces count in the Streptomyces loucheri seed solution in step S1 is 2 × 10⁻⁶. 9 CFU / mL ~ 3 × 10 9 CFU / mL; The viable Bacillus subtilis count in the Bacillus subtilis seed culture was 5 × 10⁻⁶. 9 CFU / mL ~ 6 × 10 9 CFU / mL; the viable count of *Trichoderma harzianum* in the *Trichoderma harzianum* seed culture was 0.1 × 10⁻⁶. 9 CFU / mL ~ 0.2 × 10⁻⁶ 9 CFU / mL.

[0023] Furthermore, in step S21, the mass ratio of carboxymethyl cellulose to β-cyclodextrin is 6–8:1, and 1–1.5 mL of 1,4-butanediol diglycidyl ether is used for every 10 g of carboxymethyl cellulose.

[0024] Furthermore, in step S22, the acetic acid solution contains 2%–3% acetic acid by mass, and the CaCl2 solution A contains 4.5%–5.5% CaCl2 by mass. Each 10 grams of carboxymethyl chitosan corresponds to 400 mL–600 mL of acetic acid solution and 200 mL–300 mL of CaCl2 solution, respectively.

[0025] Furthermore, in step S31, the volume ratio of Streptomyces seed solution to CaCO3 microspheres is 1:3.5 to 4.5, and the amount of glutaraldehyde solution used is 0.6 to 1.0 times that of CaCO3 microspheres.

[0026] Furthermore, in step S32, the Bacillus subtilis seed solution and humic acid buffer are mixed at a volume ratio of 1:1.5 to 2.0, and the Trichoderma harzianum seed solution is centrifuged at 5000 r / min to 6000 r / min for 12 min to 15 min to obtain Trichoderma harzianum spore mud. The mass ratio of Trichoderma harzianum spore mud to citric acid microcrystals is 1:1.0 to 1.2.

[0027] Furthermore, in step S32, the mass fraction of CaCl2 in CaCl2 solution B is 2.5%–3.5%, and the volume ratio of CaCl2 solution B to CMCHT network gel is 1:2.0–2.5.

[0028] Furthermore, in step S33, the mass ratio of alkaline bacterial gel to acidic bacterial gel is 2.5 to 3.5:1, and the added water is 4 to 5 times the total gel volume.

[0029] Optimally, the above-mentioned waste bleaching clay is oil refining waste bleaching clay with an oil content of 12% to 15%, which is inoculated with Bacillus subtilis and fermented at 30℃ to 32℃ for 12 to 15 days, resulting in a residual rate of 2% to 5%; CaCO3 microspheres are prepared by mixing polyethylene glycol 8000 solution and Na2CO3 solution to form a two-phase aqueous emulsion, then adding CaCl2 solution, reacting at 22℃ to 28℃ for 1 min to 2 min, and then aging, centrifuging, drying, and sieving; citric acid microcrystals are prepared by cooling citric acid solution to 4℃ to 5℃ for crystallization, centrifuging, and then spray drying.

[0030] The present invention discloses the following technical effects:

[0031] This invention solves the core problems of low microbial survival rate, unsustainable soil improvement, and poor crop resistance in saline-alkali land treatment through a three-pronged technical system: intelligent response carrier construction, microbial community functional division design, and solid waste resource utilization.

[0032] The dual-network gel system of this invention achieves pH-responsive release and stability in acidic environments. It precisely activates the bacterial strain under alkaline conditions, as demonstrated by the gel swelling rate exceeding 92.8% within 24 hours at pH 10.5. + The release rate is >89%, significantly higher than traditional embedding materials, ensuring the activation of the bacterial community in the target saline-alkali region. Regarding the damage to the bacterial strain caused by the acidic environmental barrier, this invention exhibits a swelling rate of <11.2% at pH 6.5. +The release rate of <5.1% demonstrates that the waste clay coating layer effectively blocks acid rain erosion and prevents unintended release. This invention achieves salt stress protection; after 90 days in a 5% NaCl simulated environment, the viable bacterial retention rate is >85%, while the control group's survival rate is less than 5% due to salt ion penetration. Furthermore, the dual-network gel system also exhibits sustained-release properties, as evidenced by the rhizosphere Streptomyces colonization remaining at 2.35 × 10⁻⁶ after 90 days. 9 The CFU / g was significantly higher than that of the conventional embedding group.

[0033] This invention achieves long-term soil remediation based on the synergistic metabolic mechanism of acidic and alkaline bacteria, specifically by the acidic bacteria secreting organic acids to neutralize OH-. - It activates insoluble phosphorus; alkaline bacteria degrade Na2CO3, reducing soil alkalinity. The synergistic effect of the two is evident in the application to severely saline-alkali soils, where the soil pH drops to 7.8, the Na2CO3 residue is only 0.09%, the salinity is reduced by 57.3%, and the soybean yield increases to 210 kg / mu (133.3% higher than the control group), verifying the synergistic effect of the bacterial community's metabolic function.

[0034] Furthermore, the coating material of this invention, waste bleaching clay, is itself a waste raw material, achieving "waste treatment with waste" through resource utilization. This invention utilizes the waste bleaching clay to form a hydrophobic microporous structure after fermentation treatment, which works together with the dual-network gel system to exert the dual effects of physical salt barrier and slow release of microbial flora.

[0035] In summary, this invention integrates three technologies: the intelligent response and slow-release properties of the dual-network gel to solve the problem of microbial survival, the synergistic metabolism of acid / alkali microorganisms to achieve deep soil remediation, and the resource utilization of waste bleaching soil to construct a low-cost engineering solution. It systematically overcomes the core bottlenecks of difficult microbial colonization, short improvement time, and high application cost in saline-alkali land treatment. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] The Bacillus subtilis, Trichoderma harzianum, and Streptomyces louchei used in the embodiments of this invention are all conventionally available commercially available mycelial powders, specifically:

[0042] Bacillus subtilis comes from Shandong Yihao Biotechnology Co., Ltd.;

[0043] Trichoderma harzianum was obtained from Jiaozuo Yuntai Dahua Biotechnology Co., Ltd.;

[0044] Streptomyces loucheri comes from Shanghai Xuanke Biotechnology Co., Ltd.

[0045] Example 1

[0046] In this embodiment, the raw materials for the microbial agent, waste clay, CaCO3 microspheres, and citric acid microcrystals, are prepared. The specific process is as follows:

[0047] Waste clay from oil refining with an initial oil content of 13.5% was selected, inoculated with Bacillus subtilis, and fermented at 31°C for 13 days to prepare waste clay sample 1 with an oil residue content of 3.2%.

[0048] A 33% polyethylene glycol 8000 solution and a 7% Na2CO3 solution were mixed at a volume ratio of 15:1 to form an aqueous two-phase emulsion. Then, 0.5M CaCl2 solution was added, and the volume ratio of CaCl2 solution to the aqueous two-phase emulsion was 1:15. The mixture was reacted at 5℃ for 1.5 min, and then aged, centrifuged, dried, and sieved to obtain CaCO3 microsphere sample 1. The particle size of the CaCO3 microspheres was measured to be 35.2 μm.

[0049] Citric acid microcrystals were prepared by cooling a 40% citric acid solution to 4.5℃ at a rate of 2℃ / min for crystallization, growing the crystals for 20 min, centrifuging, and then spray drying at an inlet air temperature of 80℃. This sample was designated as citric acid microcrystal 1, and the particle size of the citric acid microcrystals was measured to be 41.3 μm.

[0050] Example 2

[0051] In this embodiment, the raw materials for the microbial agent, waste clay, CaCO3 microspheres, and citric acid microcrystals, are prepared. The specific process is as follows:

[0052] Waste clay from oil refining with an initial oil content of 12.0% was selected, inoculated with Bacillus subtilis, and fermented at 30℃ for 15 days to prepare waste clay sample 2 with an oil residue of 2.0%.

[0053] A 34% (w / w) polyethylene glycol 8000 solution and a 6% (w / w) Na2CO3 solution were mixed at a volume ratio of 14:1 to form an aqueous two-phase emulsion. Then, 0.5 M CaCl2 solution was added, and the volume ratio of CaCl2 solution to the aqueous two-phase emulsion was 1:16. The mixture was reacted at 28℃ for 1 min, and then aged, centrifuged, dried, and sieved to obtain CaCO3 microsphere sample 2. The particle size of the CaCO3 microspheres was measured to be 37.9 μm.

[0054] Citric acid microcrystals were prepared by cooling a 42% citric acid solution to 5℃ at a rate of 2℃ / min for crystallization, growing the crystals for 25 min, centrifuging, and then spray drying at an inlet air temperature of 85℃. This sample was designated as citric acid microcrystal sample 2, and the particle size of the citric acid microcrystals was measured to be 49.8 μm.

[0055] Example 3

[0056] In this embodiment, the raw materials for the microbial agent, waste clay, CaCO3 microspheres, and citric acid microcrystals, are prepared. The specific process is as follows:

[0057] Waste clay from oil refining with an initial oil content of 14.9% was selected, inoculated with Bacillus subtilis, and fermented at 32℃ for 12 days to prepare waste clay sample 3 with an oil residue of 5.0%.

[0058] A 32% polyethylene glycol 8000 solution and an 8% Na2CO3 solution were mixed at a volume ratio of 16:1 to form an aqueous two-phase emulsion. Then, 0.5M CaCl2 solution was added, and the volume ratio of CaCl2 solution to the aqueous two-phase emulsion was 1:14. The mixture was reacted at 22℃ for 2 min, and then aged, centrifuged, dried, and sieved to obtain CaCO3 microsphere sample 3. The particle size of the CaCO3 microspheres was measured to be 33.1 μm.

[0059] Citric acid microcrystals were prepared by cooling a 38% citric acid solution to 4℃ at a rate of 2℃ / min for crystallization, growing the crystals for 15 min, centrifuging, and then spray drying at an inlet air temperature of 75℃. This sample was designated as citric acid microcrystal sample 3, and the particle size of the citric acid microcrystals was measured to be 30.2 μm.

[0060] Example 4

[0061] In this embodiment, the preparation of microbial inoculants is carried out, and the specific steps are as follows:

[0062] S1 strain activation:

[0063] Streptomyces loucherei, Bacillus subtilis, and Trichoderma harzianum were activated separately, and viable bacterial counts of 2.6 × 10⁻⁶ were obtained for each species. 9 Streptomyces seed culture with CFU / mL and a viable count of 5.4 × 10⁻⁶ 9 CFU / mL Bacillus subtilis seed culture and viable count of 0.15 × 10⁻⁶ cells / mL 9 CFU / mL Trichoderma harzianum seed solution;

[0064] Preparation of the S2 dual-network gel system:

[0065] Preparation of S21 CMC-β-CD network gel:

[0066] Carboxymethyl cellulose and β-cyclodextrin were dissolved in water, and 1,4-butanediol diglycidyl ether was added to adjust the pH to 10.6. The reaction was carried out at 31°C for 1.5 h. After the reaction was completed, the mixture was precipitated with anhydrous ethanol and dried to obtain CMC-β-CD network gel. The mass ratio of carboxymethyl cellulose to β-cyclodextrin was 7:1, and 1.2 mL of 1,4-butanediol diglycidyl ether was used for every 10 g of carboxymethyl cellulose.

[0067] Preparation of S22 CMCHT network gel:

[0068] Carboxymethyl chitosan was dissolved in acetic acid solution, and CaCl2 solution A was added dropwise to produce a precipitate. The precipitate was collected by centrifugation, washed with deionized water until the washing solution was neutral, and dried to obtain CMCHT network gel. The mass fraction of acetic acid in the acetic acid solution was 2.5%, and the mass fraction of CaCl2 in CaCl2 solution A was 5.0%. Each 10 grams of carboxymethyl chitosan corresponds to 500 mL of acetic acid solution and 250 mL of CaCl2 solution, respectively.

[0069] S3 microbial community embedding:

[0070] Preparation of S31 alkaline bacterial colony gel:

[0071] Streptomyces seed culture was mixed with CaCO3 microsphere sample 1 at a volume ratio of 1:4, and 0.1% glutaraldehyde solution was added dropwise. The reaction was carried out for 35 min. The amount of glutaraldehyde solution used was 0.8 times that of CaCO3 microsphere sample 1. After the reaction was completed, the mixture was filtered and freeze-dried to obtain CaCO3 microspheres loaded with Streptomyces. CMCHT network gel was added to a solid content of 15% to obtain an alkaline bacterial colony gel.

[0072] Preparation of S32 acidic bacteria:

[0073] Bacillus subtilis seed culture and humic acid buffer were mixed at a volume ratio of 1:1.8 to obtain a bacterial humic acid mixture.

[0074] Trichoderma harzianum seed liquid was centrifuged at 5500 r / min for 14 min to obtain Trichoderma harzianum spore mud. Trichoderma harzianum spore mud and citric acid microcrystal sample 1 were mixed at a mass ratio of 1:1.1 to obtain Trichoderma citric acid mixture.

[0075] A mixture of bacterial humic acid, Trichoderma citric acid, and CMCHT network gel was prepared by mixing them in a mass ratio of 1:1:7, and CaCl2 solution B was added dropwise. The mass fraction of CaCl2 in CaCl2 solution B was 3.0%. The mixture was reacted at 25℃ for 15 min to obtain an acidic bacterial colony gel. The volume ratio of CaCl2 solution B to CMCHT network gel was 1:2.2.

[0076] Composite of S33 dual-network gel system:

[0077] Alkaline bacterial colony gel and acidic bacterial colony gel were mixed at a mass ratio of 3.0:1, and water was added at a volume of 4.5 times the total gel volume. The mixture was heated to 58°C and shaken at 190 r / min for 1.5 h. After the reaction was completed, the mixture was filtered and freeze-dried to obtain composite microbial sclerotia.

[0078] S4 waste clay coating layer:

[0079] Waste clay sample 1 and composite microbial sclerotia were mixed at a mass ratio of 3.5:1 and granulated by drum granulation at a speed of 30 r / min. After steam curing at 96℃ for 3.5 h and drying, the microbial agent was obtained and recorded as agent sample 1.

[0080] Example 5

[0081] In this embodiment, the preparation of microbial inoculants is carried out, and the specific steps are as follows:

[0082] S1 strain activation:

[0083] Streptomyces loucherei, Bacillus subtilis, and Trichoderma harzianum were activated separately, and viable bacterial counts of 3.0 × 10⁻⁶ were obtained for each species. 9Streptomyces seed culture with CFU / mL and a viable count of 6.0 × 10⁻⁶ 9 CFU / mL Bacillus subtilis seed culture and viable count of 0.2 × 10⁻⁶ 9 CFU / mL Trichoderma harzianum seed solution;

[0084] Preparation of the S2 dual-network gel system:

[0085] Preparation of S21 CMC-β-CD network gel:

[0086] Carboxymethyl cellulose and β-cyclodextrin were dissolved in water, and 1,4-butanediol diglycidyl ether was added to adjust the pH to 11.0. The reaction was carried out at 32°C for 1 hour. After the reaction was completed, the mixture was precipitated with anhydrous ethanol and dried to obtain CMC-β-CD network gel. The mass ratio of carboxymethyl cellulose to β-cyclodextrin was 6:1, and 1.5 mL of 1,4-butanediol diglycidyl ether was used for every 10 g of carboxymethyl cellulose.

[0087] Preparation of S22 CMCHT network gel:

[0088] Carboxymethyl chitosan was dissolved in acetic acid solution, and CaCl2 solution A was added dropwise to produce a precipitate. The precipitate was collected by centrifugation, washed with deionized water until the washing solution was neutral, and dried to obtain CMCHT network gel. The mass fraction of acetic acid in the acetic acid solution was 3.0%, and the mass fraction of CaCl2 in CaCl2 solution A was 4.5%. Each 10 g of carboxymethyl chitosan corresponds to 400 mL of acetic acid solution and 300 mL of CaCl2 solution, respectively.

[0089] S3 microbial community embedding:

[0090] Preparation of S31 alkaline bacterial colony gel:

[0091] Streptomyces seed culture was mixed with CaCO3 microsphere sample 2 at a volume ratio of 1:3.5, and 0.1% glutaraldehyde solution was added dropwise. The reaction was carried out for 40 min. The amount of glutaraldehyde solution used was 0.6 times that of CaCO3 microsphere sample 2. After the reaction was completed, the mixture was filtered and freeze-dried to obtain CaCO3 microspheres loaded with Streptomyces. CMCHT network gel was added to a solid content of 12.0% to obtain an alkaline bacterial colony gel.

[0092] Preparation of S32 acidic bacteria:

[0093] Bacillus subtilis seed culture and humic acid buffer were mixed at a volume ratio of 1:2 to obtain a bacterial humic acid mixture.

[0094] Trichoderma harzianum seed liquid was centrifuged at 6000 r / min for 12 min to obtain Trichoderma harzianum spore mud. Trichoderma harzianum spore mud and citric acid microcrystal sample 2 were mixed at a mass ratio of 1:1.0 to obtain Trichoderma citric acid mixture.

[0095] A mixture of bacterial humic acid, Trichoderma citric acid, and CMCHT network gel was prepared by mixing them in a mass ratio of 1:1:8, and CaCl2 solution B was added dropwise. The mass fraction of CaCl2 in CaCl2 solution B was 2.5%. The mixture was reacted at 22℃ for 20 min to obtain an acidic bacterial colony gel. The volume ratio of CaCl2 solution B to CMCHT network gel was 1:2.0.

[0096] Composite of S33 dual-network gel system:

[0097] Alkaline bacterial colony gel and acidic bacterial colony gel were mixed at a mass ratio of 3.5:1, and water was added in a volume of 5 times the total gel volume. The mixture was heated to 60°C and shaken at 180 r / min for 2 hours. After the reaction was completed, the mixture was filtered and freeze-dried to obtain composite microbial sclerotia.

[0098] S4 waste clay coating layer:

[0099] Waste clay sample 2 and composite microbial sclerotia were mixed at a mass ratio of 3:1 and granulated by drum granulation at a speed of 32 r / min. After steam curing at 98℃ for 3 h and drying, the microbial agent was obtained and recorded as agent sample 2.

[0100] Example 6

[0101] In this embodiment, the preparation of microbial inoculants is carried out, and the specific steps are as follows:

[0102] S1 strain activation:

[0103] Streptomyces loucherei, Bacillus subtilis, and Trichoderma harzianum were activated separately, and viable bacterial counts of 2.0 × 10⁻⁶ were obtained for each species. 9 Streptomyces seed culture with CFU / mL and a viable count of 5.0 × 10⁻⁶ cells / mL 9 CFU / mL Bacillus subtilis seed culture and viable count of 0.1×10⁻⁶ 9 CFU / mL Trichoderma harzianum seed solution;

[0104] Preparation of the S2 dual-network gel system:

[0105] Preparation of S21 CMC-β-CD network gel:

[0106] Carboxymethyl cellulose and β-cyclodextrin were dissolved in water, and 1,4-butanediol diglycidyl ether was added to adjust the pH to 10.0. The reaction was carried out at 30°C for 2 hours. After the reaction was completed, the mixture was precipitated with anhydrous ethanol and dried to obtain CMC-β-CD network gel. The mass ratio of carboxymethyl cellulose to β-cyclodextrin was 8:1, and 1 mL of 1,4-butanediol diglycidyl ether was used for every 10 g of carboxymethyl cellulose.

[0107] Preparation of S22 CMCHT network gel:

[0108] Carboxymethyl chitosan was dissolved in acetic acid solution, and CaCl2 solution A was added dropwise to produce a precipitate. The precipitate was collected by centrifugation, washed with deionized water until the washing solution was neutral, and dried to obtain CMCHT network gel. The mass fraction of acetic acid in the acetic acid solution was 2.0%, and the mass fraction of CaCl2 in CaCl2 solution A was 5.5%. Each 10 g of carboxymethyl chitosan corresponds to 600 mL of acetic acid solution and 200 mL of CaCl2 solution, respectively.

[0109] S3 microbial community embedding:

[0110] Preparation of S31 alkaline bacterial colony gel:

[0111] Streptomyces seed culture was mixed with CaCO3 microsphere sample 3 at a volume ratio of 1:4.5, and 0.1% glutaraldehyde solution was added dropwise. The reaction was carried out for 30 min. The amount of glutaraldehyde solution used was 1.0 times that of CaCO3 microsphere sample 3. After the reaction was completed, the mixture was filtered and freeze-dried to obtain CaCO3 microspheres loaded with Streptomyces. CMCHT network gel was added until the solid content reached 18.0% to obtain alkaline bacterial colony gel.

[0112] Preparation of S32 acidic bacteria:

[0113] Bacillus subtilis seed culture and humic acid buffer were mixed at a volume ratio of 1:1.5 to obtain a bacterial humic acid mixture.

[0114] Trichoderma harzianum seed liquid was centrifuged at 5000 r / min for 15 min to obtain Trichoderma harzianum spore mud. Trichoderma harzianum spore mud and citric acid microcrystal sample 3 were mixed at a mass ratio of 1:1.2 to obtain Trichoderma citric acid mixture.

[0115] A mixture of bacterial humic acid, Trichoderma citric acid, and CMCHT network gel was prepared by mixing them in a mass ratio of 1:1:6, and CaCl2 solution B was added dropwise. The mass fraction of CaCl2 in CaCl2 solution B was 3.5%. The mixture was reacted at 28℃ for 10 min to obtain an acidic bacterial colony gel. The volume ratio of CaCl2 solution B to CMCHT network gel was 1:2.0.

[0116] Composite of S33 dual-network gel system:

[0117] Alkaline bacterial colony gel and acidic bacterial colony gel were mixed at a mass ratio of 2.5:1, and water was added in a volume of 4 times the total gel volume. The mixture was heated to 55°C and shaken at 200 r / min for 1 h. After the reaction was completed, the mixture was filtered and freeze-dried to obtain composite microbial sclerotia.

[0118] S4 waste clay coating layer:

[0119] Waste clay sample 3 and composite microbial sclerotia were mixed at a mass ratio of 4:1 and granulated by drum granulation at a speed of 28 r / min. After steam curing at 95℃ for 4 h and drying, the microbial agent was obtained and recorded as agent sample 3.

[0120] Comparative Example 1

[0121] In this comparative example, the seed liquid obtained from the activation of the S1 strain in Example 4 was used to prepare the control. The main difference is that the preparation of the S2 double-network gel system, the S3 separate bacterial group embedding, and the S4 waste clay coating layer are not performed. Instead, sodium alginate is used for single embedding. The specific preparation process is as follows:

[0122] Step 1: Centrifuge the Streptomyces seed liquid, Bacillus subtilis seed liquid and Trichoderma harzianum seed liquid obtained by activating the S1 strain in Example 4 at 6000 r / min for 12 min to obtain Streptomyces seed mud, Bacillus subtilis seed mud and Trichoderma harzianum seed mud respectively.

[0123] Step 2: Mix Streptomyces seed mud, Bacillus subtilis seed mud and Trichoderma harzianum seed mud in a mass ratio of 1:1:2 to obtain mixed seed mud. Add 5% trehalose and 10% water by mass of the mixed seed mud for emulsification, and vacuum dry to obtain core material.

[0124] Step 3: Mix sodium alginate and dextrin at a mass ratio of 3:1, add water (1L of water for every 10g of dextrin) to form a paste mixture, add the core material prepared above (mass ratio of core material to paste mixture is 1:5), add 2% CaCl2 solution, react for 30min, the volume of CaCl2 solution is twice that of the paste mixture, vacuum dry after reaction to obtain control sclerotium 1;

[0125] Step 4: Mix control sclerotium 1 and bentonite at a mass ratio of 1:1, granulate by drum granulation at a speed of 30 r / min, steam cure at 96℃ for 3.5 h, and dry to obtain microbial inoculant control standard, denoted as inoculant control standard 1.

[0126] Comparative Example 2

[0127] In this comparative example, the control standard was prepared according to the process of Example 4. The main difference is that the waste white clay coating layer of S4 is not performed, but bentonite is used instead. The specific preparation process is as follows: the composite microbial sclerotia prepared in step S33 of Example 4 and bentonite are mixed at a mass ratio of 1:3, granulated by drum granulation at a speed of 30 r / min, steam cured at 96°C for 3.5 h, and dried to obtain the microbial inoculant control standard, which is denoted as inoculant control standard 2.

[0128] Comparative Example 3

[0129] In this comparative example, the control standard was prepared according to the process of Example 4. The main difference is that CaCO3 microsphere sample 1 was not used in step S31, and citric acid microcrystal sample 1 was not used in step S32, which was instead replaced by commercially available CaCO3 and citric acid of equal mass. In the subsequent step of Example 4, a microbial inoculant control standard was prepared and was designated as inoculant control standard 3.

[0130] Implementation effect verification

[0131] (I) pH responsiveness verification of dual-network gel

[0132] Add bacterial agent samples 1-3 and bacterial agent control standards 1-3 to buffer solutions of different pH values ​​(where the buffer solution at pH 6.5 is a 0.1M NaH2PO4-Na2HPO4 buffer system, the buffer solution at pH 8.0 is a 0.1M Tris-HCl buffer system, and the buffer solution at pH 10.5 is a 0.1M NaHCO3-Na2CO3 buffer system), with each gram of bacterial agent corresponding to 100 mL of buffer solution;

[0133] Sampling and analysis were conducted at 0h, 12h, and 24h. The wet weight after surface moisture was absorbed was determined by gravimetric method, and the swelling ratio was calculated. H was determined by titration. + The concentration was determined and the release rate was calculated. The results are shown in Tables 1 to 3.

[0134] The formula for calculating the swelling ratio is: Swelling ratio (%) = [(W t -W0) / W0]×100, where W0 represents the initial dry weight of the inoculum, in g; W t The wet weight, in grams, is the weight after the surface moisture has been absorbed at time t.

[0135] H + The formula for calculating the release rate is: H + Release rate (%) = [(C t [×V) / M0]×100, where C t This indicates the H measured by titration. + Concentration, mol / L; V represents the buffer volume, L; M0 represents the theoretical H₂O of citric acid in the bacterial agent. + Total amount, in mol.

[0136] The calculation method for M0 is as follows: Citric acid is a tribasic weak acid, and theoretically, 1 mol of citric acid can completely dissociate to release 3 mol of H₂. + The citric acid in this invention is entirely derived from the added citric acid microcrystals, i.e., M0 = mass of added citric acid microcrystals / molar mass of citric acid × 3.

[0137] Table 1: pH responsiveness validation results of the dual-network gel at pH 6.5

[0138]

[0139] As shown in Table 1, the swelling rate and H2O of the bacterial agent samples of the present invention under acidic conditions are... + The release rates were all low, indicating good stability of the encapsulation layer; the bacterial agent control 1, due to its lack of pH responsiveness, showed low swelling rate and H₂O. + The release rate is significantly higher than expected.

[0140] Table 2: pH responsiveness validation results of the dual-network gel at pH 8.0

[0141]

[0142] As shown in Table 2, the bacterial agent sample of this invention, when activated in a weakly alkaline environment with a pH-responsive matrix, exhibits a swelling rate >68% after 24 hours. + Release rate >56%; the bacterial agent control 2 without waste clay coating responded too quickly due to the lack of a protective layer; the bacterial agent control 3 had a lower release efficiency due to the uneven particle size of the raw materials used.

[0143] Table 3: pH responsiveness validation results of the dual-network gel at pH 10.5

[0144]

[0145] As shown in Table 3, the bacterial agent sample of the present invention showed almost complete response within 24 hours under strongly alkaline conditions. + Release rate >89%, demonstrating the high pH sensitivity of the dual-network gel; H 24h of bacterial agent control standards 1-3 + The release rate decreased instead of increased, which may be due to reasons such as the disintegration of the bacterial agent structure, the influence of miscellaneous bacteria and bacterial metabolites.

[0146] In summary, bacterial control 1, which uses sodium alginate encapsulation and lacks a pH-responsive matrix, exhibits rapid swelling and H+ ionization under all pH conditions. + Release was not achieved in alkaline soil; the bacterial agent control 2 showed a swelling rate of 45.1% in an acidic environment, indicating insufficient sealing of the bentonite, which could not resist acidic rainwater / soil erosion. In a weakly alkaline environment of pH 8.0, H... +The release rate was 72.1%, indicating an premature response, which may be due to leakage of the coating layer leading to premature activation of the sclerotia. The bacterial agent control 3 used commercially available CaCO3 and citric acid, but the particle size was not accurately controlled, resulting in premature activation of the sclerotia in a strongly alkaline environment at pH 10.5. + The release rate was only 79.4%, the kinetics were significantly delayed, and it also showed a swelling rate that was related to H. + The release mismatch may be due to the large citric acid particles not being fully dissolved.

[0147] (II) Verification of microbial community survival rate and functional expression

[0148] Samples 1-3 and control samples 1-3 were placed in simulated saline-alkali culture medium, with 10g of bacterial agent added per 1L of culture medium. Samples were taken at 0 and 90 days, and the viable counts of *Streptomyces loucheri*, *Bacillus subtilis*, and *Trichoderma harzianum* were determined by plate counting. *Streptomyces loucheri* was counted using Gao's No. 1 medium, *Bacillus subtilis* was counted using LB medium containing 5% NaCl, and *Trichoderma harzianum* was counted using the PDA plate spore germination method. The Na2CO3 degradation rate was determined by acid-base titration, and the amount of organic acid secreted was determined by high performance liquid chromatography. The results are shown in Table 4.

[0149] The formula for calculating the degradation rate of Na2CO3 is:

[0150] Degradation rate (%) = [1-(C)] t [×V) / (C0×V0)]×100, where C0 represents the initial Na2CO3 concentration, mol / L; C t V represents the concentration of Na2CO3 at time t, in mol / L; V represents the volume of the culture medium, in L; V0 represents the initial volume of the culture medium, in L.

[0151] The formula for the simulated saline-alkali culture medium is as follows: 0.2 mol / L Na2CO3, 5% (w / v) NaCl, 50 g / L NaHCO3, 0.01 mol / L K2HPO4, 0.005 mol / L MgSO4·7H2O, 0.1% (w / v) yeast extract and trace elements.

[0152] Table 4: Validation results of bacterial community survival rate and functional expression

[0153]

[0154] As shown in Table 4, the bacterial agent samples of this invention, due to the use of waste clay coating to resist salt penetration and dual-network gel grouping for slow release, exhibited a viable bacterial retention rate >85%, Na2CO3 degradation rate >82%, and organic acid secretion >30 mg / g after 90 days. This demonstrates that the bacterial agent of this invention possesses sustained and efficient remediation capabilities in saline-alkali environments. Although bacterial agent control 1 was coated with bentonite and embedded in sodium alginate, the viable bacterial count rapidly decreased to below 5% of the initial value, resulting in complete loss of function. This was because the insufficient salt and alkali resistance of the embedding material led to structural disintegration, indirectly exposing and killing the bacterial community. Bacterial agent control 2, due to the hydrophilicity of the coating layer leading to salt ion penetration, not only had a low viable bacterial retention rate but also a significant decrease in function. Bacterial agent control 3, due to uneven particle size disrupting the synergistic release of the bacterial community, showed a significantly lower degradation rate and organic acid secretion than the sample group. In summary, the bacterial agent samples of the present invention exhibit long-term bacterial survival rate and stable functional expression under extreme saline-alkali environments due to the triple protection mechanism of waste clay physical barrier, intelligent release of dual-network gel and synergistic effect of uniform microcarrier.

[0155] (III) Field Experiments

[0156] The experiment was conducted using severely soda-type saline-alkali soil with a pH of 8.7, a Na2CO3 content of 0.38%, and a salinity of 7.5 mS / cm.

[0157] The experimental group used microbial agent sample 1; control group 1 used microbial agent control standard 1; control group 2 used microbial agent control standard 2; control group 3 used microbial agent control standard 3; control group 4 used physical amendment, i.e., 200 kg / mu of citric acid sulfur gypsum alone; the blank control did not apply microbial agent / amendment.

[0158] Except for control group 4, which was treated with a mixture of microbial agent (50 kg / mu) and decomposed straw powder (300 kg / mu) and rotary tilled to a soil layer of about 20 cm; control group 4 was treated with a mixture of citric acid sulfur gypsum (200 kg / mu) and decomposed straw powder (300 kg / mu) and rotary tilled to a soil layer of about 20 cm.

[0159] Sorghum, rice, and soybeans were planted in the experimental soil. The average yield per acre of crops was recorded at the harvest time, and the soil pH, Na2CO3 content, and salinity were measured after harvest. The results are shown in Table 5.

[0160] Table 5: Results of the field experiment

[0161]

[0162] As shown in Table 5, the experimental groups using the microbial agent of this invention achieved significant crop yield increases and deep improvement of saline-alkali soil. Control group 1 showed only weak improvement due to the inactivation of microorganisms in the saline-alkali environment. Control group 2 experienced a significant yield reduction due to decreased microbial survival rate caused by salt ion infiltration. Control group 3 suffered from delayed microbial release and insufficient activation of soybean nitrogen fixation due to uneven particle size. Control group 4 experienced a significant reduction in rice yield due to short-term deterioration of soil structure caused by a lack of microbial metabolic support. Therefore, the microbial agent of this invention solves the problems of low microbial survival rate, unsustainable soil improvement, and poor crop resistance in severely saline-alkali land, providing a reliable solution for the engineering remediation of soda-type saline-alkali land.

[0163] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microbial inoculant for improving saline-alkali land, characterized in that, The microbial inoculant comprises acidic and alkaline bacterial groups, a dual-network gel system, and a waste clay coating layer. The alkaline bacterial group consists of an alkaline buffer core of *Streptomyces loucheretia* and CaCO3 microspheres, with a viable count of 5 × 10⁻⁶ *Streptomyces loucheretia*. 9 CFU / g ~ 8×10 9 CFU / g; the acidic bacterial community comprises a complex of Bacillus subtilis and humic acid buffer and a core composed of Trichoderma harzianum and citrate microcrystals, with a viable Bacillus subtilis count of 2 × 10⁻⁶. 10 CFU / g ~ 3×10 10 CFU / g, viable count of Trichoderma harzianum is 3×10 9 CFU / g ~5×10 9 CFU / g; The dual-network gel system is a composite gel assembled by hydrogen bonds from a CMC-β-CD network gel containing β-cyclodextrin-modified carboxymethyl cellulose that embeds the middle layer of bacteria and a CMCHT network gel containing calcium-crosslinked carboxymethyl chitosan that embeds the inner and outer layers of bacteria; CaCO3 microspheres are CaCO3 particles with a particle size of 33μm to 38μm, and citric acid microcrystals are citric acid crystals with a particle size of 30μm to 50μm.

2. The microbial inoculant for improving saline-alkali land according to claim 1, characterized in that, The method for preparing the microbial inoculant includes: S1 strain activation: Streptomyces loucheri, Bacillus subtilis and Trichoderma harzianum were activated separately to prepare Streptomyces seed liquid, Bacillus subtilis seed liquid and Trichoderma harzianum seed liquid; Preparation of the S2 dual-network gel system: Preparation of S21 CMC-β-CD network gel: Carboxymethyl cellulose and β-cyclodextrin were dissolved in water, 1,4-butanediol diglycidyl ether was added, the pH was adjusted to 10-11, and the reaction was carried out at 30℃-32℃ for 1-2 hours. After the reaction was completed, the gel was precipitated with anhydrous ethanol and dried to obtain CMC-β-CD network gel. Preparation of S22 CMCHT network gel: Carboxymethyl chitosan was dissolved in acetic acid solution, CaCl2 solution A was added dropwise to produce a precipitate, the precipitate was collected by centrifugation, washed with deionized water until the washing solution was neutral, and dried to obtain CMCHT network gel; S3 microbial community embedding: Preparation of S31 alkaline bacterial colony gel: Streptomyces seed liquid was mixed with CaCO3 microspheres, and 0.1% glutaraldehyde solution was added dropwise. The reaction was carried out for 30 min to 40 min. After the reaction was completed, the mixture was filtered and freeze-dried to obtain CaCO3 microspheres loaded with Streptomyces. CMCHT network gel was added to a solid content of 12% to 18% to obtain alkaline bacterial colony gel. Preparation of S32 acidic bacteria: Bacillus subtilis seed culture was mixed with humic acid buffer to obtain a bacterial humic acid mixture; Trichoderma harzianum seed liquid was centrifuged to obtain Trichoderma harzianum spore mud, and Trichoderma harzianum spore mud was mixed with citric acid microcrystals to obtain Trichoderma citric acid mixture; Acidic bacterial colony gel was prepared by mixing bacterial humic acid mixture, Trichoderma citric acid mixture and CMCHT network gel at a mass ratio of 1:1:6-8, adding CaCl2 solution B dropwise, and reacting at a temperature of 22℃-28℃ for 10-20 minutes. Composite of S33 dual-network gel system: Alkaline bacterial gel and acidic bacterial gel are mixed, water is added, heated to 55℃~60℃ and shaken at 180r / min~200r / min for 1h~2h. After the reaction is completed, the composite microbial sclerotia are obtained by filtration and freeze drying. S4 waste clay coating layer: Waste bleaching clay and composite microbial sclerotia are mixed at a mass ratio of 3 to 4:1 and granulated. The mixture is then subjected to drum granulation at a speed of 28 to 32 r / min, steam curing at 95 to 98℃ for 3 to 4 hours, and drying to obtain the microbial agent.

3. The microbial inoculant for improving saline-alkali land according to claim 2, characterized in that, In step S1, the viable Streptomyces count in the Streptomyces louchei seed culture is 2 × 10⁻⁶. 9 CFU / mL ~ 3 × 10 9 CFU / mL; The viable Bacillus subtilis count in the Bacillus subtilis seed culture was 5 × 10⁻⁶. 9 CFU / mL ~ 6 × 10 9 CFU / mL; the viable count of *Trichoderma harzianum* in the *Trichoderma harzianum* seed culture was 0.1 × 10⁻⁶. 9 CFU / mL ~ 0.2 × 10⁻⁶ 9 CFU / mL.

4. The microbial inoculant for improving saline-alkali land according to claim 2, characterized in that, In step S21, the mass ratio of carboxymethyl cellulose to β-cyclodextrin is 6–8:1, and 1–1.5 mL of 1,4-butanediol diglycidyl ether is used for every 10 g of carboxymethyl cellulose.

5. The microbial inoculant for improving saline-alkali land according to claim 2, characterized in that, In step S22, the acetic acid solution contains 2%–3% acetic acid by mass, and the CaCl2 solution A contains 4.5%–5.5% CaCl2 by mass. Each 10 grams of carboxymethyl chitosan corresponds to 400 mL–600 mL of acetic acid solution and 200 mL–300 mL of CaCl2 solution, respectively.

6. The microbial inoculant for improving saline-alkali land according to claim 2, characterized in that, In step S31, the volume ratio of Streptomyces seed liquid to CaCO3 microspheres is 1:3.5 to 4.5, and the amount of glutaraldehyde solution used is 0.6 to 1.0 times that of CaCO3 microspheres.

7. The microbial inoculant for improving saline-alkali land according to claim 2, characterized in that, In step S32, the Bacillus subtilis seed solution and humic acid buffer are mixed at a volume ratio of 1:1.5 to 2.

0. The Trichoderma harzianum seed solution is centrifuged at 5000 r / min to 6000 r / min for 12 min to 15 min to obtain Trichoderma harzianum spore mud. The mass ratio of Trichoderma harzianum spore mud to citric acid microcrystals is 1:1.0 to 1.

2.

8. The microbial inoculant for improving saline-alkali land according to claim 2, characterized in that, In step S32, the mass fraction of CaCl2 in CaCl2 solution B is 2.5% to 3.5%, and the volume ratio of CaCl2 solution B to CMCHT network gel is 1:2.0 to 2.

5.

9. A microbial inoculant for improving saline-alkali land according to claim 2, characterized in that, In step S33, the mass ratio of alkaline bacterial gel to acidic bacterial gel is 2.5 to 3.5:1, and the added water is 4 to 5 times the total gel volume.

10. A microbial inoculant for improving saline-alkali land according to any one of claims 1-9, characterized in that, The waste bleaching clay is oil refining waste bleaching clay with an oil content of 12% to 15%. It is inoculated with Bacillus subtilis and fermented at 30℃ to 32℃ for 12 to 15 days, resulting in a residual rate of 2% to 5%. The CaCO3 microspheres are prepared by mixing polyethylene glycol 8000 solution and Na2CO3 solution to form a two-phase aqueous emulsion, then adding CaCl2 solution, reacting at 22℃ to 28℃ for 1 to 2 minutes, and then aging, centrifuging, drying, and sieving. The citric acid microcrystals are prepared by cooling citric acid solution to 4℃ to 5℃ for crystallization, centrifuging, and then spray drying.

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