Development of biopolymer polyhydroxyalkanoates for controlled release fertilizers

Abstract

Disclosed herein is a sustained-release fertilizer formulation comprising a core portion containing one or more fertilizer compounds and a shell portion containing a polyhydroxyalkanoate copolymer. The polyhydroxyalkanoate copolymer has a melting point at least 10°C lower than the lowest melting point of the one or more fertilizer compounds and is biodegradable. Also disclosed is a method for producing the sustained-release fertilizer formulation.

Description

[Technical Field]

[0001] The present disclosure relates generally to controlled release fertilizers (or controlled release fertilizers), and more specifically to biopolymer polyhydroxyalkanoate (PHA) controlled release fertilizers. [Background technology]

[0002] The mention or citation of any prior-published document in this specification does not imply an admission that the document is part of the state of the art or is publicly known.

[0003] Demand for food is constantly increasing with the growth of the world's population. Therefore, sustainable agriculture plays an important role in maintaining the sustainability of humankind. Currently, large amounts of fertilizers are used in the agricultural industry. As the application of fertilizers increases, fertilizer research has attracted increasing attention. However, the nutrients in uncoated fertilizers cannot be fully absorbed by plants due to leaching, runoff, and volatilization. Fertilizer loss increases agricultural production costs and also causes environmental pollution.

[0004] Therefore, innovative coated fertilizers with controlled release have been developed to provide appropriate nutrients for different plant growth stages. Controlled-release fertilizers (CRFs) gradually release nutrients into the soil over a controlled release period according to the specific plant needs (Figure 1; 101: Water penetrates through the coating; 102: N dissolves in the particles; 103: N migrates out through the polymer). However, commercially available CRFs with polymer coatings are primarily made from thermoplastic resins such as polyolefins, polyurethanes, polyvinylidene chloride, and copolymers. These polymer-coated CRFs are difficult to degrade in soil and can accumulate as microplastics over time, posing serious environmental problems. Furthermore, these commercial CRFs are toxic to aquatic organisms, potentially causing long-lasting effects.

[0005] Therefore, there is a need for new slow-release fertilizers that can overcome the above problems. Summary of the Invention

[0006] Aspects and embodiments of the present invention are summarized with reference to the following numbered paragraphs. 1. a core portion containing one or more fertilizer compounds; and a shell portion comprising a polyhydroxyalkanoate copolymer; the polyhydroxyalkanoate copolymer has a melting point at least 10° C. lower than the lowest melting point of the one or more fertilizer compounds; A sustained release fertilizer formulation, wherein the polyhydroxyalkanoate copolymer is biodegradable. 2. The controlled-release fertilizer formulation according to Item 1, wherein the one or more fertilizer compounds are one or more selected from the group consisting of urea, metal phosphates, metal nitrates, ammonium halides, superphosphates, ammonium phosphates, ammonium sulfates, ammonia, metal sulfates, potassium chloride, ammonium phosphate, ammonium nitrate, calcium ammonium nitrate, potassium sulfate, phosphoric acid, and potassium magnesium sulfate. 3. The controlled-release fertilizer formulation according to item 1, wherein the one or more fertilizer compounds include urea. 4. The controlled-release fertilizer formulation according to item 1, wherein the shell portion comprises a poly-3-hydroxybutyrate (3HB) copolymer. 5. The controlled-release fertilizer formulation according to item 1, wherein the shell portion comprises poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). 6. The controlled-release fertilizer formulation according to Item 5, wherein the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) contains 18 to 30 mol % of 3-hydroxyhexanoate. 7. The poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) contains 23 to 25 mol% of 3-hydroxyhexanoate; Item 7. The controlled release fertilizer formulation according to item 6, which may contain about 24 mol% of 3-hydroxyhexanoate. 8. The melting point of the polyhydroxyalkanoate copolymer is 120°C or less; It may be 110°C or less or 100°C or less, Item 1. The slow-release fertilizer composition according to Item 1, which may further have a temperature of 90°C or lower. 9. The controlled-release fertilizer composition according to Item 1, wherein the core portion has a minimum diameter of 3.5 to 4.2 mm. 10. The controlled-release fertilizer composition according to Item 1, wherein the shell portion has a thickness of 80 to 180 μm. 11. The controlled-release fertilizer composition according to Item 1, wherein the polyhydroxyalkanoate copolymer is a biodegradable material, and biodegradation of the polyhydroxyalkanoate copolymer is detectable by one or more of scanning electron microscopy, formation of clear zones in the polyhydroxyalkanoate agar, weight loss of the polyhydroxyalkanoate, and molecular weight loss by gel permeation chromatography. 12. The controlled-release fertilizer composition according to item 1, wherein the polyhydroxyalkanoate copolymer is synthesized by Capricornis necator and isolated from mealworm excrement. 13. The controlled release fertilizer formulation of paragraph 12, wherein the polyhydroxyalkanoate copolymer is a substrate for soil microorganisms. 14. The controlled-release fertilizer formulation according to Item 1, wherein the core portion is formed by dip-coating in a solution containing the polyhydroxyalkanoate copolymer. 15. (i) providing solid granules comprising one or more fertilizer compounds; and (ii) A method for producing the controlled-release fertilizer formulation according to Item 1, comprising dip-coating the solid granules in a solution containing a polyhydroxyalkanoate copolymer. 16. The method according to item 15, wherein the concentration of the polyhydroxyalkanoate copolymer in the solution is 10% to 30% (w / v) (or, may be 13% to 20% (w / v), for example, about 15% (w / v). 17. The method of claim 15, wherein the polyhydroxyalkanoate copolymer is soluble in a halogen-free, environmentally safe solvent, and the solvent may be acetone. [Brief explanation of the drawings]

[0007] [Figure 1] Figure 1 shows the effect of polymer-coated urea. [Figure 2] Figure 2 shows the preparation of the preculture for the fermentation experiments. [Figure 3] FIG. 3 shows the fermentation process repeated using the terminal logarithmic phase culture from the previous experiment as a seed culture. [Figure 4] FIG. 4 shows the preparation of PHA-based CRF. [Figure 5] Figure 5 shows the physical appearance of uncoated urea (left) and urea coated with P(3HB-co-24mol% 3HHx) (right). [Figure 6] Figure 6 shows scanning electron microscope (SEM) images of urea coated with multiple layers of P(3HB-co-24mol% 3HHx). As the number of coating layers increased, the coating thickness and release duration in water increased. Magnification: ×30. Scale bar: 1 mm. [Figure 7] FIG. 7 shows PHA coated urea at different concentrations. [Figure 8] FIG. 8 shows the urea release profile of PHA-CRF in soil. [Figure 9] Figure 9 shows the biodegradation of PHA coating under soil conditions, removing microplastic residues in soil. The left panel was acquired using a dissecting microscope (Olympus SZX16, Japan), and the right panel was acquired using SEM. DETAILED DESCRIPTION OF THE INVENTION

[0008] In this embodiment, the word "comprising" may be interpreted as requiring the stated features, but not limiting the presence of other features. Alternatively, the word "comprising" may relate to cases where only the stated components / features are intended to be present (e.g., the word "comprising" may be replaced with the words "consists of" or "consists essentially of"). It is expressly contemplated that both broader and narrower interpretations are applicable to all aspects and embodiments of the present invention. That is, the word "comprising" and its equivalents may be replaced with the words "consists of" or "consists essentially of," or their equivalents, and vice versa.

[0009] It has surprisingly been discovered that a core-shell structure can be used to form a controlled release fertilizer formulation, with the core comprising a fertilizer and the shell comprising a biodegradable polymer. Accordingly, in a first aspect of the present invention there is provided a controlled release fertilizer formulation comprising: a core portion containing one or more fertilizer compounds; and a shell portion comprising a polyhydroxyalkanoate copolymer; where The polyhydroxyalkanoate copolymer has a melting point at least 10° C. lower than the lowest melting point of the one or more fertilizer compounds; and The polyhydroxyalkanoate copolymer is biodegradable.

[0010] In this embodiment, the word "comprising" may be interpreted as requiring the stated features, but not limiting the presence of other features. Alternatively, the word "comprising" may relate to cases where only the stated components / features are intended to be present (e.g., the word "comprising" may be replaced with the words "consists of" or "consists essentially of"). It is expressly contemplated that both broader and narrower interpretations are applicable to all aspects and embodiments of the present invention. That is, the word "comprising" and its equivalents may be replaced with the words "consists of" or "consists essentially of," or their equivalents, and vice versa.

[0011] The phrase "consists essentially of" and its cognates may be interpreted herein to refer to a material that includes trace amounts of impurities. For example, the material may be 90% or more pure, such as 95% or more pure, such as 97% or more pure, such as 99% or more pure, such as 99.9% or more pure, such as 99.99% or more pure, such as 99.999% or more pure, such as 100% pure.

[0012] Any suitable fertilizer compound (or combination of fertilizer compounds) may be used herein. Examples of suitable fertilizer compounds include, but are not limited to, urea, metal phosphates, metal nitrates, ammonium halides, superphosphates, ammonium phosphates, ammonium sulfates, ammonia, metal sulfates, potassium chloride, ammonium phosphates, ammonium nitrates, calcium ammonium nitrates, potassium sulfates, phosphoric acid, potassium magnesium sulfates, and combinations thereof. In certain embodiments that may be mentioned herein, the fertilizer compound may be urea. Additionally or alternatively, there may be one or more fertilizer compounds, and the one or more fertilizer compounds may include urea.

[0013] The polyhydroxyalkanoate (PHA) copolymer may be any suitable PHA copolymer. For example, the PHA may be a non-halogenated solvent-soluble PHA (e.g., a PHA copolymer soluble in a non-halogenated solvent). For example, the PHA copolymer may be a poly-3-hydroxybutyrate (3HB) copolymer. For example, the poly-3-hydroxybutyrate (3HB) copolymer may be poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). In certain embodiments that may be mentioned herein, the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) may contain 18 to 30 mol% 3-hydroxyhexanoate (e.g., 23 to 25 mol% of 3-hydroxyhexanoate, e.g., about 24 mol% 3-hydroxyhexanoate). Thus, the shell portion may include a poly-3-hydroxybutyrate (3HB) copolymer. For example, the shell portion may comprise a poly-3-hydroxybutyrate (3HB) copolymer. In certain embodiments that may be mentioned herein, the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) may comprise 18-30 mol% 3-hydroxyhexanoate (e.g., 23-25 ​​mol% 3-hydroxyhexanoate, e.g., about 24 mol% 3-hydroxyhexanoate).

[0014] The PHA copolymer used herein may have any suitable melting point. For example, the polyhydroxyalkanoate copolymer may have a melting point of 120°C or less, such as 110°C or less, such as 100°C or less, such as 90°C or less. The minimum melting point may be 80°C or more, such as 88°C or more. Alternatively, the melting point of the PHA copolymer may be 80°C to 120°C, such as 85°C to 110°C, such as 88°C to 110°C, for example, about 100°C.

[0015] The compositions described herein are provided in particulate form and have a core portion and a shell portion. The core portion may have any suitable diameter, as long as the resulting composition functions as a controlled-release composition and is easily disposable on the ground. For example, the core portion may have a minimum diameter of 3.5 to 4.2 mm. The core portion may have a maximum diameter of 4.5 to 5 mm. Thus, the diameter may be 3.5 to 5 mm, e.g., 4.2 to 4.6 mm.

[0016] The shell portion may have any suitable thickness. Ideally, the shell portion has a thickness that can withstand transportation, storage, and application to / on soil at the final use site. Examples of suitable thicknesses for the shell portion include, but are not limited to, thicknesses of 80 to 180 μm.

[0017] As mentioned above, PHA copolymers are biodegradable materials. This means that the polymeric material will break down in the environment in which it is found. This may be caused by microorganisms or animals in soil / water, or by environmental influences over time. Biodegradation of polyhydroxyalkanoate copolymers can be detected by one or more of the following: scanning electron microscopy, the formation of clear zones in polyhydroxyalkanoate agar, weight loss of the polyhydroxyalkanoate, and molecular weight reduction by gel permeation chromatography.

[0018] PHA copolymers may be obtained from suitable synthetic or biological sources. For example, polyhydroxyalkanoate copolymers may be synthesized by Cupriavidus necator or isolated from mealworm excrement.

[0019] As noted above, the polyhydroxyalkanoate copolymer may provide a suitable substrate for microorganisms.

[0020] The controlled release fertilizer formulations described herein may be formed by dip coating a core portion in a solution comprising a polyhydroxyalkanoate copolymer. Thus, as a further aspect of the present invention, there is provided a method of preparing a controlled release fertilizer formulation comprising: (i) providing solid granules containing one or more fertilizer compounds; and (ii) Dip-coating solid granules in a solution containing a polyhydroxyalkanoate copolymer.

[0021] In the above method, the PHA copolymer may be contained in the solution at any suitable concentration, for example, the concentration of the polyhydroxyalkanoate copolymer in the solution may be 10% to 30% (w / v), for example, 13% to 20% (w / v), for example, about 15% (w / v).

[0022] In the above method, the polyhydroxyalkanoate copolymer may be soluble in a halogen-free, environmentally safe solvent, and the solvent may be acetone.

[0023] Further aspects and embodiments of the invention are described with reference to the following non-limiting embodiments.

[0024] [Example]

[0025] [Table 1]

[0026] Unless otherwise stated, materials were either commercially available from Sigma-Aldrich, Fisher, etc., or obtained from commercial kitchens (e.g., waste cooking oil).

[0027] Analysis method SEM The surface morphology and thickness of the CRF coating were examined using a scanning electron microscope (TM-4000, Hitachi, Japan).

[0028] Example 1. Cost-effective production of a suitable PHA for use as a biodegradable coating for urea granules PHAs are approved as plastic food contact materials (FCMs) in the European Union (EU). They are biopolyesters produced by microorganisms (bacteria and archaea) as storage compounds (carbon reservoirs) under nutrient-limited or stress conditions. While they have similar physical properties to petroleum-derived plastics, their main advantages over synthetic plastics are their biodegradability, biocompatibility, and sustainability. Bacterial PHAs can be classified into three major types based on the number of carbon atoms in the monomer unit: short-chain (scl), medium-chain (mcl), and scl-mcl combinations. scl-PHAs contain 3–5 carbon atoms, mcl-PHAs contain 6–14 carbon atoms, and scl-mcl-PHAs contain 3–14 carbon atoms per monomer. PHAs composed primarily of scl monomers can be rigid and brittle, while PHAs composed primarily of mcl monomers exhibit elastic properties. scl-mcl PHA copolymers exhibit intermediate properties between the two, depending on the ratio of scl and mcl monomers in the copolymer. scl-mcl PHA copolymers, such as poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) random copolymer P(3HB-co-3HHx), are suitable as coating materials for fertilizers used in CRF production. Because PHAs are biodegradable by soil microorganisms, they are gradually decomposed by microorganisms, releasing the fertilizer after a certain period of time. The fertilizer release time can be adjusted by changing the type of PHA coated or the thickness of the PHA coated on the fertilizer.

[0029] 3-Hydroxyhexanoate (3HHx) belongs to the mcl monomer family and consists of 6 to 14 carbon units. The longer the monomer length, the more soluble it is in a wide range of solvents, allowing for the use of inexpensive, less toxic solvents (e.g., acetone and dimethyl carbonate) for industrial applications. Therefore, P(3HB-co-3HHx) with a high 3HHx monomer content of over 18 mol% is soluble in acetone, a halogen-free solvent.

[0030] Example 2. Preparation of PHA This paper describes a cost-effective method for producing PHA as a biodegradable coating material for controlling the release of plant nutrients. The carbon source of PHA is different types of oil, and the nitrogen source is urea. Oil and urea are fed slowly and continuously depending on the growth of the bacterial strain. To shorten the fermentation process, the culture at the end of the logarithmic growth phase was harvested, and 0.1–30% of the culture was used as the seed culture for the next fermentation run (Run 2). Because the seed culture maintained its active phase and had a much higher cell number than the first run, growth was faster, shortening the duration of the fermentation run. Due to the faster cell growth, the urea and oil feed rates were also faster than in Run 1. When the culture reached the end of the logarithmic growth phase, the culture was harvested, and 0.1–30% of the culture was used as the seed culture for the next fermentation run (Run 3). This cycle was repeated until the desired culture population was achieved.

[0031] High cell density fermentation The Caprifoliidus necator transformant was first precultured in tryptic soy broth (TSB) at 200 rpm and 30°C. 600nmWhen the pH reached approximately 3.5–4.5, 3–4% (v / v) of the preculture was transferred to 2.9 L of mineral medium (MM) in a 13 L bioreactor. The mineral medium consisted of 4.0 g / L NaH2PO4, 4.6 g / L Na2HPO4, 0.45 g / L K2SO4, 0.39 g / L MgSO4, 0.062 g / L CaCl2, and 1 mL / L trace elements. The trace element solution consisted of 15 g / L FeSO4·7H2O, 2.4 g / L MnSO4·H2O, 2.4 g / L ZnSO4·7H2O, and 0.48 g / L CuSO4·5H2O dissolved in 0.1 M hydrochloric acid. The bioreactor temperature was set at 30 °C. Urea was used as the nitrogen source, and a mixture of 40% crude palm kernel oil (CPKO) and 60% palm olein (PO) was used as the carbon source. The urea and oil addition strategy is shown in Example 3.

[0032] To measure the optical density, 1 mL of the preculture was transferred to a microcentrifuge tube and centrifuged at 12,000 rpm for 2 minutes, and the supernatant was discarded. The remaining pellet was then suspended in 1 mL of distilled water, and centrifuged again at 12,000 rpm for 2 minutes, and the supernatant was again discarded. The pellet was then resuspended in 1 mL of distilled water. 1 mL of the suspension was transferred to a clean test tube, and 9 mL of distilled water was added to the test tube for a 10-fold dilution (Note: Dilution is not necessary if the cell count is low). 1 mL of the suspension was transferred to a cuvette. A 1 mL blank was prepared using distilled water. Measure the OD at 600 nm using a spectrophotometer. (*Note: Because a 10-fold dilution was performed, the OD reading should be multiplied by 10.)

[0033] Cultivation in 13 L bioreactor (6 L working volume) - Experiment 1 Figure 2 shows the cultivation of Capriavidus necator transformants in a 13 L bioreactor. As shown in Figure 2, there is a method 200 for preparing a preculture medium for fermentation experiments. Method 200 includes the following steps: 201: Glycerol stock stored at -80°C (Capriavidus necator transformant); 202: Activated on TSA agar plates (gentamicin (Gm) + kanamycin (Km)). Incubated at 30°C for 2-3 days. 203: Subculture of a single colony onto a TSA agar plate (Gm+Km). Fourth quadrant streaking; 204: TSA agar plate (Gm+Km). Incubated at 30°C for 24 hours. 205: Two TSA agar plates (Gm+Km). Incubate at 30°C for 24 hours. 206: 4 loops of vaccine were administered. 207: 2 x 50 mL TSB (Gm + Km). Cap a 250 mL Erlenmeyer flask with a cotton stopper. Incubate for 4-5 hours. 208: Transfer 100 mL of TSB culture to a 13 L bioreactor. 209: Preculture in a 13 L bioreactor. ·2.9L MM (3L concentration). 1.5% CPKO (0 hours = 0.75%, 22.5g, 6 hours = 0.75%, 22.5g). ·30mM urea (0 hours = 15mM, 6 hours = 15mM). -Set the temperature to 30°C. · Set pH to 6.8. · Set the stirring speed to 300 rpm. -Aeration rate set to 0.5wm. Dissolved oxygen (DO) setting: >40% (cascade mode). Incubate for 12 hours.

[0034] Activation of microbial strains Recombinant C. necator strain Re2058 / pCB113 was thawed from a glycerol stock stored at -80°C and streaked in four aliquots onto tryptic soy agar (TSA) plates supplemented with 50 μg / mL Km. The TSA plates were incubated at 30°C for 48 hours to obtain single colonies. A single colony was picked from the activation plate and re-plated onto a new TSA + Km plate as a full plate streak. The plate was incubated at 30°C for 24 hours.

[0035] Preparation of preculture medium for biosynthesis TSB (50 mL) was supplemented with 50 μg / mL Km and placed in a 250 mL Erlenmeyer flask. Approximately four loopfuls of bacterial culture medium from each smeared plate were aseptically transferred to the TSB+Km flask. The flask was cultured in an incubator shaker at 30°C and 200 rpm for 4-5 hours, and the optical density (OD) of the culture medium was measured. 600nm The culture was incubated until the OD reached 4-5. 600nm The culture was considered to have reached active mid-logarithmic phase when the value of β was between 4 and 5. At this point, the culture was ready to be transferred to PHA biosynthesis medium.

[0036] Seeds for PHA biosynthesis in shake flasks For PHA biosynthesis, we used the minimal medium (MM) reported by C.F. Budde et al., J. Bacteriol. 2010, 192, 5319–5328. Solution A (4.0 g / L NaH2PO4, 4.6 g / L Na2HPO4, and 0.45 g / L K2SO4) was dissolved in 800 mL of distilled water. The pH of the medium was then adjusted to 6.8 with 1 M HCl or 1 M NaOH, and the volume of the medium was adjusted to 1 L. Solution A, urea [CO(NH2)2] stock solution, MgSO4 7H2O stock solution, CaCl2 stock solution, and carbon source were each sterilized by autoclaving, and the trace element solution was filter-sterilized using a 0.2 μm cellulose acetate membrane filter. A trace element solution was prepared by dissolving 15 g of FeSO4·7H2O, 2.4 g of MnSO4·H2O, 2.4 g of ZnSO4·7H2O, and 0.48 g of CuSO4·5H2O in 1 L of 0.1 M HCl. This solution was stored in a Schott bottle covered with aluminum foil in a refrigerator at 4 °C. A 5 L Erlenmeyer flask was prepared by adding 800 mL of MM, 2.25 g / L urea, MgSO4·7H2O, 0.062 g / L CaCl2, 1 mL / L of trace elements, and the specified amount of carbon source (10 g / L CPKO). 50 μg / mL Km and 10 μg / mL Gm were added. As a final step, 3% by volume of the bacterial culture from the TSB+Km flask was added. The flasks were cultured in an incubator shaker at 30°C and 200 rpm for 10-15 hours, and the OD600nm The culture was continued until the pH reached 4.5 to 5.5.

[0037] PHA biosynthesis in a 13L bioreactor To obtain a final total culture volume of 7 L, MM was prepared as described above, but with 2.0 g / L urea. 10% by volume of the bacterial inoculum in MM was inoculated into the bioreactor from an Erlenmeyer flask. The bioreactor was set to pH 6.8, 30°C, 200 rpm agitation, and 0.5 vvm aeration (cascade mode). Samples were taken every 6 hours to monitor bacterial growth. A feeding strategy was established, adding additional 2.75 g / L urea and 50 g / L CPKO depending on the bacterial growth status. Biosynthesis was stopped when the oil-to-cell conversion rate reached nearly 100% (approximately 48 hours).

[0038] Cell harvesting and freeze-drying The culture medium was collected by centrifugation at 8000 rpm at 4°C for 10 minutes using a refrigerated centrifuge. The initial supernatant was discarded, and the cell pellet was resuspended in distilled water to remove any remaining fermentation medium between the cells. The suspension was centrifuged under the same conditions as in the first step, and the conductivity of the supernatant was measured by adding 120 μL to a conductivity meter (EC-33, HORIBA Scientific, Japan). This washing process was repeated until the conductivity was less than 3000 μS / cm. The washed cells were stored overnight at -80°C. The frozen cells were lyophilized for 72 hours in a freeze-dryer. The resulting dried cells were weighed, and the cell dry weight (CDW) was determined and used for subsequent analysis in the following examples.

[0039] For PHA extraction, we employed the biological recovery method using mealworms described by I. Zainab-L&K. Sudesh, J. Biotechnol. 2019, 305, 35-42. See also Murugan, P., L. Han, C.-Y. Gan, FHJ Maurer, K. Sudesh, (2016), A Novel Biological Recovery Method for Phosphate Using Mealworms, Tenebrio molitor, J. Biotechnol. 239:98-105; and Ong, S.Y., H.-P. Kho, S.L. Liedel, S.-W. Kim, C.-Y. Gan, T.D. Taylor, K. Sudesh, (2018), An Integrated Study of Biologically Recovered Polyhydroxyalkanoates (PHAs) and Simultaneous Assessment of the Gut Microbiota of Yellow Mealworms, J. Biotechnol. 265:31-39.

[0040] Biological Recovery The biological agents used in this experiment were approximately 1.5-month-old Tenebrio molitor mealworms, obtained from a local farm and reared on a conventional diet (wheat bran) prior to the experiment.

[0041] Preparing mealworms for biological recovery Mealworms aged 1.5 months (average body length 1-1.5 cm) were fasted for 48 hours to remove residual gastrointestinal debris from the previous feeding. Prior to the start of the fast, mealworms were fed oats or wheat bran ad libitum and periodically supplemented with carrots as a hydration source to maintain overall growth and health. Mealworms were reared in a low-light environment at room temperature of approximately 28-30°C, relative humidity of approximately 50-60%.

[0042] Feeding freeze-dried bacterial cells containing PHA Starved mealworms were weighed and sorted into containers. Mealworm feces and impurities were sieved to minimize intervention during the biological recovery process. The cell feeding ratio was 10 wt% of the mealworm's total body weight. For example, 10 g of mealworms were fed 1 g of bacterial cells. Large clumps of dried cells were broken into small particles by hand or gently tapping with a mortar and pestle to facilitate mealworm ingestion. The cells were evenly distributed throughout the container to ensure equal access for all mealworms. The mealworms were left for 24–48 hours to allow for complete ingestion of the freeze-dried bacterial cells. The excrement containing the PHA granules was then collected.

[0043] Collection of excrement containing PHA granules After 24-48 hours of feeding, the containers were inspected to ensure no bacterial cells remained. In containers with residual cells, the mealworms were left for an extended period before frass collection. To collect the PHA frass, the mealworms were transferred to a standard sieve with 0.5 mm pore size and gently shaken to separate the frass into a holding container at the bottom. The weight of the mealworms after feeding with bacterial cells was recorded. Similarly, the weight of the feces was recorded and stored in Ziploc® bags for subsequent purification.

[0044] Purification of PHA fecal pellets Approximately 200 g of fecal pellets were transferred to a 5 L plastic container and washed with tap water until the water was translucent. The water was removed, and the washed pellets were crushed into granules. The granules were washed until no black particles remained. The washed granules were purified by stirring with 0.25 M NaOH for 1 hour. The solution was separated, and the granules were washed until no black particles remained. This purification and washing with 0.25 M NaOH was repeated. The purified PHA granules were bleached with 10 vol% Clorox. The solution was separated, and the granules were washed at least six times or until the odor of Clorox disappeared. The water was drained through a 212 μm fine mesh sieve. The granules were spread on a wide tray and dried at 60 °C for 24 hours to a moisture content of ≤1%. The dried granules were ground into a powder, packed into pre-weighed Ziploc bags, and appropriately labeled.

[0045] Cultivation in a 13 L bioreactor (6 L working volume) - Experiments 1 to 3 As shown in Figure 3, the fermentation process 300 was repeated using the terminal logarithmic phase culture from the previous experiment as a seed culture. The fermentation process 300 included the following steps: 301: Experiment 1; After a 12-hour incubation period, MM (at a concentration of 2L:3L) and minerals were added. See Example 3 for feeding strategy. The cells were harvested approximately 33 hours after the start of culture. After collection, 300 mL of culture medium was left in the container. 302: Experiment 2; and Add MM (4.8L:6L concentration) and minerals to the container, and use 300mL of the culture from the previous experiment as the seed culture for this experiment. See Example 3 for feeding strategies. The cells were harvested approximately 30 hours after cultivation. 303: Experiment 3. ·MM (4.8L:6L concentration) and minerals were added to the container, and 300mL of the culture medium from the previous experiment was used as the inoculum for this experiment. See Example 3 for feeding strategies. The cells were harvested approximately 30 hours after cultivation.

[0046] Results and Discussion The PHA used herein refers to mealworm-recovered polymer granules recovered by the method described in the Biological Methods for Extraction and Purification of Polyester Granules from Bacterial Cells (Malaysia Patent Application No. PI20092081 (2009) and WO2010134798 (see the Examples therein, incorporated herein by reference). This is a more environmentally friendly polymer extraction method compared to solvent extraction methods. Furthermore, partially purified PHA significantly reduces production costs. This partially purified PHA is the only component required to produce the water-insoluble coating that covers the urea granules. The partially purified PHA exists in the form of a coarse powder.

[0047] Example 3. Fermentation Feeding Strategy (for 6 L Working Volume) Nitrogen source (urea) Experiment 1 Total amount added at 0 hours (pre-culture) = 15 mM urea 0 to 10 hours = 5 mM / h = 50 mM urea, total amount added at 15 hours = 65 mM urea 10 hours to 28 hours = 10 mM / h = 180 mM urea, total amount added at 30 hours = 245 mM urea Experiments 2 and 3 onwards 0 hours to 24 hours = 10 mM / h = 240 mM urea, total amount added in 24 hours = 240 mM urea

[0048] Carbon source (oil = mixture of 60% PO and 40% CPKO) Experiment 1 Total feeding amount at 0 hours (pre-culture) = 45g of oil 0 hours to 10 hours = 15g / h oil = 150g oil, total amount = 195g oil 10 hours to 30 hours = 20g / h oil = 400g oil, total = 595g oil Experiments 2 and 3 onwards 0-24 hours = 25g / h of oil, total dose at 25 hours = 600g of oil

[0049] Example 4. Determination of PHA content and its monomer composition The PHA content and its monomer composition were determined by gas chromatography (GC) analysis according to the report of G. Braunegg, B. Sonnleitner, and R.M. Lafferty (European J. Appl. Microbiol. Biotechnol. 1978, 6, 29-37). The PHA content was measured using a Shimadzu GC-2010 system equipped with an SPB-1 column (Supelco, USA). The column temperature started at 70 °C and was increased to 280 °C in successive steps of 10 °C / min. PHA content and composition were quantified using caprylic acid methyl ester (CME) as an internal standard.

[0050] Preparation of methanolysis solution Before running the GC, the methanolysis solution and CME solution were prepared. The methanolysis solution consisted of a mixture of concentrated sulfuric acid and methanol in a 15:85 (v / v) ratio. This solution was prepared in a fume hood. In an ice bath, 425 mL of methanol was added to a Schotten bottle. Next, 75 mL of concentrated sulfuric acid was added dropwise to the Schotten bottle and slowly stirred with a magnetic stirrer. The methanolysis solution was stored at 4 °C for later use.

[0051] Preparation of CME solution CME was used as an internal standard for PHA quantification in GC analysis. The CME solution was prepared in a fume hood. In a 100 mL volumetric flask, 0.2 mL of CME stock solution was diluted to 100 mL with chloroform. The ratio of CME stock solution to chloroform was 1:500. This working solution was mixed thoroughly, transferred to a Schott bottle wrapped in aluminum foil, and stored at 4 °C.

[0052] Methanolysis of samples Approximately 15.0–20.0 mg of lyophilized cells (or 3.0–9.0 mg of polymer) were transferred to a test tube with a screw cap. Next, 2 mL of methanolysis solution and 2 mL of chloroform were added to the test tube. The tube was sealed with PTFE tape to prevent evaporation. The test tube was then incubated (or cultured) in a heating block at 100°C for 140 minutes. The tube was gently tapped every 20 minutes during heating. The sample was then cooled to room temperature (25°C).

[0053] GC sample preparation One mL of distilled water was added to the cooled sample and vigorously stirred for one minute. After several minutes, two distinct layers formed: an upper aqueous phase and a lower organic phase. The lower organic phase (containing the hydroxyacyl methyl esters) was transferred to a clean flat-bottom tube containing anhydrous sodium sulfate (Na2SO4) to absorb any traces of water in the sample. Next, 0.5 mL of the lower organic layer was transferred to a GC vial containing 0.5 mL of CME. The GC sample was then ready for GC analysis.

[0054] GC analysis GC analysis was performed using a Shimadzu GC-2010 Plus. The GC was equipped with an AOC-20i autoinjector, a Supelco SPB®-1 capillary GC column, and a flame ionization detector. 2.0 μL of sample was injected. After injection, the syringe was automatically washed with chloroform. The sample injection process and analysis were programmed using GC Solution Version 2.30.00 SU3. The GC conditions were set as follows: 1.AOC-20i autoinjector; Carrier gas: Nitrogen gas ·Temperature: 270℃ Pressure: 84.2kPa Total flow rate: 14mL / min 2. Supelco SPB®-1 capillary GC column; and ·Initial temperature: 70℃ ·Final temperature: 280℃ 3. Flame ionization detector, ·Temperature: 280℃ Hydrogen gas flow rate: 40 mL / min Air flow rate: 400mL / min

[0055] Calculation of PHA content and its monomer composition The PHA content and its monomer composition were calculated based on the peak area at a specific retention time in the GC chromatogram. The retention time of each monomer was determined using the identified PHA polymer. The PHA content and its monomer composition were calculated based on the following formula:

[0056]

number

[0057] where 3HB retention time = 3.2-3.3 min 3HHx retention time = 5.9-6.0 min CME retention time = 7.1-7.2 min K=GC constant=11.2 k 3HB =3HB Monomer Constant = 1 k 3HHx =3HHxMonomer constant=0.4 A 3HB = area under the 3HB monomer peak A 3HHx = 3HH x area under the monomer peak A CME = area under the CME peak W = weight of freeze-dried cells subjected to methanolysis (mg)

[0058] Example 5. Preparation of PHA-based controlled release fertilizer (CRF) The PHA coarse flour obtained in Example 2 is readily soluble in non-halogenated solvents at room temperature, and the resulting solution can be used to coat plant nutrients such as water-soluble urea granules. The PHA coating forms a water-insoluble layer whose thickness can be easily controlled by adjusting the concentration of the PHA solution. The coating process is simple and rapid, and no additional components are required to stabilize the coating.

[0059] PHA-based CRF was prepared by the dip-coating method (Figure). Briefly, a 15% (w / v) solution of P(3HB-co-24 mol%3HHx) prepared in Example 2 was prepared in acetone as the solvent. The polymer solution was stirred at room temperature (25 °C) for 24 hours to completely dissolve the PHA.

[0060] Then, the urea granules were dip-coated in the PHA solution and dried at room temperature. Coated PHA-based CRFs (CRF20, CRF25, and CRF30) were prepared according to the above protocol using PHA concentration solutions of 20%, 25%, and 30%, respectively.

[0061] Example 6. Characterization of the produced PHA The properties of P(3HB-co-24mol% 3HHx) produced in Example 2 were evaluated.

[0062] Chemical extraction and purification of PHAs Approximately 1 g of lyophilized cells was mixed with 50 mL of chloroform and stirred at room temperature for 3 days. The mixture was filtered through Whatman No. 1 filter paper to remove cell debris. The resulting clear solution was added dropwise to vigorously stirred ice-cold methanol to precipitate the PHA polymer. The precipitated polymer was separated from the methanol solution by vacuum filtration and allowed to dry overnight at room temperature.

[0063] Molecular weight measurement of PHA The number-average molecular weight (Mn) and weight-average molecular weight (Mw) of the obtained polymer were measured using gel permeation chromatography (GPC) using an Agilent Technologies 1200 Series GPC (USA) equipped with a TSK guard column HHR-H and a TSKgel GMH HR-H (Tosoh, Japan). Chloroform was used as the mobile phase solvent, and was run at a flow rate of 0.8 mL / min at 40° C. The PHA polymer was dissolved in chloroform, adjusted to a final concentration of approximately 1.0 mg / mL, and then filtered through a PTFE membrane (0.22 μm).

[0064] Measurement of thermophysical properties Differential scanning calorimetry (DSC) analysis was performed to measure the glass transition temperature (Tg), melting point (Tm), and enthalpy of fusion (ΔHm) of the PHA polymer. DSC analysis was performed using a DSC-60 (Shimadzu, Japan) equipped with a liquid nitrogen cooling system under a nitrogen atmosphere. The temperature range of the DSC was -40 to 200 °C, and the heating rate was 10 °C / min.

[0065] Results and Discussion Figure 5 shows the physical appearance of uncoated urea and urea coated with P(3HB-co-24mol% 3HHx). Figure 6 shows the SEM microstructure of urea coated with multiple layers of P(3HB-co-24mol% 3HHx). Urea coated with PHA at different concentrations is shown in Figure 7.

[0066] [Table 2]

[0067] [Table 3] Reported values ​​are the mean ± standard deviation (SD) of triplicate cultures. Abbreviations: 3HB, 3-hydroxybutyrate; 3HHx, 3-hydroxyhexanoate; Mn, number average molecular weight; Mw, weight average molecular weight; PDI, polydispersity index; Tg, glass transition temperature; Tm, melting point; ΔHm, enthalpy of fusion.

[0068] The PHA used herein is P(3HB-co-24mol% 3HHx). Saettone et al. (US2021387925A1) used a P(3HB) homopolymer with a melting point of approximately 180°C, which is much higher than that of urea. The melting point of P(3HB-co-24mol% 3HHx) is 88°C, lower than that of urea (135°C). The low melting point of P(3HB-co-24mol% 3HHx) facilitates most industrial processing steps, such as melt coating, melt pressing, and extrusion, which require the polymer to melt without melting the urea granules. Furthermore, P(3HB) is a homopolymer of the 3-hydroxybutyrate monomer, a four-carbon compound. On the other hand, 3-hydroxyhexanoic acid (3HHx) is a larger monomer with six carbon atoms. This larger monomer makes the PHA copolymer more amorphous (less crystalline) and therefore soluble in a wider range of solvents, allowing the use of cheaper, less toxic solvents (such as acetone and dimethyl carbonate) for industrial applications.

[0069] Example 7. Evaluation of urea release in the aquatic environment CRF20, CRF25, and CRF30 prepared in Example 5 were used in the urea release experiments. Commercial urea CRF (SmartGro) was used as a control.

[0070] Release of urea Fifteen pots, each containing 40 g of soil (20% moisture content), were prepared to simulate the release of urea into the soil over a 15-week period (one pot per week). Four PHA-based CRF granules were placed in each pot. The initial weight of the PHA-based CRF was recorded, and 3 mL of water was added daily. Each week, four PHA-based CRF granules were removed from one pot (the pot was discarded), dried at 50°C to a constant weight, and weighed. Commercially available urea CRF (SmartGro) was purchased from Smart Fert Sdn Bhd and used as a positive control.

[0071] Results and Discussion <80% of the urea was released throughout the 60-day soil life test period, meeting the basic prototype requirements for CRF (Figure 8). Additionally, CRF20, CRF25, and CRF30 released their urea completely in 42, 56, and 105 days, respectively, while SmartGro released 67% of the urea in 105 days. 100% weight loss is an indication of complete urea release.

[0072] Example 8. Degradation of CRF in a coated PHA system The microbial degradation of the coated PHA-based CRF prepared in Example 5 was investigated.

[0073] Physical observation of coated PHA-based CRF The physical changes of PHA-CRF at 20%, 25%, and 30% (w / v) were observed. Physical observation by SEM (following the procedure described above). The formation of holes in the coating layer indicates the degradation of PHA.

[0074] Results and Discussion The PHA coating layers of CRF20, CRF25, and CRF30 began to decompose in weeks 6, 8, and 9, respectively, and then burst into small fragments. SEM images confirmed the morphological changes associated with the decomposition of PHA-CRF by enzymes secreted by the microbial community in the soil (Figure 9). This indicates that the coated PHA-based CRFs are completely biodegraded in soil without any accumulation of residues.

[0075] We developed a CRF that can be completely decomposed into carbon dioxide and water by microbial action using the biodegradable polymer polyhydroxyalkanoate (PHA), specifically poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), as a coating material. Furthermore, to overcome the high cost of PHA, we described a fermentation process, a polymer extraction method, and finally, a cost-effective method for producing PHA-based CRF.

[0076] Comparative Example 1 A comparison of coated PHA-based CRF and uncoated fertilizers is shown in Table 3 below.

[0077] [Table 4]

Claims

1. a core portion comprising one or more fertilizer compounds; and a shell portion comprising a polyhydroxyalkanoate copolymer; the polyhydroxyalkanoate copolymer has a melting point at least 10° C. lower than the lowest melting point of the one or more fertilizer compounds; A controlled release fertilizer formulation, wherein the polyhydroxyalkanoate copolymer is biodegradable.

2. 2. The controlled release fertilizer formulation of claim 1, wherein the one or more fertilizer compounds are one or more selected from the group consisting of urea, metal phosphates, metal nitrates, ammonium halides, superphosphates, ammonium phosphates, ammonium sulfates, ammonia, metal sulfates, potassium chloride, ammonium phosphate, ammonium nitrate, calcium ammonium nitrate, potassium sulfate, phosphoric acid, and potassium magnesium sulfate.

3. 10. The controlled release fertilizer formulation of claim 1, wherein the one or more fertilizer compounds comprises urea.

4. 10. The controlled release fertilizer formulation of claim 1, wherein the shell portion comprises a poly-3-hydroxybutyrate (3HB) copolymer.

5. 2. The controlled release fertilizer formulation of claim 1, wherein the shell portion comprises poly(3-hydroxybutyrate-co-3-hydroxyhexanoate).

6. 6. The controlled release fertilizer formulation of claim 5, wherein the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) contains 18 to 30 mole % of 3-hydroxyhexanoate.

7. the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) contains 23 to 25 mol % of 3-hydroxyhexanoate; 7. The controlled release fertilizer formulation of claim 6, which may contain about 24 mole % 3-hydroxyhexanoate.

8. the melting point of the polyhydroxyalkanoate copolymer is 120°C or less; It may be 110°C or less or 100°C or less, The slow-release fertilizer formulation according to claim 1, which may further be at 90°C or lower.

9. 2. The controlled release fertilizer formulation according to claim 1, wherein the core portion has a minimum diameter of 3.5 to 4.2 mm.

10. 2. The controlled release fertilizer formulation of claim 1, wherein the shell portion has a thickness of 80 to 180 μm.

11. 2. The controlled release fertilizer formulation of claim 1, wherein the polyhydroxyalkanoate copolymer is a biodegradable material, and biodegradation of the polyhydroxyalkanoate copolymer is detectable by any one or more of scanning electron microscopy, formation of clear zones in polyhydroxyalkanoate agar, weight loss of the polyhydroxyalkanoate, and molecular weight reduction by gel permeation chromatography.

12. 10. The controlled release fertilizer formulation of claim 1, wherein said polyhydroxyalkanoate copolymer is synthesized by Capriavidus necator and isolated from mealworm excrement.

13. 13. The controlled release fertilizer formulation of claim 12, wherein said polyhydroxyalkanoate copolymer is a substrate for soil microorganisms.

14. 2. The controlled release fertilizer formulation according to claim 1, wherein said core portion is formed by dip-coating in a solution containing said polyhydroxyalkanoate copolymer.

15. (i) providing solid granules comprising one or more fertilizer compounds; and 10. A method for producing the controlled release fertilizer formulation of claim 1, comprising: (ii) dip-coating the solid granules in a solution comprising a polyhydroxyalkanoate copolymer.

16. 16. The method of claim 15, wherein the concentration of the polyhydroxyalkanoate copolymer in the solution is 10% to 30% (w / v), may be 13% to 20% (w / v), for example about 15% (w / v).

17. 16. The method of claim 15, wherein the polyhydroxyalkanoate copolymer is soluble in a halogen-free, environmentally safe solvent, which may be acetone.

Images