Microbial organic compound fertilizer for soil remediation

Abstract

The invention relates to the technical field of organic fertilizers and soil remediation. The invention provides a microbial organic compound fertilizer for soil remediation. The microbial organic compound fertilizer is characterized in that a complex formed by covalent bonding of modified UiO-66 (Zr) bifunctional ligand MOF nanoparticles and low-temperature activated wood biochar through an epoxy silane coupling agent is taken as a core; a PEC microcapsule shell formed by chitosan salt and sodium carboxymethyl cellulose under the near-neutral and low-salt condition is coated outside the PEC microcapsule; phosphorus solubilizing bacteria with metal response regulation and control are packaged inside, trehalose, skimmed milk powder and glycerin are matched for keeping alive, and low-temperature drying and granulation are carried out. The compound fertilizer can release phosphorus as required at a pollution peak value, stabilize and passivate heavy metals such as Cd, Pb and the like, the availability of available phosphorus and crops is improved, the finished product viable bacteria is greater than or equal to 1 * 10 CFU / g, and the water content is 1t; the structure is stable, and the storage and transportation performance is excellent.

Description

Technical Field

[0001] This invention relates to the field of organic fertilizer and soil remediation technology, specifically to a microbial organic compound fertilizer for soil remediation. Background Technology

[0002] With the development of industry and intensive agriculture, some arable land and orchards face the typical problem of simultaneous accumulation of heavy metals such as Cd, Pb, As, and Hg, and insufficient soil phosphorus availability. On the one hand, heavy metals increase soil ecological risks and inhibit root and indigenous microbial activity; on the other hand, a large amount of phosphorus is fixed in the form of insoluble inorganic phosphorus Ca-P, Fe / Al-P, or organic phosphorus, resulting in low available phosphorus for crops and poor fertilizer utilization. How to reduce the bioavailability of heavy metals while improving phosphorus availability is a core issue facing soil remediation and the synergistic effect of organic fertilizers.

[0003] Existing repair and improvement methods mainly include: Chemical fixation methods, which use lime, phosphates, iron and manganese oxides, silicates, etc., have the advantage of being fast-acting, but they also pose risks of large pH changes, potential secondary salinization, or phosphorus loss. Physical engineering methods, such as topsoil replacement, soil replacement, and soil washing, are costly, cause significant disturbance, and have limited applicability. Phytoremediation and combined remediation methods utilize hyperaccumulating plants and rhizosphere regulation, but have long cycles and are subject to seasonal and ecological conditions. Microbial remediation utilizes microbial organic fertilizers for phosphorus solubilization, growth promotion, and toxicity-resistant strains, which are characterized by being green and long-lasting. However, environmental adaptability and stability are bottlenecks in its application.

[0004] Regarding microbial organic fertilizers, existing technologies generally suffer from the following common problems: It is difficult to keep live bacteria alive: common chitosan dissolution uses strong acids such as 1% acetic acid, and gelation often uses high ionic strength; at the same time, processes such as spray drying involve heat, shear and osmotic stress, which causes the bacteria to be greatly inactivated during the preparation process, resulting in insufficient live bacteria count and shelf-life stability of the finished product.

[0005] The release lacks an "on-demand" response: Most mainstream microcapsules rely on ion exchange or diffusion, which cannot make targeted releases when there is a "sudden increase" in heavy metal concentration. For example, local dissolution increases after rainfall or irrigation, surface concentration is caused by drought evaporation, or re-migration is triggered by changes in salinity due to fertilization. These situations often only occur at that time and in certain local areas of the field. Existing microcapsules cannot "know when and where to release".

[0006] The carrier-function combination is weak: functional powders such as adsorbents, minerals, and functional carbon materials are mostly physical mixtures or weakly interacting loads, which are easily eroded and lost during transportation, application, extraction, and freeze-thaw cycles, affecting long-term performance and safety.

[0007] Insufficient synergistic effect: A single "phosphorus promotion" or "toxicity fixation" pathway is difficult to take into account complex scenarios; there is a lack of stable and adjustable material-microbe-soil three-way synergistic mechanism between heavy metal passivation and sparingly soluble phosphorus activation.

[0008] In terms of material carriers, biochar is widely used as a carrier and amendment due to its abundant pores, large specific surface area, and oxygen-containing functional groups on its surface; while metal-organic frameworks (MOFs) have ultra-high specific surface area and designable ligand sites, showing potential in adsorbing / complexing heavy metals and carrying functional groups. However, MOFs in fertilizer systems are mostly physically combined with the substrate, and are prone to dispersion, migration, or decreased stability in soil solutions and humic media. Insufficient interfacial covalent anchoring has become a key factor affecting field efficacy.

[0009] In terms of encapsulation and gelation, common routes include alginate-Ca²⁺ gel, chitosan coating, spray-dried microcapsules, and extrusion-ionic crosslinking. In actual production, to achieve film / gel strength, a combination of acidic dissolution-high salt crosslinking and high drying temperature is often used. This contradicts the tolerance window of microorganisms to pH, osmotic pressure, temperature, and shear, resulting in problems such as reduced bacterial count in the finished product, slow start-up, and large fluctuations in field performance.

[0010] From a functional mechanism perspective, phosphate-solubilizing bacteria (such as Bacillus subtilis) activate insoluble phosphorus through pathways such as phytase, PQQ-GDH phosphate-solubilizing modules, and secreted alkaline phosphatase. However, under heavy metal stress and low pH / high salinity conditions, extracellular enzyme secretion and metabolic pathways are inhibited, easily reducing the actual phosphorus release effect. The key to improving integrated remediation performance lies in how to complete encapsulation and gelation under near-neutral, isotonic, and low-salt conditions, and trigger metabolism and release at the peak of pollution.

[0011] In summary, there is an urgent need for a new microbial organic compound fertilizer system that is structurally stable, near-neutral isotonic, and capable of responding to the concentration of heavy metals while simultaneously enhancing phosphorus release and passivating heavy metals. Furthermore, its preparation process should be scalable, with adjustable parameters and controllable quality to meet the application needs of different regions, pollution spectra, and soil types. Summary of the Invention

[0012] To address the shortcomings of existing technologies, this invention provides a microbial organic compound fertilizer for soil remediation, thereby solving the problems mentioned in the background section.

[0013] Its key technical points are as follows: On the one hand, the invention provides a compositional structure for a microbial organic compound fertilizer for soil remediation; (1) MOF-biochar covalent complex core, wherein the MOF-biochar covalent complex is modified UiO-66(Zr) bifunctional ligand MOF nanoparticles covalently bonded to low-temperature activated wood biochar through epoxy silane coupling agent; (2) The polyelectrolyte composite (PEC) microcapsule shell covering the core surface is formed by cationic chitosan salt and anionic polysaccharide under near-neutral pH and low salinity Ca²⁺ and Mg²⁺ crosslinking conditions; (3) Genetically engineered phosphate-solubilizing bacteria encapsulated in a shell, which carry a metal-responsive promoter and genes that enhance the phosphate solubility pathway; The epoxy silane coupling agent is (3-glycidylpropoxy)trimethoxysilane (GPTMS).

[0014] The cationic chitosan salt is chitosan lactate, and the anionic polysaccharide is sodium carboxymethyl cellulose (CMC-Na).

[0015] The engineered phosphate-solubilizing bacterium is Bacillus subtilis, and the gene that enhances the phosphate solubility pathway is a gene encoding phytase (EC 3.1.3.8).

[0016] The modified UiO-66(Zr) bifunctional ligand MOF nanoparticles are obtained by solvothermal reaction of Zr precursor, terephthalic acid (H2BDC) and a mixed ligand of thiophene phenol and imidazole.

[0017] The PEC microcapsule shell further contains a microbial protectant system, including one or more of trehalose, milk protein, skim milk powder, and glycerin.

[0018] On the other hand, this invention provides a method for preparing a microbial organic compound fertilizer for soil remediation, comprising: (S1) Preparation of MOF-biochar composite: Modified UiO-66(Zr) bifunctional ligand MOF nanoparticles and low-temperature activated wood biochar were covalently coupled in the presence of an epoxy silane coupling agent; (S2) Preparation of PEC microcapsule precursor solution: Cationic chitosan salt and anionic polysaccharide solutions were prepared and compounded in a near-neutral buffer system; (S3) Gel encapsulation: The composite from step S1 is dispersed in the precursor solution from step S2, mixed with an isotonic suspension containing engineered phosphate-solubilizing bacteria and a protectant, and then added dropwise into an isotonic crosslinking bath with low salinity Ca²⁺ and Mg²⁺ to form a gel. The gel is then washed with an isotonic near-neutral buffer to obtain wet microcapsules. (S4) Low-temperature drying and granulation: The wet microcapsules are freeze-dried or low-temperature fluidized bed dried to obtain composite particles with controlled moisture content.

[0019] In step S2, the near-neutral buffer system uses MES buffer, and in step S4, the polysaccharide-type desiccant maltodextrin is added to the wet microcapsules before low-temperature drying to regulate water activity and improve cell survival rate.

[0020] This invention provides a microbial organic compound fertilizer for soil remediation. It has the following beneficial effects: 1. Strong and stable heavy metal passivation: Modified UiO-66(Zr) bifunctional ligand MOF nanoparticles and biochar are covalently anchored by GPTMS, providing high-density, long-term stable metal binding / complexation sites; compared with physical mixing, it has better resistance to leaching and mechanical disturbance, and can continuously reduce the migration and bioavailability of Cd and Pb.

[0021] 2. Near-neutral isotonic encapsulation significantly preserves cell viability. Chitosan lactate and CMC-Na form PEC microcapsule shells under low-salt Ca²⁺ and Mg²⁺ conditions, resulting in a mild and pH-friendly preparation that significantly reduces acid, osmotic, and thermal shear stress. Combined with a protective system and low-temperature drying, the finished product maintains excellent cell viability and stable storage period.

[0022] 3. Heavy metal-triggered phosphorus release: Under the regulation of metal-responsive promoters, engineered phosphate-solubilizing bacteria automatically enhance the expression of the phytase pathway when heavy metal signals are present; they maintain low background release under normal circumstances and only release phosphorus strongly when needed, thereby improving nutrient utilization efficiency and reducing unnecessary consumption.

[0023] 4. Adjustable shell and scene adaptability: By adjusting the chitosan:CMC mass ratio and the Ca²⁺:Mg²⁺ ratio, a customized balance can be achieved between permeability, mechanical strength and release behavior, adapting to different soil types and management systems. Attached Figure Description

[0024] Figure 1 This is a schematic cross-sectional view of a microbial organic compound fertilizer composition for soil remediation according to the present invention. Detailed Implementation

[0025] To enable those skilled in the art to understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0026] The present invention will now be described in detail with reference to the accompanying drawings: Example 1 (refer to) Figure 1This embodiment provides a method for preparing a microbial organic compound fertilizer composition for soil remediation, the specific implementation of which is as follows: 1. Raw material preparation Substance Name Specifications / Model supplier Low-temperature activated lignocarbon Particles Merck Modified UiO-66(Zr) bifunctional ligand MOF nanoparticles Powder, specific surface area 1200 m² / g Laboratory synthesis (3-Glycidylpropoxy)trimethoxysilane (GPTMS) Purity ≥ 98% Gelest Chitosan lactate Deacetylation degree 90%, Aladdin Sodium carboxymethyl cellulose (CMC-Na) solid powder Alfa Aesar Engineering phosphate-solubilizing bacteria suspension <![CDATA[1×10 9 CFU / mL]]> Laboratory culture Trehalose Analytical Pure Merck Skim milk powder (for microbiological purposes) Bacteriological grade Merck Microbiology glycerin Analytical Pure Sinopharm Chemical Reagent Co., Ltd. MES Analytical Pure Merck <![CDATA[Calcium chloride dihydrate (CaCl2·2H2O)]]> Analytical Pure Sinopharm Chemical Reagent Co., Ltd. <![CDATA[Magnesium chloride hexahydrate (MgCl2·6H2O)]]> Analytical Pure Sinopharm Chemical Reagent Co., Ltd. Sodium chloride (NaCl) Analytical Pure Sinopharm Chemical Reagent Co., Ltd. maltodextrin Biochemical grade Aladdin Table 1 The laboratory synthesis steps for modified UiO-66(Zr) bifunctional ligand MOF nanoparticles are as follows: Bifunctional ligands used: mixed ligands containing thiophene groups and imidazole groups, in a molar ratio of 1:1. Solvent and reagent preparation Take 1.17 g of ZrCl4 (zirconia tetrachloride), which is 4 mmol. Ligand terephthalic acid (H2BDC) 0.83 g, which is equivalent to 5 mmol; Thiophene-imidazol mixed ligand 1.0 g, i.e., total molar amount 5 mmol; Solvents: 50 mL DMF (N,N-dimethylformamide), 5 mL ethanol.

[0027] Except for the "thiophene-imidazolium mixed ligand," all the solvents and reagents mentioned above can be purchased directly from chemical suppliers. All reagents and solvents used are of analytical grade.

[0028] Dissolution and Mixing At 25°C, 1.17 g ZrCl4 was first dissolved in 20 mL DMF and sonicated for 10 min until completely dissolved. Simultaneously, in another flask, 0.83 g H2BDC and 1.0 g thiophenephenol-imidazolium mixed ligand were placed in 20 mL DMF and sonicated for 10 min. The two solutions were then combined, stirred thoroughly, and the remaining 10 mL DMF and 5 mL ethanol were added. Stirring continued at room temperature for 30 min. The resulting mixture was transferred to a 100 mL high-pressure reactor, sealed, and subjected to a solvothermal reaction at 120°C for 24 h under nitrogen protection. After the reaction was completed, the mixture was allowed to cool naturally to room temperature. The precipitate was collected by centrifugation at 8000 rpm for 10 min and washed three times with 20 mL DMF and twice with 20 mL ethanol to remove unreacted ligands and solvent residues. Finally, the precipitate was dried under vacuum at 60°C for 12 h to obtain yellow powder-like modified UiO-66(Zr), which is modified UiO-66(Zr) bifunctional ligand MOF nanoparticles.

[0029] BET specific surface area was tested using a nitrogen adsorption-desorption apparatus: 1200 m² / g; The preparation process of thiophene-imidazol mixed ligands is as follows: Experimental materials: 4-mercaptophenol (CAS 637-89-8); imidazole (CAS 288-32-4); 1,3-dibromopropane (CAS 109-64-8); potassium carbonate (K2CO3); anhydrous ethanol; DMF.

[0030] Synthesis steps Step 1: Preparation of N-(3-bromopropyl)imidazolium In a 250 mL three-necked flask, 0.68 g (10 mmol) of imidazole and 1.66 g (12 mmol) of potassium carbonate were weighed out. 50 mL of anhydrous ethanol was added, and the mixture was magnetically stirred to disperse the solids thoroughly. Then, 1.90 g (11 mmol) of 1,3-dibromopropane was added dropwise, and the mixture was stirred at room temperature for 6 h. After the reaction was complete, the unreacted potassium carbonate was removed by filtering with filter paper. The filtrate was then transferred to a rotary evaporator and concentrated to one-third of its original volume under reduced pressure in a 45°C water bath. 50 mL of water was then added. 30 mL of dichloromethane (DCM) was added, and the mixture was vigorously shaken and allowed to separate into layers. The lower dichloromethane phase was collected. Then, another 30 mL of dichloromethane (DCM) was added, and the shaking and layering process was repeated. The two dichloromethane phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the yellow oily product N-(3-bromopropyl)imidazolium.

[0031] Step 2: Coupling reaction – Thiophene with N-(3-bromopropyl)imidazolium In a 100 mL flask, 10 mmol (1.24 g) of 4-mercaptophenol and 12 mmol (1.66 g) of potassium carbonate were added, followed by 30 mL of DMF and sonication for 10 min to ensure thorough dispersion of the solid. Then, 10 mmol (2.0 g) of N-(3-bromopropyl)imidazole obtained in step 1 was added, and the mixture was stirred at 80 °C for 12 h under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature, and 100 mL of water was added. The mixture was extracted three times with 30 mL of dichloromethane (DCM). The lower dichloromethane phase was collected, and the two phases were combined and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the solvent was removed by rotary evaporation. The residue was separated by silica gel column chromatography, and the eluent containing the target product was collected and concentrated to obtain a thiophenephenol-imidazole mixed ligand, which was a pale yellow solid.

[0032] The silica gel column is made of 200 mesh silica gel; the mobile phase is PE:EA = 3:1.

[0033] Step 3: Purification and Characterization The product was dissolved in a small amount of ethanol, heated, and then cooled to crystallize, thereby improving its purity. It was then used to prepare bifunctional ligand MOF nanoparticles.

[0034] Laboratory culture steps for genetically engineered phosphate-solubilizing strains: The strain used was Bacillus subtilis as the host, and the metal-responsive promoter (PcadA) and the gene encoding phytase (EC 3.1.3.8-3) were knocked into to enhance the phosphate dissolution pathway.

[0035] The modified phosphate-solubilizing strain was retrieved from a -80°C glycerol storage tube, thawed, and transferred to an LB agar plate containing 50 μg / mL ampicillin. The plate was incubated overnight at 37°C. Single colonies were then picked and inoculated into 5 mL of LB liquid medium containing the same concentration of ampicillin, and pre-cultured at 37°C and 200 rpm for 12 hours. After pre-culture, 100 μL of the bacterial culture was inoculated into 500 mL of LB liquid medium containing 50 μg / mL ampicillin for large-scale culture. The culture was continued at 37°C with shaking at 200 rpm, and the OD (octane rating) of the bacterial culture was monitored. 600 Value. OD pending. 600 Sampling was performed when the concentration reached 0.8. The bacterial cells were then collected by centrifugation at 4000 rpm for 10 minutes, the supernatant was discarded, and the cells were resuspended in physiological saline buffer. The bacterial concentration was adjusted to OD0.8. 600 =1.0, equivalent to 10 9 CFU / mL.

[0036] 2. The preparation process of microbial organic compound fertilizer for soil remediation is as follows: 2.1 By weighing 50 g of low-temperature activated wood biochar and 20 g of modified UiO-66(Zr) bifunctional ligand MOF nanoparticles and mixing them at a mass ratio of 5:2, adding 1.4 g of (3-glycidylpropoxy)trimethoxysilane, and stirring for 30 min, a MOF-biochar covalent complex was obtained. 2.2 Dissolve 9.0 g (1.5%) of chitosan lactate in 600 mL of MES in portions, stirring magnetically at 400 rpm. After complete swelling and clarification, allow to stand for 30 min to degas, obtaining solution A. Dissolve 6.0 g (1.5%) of sodium carboxymethyl cellulose in 400 mL of MES in portions, degassing for 30 min using the same method, obtaining solution B. Add solution B to solution A while stirring at 300 rpm to obtain 1000 mL of PEC microcapsule shell precursor solution, which serves to form films under neutral / low ionic strength and avoid death by strong acids.

[0037] 2.3 Prepare a 2.0 L crosslinking bath. Use 10 mM MES, pH 6.2 as a solvent to dissolve: 6.0 g of CaCl2·2H2O (0.3% w / v), 2.0 g of MgCl2·6H2O (0.1% w / v), and 17.0 g of NaCl (0.85% w / v). Pre-cool the crosslinking bath to 10°C and stir until homogeneous.

[0038] The MOF-biochar complex obtained in step 2.1 was fully dispersed into 1000 mL of the PEC microcapsule shell precursor solution from step 2.2, and stirred at 300 rpm for 15 min to obtain suspension system S. Separately, 10.0 g of trehalose (5% w / v), 10.0 g of skim milk powder (5% w / v), and 4.0 mL of glycerol (2% v / v) were dissolved in 200 mL of 0.85% NaCl solution. Engineered phosphate-solubilizing bacteria were then added and the solution was adjusted to 1×10⁻⁶. 9 CFU / mL, pre-cooled at 4℃ for 10 min, to obtain bacterial-protectant suspension B.

[0039] Add B to S at 200 rpm, maintain 10℃ and pH 6.2, and stir at 200 rpm for 5 min to make it uniform. Record this as gelation feed F. Immediately use a dripping device to drop F into the pre-cooled crosslinking bath at 25 mL / min, and let it stand for 12 min to form a gel. After gelation, wash twice with MES containing 0.85% NaCl at pH 6.2 to obtain responsive wet microcapsules.

[0040] 2.4 Add 3% maltodextrin by wet weight to the microcapsules and mix well – Example: 18 g maltodextrin for 600 g of wet capsules; spread the material evenly on a tray and quick-freeze at -40℃ for 3 h; place it in a freeze dryer with a chamber pressure ≤100 mTorr and a rack temperature gradually increased from -40 to -10 to 0℃ for 24 h; dry until the moisture content is <8% and then sieve to obtain 1–3 mm composite particles.

[0041] This composite particle consists of a core, an inner phase, and a shell, which work together in a coordinated manner. Figure 1 The core is a porous composite formed by covalently anchoring modified UiO-66(Zr) bifunctional ligand MOF and low-temperature activated lignocellulosic char with GPTMS. The inner phase is an isotonic aqueous phase located inside the shell, surrounding the core, containing engineered phosphate-solubilizing bacteria and trehalose, skim milk powder, and glycerol protectant. The outermost PEC microcapsule shell is formed by crosslinking chitosan lactate and CMC-Na with Ca²⁺ and Mg²⁺ in low salt concentrations; it is nearly neutral, slightly swellable, and has controllable microporous channels.

[0042] After the granules are applied to the soil, the core first "passivates" heavy metals in situ, creating a mild microenvironment for microorganisms. When the available state of heavy metals in the surrounding environment increases, the metal response regulation module of the engineered bacteria is triggered, promoting the expression of phytase-related pathways. The resulting enzymes and organic acids diffuse outward through the shell micropores, activating the fixed Ca-P and Al-P and some organic phosphorus, converting them into available phosphorus that crops can absorb. This forms a closed loop of "continuous core detoxification - selective shell permeability - on-demand phosphorus release in the inner phase": heavy metals are stably retained, and available phosphorus is released on demand, with the two working synergistically in the vicinity of the rhizosphere for a long time.

[0043] Compared to conventional physical compounding and acidic, high-salt gelation-high-temperature drying routes, this design offers higher structural and functional stability: the covalent bonds of the MOF-biochar covalent complex are less prone to loss; encapsulation under near-neutral, isotonic, and low-salt conditions significantly reduces acid, permeation, and thermal shear stress; and the low-temperature drying and protective agent system ensures high product viability and shelf stability.

[0044] Comparative Example 1 aims to verify the effect of microbial organic compound fertilizer on soil remediation without the addition of modified UiO-66(Zr) bifunctional ligand MOF nanoparticles. The specific implementation method is as follows: 1. Preparation steps The same process formulation as in Example 1 was used, but without the addition of modified UiO-66(Zr) bifunctional ligand MOF nanoparticles. Other steps and parameters were the same.

[0045] 1.1 Biochar core preparation, replacing "2.1" in Example 1: Weigh 50 g of low-temperature activated lignocarbon, pass it through a 60-mesh sieve to remove coarse particles, and set it aside for later use; do not add MOF-biochar covalent complex and GPTMS, and do not perform coupling treatment.

[0046] 1.2 Preparation of PEC shell precursor solution Same as 2.2 of Example 1.

[0047] 1.3 Assembly and Gluing The biochar from step 1.1 was fully dispersed into 1000 mL of the PEC microcapsule shell precursor solution obtained in step 1.2, and stirred for 15 min to obtain a homogeneous suspension system. The preparation of the bacterial-protectant composite suspension, the preparation and parameters of the cross-linking bath, the drop formation into a gel, the residence time, and the washing conditions were all the same as in Example 1, section 2.3.

[0048] 1.4 Drying and granulation Same as 2.4 of Example 1.

[0049] Example 2 provides a method for preparing a microbial organic compound fertilizer composition for soil remediation, the specific implementation of which is as follows: Based on Example 1, the mass ratio of chitosan to carboxymethyl cellulose was adjusted to 4:1, the molar ratio of Ca²⁺ to Mg²⁺ was adjusted to 3:2, and the rest were the same as in Example 1.

[0050] Based on Example 1, only the shell ratio and crosslinking ion ratio were adjusted: the mass ratio of chitosan to carboxymethyl cellulose (CMC-Na) was changed from 3:2 to 4:1, and the molar ratio of Ca²⁺ to Mg²⁺ was changed from 4:1 to 3:2; the other raw materials, equipment and operating conditions were the same as in Example 1.

[0051] 1. Preparation of precursors and strains Synthesis of modified UiO-66(Zr) bifunctional ligand MOF nanoparticles: same as in Example 1.

[0052] Synthesis of thiophene-imidazol mixed ligands: Same as in Example 1.

[0053] Cultivation of engineered phosphorus-solubilizing strains: Same as in Example 1.

[0054] 2. Compound Fertilizer Preparation Process 2.1 Preparation of MOF-biochar covalent complex Same as Example 1.

[0055] 2.2 PEC microcapsule shell precursor solution (ratio adjusted to 4:1) Solution A: Dissolve 12.0 g of chitosan lactate (1.5% w / v) in 800 mL of MES in portions, stir magnetically at 400 rpm until clear, and let stand for 30 min to remove bubbles.

[0056] Solution B: Dissolve 3.0 g of sodium carboxymethyl cellulose (1.5% w / v) in 200 mL of MES in portions, and degas using the same method.

[0057] Compounding: Add B to A while stirring at 300 rpm to obtain 1000 mL of PEC microcapsule shell precursor solution.

[0058] 2.3 Encapsulation with adhesive (Ca²⁺∶Mg²⁺ adjusted to 3∶2) Prepare a 2.0 L crosslinking bath: Dissolve 6 g of CaCl2·2H2O, 5.5 g of MgCl2·6H2O, and 17.0 g of NaCl (0.85% w / v) in 10 mM MES (pH 6.2) as solvent, pre-cool to 10°C, and stir until homogeneous.

[0059] The MOF-biochar complex obtained in step 2.1 was fully dispersed into 1000 mL of the PEC microcapsule shell precursor solution from step 2.2, and stirred at 300 rpm for 15 min to obtain suspension system S. Separately, 10.0 g of trehalose (5% w / v), 10.0 g of skim milk powder (5% w / v), and 4.0 mL of glycerol (2% v / v) were dissolved in 200 mL of 0.85% NaCl solution. Engineered phosphate-solubilizing bacteria were then added and the solution was adjusted to 1×10⁻⁶. 9 CFU / mL, pre-cooled at 4℃ for 10 min, to obtain bacterial-protectant suspension B.

[0060] Add B to S at 200 rpm, maintain 10℃ and pH 6.2, and stir at 200 rpm for 5 min to make it uniform. Record this as gelation feed F. Immediately use a dripping device to drop F into the pre-cooled crosslinking bath at 25 mL / min, and let it stand for 12 min to form a gel. After gelation, wash twice with MES containing 0.85% NaCl at pH 6.2 to obtain responsive wet microcapsules.

[0061] Explanation: Under the premise of maintaining "low salt and isotonicity", the Ca²⁺∶Mg²⁺ molar ratio of 3∶2 is achieved by adjusting CaCl₂·2H₂O and maintaining MgCl₂·6H₂O.

[0062] 2.4 Low-temperature drying and granulation Same as Example 1.

[0063] To further verify the advantages of the microbial organic compound fertilizer for soil remediation prepared in this invention in terms of heavy metal passivation ability, phosphorus release enhancement ability, and on-demand response, fertilizer samples prepared in Example 1, Example 2, and Comparative Example 1 were selected for the following tests: 1. Heavy metal passivation capability A simulation system was established using sandy loam soil. Cd (1.0 mg / kg) and Pb (100 mg / kg) were added separately by mass and mixed thoroughly. Each treatment granule was then applied at 1% of the soil dry weight, and water was added to adjust the solution to 60% field capacity. The mixture was incubated at 25℃ in the dark for 28 days. Samples were taken at the end of the incubation period, and Cd and Pb were extracted from the soil using DTPA solution. The metal content of the extract was determined by ICP-OES. Unfertilized blank soil was used as a control, and the DTPA-available metal reduction rate (%) for each treatment was calculated.

[0064] 2. Phosphorus release enhancement ability In a 10 mM MES buffer system containing 1.0 g / L sodium phytate and pH 6.2, granules for each treatment were added at the same dosage, 1% w / v on a system basis, and cultured in shake flasks at 25℃ and 180 rpm for 48 h. After culture, the supernatant was collected, centrifuged at 10,000 g for 10 min, filtered through a 0.45 μm filter, and the orthophosphate (PO4³⁻-P) was determined using the molybdenum blue colorimetric method. The concentration was converted using a standard curve prepared with KH2PO4, and the results were expressed as mg / L (PO4-P). Unfertilized blank soil was used as a control, and blank subtraction was performed to calculate the phosphorus release increment for each treatment. Each treatment was replicated in n=3.

[0065] 3. Heavy metal-triggered phosphorus release capability Microcapsules containing bacteria were placed in 10 mM MES buffer (0.85% NaCl, pH 6.2) at 25°C and equilibrated by gentle shaking at 100 rpm for 30 min. At 0 h, the concentration was instantaneously pulsed to Cd²⁺ = 0.5 mg / L using a CdCl₂ standard solution. Samples were taken at 0, 6, 12, 18, 24, 30, and 36 h. The supernatant was centrifuged, and the amount of free inorganic phosphorus generated was determined using phytic acid as a substrate and the molybdenum blue method. Extracellular phytase activity was then calculated. For ease of comparison, the activity at t=0 was normalized to 1, and the peak activity doubling time and the time to reach 50% of the peak value (T0) were recorded within 36 h. 50 A control without Cd²⁺ was set up to characterize baseline variability. Each treatment was paralleled (n=3), and results are expressed as mean ± standard deviation.

[0066] The microcapsules containing bacteria are wet capsules that have not undergone the final low-temperature drying step. Taking Example 1 as an example, the microcapsules containing bacteria are responsive wet capsules prepared in step 2.3.

[0067] Heavy metal passivation capability Table 2 Phosphorus release enhancement ability deal with <![CDATA[48h phosphorus release amount (mg / L, PO4-P)]]> Comparative Example 1 30.4 Example 1 65.2 Example 2 58.1 Table 3 Heavy metal-triggered phosphorus release capability verification deal with Peak activity multiplied (× starting point) <![CDATA[T 50 (h)]]> Comparative Example 1 1.2 48 Example 1 2.4 24 Example 2 2.1 30 The passivation capacity of heavy metals is assessed by the DTPA-available reduction rate: using DTPA extraction results 28 days after application as a criterion, the percentage reduction in Cd and Pb in the soil relative to the control is observed. A higher index indicates less "available" metal that can be absorbed by the roots, resulting in a lower risk of it entering plants and agricultural products, and making it easier to achieve planting safety goals. In application judgment, ≥50% can be considered strong passivation, suitable for light to moderately polluted fields and prioritized for sensitive crops or rapid risk reduction; 30–50% is moderate passivation, which can meet most scenarios when combined with crop rotation or soil conditioners; <30% is mostly for auxiliary improvement and should be used in conjunction with topsoil or pH adjustment and other comprehensive measures.

[0068] The phosphorus release enhancement capacity is assessed by measuring the PO4-P released over 48 hours: Under standard substrate and near-neutral conditions, the amount of orthophosphate (PO4-P, mg / L) produced over 48 hours is measured. Higher values ​​indicate that the engineered bacteria and the shell environment can rapidly convert insoluble phosphorus into usable phosphorus for crops, resulting in faster onset of action and stronger phosphorus supply in the field, reducing the amount of chemical phosphate fertilizer used and improving fertilizer efficiency. In practical interpretation, high values ​​significantly increase phosphorus supply compared to the control, meaning less pressure from topdressing and better early seedling vigor; medium values ​​correspond to steady-state phosphorus supply and are suitable for conventional fields; low values ​​suggest weak phosphorus supply, requiring appropriate increases in application rate or combination with some chemical phosphate fertilizers.

[0069] Heavy metal-triggered phosphorus release capability: Examine "Activity peak doubling × starting point" and T. 50 Normalization of phytase activity after Cd²⁺ pulse induction was performed; the doubling of the activity peak reflected the induction intensity. 50 The time to reach peak value is 50% of the time, reflecting response speed. The greater the multiplication, the higher the T... 50 The smaller the value, the faster the system can release phosphorus and stabilize its function during short-term metal surges caused by heavy rain, irrigation, or fertilization, thus reducing fluctuations in yield and quality. Empirically, a doubling of ≥2 and T... 50 ≤24–30 h can cover the common 48-hour risk window, suitable for plots or seasons with high volatility; doubled by 1.5–2 or T 50 30–48 h indicates that general fluctuations are controllable; if doubling ≈ 1 and T 50 It has a long diffusion time, almost no triggering, and near-constant diffusion, resulting in poor efficiency and economy.

[0070] Analysis of the test data from Example 1, Example 2, and Comparative Example 1 yields the following results: 1. Heavy metal passivation capability The Cd reduction rates in Examples 1 and 2 were 56.8% and 54.1%, respectively, which were comparable, while the reduction rate in Comparative Example 1 was only 28.4%. Compared with Comparative Example 1, the reduction rates in Examples 1 and 2 increased by 1.99 times and 1.90 times, respectively, indicating that the MOF-biochar covalent composite core significantly enhanced Cd passivation.

[0071] The Pb reduction rates in Examples 1 and 2 were 45.2% and 43.0%, respectively, which were similar and significantly higher than the 18.6% reduction rate in Comparative Example 1. The increases relative to Comparative Example 1 were 26.6 and 24.4 percentage points, respectively.

[0072] The results show that, in terms of Cd and Pb, the passivation capabilities of Example 1 and Example 2 are comparable and significantly better than those of Comparative Example 1. This directly indicates that Comparative Example 1, which lacks a MOF core and covalent anchoring, has insufficient effective passivation sites that are easily lost, making it difficult to achieve high-strength and stable heavy metal fixation.

[0073] 2. Phosphorus release enhancement ability Example 1 showed a concentration of 65.2 mg / L, and Example 2 showed a concentration of 58.1 mg / L, both significantly higher than Comparative Example 1's 30.4 mg / L. Compared to Comparative Example 1, Examples 1 and 2 showed increases of 114% and 91%, respectively.

[0074] This indicates that both examples demonstrate strong phosphorus supply capabilities; however, Comparative Example 1 shows a significantly lower level, suggesting that without the support of "detoxification + survival preservation," the engineered bacteria cannot fully express and secrete phosphorus-releasing enzymes, and the efficiency of activating insoluble phosphorus also decreases.

[0075] Heavy metal-triggered phosphorus release capability The peak activity was doubled: Example 1 = 2.4 × 10⁻⁶, Example 2 = 2.1 × 10⁻⁶, and Comparative Example 1 was only 1.2 × 10⁻⁶. This indicates that Examples 1 and 2 showed significant induction enhancement under metal pulse stimulation, while Comparative Example 1 had almost no effective triggering. Response speed (T) 50 Example 1 = 24 h, Example 2 = 30 h, Comparative Example 1 = 48 h; Example 1 and Example 2 showed similar performance in terms of induction amplitude and reaction time, while Comparative Example 1 had weak and delayed triggering, and was closer to a constant passive diffusion mode, making it difficult to respond in time to the high heavy metal value stage in the first two days after rainfall or irrigation.

[0076] In summary, compared with Comparative Example 1, which does not contain a MOF core and does not employ near-neutral isopermeation encapsulation and system keep-alive, the two embodiments of the present invention are significantly superior to the control in all three indicators, clearly demonstrating the effectiveness and advancement of the overall technical approach of the present invention.

[0077] It should be particularly noted that the various embodiments listed in this specification and accompanying drawings are intended to illustrate the technical solutions and advantages of the present invention, and not to limit the scope of protection of the present invention. Without departing from the core ideas and technical effects of the present invention, those skilled in the art can make any form of improvement, substitution, combination, or modification to the structural arrangement, process parameters, material selection, control logic, etc., of the described embodiments; any obvious changes based on the same concept should be considered equivalent solutions of the present invention and should be included within the scope of protection defined by the claims of the present invention. The actual scope of protection of the present invention is determined by the appended claims and should be correctly understood in conjunction with the specification and accompanying drawings.

Claims

1. A microbial organic compound fertilizer for soil remediation, characterized in that, The organic compound fertilizer is composed of compound granules, each compound granule comprising: (1) MOF-biochar covalent complex core, wherein the MOF-biochar covalent complex is modified UiO-66(Zr) bifunctional ligand MOF nanoparticles covalently bonded to low-temperature activated wood biochar through epoxy silane coupling agent; (2) The polyelectrolyte composite (PEC) microcapsule shell covering the core surface is formed by cationic chitosan salt and anionic polysaccharide under near-neutral pH and low salinity Ca²⁺ and Mg²⁺ crosslinking conditions; (3) Genetically engineered phosphate-solubilizing bacteria encapsulated in a shell, which carry a metal-responsive promoter and genes that enhance the phosphate solubility pathway.

2. The microbial organic compound fertilizer for soil remediation according to claim 1, characterized in that, The epoxy silane coupling agent is (3-glycidylpropoxy)trimethoxysilane (GPTMS).

3. The microbial organic compound fertilizer for soil remediation according to claim 1, characterized in that, The cationic polysaccharide is chitosan lactate, and the anionic polysaccharide is sodium carboxymethyl cellulose (CMC-Na).

4. The microbial organic compound fertilizer for soil remediation according to claim 1, characterized in that, The engineered phosphate-solubilizing bacteria is Bacillus subtilis, and the gene that enhances the phosphate solubility pathway is a gene encoding phytase.

5. The microbial organic compound fertilizer for soil remediation according to claim 1, characterized in that, The modified UiO-66(Zr) bifunctional ligand MOF nanoparticles are obtained by solvothermal reaction of Zr precursor, terephthalic acid (H2BDC) and a mixed ligand of thiophene phenol and imidazole.

6. The microbial organic compound fertilizer for soil remediation according to claim 1, characterized in that, The PEC shell further contains a microbial protectant system, including one or more of trehalose, milk protein, skim milk powder, and glycerin.

7. A method for preparing a microbial organic compound fertilizer for soil remediation, characterized in that, include: (S1) Preparation of MOF-biochar composite: Modified UiO-66(Zr) bifunctional ligand MOF nanoparticles and low-temperature activated wood biochar were covalently coupled in the presence of an epoxy silane coupling agent; (S2) Preparation of PEC microcapsule shell precursor solution: Cationic chitosan salt and anionic polysaccharide solutions were prepared and compounded in a near-neutral buffer system; (S3) Gel encapsulation: The composite from step S1 is dispersed in the precursor solution from step S2, mixed with an isotonic suspension containing engineered phosphate-solubilizing bacteria and a protectant, and then dropped into an isotonic crosslinking bath with low salinity Ca²⁺ and Mg²⁺ to form a gel. The gel is then washed with an isotonic near-neutral buffer to obtain wet microcapsules. (S4) Low-temperature drying and granulation: The wet microcapsules are freeze-dried or low-temperature fluidized bed dried to obtain composite particles with controlled moisture content.

8. The method according to claim 7, characterized in that, In step S2, the near-neutral buffer system uses MES buffer. In step S4, before low-temperature drying, maltodextrin, a polysaccharide-type desiccant, is added to the wet microcapsules to regulate water activity and improve cell survival.

9. A microbial organic compound fertilizer for soil remediation according to claim 1, characterized in that, The viable count of the finished composite granules is not less than 1×10⁻⁶. 8 CFU / g and moisture content less than 8%.

Images