Method for purifying polyhydroxyalkanoate

Abstract

The present invention relates to a method for purifying polyhydroxyalkanoate and polyhydroxyalkanoate purified by the purification method, wherein the purification method comprises the steps of: obtaining a suspension containing polyhydroxyalkanoate (PHA) from a microbial fermentation liquid; and adding hydrogen peroxide and an alkali component to the suspension to cause a decolorization reaction.

Description

Technology Field

[0001] The present invention relates to a method for efficiently purifying polyhydroxyalkanoate and to polyhydroxyalkanoate obtained through said purification method. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are thermoplastic natural polyester polymers that accumulate within microbial cells and possess environmentally friendly characteristics, exhibiting biodegradability not only in soil but also in the ocean. These polyhydroxyalkanoates can be obtained through cell disruption and separation of fermentation liquids derived from microorganisms.

[0003] The polyhydroxyalkanoate obtained through the aforementioned cell crushing and separation contains impurities such as proteins, phospholipids, and polypeptides; therefore, it is necessary to remove these impurities through purification processes such as decolorization and deproteinization. This is because if the polyhydroxyalkanoate containing these impurities undergoes processing, these impurities act as a cause for the degradation of the polymer's color and physical properties.

[0004] The decolorization reaction of the above polyhydroxyalkanoate can be induced by introducing hydrogen peroxide, an oxidizing agent, under conditions where alkaline conditions are continuously maintained to generate hydrogen peroxide-derived radicals. However, the generation efficiency of the above hydrogen peroxide-derived radicals varies significantly depending on surrounding environmental conditions such as metal ion content, pH, and temperature, so there are limitations in increasing the decolorization reaction efficiency. Prior art literature

[0005] Republic of Korea Published Patent Application No. 2001-0009719 The problem to be solved

[0006] In order to solve the above problem, the inventors conducted various studies and confirmed that it is more efficient to proceed with the decolorization reaction by optimally setting the amount of hydrogen peroxide and the ratio of hydrogen peroxide to alkali components, compared to conventional techniques in which the initial pH (alkaline range) of the reaction is adjusted, hydrogen peroxide is added, and the pH decrease accompanying the decolorization reaction is not corrected, or additional alkali is added continuously or in stages to maintain / correct the initial pH of the reaction.

[0007] Therefore, the objective of the present invention is to provide a method for purifying polyhydroxyalkanoate with significantly improved decolorization reaction efficiency.

[0008] In addition, another objective of the present invention is to provide a polyhydroxyalkanoate purified by the above purification method. means of solving the problem

[0009] To achieve the above objective, the present invention provides a method for purifying polyhydroxyalkanoate, comprising the steps of: obtaining a suspension containing polyhydroxyalkanoate (PHA) from a microbial fermentation liquid; and adding hydrogen peroxide and an alkaline component to the suspension to cause a decolorization reaction.

[0010] According to one embodiment of the present invention, the alkali component may include one or more selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium acetate, potassium acetate, trisodium phosphate, sodium hydrogen phosphate, tripotassium phosphate, and potassium phosphate.

[0011] According to another embodiment of the present invention, the input ratio of the hydrogen peroxide and the alkali component may be a weight ratio of 1:0.03 to 0.11.

[0012] According to another embodiment of the present invention, the hydrogen peroxide and the alkali component may be added to the suspension such that the weight ratio of the hydrogen peroxide and the OH derived from the alkali component is 1:0.01 to 0.05.

[0013] According to another embodiment of the present invention, the amount of hydrogen peroxide added may be 2% to 7% by weight based on 100% by weight of the PHA solid content contained in the suspension.

[0014] According to another embodiment of the present invention, the hydrogen peroxide and the alkaline component may be simultaneously introduced into the suspension.

[0015] According to another embodiment of the present invention, the temperature at which the decolorization reaction is performed may be 35°C to less than 70°C.

[0016] According to another embodiment of the present invention, the polyhydroxyalkanoate (PHA) may comprise a repeating unit derived from 3-hydroxybutyrate; and a repeating unit derived from one or more selected from the group consisting of 4-hydroxybutyrate, 3-hydroxypropionate, 3-hydroxyvalerate and 3-hydroxyhexanoate.

[0017] According to another embodiment of the present invention, the purification method may further include a step of deproteinizing the product obtained through the decolorization reaction.

[0018] According to another embodiment of the present invention, the purified polyhydroxyalkanoate (PHA) (PHA obtained by the purification method) may have a color-b of 25 or less when measured using a colorimeter.

[0019] Meanwhile, the present invention provides a polyhydroxyalkanoate purified by the above purification method.

[0020] According to one embodiment of the present invention, the polyhydroxyalkanoate (PHA) may have a color-b of 25 or less as measured using a colorimeter. Effects of the invention

[0021] The purification method according to the present invention optimizes the amount of hydrogen peroxide input and the ratio of hydrogen peroxide to alkali component input to perform a decolorization reaction in a state where the generation reaction of hydrogen peroxide-derived radicals is maximized. Therefore, compared to the conventional technology in which a decolorization reaction by hydrogen peroxide (specifically, starting at pH 9 or maintaining pH 9) is performed after achieving a target pH by adding an alkali component, it can exhibit the effects of improved color and cost reduction. In addition, compared to the conventional technology, the decolorization reaction can be performed at a relatively low temperature (e.g., 45°C).

[0022] Specifically, the present invention can solve the color issue by maximizing the generation reaction of hydrogen peroxide-derived radicals through controlling the ratio of alkaline components under the same hydrogen peroxide input conditions.

[0023] In addition, in the case of the above-mentioned conventional technology, hydrogen peroxide undergoes self-decay under alkaline conditions, requiring the addition of an excess amount of hydrogen peroxide. However, the present invention can effectively induce the generation of hydrogen peroxide-derived radicals by reacting hydrogen peroxide with an alkaline component of a certain level or higher at the beginning of the reaction, thereby allowing the same effect as the conventional technology to be expected even at a low reaction temperature and with a reduced amount of hydrogen peroxide added.

[0024] In addition, by controlling the input ratio of hydrogen peroxide and alkali components, the present invention can simplify the decolorization reaction process compared to conventional technology, and can also ensure process stability because there is no need to adjust the pH during the decolorization reaction. Brief explanation of the drawing

[0025] Figure 1 shows the results of evaluating the generation rate A of hydrogen peroxide-derived radicals according to reaction time using the methylene blue reaction in Evaluation Example 1. Figure 2 shows the results of evaluating the generation rate B of hydrogen peroxide-derived radicals according to the reaction temperature using the methylene blue reaction in Evaluation Example 2. Specific details for implementing the invention

[0026] The present invention will be described in detail below. However, the present invention is not limited to the contents disclosed below and may be modified in various forms as long as the essence of the invention is not altered.

[0027] In this specification, the use of the word “comprising” is intended to specify certain characteristics, regions, steps, processes, elements, and / or components, and unless specifically stated otherwise, it does not exclude the presence or addition of other characteristics, regions, steps, processes, elements, and / or components.

[0028] All numbers and expressions indicating the amounts of components, reaction conditions, etc. described in this specification shall be understood to be modified by the term "about" in all cases unless otherwise specified.

[0030] The decolorization reaction of polyhydroxyalkanoate using hydrogen peroxide is carried out by generating hydrogen peroxide-derived radicals according to the following reaction scheme 1, and the generated hydrogen peroxide-derived radicals remove impurities present in the polyhydroxyalkanoate.

[0031] [Reaction Equation 1]

[0032] HOOH + OH - → HOO - + H2O

[0033] However, in the case of conventional technology in which a decolorization reaction was performed by adding hydrogen peroxide while maintaining a constant pH by adding an alkaline component, metal ions (metal +)(e.g., Mn 2+ , Fe 3+ The decomposition of hydrogen peroxide occurs due to ) etc., which reduces the generation efficiency of hydrogen peroxide-derived radicals, and consequently lowers the decolorization reaction efficiency of polyhydroxyalkanoates.

[0034] [Reaction Equation 2]

[0035] HOO - + H2O2 → H2O + OH - + O2

[0036] Accordingly, the inventors conducted various studies to increase the decolorization reaction efficiency of polyhydroxyalkanoates. As a result, unlike conventional technology in which hydrogen peroxide is added to perform a decolorization reaction while maintaining a constant pH, by optimizing the amount of hydrogen peroxide added and the ratio of hydrogen peroxide to alkaline components, a reaction for the generation of hydrogen peroxide-derived radicals as shown in Reaction Scheme 3 below is induced, and through this, the generation efficiency of hydrogen peroxide-derived radicals (e.g., hydroperoxyl radical (HOO·), hydroxyl radical (·OH)) is maximized, and it was found that the decolorization reaction efficiency of polyhydroxyalkanoates is significantly improved.

[0037] [Reaction Equation 3]

[0038] HOO - + H2O2→·OH + HOO·+ HO -

[0039] HOO· + H2O2→·OH + O2+ H2O

[0040] That is, the present invention relates to a method for purifying polyhydroxyalkanoate in which the decolorization reaction efficiency of the polyhydroxyalkanoate is significantly improved, and to polyhydroxyalkanoate purified by said purification method having excellent color and minimized impurity content.

[0041] In particular, the present invention has technical significance in that it confirms that the generation efficiency of hydrogen peroxide-derived radicals varies depending on the input ratio of hydrogen peroxide and alkali components through an analysis of the substrate consumption rate using Methylene Blue (MB), rather than simply adjusting the input ratio of hydrogen peroxide and alkali components repeatedly, and furthermore, based on the above confirmation results, predicts the reaction rate constant of radical generation according to the input ratio of hydrogen peroxide and alkali components, thereby optimizing the input amount of hydrogen peroxide and the input ratio of hydrogen peroxide and alkali components to enable the decolorization reaction of polyhydroxyalkanoate (PHA) to proceed economically and efficiently.

[0042] The present invention will be described in detail as follows.

[0044] Method for purifying polyhydroxyalkanoates

[0045] A method for purifying polyhydroxyalkanoate according to the present invention comprises the step (S-1) of obtaining a suspension containing polyhydroxyalkanoate (PHA) from a microbial fermentation liquid; and the step (S-2) of adding hydrogen peroxide and an alkaline component to the suspension to cause a decolorization reaction. Additionally, the method for purifying polyhydroxyalkanoate according to the present invention may optionally further include the step (S-3) of deproteinizing the product obtained through the decolorization reaction as needed.

[0047] S-1: Obtaining PHA suspension

[0048] According to the present invention, step S-1 is a step of obtaining a suspension containing polyhydroxyalkanoate (PHA) from a microbial fermentation liquid.

[0049] The above microbial fermentation liquid is obtained through the fermentation of microorganisms, and PHA is accumulated (produced) inside the cells of the microorganisms through said fermentation. For example, a microbial fermentation liquid containing PHA can be obtained through fermentation using wild-type or transgenic microorganisms cultured on a specific substrate.

[0050] The method for obtaining a suspension containing PHA from the above microbial fermentation liquid is not particularly limited. Specifically, a suspension containing PHA can be obtained by separating biomass containing PHA from the above microbial fermentation liquid, adding a surfactant or the like, and performing cell lysis of the microorganisms (biomass).

[0051] For example, the S-1 step for obtaining a suspension (PHA suspension) containing the above PHA (solid content) may include the step of obtaining biomass containing PHA by separating the solid and liquid of a microbial fermentation liquid (primary solid-liquid separation) (S-1-1); the step of obtaining a mixture by mixing an alkali component and a surfactant with the biomass (S-1-2); the step of obtaining a slurry liquid by feeding the mixture into a high-pressure homogenizer and performing cell crushing (S-1-3); and the step of separating the solid and liquid of the slurry liquid (secondary solid-liquid separation) (S-1-4). Any method capable of obtaining a suspension (PHA suspension) containing the above PHA (solid content) is not limited to the above method.

[0052] In the above S-1-1 step, solid-liquid separation may be performed using mechanical separation methods such as centrifugation or membrane separation, or other known separation methods.

[0053] In the above S-1-2 step, a solvent may be introduced to the biomass before the alkali component and the surfactant are introduced, so that the solid content concentration of the biomass can be controlled. Sodium hydroxide may be used as the alkali component, and as the alkali component is mixed, the pH of the biomass may be controlled to 8 to 14 or 10 to 12. Anionic surfactants or nonionic surfactants may be used as the surfactant. Specifically, the surfactant may be sodium laureth sulfate, sodium lauryl sulfate, dodecyl sodium sulfate, dodecyl sodium benzene sulfonate, etc., but is not limited thereto.

[0054] In the above S-1-3 step, cell crushing may be performed by physical crushing (mechanical crushing) or chemical crushing (non-mechanical crushing). Specifically, the cell crushing may be performed by ultrasonic crushing, high-pressure crushing, or milling crushing, but is not limited thereto. For example, the ultrasonic crushing may be performed for 10 to 60 minutes with an energy amount of 20 Hz or more. In addition, the high-pressure crushing may be performed for 1 to 60 minutes with a pressure of 10 bar or more. In addition, the milling crushing may be performed for 1 to 60 minutes using a colloid mill, bead mill, ball mill, etc.

[0055] In the above S-1-4 step, solid-liquid separation may be achieved using mechanical separation methods such as centrifugation or membrane separation, or other known separation methods.

[0056] The PHA suspension obtained through the above steps may have a solid content of 5% to 20% by weight. Specifically, the PHA suspension may have a solid content of 6% to 18% by weight, 7% to 15% by weight, 8% to 12% by weight, 10% to 11% by weight, 9% to 11% by weight, or 8% to 11% by weight based on the total weight of the PHA suspension, but is not limited thereto. The solid content may refer to the content of PHA contained in the PHA suspension.

[0057] Meanwhile, the PHA included in the above PHA suspension may be a homopolymer or a copolymer. Specifically, the PHA is one selected from the group consisting of 2-hydroxybutyrate (2-HB), 3-hydroxybutyrate (3HB), 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HH), 4-hydroxybutyrate (4HB), 4-hydroxyvalerate (4HV), 4-hydroxyhexanoate (4HH), 5-hydroxyvalerate (5HV), and 6-hydroxyhexanoate (6HH). It may be a polymer (copolymer) comprising repeating units derived from the above (from one or more monomers), but is not limited thereto.

[0058] The above PHA can be classified into crystalline PHA (cPHA), semicrystalline PHA (scPHA), or amorphous PHA (aPHA) depending on the type of monomer and the content of repeating units derived therefrom. Specifically, the crystallinity of the above PHA can be controlled according to the content of repeating units derived from the 4-hydroxybutyrate (4HB) and classified into cPHA, scPHA, or aPHA.

[0059] According to the present invention, the PHA may be a copolymer comprising repeating units derived from 3-hydroxybutyrate (3-HB); and repeating units derived from one or more selected from the group consisting of 4-hydroxybutyrate (4-HB), 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HH). For example, the PHA may be a poly-3-hydroxybutyrate-co-4-hydroxybutyrate (P-3-HB-co-4-HB) copolymer comprising repeating units derived from 3-hydroxybutyrate (3-HB); and repeating units derived from 4-hydroxybutyrate (4-HB). The present invention optimizes the purification process of a PHA having such a specific composition, thereby providing a PHA with excellent color and minimized impurity content.

[0060] The content of the 4-HB repeating unit included in the above PHA is not particularly limited, but based on the total weight of the PHA, 1 wt% to 60 wt%, 2 wt% to 55 wt%, 3 wt% to 50 wt%, 4 wt% to 45 wt%, 5 wt% to 40 wt%, 6 wt% to 35 wt%, 1 wt% to 30 wt%, 1.5 wt% to 25 wt%, 2 wt% to 20 wt%, 2.5 wt% to 18 wt%, 3 wt% to 15 wt%, 3.5 wt% to 13 wt%, 4 wt% to 10 wt%, 4.5 wt% to 9 wt%, 5 wt% to 8.5 wt%, 6 wt% to 8 wt%, 6.5 wt% to 7.5 wt%, 15 wt% to 60 wt%, 18 wt% to It may be 55 wt%, 20 wt% to 50 wt%, 22 wt% to 45 wt%, 24 wt% to 40 wt%, 25 wt% to 38 wt%, 26 wt% to 37 wt%, 27 wt% to 36 wt%, 28 wt% to 35 wt%, 29 wt% to 34 wt%, or 30 wt% to 32 wt%.

[0061] Meanwhile, the above PHA may include isomers. Specifically, the above PHA may include structural isomers, enantiomers, or geometric isomers.

[0062] In addition, the above PHA may include one or two or more types of PHA. Specifically, the above PHA may be a single type of PHA, or a PHA in which a first PHA and a second PHA having different compositions are mixed. When the first PHA and the second PHA are mixed, their mixing ratio is not particularly limited, but may be a weight ratio of 20:80 to 80:20, or a weight ratio of 30:70 to 70:30.

[0064] S-2: Decolorization reaction

[0065] According to the present invention, step S-2 is a step of decolorizing the PHA suspension obtained in step S-1 by adding hydrogen peroxide and an alkali component. The present invention can maximize the generation reaction of hydrogen peroxide-derived radicals by controlling the amount of hydrogen peroxide added and the ratio of hydrogen peroxide to alkali component added during the decolorization reaction. Through this decolorization reaction, impurities present in the PHA contained in the suspension can be removed, thereby improving the color of the PHA.

[0066] The above alkali component is not particularly limited as long as it is an alkali component that is commonly known. Specifically, according to the present invention, the alkali component may include one or more selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium acetate, potassium acetate, trisodium phosphate, sodium hydrogen phosphate, tripotassium phosphate, and potassium phosphate. Preferably, the alkali component is sodium hydroxide, which can increase the generation efficiency of hydrogen peroxide-derived radicals.

[0067] According to the present invention, the input ratio (a:b) of hydrogen peroxide (a) and alkali component (b) added to the PHA suspension may be a weight ratio of 1:0.03 to 0.11. Specifically, the input ratio (a:b) may be a weight ratio of 1:0.03 to 0.11, a weight ratio of 1:0.04 to 0.11, a weight ratio of 1:0.04 to 0.10, a weight ratio of 1:0.05 to 0.10, a weight ratio of 1:0.05 to 0.09, or a weight ratio of 1:0.06 to 0.09, but is not limited thereto.

[0068] In addition, according to the present invention, the hydrogen peroxide and the alkali component may be added to the PHA suspension such that the weight ratio of the hydrogen peroxide and the OH (hydroxyl group) derived from the alkali component is 1:0.01 to 0.05. Specifically, the weight ratio may be 1:0.01 to 0.05, 1:0.011 to 1:0.049, 1:0.011 to 1:0.048, or 1:0.012 to 1:0.047, but is not limited thereto.

[0069] In addition, according to the present invention, the amount of hydrogen peroxide added to the PHA suspension may be 2% to 7% by weight based on 100% by weight of the solid content of the PHA suspension. Specifically, the amount of hydrogen peroxide added may be 2% to 7%, 2% to 6.8%, 2.1% to 6.6%, 2.1% to 6.5%, 2.2% to 6.4%, 2.2% to 6.2%, 2.3% to 6%, 2.3% to 5.8%, 2.4% to 5.6%, 2.4% to 5.5%, 2.5% to 5.4%, 2.5% to 5.2%, or 2.5% to 5%, based on 100% by weight of the PHA solid content contained in the PHA suspension, but is not limited thereto.

[0070] As the amount of hydrogen peroxide added, the ratio of hydrogen peroxide to the alkali component added, and the weight ratio of hydrogen peroxide to OH are each within the above ranges, the generation reaction of hydrogen peroxide-derived radicals is optimized, thereby maximizing the decolorization reaction efficiency of the PHA, and as a result, a PHA with a low Color-b value, excellent color, and minimized impurity content can be provided.

[0071] According to the present invention, the hydrogen peroxide and the alkali component can be introduced simultaneously into the PHA suspension. Specifically, after setting the amount of hydrogen peroxide to be introduced and the ratio of hydrogen peroxide to the alkali component to be introduced, these components are introduced simultaneously into the PHA suspension to perform the decolorization reaction of the PHA. This is differentiated from the conventional technology in which the decolorization reaction is carried out by introducing hydrogen peroxide while the pH is adjusted by first introducing the alkali component into the PHA suspension.

[0072] That is, the purification method according to the present invention may not include a step of first adding an alkaline component to the PHA suspension to adjust the pH of the PHA suspension. Furthermore, the purification method according to the present invention may not include a step of continuously adding an alkaline component to maintain a constant pH of the PHA suspension while the decolorization reaction is performed. By not including the above steps, the present invention can perform the purification process of PHA economically and stably.

[0073] According to the present invention, the decolorization reaction may be performed at a temperature of 35°C to less than 70°C. Specifically, the temperature at which the decolorization reaction is performed may be 35°C to less than 70°C, 35°C to 65°C, 36°C to 63°C, 37°C to 60°C, 37°C to 59°C, 38°C to 58°C, 40°C to 57°C, 42°C to 56°C, 43°C to 55°C, 44°C to 53°C, or 45°C to 50°C, but is not limited thereto. As the decolorization reaction is performed at the above temperature, energy saving can be achieved while securing decolorization reaction efficiency.

[0074] Meanwhile, the time for performing the above decolorization reaction is not particularly limited and can be appropriately set according to the weight of the PHA suspension, the amount of hydrogen peroxide and sodium hydroxide added, etc. Specifically, the above decolorization reaction may be performed for 0.5 to 3 hours, 1 to 2.5 hours, or 1.5 to 2 hours.

[0076] S-3: Deproteinization

[0077] According to the present invention, a step (step S-3) of deproteinizing the product (1) obtained through the decolorization reaction of step S-2 may be additionally performed. The deproteinization reaction removes protein components present in the product (1) obtained through the decolorization reaction and may be performed using a protease, but is not limited thereto.

[0078] The above proteolytic enzyme is not particularly limited as long as it is an enzyme that is commonly known. Specifically, the above proteolytic enzyme may be an alkalase, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, carboxypeptidase, or a combination thereof.

[0079] The amount of the above proteolytic enzyme used is not particularly limited, but based on the total weight of the above product (1), it may be 0.001 wt% or more, 0.003 wt% or more, 0.005 wt% or more, 0.006 wt% or more, 0.008 wt% or more, 0.01 wt% or more, 0.015 wt% or more, 0.02 wt% or more, or 0.025 wt% or more, and 0.2 wt% or less, 0.15 wt% or less, 0.1 wt% or less, 0.05 wt% or less, 0.045 wt% or less, or 0.04 wt% or less (e.g., 0.001 wt% to 0.1 wt%, or 0.005 wt% to 0.1 wt%).

[0080] In addition to the proteolytic enzyme, lipid-degrading enzymes, cell wall-degrading enzymes, DNA-degrading enzymes, etc. may be additionally added to the above deproteinization reaction. Lipase, phospholipase, cholinesterase, or phosphatase may be used as the lipid-degrading enzyme, lysozyme, amylase, cellulase, maltase, saccharase, α-glycosidase, β-glycosidase, or N-glycosidase may be used as the cell wall-degrading enzyme, and ribonuclease may be used as the DNA-degrading enzyme.

[0081] The above deproteinization reaction may be carried out under basic conditions. Specifically, the above deproteinization reaction may be carried out at a pH greater than 7, pH 7.5 or higher, pH 8 or higher, or pH 8.5 or higher (e.g., pH 7.5 to 12, pH 8 to 10, or pH 8.5 to 9).

[0082] In order to perform the above deproteinization reaction under the above basic conditions, a commonly known alkaline component (e.g., sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, etc.) may be added to the product (1) to raise the pH to a required level, or a commonly known acidic component may be added to the product (1) to lower the pH to a required level.

[0083] Specifically, the temperature at which the above deproteinization reaction is performed may be 40 ℃ or higher, 45 ℃ or higher, 50 ℃ or higher, 55 ℃ or higher, or 60 ℃ or higher, and may be 90 ℃ or lower, 80 ℃ or lower, 75 ℃ or lower, 70 ℃ or lower, or 65 ℃ or lower (e.g., 40 ℃ to 90 ℃, 50 ℃ to 80 ℃, or 60 ℃ to 70 ℃).

[0084] Afterwards, PHA solids can be recovered from the product (2) obtained through the above deproteinization, and PHA (PHA solids) can be obtained by washing and drying the recovered PHA solids in a conventional way.

[0086] Polyhydroxyalkanoate (PHA)

[0087] The polyhydroxyalkanoate (PHA) according to the present invention is a purified polyhydroxyalkanoate (PHA) obtained through the purification method described above. As this PHA is purified by the purification method described above, it can have excellent color and minimize the content of impurities.

[0088] Specifically, according to the present invention, the PHA may have a color-b value of 25 or less as measured using a colorimeter. More specifically, the PHA may have a color-b value of 25 or less, 23 or less, 20 or less, 19 or less, 17 or less, 16.5 or less, 15 or less, 14.5 or less, 14 or less, 13.5 or less, 13 or less, 12.5 or less, 12 or less, 11.5 or less, 11 or less, 10.5 or less, or 10 or less (e.g., 0 to 25, 0 to 21, 1 to 17, 1 to 15, 5 to 13, or 9 to 11).

[0089] Additionally, according to the present invention, the PHA may have a crude protein content of less than 0.35 wt% and a crude fat content of less than 0.25 wt%. Specifically, based on the total weight of the PHA, the crude protein content of the PHA may be less than 0.35 wt%, 0.34 wt% or less, 0.33 wt% or less, 0.32 wt% or less, 0.31 wt% or less, or 0.3 wt% or less (e.g., 0 wt% to 0.34 wt%, or 0.1 wt% to 0.32 wt%). In addition, the above PHA may have a crude fat content of less than 0.25 wt%, 0.24 wt% or less, 0.23 wt% or less, 0.22 wt% or less, 0.21 wt% or less, or 0.2 wt% or less (e.g., 0 wt% to 0.24 wt%, or 0.1 to 0.22 wt%) based on the total weight of the PHA.

[0090] Such PHA is identical to the PHA contained in the above-mentioned PHA suspension and may include the above-described details. Specifically, the PHA may be a homopolymer or a copolymer. For example, the above PHA is one selected from the group consisting of 2-hydroxybutyrate (2-HB), 3-hydroxybutyrate (3HB), 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HH), 4-hydroxybutyrate (4HB), 4-hydroxyvalerate (4HV), 4-hydroxyhexanoate (4HH), 5-hydroxyvalerate (5HV), and 6-hydroxyhexanoate (6HH). It may be a polymer (copolymer) comprising repeating units derived from the above (from one or more monomers), but is not limited thereto.

[0091] The above PHA may have a weight-average molecular weight (Mw) of 50,000 g / mol or more, 100,000 g / mol or more, 200,000 g / mol or more, 300,000 g / mol or more, 400,000 g / mol or more, or 500,000 g / mol or more, and may have a weight-average molecular weight (Mw) of 1,200,000 g / mol or less, 900,000 g / mol or less, 800,000 g / mol or less, 700,000 g / mol or less, or 600,000 g / mol or less.

[0092] The above PHA may have a purity of 95% or more, 97% or more, 98% or more, or 99% or more.

[0093] The PHA according to the present invention is obtained through the purification method described above and has excellent color and purity, so it can be used in the manufacture of various biodegradable articles (e.g., films, containers, etc.).

[0095] The present invention will be explained in more detail through the following examples. However, the scope of the present invention is not limited by these examples.

[0097] [Experimental Example]

[0098] S-1: Obtaining PHA suspension

[0099] The first solid-liquid separation of the PHA fermentation liquid was performed using a mechanical separator, thereby separating impurities from the PHA fermentation liquid and the PHA-containing biomass. Subsequently, the total solid content of the separated PHA-containing biomass was adjusted to approximately 13 wt%, and the pH of the PHA-containing biomass was adjusted to 11 using sodium hydroxide (concentration: 40%, Deoksan Science). Then, sodium laureth sulfate (concentration: 30%, Miwon Commercial Co.) was added as a surfactant to a concentration of 1 wt% relative to the total solid content of the PHA-containing biomass. After thoroughly stirring the PHA-containing biomass with the added surfactant, physical cell fragmentation was performed using a high-pressure homogenizer (manufacturer: Bertoli) (400–800 bar, 1 pass). After treatment with the above-mentioned high-pressure homogenizer, the obtained PHA slurry was subjected to a second solid-liquid separation using a mechanical separator, and as a result, a PHA suspension (PHA (P-3-HB-co-4-HB) solid content 10 wt%) was obtained.

[0100] S-2: Decolorization reaction

[0101] Hydrogen peroxide and sodium hydroxide were added to the PHA suspension by adjusting the amount of hydrogen peroxide and the ratio of hydrogen peroxide to sodium hydroxide, and the reaction was carried out by reacting at a temperature of 45°C for 2 hours to perform a decolorization reaction of the PHA suspension.

[0102] S-3: Deproteinization

[0103] A protease (0.4% Protease) was added to the product obtained through the above decolorization reaction, and a deproteinization reaction was performed for 2 hours in a shaking incubator (IS-971R, JEIO TECH) at a temperature of 60 ℃.

[0104] Subsequently, the solid was recovered from the product obtained through the above deproteinization reaction, and the recovered PHA solid was washed with water and dried to obtain the final product, PHA.

[0106] [Comparison Example]

[0107] In the above experimental example, the final product, PHA, was obtained through the same process as the above experimental example (i.e., only the decolorization reaction process was performed differently, while the processes of obtaining the PHA suspension, deproteinization, and solid recovery were performed identically), except that sodium hydroxide was added to the PHA suspension (PHA solid content 10 wt%) obtained through the 'S-1: Obtaining PHA suspension' process to adjust the pH to 9, hydrogen peroxide was added, and a decolorization reaction was performed at 60°C for 2 hours. Specifically, in the case of the control example, unlike the experimental example, after adjusting the pH of the PHA suspension to 9, sodium hydroxide was added to the PHA suspension during the decolorization reaction to control the amount of hydrogen peroxide added or to maintain the initial pH of the reaction after adding hydrogen peroxide.

[0109] In the above 'S-2: Decolorization reaction', experimental examples and control examples were classified according to the amount of hydrogen peroxide (H2O2) added and the ratio of hydrogen peroxide (H2O2) to sodium hydroxide (NaOH) added, as shown in Table 1 below.

[0111] H2O2 input amount (weight%) H2O2:NaOH input ratio (weight ratio) k obs Experimental Example 1 7 wt% 1:0.05 0.0219 Experimental Example 2 7 wt% 1:0.09 0.0394 Experimental Example 3 7 wt% 1:0.11 0.0482 Experimental Example 4 5 wt% 1:0.05 0.0157 Experimental Example 5 5 wt% 1:0.09 0.0282 Experimental Example 6 3.5 wt% 1:0.03 0.0066 Experimental Example 7 3.5 wt% 1:0.05 0.0110 Experimental Example 8 3.5 wt% 1:0.07 0.0153 Experimental Example 9 3.5 wt% 1:0.09 0.0197 Experimental Example 10 3.5 wt% 1:0.11 0.0241 Experimental Example 11 3.5 wt% 1:0.12 0.0263 Experimental Example 12 3.5 wt% 1:0.14 0.0307 Experimental Example 13 3 weight% 1:0.06 0.0113 Experimental Example 14 3 weight% 1:0.09 0.0169 Experimental Example 15 2.5 wt% 1:0.07 0.0110 Experimental Example 16 2 wt% 1:0.06 0.0075 Experimental Example 17 2 wt% 1:0.09 0.0113 Experimental Example 18 1.5 wt% 1:0.03 0.0028 Experimental Example 19 1.5 wt% 1:0.05 0.0047 Experimental Example 20 1.5 wt% 1:0.12 0.0113 Comparative Example 1 7 wt% cf. pH adjusted to 9 by adding NaOH, then no additional NaOH added (pH not adjusted). H2O2:NaOH addition ratio = 1:0.005~0.013 by weight. Comparison Example 2 3.5 wt% cf. pH adjusted to 9 by adding NaOH, then no additional NaOH added (pH not adjusted). H2O2:NaOH addition ratio = 1:0.005~0.013 by weight. Comparison Example 3 7 wt% cf. After adjusting the pH to 9 by adding NaOH, NaOH is continuously added to maintain the initial reaction pH. The weight ratio of H2O2 to NaOH is 1:0.01. Hydrogen peroxide input amount -> Based on 100 wt% of PHA solids contained in the PHA suspension Hydrogen peroxide -> Use a 30% concentration aqueous hydrogen peroxide solution Sodium hydroxide -> Use a 20% concentration aqueous sodium hydroxide solution k obs = Reaction rate constant for the generation of hydrogen peroxide-derived radicals

[0113] Meanwhile, k in Table 1 above obs represents the reaction rate constant for the generation of hydrogen peroxide-derived radicals, which can be predicted through the results of evaluating how much hydrogen peroxide-derived radical is generated via the methylene blue reaction. The evaluation using the methylene blue reaction can be performed by conducting a decolorization reaction at 45°C for 2 hours while controlling the input ratio of hydrogen peroxide and sodium hydroxide in a 20 μM methylene blue solution, and measuring the absorbance at 664 nm using a UV spectrophotometer before and after the decolorization reaction (see Evaluation Example 1 below).

[0114] Based on the evaluation results using the above methylene blue reaction, the reaction rate constant (k) for the generation of hydrogen peroxide-derived radicals obs The value can be predicted by calculating ) according to the following process, and the predicted value (k obs ) was summarized and shown in Table 2 below.

[0115] The decolorization reaction using a methylene blue substrate can be represented by the following detailed reaction (A. Mohey El-Dein et al., 2003).

[0116]

[0117] Here, since the reactions in Equation 1 (autodecomposition of hydrogen peroxide) and Equation 3 (autodecomposition under alkaline conditions) are minor reactions and are judged to have a negligible effect on the main decolorization reaction, if Equations 2 and 4 to 10 are considered as the main decolorization reaction of the methylene blue substrate, the hydroxyl radical concentration (C OH- ) can be expressed as follows.

[0118]

[0119] The decomposition reaction of methylene blue is known to be a first-order reaction, and the rate equation including radical concentration is as follows.

[0120]

[0121] Here, the reaction rate constant for the generation of hydrogen peroxide-derived radicals (apparent decolorization reaction rate) (k obs ) can be re-expressed as follows.

[0122]

[0123] Under the methylene blue substrate concentration conditions (20 μM) used in this test, the denominator's Since it is evaluated to be at a negligible level, the reaction rate constant (k) for the generation of hydrogen peroxide-derived radicals obs ) ultimately depends on the OH- concentration added as follows.

[0124]

[0125] Considering this, a relationship equation for the change in k value according to various input conditions of hydrogen peroxide and sodium hydroxide was derived, and finally k obs The values ​​were predicted as shown in Table 2 below.

[0127]

[0129] [Evaluation Method]

[0130] a. Methylene Blue Reaction Analysis

[0131] Methylene blue was used as a substrate to evaluate the radical generation efficiency according to the ratio of hydrogen peroxide and sodium hydroxide. Hydrogen peroxide and sodium hydroxide were added to a 20 μM methylene blue solution, and the absorbance at 664 nm was measured using a UV spectrophotometer (HACH, DR6000).

[0132] b. Measurement of chemical input amount

[0133] The chemicals used during PHA purification refer to acidic and basic components for adjusting the reaction pH; the amount of chemical input was measured by calculating the solid content of the acidic and basic components introduced into processes such as decolorization and deproteinization, and determining the amount input relative to the PHA content.

[0134] c. Color-b measurement

[0135] The Color-b value of PHA was measured using a colorimeter (spectrophotometer, Konica Minolta). Approximately 8 g of PHA was placed on a petri dish dedicated to the colorimeter, and the color was measured.

[0136] d. Measurement of impurity content

[0137] The content of crude protein and crude fat as impurities contained in PHA was measured. The content of crude protein was confirmed by the Dumas Nitrogen / Protein Analysis method. The content of crude fat was confirmed through a process of extracting crude fat using diethyl ether.

[0138] e. Potential for Process Application

[0139] The feasibility of process utilization was evaluated based on the Color-b value of PHA and the amount of chemical input, according to the following criteria.

[0140] ○: When the difference between the Color-b value of the PHA in the experimental example and the Color-b value of the PHA in the control example is less than 1.5, and the chemical input amount of the experimental example is less than 2 times the chemical input amount of the control example.

[0141] △: When the difference between the Color-b value of the PHA in the experimental example and the Color-b value of the PHA in the control example is 1.5 or more, and the chemical input amount of the experimental example is 2 times or more compared to the chemical input amount of the control example.

[0143] [Evaluation Example 1]

[0144] The generation rate A of hydrogen peroxide-derived radicals according to reaction time was evaluated using the methylene blue reaction, and the results are shown in Figure 1.

[0145] Specifically, using a 20 μM methylene blue solution, the absorbance at 664 nm was measured using a UV spectrophotometer for reaction sample (a mixture of methylene blue solution, hydrogen peroxide, and sodium hydroxide) at the time of reaction before, 0.5 hours, 1 hour, 1.5 hours, and 2 hours. Next, the generation rate A (%) of hydrogen peroxide-derived radicals was calculated according to Equation 2 below using the above measurement results. The value according to Equation 2 below represents the relative consumption rate (%) of methylene blue. That is, as radicals are generated from hydrogen peroxide and methylene blue is consumed, the color of the methylene blue solution becomes lighter and the relative consumption rate (%) of methylene blue increases, thereby confirming the generation rate A (%) of hydrogen peroxide-derived radicals.

[0146] [Equation 2]

[0147] Generation rate of hydrogen peroxide-derived radicals A(%) = {(A0- A1) / A0} × 100

[0148] In Equation 2,

[0149] A0 is the absorbance of the reaction solution sample measured at 664 nm before starting the reaction, and

[0150] A1 is the absorbance of the reaction sample measured at 664 nm after extracting the reaction sample while performing the reaction over time.

[0151] Referring to Fig. 1, it can be seen that the generation rate A of hydrogen peroxide-derived radicals varies significantly depending on the input ratio of hydrogen peroxide and sodium hydroxide during the reaction. Specifically, it can be seen that the reaction is carried out by simultaneously inputting hydrogen peroxide and sodium hydroxide and controlling the input ratio ((a), (b), and (c)) as in the present invention, rather than starting the reaction at pH 9 (pH start) or carrying out the reaction while maintaining pH 9 (pH maintenance).

[0153] [Evaluation Example 2]

[0154] The generation rate B of hydrogen peroxide-derived radicals according to the decolorization reaction temperature was evaluated using the methylene blue reaction, and the results are shown in Figure 2.

[0155] Specifically, reaction samples (a mixture of methylene blue solution, hydrogen peroxide, and sodium hydroxide) were reacted with a 20 μM methylene blue solution at temperatures of 25 ℃, 45 ℃, 50 ℃, 55 ℃, and 60 ℃, and the absorbance at 664 nm was measured for the reaction samples at each reaction temperature using a UV spectrophotometer. Next, the generation rate B (%) of hydrogen peroxide-derived radicals was calculated according to Equation 3 below using the above measurement results. The value according to Equation 3 below also represents the relative consumption rate (%) of methylene blue.

[0156] [Equation 3]

[0157] Generation rate of hydrogen peroxide-derived radicals B(%) = {(A0- A2) / A0} × 100

[0158] In Equation 3,

[0159] A0 is the absorbance of the reaction solution sample measured at 664 nm before starting the reaction, and

[0160] A2 is the absorbance of the reaction sample measured at 664 nm after extracting the reaction sample while performing the reaction according to the set temperature.

[0161] Referring to FIG. 2, when the reaction is carried out by controlling the input ratio of hydrogen peroxide and sodium hydroxide as in the present invention, the generation rate B of hydrogen peroxide-derived radicals is high even at a relatively low temperature (e.g., 45°C), and it can be seen that the reaction is carried out efficiently at a low temperature in the present invention compared to the conventional technology in which the reaction was carried out at 60°C.

[0163] [Evaluation Example 3]

[0164] The amount of chemicals consumed in the process of obtaining PHA for Experimental Examples 3, 6 to 10 and Control Examples 1 and 2, as well as the Color-b and impurity content of the PHA, were evaluated, and the results are shown in Table 3 below.

[0166] H2O2 input amount (weight%) H2O2:NaOH input ratio (weight ratio) k obs Color-b Chemical input amount impurities Crude protein (wt%) Crude fat (wt%) Experimental Example 3 7 wt% 1:0.11 0.0482 11.4 3.2 0.31 0.11 Experimental Example 6 3.5 wt% 1:0.03 0.0066 16 0.92 - - Experimental Example 7 3.5 wt% 1:0.05 0.0110 15.2 1.07 0.31 0.17 Experimental Example 8 3.5 wt% 1:0.07 0.0153 14.7 1.37 - - Experimental Example 9 3.5 wt% 1:0.09 0.0197 14.6 1.74 - - Experimental Example 10 3.5 wt% 1:0.11 0.0241 12.5 2.08 - - Comparative Example 1 7 wt% cf. Adjusted pH to 9 by adding NaOH, then no additional NaOH added (pH not adjusted) 16.6 1.15 0.35 0.25 Comparison Example 2 3.5 wt% cf. Adjusted pH to 9 by adding NaOH, then no additional NaOH added (pH not adjusted) 17.6 0.96 - -

[0167] Referring to Table 3 above, it can be confirmed that by controlling the input ratio of hydrogen peroxide and sodium hydroxide as in the present invention, a PHA with a relatively low Color-b value and reduced impurity content is obtained. Specifically, it can be seen that even if the input amount of hydrogen peroxide is reduced from 7 wt% to 3.5 wt% (see Experimental Examples 3 and 7), by controlling the input amount of sodium hydroxide, a PHA with a reduced impurity content compared to Control Example 1 is obtained. This supports the fact that by optimizing the input ratio of hydrogen peroxide and sodium hydroxide, the generation of hydrogen peroxide-derived radicals is activated (maximized), and the decolorization reaction is carried out efficiently.

[0168] Meanwhile, when the amount of hydrogen peroxide added is 3.5 wt%, increasing the amount of sodium hydroxide added lowers the Color-b value of PHA and improves color, but the amount of chemical added increases. Considering the Color-b value and the efficiency of the downstream process, it can be determined that the case of Experimental Example 7, in which 3.5 wt% of hydrogen peroxide is added and the ratio of hydrogen peroxide to sodium hydroxide added is 1:0.05, will most significantly improve economic efficiency and decolorization reaction efficiency.

[0170] [Evaluation Example 4]

[0171] The decolorization reaction efficiency was evaluated based on Experimental Example 7, which showed excellent results in Evaluation Example 3 above. Specifically, the reaction rate constant (k) of the generation of hydrogen peroxide-derived radicals in Experimental Example 7 obs Based on 0.011, in order to design economical and high-efficiency decolorization reaction conditions, the reaction rate constant (k) formed under the condition where the input amount of hydrogen peroxide is 3.5 wt% or less obs Experimental examples corresponding to the range of 0.003 to 0.026 were selected to evaluate the chemical input amount, Color-b of PHA, recovery rate of PHA, and process applicability, and the results are shown in Table 4 below.

[0173] H2O2 input amount (weight%) H2O2:NaOH input ratio (weight ratio) k obs Color-b recovery rate Chemical input amount Whether process utilization is feasible Experimental Example 7 3.5 wt% 1:0.05 0.0110 11.54 100.14 1.01 ○ Experimental Example 11 3.5 wt% 1:0.12 0.0263 11.78 94.78 2.15 △ Experimental Example 12 3.5 wt% 1:0.14 0.0307 12.04 97.02 2.49 △ Experimental Example 13 3 weight% 1:0.06 0.0113 11.62 94.83 1.09 ○ Experimental Example 14 3 weight% 1:0.09 0.0169 11.29 98.17 1.51 ○ Experimental Example 15 2.5 wt% 1:0.07 0.0110 11.78 101.82 1.35 ○ Experimental Example 16 2 wt% 1:0.06 0.0075 14.5 98.76 1.01 △ Experimental Example 17 2 wt% 1:0.09 0.0113 13.76 95.42 1.16 △ Experimental Example 18 1.5 wt% 1:0.03 0.0028 15.16 96.33 0.82 △ Experimental Example 19 1.5 wt% 1:0.05 0.0047 13.05 99.64 0.85 △ Experimental Example 20 1.5 wt% 1:0.12 0.0113 12.76 101.40 1.23 △ Comparative Example 1 7 wt% cf. Adjusted pH to 9 by adding NaOH, then no additional NaOH added (pH not adjusted) 11.2 97.43 0.95 ○

[0174] Referring to Table 4 above, it can be seen that when the amount of hydrogen peroxide added is 2 wt% or less, the Color-b value of the recovered PHA tends to increase, and when the ratio of hydrogen peroxide to sodium hydroxide added is 1:0.12 or higher, the amount of chemical added tends to increase. Accordingly, it was confirmed that it is advantageous to control the amount of hydrogen peroxide added to 2.5 wt% or more and the ratio of hydrogen peroxide to sodium hydroxide added to 1:0.05 to 0.09.

[0176] [Evaluation Example 5]

[0177] When the input ratio of hydrogen peroxide to sodium hydroxide was 1:0.05 to 0.09, Experimental Examples 1, 2, 4, 5, 7, and 9 were selected to optimize the input amount of hydrogen peroxide, and the chemical input amount, Color-b of PHA, recovery rate of PHA, and process applicability were evaluated, and the results are shown in Table 5 below.

[0179] H2O2 input amount (weight%) H2O2:NaOH input ratio (weight ratio) k obs Color-b recovery rate Chemical input amount Whether process utilization is feasible Experimental Example 1 7 wt% 1:0.05 0.0219 10.5 96.20 1.90 △ Experimental Example 2 7 wt% 1:0.09 0.0394 10.2 98.00 2.89 △ Experimental Example 4 5 wt% 1:0.05 0.0157 11.3 97.22 1.14 ○ Experimental Example 5 5 wt% 1:0.09 0.0282 11.1 97.89 1.74 ○ Experimental Example 7 3.5 wt% 1:0.05 0.0110 11.54 100.14 1.01 ○ Experimental Example 9 3.5 wt% 1:0.09 0.0197 11.5 97.65 1.42 ○ Comparative Example 1 7 wt% cf. Adjusted pH to 9 by adding NaOH, then no additional NaOH added (pH not adjusted) 11.2 97.43 0.95 ○

[0180] Referring to Table 5 above, considering the Color-b value and the downstream process efficiency according to the amount of chemical input, it can be confirmed that Experimental Examples 4, 5, 7, and 9 have superior decolorization reaction efficiency. Through this, it was confirmed that it is more advantageous to control the amount of hydrogen peroxide input to 2.5 wt% to 5 wt% and the input ratio of hydrogen peroxide to sodium hydroxide to 1:0.05 to 0.09.

Claims

Claim 1 A method for purifying polyhydroxyalkanoate, comprising the steps of: obtaining a suspension containing polyhydroxyalkanoate (PHA) from a microbial fermentation liquid; and adding hydrogen peroxide and an alkali component to the suspension to cause a decolorization reaction, wherein the weight ratio of the hydrogen peroxide and the alkali component is 1:0.03 to 0.

11. Claim 2 A method for purifying polyhydroxyalkanoate according to claim 1, wherein the alkali component comprises one or more selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium acetate, potassium acetate, trisodium phosphate, sodium hydrogen phosphate, tripotassium phosphate, and potassium phosphate. Claim 3 delete Claim 4 A method for purifying polyhydroxyalkanoate according to claim 1, wherein the hydrogen peroxide and the alkali component are added to the suspension such that the weight ratio of the hydrogen peroxide and the OH derived from the alkali component is 1:0.01 to 0.

05. Claim 5 A method for purifying polyhydroxyalkanoate according to claim 1, wherein the amount of hydrogen peroxide added is 2% to 7% by weight based on 100% by weight of the PHA solid content contained in the suspension. Claim 6 A method for purifying polyhydroxyalkanoate according to claim 1, wherein the hydrogen peroxide and the alkali component are simultaneously introduced into the suspension. Claim 7 A method for purifying polyhydroxyalkanoate according to claim 1, wherein the temperature at which the decolorization reaction is performed is 35 ℃ to less than 70 ℃. Claim 8 A method for purifying a polyhydroxyalkanoate according to claim 1, wherein the polyhydroxyalkanoate (PHA) comprises a repeating unit derived from 3-hydroxybutyrate; and a repeating unit derived from one or more selected from the group consisting of 4-hydroxybutyrate, 3-hydroxypropionate, 3-hydroxyvalerate and 3-hydroxyhexanoate. Claim 9 A method for purifying polyhydroxyalkanoate according to claim 1, further comprising the step of deproteinizing the product obtained through the decolorization reaction. Claim 10 A method for purifying polyhydroxyalkanoate according to claim 1, wherein the purified polyhydroxyalkanoate (PHA) has a color-b of 25 or less as measured using a colorimeter. Claim 11 Polyhydroxyalkanoate purified by a purification method according to any one of claims 1, 2, and 4 to 9. Claim 12 In claim 11, the polyhydroxyalkanoate (PHA) is a polyhydroxyalkanoate having a color-b of 25 or less as measured using a colorimeter.

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