Low-temperature-resistant biological functional preparation for saline-alkali soil treatment, and preparation method and application thereof

Abstract

The application discloses a low-temperature-resistant biological functional preparation for saline-alkali soil treatment and a preparation method and application thereof, and belongs to the technical field of microorganisms.The polyvinyl alcohol solution, the sodium alginate solution, the 4-carboxyphenylboronic acid solution and the chitosan solution are mixed in proportion, magnetized to obtain a glue solution, and then the low-temperature-resistant and saline-alkali-resistant bacterial agent A or the bacterial agent B composed of sulfonated humic acid powder, mixed sugar sources, nutrient salts and specific bacterial strains is added to the bacterial suspension, stirred uniformly, dripped into a calcium chloride aqueous solution, and then shaped to obtain the biological functional preparation.The application constructs an "intelligent micro-ecological factory" which integrates carbon source slow release, microbial immobilization, pollutant adsorption and biological catalysis enhancement, and realizes precise treatment of different degrees of saline-alkali soil.Different from traditional simple bacterial embedding technology, the system is helpful to improve the soil ecological environment and improve crop yield, and embodies the advanced concept that the treatment strategy develops from single function to compounding and systematization.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a low-temperature resistant biological functional agent for saline-alkali land remediation, its preparation method, and its application. Background Technology

[0002] Soil salinization is a significant cause of the decline in soil quality in Northeast China's black soil region. The western Songnen Plain (Daqing and Qiqihar in Heilongjiang Province, and Baicheng and Songyuan in Jilin Province) is my country's largest concentration of soda-alkali soil, accounting for over 70% of the total saline-alkali land in the black soil region. Additionally, the lower reaches of the Liaohe Plain (Panjin and Yingkou in Liaoning Province) are significantly affected by seawater intrusion, resulting in predominantly coastal saline-alkali soil. Severe soil salinization deteriorates soil physical properties, reduces soil fertility, disrupts soil aggregate structure, and decreases soil aeration and water retention. It causes soil compaction and dryness, affecting seed germination, seedling emergence, crop nutrient absorption, and water balance, ultimately reducing crop yield per unit area. Currently, several research institutions and technical teams are studying saline-alkali land remediation, primarily focusing on chemical treatment and water conservancy projects, which are costly. Biological treatment is safe and environmentally friendly, mainly using Bacillus, Halomonas, and Pseudomonas. These bacteria are not cold-resistant, so the effective time for biological treatment is only from after spring plowing (late May) to autumn harvest (early October), and there is no specific formula applicable to different salinity levels. Summary of the Invention

[0003] To address the problems of insufficient low-temperature resistance and short treatment time of microbial agents in the treatment of saline-alkali land in northern China, the present invention aims to provide a low-temperature resistant biological functional agent for saline-alkali land treatment, its preparation method, and its application. This invention constructs an "intelligent micro-ecological factory" integrating slow-release carbon source, microbial immobilization, pollutant adsorption, and biocatalytic enhancement. By optimizing the agent formulation, it achieves precise treatment of soils with varying degrees of salinization. Unlike traditional simple bacterial encapsulation technology, this system helps improve the soil ecological environment and increase crop yield, reflecting the advanced concept of developing treatment strategies from single-function to multi-functional and systematic approaches.

[0004] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing a low-temperature resistant biological functional agent for saline-alkali land remediation, comprising the following steps: (1) Prepare polyvinyl alcohol (PVA) solution with a mass concentration of 10-30%, sodium alginate (SA) solution with a mass concentration of 1-10%, 4-carboxyphenylboronic acid (PBA) solution with a mass concentration of 0.5-5% and chitosan (CS) solution with a mass concentration of 1-5% respectively. (2) Mix PVA solution, SA solution, PBA solution and CS solution in a volume ratio of 2~4:4~6:0.5~2:0.5~2, stir evenly, and place the resulting mixed solution in a magnetic field for magnetization treatment, or flow it through a magnetically attached pipe at a constant speed for magnetization treatment to obtain the adhesive solution. (3) Prepare low-temperature and salt-alkali resistant bacterial agents A and B. Bacterial agent A consists of Flavobacterium frigoritolerans, Algoriphagus halophilus, Bacillus subtilis, and Bacillus licheniformis, with a ratio of Flavobacterium frigoritolerans: Algoriphagus halophilus: Bacillus subtilis: Bacillus licheniformis of 1~3:3~5:1~3:1~3. Bacterial agent B consists of Flavobacterium frigoritolerans, Bacillus daqingensis, Bacillus suaedaesalsae, and Bacillus subtilis. The bacterial suspension is composed of *Bacillus subtilis*, *Bacillus daqingensis*, *Bacillus salsa*, and *Bacillus subtilis* in a ratio of 1-3:2-4:2-4:1-3; bacterial agent A or bacterial agent B is prepared into a bacterial suspension with a concentration of 1×10⁻⁶. 7 ~1×10 10 CFU / mL; (4) Add sulfonated humic acid powder, mixed sugar source, nutrients and bacterial suspension obtained in step (3) to the gel obtained in step (2), stir evenly to obtain mixed hydrogel; (5) The mixed hydrogel is dropped into an aqueous solution of calcium chloride (CaCl2) and the biological functional preparation is obtained after it is formed.

[0005] Based on the above technical solution, further, the polyvinyl alcohol mentioned in step (1) is polyvinyl alcohol 1788, the solvent of polyvinyl alcohol (PVA) solution and sodium alginate (SA) solution is water, and the solvent of 4-carboxyphenylboronic acid (PBA) solution and chitosan (CS) solution is a mixed solvent of acetic acid and water with a volume ratio of 1:5 to 1:20.

[0006] Based on the above technical solution, further, the mass concentration of the polyvinyl alcohol (PVA) solution in step (1) is 10~20%, the mass concentration of the sodium alginate (SA) solution is 1~5%, the mass concentration of the 4-carboxyphenylboronic acid (PBA) solution is 0.5~2%, and the mass concentration of the chitosan (CS) solution is 1~5%.

[0007] Based on the above technical solution, further, the PVA solution, SA solution, PBA solution and CS solution mentioned in step (2) are in a volume ratio of 3:5:1:1.

[0008] Based on the above technical solution, further, in step (2), the magnetic field strength is 250~350 mT and it is left to stand for 10~60 min; the magnetic field strength of the magnetically attached pipe is 250~350 mT and the flow rate is 0.01~0.5 m / s.

[0009] Based on the above technical solution, further, in step (3), the CGMCC numbers of the following bacteria are as follows: Flavobacterium tumefaciens: CGMCC1.11577, halophilic psychrophilic bacteria: CGMCC1.10158, Bacillus subtilis: CGMCC1.2416, Bacillus licheniformis: CGMCC1.4401, Bacillus daqingensis: CGMCC1.12295, and Bacillus salsa: CGMCC1.18659.

[0010] Based on the above technical solution, the preparation process of the bacterial suspension in step (3) is as follows: Add bacterial agent A or bacterial agent B to water to activate and form a concentrated bacterial slurry, inoculate it onto a Sehgal-Gibbons medium plate, and incubate it upside down in an incubator at 20~25℃; according to the Sehgal-Gibbons medium formula, only prepare the basic salt solution, dilute it to a salt concentration of 5~6%, adjust the pH to 7.0~7.5, sterilize it, gently scrape the bacterial moss off the Sehgal-Gibbons medium plate with a sterile inoculation loop or scraper, transfer it to the sterilized basic salt solution, disperse the bacterial cells evenly to form an initial bacterial suspension, centrifuge to obtain the precipitate bacterial cells, add water to prepare a bacterial suspension.

[0011] Based on the above technical solution, further, the formula of Sehgal-Gibbons culture medium in step (3) is as follows: sodium chloride 40.0 g / L, potassium chloride 2.0 g / L, magnesium sulfate heptahydrate 20.0 g / L, yeast extract 10.0 g / L, tryptone 5.0 g / L, sodium citrate dihydrate 3.0 g / L, casein hydrolysate 5.0 g / L, and the remainder is water.

[0012] Based on the above technical solution, further, in step (4), based on 100ml of glue solution, 1~10g of sulfonated humic acid powder, 1~5g of mixed sugar source, 0.1~2.0g of nutrient salt and 5~25ml of bacterial suspension obtained in step (3) are added to the glue solution.

[0013] Based on the above technical solution, further, in step (4), the particle size of the sulfonated humic acid powder is ≤100 mesh, the sulfonated humic acid is composed of sulfonated humic acid and sulfonated fulvic acid, and the mass ratio of sulfonated humic acid and sulfonated fulvic acid is 1:1~3; the mixed sugar source is composed of glucose, sucrose and starch, and the weight ratio of glucose, sucrose and starch is 1:1:1; the nutrient salt is composed of potassium dihydrogen phosphate, magnesium sulfate, superphosphate and ammonium sulfate, and the weight ratio of potassium dihydrogen phosphate, magnesium sulfate, superphosphate and ammonium sulfate is 1:1:1:1.

[0014] Based on the above technical solution, the preparation process of sulfonated humic acid in step (4) is as follows: Add 25-35 times the mass of sulfuric acid aqueous solution with a concentration of 12-16% to humic acid, stir and react at 70-80℃ for 3-4 hours, let stand until the solution separates into layers, filter, add 40-60 times the mass of sodium hydroxide aqueous solution with a concentration of 20-30% to the obtained solid, stir and dissolve at room temperature for 1-5 hours, separate, slowly add dilute sulfuric acid to the supernatant, adjust the pH to 1.0-1.5, let stand at room temperature for 2-6 hours, precipitate black sulfonated humic acid, filter, wash with water until neutral, and dry to obtain the product.

[0015] Based on the above technical solution, the preparation process of sulfonated fulvic acid in step (4) is as follows: Fuming sulfuric acid (SO3 content 10-30%) is added to the reaction flask, cooled to 0-5℃ in an ice bath, fulvic acid powder is added in batches, each addition amount is <10%, the temperature is controlled <20℃, the addition time is 1-2 hours, the mass ratio of fulvic acid to fuming sulfuric acid is 1:4, after the addition is completed, the ice bath is removed and the reaction is carried out at room temperature for 4-8 hours, the obtained reaction solution is slowly dripped into ice water, the volume ratio of reaction solution to ice water is 1:10-1:20, the temperature is controlled <20℃, neutralized to pH 5-6 with Ca(OH)2 or CaCO3, CaSO4 precipitate is removed by filtration, the filtrate is purified by dialysis (MWCO =1000Da membrane), and then freeze-dried to obtain the product.

[0016] Based on the above technical solution, further, the mass concentration of the calcium chloride (CaCl2) aqueous solution in step (5) is 2~5%.

[0017] Based on the above technical solution, further, in step (5), the particle size of the biological functional preparation containing bacterial agent A is controlled at 4~8mm, and the particle size of the biological functional preparation containing bacterial agent B is controlled at 1~3mm.

[0018] Based on the above technical solution, the biofunctional preparation obtained in step (5) is further processed and matured. The specific process is as follows: the formed biofunctional preparation pellets are soaked in cold water for 20-30 minutes to stabilize their internal structure. Then, the surface of the pellets is gently rinsed with clean water or sterile water and transferred to a moist, sterile container. The pellets are then cultured at 20-25°C for 24-48 hours. This process allows the embedded microorganisms to recover from stress, begin to reproduce and colonize, and activate the function of the pellets.

[0019] Secondly, the present invention provides a biological functional preparation obtained by the above preparation method.

[0020] Thirdly, the present invention provides the application of the above-mentioned biological functional agents in the treatment of saline-alkali land.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention selects low-temperature resistant, salt-alkali resistant or halophilic bacteria, which can increase the action time of the bacterial agent in cold regions.

[0022] 2. The microspheres made from a gel matrix (polyvinyl alcohol and sodium alginate) in this invention act as a cold buffer for the microorganisms within them. The gel matrix (especially hydrogel) has a high specific heat capacity, which can buffer drastic fluctuations in external temperature, creating a relatively stable thermal environment for the internal bacteria and reducing cold shock. The gel network can limit the growth size and morphology of ice crystals, reducing mechanical damage to the embedded cells.

[0023] 3. The humic acid used in this invention undergoes sulfonation treatment to introduce the -SO3H group, enhancing its water solubility and salt resistance. -SO3H is a strongly hydrophilic and strongly ionizing group. Its introduction significantly increases the total hydrophilic point of the molecule. Furthermore, the ionization constant of the sulfonic acid group is much higher than that of the carboxyl group, and the sulfonate (-SO3Na) formed by it has extremely high solubility in hard water or salt water and is not prone to precipitation with calcium, magnesium, or other ions. -SO3H is a strong hydration group, which can combine with more water molecules through hydrogen bonds to form a strong hydration film. This strong solvation effect interferes with the process by which water molecules themselves form regular ice crystal structures, thereby lowering the freezing point of the solution.

[0024] 4. In the early stages of remediation, when the soil salinity is high, it is advisable to use biological agents containing microbial agent B to quickly adapt to the extreme environment and initiate the improvement process. After 1-2 years of remediation, once the soil salinity has initially decreased and the physicochemical properties have gradually improved, the biological agents containing microbial agent A can be used to further enhance the soil's ecological function, consolidate the remediation results, and achieve a smooth transition from "emergency remediation" to "systematic restoration".

[0025] 5. The biological functional formulation of the present invention contains different carbon sources, which can provide nutrient resources for different types of soil organisms after application and meet the needs of most organisms. Among them, glucose is easily utilized and released quickly; sucrose and starch are released more slowly. A mixed sugar source of "fast and slow combination" is used to achieve gradient release of carbon source.

[0026] 6. The biological functional preparation of the present invention delivers nitrogen, potassium, calcium and magnesium elements into the soil by adding nutrients.

[0027] 7. This invention places the PVA / SA mixed adhesive in a magnetic field for solvent magnetization treatment, which can significantly enhance the freeze-thaw cycle resistance of the prepared biological agent, thereby better adapting to the harsh environmental conditions of large diurnal temperature differences and low average temperature in northern my country; the magnetized solvent can also change the stability of biological colloidal particles, enabling them to form a stable three-dimensional gel network through charge neutralization and bridging. Attached Figure Description

[0028] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0029] Figure 1 This is a schematic diagram of the structure of the smallest unit (microsphere) of the biofunctional formulation prepared in Example 1.

[0030] Figure 2 This is a description of the sample information and test items in the test report of Example 2.

[0031] Figure 3 The graph shows the performance verification results of the biofunctional formulation in Example 2. Detailed Implementation

[0032] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0033] Example 1 This embodiment provides a method for preparing a low-temperature resistant biological functional agent for saline-alkali land remediation, comprising the following steps: (1) Preparation of low-temperature and salt-alkali resistant bacterial agent A and bacterial agent B: Inoculant A (suitable for mild to moderate saline-alkali soils, pH < 9, alkalinity < 15) contains: Flavobacterium frigoritolerans (CGMCC No.: CGMCC 1.11577), Algoriphagus halophilus (CGMCC No.: CGMCC 1.10158), Bacillus subtilis (CGMCC No.: CGMCC 1.2416), and Bacillus licheniformis (CGMCC No.: CGMCC 1.4401). The ratio of Flavobacterium frigoritolerans: Algoriphagus halophilus: Bacillus subtilis: Bacillus licheniformis is 2:4:2:2.

[0034] Inoculant B (suitable for severely saline-alkali land, pH>9, alkalinity>15) contains: Flavobacterium frigoritolerans (CGMCC No.: CGMCC 1.11577), Bacillus daqingensis (CGMCC No.: CGMCC 1.12295), Bacillus suaedaesalsae (CGMCC No.: CGMCC 1.18659), and Bacillus subtilis (CGMCC No.: CGMCC 1.2416). The ratio of Flavobacterium frigoritolerans: Bacillus daqingensis: Bacillus suaedaesalsae: Bacillus subtilis is 2:3:3:2.

[0035] (2) Preparation of bacterial agent solution Add bacterial agent A or bacterial agent B to a small amount of water at 20-25℃, let stand for 0.5-1 hour to activate, and gently stir until completely dissolved / dispersed to form a concentrated bacterial slurry. Inoculate the concentrated bacterial slurry onto Sehgal-Gibbons medium plates at an inoculation rate of 2% and incubate upside down in a 25℃ incubator.

[0036] Prepare the basic salt solution according to the Sehgal-Gibbons medium formula (40.0 g / L sodium chloride, 2.0 g / L potassium chloride, 20.0 g / L magnesium sulfate heptahydrate, 10.0 g / L yeast extract, 5.0 g / L tryptone, 3.0 g / L sodium citrate dihydrate, 5.0 g / L casein hydrolysate, balance water), dilute to a salt concentration of 5%, adjust the pH to 7.0-7.5, and sterilize for later use. Add 2-5 ml of basal salt solution to a sterile centrifuge tube. Gently scrape the bacterial colony from the Sehgal-Gibbons agar plate using a sterile inoculation loop or spatula. Transfer the scraped bacterial cells to the basal salt solution and gently vortex or repeatedly pipette to disperse the cells evenly, forming an initial bacterial suspension. Centrifuge the initial bacterial suspension at 4500 rpm for 15 min to obtain the precipitate of bacterial cells. Take an appropriate amount of the bacterial cells and dissolve them in distilled water to prepare a bacterial suspension with a concentration of 1×10⁻⁶. 9 CFU / mL.

[0037] (3) Humic acid production: Raw material ratio: Fresh soybean residue : Fresh vinegar residue = 1:2 mixture, so that the C / N ratio reaches the ideal composting range of 20:1; Adjusting auxiliary material: wheat bran, added at 5% of the total mass, with a particle size of 1-3 mm (to improve porosity); Preliminary composting: Crush vinegar residue into 2cm particles, adjust the pH to 5.5 with lime water (10%), mix with soybean residue, and adjust the water content to about 60% (it should clump together when squeezed by hand, dripping water between the fingers without forming a thread, and crumble easily when released); add auxiliary materials and mix thoroughly to obtain fermentation raw materials; add RW enzyme agent (Hebi Renyuan Biotechnology Development Co., Ltd.), at a dosage of 250g RW enzyme agent / 400kg of raw materials to be fermented, turn and turn every 3 days, continue for 7 days until the material temperature drops, and obtain pre-treated raw materials. Signs of maturity: The material is loose, has no original odor, has a slight ammonia smell, and white mycelium is present in the pile.

[0038] (4) Alkaline oxidation extraction: Add the pretreated raw materials to water at a solid-liquid ratio of 1:8 (g / g), and add potassium hydroxide at a concentration of 3g / 100g (total solid and liquid). Start stirring, stirring speed: 200 rpm, temperature: 65±2℃, time: 2h; Centrifugation was performed using a high-speed centrifuge at 10,000 r / min for 10 min to completely separate the remaining solid residue from the supernatant. The supernatant was the crude humic acid extract.

[0039] (5) Fractional purification: The crude humic acid extract was acidified with dilute HCl to pH 1.0 and allowed to stand for 10-12 hours. The resulting precipitate was humic acid. It was washed 2-3 times with dilute sulfuric acid at pH 1.0 to remove surface impurities and then dried in an oven at 70°C. The resulting supernatant was fulvic acid. 0.1M NaOH was slowly added dropwise to pH 5.0-6.0 to prevent localized over-alkalinity from causing precipitation. The supernatant was then filtered through a membrane (MWCO = 500-1000 Da) and freeze-dried to obtain high-purity fulvic acid.

[0040] (6) Humic acid sulfonation (to improve low temperature resistance) Humic acid sulfonation (1g as an example): Set the water bath temperature to 70-80℃ in a stirred water bath. Weigh 1.00g of humic acid and add it to a 100ml three-necked flask. After the water bath temperature reaches the set value, add 30ml of 15% sulfuric acid aqueous solution to the three-necked flask and stir well. After reacting for 3-4 hours, remove the three-necked flask from the water bath and let it stand until the solution separates into layers. Filter the solution. When the solid on the filter paper shows micro-cracks, transfer the solid to another 100ml three-necked flask. Wash with water until the filtrate is neutral. Add 50ml of 25% sodium hydroxide solution and stir at 500 r / min at room temperature for 2 hours. Separate using a centrifuge, retaining the upper blackish-brown supernatant (containing the target sulfonated sodium humate), and discard the insoluble impurities at the bottom. Slowly add dilute sulfuric acid dropwise to the supernatant to adjust the pH to 1.0-1.5. Let it stand at room temperature for 4 hours to precipitate a black sulfonated humic acid precipitate. Vacuum filter again, wash with deionized water until neutral, place the filter cake in an oven; dry at low temperature of 50-60 ℃ to constant weight, remove and place in a desiccator to cool to room temperature, pulverize and store for testing.

[0041] (7) Fulvic acid sulfonation: Pulverized fulvic acid was pulverized and passed through a 100-mesh sieve (particle size <150μm). Fuming sulfuric acid (SO3 content 10-30%) was prepared, with a fulvic acid:fuming sulfuric acid mass ratio of 1:4. The fuming sulfuric acid was added to the reaction flask and cooled to 0-5℃ in an ice bath. The fulvic acid powder was slowly added in batches, each batch <10%, with the temperature controlled <20℃ and the addition time: 1-2 hours. After the addition was complete, the ice bath was removed, and the reaction was carried out at room temperature (20-30℃) for 4-8 hours with a stirring speed of 200-400 rpm to obtain the reaction solution. The reaction solution was slowly added dropwise to ice water, with a reaction solution to ice water volume ratio of 1:10-20, with the temperature controlled <20℃. The solution was neutralized to pH 5-6 with Ca(OH)2 or CaCO3, and the CaSO4 precipitate was removed by filtration. The filtrate was purified by dialysis (MWCO = 1000Da membrane) until the conductivity was <100μS / cm, and then freeze-dried.

[0042] (8) Preparation of biological functional preparations 1) Add polyvinyl alcohol 1788 (PVA) to deionized water and stir at 90°C for 3 hours to obtain a 15% PVA solution.

[0043] 2) Add sodium alginate (CAS: 9005-38-3) (SA) to deionized water, heat and stir in a water bath at 50-60℃ to obtain a 3% SA solution.

[0044] 3) Add 1g of 4-carboxyphenylboronic acid (PBA) to a mixed solution of 9ml acetic acid and 90ml water, and stir magnetically until completely dissolved to obtain a 1% PBA solution.

[0045] 4) Add 2g of chitosan (CS, CAS: 9012-76-4) to a mixed solution of 8 ml acetic acid and 90 ml water, and stir magnetically until completely dissolved to obtain a 2% CS solution.

[0046] Cool the above solution to 30-40℃, mix PVA solution, SA solution, PBA solution and CS solution in a volume ratio of 3:5:1:1, stir to make them evenly mixed, and let the mixed solution stand in a magnetic field (magnetic field strength 290-320 mT) for 30 minutes or flow through a magnetically attached pipe at a constant speed for magnetization treatment (magnetic field strength 290-320 mT, flow rate 0.1m / s) to obtain the adhesive solution.

[0047] (9) Addition of solid-phase functional materials (based on 100ml of adhesive solution): Add the following to the above mixed adhesive solution in sequence: Humic acid powder (passed through a 100-mesh sieve): 4g (accounting for 4% of the total liquid mass), sulfonated humic acid: sulfonated fulvic acid mass ratio is 2:3; Mixed sugar source: 3.0g (3.0%, glucose:sucrose:starch weight ratio of 1:1:1); Nutrient salt: 1.0g (potassium dihydrogen phosphate: magnesium sulfate: superphosphate: ammonium sulfate weight ratio = 1:1:1:1); Add bacterial agent: Add 15ml of the bacterial suspension prepared above, and stir quickly and gently until homogeneous to obtain a mixed hydrogel.

[0048] (10) Droplet Use a syringe or dropper to draw up the mixed hydrogel and drop it into a 4% calcium chloride (CaCl2) aqueous solution to form a calcium alginate shell, making the spheres more robust. The particle size of the spheres can be adjusted according to needs. For mild to moderate saline-alkali land, larger spheres (6mm) can be selected, while for severely saline-alkali land, smaller spheres (2mm) can be used.

[0049] (11) Post-processing and ripening: Curing: Soak the molded spheres in cold water for 20-30 minutes to stabilize their internal structure.

[0050] Cleaning: Gently rinse the surface of the ball with clean or sterile water.

[0051] Curing: Transfer the microspheres to a moist, sterile container and incubate them at 25°C for 36 hours. This process allows the encapsulated microorganisms to recover from stress, begin to multiply and colonize, and activate the microspheres' function.

[0052] Example 2 The performance of the biofunctional formulation prepared in Example 1 was verified using indoor experiments. Dates: February 23 - April 23, 2026; Room temperature: 15-16℃ (maintain a low temperature to verify the effect of low temperature); Lighting: Simulate sunlight with fluorescent tubes, 12 hours during the day and 12 hours at night; Experimental design: Using an area of ​​1m² 2 Soil was placed in plastic boxes with holes at the bottom to ensure the spray solution could flow out. The soil depth was 20cm, and each plastic box represented one sample. Four groups were set up: original soil A (mild to moderately saline-alkali soil, pH≈8.5, alkalinity <15, no treatment), original soil B (severely saline-alkali soil, pH≈10, alkalinity >15, no treatment), modified soil-A, and modified soil-B. Modified soil-A was treated with a bio-functional agent containing microbial agent A, and modified soil-B was treated with a bio-functional agent containing microbial agent B. The treatment with the bio-functional agent was performed in triplicate. The amount added to modified soil-A was 4kg, and the amount added to modified soil-B was 8kg, both thoroughly mixed with the soil. Rainfall was simulated using a watering can. Due to reduced rainfall in northern regions during winter, watering was conducted every 7 days in the first month and every 10 days in the second month, with 3 liters of water each time.

[0053] Testing: After the experiment, five samples were collected from each plastic box using the plum blossom method to make a mixed sample and put into a sealed bag, with each sample weighing 1 kg; the test indicators included soil pH, electrical conductivity, soluble salts and organic matter.

[0054] The results are as follows Figure 2-3 As shown in the figure, after two months of treatment, the biological functional microbial agent can reduce soil pH, electrical conductivity and soluble salt content, while significantly increasing soil organic matter content, and has the potential for widespread application.

[0055] Example 3 Implementation Plan - Mild to Moderate Saline-Alkali Land Core objective: Through the synergistic effects of physicochemical and biological processes, reduce soil pH from 8.0-9.0 to 7.0-7.5 and total salt content by 10%-30% within 2-3 years.

[0056] Tillage depth: 20-30cm; Implementation method: Place the bio-functional agent containing microbial agent A into a rotary tiller and fertilizer applicator, and spread the bio-functional agent while performing rotary tillage, ensuring thorough mixing with the topsoil. If a rotary tiller and fertilizer applicator is unavailable, first create ridges in the field using a ridging machine, then spread the bio-functional agent containing microbial agent A into the ridges, and then perform two more rotary tillage operations to evenly distribute the bio-functional agent into the soil.

[0057] Implementation time: The application time is from April to June in spring and from October to November in autumn.

[0058] Dosage of formulation: 2-3 tons / mu for the first time, and 1-2 tons / mu for the second time.

[0059] Implementation Plan - Severely Saline-Alkali Land Core objective: To achieve rapid improvement of severely saline-alkali wasteland through the synergistic effect of physical, chemical and biological methods, so as to generate economic benefits in the same year, achieve outstanding improvement results, and be green, environmentally friendly and pollution-free.

[0060] Tillage depth: 50cm to ensure effective breaking of the obstructing soil layer, improve soil structure, and create favorable conditions for subsequent improvement measures.

[0061] Implementation method: Place the bio-functional preparation containing microbial agent B into a rotary tiller and fertilizer applicator, and spread the bio-functional preparation while performing rotary tillage, ensuring thorough mixing with the topsoil. If a rotary tiller and fertilizer applicator is unavailable, first create raised beds in the field using a ridging machine, then spread the bio-functional preparation containing microbial agent B into the raised beds, and then perform multiple rotary tillage operations until the bio-functional preparation is evenly distributed into the soil.

[0062] Implementation time: The application time is from April to June in spring and from October to November in autumn.

[0063] Dosage of formulation: 5-7 tons / mu for the first time, and 3-5 tons / mu for the second time.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; modifications or equivalent substitutions may be made to the technical solutions described in the foregoing embodiments, and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a low-temperature resistant biological functional agent for saline-alkali land remediation, characterized in that, Includes the following steps: (1) Prepare polyvinyl alcohol (PVA) solution with a mass concentration of 10-30%, sodium alginate (SA) solution with a mass concentration of 1-10%, 4-carboxyphenylboronic acid (PBA) solution with a mass concentration of 0.5-5% and chitosan (CS) solution with a mass concentration of 1-5% respectively. (2) Mix PVA solution, SA solution, PBA solution and CS solution in a volume ratio of 2~4:4~6:0.5~2:0.5~2, stir evenly, and place the resulting mixed solution in a magnetic field for magnetization treatment, or flow it through a magnetically attached pipe at a constant speed for magnetization treatment to obtain the adhesive solution. (3) Prepare low-temperature and salt-alkali resistant bacterial agents A and B. Bacterial agent A consists of Flavobacterium frigoritolerans, Algoriphagus halophilus, Bacillus subtilis, and Bacillus licheniformis, with a ratio of Flavobacterium frigoritolerans: Algoriphagus halophilus: Bacillus subtilis: Bacillus licheniformis of 1~3:3~5:1~3:1~3. Bacterial agent B consists of Flavobacterium frigoritolerans, Bacillus daqingensis, Bacillus suaedaesalsae, and Bacillus subtilis. The bacterial suspension is composed of *Bacillus subtilis*, *Bacillus daqingensis*, *Bacillus salsa*, and *Bacillus subtilis* in a ratio of 1-3:2-4:2-4:1-3; bacterial agent A or bacterial agent B is prepared into a bacterial suspension with a concentration of 1×10⁻⁶. 7 ~1×10 10 CFU / mL; (4) Add sulfonated humic acid powder, mixed sugar source, nutrients and bacterial suspension obtained in step (3) to the gel obtained in step (2), stir evenly to obtain mixed hydrogel; (5) The mixed hydrogel is dropped into an aqueous solution of calcium chloride (CaCl2) and the biological functional preparation is obtained after it is formed.

2. The preparation method according to claim 1, characterized in that, The polyvinyl alcohol mentioned in step (1) is polyvinyl alcohol 1788, the solvent for polyvinyl alcohol (PVA) solution and sodium alginate (SA) solution is water, and the solvent for 4-carboxyphenylboronic acid (PBA) solution and chitosan (CS) solution is a mixed solvent of acetic acid and water with a volume ratio of 1:5 to 1:

20.

3. The preparation method according to claim 1, characterized in that, In step (2), the magnetic field strength is 250~350 mT and the magnetic field is left to stand for 10~60 min; the magnetic field strength of the magnetically attached pipe is 250~350 mT and the flow rate is 0.01~0.5 m / s.

4. The preparation method according to claim 1, characterized in that, In step (3), the CGMCC numbers for the following bacteria are as follows: Flavobacterium frutescens: CGMCC 1.11577; halophilic psychrophilic bacteria: CGMCC 1.10158; Bacillus subtilis: CGMCC 1.2416; Bacillus licheniformis: CGMCC 1.4401; Bacillus daqingensis: CGMCC 1.12295; and Bacillus salsa: CGMCC 1.18659.

5. The preparation method according to claim 1, characterized in that, In step (4), based on 100ml of gel solution, add 1~10g of sulfonated humic acid powder, 1~5g of mixed sugar source, 0.1~2.0g of nutrient salt and 5~25ml of bacterial suspension obtained in step (3) to the gel solution.

6. The preparation method according to claim 1, characterized in that, In step (4), the particle size of the sulfonated humic acid powder is ≤100 mesh. The sulfonated humic acid is composed of sulfonated humic acid and sulfonated fulvic acid, with a mass ratio of 1:1 to 1:

3. The mixed sugar source is composed of glucose, sucrose and starch, with a weight ratio of 1:1:

1. The nutrient salt is composed of potassium dihydrogen phosphate, magnesium sulfate, superphosphate and ammonium sulfate, with a weight ratio of 1:1:1:

1.

7. The preparation method according to claim 1, characterized in that, In step (5), the mass concentration of calcium chloride (CaCl2) aqueous solution is 2-5%; the particle size of the biofunctional preparation containing bacterial agent A is controlled at 4-8 mm, and the particle size of the biofunctional preparation containing bacterial agent B is controlled at 1-3 mm.

8. The preparation method according to claim 1, characterized in that, The biofunctional preparation obtained in step (5) is post-processed and matured. The specific process is as follows: the formed biofunctional preparation pellets are soaked in cold water for 20-30 minutes to stabilize their internal structure. Then, the surface of the pellets is rinsed with clean water or sterile water and transferred to a moist, sterile container. The pellets are then incubated at 20-25°C for 24-48 hours.

9. A biofunctional preparation obtained by the preparation method according to any one of claims 1-8.

10. The application of the biofunctional agent according to claim 9 in the treatment of saline-alkali land.

Images