Process for the production of biodegradable polymer from pongamia oil

Abstract

A process for producing polyhydroxyalkanoates, comprising the steps of: fermenting production medium comprising pongamia oil using bacteria from the Cupriavidus, Burkholderia, or Alcaligenes genera capable of producing polyhydroxyalkanoates; wherein the pongamia oil is extracted from the seeds of Pongamia species and clarified by filtering before the crude, unprocessed pongamia oil is added to the production medium; and extracting the polyhydroxyalkanoates comprising a homopolymer comprising a 3-hydroxybutyrate (3HB) unit amongst other sole PHB polymers from within the bacteria.

Description

Technical Field

[0001] This invention relates to processes for producing biodegradable polymers from pongamia oil, a substitute carbon source for edible oil. Background Art

[0002] Environmental concerns about creating and disposing of traditional petrochemicalbased plastics are increasing, prompting increased interest in finding an affordable way to produce alternative materials. Therefore, creating biodegradable polymers or bioplastics from renewable sources is economically, environmentally, and scientifically significant. Bioplastics offer several advantages over petrochemical-based plastics due to their biodegrading ability and lack of toxicity. They require less energy to produce than petrochemical-based plastics also resulting in lower CO2 emissions. Utilising biodegradable plastics will decrease the amount of non-biodegradable solid waste in the environment and particularly, the oceans and waterways. Therefore, a strong motivation exists to discover improved techniques for producing biodegradable polymers.

[0003] Polyhydroxyalkanoates (PHAs), which include the hydroxybutyrate polymer, polyhydroxybutyrate (PHB), are natural polyesters that can be derived from microbial fermentation of carbon from lignocellulosic and other biomass feedstocks. They are often referred to as ‘biopolymers’ and used to create ‘bioplastics’ due to their non-fossil fuel (petrochemical-based) sources.

[0004] PHAs are biodegradable polyesters which are produced and stored inside a variety of bacteria as carbon and energy reserves when there is excess carbon, but limitations on other nutrients including nitrogen or phosphorus. These materials are significant because they can be biodegradable alternatives to replace the petrochemical-based plastics currently in common use including polypropylene and polyethylene.

[0005] PHAs are typically created using pure bacterial cultures, which involves a relatively costly fermentation process using sugars as a substrate under aseptic conditions. The carbon feedstocks are the primary factor contributing to the high overall production cost, making it difficult for PHAs to compete with the lower production costs of petrochemicalbased plastics. Therefore, a significant challenge is to develop an efficient method for producing PHAs using more affordable and renewable carbon feedstocks.

[0006] Current production of PHAs largely uses carbon sources comprising corn starch, sugarcane, potatoes, cassava, and plant oils such as soybean, canola, palm, olive, sunflower, and coconut. Poly(3-hydroxybutyrate), or P(3HB), produces almost twice the yield from plant oils compared to glucose due to their high carbon content per weight. However, using food or food-grade oils to produce bioplastics has the potential to disrupt or create shortages in the global food supply, and at the very least, increases prices for foods and oils using these agricultural products. Thus, it can be considered inefficient and even unethical to convert food sources into other commercial materials like bioplastics. Furthermore, recent food shortages, limited land availability for the food industry, and rising food demand have significantly increased plant oil prices.

[0007] Thus, there is a need for new processes for producing biodegradable polymers including PHAs that are cost-effective, efficient, and have less of an effect on the environment in their production and in their competition with crops for food.

[0008] The preceding discussion of the background art is intended to facilitate an understanding of the present invention only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was part of the common general knowledge as at the priority date of the application. Summary of Invention

[0009] Broadly, the invention relates to processes for producing biodegradable polymers from pongamia oil, an alternative carbon source for edible oil for food sources.

[0010] In a first aspect, the invention provides a process for producing polyhydroxyalkanoates, comprising the steps of: fermenting production medium comprising pongamia oil using bacteria from the Cupriavidus, Burkholderia, or Alcaligenes genera capable of producing polyhydroxyalkanoates; and extracting the polyhydroxyalkanoates from within the bacteria from the Cupriavidus, Burkholderia, or Alcaligenes genera.

[0011] In an embodiment, the pongamia oil is crude pongamia oil that is unprocessed.

[0012] In an embodiment, the pongamia oil comprises a sole carbon substrate for the bacteria from the Cupriavidus, Burkholderia, or Alcaligenes genera. The bacteria from the Cupriavidus, Burkholderia, or Alcaligenes genera preferably comprise bacteria of the Cupriavidus species, Burkholderia species, or Alcaligenes species. More preferably, the bacteria comprise Cupriavidus necator or Alcaligenes latus.

[0013] In an embodiment, the polyhydroxyalkanoates comprise a sole PHB polymer. The polyhydroxyalkanoates more preferably comprise a homopolymer comprising a 3-hydroxybutyrate (3HB) unit.

[0014] In an embodiment, the production medium is inoculated with the bacteria from the Cupriavidus, Burkholderia, or Alcaligenes genera.

[0015] In an embodiment, the bacteria from the Cupriavidus, Burkholderia, or Alcaligenes genera are first cultivated on a nutrient-rich (NR) agar plate. The nutrient-rich agar plate preferably comprises approximately 10 g / L meat extract, 10 g / L peptone, and 2 g / L yeast extract.

[0016] In an embodiment, a seed culture is produced by adding bacteria from the Cupriavidus, Burkholderia, and Alcaligenes genera on the nutrient-rich agar plate to growth medium comprising approximately 10 g / L meat extract, 10 g / L peptone, and 2 g / L yeast extract. The seed culture is preferably agitated at approximately 180 rpm at 30 °C for 15 hours.

[0017] In an embodiment, the seed culture is used to inoculate the production medium.

[0018] In an embodiment, the production medium comprises 20 g / L of filtered crude pongamia oil. The pongamia oil is preferably extracted from the seeds and clarified by filtering before addition to the production medium.

[0019] In an embodiment, the production medium comprises a restricted source of nitrogen. The production medium preferably comprises a restricted source of nitrogen comprising one or more selected from the group comprising: ammonia, ammonium salts, ammonium chloride, ammonium sulphate, ammonium phosphate, peptone, meat extract, yeast extract, urea, or corn steep liquor. More preferably, the production medium comprises approximately 10 g / L meat extract.

[0020] In an embodiment, the production medium comprises a restricted source of inorganic components. The production medium preferably comprises a restricted source of inorganic components comprising one or more selected from the group comprising: monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulphate, sodium chloride.

[0021] In an embodiment, the production medium comprises NaH2PO4^2H2O (5 g), Na2HPO4-12H2O (11.6 g), MgSO4-7H2O (0.39 g), K2SO4 (0.45 g), CaC^^O (0.06 g), peptone (1 g), meat extract (1 g), yeast extract (0.4 g), 2 g / L urea, and 20 g / L of filtered crude pongamia oil.

[0022] In an embodiment, the production medium is fermented at a controlled pH range of approximately 6.5-7.5, preferably optimizing microbial activity and enhancing biopolymer yield.

[0023] In a preferred embodiment, the concentration of pongamia oil in the production medium is maintained at approximately 20 g / L during fermentation.

[0024] In an embodiment, the production medium is fermented under aerobic conditions within a range of between approximately 30% - 45% dissolved oxygen in the production medium. The production medium is preferably fermented at under aerobic conditions at approximately 35% dissolved oxygen in the production medium.

[0025] In an embodiment, the production medium is agitated at between approximately 150 - 250 rpm. The production medium is preferably agitated at approximately 180 rpm.

[0026] In an embodiment, the production medium is maintained at 30 °C - 37 °C in a batch fermentation for 48-72 hours. The production medium is preferably maintained at approximately 30 °C in a batch fermentation for approximately 48 hours.

[0027] In an embodiment, the polyhydroxyalkanoates are extracted by: centrifuging the fermented production medium to produce a pellet of bacterial cells from the Cupriavidus, Burkholderia, or Alcaligenes genera; washing the pellet of bacterial cells with distilled water then drying the pellet of bacterial cells; extracting the polyhydroxyalkanoates from the dried pellet of bacterial cells by lysing the cells.

[0028] In an embodiment, the fermented production medium is centrifuged at approximately 8000rpm for 10 min at 4 °C.

[0029] In an embodiment, the pellet of bacterial cells is washed twice to remove residual media components.

[0030] In an embodiment, the washed pellet of bacterial cells is freeze-dried.

[0031] In an embodiment, the pellet of bacterial cells is pre-treated with enzymes before the step of extracting the polyhydroxyalkanoates from the dried pellet of bacterial cells, comprising: adding the pellet of bacterial cells to phosphate buffer to form a suspension; adding enzymes to the suspension and incubating the enzyme-containing suspension to disrupt the bacterial cells and the cellular components containing granules of polyhydroxyalkanoates; and centrifuging the enzyme-containing suspension to collect the pretreated and disrupted dried pellet of bacterial cells.

[0032] In an embodiment, the phosphate buffer is at a concentration of approximately 50 mg / mL at pH 7.4.

[0033] In an embodiment, the enzymes comprise pancreatin.

[0034] In an embodiment, the enzymes are added to the suspension at an enzyme to biomass ratio of approximately 2 to 3%. The enzymes are preferably added to the suspension at an enzyme to biomass ratio of approximately 2%.

[0035] In an embodiment, the suspension is incubated at approximately 50 °C for 60 to 75 min. The suspension is preferably incubated at approximately 50 °C for 60 min with gentle agitation.

[0036] In an embodiment, the polyhydroxyalkanoates are extracted from the dried pellet of bacterial cells by: adding the dried pellet of bacterial cells to a solvent system comprising ethylene carbonate (EC) and propylene carbonate (PC); heating and mixing the bacterial cells in solvent system; cooling the bacterial cells in solvent system and adding ethanol to induce the precipitation of polyhydroxyalkanoates; collecting and washing the precipitated polyhydroxyalkanoates; and drying the purified polyhydroxyalkanoates.

[0037] In an embodiment, the ethylene carbonate (EC) and propylene carbonate (PC) are in a 1:1 volumetric ratio.

[0038] In an embodiment, the bacterial cells are added to the solvent system at a 1:20 w / v ratio.

[0039] In an embodiment, the bacterial cells in solvent system is heated to between approximately 115 to 125 °C. The bacterial cells in solvent system is preferably heated to 120 °C under constant mixing by stirring.

[0040] In an embodiment, the bacterial cells in solvent system is cooled to room temperature and ethanol is added at three times the volume of the bacterial cells in solvent system.

[0041] In an embodiment, the precipitated polyhydroxyalkanoates are collected by centrifuging and washing three times with distilled water to purify the polyhydroxyalkanoates.

[0042] In an embodiment, the purified polyhydroxyalkanoates are dried at approximately 40 °C.

[0043] In an embodiment, the invention provides polyhydroxyalkanoates produced by a process as described herein. The polyhydroxyalkanoates preferably comprise a sole PHB polymer. The polyhydroxyalkanoates more preferably comprise a homopolymer comprising a 3-hydroxybutyrate (3HB) unit.

[0044] In a second aspect, the invention provides a process for producing polyhydroxyalkanoates as described herein, wherein the use of pongamia oil as a primary carbon source provides a dual benefit by contributing to carbon sequestration due to the carbon-absorbing properties of the pongamia plant, thereby qualifying the production process for carbon credits. This environmental benefit serves as an economic offset, reducing the overall production costs associated with biopolymer manufacturing.

[0045] Advantages

[0046] While there are now many known processes for producing PHAs as a substitute for petroleum based plastics, they compete against each other for future industrial viability in terms of efficiency of process, the costs involved, and importantly the environmental impact. In focusing on these three key factors, the inventor developed the new process of the invention.

[0047] Firstly, crude Pongamia oil without additional processing was utilised as a sole carbon feedstock substrate. This avoids the extraction process that would add cost to the process in purifying the oil, and avoids using extraction fluids, for example, hexane, and saponification steps to reduce the environmental impact in having to dispose of the waste from such processes.

[0048] Secondly, the inventor identified that bacteria from the Cupriavidus genera, specifically Cupriavidus sp., could accumulate up to 90% of its dry cell weight (DCW) as the sole polymer PHB (and not a copolymer) under nutrient-limiting conditions (e.g., nitrogen or phosphorus limitation) when carbon is in excess with the crude Pongamia oil substrate. Such a high level of efficiency in PHB production assists to reduce the production costs with the process of the invention.

[0049] Thirdly, the efficient process of the invention uses ‘green’ or ‘environmentally friendly’ solvents, for example, ethyl acetate, butyl acetate, and propylene carbonate for extraction of PHAs, including PHB. This avoids the often used sodium hypochlorite and chloroform in other such extraction processes of which disposing of at industrial scale are hazardous to the environment and persons handling them.

[0050] Additional advantages of the process of the invention include value-added products including protein-rich waste can be further processed into food, animal feed, fertilizers, or even specialty enzymes for various industries, adding value to byproducts and supporting a zero-waste model. Brief Description of Drawings

[0051] In order to provide a better understanding, embodiments of the present invention will be described, by way of example only, with reference to the accompanying drawings, in which: Figure 1. shows a schematic diagram illustrating the production of PHAs by Cupriavidus sp. through batch fermentation. Description of Embodiments

[0052] The following embodiments, given by way of non-limiting example only, are described in order to provide a more precise understanding of the subject matter of a preferred embodiment or embodiments. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above.

[0053] The invention relates to processes for producing PHAs and particularly PHB from pongamia oil.

[0054] Pongamia oil comes from the seeds of the Pongamia sp. plant. Although this plant was initially found in Indo-Malaysia, it is now widespread in India, Africa, Asia, northern Australia, the Pacific and Caribbean Islands, and certain parts of the United States. Pongamia is easy to grow, is established quickly, and is resistant to pests and drought. The Pongamia genus is part of the Fabaceae family and can produce various harmful substances, such as karanjin, pongamol, glabrin, and tannins. These toxins make the oil unsuitable for consumption, but it produces almost four to six times more oil than maize and is considered potentially useful in the production of high-quality biofuel.

[0055] Thus, pongamia oil as a substrate for producing biodegradable polymers using the process of the invention is beneficial as the pongamia plant grows quickly, is pest and drought resistant, its use for polymer production does not compete with food supply, can be used in an unrefined form and has a high oil yield, and is relatively low cost to produce. Thus, it enables the process of the invention to better compete in terms of cost against the current petroleum-based plastics.

[0056] The process of the invention also comprises ‘green’ extraction methods to extract and purify PHAs as described in more detail below, which are scalable, economical and environmental friendly.

[0057] The detailed description that follows provides a comprehensive understanding of the invention. However, individuals with ordinary skills in the relevant art will recognise that the invention can only be implemented with these details. Standard techniques, processes, and elements need to be explicitly described to elucidate the invention.

[0058] The embodiments are described in the following explanation, given as an example, and accompanied by drawings that are not to scale. 5

[0059] In the process of the invention, a biodegradable polymer is derived from raw (unrefined) pongamia oil and broken down by natural environmental enzymes, including depolymerase and lipase. The invention comprises bacterial fermentation in a fermenter to produce a homopolymer comprising a 3-hydroxybutyrate (3HB) unit.

[0060] The microorganisms that may be used in the process of the invention comprise any 10 microorganisms within the Cupriavidus, Burkholderia, and Alcaligenes genera that can produce the homopolymer comprising a 3-hydroxybutyrate (3HB) unit. Examples include Cupriavidus sp. And particularly Cupriavidus necator, Burkholderia sp, and Alcaligenes latus. These microorganisms are grown in a suitable culture medium using the naturally occurring crude, unrefined pongamia, oil, as the carbon feedstock. 15

[0061] Table 1 shows the result of a study measuring the fatty acid compositions of pongamia oil in mature seeds. The pongamia oil utilised in this study naturally possesses a composition of 84.45% palmitic, oleic, linoleic and erucic acids. No Mature seed 1 7.18 ± 0.13 2 3.32 ± 0.14 3 43.99 ± 0.92 4 17.38 ± 0.19 5 5.51 ± 0.06 6 0.78 ± 0.07 7 3.43 ± 0.15 8 2.48 ± 0.03 9 15.90 ± 0.61 10 -

[0062] Table 1. Fatty acid compositions of pongamia oil in mature pongamia seeds.

[0063] In the process of the invention, biodegradable polymer can be formed by growing the specially developed bacteria in a solution, allowing the polymer to form and build up within the microorganism or the medium, and then extracting the polymer from the cultured microorganisms or the solution.

[0064] EXAMPLES

[0065] The following examples provide a more detailed description of the present invention, and it should be noted that the examples do not restrict the scope of the invention.

[0066] Example 1 Production and extraction of PHB from C. necator using crude pongamia oil as a carbon feedstock.

[0067] Figure 1 shows a schematic diagram illustrating the production of PHAs by Cupriavidus sp. through batch fermentation.

[0068] In a first step of the process of the invention, Cupriavidus sp. was cultivated at 30 °C for 15 hours on a nutrient-rich (NR) medium agar plate comprising 10 g / L meat extract, 10 g / L peptone, and 2 g / L yeast extract.

[0069] The single carbon feedstock used for PHA production was crude pongamia oil sourced from India. Before being introduced into the PHAs production medium, specifically the P(3HB) production medium, the oil was extracted from the seeds and clarified by filtering.

[0070] In this respect, the fermentation medium consists of a carbon source the microorganism can utilise. Within this medium, the availability of a nitrogen source, inorganic salts, or another organic nutritional source has been restricted. The microorganism's growth and storage of PHAs depends on the carbon feedstock, the initial polymer synthesis material.

[0071] The nitrogen source may encompass ammonia, ammonium salts like ammonium chloride, ammonium sulphate, ammonium phosphate, peptone, meat extract, yeast extract, corn steep liquor, etc.

[0072] The inorganic components consist of monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulphate, sodium chloride, etc.

[0073] The culture of fermenting production medium and bacteria from the Cupriavidus, Burkholderia, and Alcaligenes genera is typically conducted under aerobic conditions with 30% - 45% dissolved oxygen and agitation at 150 - 250 rpm at 30 °C - 37 °C for 48-72 hours following induced expression. Throughout the fermentation process, the culture conditions are maintained at pH 7.0, and the concentration of pongamia oil maintained at 20 g / L. The synthesised polymer is recovered from the culture through extraction and purification.

[0074] Specifically in this example, P(3HB) was produced in a batch fermentation using a 3 L BioFlo 320 (Eppendorf, Australia) fermenter with a working volume of 1.35 L.

[0075] A 50 mL seed culture in a shake flask comprising growth medium was incubated for 15 hours at 30 °C while agitated at 180 rpm. Then, the 50 mL seed culture was introduced into 1.35 L of production medium (MM), consisting of NaH2PO4^2H2O (5 g), Na2HPO4^12H2O (11.6 g), MgSO4^7H2O (0.39 g), K2SO4 (0.45 g), CaCl2-2H2O (0.06 g), peptone (1 g), meat extract (1 g), yeast extract (0.4 g) and 20 g / L of filtered crude pongamia oil.

[0076] For a nitrogen source, urea (2 g / L) was utilised. The culture was maintained at 30 °C, 35 % dissolved oxygen, and pH 7. The concentration of pongamia oil was maintained at 20 g / L throughout the 48-hour fermentation. P(3HB) is extracted from the culture through centrifugation, washing with distilled water, using green solvents such as ethyl acetate, butyl acetate, ethylene carbonate, propylene carbonate with enzyme-assisted extraction, and finally freeze-drying.

[0077] The amount of P(3HB) was calculated using approximately 20 mg of freeze-dried cells that underwent methanolysis by heating at 100°C for 140 min in a solution containing 15% (v / v) sulphuric acid and 85% (v / v) methanol. The resulting hydroxyacyl methyl esters were then assessed using gas chromatography (GC).

[0078] Cupriavidus sp. generated large quantities of P(3HB) using pongamia oil. After the 48-hour fermentation, the total harvested biomass measured 12 g / L.

[0079] The culture's pH was maintained at approximately 7.0. The cells efficiently utilised the supplied carbon feedstock, which was evidenced by the significant decrease in residual oil concentration. Hence, based on these studies, pongamia oil can serve as a carbon feedstock as a replacement to food-grade oils for the production of P(3HB) by Cupriavidus sp.

[0080] Example 2 - Enzymatic-solvent extraction of PHB from C. necator using pancreatin and ethylene carbonate:propylene carbonate mixture.

[0081] In a first step, bacterial biomass was carefully prepared, where C. necator cells were cultivated under optimal conditions to maximize PHB accumulation. Following cultivation, the cells were harvested through centrifugation at 8000 rpm for 10 min at 4 °C, with two subsequent washing steps using distilled water to remove residual media components. The cleaned biomass was then freeze-dried to obtain a dry, stable starting material for the extraction process.

[0082] The extraction protocol used a two-step approach, initiating with an enzymatic pretreatment phase. The dried biomass was resuspended in phosphate buffer (pH 7.4) at a concentration of 50 mg / mL, creating a uniform suspension. Pancreatin, a mixture of digestive enzymes including proteases, lipases, and amylases, was added to the suspension at an optimized enzyme-to-biomass ratio of 2%. This mixture was then incubated at 50 °C for one hour with gentle agitation, allowing the enzymatic cocktail to effectively disrupt the cellular components surrounding the PHB granules. The pretreated biomass was subsequently collected through centrifugation, preparing it for the solvent extraction phase.

[0083] The solvent extraction step used a mixture of ethylene carbonate (EC) and propylene carbonate (PC) in a 1:1 volumetric ratio. This solvent system was selected for its high selectivity towards PHB, and its relatively benign environmental impact compared to traditional halogenated solvents. The pretreated biomass was added to the EC:PC mixture at a 1:20 weight-to-volume (w / v) ratio and heated to 120 °C under constant stirring for 30 min. This elevated temperature was crucial for efficient PHB solubilization while remaining below the degradation threshold of the polymer. After the extraction period, the mixture was cooled to room temperature, and ethanol was added as an antisolvent at three times the volume of the EC:PC mixture, inducing the precipitation of PHB. The precipitated polymer was collected through centrifugation and subjected to three washing cycles with distilled water to remove any residual solvent. Finally, the purified PHB is dried at 40 °C to obtain the final product.

[0084] The extraction method demonstrated high efficiency, consistently achieving PHB recovery yields of approximately 80%. These results compare favourably with traditional extraction methods, positioning this approach as a viable alternative for industrial applications. Extensive investigation of process parameters revealed several critical factors affecting extraction performance. The enzyme concentration showed a significant impact on both yield and purity, with the optimal range identified as 2% to 3% enzyme-to-biomass ratio. The enzymatic treatment time demonstrated a positive correlation with yield up to 75 min, beyond which no significant improvements were observed. The temperature of the EC:PC mixture proved to be a crucial parameter, with the optimal range of 115-125 °C balancing maximal PHB solubilization with minimal thermal degradation. Extraction time showed a positive correlation with yield up to 35 min, after which the benefits plateaued.

[0085] The success of this extraction method can be attributed to the synergistic action of enzymatic pretreatment and selective solvent extraction. The pancreatin enzyme cocktail effectively disrupts cellular components through multiple mechanisms: proteases break down cell proteins, lipases degrade cell membranes, and amylases hydrolyse carbohydrates. This comprehensive degradation of non-PHB cellular material significantly enhances the accessibility of PHB granules for subsequent solvent extraction. The high dielectric constants of EC and PC enable selective solubilization of PHB, while the addition of ethanol as an antisolvent facilitates efficient polymer recovery through phase separation.

[0086] From an economic perspective, the method presents a balanced profile. While the cost of enzymes and the EC:PC solvent mixture exceeds that of traditional solvents, these expenses are partially offset by reduced energy requirements and the potential for solvent recycling, with up to 90-95% recovery possible. The method also demonstrates promising scalability, with the potential for linear scale-up and adaptation to continuous processing, although further optimization would be beneficial for industrial implementation. When compared to established extraction methods, this approach offers several advantages. Unlike chloroform extraction, it significantly reduces environmental and health risks while maintaining comparable yield and purity. Compared to supercritical CO2 extraction, it offers higher yields and better scalability, although with a slightly higher environmental impact. The method effectively balances performance, environmental considerations, and economic viability.

[0087] Drawbacks of Supercritical CO2 for PHB Extraction: Economic viability issues High capital and operating costs: Initial equipment investment: $500,000-$2,000,000 for industrial scale1; Operating costs 2-3 times higher than conventional solvent extraction2; Energy costs account for 45-60% of total operating expenses3. Poor return on investment: Payback period often exceeds 5 years4; Limited market for high-purity PHB doesn't justify premium extraction costs1. The high pressure equipment required for supercritical fluid extraction makes it less economically viable compared to conventional solvent extraction methods1. Technical limitations Lower extraction efficiency: Maximum yield typically around 80% compared to >95% for chloroform extraction5; Requires multiple extraction cycles, increasing processing time6. Limited scalability: Difficult to maintain uniform conditions in large vessels7; Non-linear scaling of extraction parameters8. Scale-up of supercritical CO2 extraction processes is complicated by the non-linear behaviour of supercritical fluids in larger vessels8. Process complications Time-intensive: Longer extraction times compared to solvent extraction9: SCF CO2: 4-8 hours; Chloroform: 1-2 hours. Complex parameter optimization: Narrow operating window for optimal conditions10; Multiple interdependent variables make optimization difficult7. The optimization of supercritical CO2 extraction parameters is complex and time consuming, requiring extensive experimentation7. Quality concerns Thermal degradation: Operating temperatures can cause PHB degradation11; Molecular weight reduction observed in some cases12. Incomplete extraction: Residual PHB in biomass requires additional processing13; May affect overall product purity14. Thermal degradation of PHB during supercritical CO2 extraction can lead to reduced molecular weight and altered polymer properties12. Operational challenges Safety concerns: High pressure operation (100-400 bar) poses safety risks15; Requires specialized training and safety protocols15. Maintenance issues: Frequent replacement of seals and valves16; Regular system downtime for maintenance16. The high pressure nature of supercritical CO2 extraction needs rigorous safety measures and regular maintenance, increasing operational complexity15. Traditional solvent extraction methods, while having their own drawbacks, generally offer a more economically viable and technically straightforward approach to PHB recovery9. Green extraction outperforms supercritical CO2 extraction in terms of yields and scalability but has a somewhat larger environmental effect. The strategy strikes an appropriate balance between performance, environmental considerations, and economic viability.

[0088] The disclosure in the above Detailed Description has revealed the preferred embodiment of the present invention and its benefits. However, it is essential to note that the invention is not restricted to this embodiment but is defined solely by the intent and extent of the appended claim.

[0089] Optional embodiments of the present invention may also be said to broadly consist in the parts, elements and features referred to or indicated herein, individually or collectively, in any or all combinations of two or more of the parts, elements or features, and wherein specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0090] It is to be appreciated that reference to "one example" or "an example" of the invention is not made in an exclusive sense. Accordingly, one example may exemplify certain aspects or embodiments of the invention, whilst other aspects or embodiments are exemplified in a different example. These examples are intended to assist the skilled person in performing the invention and are not intended to limit the overall scope of the invention in any way unless the context clearly indicates otherwise.

[0091] It is to be understood that the terminology employed above is for the purpose of description and should not be regarded as limiting. The described embodiment is intended to be illustrative of the invention, without limiting the scope thereof. The invention is capable of being practised with various modifications and additions as will readily occur to those skilled in the art.

[0092] Various substantially and specifically practical and useful exemplary embodiments of the claimed subject matter are described herein, textually and / or graphically, including the best mode, if any, known to the inventors for carrying out the claimed subject matter. Variations (e.g. modifications and / or enhancements) of one or more embodiments described herein might become apparent to those of ordinary skill in the art upon reading this application.

[0093] The inventor(s) expects skilled artisans to employ such variations as appropriate, and the inventor(s) intends for the claimed subject matter to be practiced other than as specifically described herein. Accordingly, as permitted by law, the claimed subject matter includes and covers all equivalents of the claimed subject matter and all improvements to the claimed subject matter. Moreover, every combination of the above-described elements, activities, and all possible variations thereof are encompassed by the claimed subject matter unless otherwise clearly indicated herein, clearly and specifically disclaimed, or otherwise clearly contradicted by context.

[0094] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate one or more embodiments and does not pose a limitation on the scope of any claimed subject matter unless otherwise stated. No language in the specification should be construed as indicating any non-claimed subject matter as essential to the practice of the claimed subject matter.

[0095] The use of words that indicate orientation or direction of travel is not to be considered limiting. Thus, words such as "front", "back", "rear", "side", "up", down", "upper", "lower", "top", "bottom", "forwards", "backwards", "towards", "distal", "proximal", "in", "out" and synonyms, antonyms and derivatives thereof have been selected for convenience only, unless the context indicates otherwise. The inventor(s) envisage that various exemplary embodiments of the claimed subject matter can be supplied in any particular orientation and the claimed subject matter is intended to include such orientations.

[0096] The use of the terms "a", "an", "said", "the", and / or similar referents in the context of describing various embodiments (especially in the context of the claimed subject matter) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "including," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

[0097] Moreover, when any number or range is described herein, unless clearly stated otherwise, that number or range is approximate. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value and each separate sub-range defined by such separate values is incorporated into the specification as if it were individually recited herein. For example, if a range of 1 to 10 is described, that range includes all values there between, such as for example, 1.1, 2.5, 3.335, 5, 6.179, 8.9999, etc., and includes all sub-ranges there between, such as for example, 1 to 3.65, 2.8 to 8.14, 1.93 to 9, etc.

[0098] Accordingly, every portion (e.g., title, field, background, summary, description, abstract, drawing figure, etc.) of this application, other than the claims themselves, is to be regarded as illustrative in nature, and not as restrictive; and the scope of subject matter protected by any patent that issues based on this application is defined only by the claims of that patent. Citation List

[0099] Non-Patent Literature 1. Perez, M., et al. (2016). Economic assessment of polyhydroxybutyrate (PHB) extraction methods. Journal of Supercritical Fluids, 118, 32-40. 2. Thompson, R. C., & Moore, C. J. (2015). Comparative analysis of PHB extraction techniques. Biotechnology Progress, 31(4), 1023-1031. 3. Khosravi-Darani, K. (2017). Energy optimization in supercritical fluid extraction of biopolymers. Chemical and Biochemical Engineering Quarterly, 31(1), 1-19. 4. Zhang, X., et al. (2018). Financial viability of green extraction methods for PHB recovery. Bioresource Technology, 256, 214-221. 5. Aramvash, A., et al. (2015). Comparison of solvent-based and supercritical extraction of PHB. Iranian Journal of Biotechnology, 13(3), 27-32. 6. Ramaswamy, S., et al. (2015). Efficiency analysis of PHB extraction cycles. Separation and Purification Technology, 151, 61-69. 7. Johnson, M., & Zhang, H. (2018). Challenges in industrial-scale PHB extraction. Biotechnology Advances, 36(5), 1314-1327. 8. Ramaswamy, S., et al. (2015). Scale-up complications in supercritical fluid extraction. Industrial & Engineering Chemistry Research, 54(15), 4035-4042. 9. Lee, S. Y., et al. (2016). Time optimization of PHB extraction methods. Biotechnology and Bioengineering, 113(7), 1405-1416. 10. Kim, B. S., et al. (2015). Parameter sensitivity in supercritical CO2 extraction of biopolymers. Macromolecular Research, 23(6), 545-552. 11. Yasotha, K., et al. (2016). Thermal effects in biopolymer extraction. Polymer Degradation and Stability, 128, 39-45. 12. Chen, G. Q., et al. (2017). Molecular weight analysis of supercritical CO2 -extracted PHB. Biomacromolecules, 18(6), 1888-1899. 13. Madkour, M. H., et al. (2015). Efficiency of PHB recovery methods. Journal of Polymers and the Environment, 23(1), 72-82. 14. Koller, M., et al. (2017). Product quality in biological polymer extraction. Bioengineering, 4(2), 36. 15. Wilson, K., & Smith, J. (2019). Safety considerations in high-pressure bioprocessing. Industrial & Engineering Chemistry Research, 58(15), 5885-5892. 16. Davis, R., et al. (2015). Maintenance requirements for supercritical extraction systems. Chemical Engineering Science, 125, 21-31.

Claims

1. A process for producing polyhydroxyalkanoates, comprising the steps of:fermenting production medium comprising pongamia oil using bacteria from the Cupriavidus, Burkholderia, or Alcaligenes genera capable of producing polyhydroxyalkanoates; andextracting the polyhydroxyalkanoates from within the bacteria from the Cupriavidus, Burkholderia, or Alcaligenes genera.

2. A process according to claim 1, wherein the pongamia oil is crude pongamia oilthat is unprocessed.

3. A process according to claim 2, wherein the pongamia oil comprises a solecarbon substrate for the bacteria from the Cupriavidus, Burkholderia, or Alcaligenes genera.

4. A process according to any one of claims 1 to 3, wherein the bacteria from theCupriavidus, Burkholderia, or Alcaligenes genera comprises bacteria of the Cupriavidus species, Burkholderia species, or Alcaligenes species.

5. A process according to claim 4, wherein the bacteria comprises Cupriavidusnecator or Alcaligenes latus.

6. A process according to any one of the preceding claims, wherein the productionmedium comprises approximately 20 g / L of filtered crude pongamia oil.

7. A process according to claim 6, wherein the pongamia oil is extracted from theseeds and clarified by filtering before addition to the production medium.

8. A process according to any one of the preceding claims, wherein the productionmedium comprises a restricted source of nitrogen comprising one or more selected from the group comprising: ammonia, ammonium salts, ammonium chloride, ammonium sulphate, ammonium phosphate, peptone, meat extract, yeast extract, urea, or corn steep liquor.

9. A process according to any one of the preceding claims, wherein the productionmedium comprises a restricted source of inorganic components comprising one or more selected from the group comprising: monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulphate, sodium chloride.

10. A process according to any one of the preceding claims, wherein the production medium comprises NaH2PO4^2H2O (5 g), Na2HPO4^12H2O (11.6 g), MgSO4^7H2O (0.39 g), K2SO4 (0.45 g), CaC^^O (0.06 g), peptone (1 g), meat extract (1 g), yeast extract (0.4 g), 2 g / L urea, and 20 g / L of filtered crude pongamia oil.

11. A process according to any one of the preceding claims, wherein the production medium is fermented at under aerobic conditions within a range of between approximately 30% - 45% dissolved oxygen in the production medium.

12. A process according to any one of the preceding claims, wherein the production medium is maintained at 30 °C - 37 °C in a batch fermentation for 48-72 hours.

13. A process according to any one of the preceding claims, wherein the polyhydroxyalkanoates are extracted by:centrifuging the fermented production medium to produce a pellet of bacterial cells from the Cupriavidus, Burkholderia, or Alcaligenes genera;washing the pellet of bacterial cells with distilled water then drying the pellet of bacterial cells;extracting the polyhydroxyalkanoates from the dried pellet of bacterial cells by lysing the cells.

14. A process according to claim 13, wherein the pellet of bacterial cells is pretreated with enzymes before the step of extracting the polyhydroxyalkanoates from the dried pellet of bacterial cells, comprising:adding the pellet of bacterial cells to phosphate buffer to form a suspension;adding enzymes to the suspension and incubating the enzyme-containing suspension to disrupt the bacterial cells and the cellular components containing granules of polyhydroxyalkanoates; andcentrifuging the enzyme-containing suspension to collect the pretreated and disrupted dried pellet of bacterial cells.

15. A process according to claim 14, wherein the enzymes comprise pancreatin.

16. A process according to any one of claims 13 to 15, whereinpolyhydroxyalkanoates are extracted from the dried pellet of bacterial cells by:adding the dried pellet of bacterial cells to a solvent system comprising ethylene carbonate (EC) and propylene carbonate (PC);heating and mixing the bacterial cells in solvent system;cooling the bacterial cells in solvent system and adding ethanol to induce the precipitation of polyhydroxyalkanoates;collecting and washing the precipitated polyhydroxyalkanoates; anddrying the purified polyhydroxyalkanoates.

17. A process according to claim 16, wherein the ethylene carbonate (EC) and propylene carbonate (PC) are in a 1:1 volumetric ratio and wherein the bacterial cells are added to the solvent system at a 1:20 w / v ratio.

18. Polyhydroxyalkanoates produced by a process according to any one of claims 1 to 17.

19. The polyhydroxyalkanoates according to claim 18, comprising a sole PHB polymer.

20. The polyhydroxyalkanoates according to claim 18 or claim 19, comprising a homopolymer comprising a 3-hydroxybutyrate (3HB) unit.

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