A ternary synergistic functionalized active composite membrane of enzyme, bacteria, and fertilizer, its preparation method and application

Abstract

This invention relates to a synergistic functionalized active composite membrane of enzymes, bacteria, and fertilizer adapted to crop growth, its preparation method, and its application. The preparation method includes the following steps: dispersing carboxylated cellulose nanocrystals in water, adding proteinase K, Bacillus subtilis suspension, and slow-release fertilizer, stirring evenly at room temperature, followed by dialysis and freeze-drying to obtain BioFert@CNC; dissolving PLA in chloroform to form a matrix solution, dispersing BioFert@CNC in chloroform and adding it to the matrix solution, mixing, coating, and drying to obtain the composite membrane. This invention utilizes the interfacial interaction between proteinase K and the PLA matrix to improve the compatibility of the filler and the matrix. Simultaneously, Bacillus subtilis can colonize the soil for a short period after membrane degradation, inhibiting soil-borne pathogens and improving the rhizosphere microecology. The slow-release fertilizer gradually releases nutrients during the degradation process, matching the crop's nutrient requirement cycle and improving fertilizer utilization, making it effective in the field of agricultural films.

Description

Technical Field

[0001] This invention belongs to the field of biomass material utilization technology, specifically relating to a synergistic functionalized active composite membrane of enzymes, bacteria, and fertilizers that can be adapted to crop growth, its preparation method, and its application. Background Technology

[0002] Plastics are widely used in various industrial sectors due to their versatility and excellent physicochemical properties. However, the inherent resistance to degradation of plastics leads to their long-term presence in the environment after use, causing serious pollution problems. The continuous accumulation of traditional petroleum-based plastics such as polyethylene (PE) and polypropylene (PP) further exacerbates the plastic pollution problem. These materials gradually break down into microplastics in the environment, entering the food chain and posing a potential threat to ecosystems and human health. Faced with increasingly severe plastic pollution, developing environmentally friendly, biodegradable alternative materials has become an urgent need to promote sustainable development.

[0003] PLA, as a typical biodegradable material, is considered one of the important directions for alleviating plastic pollution. However, PLA still has several obvious drawbacks, such as a degradation cycle of several years in the natural environment, low crystallinity, and the tendency to generate a lot of microplastics during use, which limits its widespread application in practical scenarios.

[0004] In recent years, enzyme-catalyzed degradation technology has provided a new approach to improving the degradation performance of PLA. Proteinase K, due to its high catalytic activity over a wide temperature range and under weakly acidic to neutral conditions, has shown strong potential in PLA degradation. However, natural enzymes are easily inactivated during processing and have poor affinity for the hydrophobic PLA matrix, making it difficult to precisely control the degradation rate. To overcome these shortcomings, functional bacterial strains, such as Bacillus subtilis, can be introduced to construct an "enzyme-bacterium" synergistic system. This bacterium can secrete multiple extracellular enzymes to synergistically hydrolyze PLA intermediates and inhibit soil-borne pathogens by producing antibiotics, thereby improving the rhizosphere microecology. Further introduction of slow-release fertilizers can then gradually release nutrients during membrane degradation, matching the crop's nutrient requirements and improving fertilizer utilization. This forms a "enzyme-bacterium-fertilizer" ternary synergistic strategy, achieving integrated functionality of plastic degradation, soil improvement, and crop growth promotion.

[0005] It is noteworthy that cellulose nanocrystals (CNCs), as a naturally derived nanomaterial, possess high strength, high aspect ratio, and abundant surface hydroxyl groups, making them an ideal reinforcing material for improving PLA properties. CNCs can enhance the crystallinity of PLA through heterogeneous nucleation, thereby strengthening the material's mechanical strength and thermal stability. Therefore, combining the synergistic characteristics of the "enzyme-bacteria-fertilizer" ternary system with the structural advantages of CNCs, and effectively addressing the interfacial compatibility issues between both and the PLA matrix, has become a key pathway to overcome the performance bottlenecks of biodegradable materials and promote their practical application. Summary of the Invention

[0006] Based on the aforementioned shortcomings and deficiencies in the prior art, one of the objectives of this invention is to at least solve one or more of the aforementioned problems in the prior art. In other words, one of the objectives of this invention is to provide an enzyme-bacterial fertilizer ternary synergistic functionalized active composite membrane that can be adapted to crop growth, which meets one or more of the aforementioned needs, as well as its preparation method and application.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for preparing a ternary synergistic functionalized active composite membrane of enzyme, bacteria, and fertilizer includes the following steps: (1) Disperse carboxylated cellulose nanocrystals in water, adjust the pH to 7.5-8.0, add proteinase K, Bacillus subtilis suspension and slow-release fertilizer, stir evenly at room temperature, and then obtain BioFert@CNC by dialysis and freeze drying; (2) Dissolve PLA in chloroform to form a matrix solution, disperse BioFert@CNC in chloroform and add it to the matrix solution. After stirring and mixing, coating and drying, a composite film is obtained.

[0008] As a preferred embodiment, in step (1), the solid-liquid ratio of carboxylated cellulose nanocrystals to water is 1 g: (100-150) mL.

[0009] As a preferred embodiment, in step (1), the concentration of proteinase K added is 0.5 to 1 mg / mL, based on the volume of water.

[0010] As a preferred embodiment, in step (1), the concentration of the Bacillus subtilis suspension is 0.5–1 mg / mL; Based on the volume of water, the volume ratio of Bacillus subtilis suspension to water is 1:(4-6).

[0011] As a preferred embodiment, in step (1), the slow-release fertilizer is a mixture of urea and potassium dihydrogen phosphate, and the amount added is 15-20% of the mass of carboxylated cellulose nanocrystals; wherein the mass ratio of urea to potassium dihydrogen phosphate is (1-3):1.

[0012] As a preferred embodiment, in step (1), the preparation process of carboxylated cellulose nanocrystals includes: Add straw powder to sodium hypochlorite solution and heat at 95-100℃ for 2-5 hours. After the reaction is complete, filter until neutral to obtain pretreated straw. Pretreated straw was added to a sulfuric acid / oxalic acid mixed acid system and stirred at 75-85℃ for 2-5 hours. Then, it was centrifuged, washed, and freeze-dried to obtain carboxylated cellulose nanocrystals (CNC). As a preferred embodiment, the solid-liquid ratio of the straw powder to the sodium hypochlorite solution is 1g:(100-150)mL, and the mass fraction of the sodium hypochlorite solution is 5-7%. The solid-liquid ratio of the pretreated straw to the sulfuric acid / oxalic acid mixed acid system is 1 g: (100-150) mL; In the sulfuric acid / oxalic acid mixed acid system, the mass ratio of sulfuric acid to oxalic acid is (1-3):1, and the sulfuric acid concentration is 30-40 wt%.

[0013] As a preferred embodiment, in step (2), the mass fraction of PLA in the matrix solution is 8-12 wt%. The amount of BioFert@CNC added is 1 to 5% of the mass fraction of PLA.

[0014] The present invention also provides an enzyme-bacterial-fertilizer ternary synergistic functionalized active composite membrane prepared by the preparation method described in any of the preceding embodiments.

[0015] The present invention also provides the application of the composite film described above in agricultural films to promote plant growth.

[0016] Compared with the prior art, the beneficial effects of this invention are: This invention adopts a ternary synergistic functionalization strategy of "enzyme-bacteria-fertilizer" to synthesize carboxylated cellulose nano-CNCs using agricultural waste straw as raw material. Through co-immobilization technology, proteinase K, Bacillus subtilis and slow-release fertilizer are stably loaded onto the surface of nano-cellulose, and then combined with polylactic acid to prepare a multifunctional composite membrane.

[0017] Compared to traditional methods that rely solely on physical blending or single-component modification, this invention utilizes the interfacial interaction between proteinase K and the PLA matrix to improve the compatibility of the filler and matrix, optimizing the mechanical properties and thermal stability of the composite material. Simultaneously, Bacillus subtilis can colonize the soil briefly after membrane degradation, inhibiting soil-borne pathogens and improving the rhizosphere microecology; slow-release fertilizer gradually releases nutrients during degradation, matching the crop's nutrient requirements and improving fertilizer utilization. This composite membrane achieves a synergistic integration of controlled plastic degradation, continuous soil improvement, and crop growth promotion during use.

[0018] This design not only realizes the high-value utilization of agricultural waste straw, but also provides a new technical path for developing bio-based composite materials with functions of plastic pollution system remediation and agricultural growth adaptation, and has broad application prospects in fields such as green packaging and agricultural mulch film. Attached Figure Description

[0019] Figure 1 These are scanning electron microscope images of carboxylated cellulose nanocrystals (CNC) and BioFert@CNC from Example 1 of this invention. Figure 2 Laser confocal microscope images of the PCBioFert composite film of Embodiment 3 and the PC composite film of Comparative Example 1 of the present invention; Figure 3 The diagram shows the water absorption and barrier properties of the PCBioFert composite membrane of Examples 1-3, the PC composite membrane of Comparative Example 1, and the PLA membrane. Figure 4 The mechanical properties of the PCBioFert composite film of Examples 1-3 of the present invention, the PC composite film of Comparative Example 1, and the PLA film are shown in the figure. Figure 5 The graphs show the degradation rates of the PCBioFert composite membranes of Examples 1-3, the PC composite membrane of Comparative Example 1, and the PLA membrane in soil. Detailed Implementation

[0020] The following provides a detailed description of the enzyme-bacterial-fertilizer ternary synergistic functionalized active composite membrane of the present invention, its preparation method, and its application.

[0021] The preparation method of the enzyme-bacterial-fertilizer ternary synergistic functionalized active composite membrane of the present invention includes the following steps: (1) Disperse carboxylated cellulose nanocrystals in water, adjust the pH to 7.5-8.0, add proteinase K, Bacillus subtilis suspension and slow-release fertilizer, stir evenly at room temperature, and then obtain BioFert@CNC by dialysis and freeze drying; The preparation process of the above-mentioned carboxylated cellulose nanocrystals includes: Add straw powder to sodium hypochlorite solution and heat at 95-100℃ for 2-5 hours. After the reaction is complete, filter until neutral to obtain pretreated straw. The specific reaction temperature and time can be determined according to the actual application requirements. Pretreated straw is added to a sulfuric acid / oxalic acid mixed acid system and stirred at 75-85℃ for 2-5 hours. Then, it is centrifuged, washed, and freeze-dried to obtain carboxylated cellulose nanocrystals (CNC). The specific reaction temperature and time can be determined according to the actual application requirements. The solid-liquid ratio of straw powder to sodium hypochlorite solution is 1g:(100-150)mL, and the mass fraction of sodium hypochlorite solution is 5-7%; the specific solid-liquid ratio and mass fraction can be determined according to actual application requirements. The solid-liquid ratio of the pretreated straw to the sulfuric acid / oxalic acid mixed acid system is 1g:(100-150)mL; wherein, the mass ratio of sulfuric acid to oxalic acid in the sulfuric acid / oxalic acid mixed acid system is (1-3):1, and the sulfuric acid concentration is 30-40wt%; the specific solid-liquid ratio, mass ratio and concentration can be determined according to the actual application requirements. The solid-liquid ratio of the above-mentioned carboxylated cellulose nanocrystals to water is 1g:(100-150)mL. The specific solid-liquid ratio can be determined according to the actual application requirements. The proteinase K concentration mentioned above, based on the volume of water, is 0.5–1 mg / mL. The specific concentration can be determined according to the actual application requirements. The concentration of the above-mentioned Bacillus subtilis suspension is 0.5–1 mg / mL, and the specific concentration can be determined according to the actual application requirements; the Bacillus subtilis suspension is obtained by dispersing Bacillus subtilis in water; In step (1) above, based on the volume of water, the volume ratio of Bacillus subtilis suspension to water is 1:(4-6). The specific volume ratio can be determined according to the actual application requirements. The slow-release fertilizer in this embodiment of the invention is a mixture of urea and potassium dihydrogen phosphate, and the amount added is 15-20% of the mass of carboxylated cellulose nanocrystals; wherein, the mass ratio of urea to potassium dihydrogen phosphate is (1-3):1; the specific amount added and the mass ratio can be determined according to the actual application requirements.

[0022] (2) Dissolve PLA in chloroform to form a matrix solution, disperse BioFert@CNC in chloroform and add it to the matrix solution, mix by stirring, coat and dry to obtain a composite film; The mass fraction of PLA in the matrix solution is 8-12 wt%, and the specific mass fraction can be determined according to the actual application requirements. The amount of BioFert@CNC added is 1-5% of the mass fraction of PLA, and the specific mass fraction can be determined according to the actual application requirements. The present invention also provides an enzyme-bacterial fertilizer ternary synergistic functionalized active composite film prepared by the above preparation method, which can be used as an agricultural film, is biodegradable, and promotes plant growth.

[0023] The following specific examples and comparative examples further explain and illustrate the enzyme-bacterial-fertilizer ternary synergistic functionalized active composite membrane of the present invention, its preparation method, and its application.

[0024] Example 1: The method for preparing the PCBioFert composite film in this embodiment includes the following steps: (1) 4 g of straw powder was immersed in 400 mL of 5 wt% sodium hypochlorite solution and heated at 98 °C for 2 hours to remove lignin from the straw. After the reaction, the reactants were filtered by suction to remove impurities until the solution was neutral, and pretreated straw was obtained. The pretreated straw was added to a 35 wt% sulfuric acid / oxalic acid mixed solution (mass ratio 7:3) and stirred and heated at 80 °C for 2 hours. Subsequently, the solid product was separated by centrifugation three times at 11000 rpm. The solid product was freeze-dried to form a powder, and carboxylated cellulose nanocrystals (CNC) were obtained. (2) 0.4 g of carboxylated cellulose nanocrystals (CNC) were dispersed in 50 mL of deionized water using an ultrasonic machine. The pH was adjusted to 7.5 with NaOH solution. 30 mg of proteinase K, 10 mL of 0.5 mg / mL Bacillus subtilis suspension, 0.0533 g of urea and 0.0267 g of potassium dihydrogen phosphate were added to the solution. The mixture was stirred at room temperature for 2 hours. The mixture was dialyzed with ultrapure water for 48 hours using a 3.5 kDa dialysis bag. After dialysis, the mixture was freeze-dried into powder to obtain BioFert@CNC. (3) 0.02g BioFert@CNC was uniformly dispersed in 2g PLA chloroform solution, mixed, coated and dried at room temperature to obtain PCBioFert composite film; wherein, the mass of BioFert@CNC accounted for 1% of the total mass of PCBioFert composite film.

[0025] Example 2: The method for preparing the PCBioFert composite film in this embodiment differs from that in Example 1 in that: The BioFert@CNC component in the PCBioFert composite film accounts for 3% of the total mass of the PCBioFert composite film. Other steps can be found in Example 1.

[0026] Example 3: The method for preparing the PCBioFert composite film in this embodiment differs from that in Example 1 in that: The BioFert@CNC component in the PCBioFert composite film accounts for 5% of the total mass of the PCBioFert composite film. Other steps can be found in Example 1.

[0027] Comparative Example 1: The preparation method of the PC composite film in this comparative example differs from that in Example 3 in that: CNC, proteinase K, Bacillus subtilis suspension and slow-release fertilizer were directly mixed and then evenly dispersed in PLA chloroform solution. After mixing and coating, PCBioFert composite membrane was obtained. Other conditions are the same as in Example 3.

[0028] Comparative Example 2: The preparation method of the PCBioFert composite film in this comparative example differs from that in Example 3 in that: In step (2), the pH is controlled to 7 using NaOH solution; Other steps and process parameters are the same as in Example 3.

[0029] Comparative Example 3: The preparation method of the PCBioFert composite film in this comparative example differs from that in Example 1 in that: In step (2), the pH is controlled to 9 using NaOH solution; Other steps and process parameters are the same as in Example 3.

[0030] The appearance of the product was evaluated using scanning electron microscopy. SEM images of the CNC prepared with mixed acid and the BioFert@CNC after loading are shown below. Figure 1 As shown, CNC ( Figure 1 a) exhibits a rod-shaped single-crystal morphology, in contrast to BioFert@CNC ( Figure 1 b) After grafting, the surface and shape change from smooth to rough.

[0031] like Figure 2 As shown, the PCBioFert composite membrane of Example 3 of this invention exhibits significant green fluorescence after Calcein-AM staining, and both the fluorescence intensity and the density of positive areas are significantly higher than those of the PC composite membrane in Comparative Example 1. Calcein-AM can only enter living cells and emit fluorescence under the action of esterases; therefore, the fluorescence intensity directly reflects the enzyme activity. This indicates that, compared with PC, the PCBioFert composite membrane of this invention has higher enzyme activity, which is beneficial to the performance of the material in biodegradation or bioactivity-related applications.

[0032] like Figure 3 As shown, the water absorption rate and water vapor transmission rate of the PCBioFert composite membranes of Examples 1-3 of the present invention are lower than those of pure PLA membranes and PC composite membranes of Comparative Example 1, indicating that the PCBioFert composite membranes of the present invention have excellent water vapor barrier performance.

[0033] like Figure 4 As shown, the tensile strength of the PCBioFert composite film is improved after BioFert@CNC filling, which is due to the stiffness generated by the reinforcing effect of the filler on the PLA matrix.

[0034] like Figure 5As shown, in the 120-day soil degradation experiment, the degradation rate of the PCBioFert composite membrane in Example 3 was greater than that of the traditional PLA membrane and the PC composite membrane in Comparative Example 1. Therefore, the present invention can effectively improve the degradation rate of the composite membrane, thereby preparing a biomaterial with growth cycle adaptability.

[0035] The mechanical properties, thermogravimetric (TG) properties, water vapor transmission rate, and water absorption rate of Example 3, Comparative Examples 2 and 3 were tested below. The test process is as follows: 1. Mechanical testing: The tensile strength of the composite film was determined using a universal testing machine according to GB / T 1040.3-2006 standard at a tensile rate of 5 mm / min. 2. TG test: Using a thermogravimetric analyzer, the material was heated from room temperature to 600 °C at a heating rate of 10 °C / min under a nitrogen atmosphere, and the maximum thermal decomposition temperature of the material was recorded. 3. Water absorption rate test: Immerse the sample in deionized water for 24 hours, remove it, wipe off the surface moisture, weigh it, and calculate the water absorption rate; The test results are shown in Table 1: Table 1 Test Results ; As shown in Table 1, the tensile strength and thermal decomposition temperature of Example 3 are significantly higher than those of Comparative Example 2 and Comparative Example 3, while the water absorption rate is lower than that of the two comparative examples, indicating that the PCBioFert composite film of Example 3 has better mechanical properties and thermal stability.

[0036] The mechanism is as follows: Under weakly alkaline conditions (pH 7.5–8), the intermolecular interactions of the components within the PCBioFert composite membrane are stronger, and the cross-linked network structure is more dense and uniform, which is beneficial for stress transfer and improved thermal stability. However, at pH 7, the cross-linking is incomplete, and at pH 9, the excessively alkaline environment may lead to hydrolysis of some components or structural defects, thus reducing mechanical and thermal properties. Furthermore, the membrane structure formed under weakly alkaline conditions is denser, with reduced free volume, limiting adsorption and permeation channels; while at pH 7 or pH 9, the membrane structure is relatively loose or contains microporous defects, making it easier for water molecules to enter and permeate.

[0037] As mentioned earlier, a pH of 7.5–8 is most conducive to the activity of enzymes and bacteria, and higher enzyme and bacteria activity can promote the degradation rate of materials. However, under pH conditions of 7 or 9, enzyme and bacteria activity is lower, and the degradation rate is significantly reduced.

[0038] In summary, the BioFert@CNC of this invention forms a denser structure compared to traditional PLA films, resulting in better barrier and mechanical properties, as well as a faster degradation rate, which can effectively promote plant growth.

[0039] Given that there are numerous embodiments of the present invention, and that the raw materials and dosages can be adjusted within the appropriate range according to actual application requirements, and that the experimental data for each embodiment are extensive and numerous, it is not suitable to list and describe them one by one here. However, the content to be verified and the final conclusions obtained in each embodiment are similar. Therefore, the verification content of each embodiment will not be described one by one here.

[0040] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A preparation method of an enzyme bacteria fertilizer ternary synergistic functional active composite film, characterized in that, Includes the following steps: (1) Disperse carboxylated cellulose nanocrystals in water, adjust the pH to 7.5-8.0, add proteinase K, Bacillus subtilis suspension and slow-release fertilizer, stir evenly at room temperature, and then obtain BioFert@CNC by dialysis and freeze drying; (2) Dissolve PLA in chloroform to form a matrix solution, disperse BioFert@CNC in chloroform and add it to the matrix solution. After stirring and mixing, coating and drying, a composite film is obtained.

2. The production method according to claim 1, characterized by, In step (1), the solid-liquid ratio of carboxylated cellulose nanocrystals to water is 1g:(100-150)mL.

3. The production method according to claim 2, characterized by, In step (1), the concentration of proteinase K added is 0.5 to 1 mg / mL, based on the volume of water.

4. The production method according to claim 2, characterized by, In step (1), the concentration of Bacillus subtilis suspension is 0.5–1 mg / mL; Based on the volume of water, the volume ratio of Bacillus subtilis suspension to water is 1:(4-6).

5. The preparation method according to claim 1, characterized in that, In step (1), the slow-release fertilizer is a mixture of urea and potassium dihydrogen phosphate, and the amount added is 15-20% of the mass of carboxylated cellulose nanocrystals; wherein the mass ratio of urea to potassium dihydrogen phosphate is (1-3):

1.

6. The preparation method according to claim 1, characterized in that, In step (1), the preparation process of carboxylated cellulose nanocrystals includes: Add straw powder to sodium hypochlorite solution and heat at 95-100℃ for 2-5 hours. After the reaction is complete, filter until neutral to obtain pretreated straw. Pretreated straw was added to a sulfuric acid / oxalic acid mixed acid system and stirred at 75-85℃ for 2-5 hours. Then, it was centrifuged, washed, and freeze-dried to obtain carboxylated cellulose nanocrystals (CNC).

7. The preparation method according to claim 1, characterized in that, The solid-liquid ratio of the straw powder to the sodium hypochlorite solution is 1g:(100-150)mL, and the mass fraction of the sodium hypochlorite solution is 5-7%. The solid-liquid ratio of the pretreated straw to the sulfuric acid / oxalic acid mixed acid system is 1 g: (100-150) mL; In the sulfuric acid / oxalic acid mixed acid system, the mass ratio of sulfuric acid to oxalic acid is (1-3):1, and the sulfuric acid concentration is 30-40 wt%.

8. The preparation method according to claim 1, characterized in that, In step (2), the mass fraction of PLA in the matrix solution is 8-12 wt%. The amount of BioFert@CNC added is 1 to 5% of the mass fraction of PLA.

9. A ternary synergistic functionalized active composite membrane of enzyme, bacteria, and fertilizer prepared by the preparation method according to any one of claims 1-8.

10. The application of the composite membrane as described in claim 9, characterized in that, Used in agricultural films.

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