Liquid peptidase biological organic fertilizer for soil remediation and preparation process of liquid peptidase biological organic fertilizer

Abstract

The invention discloses a liquid peptidase biological organic fertilizer for soil remediation and a preparation process of the liquid peptidase biological organic fertilizer, and particularly relates to the technical field of agricultural environment remediation, the fertilizer comprises a liquid peptidase compound system, a multifunctional carrier matrix loading the system and a synergistic microbial flora compounded with the multifunctional carrier matrix, the carrier matrix is prepared from Fe3O4 modified biochar, a calcium alginate-chitosan microcapsule and modified attapulgite clay. The preparation process comprises the steps of carrier pre-activation, enzyme compound immobilization, staged temperature-controlled fermentation and magnetization treatment. By constructing the enzyme-carrier-flora synergistic remediation micro-unit, synchronous and efficient removal and passivation of protein organic pollutants and heavy metals in soil are realized, the environmental stability and action durability of remediation factors are remarkably improved, the soil agglomeration structure can be effectively improved, and micro-ecological health can be recovered.

Description

Technical Field

[0001] This invention relates to the field of agricultural environmental remediation technology, and more specifically, to a liquid peptidase for soil remediation and its preparation process. Background Technology

[0002] With the rapid development of industry and agriculture, soil pollution has become increasingly serious, especially the compound pollution of organic pollutants and heavy metals, which has become an urgent problem to be solved in the field of soil remediation. Bio-organic fertilizers have shown great potential in soil remediation due to their advantages such as environmental friendliness and low cost.

[0003] However, existing bio-organic fertilizers and their remediation technologies still have significant limitations: 1. The functions are highly singular. Commercially available products or research mainly focus on degrading specific organic pollutants (such as pesticides and petroleum hydrocarbons) or passivating heavy metals, lacking a multi-functional integrated system that can simultaneously and efficiently treat complex pollution of protein-based organic matter and heavy metals. 2. Core active ingredients (such as enzyme preparations and functional microorganisms) are easily deactivated and have short half-lives in complex soil environments. Conventional physical adsorption or encapsulation methods have limited protective effects and are difficult to maintain long-term remediation efficacy. 3. Existing technologies mostly involve simple physical mixing of enzymes, microorganisms, and carriers, lacking effective synergy and functional coupling between components, resulting in limited improvement in remediation efficiency. In addition, most products fail to address both pollution removal and the restoration of the soil's own microecological structure and physical properties, leading to incomplete remediation effects.

[0004] Therefore, developing a bio-organic fertilizer and its preparation method that can synergistically, efficiently, and sustainably remediate complex polluted soils and simultaneously improve soil ecological functions is of great practical significance and application value. In view of this, the present invention provides a bio-organic fertilizer for soil remediation using liquid peptidase and its preparation process. Summary of the Invention

[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides a liquid peptidase for soil remediation and its preparation process, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a liquid peptidase-based bio-organic fertilizer for soil remediation, comprising the following components: Liquid peptidase compound system; A multifunctional carrier matrix for loaded liquid peptidase complex systems; Synergistic microbial community in combination with liquid peptidase complex system and multifunctional carrier matrix; The multifunctional carrier matrix comprises biochar modified with Fe3O4 nanoparticles, calcium alginate-chitosan microcapsules, and modified attapulgite clay.

[0007] Preferably, the liquid peptidase compound system comprises: Thermostable liquid peptidase, which is a commercially available protease derived from the thermophilic mold *Thermophilus spp.* or an engineered variant thereof; Heavy metal chelating peptidase, which is a recombinant protease obtained by introducing a cysteine-rich metal-binding region into the Bacillus licheniformis protease sequence; The hydrophobic substrate-penetrating enhanced peptidase is a fusion protease obtained by fusing a protease with a hydrophobic peptide. The host for fusion expression is Bacillus subtilis WB600.

[0008] Preferably, the multifunctional carrier matrix is ​​composed of the following components in parts by weight: 30-40 parts of decomposed straw biochar modified with Fe3O4 nanoparticles; Calcium alginate-chitosan microcapsules, 20-25 parts; Modified attapulgite clay loaded with Fe3O4 nanoparticles, 15-20 parts; Humic acid-amino acid chelate, 10-15 parts; Among them, the specific surface area of ​​the decomposed straw biochar modified with Fe3O4 nanoparticles is greater than 400m² / g, the magnetic saturation strength is greater than 35emu / g, the chelation rate of humic acid-amino acid chelate is ≥85%, and the enzyme encapsulation rate of calcium alginate-chitosan microcapsules is ≥80%.

[0009] Preferably, the synergistic microbial community is composed of functionally complementary strains, and its composition, based on the total number of colony-forming units, includes: The enzyme activity protection bacteria group, consisting of 40-50% of the bacteria, is composed of strains of Pseudomonas that can secrete extracellular polysaccharides to stabilize liquid peptidase activity. 30-40% of the heavy metal passivating bacteria group consisted of heavy metal resistant strains selected from the genera *Copper-loving Bacteria* or *Rhizobium* that carry heavy metal resistance and transformation gene clusters and have Cd resistance concentration ≥100 mg / L and Pb resistance concentration ≥500 mg / L. 20-30% of the soil structure improving microbial group consists of strains selected from the genus Streptomyces that can secrete extracellular polysaccharides and plant hormone analogs to promote the formation of soil aggregates; The strains mentioned are those preserved in public culture centers or those isolated and identified from contaminated soil or plant rhizosphere using conventional screening methods and possessing the corresponding functions.

[0010] This invention also provides a preparation process for preparing the above-mentioned liquid peptidase-based bio-organic fertilizer for soil remediation, specifically including the following steps: S1, Carrier pre-activation: S1.1. The decomposed straw biochar was impregnated in 0.1M FeCl3 solution, ultrasonically treated at 40kHz for 30min, and then vacuum dried at 60℃. S1.2 The dried biochar was heat-treated in a N2 atmosphere at a temperature of 5℃ / min to 450℃ for 2h to obtain Fe3O4@biochar composite carrier; S1.3. Attapulgite clay and FeCl3 solution are mixed at a mass ratio of 1:3, stirred for 2 hours, dried, and calcined at 400℃ for 1.5 hours to obtain modified attapulgite clay. S1.4. Humic acid and amino acids were mixed in a molar ratio of 1:1.2 and reacted at pH 7 and 60℃ for 3 hours. The mixture was then spray-dried to obtain a humic acid-amino acid chelate. S2, Construction of multifunctional carrier matrix: S2.1 Preparation of liquid peptidase complex system: Heat-stable liquid peptidase, heavy metal chelating peptidase and hydrophobic substrate penetration-enhancing peptidase are mixed at an enzyme activity unit ratio of 2:1:1.5 and prepared into an enzyme solution with a total protein concentration of 2.5 mg / mL using phosphate buffer at pH 5.5. S2.2 Mix the enzyme solution with an equal volume of 1% (w / v) chitosan acetate solution and stir at 4℃ and 150 rpm for 2 hours for electrostatic adsorption. S2.3 Add 2% (w / v) sodium alginate solution and 0.1M CaCl2 solution dropwise to the mixture to form gel microcapsules with a particle size of 50-100μm; S2.4 Mix Fe3O4@biochar composite carrier, calcium alginate-chitosan microcapsules, modified attapulgite clay and humic acid-amino acid chelate in a weight ratio of 30-40:20-25:15-20:10-15. S2.5. In a fluidized bed, the mixture is treated at 40°C and a fluidization number of 1.5 for 1 hour to ensure uniform compounding of the components and obtain a multifunctional carrier matrix for loading enzyme complexes. S3, Staged Temperature-Controlled Fermentation: S3.1 Preparation of synergistic microbial community: Mix enzyme-protecting bacteria, heavy metal-passivating bacteria and soil structure-improving bacteria at a ratio of 40-50%:30-40%:20-30% of colony-forming units; S3.2. The multifunctional carrier matrix of the enzyme complex is added to the fermentation medium at a concentration of 30 g / L, and 10% (v / v) of synergistic microbial community is inoculated. The fermentation medium contains: glucose 8-12 g / L, peptone 4-6 g / L, yeast extract 2-4 g / L, potassium dihydrogen phosphate 1.5-2.5 g / L, magnesium sulfate heptahydrate 0.4-0.6 g / L, and trace element solution 0.5-1.5 mL / L. The specific components of the trace element solution are: zinc sulfate heptahydrate 0.1 g / L, manganese sulfate monohydrate 0.05 g / L, copper sulfate pentahydrate 0.01 g / L, cobalt chloride hexahydrate 0.005 g / L, and sodium molybdate crystals 0.005 g / L. S3.3, First stage aerobic fermentation: cultured at 35-38℃, 180rpm, and pH 6.5-7 for 48h; S3.4, Second stage microaerobic fermentation: Cultivate for 48 hours at 30-32℃, 50 rpm, and dissolved oxygen concentration of 2-3 mg / L; S3.5, Third stage low-temperature ripening: Cultivate at 15-18℃ for 48 hours; S4. Magnetization and Drying: S4.1 Apply an alternating magnetic field with a frequency of 50 Hz and an intensity of 10 mT for 6 hours at the end of fermentation. S4.2 The fermentation product was vacuum freeze-dried at -45℃ and 0.1Pa to constant weight to obtain bio-organic fertilizer.

[0011] Preferably, in step S1.3, the concentration of the FeCl3 solution is 0.15M, and the calcination process is carried out under N2 protection.

[0012] Preferably, in step S2.4, the specific weight ratio of each component is as follows: 35 parts Fe3O4@biochar composite carrier, 22 parts calcium alginate-chitosan microcapsules, 18 parts modified attapulgite clay, and 12 parts humic acid-amino acid chelate.

[0013] Preferably, in step S3.1, the specific composition of the synergistic microbial community is: 45% enzyme-protecting bacteria, 35% heavy metal passivating bacteria, and 20% soil structure improving bacteria.

[0014] Preferably, in the microaerobic fermentation stage of step S3.4, microorganisms form a biofilm complex on the carrier surface with a membrane thickness of 50-100 μm and a biofilm viable cell density ≥1×10⁻⁶. 8 CFU / cm², extracellular polysaccharide content ≥8g / L.

[0015] The technical effects and advantages of this invention are as follows: 1. This invention constructs a three-in-one synergistic remediation system consisting of a liquid peptidase compound system, a multifunctional carrier matrix, and a synergistic microbial community. This system enables the simultaneous and efficient removal and passivation of protein-based organic pollutants and heavy metal pollutants in soil, effectively solving the technical problem of the limited functionality of single remediation materials and their inability to cope with complex pollution. 2. The specific multifunctional carrier matrix and staged temperature-controlled fermentation process provided by this invention can significantly enhance the activity stability and functional durability of liquid peptidase and functional microorganisms in the soil environment. Through physical protection and slow release, it prolongs the effective action period of the repair factors and improves the long-term efficacy of the product. 3. The synergistic remediation micro-units formed by this invention have environmental responsiveness characteristics and can be intelligently activated under specific soil conditions. While efficiently removing pollutants, they simultaneously promote the formation of soil aggregate structures and the restoration of the health of the micro-ecosystem, realizing integrated remediation from pollution control to soil ecological function enhancement. Attached Figure Description

[0016] Figure 1 This is a flowchart illustrating the overall process flow of the present invention. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0018] Example 1: Preparation of the bio-organic fertilizer of the present invention S1, Carrier Pre-activation S1.1 Take 100g of well-rotted straw biochar that has passed through a 100-mesh sieve, immerse it in 500mL of 0.1M FeCl3 solution, sonicate it at a frequency of 40kHz for 30min to allow the solution to fully penetrate the pores of the biochar, and then dry it in a vacuum drying oven at 60℃ for 12h. S1.2. The dried material was placed in a tube furnace and heated to 450℃ at a rate of 5℃ / min under a N2 atmosphere, and held for 2 hours for heat treatment. After natural cooling, a magnetic Fe3O4@biochar composite carrier was obtained. The specific surface area was determined to be 435 m² / g by the BET method, and the magnetic saturation intensity was measured to be 38.2 emu / g by a vibrating sample magnetometer. S1.3. Mix 100g of attapulgite clay with 300mL of 0.15M FeCl3 solution, stir mechanically for 2h, filter and dry at 105℃, then calcine at 400℃ for 1.5h under N2 protection to obtain modified attapulgite clay loaded with Fe3O4 nanoparticles. S1.4. Humic acid and L-glutamic acid were mixed in a molar ratio of 1:1.2 and dissolved in deionized water. The pH was adjusted to 7 with NaOH and the mixture was reacted in a water bath at 60°C for 3 hours. The reaction solution was spray-dried (inlet temperature 180°C, outlet temperature 80°C) to obtain humic acid-amino acid chelate with a chelation rate of 87.3%.

[0019] S2, Construction of Multifunctional Carrier Matrix S2.1 Preparation of liquid peptidase complex system: Heat-stable liquid peptidase (commercial protease derived from *Thermophilus licheniformis*), heavy metal chelating peptidase (obtained by genetically modifying Bacillus licheniformis alkaline protease to introduce a metal-binding region enriched with cysteine), and hydrophobic substrate penetration-enhancing peptidase (obtained by fusing Bacillus subtilis protease A with the hydrophobic peptide Leu-Leu-Val-Ile-Ala) were mixed at an enzyme activity ratio of 2:1:1.5 and prepared into an enzyme solution with a total protein concentration of 2.5 mg / mL and a total enzyme activity of 8500 U / mL using phosphate buffer at pH 5.5. S2.2. Mix the above enzyme solution with an equal volume of 1% (w / v) chitosan acetate solution (degree of deacetylation ≥90%) and stir at 4℃ and 150 rpm for 2 h for electrostatic adsorption. S2.3. Slowly add 2% (w / v) sodium alginate solution (volume ratio 1:1) dropwise to the above mixture, while simultaneously adding 0.1M CaCl2 solution dropwise at a uniform rate and stirring gently to form uniform gel microspheres. After standing and solidifying for 30 min, filter and collect the microspheres, wash with deionized water to obtain enzyme-loaded calcium alginate-chitosan microcapsules with a particle size of 50-100 μm. The encapsulation efficiency was determined to be 89.2% by the Coomassie Brilliant Blue method. S2.4. Accurately weigh and premix 35 parts by weight of Fe3O4@biochar composite carrier, 22 parts by weight of calcium alginate-chitosan microcapsules, 18 parts by weight of modified attapulgite clay, and 12 parts by weight of humic acid-amino acid chelate. S2.5. Place the premixed material in a small fluidized bed device and treat it for 1 hour at 40°C and a fluidization number of 1.5 (fluidization number = actual gas velocity / minimum fluidization gas velocity, the minimum fluidization gas velocity is calculated according to the Ergun equation) to make the components uniformly compounded and obtain a multifunctional carrier matrix for loading enzyme complex.

[0020] S3, staged temperature-controlled fermentation S3.1 Preparation of synergistic microbial communities: Enzyme-protected bacterial group: Pseudomonas putida CGMCC 1.3136 was cultured, and the viable count reached 3 × 10⁻⁶ in the late logarithmic phase. 9 CFU / mL or higher, extracellular polysaccharide secretion 5.8 g / L; Heavy metal passivation bacteria group: The heavy metal-tolerant copper-loving bacterium *Cupriavidus metallidurans* ATCC 43123 carrying the *czc* gene cluster was cultured, and the viable count reached 2.5 × 10⁻⁶ at the late logarithmic phase. 9 CFU / mL or higher, Cd resistance concentration 120 mg / L, Pb resistance concentration 550 mg / L; Soil structure improving microbial group: Streptomyces fulvissimus DSM 40593 was used for culture, and the spore concentration reached 1.5 × 10⁻⁶ at the spore maturity stage. 9 CFU / mL or higher; The three bacterial suspensions were mixed at a viable count ratio of 45%:35%:20% to serve as a synergistic microbial inoculum, with a total viable count of 2.5 × 10⁻⁶. 9 CFU / mL; S3.2. Add 30g of the multifunctional carrier matrix of the enzyme-loaded complex obtained in step S2 into a fermenter containing 1L of liquid fermentation medium. The fermentation medium consists of: 10g / L glucose, 5g / L peptone, 3g / L yeast extract, 2g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate heptahydrate, and 1mL / L trace element solution. Inoculate the above-mentioned synergistic microbial inoculum at an inoculation rate of 10% (v / v). The specific components of the trace element solution are: zinc sulfate heptahydrate 0.1 g / L, manganese sulfate monohydrate 0.05 g / L, copper sulfate pentahydrate 0.01 g / L, cobalt chloride hexahydrate 0.005 g / L, and sodium molybdate crystals 0.005 g / L. S3.3, First stage aerobic fermentation: control the temperature at 37±1℃, stir at 180rpm, introduce sterile air to maintain dissolved oxygen saturation >80%, use 2M NaOH to automatically adjust the pH to maintain at 6.8±0.2, and incubate for 48h; S3.4 Second Stage Microaerobic Fermentation: The temperature was lowered to 31±1℃, and the stirring speed was reduced to 50 rpm. The dissolved oxygen concentration was controlled at 2.5±0.5 mg / L by adjusting the ratio of N2 / air mixture. The culture was carried out for 48 hours. After this stage, samples were taken and observed using a laser confocal microscope. This confirmed that a dense biofilm had formed on the carrier surface, with a thickness of 65-85 μm and a viable cell density of 1.2 × 10⁻⁶. 8 CFU / cm², extracellular polysaccharide content 8.5 g / L; S3.5, Third stage low-temperature maturation: Stop stirring and heating, and let the fermentation system stand at 16±2℃ for 48 hours to mature.

[0021] S4, Magnetization and Drying An alternating magnetic field generator was placed outside the fermentation tank, and an alternating magnetic field with a frequency of 50 Hz and an intensity of 10 mT was applied to continuously treat the material inside the tank for 6 hours. After treatment, the material was removed and placed in a vacuum freeze dryer, where it was dried to constant weight at -45°C and 0.1 Pa (approximately 36 hours) to obtain the powdered bio-organic fertilizer of this invention. The product was tested and found to have: a moisture content of 7.2%, a liquid peptidase activity retention rate of 86.5%, and a viable bacteria count of 2.3 × 10⁻⁶. 9 CFU / g.

[0022] Example 2: Implementation of different functional strains The preparation process in this embodiment is the same as in Example 1, except that the strain source of the synergistic microbial community in step S3 is changed: Enzyme-protected bacterial group: Pseudomonas fluorescens ATCC 13525 was selected, with an extracellular polysaccharide secretion of 5.2 g / L; Heavy metal passivating bacteria group: A strain of Rhizobium, which is isolated from heavy metal contaminated soil through conventional screening methods and has high tolerance and adsorption capacity to Cd and Pb, was selected. The Cd resistance concentration was 110 mg / L and the Pb resistance concentration was 520 mg / L. Soil structure improving microbial group: Streptomyces griseus ATCC 10137 was selected.

[0023] Three bacterial strains were combined and inoculated at the same CFU ratio of 45%:35%:20%, resulting in a viable count of 2.1 × 10⁻⁶. 9 CFU / g, enzyme activity retention rate 84.8%.

[0024] Comparative Example 1: Lack of synergistic microbial community, enzyme-carrier system only. The preparation process in this embodiment is the same as in Example 1, but in step S3, no microbial flora is inoculated. Instead, the multifunctional carrier matrix of the enzyme complex is soaked in liquid culture medium under the same conditions and subjected to the same time and temperature treatment.

[0025] Comparative Example 2: Incomplete functional flora, lacking heavy metal passivation bacteria. The preparation process was the same as in Example 1, but in step S3.1, only the enzyme-activating protective bacteria group and the soil structure-improving bacteria group were combined, while the heavy metal passivating bacteria group was omitted. The ratio of the two bacteria was adjusted to approximately 56%:44% according to CFU.

[0026] Comparative Example 3: Simplified carrier system, physical hybridization substitution The preparation process in this embodiment is the same as in Example 1, but with the following modifications: The modification treatment in step S1 is omitted, and ordinary commercial activated carbon and unmodified attapulgite clay are used directly. The microcapsules formed in step S2.3 are not compounded in a fluidized bed, but are instead mixed with ordinary activated carbon, unmodified attapulgite clay, and humic acid by simple mechanical stirring.

[0027] Comparative Example 4: Simplified fermentation process, single-stage constant temperature The preparation process in this embodiment is the same as in embodiment 1, but step S3 is modified as follows: after inoculating the synergistic microbial community, ferment continuously for 144 hours under constant temperature and aerobic conditions at 35°C and 150 rpm, without performing microaerobic and low-temperature ripening stages.

[0028] Comparison Example 5: Comparison with similar products on the market We selected a commercially available compound microbial fertilizer that claims to have soil remediation functions as a comparison. Its main components are Bacillus subtilis, Bacillus spp., and humic acid.

[0029] The soil remediation efficacy and synergistic effect of the products in the above embodiments and comparative examples are evaluated and verified respectively. The specific test methods are as follows: 1. Test soil: Soil from an industrial site with complex pollution was collected, air-dried, and sieved through a 2mm sieve. Basic properties: pH 7.3, organic matter 1.8%, protein pollutants 312mg / kg, available Cd 3.2mg / kg, available Pb 112mg / kg, water-stable aggregates (>0.25mm) content 15.3%; Pot experiment: The product of each example and comparative example was mixed with soil at a dosage of 1.5% (w / w), with 4 replicates, maintaining field water holding capacity of 60%, and cultured for 60 days. Soil without added product was used as a blank control. Indicator detection: After 60 days of cultivation, the protein degradation rate, the passivation rate of bioavailable heavy metals (Cd, Pb), the increase rate of water-stable macroaggregates (>0.25 mm), and the residual rate of soil protease activity were measured. The changes in the Shannon diversity index of soil microorganisms were analyzed by high-throughput sequencing.

[0030] The test results are shown in the table below: From the data in the table above, we can see that: 1. Comparing the data of Example 1 and Comparative Example 1 (without microorganisms), it can be seen that the addition of microbial communities is a necessary condition for achieving efficient remediation. Further comparison of Example 1 and Comparative Example 2 (without heavy metal passivation bacteria) shows that the two have similar effects in protein degradation and soil aggregate improvement. However, the heavy metal passivation rate of Comparative Example 2 drops sharply to about 25%, which is less than one-third of that of Example 1. This result clearly proves that in the synergistic microbial community, the heavy metal passivation bacteria group undertakes a specific and irreplaceable heavy metal remediation function, while the enzyme activity protection bacteria group and the soil structure improvement bacteria group mainly synergistically ensure enzyme activity persistence and soil physical improvement. The three types of complementary bacteria groups and the specific liquid peptidase system on the modified carrier successfully constitute a synergistic remediation micro-unit, realizing the simultaneous and efficient remediation of organic pollution and heavy metal pollution. The overall effect is far beyond what can be achieved by the simple superposition of individual functional components. 2. The performance indicators of Comparative Example 3 (simplified carrier) all decreased significantly, proving the key role of the Fe3O4@biochar composite carrier modified by the specific process, the modified attapulgite clay, and the fluidized bed composite process in the present invention for immobilizing enzyme activity, adsorbing heavy metals, and promoting microbial colonization. Although the remediation effect of Comparative Example 4 (single-stage fermentation) was better than that of the simplified carrier, it was still significantly lower than that of Example 1. Combined with microscopic observation, it can be seen that the biofilm formed by the microorganisms on the carrier was thinner and more uneven. This indicates that the staged temperature-controlled fermentation process defined in the present invention (especially the microaerobic stage to induce the formation of a uniform thick biofilm and the low-temperature maturation and stabilization system) is crucial for constructing stable and efficient synergistic remediation micro-units. The selection of this specific process condition brought about a significant improvement in remediation performance. 3. Compared with the commercially available Comparative Example 5, the product of Example 1 of the present invention shows a multiple-level improvement in all core repair indicators. For example, the protein degradation rate is increased by about 1 time and the Cd passivation rate is increased by about 1.5 times, which fully demonstrates the outstanding technical progress and significant practical advantages of the technical solution of the present invention.

[0031] In summary: 1. The liquid peptidase compound system of the present invention, the multifunctional carrier matrix and the synergistic microbial community together form a synergistic repair microunit, which produces an unexpected synergistic repair effect and effectively solves the technical problems of single function and low efficiency in the remediation of compound contaminated soil. 2. The specific preparation process adopted in this invention, including carrier modification, three-step fixation method, staged temperature-controlled fermentation and magnetization treatment, is an indispensable process guarantee for achieving the above-mentioned synergistic effect and achieving excellent repair effect; 3. This technical solution does not depend on any specific strain; it can be implemented with any microorganism that meets the functional requirements, and has good universality and industrial reproducibility.

[0032] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A liquid peptidase-based bio-organic fertilizer for soil remediation, characterized in that: It contains the following components: Liquid peptidase compound system; A multifunctional carrier matrix for loaded liquid peptidase complex systems; Synergistic microbial community in combination with liquid peptidase complex system and multifunctional carrier matrix; The multifunctional carrier matrix comprises biochar modified with Fe3O4 nanoparticles, calcium alginate-chitosan microcapsules, and modified attapulgite clay.

2. The bio-organic fertilizer according to claim 1, characterized in that: The liquid peptidase complex system comprises: Thermostable liquid peptidase, which is a commercially available protease or an engineered variant of a thermophilic mold derived from cottony mildew; Heavy metal chelating peptidase, which is a recombinant protease obtained by introducing a cysteine-rich metal-binding region into the Bacillus licheniformis protease sequence; The hydrophobic substrate-penetrating enhanced peptidase is a fusion protease obtained by fusing a protease with a hydrophobic peptide. The host for fusion expression is Bacillus subtilis WB600.

3. The bio-organic fertilizer according to claim 1, characterized in that: The multifunctional carrier matrix is ​​composed of the following components in parts by weight: 30-40 parts of decomposed straw biochar modified with Fe3O4 nanoparticles; Calcium alginate-chitosan microcapsules, 20-25 parts; Modified attapulgite clay loaded with Fe3O4 nanoparticles, 15-20 parts; Humic acid-amino acid chelate, 10-15 parts; Among them, the specific surface area of ​​the decomposed straw biochar modified with Fe3O4 nanoparticles is greater than 400m² / g, the magnetic saturation strength is greater than 35emu / g, the chelation rate of humic acid-amino acid chelate is ≥85%, and the enzyme encapsulation rate of calcium alginate-chitosan microcapsules is ≥80%.

4. The bio-organic fertilizer according to claim 1, characterized in that: The synergistic microbial community is composed of complementary strains and, based on the total number of colony-forming units, includes: The enzyme activity protection bacteria group, consisting of 40-50% of the bacteria, is composed of strains of Pseudomonas that can secrete extracellular polysaccharides to stabilize liquid peptidase activity. 30-40% of the heavy metal passivating bacteria group consisted of heavy metal resistant strains selected from the genera *Copper-loving Bacteria* or *Rhizobium* that carry heavy metal resistance and transformation gene clusters and have Cd resistance concentration ≥100 mg / L and Pb resistance concentration ≥500 mg / L. 20-30% of the soil structure improving microbial group consists of strains selected from the genus Streptomyces that can secrete extracellular polysaccharides and plant hormone analogs to promote the formation of soil aggregates; The strains mentioned are those preserved in public culture centers or those isolated and identified from contaminated soil or plant rhizosphere using conventional screening methods and possessing the corresponding functions.

5. A preparation process for preparing the liquid peptidase for soil remediation as described in any one of claims 1-4, characterized in that: Specifically, the following steps are included: S1, Carrier pre-activation: S1.

1. The decomposed straw biochar was impregnated in 0.1M FeCl3 solution, ultrasonically treated at 40kHz for 30min, and then vacuum dried at 60℃. S1.2 The dried biochar was heat-treated in a N2 atmosphere at a temperature of 5℃ / min to 450℃ for 2h to obtain Fe3O4@biochar composite carrier; S1.

3. Attapulgite clay and FeCl3 solution are mixed at a mass ratio of 1:3, stirred for 2 hours, dried, and calcined at 400℃ for 1.5 hours to obtain modified attapulgite clay. S1.

4. Humic acid and amino acids were mixed in a molar ratio of 1:1.2 and reacted at pH 7 and 60℃ for 3 hours. The mixture was then spray-dried to obtain a humic acid-amino acid chelate. S2, Construction of multifunctional carrier matrix: S2.1 Preparation of liquid peptidase complex system: Heat-stable liquid peptidase, heavy metal chelating peptidase and hydrophobic substrate penetration-enhancing peptidase are mixed at an enzyme activity unit ratio of 2:1:1.5 and prepared into an enzyme solution with a total protein concentration of 2.5 mg / mL using phosphate buffer at pH 5.

5. S2.2 Mix the enzyme solution with an equal volume of 1% (w / v) chitosan acetate solution and stir at 4℃ and 150 rpm for 2 hours for electrostatic adsorption. S2.3 Add 2% (w / v) sodium alginate solution and 0.1M CaCl2 solution dropwise to the mixture to form gel microcapsules with a particle size of 50-100μm; S2.4 Mix Fe3O4@biochar composite carrier, calcium alginate-chitosan microcapsules, modified attapulgite clay and humic acid-amino acid chelate in a weight ratio of 30-40:20-25:15-20:10-15. S2.

5. In a fluidized bed, the mixture is treated at 40°C and a fluidization number of 1.5 for 1 hour to ensure uniform compounding of the components and obtain a multifunctional carrier matrix for loading enzyme complexes. S3, Staged Temperature-Controlled Fermentation: S3.1 Preparation of synergistic microbial community: Mix enzyme-protecting bacteria, heavy metal-passivating bacteria and soil structure-improving bacteria at a ratio of 40-50%:30-40%:20-30% of colony-forming units; S3.

2. The multifunctional carrier matrix of the enzyme complex is added to the fermentation medium at a concentration of 30 g / L, and 10% (v / v) of synergistic microbial community is inoculated. The fermentation medium contains: glucose 8-12 g / L, peptone 4-6 g / L, yeast extract 2-4 g / L, potassium dihydrogen phosphate 1.5-2.5 g / L, magnesium sulfate heptahydrate 0.4-0.6 g / L, and trace element solution 0.5-1.5 mL / L. The specific components of the trace element solution are: zinc sulfate heptahydrate 0.1 g / L, manganese sulfate monohydrate 0.05 g / L, copper sulfate pentahydrate 0.01 g / L, cobalt chloride hexahydrate 0.005 g / L, and sodium molybdate crystals 0.005 g / L. S3.3, First stage aerobic fermentation: cultured at 35-38℃, 180rpm, and pH 6.5-7 for 48h; S3.4, Second stage microaerobic fermentation: Cultivate for 48 hours at 30-32℃, 50 rpm, and dissolved oxygen concentration of 2-3 mg / L; S3.5, Third stage low-temperature ripening: Cultivate at 15-18℃ for 48 hours; S4. Magnetization and Drying: S4.1 Apply an alternating magnetic field with a frequency of 50 Hz and an intensity of 10 mT for 6 hours at the end of fermentation. S4.2 The fermentation product was vacuum freeze-dried at -45℃ and 0.1Pa to constant weight to obtain bio-organic fertilizer.

6. The preparation process according to claim 5, characterized in that: In step S1.3, the concentration of the FeCl3 solution is 0.15M, and the calcination process is carried out under N2 protection.

7. The preparation process according to claim 5, characterized in that: In step S2.4, the specific weight proportions of each component are as follows: 35 parts Fe3O4@biochar composite carrier, 22 parts calcium alginate-chitosan microcapsules, 18 parts modified attapulgite clay, and 12 parts humic acid-amino acid chelate.

8. The preparation process according to claim 5, characterized in that: In step S3.1, the specific composition of the synergistic microbial community is as follows: 45% enzyme-activating bacteria, 35% heavy metal-passivating bacteria, and 20% soil structure-improving bacteria.

9. The preparation process according to claim 5, characterized in that: In step S3.4, during the microaerobic fermentation stage, microorganisms form a biofilm complex on the carrier surface with a membrane thickness of 50-100 μm and a viable cell density ≥1×10⁻⁶. 8 CFU / cm², extracellular polysaccharide content ≥8g / L.

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