Method for producing 3-hydroxypropionic acid polymer using co-culturing process and use thereof

Abstract

A method for producing a 3-hydroxy propionic acid polymer includes fermenting glycerol with a first strain to produce 3-hydroxypropionic acid, and polymerizing the 3-hydroxypropionic acid with a second strain to produce a 3-hydroxypropionic acid polymer. and controlling reaction rates of the fermenting and the polymerizing by adjusting amounts of the substrate and the carbon source supplied to a bioreactor, thereby simplifying the 3-hydroxypropionic acid polymer production process, significantly reducing process costs, and improving process stability by reducing the amounts of acidic and basic solutions used. The fermenting and the polymerizing are performed in a single bioreactor.

Description

[0001] METHOD FOR PRODUCING 3-HYDROXYPROPIONIC ACID POLYMER USING CO-CULTURING PROCESS AND USE THEREOF

[0002] BACKGROUND

[0003] 1 Technical Field

[0004]

[0001] The present invention relates to a method for producing a 3 -hydroxy propionic acid polymer using a co-culturing process and the use thereof.

[0005] 2. Background Art

[0006]

[0002] 3-Hydroxypropionic acid (3-HP) is an isomer of lactic acid (2-hydroxypropionic acid) and has a carboxyl group and a hydroxyl group at opposite ends, making it a useful material that can be converted into various chemicals, such as 1,3 -propanediol, acrylic acid, acry lamide, and polymers. For this reason, in 2004, the United States Department of Energy' (DoE) identified 3-hydroxypropionic acid as one of the promising platform chemicals that can be produced from biomass. Among the 12 selected platform chemicals by DoE, 3-HP was ranked at 3rd position as it can be used as substrate / raw material to produce high impact commercially important chemicals. Acrylic acid, one of the derivatives of 3-HP, has high market value due to its applications in coatings, adhesives, additives, and diapers. Like acrylic acid, poly 3 -hydroxy propionate (P3HP) is another derivative of 3-HP, which is a promising homopolymer with unique properties. This polymer is highly degradable in various natural environments like soil, fresh or marine water, home compost, etc. Therefore, P3HP production has the highest importance for commercialization to replace non-degradable conventional plastics.

[0007] [3] Few studies have been reported to produce P3HP by fermentation method using 1,3-PDO or CO2, etc., as feed stocks. However, its production is not well documented from typical feed stocks like glucose or glycerol. Generally, in bioprocesses, a single microorganism (homogeneous cultures) is used to produce specific chemicals or materials for precise process control and consistent production. There is very' limited success in bioprocess operations to produce bioproducts using multicultures or co-cultures. In specific to P3HP production, monoculture is ven difficult as the microorganisms which can produce 3-HP, monomer of P3HP, have no ability to accumulate biopolymers. On the other hand, microorganisms with the ability to produce and accumulate P3HP fail to produce 3-HP monomers in commercially viable methods (limitations in coenzyme B12 production and low' 3-HP yield, titer and productivity). Therefore, it was identified that P3HP can be produced by two different approaches. 1. Two bioreactor systems, in the first bioreactor, 3-HP is produced as monomer and used as a substrate for polymerization in the second bioreactor where specific microorganisms are cultured to accumulate P3HP polymer. 2. One bioreactor system, where two microorganisms are co-cultured in one bioreactor. One microorganism produces the monomer, and the second microorganism will readily use this monomer for polymerization.

[0008]

[0004] In two bioreactor systems after producing 3 HP monomer in first bioreactor, it should be concentrated, acidified followed by salt separation before using as feed into the second bioreactor. This long process is tedious and expensive. On the other hand, in this method, the salts generated due to acidification are not completely removed, there is a high possibility' that the spargers are blocked and limit the air supply. This will result in very low P3HP synthesis as P3HP is a typical aerobic process. In addition, the use of acidic substrate causes corrosion to the bioreactors, making the process unsafe, and results in cell death, if not, have a negative impact on the strain health. These process complications result in increasing the production cost and, making it difficult to commercialize the process using t 'o bioreactor systems. One the other hand, the second method, co-culturing the two microorganisms in one bioreactor, perhaps increase P3HP, productivity', yield, titer of target product along with significantly reducing the production cost. This will increase the possibilities of commercializing P3HP production.

[0009] [5] Accordingly, the inventors have conducted research to solve the problems existing m the conventional 3 -hydroxy propionic acid polymer production method and have completed the present invention, which enables the simultaneous production of a 3-hydroxypropionic acid monomer and a 3-hydroxypropionic acid polymer using a co¬ culture process in which a 3-hydroxypropionic acid monomer production strain and a 3-hy dr oxy propionic acid polymer production strain are rown in a single bioreactor.

[0010] [6] Co-culture strains may present several challenges and limitations during the process. For example, two strains within a single culture reactor may compete for limited nutrients, inhibiting the growth of the other strain. To overcome this limitation, medium nutrients were designed very carefully as it has immense impact on controlling the cell performance. To this end, the growth and productivity of two genetically modified strains in one bioreactor were controlled to make each strains utilize a specific and unique carbon source by each microorganism. For example, one strain utilizes glucose exclusively as a carbon source for growth and survival, while the second microorganism cannot utilize this carbon source. In this way, the growth of the strains, production of target molecules, and secretion of toxic substances can be controlled carefully, if not precisely. 3-HP production can be controlled by the first strain, and 3-HP polymer production can be controlled by the second strain through substrates, carbon sources, and initial inoculum concentrations. A comprehensive control method was developed which is highly essential in co-culture process to enhance the efficiency of target product formation.

[0011] SUMMARY

[0012] [7] An object of the present invention is to provide a method for producing a 3-hydroxypropionic acid polymer using a co-culture process.

[0008] Another object of the present invention is to provide a 3-hydroxypropionic acid polymer produced by the method using a co-culture process.

[0013]

[0009] Yet another object of the present invention is to provide an oligomer, monomer, film, or coated packaging material manufactured from a 3-hydroxypropionic acid polymer produced by a co-culture process.

[0014]

[0010] 1. A method for producing a 3-hydroxypropionic acid polymer by co-culturing a first strain and a second strain, the method including: a first step of fermenting a substrate with the first strain to produce -hydroxy propionic acid, and a second step of polymerizing the 3-hydroxypropionic acid with the second strain to produce a 3 -hydrox propionic acid polymer, wherein the first step and the second step are performed in a single bioreactor; and a step of controlling reaction rates of the first step and the second step by adjusting an amount of the substrate supplied to the bioreactor.

[0015]

[0011] 2. The method for producing a 3-hydroxypropionic acid polymer according to claim 1. further including a step of controlling the reaction rate by additionally inoculating the first strain or the second strain into the bioreactor

[0016]

[0012] 3. The method for producing a 3-hydroxypropionic acid polymer according to claim 1, further including a step of monitoring a pH in the bioreactor to increase or decrease the amount of the substrate supplied or to additionally inoculate the first strain or the second strain.

[0017]

[0013] 4. The method for producing a 3-hydroxypropionic acid polymer according to claim 1, wherein the first strain is a strain belonging to any one genus selected from the group consisting of Escherichia, Klebsiella, and Pseudomonas.

[0018]

[0014] 5. The method for producing a 3-hydroxypropionic acid polymer according to claim 1, wherein the second strain is a strain belonging to any one genus selected from the group consisting of Escherichia, Cupriavidus, and Pseudomonas.

[0015] 6. The method for producing a 3-hydroxypropionic acid polymer according to claim 1, wherein the substrate for producing 3-hydroxypropionic acid is glycerol or glucose.

[0019]

[0016] 7. The method for producing a 3-hydroxypropionic acid polymer according to claim 1, further including a step of controlling a growth rate of the first strain or the second strain by adjusting an amount of a carbon source supplied to the first strain or the second strain

[0020]

[0017] 8. The method for producing a 3-hydroxypropionic acid polymer according to claim 7, wherein the carbon source is at least one selected from the group consisting of gluconate, glucose, citric acid, and glycerol.

[0021]

[0018] 9, The method for producing a 3-hydroxypropionic acid polymer according to claim 1, wherein the second strain is a recombinant strain in which at least one gene selected from the group consisting of acs, phaC, and phaPl genes is introduced into an Escherichia strain,

[0022]

[0019] 10. The method for producing a 3-hydroxypropionic acid polymer according to claim 1, further including a step of inoculating the first strain and the second strain into the bioreactor in a ratio of 100: 1 to 1: 100 (% w' / v).

[0023]

[0020] 11. The method for producing a 3-hydroxypropionic acid polymer according to claim 7, wherein the carbon source of the first strain and the carbon source of the second strain are in a ratio of 100:1 to 1:100 (% w / v) in the bioreactor.

[0024]

[0021] 12. The method for producing a 3-hydroxypropionic acid polymer according to claim 1, further including a step of isolating and purifying the 3-hydroxypropionic acid polymer.

[0025]

[0022] 13 A 3-hydroxypropionic acid polymer having an average molecular weight of 100,000 to 1,000,000 g / mol. produced according to the method of any one of the above 1 to 12.

[0023] 14. The 3 -hydroxy propionic acid polymer according to claim 13, wherein the 3-hy dr oxy propionic acid polymer has a melting point of 70 to 140 °C, a glass transition temperature of -30 to -10 °C. a tensile strength of 10 to 50 MPa, and an elongation of 400 to 800%.

[0026]

[0024] 15. A product including the 3-hydroxypropionic acid polymer of claim 13,

[0025] 16. The product according to claim 15, wherein the product is any one selected from the group consisting of a film, a film packaging material, a coated packaging material, a fiber, and aspherical powder.

[0027]

[0026] 17. The product according to claim 15, wherein the product has a diameter of 1 pm to 100 pm.

[0028]

[0027] 18. A method for producing 3-hydroxypropionic acid, including the steps of: dissolving the 3-hydroxypropionic acid polymer of claim 13 in an aqueous solvent and mixing it with an acidic solution to prepare a mixture; and treating the mixture with a basic solution to induce a neutralization reaction,

[0029]

[0028] 19. A method for producing methyl 3-hydrox propionic acid, including the steps of: dissolving the 3-hydroxypropionic acid polymer of claim 13 in a methanol solvent and mixing it with an acidic solution to prepare a mixture; and treating the mixture with a basic solution to induce a neutralization reaction.

[0030]

[0029] 20. A method for producing ethyl 3-hydroxypropionic acid, including: dissolving the 3-hydroxypropionic acid polymer of claim 13 in an ethanol solvent and mixing it with an acidic solution to prepare a mixture; and treating the mixture with a basic solution to induce a neutralization reaction.

[0031]

[0030] The present invention may simplify the 3-hydroxypropionic acid polymer production process.

[0031] The present invention may reduce the two batch steps of the existing process to a single batch step through the use of co-cuhure. thereby simplifying the production process.

[0032]

[0032] The present invention may reduce the use of acidic and basic solutions, thereby enhancing process stability' and reducing production costs.

[0033]

[0033] Since the present invention does not use acidic solutions, it may prevent corrosion of the bioreactor and prevent the death of strains

[0034]

[0034] The present invention may reduce the amount of basic solutions used, thereby reducing the salt content in the medium, increasing the purity of the 3-hydroxypropionic acid polymer, and enhancing the possibility of water reuse.

[0035]

[0035] Since the present invention does not use acidic 3-hydroxypropionic acid used in the existing process, it may minimize the toxicity of 3-hydroxypropionic acid.

[0036]

[0036] The present invention effectively prevents contamination caused by external microorganisms, thereby enhancing process stability.

[0037]

[0037] The present invention may increase the molecular weight and productivity of 3-hydroxypropionic acid polymers.

[0038]

[0038] The present invention may optimize the production route for 3-hydroxypropionic acid polymers simply by adjusting process conditions, without requiring additional genetic modification of the strain.

[0039]

[0039] The present invention may optimize the process for producing a 3-hydroxy propionic acid polymer by controlling the carbon source supply and strain inoculation amount.

[0040]

[0040] The 3-hydroxypropionic acid polymer of the present invention may be used to produce oligomers, monomers, films, or coated packaging materials.

[0041]

[0041] The 3-hydroxypropionic acid polymer of the present invention is biodegradable and environmentally friendly, making it an eco-friendly alternative.

[0042] The 3-hydroxypropionic acid polymer of the present invention may be used as a high-performance polyester by forming a copolymer with other monomers,

[0042]

[0043] The film packaging material manufactured from the 3-hydroxypropionic acid polymer of the present invention has oxygen barrier properties, water resistance, and low-temperature stability.

[0043]

[0044] The coated packaging material manufactured from the 3-hydroxypropionic acid polymer of the present invention has water resistance and flame retardancy.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS

[0045]

[0045] FIG. 1 is an overall schematic view illustrating the production of a 3-hydroxypropionic acid polymer using co-culture.

[0046]

[0046] FIG. 2 is a view comparing the monoculture and co-culture processes.

[0047]

[0047] FIG. 3 is a graph illustrating the production of a 3-hydroxypropionic acid polymer using co-culture.

[0048]

[0048] FIGS. 4A and 4B are graphs comparing the molecular weights of 3-hydroxypropionic acid polymers produced using monoculture and co-culture.

[0049]

[0049] FIG. 5 shows the NMR analysis results of the 3-hydroxypropionic acid polymer.

[0050]

[0050] FIG. 6 is a graph comparing the tensile strength of the 3-hydrox propionic acid polymer with other polymers.

[0051]

[0051] FIGS. 7A and 7B show the oxygen permeability and moisture permeability measurements of films produced from the 3-hydroxypropionic acid polymer.

[0052]

[0052] FIG. 8 shows the results of a waterproofing test of a film packaging material manufactured from the 3-hydroxypropionic acid polymer.

[0053]

[0053] FIG. 9 shows the results of a low-temperature stability test of the film packaging material manufactured from the 3-hydroxypropionic acid polymer.

[0054] FIG. 10 shows the results of a water resistance test of a coated packaging material manufactured from the 3-hydroxypropionic acid polymer

[0054]

[0055] FIG. 11 shows the results of a flame retardancy test of the coated packaging material manufactured from the 3-hydroxypropionic acid polymer.

[0055]

[0056] FIGS. 12A and 12B show the results of a test of the physical properties of a fiber manufactured from the 3-hydroxypropionic acid polymer.

[0056]

[0057] FIGS 13A and 13B show the physical properties of a powder mixture manufactured from the 3-hydroxypropionic acid oligomer.

[0057]

[0058] FIG. 14 shows the yields of monomers manufactured from the 3-hydroxypropionic acid polymer and the 3-hydroxypropionic acid oligomer.

[0058]

[0059] FIGS. 15A to 15F show the results of a concentration analysis of monomers manufactured from the 3-hydroxypropionic acid polymer and the 3-hydroxypropionic acid oligomer. Specifically, FIG. I5A shows the HPLC peak pattern analysis of 3 -HP manufactured from the 3-HP oligomer, FIG. 15B shows the HPLC peak pattern analysis of 3-HP manufactured from the 3-HP polymer, FIG. 15C shows the GC peak pattern analysis of M3-HP manufactured from the 3-HP oligomer, FIG. 15D shows the GC peak pattern analysis of M3-HP manufactured from the 3-HP polymer, FIG. 15E shows the GC peak pattern analysis of E3-HP manufactured from the 3-HP oligomer, and FIG. 15F shows the GC peak pattern analysis of E3-HP manufactured from the 3-HP polymer.

[0059] DETAILED DESCRIPTION

[0060]

[0060] The present invention provides a method for producing a 3 -hydroxypropionic acid polymer.

[0061]

[0061] The present invention provides a method for producing a 3-hydroxypropionic acid polymer by co-culturing a first strain and a second strain, the method including a first step of fermenting a substrate with the first strain to produce 3-hydroxypropionic acid, and a second step of polymerizing the 3-hydroxypropionic acid with the second strain to produce a 3-hydroxypropionic acid polymer, wherein the first step and the second step are performed in a single bioreactor; and a step of controlling reaction rates of the first step and the second step by adjusting an amount of the substrate supplied to the bioreactor.

[0062]

[0062] The term "co-culture” refers to a culture method that enhances biological processes through interactions between two or more microbial species by growing them together in the same culture environment.

[0063]

[0063] The production method of the present invention may further include a step of controlling the reaction rate by additionally inoculating the first strain or the second strain into the bioreactor.

[0064]

[0064] The production method of the present invention may further include a step of monitoring a pH in the bioreactor to increase or decrease the amount of the substrate supplied or to additionally inoculate the first strain or the second strain.

[0065]

[0065] In the production method of the present invention, the first strain may be a strain belonging to any one genus selected from the group consisting of Escherichia, Klebsiella, and Pseudomonas.

[0066]

[0066] In one embodiment, the first strain is a Pseudomonas strain.

[0067]

[0067] Pseudomonas strains may produce vitamin B12 on their own, enabling the production of a 3-hydroxypropionic acid monomer and 1.3 -propanediol without supplementing vitamin B12 In addition. Pseudomonas strains produce fewer organic acid byproducts, including acetate and lactate, enabling the production of high-purity 3-hydroxypropionic acid monomer.

[0068]

[0068] In one embodiment, the first strain may be a Klebsiella strain that has been genetically recombined to produce vitamin B12. Genetic recombination of Klebsiella strains may be performed using methods known in the art. For example, the genetic recombination of a Klebsiella strain may be performed using the method disclosed in Somasundar Ashok et al., Biotechnol. Bioeng 2013:110: 511-524.

[0069]

[0069] In one embodiment, the first strain may be an Escherichia strain that has been genetically recombined to produce vitamin B12. Genetic recombination of Escherichia strain may be performed using methods known in the art. For example, the genetic recombination of an Escherichia strain may be performed using the method disclosed in Huan Fang et al, Nature Communications, (2018) 9:4917.

[0070]

[0070] In one embodiment, the first strain may be a genetically recombinant strain capable of producing a 3 -hydroxy propionic acid monomer using a pathway that does not utilize vitamin B12.

[0071]

[0071] In the production method of the present invention, the second strain may be a strain belonging to any one genus selected from the group consisting of Escherichia, Cupriavidus, and Pseudomonas.

[0072]

[0072] In one embodiment, the second strain may be a wild-type Escherichia strain. Wild-type Escherichia strains have a rapid growth rate and an excellent ability to accumulate 3 -hydroxy ropionic acid polymers, enabling them to polymerize 3- hydroxypropionic acid monomers into 3-hydroxypropionic acid polymers in a short period of time.

[0073]

[0073] In the production method of the present invention, the second strain may be a recombinant strain in which at least one gene selected from the group consisting of acs, phaC, and phaPi genes is introduced into an Escherichia strain.

[0074]

[0074] In one embodiment, the Escherichia strain may be introduced with an acyl coenzyme A synthase (acs) gene capable of producing polyhydroxyalkanoate (PHA). The acs gene may be, for example, a sequence represented by SEQ ID NO: 1 or a sequence having at least 85% sequence identity to the sequence represented by SEQ ID NO: 1.

[0075] In one embodiment, the genetic recombination of an Escherichia strain may be performed by introducing the acs gene in plasmid form.

[0075]

[0076] In one embodiment, the genetic recombination of an Escherichia strain may be performed by inserting the acs gene into the genome of the Escherichia strain.

[0076]

[0077] In one embodiment, the Escherichia strain may be introduced with a polyhydroxy alkanoate synthase phaC) gene capable of producing polyhydroxy alkanoate (PHA) The phaC gene may be, for example, a sequence represented by SEQ ID NO: 2 or a sequence having at least 85% sequence identity to the sequence represented by SEQ ID NO: 2.

[0077]

[0078] In one embodiment, the genetic recombination of an Escherichia strain may be performed by introducing the phaC gene in plasmid form.

[0078]

[0079] In one embodiment, the genetic recombination of an Escherichia strain may be performed by inserting the phaC gene into the genome of the Escherichia strain.

[0079]

[0080] In one embodiment, the Escherichia strain may be introduced with a phaPl (phasin proteins) gene capable of producing polyhydroxyalkanoate (PHA). The phaPl gene may be, for example, a sequence represented by SEQ ID NO: 3 or a sequence having at least 85% sequence identity to the sequence represented by SEQ ID NO: 3.

[0080]

[0081] In one embodiment, the genetic recombination of an Escherichia strain may be performed by introducing the phaPl gene in plasmid form.

[0081]

[0082] In one embodiment, the genetic recombination of an Escherichia strain may be performed by inserting the phaPl gene into the genome of the Escherichia strain.

[0082]

[0083] In one embodiment, the Escherichia strain may be recombined by introducing one to four genes selected from the group consisting of acs, phaC, and phaPl genes.

[0083]

[0084] In one embodiment, the Escherichia, strain is a recombinant strain introduced with the acs, phaC, and phaPl genes.

[0085] In one embodiment, the Escherichia strain is a recombinant strain introduced with the acs and phaC genes,

[0084]

[0086] In one embodiment, the Escherichia strain may be introduced with I to 4 copies of each of the acs, phaC. or phaPl genes. For example, the strain may be introduced with 1 to 4, 1 to 3, or 2 to 4 copies of the acs gene; 1 to 4, 1 to 3, or 2 to 4 copies of the phaC gene; and 1 to 4, 1 to 3, or 2 to 4 copies of the phaPl gene.

[0085]

[0087] Introducing the acs, phaC, and phaPl genes into an Escherichia strain may increase the production of a 3-hydroxypropionic acid polymer. As the number of introduced gene copies increases, a greater amount of the polymer may be produced.

[0086]

[0088] In one embodiment, the acs gene employed in genetic recombination may be used together with the three existing acyl coenzyme A synthase genes required for the polymer production reaction, or a single gene may be sufficient to perform the acyl coenzyme A synthesis reaction for producing a 3-hydroxypropiomc acid polymer.

[0087]

[0089] In one embodiment, the Escherichia strain may have the poxB gene deleted. In this case, blocking the acetate production pathway minimizes the production of organic acid byproducts, thereby producing a high-purity 3-hydroxypropionic acid polymer.

[0088]

[0090] In one embodiment, the Escherichia strain may have the IdhLl or IdhD gene deleted. In this case, blocking the lactate production pathway minimizes the production of organic acid byproducts, thereby producing a high-purity 3-hydroxypropionic acid polymer.

[0089]

[0091] In the production method of the present invention, the substrate may be glycerol or glucose.

[0090]

[0092] In the production method of the present invention, 3-hydroxypropionic acid may be a substrate of the second strain.

[0091] 1

[0093] The production method of the present invention may further include a step of controlling a growth rate of the first strain or the second strain by adjusting an amount of a carbon source supplied to the first strain or the second strain.

[0092]

[0094] In the production method of the present invention, the carbon source of the first strain and the second strain may be at least one selected from the group consisting of gluconate, glucose, citric acid, and glycerol.

[0093]

[0095] In one embodiment, the carbon source of the first strain is gluconate.

[0094]

[0096] In one embodiment, the carbon source of the second strain is glucose

[0095]

[0097] The production method of the present invention may further include a step of inoculating the first strain and the second strain into the bioreactor in a ratio of 100:1 to 1:100 (% w / v).

[0096]

[0098] In the production method of the present invention, the carbon source of the first strain and the carbon source of the second strain may be in a ratio of 100:1 to 1: 100 (% w / v) in the bioreactor,

[0097]

[0099] In one embodiment, the carbon source of the first strain and the carbon source of the second strain may be in a ratio of 100:1 to 1:100, 90:1 to 1:90, 80:1 to 1:80, 70:1 to 1:70, 60: 1 to 1:60, 50: 1 to 1:50, 40: 1 to 1:40, 30:1 to 1:30, or 25: 1 to 1:25 (% w / v) in the bioreactor.

[0098]

[0100] The production method of the present invention may further include a step of inoculating the first strain and the second strain into the bioreactor in a ratio of 100: 1 to 1:100 (% w / v).

[0099]

[0101] In one embodiment, the first strain and the second strain may be inoculated into the bioreactor m a ratio of 100:1 to 1:100, 90:1 to 1:90, 80: 1 to 1:80, 70:1 to 1:70, 60:1 to 1:60, 50:1 to 1:50, 40: 1 to 1:40, 30: 1 to 1:30, or 25:1 to 1:25 (% w / v).

[0100]

[0102] The production method of the present invention may prevent contaminants, such as external microorganisms, from proliferating within the culture system by allowing the two predetermined strains to preempt limited nutrient sources. In addition, the first strain of the present invention may continuously produce 3-hydroxypropionic acid, which has antimicrobial activity, thereby effectively inhibiting the growth of external contaminants throughout the culture process. Therefore, the production method of the present invention may ensure high culture stability and contamination resistance without the addition of separate antibiotics.

[0101]

[0103] The production method of the present invention may further include a step of isolating and purifying the 3-hydroxypropionic acid polymer.

[0102]

[0104] In one embodiment, the isol ation and purification of the 3 -hydroxypropioni c acid polymer may be performed by solvent extraction, and is preferably performed by surfactant extraction.

[0103]

[0105] The solvent extraction method is a method in which the 3-hydroxypropionic acid polymer is dissolved m a solvent at a high temperature and then extracted by coagulating and precipitating the 3-hydroxypropionic acid polymer using ethanol,

[0104]

[0106] The surfactant extraction method is a method in which cells are dissolved with a surfactant and the 3-hydroxypropionic acid polymer is isolated by centrifugation. First, only the 3-hydroxypropionic acid polymer-producing strain that settles is collected, and the surfactant concentration is then adjusted. When the temperature is 80 °C to 120 °C and the pH is 9 or higher, large-scale extraction of high -purity 3-hydroxypropionic acid polymers and oligomers is possible, and the cost is reduced compared to the solvent extraction method. In addition, the 3-hydroxypropionic acid monomer-producing strain present in the separated supernatant may be reused for various purposes.

[0105]

[0107] In one embodiment, the isolation and purification of the 3 -hy droxypropionic acid polymers may utilize lauric acid.

[0106]

[0108] In one embodiment, 1 to 10% (w / v) lauric acid may be added to the bioreactor to achieve mass extraction of the 3-hydroxypropionic acid polymers and reduce costs.

[0107] 1

[0109] The present invention provides a 3-hydroxypropionic acid polymer having an average molecular weight of 100,000 to 1,000,000 g / mol, produced according to the method of the present invention.

[0108]

[0110] In one embodiment, the average molecular weight of the 3-hydroxypropionic acid polymer may be at least 100,000 g / mol, 200,000 g / mol, 300,000 g / mol, 400,000 g / mol, 500,000 g / mol, 560,000 g / mol, 570,000 g / mol, 580,000 g / mol, 590,000 g / mol, 600,000 g / mol, 650,000 g / mol, 700,000 g / mol, 800,000 g / mol. or 900.000 g / mol, and at most 1,000.000 g / moi.

[0109]

[0111] The present invention provides a 3-hydroxypropionic acid polymer produced according to the production method of the present invention having an average production concentration of 10 g / L to 100 g / L.

[0110]

[0112] The present invention provides a 3-hydroxypropiomc acid polymer produced according to the production method of the present invention having an average production rate of 0.2 g / L / h to 2 g / L / h.

[0111]

[0113] The present invention provides a 3-hydroxypropionic acid polymer produced according to the production method of the present invention having an average production yield of 0.1 g / g to 0.8 g / g.

[0112]

[0114] The 3-hydroxypropionic acid polymer of the present invention may have a melting point of 70 to 140 °C, a glass transition temperature of -30 to -10 °C, a tensile strength of 10 to 50 MPa, and an elongation of 400 to 800%.

[0113]

[0115] In one embodiment, the melting point of the 3-hydroxypropionic acid polymer may be 70 to 140 °C. 70 to 130 °C, 70 to 120 °C, 70 to 110 °C, 70 to 100 °C, 70 to 90 °C, 70 to 80 °C, 75 to 140 °C, 75 to 130 °C, 75 to 120 °C, 75 to 110 °C, 75 to 100 °C, 75 to 90 °C, or 75 to 80 °C.

[0114]

[0116] The term “glass transition temperature (Tg)” refers to the temperature at which a polymer material begins to transform from a rigid glassy state to a flexible rubbery' state.

[0115] 6 At this temperature, the molecular chain mobility of the polymer increases, resulting in a material exhibiting softer and more flexible characteristics Below the glass transition temperature, the polymer maintains a hard, brittle glassy state, while above the glass transition temperature, it exhibits elastic, rubber-like properties.

[0116]

[0117] In one embodiment, the glass transition temperature of the 3-hydroxypropionic acid polymer may be -30 to -10 °C, -25 to -10 °C, -23 to -10 °C, or -20 to -10 °C.

[0117]

[0118] The term “tensile strength” refers to the maximum tension a material may withstand before breaking or fracturing. In other words, it is an indicator of how strongly a material may withstand a stretching force. The higher the tensile strength, the stronger the material and the better it may withstand stretching forces.

[0118]

[0119] In one embodiment, the tensile strength of the 3-hydroxypropionic acid polymer may be 10 to 50 MPa, 10 to 40 MPa, 10 to 30 MPa, 15 to 50 MPa, 15 to 40 MPa, 15 to 30 MPa, 20 to 50 MPa, 20 to 40 MPa, 20 to 30 MPa, 25 to 50 MPa. 25 to 40 MPa. or 25 to 30 MPa.

[0119]

[0120] The term “elongation at break” is an indicator of the degree of deformation a material may withstand w hile being stretched, expressed as a percentage of how much the material stretches before breaking. Elongation is an important characteristic for assessing the flexibility and ductility of a material. Materials with high elongation are more ductile and thus more easily deformed, while materials with low elongation are harder and more brittle.

[0120]

[0121] In one embodiment, the elongation of the 3-hydroxypropionic acid polymer may be 400 to 800%, 400 to 750%, 400 to 700%, 400 to 650%, 500 to 800%, 500 to 750%, 500 to 700%. 500 to 650%, 600 to 800%, 600 to 750%, 600 to 700%, 600 to 650%, 620 to 800%, 620 to 750%, 620 to 700%, or 620 to 650%.

[0121]

[0122] The present invention provides a product including a 3-hydroxypropionic acid polymer produced by the production method of the present invention.

[0123] In one embodiment, the product may be any one selected from the group consisting of a film, a film packaging material, a coated packaging materia], a fiber, and a spherical powder.

[0122]

[0124] In the present invention, the product may have a diameter of 1 uni to 100 um. In one embodiment, the product may be a fiber having a diameter of I um to 100 um. In one embodiment, the product may be a fiber having a diameter of 1 um to 90 um, 1 um to 80 um, 1 um to 50 um, 1 um to 30 um, 5 um to 100 um, 10 um to 100 um, or 20 um to 100 um.

[0123]

[0125] In the present invention, the product has oxygen barrier properties, waterproof properties, and flame retardancy.

[0124]

[0126] The present invention provides a method for producing 3 -hydroxy propionic aci d,

[0127] The present invention provides a method for producing 3 -hydroxypropionic acid, including the steps of: dissolving the 3-hydroxypropionic acid polymer of the present invention in an aqueous solvent and mixing it with an acidic solution to prepare a mixture; and treating the mixture with a basic solution to induce a neutralization reaction.

[0125]

[0128] In the method for producing 3-hydroxypropionic acid of the present invention, the 3-hydroxypropionic acid polymer dissolved in the aqueous solvent may be 5 to 50% (w / w).

[0126]

[0129] In the method for producing 3-hydroxypropionic acid of the present invention, the acidic solution may be H2SO4. In one embodiment, the acidic solution may be 1 to 10% (w / w).

[0127]

[0130] The method for producing 3 -hy droxypropionic acid of the present invention may further include the steps of: heating the mixture prepared above at 50 to 150 °C; and mixing a basic solution with the heated mixture to induce a neutralization reaction.

[0128]

[0131] The method for producing 3 -hydroxy propionic acid of the present invention may further include the step of centrifuging the mixture in which the neutralization reaction has been induced to isolate and purify only the filtrate, thereby obtaining 3- hy dr oxy propionic acid.

[0129]

[0132] In the method for producing 3 -hydroxy propionic acid of the present invention, 3- hydroxypropi onic acid is produced from a 3 -hydroxypropionic acid polymer or a 3-hydroxy ropionic acid oligomer, and the conversion rate may be 50% w / w to 99% w / w.

[0130]

[0133] The present invention provides a method for producing methyl 3-hydroxypropionic acid.

[0131]

[0134] The present invention provides a method for producing methyl 3-hydroxypropionic acid, including the steps of: dissolving the 3 -hydroxy propionic acid polymer of the present invention in a methanol solvent and mixing it with an acidic solution to prepare a mixture; and treating the mixture with a basic solution to induce a neutralization reaction.

[0132]

[0135] In the method for producing methyl 3 -hydroxypropionic acid of the present invention, the 3-hydroxypropionic acid polymer dissolved in methanol may be 5 to 50% (w / w).

[0133]

[0136] In the method for producing methyl 3-hydroxypropionic acid of the present invention, the acidic solution may be H2SO4. In one embodiment, the acidic solution may be 1 to 10% (w / w).

[0134]

[0137] The method for producing methyl 3-hydroxypropionic acid of the present invention may further include the steps of heating the above mixture at 50 to 150 °C; and mixing a basic solution with the heated mixture to induce a neutralization reaction.

[0135]

[0138] The method for producing methyl 3-hydroxypropionic acid of the present invention may further include the step of centrifuging the mixture in which the neutralization reaction has been induced, separating and purifying only the filtrate, and then obtaining methyl 3-hydroxypropionic acid.

[0136] 9

[0139] In the method for producing methyl 3-hydroxypropionic acid of the present invention, methyl 3-hydroxypropanoic acid is produced from a 3-hydroxypropionic acid polymer or a 3-hydroxypropionic acid oligomer, and the conversion rate may be 50% w / w to 99% w / w.

[0137]

[0140] The present invention provides a method for producing ethyl 3-hydroxypropionic acid.

[0138]

[0011] The present invention provides a method for producing ethyl 3-hydroxypropionic acid, including the steps of: dissolving the 3-hydroxyprop onic acid polymer of the present invention in an ethanol solvent and mixing it with an acidic solution to prepare a mixture; and treating the mixture with a basic solution to induce a neutralization reaction.

[0139]

[0142] In the method for producing ethyl 3-hydroxypropionic acid of the present invention, the 3-hydroxypropionic acid polymer dissolved in the ethanol solvent may be included in an amount of 5 to 50% (w / w).

[0140]

[0143] In the method for producing ethyl 3-hydroxypropionic acid of the present invention, the acidic solution may be H2SO4. In one embodiment, the acidic solution may be 1 to 10% (w / w).

[0141]

[0144] The method for producing ethyl 3-hydroxypropionic acid of the present invention may further include the steps of: heating the mixture prepared above at 50 to 150 °C; and mixing a basic solution with the heated mixture to induce a neutralization reaction.

[0142]

[0015] The method for producing ethyl 3-hydroxypropionic acid of the present invention max' further include the step of centrifuging the mixture in which the neutralization reaction has been induced to isolate and purify only the filtrate, thereby obtaining ethyl 3-hydroxypropionic acid.

[0143]

[0146] In the method for producing ethyl 3-hydroxypropionic acid of the present invention, ethyl 3-hydroxypropionic acid is produced from a 3-hydroxypropionic acid polymer or a 3-hydroxypropionic acid oligomer, and the conversion rate may be 50% w / w to 99% w / w.

[0144]

[0147] The present invention provides 3-hydroxypropionic acid produced by the method for producing 3-hydroxypropionic acid of the present invention.

[0145]

[0148] The present invention provides methyl 3-hydroxypropionic acid produced by the method for producing methyl 3-hydroxypropionic acid of the present invention.

[0146]

[0149] The present invention provides ethyl 3-hydroxypropionic acid produced by the method for producing ethyl 3-hydroxypropionic acid of the present invention.

[0147]

[0150] Hereinafter, the present invention will be described in more detail through examples.

[0148]

[0151] Examples

[0149]

[0152] Example 1. Preparation of Strain

[0150]

[0153] A Pseudomonas strain was used as the first strain for producing a 3-hydroxy propionic acid monomer, and a recombinant Escherichia strain into which the acs and phaC genes had been introduced was used as the second strain for polymerizing the 3-hydroxypropionic acid monomer. The specific method for preparing the strains is as follows.

[0151]

[0154] 1.1. Preparation of Recombinant Escherichia Strain

[0152]

[0155] The pKOV plasmid was introduced into cells using electroporation. The plasmid was then inserted into the genome of the Escherichia strain under high-temperature conditions (42 °C), after which the inserted plasmid w as removed under low-temperature conditions (37 °C) to prepare the recombinant strain. For plasmid insertion, a solid medium containing the antibiotic chloramphenicol was used. For plasmid removal, a solid medium containing sucrose was used.

[0153]

[0156] 1.2. Preculture of First and Second Strains

[0157] A single colony was inoculated into a 250-ml flask containing 50 ml of LB medium for the first preculture. After 12 hours, the culture was inoculated into a 1-L flask containing 100 ml of M9 medium for the second preculture, which was carried out for 12 hours.

[0154]

[0158] Example 2. Production of 3-Hydroxypropionic Acid Polymer Using co¬ culture

[0155]

[0159] The overall schematic diagram illustrating the production of 3-hy droxypropionic acid polymer using co-culture is shown in FIG. 1. The specific method is as follows.

[0156]

[0160] 2.1. Preparation of minimal medium

[0157]

[0161] A minimal medium containing gluconate and glucose at a ratio of 1:1 was prepared using sterilized gluconate and glucose solutions.

[0158]

[0162] 2.2. Inoculation of First and Second Strain

[0159]

[0163] The first strain. Pseudomonas, and the second strain, the recombinant Escherichia strain, were inoculated into the minimal medium at a 1: 1 ratio.

[0160]

[0164] 2.3. Additional Administration of Gluconate and Glucose as Carbon sources

[0165] Sterilized gluconate solution or sterilized glucose solution was periodically added to the minimal medium in the incubator to maintain a 1: 1 gluconate-to-glucose ratio while monitoring the pH for 12 hours (growth phase).

[0161]

[0166] 2.4.3-Hydroxypropionic Acid Polymerization

[0162]

[0167] The production phase was initiated by administering IPTG, an inducer for 3-hy droxypropionic acid polymerization, after the strain population had been maximized for 12 hours. 'Thereafter, glycerol was periodically added while monitoring the pH for up to 48 hours to maintain the 3-hy droxypropionic acid concentration above 100 mM and thereby enhance the polymerization reaction.

[0168] 2.5. Determination of Residual Amounts of Gluconate, Glucose, and 3- Hydroxypropionic Acid

[0163]

[0169] Samples were taken every three hours, and the concentrations of each substance were measured using HPLC to determine the residual amounts.

[0164]

[0170] 2.6. Extraction of the Produced 3-Hydroxypropionic Acid Polymer

[0171] The 3-hydroxypropionic acid polymer accumulated in the second strain, the recombinant Escherichia strain, was extracted. High-molecular-weight polymers (> 500,000 g / mol) were extracted using dimethyl carbonate (DMC), while low-molecular-weight polymers (< 50,000 g / mol) were extracted using a surfactant. The specific method is as follows.

[0165]

[0172] 2.6.1. DMC Solvent Extraction Method

[0166]

[0173] First, cells containing the polymer were collected as a pellet by centrifugation. Dimethyl carbonate (DMC) was then added at a ratio of 50 mL per 1 g of wet cell pellet (WCP), and the mixture was heated to a high temperature (100 °C) to dissolve the polymer. After dissolution, the remaining solids were removed by centrifugation, and the supernatant was collected. Next, 150 mL of ethanol was added to the supernatant to coagulate the polymer. The coagulated polymer was recovered by filtration and dried to obtain the solid 3-hydroxypropionic acid polymer.

[0167]

[0174] 2.6.2. Surfactant Extraction Method

[0168]

[0175] 1 L of polymer broth was adjusted to a 5% (w / v) surfactant concentration with water to a final volume of 2 L. The solution was then incubated at pH 9 and 120 °C for 3 hours to lyse the cells. The 3-hydroxypropionic acid polymer was then filtered out as a pellet by centrifugation and washed once with 1 L of water. The 3-hydroxypropionic acid polymer was filtered by centrifugation and washed once more with 1 L of ethanol. Only the aggregated 3-hydroxypropionic acid polymer was filtered and dried in a dry oven (60 °C) for one day to obtain a solid oligomer.

[0176] Example 3. Continuous Separation and Purification of 3-Hydroxypropionic Acid Polymer Using a Bioreactor

[0169]

[0177] The 3-hydroxypropionic acid polymer produced according to Example 2 was isolated and purified from the medium using a bioreactor. The specific method is as follows.

[0170]

[0178] A predissolved lauric acid solution for 5% (w / v) was added to the bioreactor. The 1 L polymer broth in the bioreactor was then diluted with water to a final volume of 2 L, adjusting the surfactant concentration to 5% (w / v). The mixture was incubated at pH 9 and 70 °C for 3 hours at 500 rpm to lyse the cells. The 3-hydroxypropionic acid polymer was collected as a pellet by centrifugation and washed once with 1 L of water. After another round of centrifugation, the polymer pellet was washed once more with 1 L of ethanol. The aggregated 3-hydroxypropionic acid polymer was then collected and dried in a dry oven at 60 °C for 24 hours, yielding the solid oligomer.

[0171]

[0179] Example 4. Physical Properties of 3-Hydroxypropionic Acid Polymer

[0180] The physical properties of the 3-hydroxypropionic acid polymer produced using the method of Example 2 were evaluated. The evaluation methods for each property are as follows.

[0172]

[0181] 4.1. Manufacture of Film

[0173]

[0182] The polymer tissue was heat-treated at 105 °C for 1 hour and then cut to manufacture a film.

[0174]

[0183] 4.2. Evaluation of Tensile Strength and Elongation

[0175]

[0184] Tensile strength and elongation were measured using a UTM analyzer (Salt, ST-1001). Specimens conforming to ISO 527-3 were prepared and tested at a rate of 500 mm / min. Five specimens were analyzed per sample, and the average of three values was calculated after excluding the highest and lowest values.

[0176]

[0185] 4.3. Evaluation of Melting Point and Glass Transition Temperature

[0186] The melting point and glass transition temperature were measured using a DSC analyzer (Mettler Toledo, DSC-1). Measurements were conducted at a heating rate of 10 K / min within a temperature range of -40 to 200 °C.

[0177]

[0187] 4.4. Evaluation of Oxygen Permeability

[0178]

[0188] For oxygen permeability testing, measurements were performed at 23 °C using an OX-TRAN Model 2 / 61 analyzer in accordance with ASTM D3985, with a measurement range of 0.5 to 10,000 cm’ / (m2-24 h- atm).

[0179]

[0189] 4.5. Evaluation of Moisture Permeability

[0180]

[0190] For moisture permeability testing, measurements were performed at 38 °C using a Permatran-W 3 / 33 MA analyzer in accordance with ASTM F 1249, with a measurement range of 0.005 to 500 g / (m2day).

[0181]

[0191] 4.6. Measured Physical Properties

[0182]

[0192] The physical properties of the 3-hydroxypropionic acid polymer were measured to be: a melting point of 75 to 80 °C: a glass transition temperature of -20 to -10 °C; a tensile strength of 25 to 30 MPa; and an elongation of 620 to 650%.

[0183]

[0193] Comparative Example 1.

[0184]

[0194] The molecular weight of the 3-hydroxypropionic acid polymer produced by a monoculture was compared with that of the 3-hydroxypropionic acid polymer produced by the method of Example 2. GPC analysis revealed that the 3-hydroxypropionic acid polymer produced by the method of Example 2 had a significantly higher molecular weight of 569,298 g / mol (FIGS. 4A and 4B).

[0195] Example 5. Manufacture and Analysis of Film Packaging Material Using 3-Hydroxypropionic Acid Polymer

[0185]

[0196] 3-FIydroxypropionic acid film packaging materials were manufactured and analyzed using the 3-hydroxypropionic acid polymer produced according to Example 2. The manufacturing and analysis methods are as follows.

[0186]

[0197] 5.1. Manufacture of 3-Hydroxypropionic Acid Film Packaging Material

[0198] The 3-hydroxypropiomc acid polymer produced according to Example 2 was dissolved in chloroform at 8% (w / w) and applied onto a glass plate to manufacture film packaging materials. The analysis results of the manufactured film packaging materials are presented in the comparative example below.

[0187]

[0199] Comparative Example 2. Comparison of Melting Point, Glass Transition Temperature, Tensile Strength, and Elongation

[0188]

[0200] The melting point, glass transition temperature, tensile strength, and elongation of the film packaging materials manufactured using PL A and the 3 -hydroxypropionic acid polymer were measured. The results are presented in Table 1.

[0189]

[0201] [TABLE 1]

[0190] PLA P(3-HP)

[0191] Tm(°C) 140 to 180 77 Tg(°C) 55 -15 Tensile Strength (MPa) 66 27

[0192]

[0193] Elongation (%) 4 634

[0194]

[0202] Comparative Example 3. Comparison of Tensile Strength

[0195]

[0203] The tensile strength of the film packaging materials manufactured from PL A, LDPE, or PBAT was compared with that of the film packaging material manufactured from the 3-hydroxypropionic acid polymer. The results are shown in FIG. 6. The film packaging material manufactured from the 3-hydroxypropionic acid polymer was found to exhibit the highest tensile strength.

[0196]

[0204] Comparative Example 4. Comparison of Oxygen Permeability and Moisture Permeability

[0197]

[0205] The oxygen permeability and moisture permeability of the film packaging materials manufactured from PLA, LDPE, PBAT, or PBS were compared with those of the film packaging material manufactured from the 3-hydroxypropionic acid polymer. The results are shown in FIGS. 7A and 7B. The oxygen permeability of the film packaging material manufactured from the 3 -hydroxypropionic acid polymer was the lowest, at 14.3 cm3 / (m2-24 h atm). The moisture permeability was measured to be 146 g / (m2-day), which was lower than that of the film packaging materials manufactured from PBAT and PBS.

[0198]

[0206] Comparative Example 5. Comparison of Waterproofing Properties of Film Packaging Materials

[0199]

[0207] The w ater resistance properties of the paper packaging material and the film packaging material manufactured from PLA, and the film packaging material manufactured from the 3-h droxypropionic acid polymer were compared by measuring the rate of change in mass after moisture treatment. The results are shown in FIG. 8. The film packaging material manufactured from the 3 -hydroxy propionic acid polymer was found to exhibit the lowest rate of change in mass at 0.0644% w / w, indicating the best waterproofing properties.

[0200]

[0208] Comparative Example 6. Comparison of Low-Temperature Stability of Film Packaging Materials

[0201]

[0202]

[0209] The film packaging materials manufactured from PLA and the film packaging material manufactured from the 3 -hydroxy propionic acid polymer were observed at -80 °C for one month to determine their low-temperature stability. The results are shown in FIG. 9. Unlike the film packaging material manufactured from PLA, the film packaging material manufactured from the 3-hydroxypropionic acid polymer did not show any breakage even after being stored at -80 °C for more than one month.

[0203]

[0210] Example 6. Manufacture and Analysis of Coated Packaging Material Using 3-Hydroxypropionic Acid Polymer

[0204]

[0211] A 3-hydroxypropionic acid-coated packaging material was manufactured and analyzed using the 3-hydroxypropionic acid polymer produced according to Example 2, The manufacturing and analysis methods are as follows.

[0205]

[0212] 6.1. Method for Manufacturing 3-Hydroxypropionic Acid Coated Packaging Material

[0206]

[0213] The 3-hydroxypropionic acid polymer produced in Example 2 was dissolved in chloroform at an 8% (w / w) ratio and then applied to the upper surface of a paper packaging material placed on a glass plate to manufacture a coated packaging material. The analysis results of the manufactured coated packaging material are presented in the Comparative Example below.

[0207]

[0214] Comparative Example 7. Comparison of Waterproofing Properties of Coated Packaging Materials

[0208]

[0215] The waterproofing properties of the paper packaging material and the coated packaging material manufactured from the 3-hydroxypropionic acid polymer were compared by measuring the rate of change in mass after moisture treatment. The results are shown in FIG. 10. While the waterproofing properties of the original paper packaging material were low, the rate of change in mass decreased to 0.4645% (w7w) after coating with the 3-hydroxypropionic acid polymer, demonstrating an increase in waterproofing properties.

[0209]

[0216] Comparative Example 8. Comparison of Flame Retardancy of Coated Packaging Materials

[0210]

[0217] The coated packaging material manufactured from an LDPE polymer and the coated packaging material manufactured from the 3-hydroxypropionic acid polymer were subjected to the same combustion reaction, and the rate of change in mass was measured to compare their flame retardancy. The results are shown in FIG. 11. While the conventional LDPE polymer-coated packaging material experienced a mass loss of 43.89%, the 3-hydroxypropionic acid polymer-coated packaging material experienced a mass loss of 2.61%, demonstrating improved flame retardancy.

[0211]

[0218] Example 7. Manufacture and Analysis of Fiber Using 3-Hydroxy propionic Acid Polymer

[0212]

[0219] 3-Hydroxypropionic acid fibers were manufactured and analyzed using the 3-hydroxypropionic acid polymer produced according to the method of Example 2.

[0213]

[0220] 7.1. Method for Manufacturing 3-Hydroxypropionic Acid Fiber

[0214]

[0221] 3-Hydroxypropionic acid fibers were manufactured by melt-spinning the 3-hydroxy propionic acid raw material alone at 155 °C for approximately 10 minutes.

[0215]

[0222] 7.2. Analysis Results of Fiber Material

[0216]

[0223] The surface morphology and size of the 3-hydroxypropionic acid fibers were analyzed using a scanning electron microscope (SEM). The results are shown in FIGS.

[0217] 12A and 12B. It was confirmed that the 3-hydroxypropionic acid fibers were formed in a fibrous form, having an average diameter of 1 um to 100 pm.

[0224] Example 8. Manufacture and Analysis of Powder Mixture Using 3-Hydroxypropionic Acid Oligomer

[0218]

[0225] A 3-hydroxypropionic acid powder mixture was manufactured and analyzed using the 3-hydroxypropionic acid oligomer produced according to the method of Example 2. The manufacturing and analysis methods are as follows.

[0219]

[0226] 8.1. Manufacture of 3-Hydroxypropionic Acid Powder Mixture

[0220]

[0227] The 3-hydroxypropionic acid oligomer produced according to the method of Example 2 was added to dimethyl carbonate or chloroform at 0.1% to 10% (w / v) and to lauric acid at 0.1% to 10% (w / v) to prepare a mixture. The mixture was then heated and then blended. Ethanol was then added to the blended mixture to induce a coagulation reaction. The coagulated mixture was applied to a substrate and dried to manufacture a powder mixture.

[0221]

[0228] 8.2. Image Analysis Results of Powder Mixture

[0222]

[0229] The surface morphology and particle size of the 3-hydroxypropionic acid powder mixture were analyzed using a scanning electron microscope (SEM). The results are shown in FIGS. 13A and 13B. When lauric acid was added to the 3-hydroxypropionic acid oligomer to produce a powder, it was confirmed that spherical particles having a size of 1 pm to 100 pm were formed.

[0223]

[0230] Example 9. Manufacture and Analysis of 3-hydroxypropionic acid Monomer

[0231] 3-Hydroxypropionic acid monomers were manufactured and analyzed using the 3-hydroxypropionic acid polymer and the 3-hydroxypropionic acid oligomer produced according to the method in Example 2. The manufacturing and analysis methods are as follows.

[0224]

[0232] 9.1. Method for manufacturing 3-Hydroxypropionic Acid Monomer

[0233] The 3-hydroxypropionic acid polymer or oligomer produced according to Example 2 was added to water at a concentration of 5% to 50% (w / w), and H2SO4 was added at a concentration of' 1% to 10% (w / w) to prepare a mixture. The mixture was then heated to 50 to 150 °C and then blended. A basic solution was added to the blended mixture to induce a neutralization reaction. The mixture was centrifuged, and only the filtrate was isolated and purified to obtain the 3-hydroxypropionic acid monomer.

[0225]

[0234] 9.2. Analysis of 3-Hydroxypropionic Acid Monomer

[0226]

[0235] The concentration of the manufactured 3-hydroxypropionic acid monomer was measured using high-performance liquid chromatography (HPLC). The results are shown in FIG. 14. According to the HPLC analysis, the use of a 3-hydroxypropionic acid oligomer resulted in a monomer conversion rate of over 90%, whereas the use of a 3-hydroxypropionic acid polymer resulted in a higher monomer conversion rate of over 95%. The higher monomer conversion rate obtained from the polymer was attributed to the influence of impurities present in the 3-hydroxypropionic acid oligomer.

[0227]

[0236] Example 10. Manufacture and Analysis of Methyl 3-Hydroxypropanoic Acid Monomer

[0228]

[0237] Methyl 3 -hydroxy propanoic acid monomers were manufactured and analyzed using the 3 -hydroxy propionic acid polymer and the 3-hydroxypropionic acid oligomer produced according to the method of Example 2. The manufacturing and analysis methods are as follows.

[0229]

[0238] 10.1. Manufacture of Methyl 3-Hydroxypropanoic Acid Monomer

[0239] The 3-hydroxypropionic acid polymer or oligomer produced according to Example 2 was added to methanol at a concentration of 5% to 50% (w / w), and H2SO4 was added at a concentration of 1 % to 10% (w / w) to prepare a mixture. The mixture was heated to 50 to 150 °C and then blended. A basic solution was then added to the blended mixture to induce a neutralization reaction. The mixture was centrifuged, and only the filtrate was isolated and purified to obtain the methyl 3-hydroxypropanoic acid monomer.

[0230]

[0240] 10.2. Analysis of Methyl 3-Hydroxypropionic Acid Monomer

[0231]

[0241] The concentration of the manufactured methyl 3-hydroxypropanoic acid monomer was measured using gas chromatography (GC). The results are shown in FIG.

[0232] 14. According to the GC analysis, the use of a 3-hydroxypropionic acid oligomer resulted in a methyl 3-hydroxypropanoic acid monomer conversion rate of over 65%, whereas the use of a 3-hydroxypropionic acid polymer resulted in a higher conversion rate of over 70%. The higher monomer conversion rate obtained from the polymer was attributed to the influence of impurities present in the 3-hydroxypropionic acid oligomer.

[0233]

[0242] Example 11. Manufacture and Analysis of Ethyl 3-Hydroxypropanoic Acid Monomer

[0234]

[0243] Ethyl 3-hydroxypropanoic acid monomers were manufactured and analyzed using the 3-hydroxypropionic acid polymer and the 3-hydroxypropionic acid oligomer produced according to the method of Example 2. The manufacturing and analysis methods are as follows.

[0235]

[0244] 11.1. Manufacture of Ethyl 3-Hydroxypropanoic Acid Monomer

[0245] The 3-hydroxypropionic acid polymer or oligomer produced according to the method of Example 2 was added to ethanol at a concentration of 5% to 50% w / w, and H2SO4 was added at 1 % to 10% w / w to prepare a mixture. The mixture was heated to 50 to 150 °C and then blended. A basic solution was added to the blended mixture to induce a neutralization reaction. The mixture w as centrifuged, and only the filtrate was isolated and purified to obtain the ethyl 3-hydroxypropanoic acid monomer.

[0236]

[0246] 11.2. Analysis of Ethyl 3-Hydroxypropionic Acid Monomer

[0237]

[0238]

[0247] The concentration of the manufactured ethyl 3 -hydroxy propionic acid monomer was measured using gas chromatography (GC). The results are shown in FIG. 14. According to the GC analysis, the use of a 3-hydroxypropionic acid oligomer resulted in a conversion rate of over 55% to ethyl 3 -hydroxy propanoic acid, whereas the use of a 3- hydroxypropionic acid polymer resulted in a higher conversion rate of over 70%. The higher monomer conversion rate obtained from the polymer was attributed to the influence of impurities present in the 3-hydroxypropionic acid oligomer.

Claims

WHAT IS CLAIMED IS:[Claim 11 A method for producing a 3 -hydroxy propionic acid polymer by co¬ culturing a first strain and a second strain, the method comprising:fermenting a substrate with the first strain to produce 3-hydroxypropionic acid, and polymerizing the 3-hydroxypropionic acid with the second strain to produce a 3-hydroxypropionic acid polymer, wherein the fermenting and the polymerizing are performed in a single bioreactor: andcontrolling reaction rates of the fermenting and the polymerizing by adjusting an amount of the substrate supplied to the bioreactor,

2. The method of claim 1, further comprising controlling the reaction rate by additionally inoculating the first strain or the second strain into the bioreactor.[Claim 31 The method of claim 1, further comprising monitoring a pH in the bioreactor to increase or decrease the amount of the substrate supplied or to additionally inoculate the first strain or the second strain.[Claim 4j The method of claim 1, wherein the first strain is a strain belonging to any one genus selected from the group consisting of Escherichia, Klebsiella, and Pseudomonas.

5. The method of claim 1, wherein the second strain is a strain belonging to any one genus selected from the group consisting of Escherichia, Cupriavidus, and Pseudomonas.

6. The method of claim 1, wherein the substrate is glycerol or glucose.[Claim 71 The method of claim 1. further comprising controlling a growth rate of the first strain or the second strain by adjusting an amount of a carbon source supplied to the first strain or the second strain.

8. The method of claim 7, wherein the carbon source is selected from the group consisting of gluconate, glucose, citric acid, glycerol, and a combination thereof,

9. The method of claim 1, wherein the second strain is a recombinant strain in which at least one gene selected from the group consisting of acs, phaC, and phaPl genes is introduced into an Escherichia strain.

10. The method of claim 1. further comprising inoculating the first strain and the second strain into the bioreactor in a ratio of 100: 1 to 1:100 (% w / v).

11. The method of claim 7, wherein the carbon source of the first strain and the carbon source of the second strain are in a ratio of 100:1 to 1:100 (% w / v) in the bioreactor.

12. The method of claim 1, further comprising isolating and purifying the 3-hydroxy propionic acid polymer.

13. A 3-hydroxypropionic acid polymer having an average molecular weight of 100,000 to 1,000,000 g / mol, produced according to the method of claim 1.

14. The 3-hydroxypropionic acid polymer according to claim 13, wherein the 3-hydroxypropionic acid polymer has a melting point of 70 to 140 °C. a glass transition temperature of -30 to -10 °C. a tensile strength of 10 to 50 MPa. and an elongation of 400 to 800%.

15. A product comprising the 3-hydroxypropionic acid polymer of claim 13.

16. The product according to claim 15. wherein the product is any one selected from the group consisting of a film, a film packaging material, a coated packaging material, a fiber, and a spherical powder.

17. The product according to claim 15, wherein the product has a diameter of 1 pm to 100 pm.

18. A method for producing 3-hydroxypropionic acid, the method comprising:dissolving the 3-hydroxypropionic acid polymer of claim 13 in an aqueous solvent and mixing it with an acidic solution to prepare a mixture; andtreating the mixture with a basic solution to induce a neutralization reaction.

19. A method for producing methyl 3-hydroxypropionic acid, the method comprising:dissolving the 3-hydroxypropionic acid polymer of claim 13 in a methanol solvent and mixing it with an acidic solution to prepare a mixture; andtreating the mixture with a basic solution to induce a neutralization reaction

20. A method for producing ethyl 3 -hydroxypropionic acid, the method comprising:dissolving the 3-hydroxypropionic acid polymer of claim 13 in an ethanol solvent and mixing it with an acidic solution to prepare a mixture; andtreating the mixture with a basic solution to induce a neutralization reaction.

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