Development of biopolymer polyhydroxyalkanoate for controlled release fertilizer

Abstract

Disclosed herein is a sustained-release fertiliser formulation comprising a core portion comprising one or more fertiliser compounds, and a shell portion comprising a polyhydroxyalkanoate copolymer, wherein the polyhydroxyalkanoate copolymer has a melting point that is at least 10°C lower than the lowest melting point of the one or more fertiliser compounds, and the polyhydroxyalkanoate copolymer is biodegradable. Also disclosed herein is a method of preparing the sustained-release fertiliser formulation.

Description

[0001] DEVELOPMENT OF BIOPOLYMER POLYHYDROXYALKANOATE FOR CONTROLLED RELEASE FERTILIZER Field of Invention The present disclosure generally relates to controlled release fertilizer, and more particularly relates to biopolymer polyhydroxyalkanoate (PHA) controlled release fertilizer. Background The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. The demand for food is constantly increasing in parallel with the ever-growing world population. Due to this, sustainable agriculture plays an important role in maintaining human sustainability. Currently, large amounts of fertilizers are being used by the agricultural industries. Therefore, more focus and emphasis are being given to fertilizer research due to its increased application. However, nutrients from uncoated fertilizers are not completely available for uptake by plants as a result of leaching, runoff, and volatilization. Fertilizer loss increases the cost of agriculture production and also causes environmental pollution. As such, innovative coated fertilizers were introduced so that they are released in a controlled manner in order to supply sufficient nutrients for plants at different growth stages. Controlled release fertilizer (CRF) gradually releases nutrients into the soil with a controlled release period as per specific plant requirements (FIG. 1; 101: water moves in through the coating. 102: N dissolves into solution inside the granule.103: N moves out through the polymer). However, commercial CRFs using polymer coatings are mostly made of thermoplastic resins such as polyolefin, polyurethane-like, polyvinylidene chloride and copolymers, which has led to serious environmental problems as they cannot degrade easily in soil and can accumulate as microplastics overtime. Further, these commercial CRFs can be toxic to aquatic life with long lasting effects. Therefore, there exists a need for new controlled release fertilizers that can overcome the problems stated above. Summary of Invention Aspects and embodiments of the invention will now be summarised by reference to the following numbered clauses. 1. A sustained-release fertiliser formulation comprising: a core portion comprising one or more fertiliser compounds; and a shell portion comprising a polyhydroxyalkanoate copolymer, wherein: the polyhydroxyalkanoate copolymer has a melting point that is at least 10°C lower than the lowest melting point of the one or more fertiliser compounds; and the polyhydroxyalkanoate copolymer is biodegradable. 2. The sustained-release fertiliser formulation according to Clause 1, wherein the one or more fertiliser compounds are selected from one or more of the group consisting of urea, a metal phosphate, a metal nitrate, an ammonium halide, a superphosphate, ammonium phosphate, ammonium sulfate, ammonia, a metal sulfate, potassium chloride, ammonium phosphate, ammonium nitrate, calcium ammonium nitrate, potassium sulfate, phosphoric acid and potassium magnesium sulfate. 3. The sustained-release fertiliser formulation according to Clause 1 or 2, wherein the one or more fertiliser compounds comprise urea. 4. The sustained-release fertiliser formulation according to any one of the preceding clauses, wherein the shell portion comprises a poly-3-hydroxybutyrate (3HB) copolymer. 5. The sustained-release fertiliser formulation according to any one of the preceding clauses, wherein the shell portion comprises poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). 6. The sustained-release fertiliser formulation according to Clause 5, wherein the poly(3- hydroxybutyrate-co-3-hydroxyhexanoate) comprises from 18 to 30 mol. % of 3- hydroxyhexanoate. 7. The sustained-release fertiliser formulation according to Clause 6, wherein the poly(3- hydroxybutyrate-co-3-hydroxyhexanoate) comprises from 23 to 25 mol. % of 3- hydroxyhexanoate, optionally about 24 mol. % of 3-hydroxyhexanoate. 8. The sustained-release fertiliser formulation according to any one of the preceding clauses, wherein the polyhydroxyalkanoate copolymer has a melting point of less than or equal to 120°C, optionally less than or equal to 110°C or less than or equal to 100°C, more optionally less than or equal to 90°C. 9. The sustained-release fertiliser formulation according to any one of the preceding clauses, wherein the core portion has a minimum diameter of from 3.5 to 4.2 mm. 10. The sustained-release fertiliser formulation according to any one of the preceding clauses, wherein the shell portion has a thickness of from 80 to 180 µm.. 11. The sustained-release fertiliser formulation according to any one of the preceding clauses, wherein the polyhydroxyalkanoate copolymer is a biodegradable material, a biodegradation of the polyhydroxyalkanoate copolymer can be detected through one or more of scanning electron microscopy, clear zone formation in polyhydroxyalkanoate agar, weight loss of polyhydroxyalkanoate, and decrease in molecular weight using gel permeation chromatography. 12. The sustained-release fertiliser formulation according to any one of the preceding clauses, wherein the polyhydroxyalkanoate copolymer is synthesised by Cupriavidus necator and isolated from the excrement of mealworms. 13. The sustained-release fertiliser formulation according to Clause 12, wherein the polyhydroxyalkanoate copolymer is a suitable substrate for microorganisms. 14. The sustained-release fertiliser formulation according to any one of the preceding clauses, which is formed by dip-coating the core portion into a solution comprising the polyhydroxyalkanoate copolymer. 15. A method of preparing a sustained-release fertiliser formulation according to any one of the preceding clauses, comprising: (i) providing a solid granule comprising the one or more fertiliser compounds; and (ii) dip-coating the solid granule in a solution comprising the polyhydroxyalkanoate copolymer. 16. The method according to Clause 15, wherein the concentration of polyhydroxyalkanoate copolymer in the solution is from 10% to 30% (w / v), optionally from 13% to 20% (w / v), such as about 15% (w / v). 17. The method according to Clause 15 or Clause 16, wherein the polyhydroxyalkanoate copolymer is soluble in a halogen free and environmentally safe solvent, optionally wherein the solvent is acetone. Drawings FIG.1 depicts how polymer-coated urea works. FIG.2 depicts the preparation of preculture for fermentation run. FIG.3 depicts the fermentation process repeated by using end log phase culture from previous run as the seed culture. FIG.4 depicts the preparation of PHA-based CRF. FIG.5 depicts the physical appearance of uncoated urea (left) and coated urea with P(3HB- co-24 mol% 3HHx) (right). FIG. 6 depicts the scanning electron microscopy (SEM) micrographs of urea coated with multiple layers of P(3HB-co-24 mol% 3HHx). The thickness of the coating and release duration in water increased when the number of coating layers increased. Magnification: × 30. Scale bar: 1 mm. FIG.7 depicts the PHA-coated urea at varying concentrations. FIG.8 depicts the urea release profile of PHA-CRF in soil. FIG.9 depicts the biodegradation of PHA coating occurring under soil condition, eliminating microplastic residue in soil. Left panels are obtained using a dissecting microscope (Olympus SZX16, Japan), while the right panels are obtained using SEM. Description In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of” or synonyms thereof and vice versa. It has been surprisingly found that a sustained-release fertiliser formulation may be formed using a core-shell arrangement, where the core contains a fertiliser and the shell contains a biodegradable polymer. Thus, in a first aspect of the invention, there is provided a sustained- release fertiliser formulation comprising: a core portion comprising one or more fertiliser compounds; and a shell portion comprising a polyhydroxyalkanoate copolymer, wherein: the polyhydroxyalkanoate copolymer has a melting point that is at least 10°C lower than the lowest melting point of the one or more fertiliser compounds; and the polyhydroxyalkanoate copolymer is biodegradable. In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of” or synonyms thereof and vice versa. The phrase, “consists essentially of” and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure. 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, a metal phosphate, a metal nitrate, an ammonium halide, a superphosphate, ammonium phosphate, ammonium sulfate, ammonia, a metal sulfate, potassium chloride, ammonium phosphate, ammonium nitrate, calcium ammonium nitrate, potassium sulfate, phosphoric acid, potassium magnesium sulfate, and combinations thereof. In particular embodiments that may be mentioned herein, the fertiliser compound may be urea. Additionally or alternatively, there may be one or more fertilizer compounds and the one or more fertilizer compounds may comprise urea. 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 that is soluble in a non-halogenated solvent). For example, the PHA copolymer may be a poly- 3-hydroxybutyrate (3HB) copolymer. For example, the a poly-3-hydroxybutyrate (3HB) copolymer may be a poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). In particular embodiments that may be mentioned herein, the poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) may comprise from 18 to 30 mol. % of 3-hydroxyhexanoate (e.g. from 23 to 25 mol. % of 3-hydroxyhexanoate, such as about 24 mol. % of 3-hydroxyhexanoate). Thus, the shell portion may comprise a poly-3-hydroxybutyrate (3HB) copolymer. For example, the shell portion may comprise a poly-3-hydroxybutyrate (3HB) copolymer. In particular embodiments that may be mentioned herein, the poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) may comprise from 18 to 30 mol. % of 3- hydroxyhexanoate (e.g. from 23 to 25 mol. % of 3-hydroxyhexanoate, such as about 24 mol. % of 3-hydroxyhexanoate). The PHA copolymer used herein may have any suitable melting point. For example, the polyhydroxyalkanoate copolymer may have a melting point of less than or equal to 120°C such as less than or equal to 110°C, such as less than or equal to 100°C, such as less than or equal to 90°C. The minimal melting point may be greater than or equal to 80°C, such as greater than or equal to 88°C. Alternatively, the melting point of the PHA copolymer may be from 80 to 120°C, such as from 85 to 110°C, such as from 88 to 110°C, such as about 100°C, The formulation described herein is provided in the form of particles, with a core portion and a shell portion. The core portion may have any suitable diameter, provided that the resulting formulation is still able to function as a controlled-release formulation and is easily placed into the ground. For example, the core portion may have a minimum diameter of from 3.5 to 4.2 mm. The core portion may have a maximal diameter of from 4.5 to 5 mm. Thus, the diameter may be from 3.5 to 5 mm, such as from 4.2 to 4.6 mm. The shell portion may have any suitable thickness. Ideally, the shell portion has a thickness that can survive transportation, storage and distribution into / onto the soil at its final site of use. Examples of suitable thicknesses for the shell portion include, but are not limited to a thickness of from 80 to 180 µm. As noted hereinbefore, the PHA copolymer is a biodegradable material. This means that the polymeric material may be broken down in the ambient environment it finds itself in. This may be by microbes in the soil / water or by animals, as well as by environmental effects over time. The biodegradation of the polyhydroxyalkanoate copolymer can be detected through one or more of scanning electron microscopy, clear zone formation in polyhydroxyalkanoate agar, weight loss of polyhydroxyalkanoate, and a decrease in molecular weight using gel permeation chromatography. The PHA copolymer may be obtained from any suitable source, whether synthetic or biological. For example, the polyhydroxyalkanoate copolymer may be synthesised by Cupriavidus necator and / or isolated from the excrement of mealworms. As noted above, the polyhydroxyalkanoate copolymer may be a suitable substrate for microorganisms. The sustained-release fertiliser formulation described herein may be formed by dip-coating the core portion into a solution comprising the polyhydroxyalkanoate copolymer. Thus, in a further aspect of the invention, there is provided a method of preparing a sustained-release fertiliser formulation as described herein, comprising: (i) providing a solid granule comprising the one or more fertiliser compounds; and (ii) dip-coating the solid granule in a solution comprising the polyhydroxyalkanoate copolymer. In the above method, the PHA copolymer may have any suitable concentration in the solution. For example, the concentration of polyhydroxyalkanoate copolymer in the solution may be from 10% to 30% (w / v), such as from 13% to 20% (w / v), such as about 15% (w / v). In the method above the polyhydroxyalkanoate copolymer may be soluble in a halogen free and environmentally safe solvent, optionally wherein the solvent is acetone. Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting embodiments. Examples Materials Materials required for PHA Production Tryptic soy broth (TSB) [seed culture] Minimial medium with carbon and nitrogen source [seed culture: 0.7 L] Waste cooking oil Urea Sodium dihydrogen phosphate Disodium hydrogen phosphate Potassium sulphate Calcium chloride Magnesium sulphate Ferrous sulphate∙7H2O Manganese sulphate∙ H2O Zinc sulphate∙7H2O Copper sulphate∙5H2O Materials required for PHA-CRF Acetone Urea PHA Unless otherwise stated, the materials were obtained from commercial sources, such as Sigma Aldrich, Fischer, etc. or were obtained from a source (such as a commercial kitchen for the waste cooking oil). Analytical techniques SEM The surface morphology and thickness of the coating of CRF were examined using a scanning electron microscope (TM-4000, Hitachi, Japan). Example 1. Production of cost-effective and suitable PHA for use as a biodegradable coating for urea granules PHA has been authorized as one of the Plastic Food Contact Materials (FCMs) in the European Union. PHAs are biopolyesters produced from microorganisms (bacteria and archaea) under nutrient limitations and stress conditions as storage compounds (carbon reserve). Besides possessing physicochemical properties like petrochemical-derived plastics, the major advantages of PHA compared to synthetic plastics are its biodegradability, biocompatibility, and sustainability. Bacterial PHA can be divided into three main types depending on the number of carbon atoms in the monomeric units: short-chain-length (scl), medium-chain-length (mcl) and a combination of scl-mcl. The scl-PHAs consist of 3-5 carbon atoms, mcl-PHAs have 6-14 carbon atoms whereas the number of carbon atoms in scl-mcl- PHAs can range from 3-14 per monomer. PHAs composed of mostly scl monomers are often stiff and brittle, whereas PHAs composed of mostly mcl monomers are elastomeric in nature. Scl-mcl PHA copolymers can have properties between the two states, dependent 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 to use for the coating of fertilizer to produce CRF. Since PHA is biodegradable by soil microorganisms, it will be slowly degraded by the microorganism and the fertilizer will be released after a certain period. The released time of the fertilizer can be controlled by either the types of the PHA coated or the thickness of the PHA coated on the fertilizer. 3-hydroxyhexanoate (3HHx) belongs to mcl monomer, which consists of 6-14 carbon units. The longer monomer length makes it able to dissolve in a much broader solvent range, so cheaper and less toxic solvents (such as acetone and dimethyl carbonate) may be used in the industrial setting. As such, P(3HB-co-3HHx) with high composition (>18 mol%) of 3HHx monomer can be dissolved in halogen-free solvent such as acetone. Example 2. Production of PHA We describe a cost-effective method of producing PHA as a biodegradable coating material to control the release of plant nutrients. The carbon sources of PHA can be different types of oil while the nitrogen source is urea. The oil and urea will be fed slowly and continuously based on the growth of the bacterial strain. To shorten the time of the fermentation process, the end log phase culture will be harvested and 0.1-30% of the culture will be used as the seed culture for the next fermentation run (second run). Since the seed culture remains at its active phase and the number of cells is much higher than the number of cells in the seed culture of the first run, the growth will be faster, and the duration of the fermentation run is reduced. The feeding of urea and oil will be faster compared to the first run as the cells grow faster. When the cultures reach the end log phase, they will be harvested and 0.1-30% of the culture will be used as the seed culture for the next fermentation run again (third run). The cycles can be repeated until desired number of cultures is obtained. High cell density fermentation The Cupriavidus necator transformant was first pre-cultured into a tryptic soy broth (TSB) at 200 rpm, 30 °C.3-4 % (v / v) of the preculture was transferred into 2.9 L mineral medium (MM) in the 13 L bioreactor when the optical density (OD600 nm) reached approximately 3.5–4.5. The composition of the mineral medium was 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 of 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 temperature of the bioreactor was set at 30 °C. Urea was used as the nitrogen source while a mixture of oil [40% crude palm kernel oil (CPKO) and 60% palm olein (PO)] was used as the carbon source. The feeding strategies of urea and oil are shown below in Example 3. The optical density was measured by transferring 1 mL of preculture into a microcentrifuge tube and it was then centrifuged at 12,000 rpm for 2 minutes, then the supernatant was discarded. Next, 1 mL of distilled water was added to the remaining pellet to resuspend the contents and this was centrifuged again at 12,000 rpm for 2 minutes, with the supernatant discarded once again. After that, 1 mL of distilled water was added to the pellet to resuspend the contents again. The suspension was transferred (1 mL) into a clean test tube and 9 mL of distilled water was added into the test tube (10 × dilution) (Note: dilution is not needed if cells are too little). 1 mL of the suspension was transferred into a cuvette. Blank will be 1 mL of distilled water. The OD was checked at 600 nm using a spectrophotometer. (*Note: As we did 10 x dilution, the O.D. reading should be multiplied by 10.) Cultivation in 13 L bioreactor (6 L working volume) – Run 1 FIG.2 depicts the cultivation of Cupriavidus necator transformant in 13 L bioreactor. As shown in FIG.2, there is a method 200 for preparation of preculture for fermentation run. The method 200 includes: 201: glycerol stock from –80 °C (Cupriavidus necator transformant); 202: activate on TSA agar plate (gentamycin (Gm) + kanamycin (Km)). Incubation at 30 °C for 2 – 3 days; 203: subculture a single colony to TSA agar plate (Gm + Km). Fourth quadrant streaking; 204: TSA agar plate (Gm + Km). Incubation at 30 °C for 24 h; 205: 2 x TSA agar plate (Gm + Km). Incubation at 30 °C for 24 h; 206: inoculate 4 loops full; 207: 2 x 50 mL TSB (Gm + Km).250 mL Erlenmeyer flask covered with cotton plug. Incubation for 4 – 5 h; 208: transfer 100 mL TSB culture into the 13 L bioreactor; and 209: preculture in 13 L bioreactor. ^ 2.9 L MM (3 L concentration). ^ 1.5% CPKO (0thh = 0.75%, 22.5 g, 6thh = 0.75%, 22.5 g). ^ 30 mM urea (0thh = 15 mM, 6thh = 15 mM). ^ Temperature set at 30 °C. ^ pH set at 6.8. ^ Stirring speed set at 300 rpm. ^ Aeration set at 0.5 wm. ^ Dissolved oxygen (DO) setting: >40% (cascade mode). ^ Cultivate for 12 h. Activation of the microbial strain The recombinant C. necator strain Re2058 / pCB113 from glycerol stock in –80 °C was thawed and streaked in a four-quadrant streak on tryptic soy agar (TSA) supplemented with 50 μg / mL Km. The TSA plate was incubated for 48 h at 30 °C to obtain single colonies. From the activated plate, single colonies were picked out and sub-cultured onto new TSA + Km plate as full-plate streaks. The plate was incubated at 30 °C for 24 h. Preparation of pre-cultures for biosynthesis TSB (50 mL) supplemented with 50 μg / mL Km was prepared in 250 mL Erlenmeyer flask. Approximately 4 loops full of bacterial culture from the full-streak plate were transferred aseptically into the TSB + Km flask. The flask was incubated at 30 °C with 200 rpm agitation in an incubator shaker for 4 – 5 h until the optical density of the cultures (OD600nm) reached between 4 – 5. The cultures were considered to have reached the active mid-log phase once the OD600nm readings are between 4 – 5. The cultures are ready to be transferred into the PHA biosynthesis medium at this point. Seed for PHA biosynthesis in shake flask For PHA biosynthesis, minimal medium (MM) as reported in C. F. Budde et al., J. Bacteriol. 2010, 192, 5319-5328, were used. Solution A (4.0 g / L of NaH2PO4, 4.6 g / L of Na2HPO4 and 0.45 g / L of K2SO4) were dissolved in 800 mL of distilled water. Then, the volume of the medium was topped up to 1 L after the pH of the medium was adjusted to 6.8 using 1 M HCl or 1 M NaOH. Solution A, urea [CO(NH2)2] stock solution, MgSO4·7H2O stock solution, CaCl2stock solution and carbon sources were sterilized separately by autoclaving while trace element solution was filter sterilized by 0.2 μm cellulose acetate membrane filter. 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. The solution was kept in a Schott bottle covered with aluminium foil in a 4 °C fridge. 800 mL MM was prepared in a 5 L Erlenmeyer flask, 2.25 g / L of urea, 0.80 g / L of MgSO4·7H2O, 0.062 g / L CaCl2, 1 mL / L of trace element and a specific amount of carbon source (10 g / L of CPKO) was added into the MM and supplemented with 50 μg / mL Km and 10 μg / mL Gm. 3 vol% of bacterial cultures from TSB + Km flasks were added as a final step. The flasks were then incubated at 30 °C with 200 rpm agitation in an incubator shaker for 10 – 15 h until OD600nm reached between 4.5 – 5.5. PHA biosynthesis in 13 L bioreactor To achieve final total volume of 7 L culture media, 5.7 L of MM was prepared as above except for urea at 2.0 g / L.10 vol% of bacterial seed from MM in Erlenmeyer flask were inoculated into the bioreactor. The bioreactor was set at pH 6.8, 30 °C, 200 rpm agitation, and 0.5 vvm aeration with cascade mode. Sampling was done every 6 h to observe the bacterial growth. The feeding strategies were set for additional 2.75 g / L of urea and 50 g / L of CPKO according to the bacterial growth. Biosynthesis was stopped once the conversion of oil to cell was almost 100% (approximately 48 h). Cell harvest and lyophilization The culture was harvested via centrifugation at 8000 rpm, 4 °C for 10 min using a refrigerated centrifuge. The first supernatant was discarded, and the cell pellets were resuspended with distilled water to remove any residual fermentation media that may have trapped between the cells. The suspension was subjected to centrifugation using the same conditions as the first step and the conductivity of the supernatant was checked by adding 120 µL of supernatant to a conductivity meter (EC-33 HORIBA Scientific, Japan) and measuring the conductivity. The washing was repeated until conductivity < 3000 μS / cm. The washed cells were stored at – 80 °C overnight. The frozen cells were then subjected to lyophilization using a freeze dryer for 72 h. The resulting dried cells were then weighed to determine the cell dry weight (CDW) and used for subsequent analyses in the following examples. Biological recovery using mealworm was utilized for the PHA extraction method as published in I. Zainab-L & K. Sudesh, J. Biotechnol.2019, 305, 35-42. See also Murugan, P., L. Han, C.-Y. Gan, F. H. J. Maurer, K. Sudesh. (2016) A new biological recovery approach for PHA using mealworm, Tenebrio molitor. J. Biotechnol.239: 98-105. and Ong, S. Y., H.-P. Kho, S. L. Riedel, S.-W. Kim, C.-Y. Gan, T. D. Taylor, K. Sudesh. (2018) An integrative study on biologically recovered polyhydroxyalkanoates (PHAs) and simultaneous assessment of gut microbiome in yellow mealworm. J. Biotechnol.265: 31-39. Biological recovery The biological agent used in this experiment was Tenebrio molitor mealworms of approximately 1.5 months old. They were obtained from a local farm and maintained on their conventional diet (wheat bran) prior to the experiment. Preparation of mealworms for biological recovery Mealworms that are 1.5 months old (1 – 1.5 cm in average length) were starved for 48 h to remove any residual frass from their gastrointestinal tract from the previous feeding. Before starvation commenced, the mealworms were well-fed with oats or wheat bran ad-libitum and periodically supplemented with carrots as a water source to maintain their overall growth and health. The worms were maintained in a low light condition room with an ambient temperature of about 28 – 30 °C and a relative humidity of about 50 – 60%. Feeding of freeze-dried bacterial cells containing PHA The starved mealworms were weighed and sorted into containers. The frass and impurities from the mealworms were sieved out to minimise interventions during the biological recovery process. The feeding ratio of the cells is 10 wt% of the total body weight of the mealworms. For instance, 10 g mealworms were used to feed 1 g of bacterial cells. Dried cells that existed in large clumps were broken down into smaller pieces either by hand or light pounding using a mortar and pestle to ease the intake of cells by the mealworms. The cells were evenly spread throughout the container to ensure that all the mealworms had equal access to the cells. The mealworms were left for 24-48 h for complete consumption of the freeze-dried bacterial cells. The excrement, which contains PHA granules, was harvested thereafter. Harvesting of frass containing PHA granules After 24-48 h of feeding, the containers were examined to ensure that no residual bacterial cells remained. For containers which still have residual cells, the mealworms were left for a longer period before the frass was harvested. To harvest the PHA frass, the mealworms were transferred onto a 0.5 mm pore sized standard sieve and gently shaken to separate the frass into a holding container at the bottom. The weight of the mealworms after feeding the bacterial cells was recorded. Similarly, the weight of the frass was also recorded and stored in Ziplock bags for subsequent purification. 

[0002] PHA faecal pellet purification Approximately 200 g of the faecal pellets were transferred into a 5 L plastic container and washed with tap water until the water turned semi-transparent. Water was removed and the washed pellet was grinded into granules. The granules were washed until there were no black particles. The washed granules were then purified using 0.25 M NaOH by stirring for 1 h. The solution was decanted, and the granules were washed until there are no black particles. The purification using 0.25 M NaOH and washing were repeated. The purified PHA granules were then bleached using 10 vol% Clorox. The solution was decanted, and the granules were washed at least six times or until there is no Clorox odour. The water was drained out using a fine mesh (212 µm) sieve. The granules were spread on a wide tray and dried at 60 °C for 24 h until moisture content ≤ 1%. The dried granules were mashed into powder form, packed into a pre-weighed Ziplock bag, and labelled accordingly. Cultivation in 13 L bioreactor (6 L working volume) – Runs 1 to 3 As shown in FIG. 3, there is a fermentation process 300 repeated by using end log phase culture from previous run as the seed culture. The fermentation process 300 includes: 301: Run 1; ^ MM (2 L : 3 L concentration) and minerals were added after 12 h cultivation period. ^ Refer to Example 3 for the feeding strategy. ^ Harvest after ~33 h of cultivation. ^ Left 300 mL of culture in the vessel after harvest. 302: Run 2; and ^ MM (4.8 L : 6 L concentration) and minerals were added to the vessel and the 300 mL of the culture from the previous run will act as the seed culture for this run. ^ Refer to Example 3 for the feeding strategy. ^ Harvest after ~30 h of cultivation. 303: Run 3. ^ MM (4.8 L : 6 L concentration) and minerals were added to the vessel and the 300 mL of the culture from the previous run will act as the seed culture for this run. ^ Refer to Example 3 for the feeding strategy. ^ Harvest after ~30 h of cultivation. Results and discussion The PHA used in the present disclosure is mealworm recovered polymer granules which was recovered by the method described in a Biological Method for the Extraction and Purification of Polyester Granules from Bacterial Cells. Malaysian Patent Application No. PI 20092081 (2009) and WO2010134798 (see Example therein, which is hereby incorporated by reference). This is an environmentally friendly polymer extraction method compared to the solvent extraction method. The partially purified PHA also significantly reduces the cost of production. This partially purified PHA is the only component that is necessary to produce a water insoluble coating surrounding the urea granules. The partially purified PHA appears in the form of coarse powder. Example 3. Fermentation feeding strategy (For 6 L working volume) Nitrogen source (Urea) Run 1 Total feeding at 0thh (in preculture) = 15 mM urea 0th– 10thh = 5 mM / h = 50 mM urea, total feeding at 15th h = 65 mM urea 10th– 28thh = 10 mM / h = 180 mM urea, total feeding at 30th h = 245 mM urea Run 2, 3 and etc. 0th– 24thh = 10 mM / h = 240 mM urea, total feeding at 24th h = 240 mM urea Carbon source (oil = mixture of 60% PO and 40% CPKO) Run 1 Total feeding at 0thh (in preculture) = 45 g of oil 0th– 10thh = 15 g / h of oil = 150 g of oil, total = 195 g of oil 10th– 30thh = 20 g / h of oil = 400 g of oil, total = 595 g of oil Run 2, 3 and etc. 0th– 24thh = 25 g / h of oil, total feeding at 25th h = 600 g of oil Example 4. Determination of PHA content and its monomer composition Gas chromatography (GC) analysis was used to determine the PHA content and its monomer composition, as reported in G. Braunegg, B. Sonnleitner & R. M. Lafferty, European J. Appl. Microbiol. Biotechnol. 1978, 6, 29–37. The PHA content was determined by gas chromatography (GC) using the Shimadzu GC-2010 system equipped with an SPB-1 column (Supelco, USA). The column temperature was initiated at 70 °C and then increased to 280 °C in continuous steps of 10 °C / min. The PHA content and composition were quantified with caprylic acid methyl ester (CME) as an internal standard. Preparation of methanolysis solution Methanolysis solution and CME solution were prepared before the GC was carried out. Methanolysis solution consists of concentrated sulfuric acid and methanol at a ratio of 15:85 (v / v). Preparation of this solution was done in the fume hood. In an ice bath, 425 mL of methanol was added into a Schott bottle. Then, 75 mL of concentrated sulfuric acid was added dropwise into the Schott bottle and stirred slowly with a magnetic stirrer. The methanolysis solution was then kept at 4 °C for further use. Preparation of CME solution CME was used as the internal standard for PHA quantification in GC analysis. Preparation of CME solution was done in the fume hood. In a 100 mL volumetric flask, 0.2 mL of CME stock solution was topped up to 100 mL with chloroform. The ratio of CME stock solution to chloroform is 1:500. This working solution was then mixed thoroughly, transferred to a Schott bottle wrapped with aluminium foil and kept at 4 °C for further use. Methanolysis of samples Approximately 15.0 to 20.0 mg of lyophilized cells (or 3.0 to 9.0 mg of polymer) was transferred to a screw capped test tube. Then, 2 mL of methanolysis solution and 2 mL of chloroform were added into the test tube. The mouth of the tube was wrapped with PTFE tape so that the test tube can be sealed firmly to avoid evaporation. The test tubes were incubated at 100 °C for 140 min using heating block. The heated test tubes were tapped gently at every 20 min interval. After that, the samples were left to cool down to room temperature (25 °C). Preparation of GC samples One mL of distilled water was added to the cooled samples and vortexed vigorously for one min. After a few minutes, there was a formation of two distinct phases, which were the upper aqueous layer and the lower organic layer. The lower organic layer which contained hydroxyacyl methyl esters was transferred to a clean flat bottom tube containing anhydrous sodium sulfate (Na2SO4) to absorb any trace amount of water within the sample. Then, 0.5 mL of the lower organic layer was transferred into GC vial containing 0.5 mL of CME. The GC sample was then ready to be subjected to GC analysis. GC analysis GC analysis was carried out using a Shimadzu GC-2010 Plus. The GC was equipped with AOC-20i Auto Injector, Supelco SPB®-1 Capillary GC Column and flame ionization detector. A total of 2.0 μL of the sample was injected. The syringe was automatically rinsed with chloroform after every injection. The sample injection process and analyzation were programmed using GC Solution Version 2.30.00 SU3. The GC conditions were programmed as below: 1. AOC-20i Auto Injector; ^ Carrier gas: Nitrogen gas ^ Temperature: 270 °C ^ Pressure: 84.2 kPa ^ Total flow: 14 mL / min 2. Supelco SPB®-1 Capillary GC Column; and ^ Initial temperature: 70 °C ^ Final temperature: 280 °C 3. flame ionization detector, ^ Temperature: 280 °C ^ Hydrogen gas flow: 40 mL / min ^ Air flow: 400 mL / min Calculation of PHA content and its monomer composition PHA content and its monomer composition were calculated based on the peak areas at certain retention time on the GC chromatogram. The retention time of each monomer was determined by using the identified PHA polymer. PHA content and its monomer composition were calculated based on the formulae shown below: 1. PHA content ^wt%^ =K × ^^A3HB× k3HB^ + ^AHA× kHA^^ACME× W× 100% 2. HA monomer content in PHA ^mol%^ =AHA× kHA^A3HB× k3HB^ + ^AHA× kHA^× 100% 3. PHA yield (g / L) = DCW (g / L) × PHA content 100 where, Retention time of 3HB = 3.2 – 3.3 min Retention time of 3HHx = 5.9 – 6.0 min Retention time of CME = 7.1 – 7.2 min K = GC constant = 11.2 k3HB= 3HB monomer constant = 1 k3HHx = 3HHx monomer constant = 0.4 A3HB = Area below 3HB monomer peak A3HHx= Area below 3HHx monomer peak ACME = Area below CME peak W = Weight of lyophilized cells subjected to methanolysis (mg) Example 5. Preparation of PHA-based controlled release fertilizer (CRF) The resulting PHA coarse powder in Example 2 can be readily dissolved in non-halogenated solvents at room temperature and the solution can subsequently be used to coat plant nutrients such as the water-soluble urea granules. The PHA coat forms a water-insoluble layer around which thickness can be easily controlled by the adjusting the concentration of the PHA solution. The coating process is both simple and rapid and does not need other components to stabilize the coat. The PHA-based CRF was prepared by dip-coating method (FIG.). Briefly, P(3HB-co-24 mol% 3HHx) solution (prepared in Example 2) with concentration of 15% (w / v) was prepared using acetone as solvent. The polymer solution was stirred at room temperature (25 °C) for 24 h to dissolve the PHA completely. After that, urea granules were dip-coated in the PHA solution and then, dried at ambient temperature. Coated PHA-based CRFs, CRF20, CRF25 and CRF30, were prepared using 20%, 25% and 30% PHA concentration solutions, respectively, by following the protocol above. Example 6. Characterization of PHA produced P(3HB-co-24 mol% 3HHx) prepared in Example 2 was characterized. Chemical extraction and purification of PHA Approximately 1 g of lyophilized cell was mixed with 50 mL of chloroform and stirred at room temperature for three days. The mixture was filtered using Whatman No. 1 filter papers to remove cell debris. The resulting clear solution was then added dropwise into vigorously stirring ice-cold methanol to precipitate the PHA polymers. The precipitated polymers were separated from the methanol solution using vacuum filtration and then dried overnight at room temperature. Determination of molecular weight of PHA The resulting polymer were used to measure the number average molecular weight (Mn) and weight average molecular weight (Mw) by gel permeation chromatography (GPC) using Agilent Technologies 1200 Series GPC (USA) equipped with TSK guard column HHR-H and TSKgel GMH HR-H (Tosoh, Japan). Chloroform was used as the solvent for the mobile phase with a flow rate of 0.8 mL / min at 40 °C. PHA polymers were dissolved in chloroform to a final concentration of approximately 1.0 mg / mL and filtered (PTFE membrane, 0.22 μm) before analysis. Determination of thermal properties Differential scanning calorimetry (DSC) analysis was performed to determine the glass transition temperature (Tg), melting temperature (Tm) and enthalpy of fusion (ΔHm) of the PHA polymers. DSC analysis was done using DSC-60 (Shimadzu, Japan) equipped with a liquid nitrogen cooling accessory under nitrogen atmosphere. The temperature range for DSC varied from −40 to 200 °C at a heating rate of 10 °C / min. Results and discussion FIG.5 depicts the physical appearance of uncoated urea and coated urea with P(3HB-co-24 mol% 3HHx). FIG. 6 depicts the SEM micrographs of urea coated with multiple layers of P(3HB-co-24 mol% 3HHx). PHA-coated urea at varying concentrations are depicted in FIG.7. Table 1. High cell density PHA production by C. necator transformant from the mixture of CPKO and PO. Working Dry cell PHA content PHA yield Run no. Duration (h) volume (L) weight (g / L) (wt%) (g L-1h-1) Run 1 5.7 45 (12 + 33) 96.1 69 ± 9 1.47 ± 0.19 Run 2 6 31 90.3 65 ± 8 1.89 ± 0.23 Run 3 Repeat cycle as the Run 2 until desired amount of PHA was obtained. Table 2. Characterization of the produced P(3HB-co-3HHx). PHA composition Run(mol %)Mn (× 105) Mw (× 105) PDI Tg Tm3HB 3HHx 1 77 ± 0 23 ± 0 3.5 ± 0.0 5.8 ± 0.0 1.7 ± 0.0 −5.0 87.2 2 77 ± 0 23 ± 0 3.3 ± 0.1 5.7 ± 0.0 1.7 ± 0.0 −4.9 87.7 The values reported are averages from triplicate cultures ± SDs. 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 temperature; and ΔHm, melting enthalpy. The PHA used in this disclosure is P(3HB-co-24 mol% 3HHx), while Saettone et al. (US2021387925A1) used P(3HB) homopolymer which has a melting temperature of about 180 °C, much higher than the melting point of urea. The melting point of P(3HB-co-24 mol% 3HHx) is 88 °C, which is lower than the melting temperature of urea (135 °C). The lower melting point of P(3HB-co-24 mol% 3HHx) facilitates most of the industrial processing processes, such as melt coating, melt pressing and extrusion, which require the melting of the polymer without melting the urea granules. In addition, P(3HB) is a homopolymer of 3- hydroxybutyrate monomers consisting of 4 carbon compounds. On the other hand, the 3- hydroxyhexanoate (3HHx) is a bulkier monomer with 6 carbons. The bulkier monomer makes the PHA copolymer more amorphous (less crystalline) and therefore it is able to dissolve in a much broader solvent range, so cheaper and less toxic solvents (such as acetone and dimethyl carbonate) may be used in the industrial setting. Example 7. Evaluation of the release of urea in water environment CRF20, CRF25 and CRF30 (prepared in Example 5) were tested in the urea release experiments and commercial urea CRF (SmartGro) served as the control. Urea release Fifteen pots, each containing 40 g of soil (moisture content of 20%) were prepared and used to represent the release of urea into soil over 15 weeks (1 pot per week). Four PHA-based CRFs granules were placed into each pot. The initial weight of PHA-based CRFs were recorded and 3 mL of water was added daily. Each week, the four PHA-based CRFs from one pot were removed (and that pot discarded) and weighed after being dried at 50 °C to a constant weight. Commercial urea CRF, SmartGro was purchased from Smart Fert Sdn Bhd to serve as positive control. Results and discussion <80% urea was released throughout the 60-day period of longevity test in soil, meeting the basic prototyping requirements for CRF (FIG.8). In addition, the results revealed that CRF20, CRF25 and CRF30 released urea completely at day 42, 56 and 105, respectively, whereas SmartGro released 67% of urea at day 105.100% weight loss is an indication for the complete release of urea. Example 8. Degradation of coated PHA-based CRFs The degradation of coated PHA-based CRFs (prepared in Example 5) through the action of microorganisms was investigated. Physical observation of the degradation of coated PHA-based CRF The physical changes on the PHA-CRF 20, 25, and 30% (w / v) were observed. Physical observation via SEM (method described above). PHA degradation is indicated by pores on the coating layer. Results and discussion The PHA coating layer of CRF20, CRF25 and CRF30 started to degrade in weeks 6, 8, and 9, respectively, then ruptured and broken into small pieces. SEM images showed morphological changes due to the degradation of PHA-CRF as a result of enzymes secreted by microbial communities in the soil (FIG.9). The coated PHA-based CRF was shown to be biodegraded completely in soil without residual material accumulating in the soil. Thus, we have developed CRF by using biodegradable polymer polyhydroxyalkanoate (PHA), specifically poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), as coating material which the coating material can be degraded completely through the action of microorganisms into carbon dioxide and water. In addition, we have described a method to produce cost-effective PHA from fermentation process, polymer extraction method and lastly PHA-based CRF, that overcomes the problem of high production cost of PHA. Comparative Example 1 The advantages of coated PHA-based CRF over uncoated fertilizer are depicted in Table 3 below.

[0003] Table 3. Advantages of PHA-CRF. Coated fertilizer Uncoated fertilizer Nutrients are not completely Nutrients are released in a available for uptake by Nutrient availability / release controlled manner plants due to leaching, runoff, and volatilization Fertilizer loss - increase in Fertilizer loss Fertilizer loss is reduced agriculture production cost Less environmental More environmental Environmental pollution population pollution

Claims

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

2. The sustained-release fertiliser formulation according to Claim 1, wherein the one or more fertiliser compounds are selected from one or more of the group consisting of urea, a metal phosphate, a metal nitrate, an ammonium halide, a superphosphate, ammonium phosphate, ammonium sulfate, ammonia, a metal sulfate, potassium chloride, ammonium phosphate, ammonium nitrate, calcium ammonium nitrate, potassium sulfate, phosphoric acid and potassium magnesium sulfate.

3. The sustained-release fertiliser formulation according to Claim 1, wherein the one or more fertiliser compounds comprise urea.

4. The sustained-release fertiliser formulation according to Claim 1, wherein the shell portion comprises a poly-3-hydroxybutyrate (3HB) copolymer.

5. The sustained-release fertiliser formulation according to Claim 1, wherein the shell portion comprises poly(3-hydroxybutyrate-co-3-hydroxyhexanoate).

6. The sustained-release fertiliser formulation according to Claim 5, wherein the poly(3- hydroxybutyrate-co-3-hydroxyhexanoate) comprises from 18 to 30 mol. % of 3- hydroxyhexanoate.

7. The sustained-release fertiliser formulation according to Claim 6, wherein the poly(3- hydroxybutyrate-co-3-hydroxyhexanoate) comprises from 23 to 25 mol. % of 3- hydroxyhexanoate, optionally about 24 mol. % of 3-hydroxyhexanoate.

8. The sustained-release fertiliser formulation according to Claim 1, wherein the polyhydroxyalkanoate copolymer has a melting point of less than or equal to 120°C, optionally less than or equal to 110°C or less than or equal to 100°C, more optionally less than or equal to 90°C.

9. The sustained-release fertiliser formulation according to Claim 1, wherein the core portion has a minimum diameter of from 3.5 to 4.2 mm.

10. The sustained-release fertiliser formulation according to Claim 1, wherein the shell portion has a thickness of from 80 to 180 µm.

11. The sustained-release fertiliser formulation to Claim 1, wherein the polyhydroxyalkanoate copolymer is a biodegradable material, a biodegradation of the polyhydroxyalkanoate copolymer can be detected through one or more of scanning electron microscopy, clear zone formation in polyhydroxyalkanoate agar, weight loss of polyhydroxyalkanoate, and a decrease in molecular weight using gel permeation chromatography.

12. The sustained-release fertiliser formulation according to Claim 1, wherein the polyhydroxyalkanoate copolymer is synthesised by Cupriavidus necator and isolated from the excrement of mealworms.

13. The sustained-release fertiliser formulation according to Claim 12, wherein the polyhydroxyalkanoate copolymer is a substrate for soil microorganisms.

14. The sustained-release fertiliser formulation according to Claim 1, which is formed by dip-coating the core portion into a solution comprising the polyhydroxyalkanoate copolymer.

15. A method of preparing a sustained-release fertiliser formulation according to Claim 1, comprising: (i) providing a solid granule comprising the one or more fertiliser compounds; and (ii) dip-coating the solid granule in a solution comprising the polyhydroxyalkanoate copolymer.

16. The method according to Claim 15, wherein the concentration of polyhydroxyalkanoate copolymer in the solution is from 10% to 30% (w / v), optionally from 13% to 20% (w / v), such as about 15% (w / v).

17. The method according to Claim 15, wherein the polyhydroxyalkanoate copolymer is soluble in a halogen free and environmentally safe solvent, optionally wherein the solvent is acetone.

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