Preparation method of cellulose nanocrystal porous PLA coated controlled release bacterial fertilizer

Abstract

The application belongs to the field of fertilizers, and particularly discloses a preparation method of cellulose nanocrystal porous PLA coated controlled-release bacterial fertilizer. The technology discards traditional petroleum-based coating materials, and uses biomass-based polylactic acid (PLA) and cellulose nanocrystal as coating layer matrix materials. The method uses fluidized bed low-temperature bottom spraying coating and supercritical CO2 foaming in sequence to produce the controlled-release bacterial fertilizer with controllable pores, and the actual use can realize degradable and residue-free, and the coating layer is stable, the pores are controllable, the bacterial fertilizer is green and environmentally friendly, and the survival rate of the bacterial fertilizer is high.

Description

Technical Field

[0001] This invention belongs to the field of fertilizers, specifically relating to a method for preparing a controlled-release bacterial fertilizer coated with cellulose nanocrystal porous PLA. Background Technology

[0002] Slow-release fertilizer technology is an important means to achieve precision fertilization in agriculture and improve nutrient utilization efficiency. By constructing a controllable release coating layer on the surface of fertilizer granules, nutrients can be continuously supplied according to the crop's growth rhythm, reducing leaching losses and environmental pollution. In recent years, combining microbial agents (such as biocontrol bacteria like *Paecilomyces lilacinus*) with slow-release fertilizers to form compound fertilizers that combine nutrient supply and biological control functions has become a research hotspot in the field of green agriculture.

[0003] *Paecilomyces lilacinus* is a fungus with broad application prospects in agricultural biological control. Live bacteria can parasitize the eggs, larvae, and adults of nematodes, penetrating the eggshell and body wall by secreting chitinases and proteases, disrupting their physiological metabolism, leading to nematode death, and effectively reducing nematode damage to crops. It can also produce indoleacetic acid-like derivatives, which, at low concentrations, can promote root development, plant growth, and seed germination, enhancing crop resistance (such as drought, cold, and waterlogging resistance), and improving crop yield and quality. However, the effectiveness of *Paecilomyces lilacinus* is greatly influenced by external factors, limiting its market development. First, it is highly dependent on the environment; its activity and effectiveness are significantly affected by environmental factors such as soil temperature, humidity, pH, and organic matter content. Under extreme or unsuitable environmental conditions, its shelf life is short, and the fungicide may not be fully effective.

[0004] In terms of coating materials, traditional petroleum-based polymer coating materials are non-degradable, and long-term use will lead to continuous accumulation in the soil, damaging soil structure and polluting the ecological environment. Polylactic acid (PLA), as a renewable and biodegradable polyester material, shows potential to replace traditional petroleum-based plastics in the field of agricultural coatings due to its good biocompatibility and environmental friendliness. For example, CN118834102A discloses a polylactic acid-biochar-based slow-release fertilizer and its preparation method and device. This technology mixes polylactic acid-biochar composite with fertilizer powder and then coats it on the surface of fertilizer particles. Biochar acts as a carrier to give the fertilizer slow-release function. However, the coating layer is a solid structure rather than a porous structure, resulting in a single nutrient release pathway and limited controllable release precision.

[0005] CN116120132A discloses a fully biodegradable plastic controlled-release fertilizer coating material and fertilizer molding process. Its weight composition includes a semi-aromatic carbon dioxide-based copolymer, regulators (including PPC, PBAT, PCL, PLA, and pore-forming agents). The core technology involves physically mixing the pore-forming agent and various biodegradable resins. However, the coating material has a complex composition, involving multiple chemical copolymers, increasing production costs and process control difficulty. Furthermore, it relies on halogenated hydrocarbon solvents such as dichloromethane, which does not meet the highest standards of green chemistry. In addition, existing technologies in the field of green, fully biodegradable coated slow-release fertilizers have the following technical defects: firstly, coatings based on pure PLA are prone to rupture; secondly, current high-temperature melt coating and organic solvent processes pose a significant risk of microbial agent inactivation. In summary, this invention aims to provide a method for preparing PLA-coated controlled-release microbial fertilizer, achieving the goals of film stability, controllable pore size, environmental friendliness, and high microbial agent survival rate. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention uses biomass-based polylactic acid (PLA) and cellulose nanocrystals as the coating matrix materials, and *Paecilomyces lilacinus* granules as the core material. The mixture is sequentially coated using a fluidized bed low-temperature bottom spraying process and then foamed with supercritical CO2 to produce a controlled-release microbial fertilizer with controllable porosity. This technology eliminates the need for traditional petroleum-based coating materials, leaving no harmful residues in the soil and achieving biodegradability and residue-free operation. Furthermore, the room-temperature process ensures high retention of heat-sensitive bacterial activity.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for preparing cellulose nanocrystal porous PLA-coated controlled-release bacterial fertilizer, comprising the following steps: (1) Dissolve PLA in an organic solvent and stir at room temperature until completely dissolved. Add plasticizer and dispersant in sequence and stir. Then add cellulose nanocrystals, disperse and mix by ultrasonication to obtain the coating solution. (2) Vacuum freeze-dried spore powder of *Paecilomyces lilacinus* is mixed with a porous carrier, nutrients and binder, and core particles are obtained by rotary granulation and extrusion rounding. (3) The core particles are put into a fluidized bed and coated with liquid bottom spraying process. After the spraying is completed, the particles are dried by blowing in the fluidized state to obtain solid particles. (4) The solid particles are placed in supercritical CO2, and after depressurization, they are dried by ventilation and vacuum to obtain cellulose nanocrystal porous PLA coated controlled-release bacterial fertilizer.

[0008] Preferably, the Mn of the PLA is 80000 to 120000.

[0009] This invention utilizes a low-toxicity, low-boiling-point organic solvent that rapidly evaporates and removes microplastics at room temperature, avoiding the risk of thermal damage to *Paecilomyces lilacinus* caused by high-temperature processing. Ultimately, residues are reduced to the ppm level through supercritical CO2 extraction. Furthermore, the addition of an environmentally friendly plasticizer effectively lowers the glass transition temperature (Tg) of PLA, giving the PLA molecular chains greater flexibility and fluidity at room temperature, thus improving the uniformity of the coating solution on the surface of the bacterial particles. In addition, the plasticizer enhances PLA's ability to dissolve and absorb CO2 during the supercritical CO2 saturation stage, allowing CO2 to dissolve more fully in the PLA shell, which is beneficial for subsequent foaming to form a uniform and fine pore structure. Compared with traditional petroleum-based coating materials, the controlled-release bacterial fertilizer provided by this system leaves no microplastic residue in the soil and requires no toxic crosslinking agents or curing agents, taking into account green raw materials, adjustable porosity, mechanical durability, and process feasibility.

[0010] In some embodiments, the molecular structure of the dispersant in step (1) is as follows: ; Where n = 20 to 25, and is an integer.

[0011] In some embodiments, the plasticizer in step (1) comprises tributyl citrate and acetylated tributyl citrate in a mass ratio of (6-9):(1-4).

[0012] The hydroxyl groups on tributyl citrate form strong hydrogen bonds with PLA ester groups, which can more effectively insert into the PLA molecular chains to weaken interchain interactions. Acetyltributyl citrate has a larger molecular weight and its acetylation modification gives it a higher free volume contribution. Both components synergistically disrupt the ordered arrangement of PLA molecular chains at different scales, resulting in a plasticizing efficiency higher than either component used alone. Under the operating requirements of supercritical CO2 foaming, compound plasticizing can reduce the Tg of PLA to 15-20℃, far below room temperature, ensuring that PLA remains in a fully rubbery state during CO2 saturation, improving gas solubility and diffusion rate, and optimizing pore size distribution.

[0013] In some embodiments, the mass ratio of PLA, plasticizer, dispersant and cellulose nanocrystals in step (1) is (10-20):(0.2-1.5):(0.1-0.9):(0.5-2.5).

[0014] Uniformly dispersed cellulose nanocrystals act as heterogeneous nucleating agents in the subsequent foaming stage, providing numerous nucleation sites. This promotes the formation of high-density, uniform micron-sized pores from CO2 during depressurization, resulting in a porous structure with uniform pore size and controllable connectivity. This avoids the localized large bubbles and pore merging defects that easily occur without nucleating agents, giving the porous coating layer superior mechanical integrity and release uniformity. Simultaneously, the cellulose nanocrystals impart a mechanical strengthening effect to the coating layer; their high modulus and high strength effectively compensate for the mechanical shortcomings of the porous PLA structure, improving tensile strength and solving the problem of easy breakage in traditional porous PLA coating layers.

[0015] In some embodiments, the viable spore content of the *Paecilomyces lilacinus* spore powder in step (2) is ≥1×10⁻⁶. 10 CFU / g.

[0016] In some embodiments, the mass ratio of *Paecilomyces lilacinus* spore powder, porous carrier, nutrients, and binder in step (2) is (8-15):(10-20):(4-10):(1-3).

[0017] Preferably, the porous carrier is diatomaceous earth; the nutrients are preferably humic acid and compound amino acid powder.

[0018] Nutrients provide early nutritional support for the rapid germination and mycelial propagation of *Paecilomyces lilacinus* after it is applied to the soil, preventing the fungi from becoming inactive due to early starvation caused by the delayed degradation of the coating layer.

[0019] In some embodiments, the particle size of the core particles in step (2) is 1.5 to 2.5 mm.

[0020] In some embodiments, the fluidized bed inlet temperature in step (3) is 25-35°C, the amount of coating liquid is 20-25% of the core particle mass, the atomization pressure is 0.01-0.20 MPa, and the spray rate is 1-5 mL / min.

[0021] This invention provides a customized safety window for thermosensitive spores: the inlet air temperature of 28-30°C is lower than the critical activity temperature of *Paecilomyces lilacinus* (approximately 40-45°C), ensuring the survival rate of spores during the coating process; the combination of atomization pressure and spray rate produces uniformly sized microdroplets, achieving uniform wetting and dense film formation on the surface of each spore in the fluidized bed, avoiding localized weak areas and early damage caused by uneven coating.

[0022] In some embodiments, the supercritical CO2 pressure in step (4) is 5-15 MPa and the holding time is 4-12 h.

[0023] With the synergistic effect of cellulose nanocrystals and plasticizers, PLA's thermal glutaraldehyde (Tg) drops below room temperature under supercritical CO2 plasticization, enabling efficient CO2 dissolution without heating. Supercritical CO2 possesses liquid-like dissolving power and gas-like diffusion capabilities, allowing it to efficiently penetrate into the amorphous region of PLA, increasing the interchain spacing of PLA molecules, achieving full plasticization and swelling of PLA, avoiding the high-temperature operation required for traditional thermoplastic foaming, and reducing damage to heat-sensitive bacterial particles.

[0024] In some embodiments, the depressurization rate in step (4) is 10 to 20 MPa / s.

[0025] When the pressure drops to atmospheric pressure, the CO2 dissolved in PLA reaches a supersaturated state. Thermodynamic instability drives the rapid precipitation and foaming of CO2 at the nucleation sites of cellulose nanocrystals. The bubbles grow and merge to form an interconnected microporous structure. The interconnected open structure provides microscopic channels for water to enter the coating layer and nutrients to diffuse outward, so that the fertilizer release rate no longer depends solely on material degradation, but can be independently controlled by the pore structure parameters to achieve controlled release.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. Using biomass-derived PLA and cellulose nanocrystals as coating material replaces traditional petroleum-based non-degradable coating materials; the final degradation products of PLA in soil are only CO2 and water, and cellulose nanocrystals can be further decomposed into small sugar molecules by microorganisms in soil and absorbed by crops; low-toxicity organic solvents are used in the process to reduce risks, and supercritical CO2 physical foaming is used to replace solid pore-forming agents, and the solvents and CO2 can be recycled and reused. The entire process has low pollution, and from raw material selection to processing technology to final degradation, it achieves environmental friendliness throughout the entire life cycle.

[0027] 2. The high melting temperature required for traditional PLA coating is significantly reduced, which improves the spore survival rate of *Paecilomyces lilacinus*. This ensures that after the product is applied in the field, sufficient live bacteria can still be released from the sclerotia and colonize the rhizosphere when the coating layer degrades, thus achieving continuous biological control of root-knot nematodes.

[0028] 3. By forming a through-type rigid skeleton in the PLA matrix through a cellulose nanocrystal nanonetwork, the compressive strength and tensile properties of the porous coating layer are improved, thus solving the problem of PLA's fragility.

[0029] 4. By compounding and plasticizing, the Tg of PLA is lower than the room temperature, ensuring that PLA is in a completely rubbery state during CO2 saturation, thereby improving gas solubility and diffusion rate and optimizing pore size distribution. Detailed Implementation

[0030] The present invention will be described below with reference to specific implementation schemes. It should be noted that the following embodiments and comparative examples are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope. It is worth noting that, unless otherwise specified, the raw materials used in the following preparation examples and embodiments are all from any commercially available manufacturer.

[0031] Example 1 A method for preparing a cellulose nanocrystal porous PLA-coated controlled-release bacterial fertilizer specifically includes the following preparation steps: (1) Dissolve PLA (Mn=100000) in ethyl acetate to a concentration of 0.15 g / mL, stir at room temperature until completely dissolved, add plasticizer (tributyl citrate and acetylated tributyl citrate in a mass ratio of 7:3) and dispersant in sequence, and stir for 20 min; then add cellulose nanocrystals, and disperse by ultrasonication (200 W, 20 min) and stirring (400 rpm, 30 min) to obtain a coating solution; wherein, the mass ratio of PLA, plasticizer, dispersant and cellulose nanocrystals is 15:0.8:0.3:1.5; (2) Mix vacuum freeze-dried *Paecilomyces lilacinus* spore powder (live spore content ≥ 1×10⁻⁶) at a mass ratio of 10:15:2:5:3. 10 The mixture of CFU / g), diatomaceous earth, potassium humate, compound amino acid powder (total amino acid content ≥45%), and sodium lignosulfonate (15000Da) was used to obtain core particles with a particle size of 2mm by rotary granulation. (3) The core particles are put into a fluidized bed (inlet air temperature 30℃), and the coating liquid is sprayed at the bottom. The amount of coating liquid is 25% of the mass of the core particles, the atomization pressure is 0.12MPa, and the spraying rate is 2.5mL / min. After the spraying is completed, the particles are dried by blowing in the fluidized state to obtain solid particles. (4) The solid particles were placed in supercritical CO2 (pressure 10MPa, holding time 8 h), and after depressurization (pressure reduction rate 12MPa / s), they were ventilated and vacuum dried at 40℃ to obtain cellulose nanocrystal porous PLA coated controlled-release bacterial fertilizer. The molecular structural formula of the dispersant is as follows, n=22: .

[0032] Example 2 A method for preparing a cellulose nanocrystal porous PLA-coated controlled-release bacterial fertilizer specifically includes the following preparation steps: (1) Dissolve PLA (Mn=80000) in ethyl acetate to a concentration of 0.15 g / mL, stir at room temperature until completely dissolved, add plasticizer (tributyl citrate and acetylated tributyl citrate in a mass ratio of 6:4) and dispersant in sequence, and stir for 20 min; then add 1.5 g of cellulose nanocrystals, and disperse by ultrasonication (200 W, 20 min) and stirring (400 rpm, 30 min) to obtain a coating solution; wherein, the mass ratio of PLA, plasticizer, dispersant and cellulose nanocrystals is 10:0.5:0.2:1.0; (2) Mix vacuum freeze-dried *Paecilomyces lilacinus* spore powder (live spore content ≥ 1×10⁻⁶) at a mass ratio of 8:18:1:3:1. 10 The mixture of CFU / g), diatomaceous earth, potassium humate, compound amino acid powder (total amino acid content ≥45%), and sodium lignosulfonate (15000Da) was used to obtain core particles with a particle size of 2mm by rotary granulation. (3) The core particles are put into a fluidized bed (inlet air temperature 30℃), and the coating liquid is sprayed at the bottom. The amount of coating liquid is 25% of the mass of the core particles, the atomization pressure is 0.20 MPa, and the spraying rate is 2.5 mL / min. After the spraying is completed, the particles are dried by blowing in the fluidized state to obtain solid particles. (4) The solid particles were placed in supercritical CO2 (pressure 12MPa, holding time 6 h), and after depressurization (pressure reduction rate 12MPa / s), they were ventilated and vacuum dried at 40℃ to obtain cellulose nanocrystal porous PLA coated controlled-release bacterial fertilizer.

[0033] Example 3 A method for preparing a cellulose nanocrystal porous PLA-coated controlled-release bacterial fertilizer specifically includes the following preparation steps: (1) Dissolve PLA (Mn=120000) in ethyl acetate to a concentration of 0.15 g / mL, stir at room temperature until completely dissolved, add plasticizer (tributyl citrate and acetylated tributyl citrate in a mass ratio of 9:1) and dispersant in sequence, and stir for 20 min; then add 1.5 g of cellulose nanocrystals, and disperse by ultrasonication (200 W, 20 min) and stirring (400 rpm, 30 min) to obtain the coating solution; wherein, the mass ratio of PLA, plasticizer, dispersant and cellulose nanocrystals is 20:1.5:0.5:2.5; (2) Mix vacuum freeze-dried *Paecilomyces lilacinus* spore powder (live spore content ≥ 1×10⁻⁶) at a mass ratio of 15:20:3:7:3. 10 The mixture of CFU / g), diatomaceous earth, potassium humate, compound amino acid powder (total amino acid content ≥45%), and sodium lignosulfonate (15000Da) was used to obtain core particles with a particle size of 2mm by rotary granulation. (3) The core particles are put into a fluidized bed (inlet air temperature 30℃), and the coating liquid is sprayed at the bottom. The amount of coating liquid is 25% of the mass of the core particles, the atomization pressure is 0.08MPa, and the spraying rate is 2.5mL / min. After the spraying is completed, the particles are dried by blowing in the fluidized state to obtain solid particles. (4) The solid particles are placed in supercritical CO2 (pressure 8MPa, holding time 10h), and after depressurization (pressure reduction rate 12MPa / s), they are ventilated and vacuum dried at 40℃ to obtain cellulose nanocrystal porous PLA coated controlled-release bacterial fertilizer.

[0034] Example 4 This embodiment provides a method for preparing cellulose nanocrystalline porous PLA-coated controlled-release bacterial fertilizer. The specific implementation method is the same as in Embodiment 1, except that acetylacetic acid tributyl ester is replaced by an equal amount of tributyl citrate.

[0035] Comparative Example 1 This comparative example provides a method for preparing a controlled-release bacterial fertilizer coated with porous PLA containing cellulose nanocrystals. The specific implementation method is the same as in Example 1, except that cellulose nanocrystals are not added.

[0036] Comparative Example 2 This comparative example provides a method for preparing a controlled-release bacterial fertilizer coated with cellulose nanocrystal porous PLA. The specific implementation method is the same as in Example 1, except that no plasticizer is added.

[0037] Performance testing The microbial fertilizers provided in Examples 1-4 and Comparative Examples 1-2 were used as samples for the following tests, and the results are shown in Table 1: 1. Porosity: The anhydrous ethanol replacement method was used.

[0038] 2. Average pore size: Cut the microbial fertilizer in half, take SEM images of the coating layer, and use ImageJ software to count at least 100 pores and calculate the average pore size.

[0039] 3. Compressive strength: Using a universal testing machine (equipped with a 2 kN sensor), place a single-particle microbial fertilizer between two plates and compress it at a rate of 1 mm / min until the particle breaks. Record the maximum load F (N). The compressive strength σ = F / (πr 2 ), where r is the particle radius (mm).

[0040] 4. Spore survival rate: Dissolve the granules before coating (solid granules in step 3) and after coating (microbial fertilizer in step 4) in sterile water (mass ratio 1:10) until the solid residue is stable. Take the supernatant, serially dilute it and spread it on PDA plates. Incubate at 28℃ for 72h, count the number of colonies, and the survival rate (%) = (number of viable bacteria after treatment / number of viable bacteria at the beginning) × 100%.

[0041] 5. Degradation half-life: The sample was buried in standard farmland soil (60% WHC, constant temperature 25℃). Five samples were taken out every 5 days, weighed and the mass retention rate was calculated. The half-life was defined as the time when the mass retention rate dropped to 50%.

[0042] 6. Cumulative spore release rate: Simulated soil solution (0.2% agar aqueous solution, pH 6.5, constant temperature shaker at 25℃ 100rpm), 1kg soil solution: 1g sample added to the sample; at 7d, 14d, 21d, 28d, and 42d, the solution was spread on PDA plates and counted, and the percentage of cumulative released spores to the total number of spores in the particles was calculated.

[0043] 7. Effectiveness Test Test nematodes: Second instar larvae (J2) and eggs of the southern root-knot nematode were isolated and purified from the rhizosphere of infected tomatoes; Test crop: Tomato (variety "Hezuo 903", transplanted at the three-leaf-one-heart stage); Test microbial fertilizers: The coated microbial fertilizers prepared in Examples 1-4 and Comparative Examples 1-2, with a blank control (no fertilizer) and a positive control (thiazophos granules of chemical pesticide, 10% active ingredient). Test procedure: Sterilized sandy loam and river sand were mixed at a ratio of 3:1, and 2.5 kg of soil was placed in each pot (20 cm in diameter). Approximately 2000 southern root-knot nematode eggs were evenly inoculated into each pot (mixed into the top 5 cm of soil). After transplanting tomato seedlings, 10 g of microbial fertilizer granules (core weight) were applied in a ring around the root zone of each pot, and the soil was gently covered. Each treatment was replicated 5 times. The temperature was 25±3℃, the light intensity was 12 h / d, and the water and fertilizer management was conventional, but the application of additional fungicides was avoided.

[0044] Investigation and calculation: Plants were harvested 45 days after transplanting, and the root knot index (0-10 grade standard) was calculated. The root knot reduction rate (%) was calculated as (control root knot index - treatment root knot index) / control root knot index × 100%. The fresh weight of the roots of each plant was taken, and nematode egg sacs in the rhizosphere soil were collected by sodium hypochlorite-centrifugation method. The number of nematode eggs per gram of root was calculated.

[0045] Table 1 Performance Test Results

[0046] As shown in Table 1, Examples 1-3 all exhibited good porosity distribution, compressive strength, and spore survival rate. In Example 2, the use of lower molecular weight PLA weakened the matrix's mechanical properties, and the larger pore size may have been caused by increased foaming pressure. In Example 3, the high molecular weight PLA melt viscosity limited the growth of foaming pores under low pressure, resulting in smaller pore sizes and insufficient porosity. Regarding the cumulative spore release rate, Example 1 showed a release rate of only 8.2% after 7 days, avoiding premature spore exposure; the peak period was reached between 14 and 28 days, with a cumulative release rate of 92.5% after 42 days. In this mode, spores gradually seeped out through interconnected pores and actively germinated and broke through under the induction of humic acid / amino acid nutrition. Example 2's larger pore size and faster shell degradation led to a large premature spore release, which, while beneficial for rapid biocontrol, may have missed the nematode control window in the later stages of crop growth, and premature spore release made the soil susceptible to chemical pesticide residues. Example 3: The pore size is too small, the pore connectivity is poor, PLA degradation is slow, and spore release mainly depends on shell degradation rather than pore diffusion, resulting in a large number of spores remaining in the core grain, which affects the biocontrol effect.

[0047] Compared to Example 1, Example 4 completely replaced acetylacetic acid tributyl citrate with tributyl citrate, resulting in a wider average pore size. This may be because the acetyl group of acetylacetic acid tributyl citrate has a stronger affinity for CO2, and its combination with tributyl citrate helps to improve CO2 solubility and pore uniformity, forming a more uniform pore structure. In addition, the wider pore size distribution may also cause some areas of pores to be non-connected, thereby hindering spore diffusion and resulting in an overall release rate lower than that of Example 1.

[0048] Based on Examples 1 and 1 (Comparative Example), the lack of heterogeneous nucleation by cellulose nanocrystals leads to coarse and merged supercritical foam pores, a significant decrease in pore wall mechanical strength, and extremely rapid spore release in the early stages of Comparative Example 1. In Comparative Example 2, without plasticizer, PLA is in a glassy state at room temperature. Although supercritical CO2 has a certain plasticizing effect, it is insufficient to fully soften PLA, resulting in low CO2 solubility and difficulty in foaming. Only a small number of tiny closed pores can be formed, making it difficult for spores to pass through the shell.

[0049] Regarding control efficacy, Example 1 showed a 67.4% reduction rate in root knots and the lowest number of rhizosphere eggs. This was because its spore release curve overlapped with the peak hatching time of second-instar nematode larvae from eggs (approximately 15-25 days), and the cumulative spore release rate reached 92.5% by day 42 of the shell degradation, ensuring continuous activity of live bacteria throughout the entire nematode infection cycle. Simultaneously, the humic acid and amino acids added to the core provided immediate nutrition for spore germination, resulting in strong mycelial colonization. While Example 2 showed higher efficacy than the control, its spore release was too early (massive release on days 5-7), leading to relatively insufficient bacterial density during the peak infection period (days 15-25), allowing nematodes to still partially invade the root system. Example 3 showed severely delayed spore release, missing the optimal window for nematode infection, and the cumulative release rate after 42 days was less than 80%, with a large number of spores remaining in the incompletely degraded shell, unable to contact the nematodes. Although Example 4's spore release was slightly slower, it still covered most of the nematode hatching period. In Comparative Example 1, when the spores released in the early stage came into contact with nematode eggs, the eggs had not yet hatched in large numbers, and the spores aged and became inactive. In the later stage, the shell layer was partially broken and blocked due to the lack of CNC enhancement, which prevented the continuous release of fresh spores, resulting in a low root knot reduction rate. In Comparative Example 2, because the spores were almost sealed inside the shell layer (the release rate was only 32.4% after 42 days), they could not exert a biocontrol effect.

[0050] The embodiments and comparative examples described above do not limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing a controlled-release bacterial fertilizer coated with cellulose nanocrystal porous PLA, characterized in that, Includes the following steps: (1) Dissolve PLA in an organic solvent and stir at room temperature until completely dissolved. Add plasticizer and dispersant in sequence and stir. Then add cellulose nanocrystals, disperse and mix by ultrasonication to obtain the coating solution. (2) Vacuum freeze-dried spore powder of *Paecilomyces lilacinus* is mixed with a porous carrier, nutrients and binder, and core particles are obtained by rotary granulation and extrusion rounding. (3) The core particles are put into a fluidized bed and coated with liquid bottom spraying process. After the spraying is completed, the particles are dried by blowing in the fluidized state to obtain solid particles. (4) The solid particles are placed in supercritical CO2, and after depressurization, they are dried by ventilation and vacuum to obtain cellulose nanocrystal porous PLA coated controlled-release bacterial fertilizer.

2. The method for preparing the cellulose nanocrystalline porous PLA-coated controlled-release bacterial fertilizer according to claim 1, characterized in that, The molecular structure of the dispersant in step (1) is as follows: ； Where n = 20 to 25, and is an integer.

3. The method for preparing the cellulose nanocrystalline porous PLA-coated controlled-release bacterial fertilizer according to claim 1, characterized in that, In step (1), the plasticizer comprises tributyl citrate and acetylated tributyl citrate in a mass ratio of (6-9):(1-4).

4. The method for preparing the cellulose nanocrystalline porous PLA-coated controlled-release bacterial fertilizer according to claim 2, characterized in that, In step (1), the mass ratio of PLA, plasticizer, dispersant, and cellulose nanocrystals is (10-20): (0.2-1.5): (0.1-0.9): (0.5-2.5).

5. The method for preparing the cellulose nanocrystalline porous PLA-coated controlled-release bacterial fertilizer according to claim 1, characterized in that, In step (2), the live spore content of the *Paecilomyces lilacinus* spore powder is ≥1×10⁻⁶. 10 CFU / g.

6. The method for preparing the cellulose nanocrystalline porous PLA-coated controlled-release bacterial fertilizer according to claim 1, characterized in that, In step (2), the mass ratio of *Paecilomyces lilacinus* spore powder, porous carrier, nutrients, and binder is (8-15):(10-20):(4-10):(1-3).

7. The method for preparing the cellulose nanocrystalline porous PLA-coated controlled-release bacterial fertilizer according to claim 1, characterized in that, The particle size of the core particles in step (2) is 1.5 to 2.5 mm.

8. The method for preparing the cellulose nanocrystalline porous PLA-coated controlled-release bacterial fertilizer according to claim 1, characterized in that, In step (3), the fluidized bed inlet temperature is 25-35℃, the amount of coating liquid is 20-25% of the core particle mass, the atomization pressure is 0.01-0.20 MPa, and the spray rate is 1-5 mL / min.

9. The method for preparing the cellulose nanocrystalline porous PLA-coated controlled-release bacterial fertilizer according to claim 1, characterized in that, In step (4), the supercritical CO2 pressure is 5-15 MPa and the holding time is 4-12 h.

10. The method for preparing the cellulose nanocrystalline porous PLA-coated controlled-release bacterial fertilizer according to claim 1, characterized in that, The depressurization rate in step (4) is 10-20 MPa / s.

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