Method for producing polyhydroxyalkanoates and their use

Abstract

The purpose of the present invention is to provide a method for producing PHA which enables efficient filtering. The above problem is solved by providing a method for producing PHA, said method comprising a step for using a filtering material having an airflow rate of 0.01-5.0 cc / cm2 / sec to perform dead-end filtration of an aqueous PHA suspension having a pH of 2.5-5.5, wherein the amount of PHA-surface-adhering protein in the aqueous PHA suspension is not more than 2,000 ppm, and the liquid density of the aqueous PHA suspension in the filtration step is 0.50-1.08 g / mL.

Description

[Technical Field]

[0001] This invention relates to a method for producing polyhydroxyalkanoates and to the use of such polyhydroxyalkanoates. [Background technology]

[0002] Polyhydroxyalkanoates (hereinafter sometimes referred to as "PHA") are known to be biodegradable.

[0003] Since PHA produced by microorganisms accumulates within the microbial cells, a process of separating and purifying PHA from the microbial cells is necessary for its use as a plastic. In the process of separating and purifying PHA, the microbial cells of PHA-containing microorganisms are crushed or other biological components are solubilized, and then PHA is extracted from the resulting aqueous suspension. At this time, separation operations such as centrifugation, filtration, and drying are performed.

[0004] As a method for producing PHA using filtration, for example, Patent Document 1 discloses a method for producing PHA that includes the steps of: inoculating a PHA-fermenting bacterial species into a fermentation medium and fermenting it; separating the fermentation liquid into solid and liquid to obtain the fermentation supernatant and bacterial cell precipitate; precipitating the bacterial cells to disrupt the cell walls, and then performing plate-frame filtration using a filter pre-coated with the disrupted cell walls to obtain PHA.

[0005] Furthermore, Patent Document 2 discloses a method for recovering and purifying PHA from cell cultures, which includes a step of performing an acid treatment as a pretreatment. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Chinese Patent No. 111500650 Specification [Patent Document 2] International Publication No. 2015 / 015395 [Overview of the project]

Problems to be Solved by the Invention

[0007] However, the above-described technology still had room for further improvement in terms of the filtrate permeation rate and the leakage rate.

[0008] Therefore, an object of the present invention is to provide a method for producing PHA that can be efficiently filtered.

Means for Solving the Problems

[0009] As a result of intensive studies to solve the above problems, the present inventor has found that by controlling the pH of the PHA aqueous suspension, the air flow rate of the filter medium, the amount of protein attached to the surface of PHA, and the liquid density of the PHA aqueous suspension in the filtration step of producing PHA, a specific filtrate permeation rate and a specific leakage rate can be achieved (that is, efficient filtration is possible), and the present invention has been completed.

[0010] Therefore, one aspect of the present invention includes a step of performing dead-end filtration on a PHA aqueous suspension having a pH of 2.5 to 5.5 using a filter medium having an air flow rate of 0.01 to 5.0 cc / cm 2 / sec, wherein the amount of protein attached to the surface of PHA in the PHA aqueous suspension is 2000 ppm or less, and the liquid density of the PHA aqueous suspension in the filtration step is 0.50 to 1.08 g / mL. This is a method for producing PHA (hereinafter referred to as "the present production method").

[0011] Another aspect of the present invention is a PHA aggregate (hereinafter referred to as "the present PHA aggregate") having a Feret diameter of 1 to 100 mm and a water content of 25.0 to 50.0% (W.B.).

Advantages of the Invention

[0012] According to one aspect of the present invention, a method for producing PHA that can be efficiently filtered can be provided.

Brief Description of the Drawings

[0013] [Figure 1] This figure shows an example of measuring the ferret diameter of a PHA aggregate according to Example 10 of the present invention. [Modes for carrying out the invention]

[0014] One embodiment of the present invention will be described in detail below. Unless otherwise specified in this specification, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B". Furthermore, all references cited in this specification are incorporated herein by reference.

[0015] [1. Outline of the present invention] PHA produced within bacterial cells has a particle size of approximately 1-2 μm, making filtration difficult. Furthermore, while centrifugation is primarily used to recover PHA, the resulting separation is in the form of an aqueous suspension, meaning the PHA is recovered in a state containing a large amount of water. To recover PHA by separating it from water, it is necessary to evaporate the water from the aqueous suspension, which requires a large amount of energy and presents other problems.

[0016] As a method for producing PHA using filtration, for example, the aforementioned Patent Documents 1 and 2 are known. In the technique of Patent Document 1, filtration is performed before purification (when a large amount of biological residue remains), resulting in a problem of high impurity content and extremely slow filtration speed. Furthermore, when the present inventor conducted further tests on the technique of Patent Document 2, it was found that the pH range was 6 to 7, and that filtration under these conditions resulted in a significant amount of resin leakage. It was also found that, under the same conditions, using finer filter cloth resulted in an extremely slow filtration speed.

[0017] Therefore, the inventors diligently studied the filtration process in the production of PHA from the viewpoint of improving the filtrate permeation rate and leakage rate. As a result, they discovered for the first time that a specific filtrate permeation rate and a specific leakage rate can be achieved (i.e., efficient filtration becomes possible) by controlling the pH of the PHA aqueous suspension, the air permeability of the filter material, the amount of protein attached to the PHA surface, and the liquid density of the PHA aqueous suspension.

[0018] This manufacturing method provides a method for efficiently filtering PHA. Therefore, this manufacturing method is extremely advantageous in the industrial production of PHA. In this specification, "efficiently filtering" means that the filtrate permeation rate is 200 L / m³. 2 This means that the data usage is above / hr and the leakage rate is 5% or less.

[0019] Furthermore, the above-described configuration reduces the amount of heat, time, and cost required in the drying process after filtration, i.e., energy. This contributes to achieving Sustainable Development Goals (SDGs), such as Goal 7, "Ensure access to affordable, reliable, sustainable and modern energy for all." The present invention will be described in detail below.

[0020] [2. Method for producing PHA] This manufacturing method includes the following steps: • A PHA aqueous suspension with a pH of 2.5 to 5.5 is subjected to an aeration rate of 0.01 to 5.0 cc / cm². 2 A dead-end filtration step using a filter medium with a density of / sec, wherein the amount of PHA surface-adhering protein in the PHA aqueous suspension is 2000 ppm or less, and the liquid density of the PHA aqueous suspension in the filtration step is 0.50 to 1.08 g / mL (hereinafter referred to as step (d)).

[0021] Furthermore, in one embodiment of the present invention, it is preferable that the manufacturing method includes at least one of the following steps in addition to step (d) above. • Step (a): A step (also referred to as the "solubilization step") that destroys and solubilizes cell-derived components other than PHA in bacterial cells containing PHA, wherein the volume median diameter of the PHA is 0.5 to 5.0 μm. • Step (b): After step (a), the PHA aqueous suspension is recovered by centrifugation (also referred to as the "recovery step"). Step (c'): A step to adjust the pH of the PHA aqueous suspension obtained in step (b) to 2.5-5.5 (also referred to as the "preparation step"). • Step (c): A step in which the PHA aqueous suspension is heated to a temperature of 60-120°C (also referred to as the "heat treatment step"). • Step (e): A step of drying the PHA obtained in step (d) at 20 to 160°C (also referred to as the "drying step"). Step (f): A step of redispersing the dried PHA in an aqueous solvent to obtain an aqueous PHA suspension containing PHA with a volume median diameter of 0.5 to 5.0 μm (also referred to as the "redispersion step").

[0022] In this manufacturing method, the above steps are preferably carried out in the order of (a), (b), (c'), (c), (d), (e), and (f), but the order can be changed as appropriate depending on the purpose. For example, the order of steps (a) and (b) can be changed to (b) and (a), and the order of steps (c') and (c) can be changed to (c) and (c'). Also, depending on the purpose, it is possible to carry out steps (a), (b), (c') and (c) two or more times. That is, for example, the steps can be carried out in the order of (b), (a), (b), or (c'), (c), (c'). For the sake of explanation, the steps will be described below in the order of (a), (b), (c'), (c), (d), (e), and (f). In this specification, an aqueous suspension containing at least PHA may be abbreviated as "PHA aqueous suspension."

[0023] (Step (a)) In step (a) of this manufacturing method, cell-derived components other than PHA in the bacterial cells containing PHA are destroyed and solubilized. By destroying and removing impurities (cell walls, proteins, etc.) derived from the bacterial cells in step (a), PHA with a volume median diameter of 0.5 to 5.0 μm can be efficiently recovered from the bacterial cells.

[0024] <pha> In this specification, "PHA" is a general term for polymers having hydroxyalkanoic acid as the monomer unit. The hydroxyalkanoic acid constituting PHA is not particularly limited, but examples include 3-hydroxybutanoic acid, 4-hydroxybutanoic acid, 3-hydroxypropionic acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, and 3-hydroxyoctanoic acid. These polymers may be homopolymers or copolymers containing two or more monomer units.

[0025] More specifically, examples of PHAs include poly(3-hydroxybutyrate) (P3HB), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P3HB3HH), poly(3-hydroxybutyrate-co-3-hydroxyvariate) (P3HB3HV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (P3HB3HO), poly(3-hydroxybutyrate-co-3-hydroxyoctadecanoate) (P3HB3HOD), poly(3-hydroxybutyrate-co-3-hydroxydecanoate) (P3HB3HD), and poly(3-hydroxybutyrate-co-3-hydroxyvariate-co-3-hydroxyhexanoate) (P3HB3HV3HH). Among these, P3HB, P3HB3HH, P3HB3HV, and P3HB4HB are preferred because they are easy to produce industrially.

[0026] Furthermore, by changing the composition ratio of the repeating units, the melting point and degree of crystallinity can be changed, and as a result, physical properties such as Young's modulus and heat resistance can be altered, and it is possible to impart physical properties between those of polypropylene and polyethylene. In addition, as mentioned above, P3HB3HH, a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, is more preferable from the viewpoint that it is easy to produce industrially and is a physically useful plastic.

[0027] In one embodiment of the present invention, the composition ratio of the repeating units of P3HB3HH is preferably such that the composition ratio of 3-hydroxybutyrate units to 3-hydroxyhexanoate units is 80 / 20 to 99.9 / 0.1 (mol / mol), and more preferably 85 / 15 to 97 / 3 (mol / mol), from the viewpoint of balancing flexibility and strength. If the composition ratio of 3-hydroxybutyrate units to 3-hydroxyhexanoate units is 99.9 / 0.01 (mol / mol) or less, sufficient flexibility is obtained, and if it is 80 / 20 (mol / mol) or more, sufficient hardness is obtained.

[0028] In step (a), the volume median diameter of the PHA is preferably 50 times or less, more preferably 20 times or less, and even more preferably 10 times or less, the volume median diameter of the primary particles of the PHA (hereinafter referred to as "primary particle diameter"). When the volume median diameter of the PHA is 50 times or less of the primary particle diameter, the aqueous suspension of PHA exhibits better fluidity, and thus the productivity of PHA tends to improve even further.

[0029] In one embodiment of the present invention, the volume median diameter of the PHA is preferably 0.5 to 5.0 μm, more preferably 1.0 to 4.5 μm, and even more preferably 1.0 to 4.0 μm, from the viewpoint of achieving excellent fluidity. The volume median diameter of the PHA is measured using a HORIBA LA-950 laser diffraction / scattering particle size distribution analyzer.

[0030] <Bacteria (microorganisms)> The microorganisms used in step (a) are not particularly limited, as long as they are microorganisms capable of producing PHA within their cells. For example, microorganisms isolated from nature and deposited in depositaries of strains (e.g., IFO, ATCC, etc.), or mutants and transformants that can be prepared from them can be used. For example, Bacillus megaterium, discovered in 1925, was the first microorganism to produce P3HB, an example of PHA, and other examples of naturally occurring microorganisms include Cupriavidus necator (formerly classified as Alcaligenes eutrophus, Ralstonia eutropha), and Alcaligenes latus. It is known that PHA accumulates within the cells of these microorganisms.

[0031] Furthermore, examples of bacterial cells that produce copolymers of hydroxybutyrate and other hydroxyalkanoates, which are examples of PHAs, include Aeromonas caviae, which produces P3HB3HV and P3HB3HH, and Alcaligenes eutrophus, which produces P3HB4HB. In particular, with respect to P3HB3HH, Alcaligenes eutrophus AC32 strain (FERM BP-6038) (T.Fukui, Y.Doi, J.Bateriol., 179, p4821-4830 (1997)), into which genes for PHA synthases have been introduced to increase the productivity of P3HB3HH, is more preferable. In addition, the bacterial cells may also be genetically modified microorganisms into which various PHA synthesis-related genes have been introduced according to the PHA to be produced.

[0032] <Destruction and solubilization of cell-derived components> The method for destroying and solubilizing cell-derived components other than PHA in the bacterial cells containing PHA in step (a) is not particularly limited.

[0033] In one embodiment of the present invention, the destruction and solubilization are carried out, for example, using a lytic enzyme, a proteolytic enzyme (for example, an alkaline proteolytic enzyme).

[0034] In this specification, "lytic enzyme" refers to an enzyme that has the activity to break down (lyse) the cell wall of a microbial organism (e.g., peptidoglycan).

[0035] In one embodiment of the present invention, the lytic enzyme is not particularly limited and examples include lysozyme, lavia, β-N-acetylglucosaminidase, endolysin, autolysin, etc. From the viewpoint of economic advantage, lysozyme is preferred. One of these may be used alone, or two or more may be used in combination.

[0036] Commercially available lysing enzymes can also be used, such as "Lysozyme" and "Achromopeptidase" manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0037] In one embodiment of the present invention, the optimal pH of the lytic enzyme is not particularly limited as long as the lytic enzyme has cell wall degradation activity, but is, for example, 5.0 to 11.0, preferably 6.0 to 9.0, and more preferably 6.0 to 8.0.

[0038] In one embodiment of the present invention, the optimal temperature of the lytic enzyme is not particularly limited, but from the viewpoint of not requiring excessive heating and preventing thermal changes (thermal decomposition) of PHA, it is preferably 60°C or lower, and more preferably 50°C or lower. The lower limit of the optimal temperature is not particularly limited, but from the viewpoint of not requiring excessive cooling and being economical, it is preferably room temperature (e.g., 25°C) or higher.

[0039] In this specification, "alkaline protease" refers to a protease that has the activity to decompose proteins in an alkaline environment (for example, in a solution with a pH of 8.5).

[0040] In one embodiment of the present invention, the alkaline protease is not particularly limited as long as it has the activity to degrade proteins in an alkaline environment, and examples include serine-specific proteases (e.g., subtilisin, chymotrypsin, trypsin), cysteine-specific proteases (e.g., papain, bromelain, cathepsin), and aspartate-specific proteases (e.g., pepsin, cathepsin D, HIV protease). From the viewpoint of economic advantage, serine-specific proteases, particularly subtilisin (e.g., alcalase), are preferred. One of these may be used alone, or two or more may be used in combination.

[0041] Commercially available alkaline proteolytic enzymes can also be used, such as Novozyme's "Alcalase 2.5L"; Amano Enzyme Co., Ltd.'s "Protin SD-AY10" and "Protease P "Amano" 3SD"; Danisco Japan Co., Ltd.'s "Multifect PR6L" and "Optimase PR89L"; Shin Nippon Chemical Industries, Ltd.'s "Sumizyme MP"; DSM Japan Co., Ltd.'s "Delborase"; Nagase ChemteX Corporation's "Bioprase OP," "Bioprase SP-20FG," and "Bioprase SP-4FG"; HBI Corporation's "Orientase 22BF"; and Yakult Pharmaceutical Co., Ltd.'s "Aloase XA-10."

[0042] In one embodiment of the present invention, the optimal pH of the alkaline protease is not particularly limited as long as the alkaline protease is active in an alkaline environment, but is for example 8.0 to 14.0, preferably 8.0 to 12.0, more preferably 8.0 to 10.0, even more preferably 8.0 to 9.0, and most preferably 8.5.

[0043] In one embodiment of the present invention, the optimal temperature of the alkaline protease is not particularly limited, but from the viewpoint of not requiring excessive heating and preventing thermal changes (thermal decomposition) of PHA, it is preferably 60°C or lower, and more preferably 50°C or lower. The lower limit of the optimal temperature is not particularly limited, but from the viewpoint of not requiring excessive cooling and being economical, it is preferably room temperature (e.g., 25°C) or higher.

[0044] In one embodiment of the present invention, the disruption and solubilization of cell-derived components in step (a) may be carried out in combination with lysozyme and alcalase.

[0045] The enzyme treatment time in step (a) may vary depending on the type of enzyme, pH, temperature, and other conditions, but is, for example, 1 to 8 hours, with 2 to 6 hours being preferred.

[0046] In this manufacturing method, the solvent constituting the PHA aqueous suspension ("solvent" is also referred to as "aqueous medium") may be water or a mixed solvent of water and an organic solvent. Furthermore, the concentration of the water-compatible organic solvent in the mixed solvent is not particularly limited as long as it is less than or equal to the solubility of the organic solvent used in water. Furthermore, while there are no particular limitations on the water-compatible organic solvent, examples include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, pentanol, hexanol, and heptanol; ketones such as acetone and methyl ethyl ketone; ethers such as tetrahydrofuran and dioxane; nitriles such as acetonitrile and propionitrile; amides such as dimethylformamide and acetamide; and dimethyl sulfoxide, pyridine, and piperidine. Among these, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, propionitrile, etc. are preferred because they are easy to remove. Furthermore, methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone, etc. are more preferred because they are readily available. In addition, methanol, ethanol, and acetone are particularly preferred. The aqueous medium constituting the PHA aqueous suspension may contain other solvents, bacterial-derived components, compounds generated during purification, etc., as long as it does not impair the essence of the present invention.

[0047] In this manufacturing method, the aqueous medium constituting the PHA aqueous suspension preferably contains water. The water content in the aqueous medium is preferably 5% by weight or more, more preferably 10% by weight or more, even more preferably 30% by weight or more, and particularly preferably 50% by weight or more.

[0048] (Other processes) In one embodiment of the present invention, the manufacturing method may include the following step before step (a).

[0049] <Process (a1)> Step (a1) is the step of culturing bacterial cells containing PHA.

[0050] In step (a1), the bacterial cells used are, for example, the bacterial cells described in the section <Bacterial cells (microorganisms)> above.

[0051] In step (a1), the method for culturing the bacterial cells is not particularly limited, but examples include the method described in paragraphs

[0041] to

[0048] of International Publication No. WO2019 / 142717.

[0052] <Process (a2)> Step (a2) is a step to inactivate the bacterial cells obtained in step (a1). In this step, the bacterial cells obtained in step (a1) are inactivated to obtain an inactivated culture medium.

[0053] In step (a2), the method of inactivation is not particularly limited, but one example is a method in which the culture medium containing P3HA is heated and stirred at an internal temperature of 60-70°C for 7 hours.

[0054] <Process (a3)> Step (a3) ​​is a step of adjusting the concentration and pH of the inactivated culture medium obtained in step (a2). Step (a3) ​​is mainly performed when the viscosity of the inactivated culture medium obtained in step (a2) is high, and the viscosity of the inactivated culture medium is reduced by adjusting the concentration and pH of the inactivated culture medium. Step (a3) ​​facilitates solubilization in step (a).

[0055] In step (a3), the method for adjusting the concentration and pH of the inactivated culture medium is not particularly limited and can be carried out by any method used in the art. For example, the concentration of the inactivated culture medium can be adjusted by adding hydrogen peroxide or the like. As a method for adjusting the pH, for example, an example is to add a basic compound to the inactivated culture medium. The basic compound is not particularly limited, but alkali metal hydroxides or alkaline earth metal hydroxides are preferred, and sodium hydroxide is more preferred. The basic compound may be used alone or two or more in combination.

[0056] (Step (b)) In step (b) of this manufacturing method, the PHA aqueous suspension is recovered by centrifugation after step (a). Step (b) removes impurities (cell walls, proteins, etc.) derived from the bacterial cells from the PHA aqueous suspension.

[0057] In step (b), the PHA aqueous suspension is recovered by any centrifugation method known in the art. The centrifugation method is not particularly limited, but examples include centrifugation using a centrifugal sedimentation machine, a centrifugal dehydrator, etc.

[0058] Examples of centrifugal sedimentation machines include separation plate type (e.g., disk type, self-cleaning type, nozzle type, screw decanter type, skimming type, etc.), cylindrical type, and decanter type centrifugal sedimentation machines. Each type can be classified into palindromic and continuous types depending on the method of discharge of the settled components. Similarly, centrifugal dehydrators can also be classified into palindromic and continuous types. By using these devices, it is possible to separate the settled material containing PHA from the culture medium components based on the difference in specific gravity.

[0059] Since steps (a) and (b) largely determine the amount of impurities remaining in the final product, it is preferable to reduce these impurities as much as possible. Naturally, depending on the application, it is acceptable for impurities to be present as long as they do not impair the physical properties of the final product. However, when high-purity PHA is required, such as for medical applications, it is preferable to reduce impurities as much as possible. An indicator of the degree of purification in this case is, for example, the amount of protein attached to the surface of the PHA in the aqueous PHA suspension. This amount of protein is 2000 ppm or less per weight of PHA, preferably 1900 ppm or less, more preferably 1800 ppm or less, and most preferably 1700 ppm or less. When the amount of protein attached to the surface of the PHA in the aqueous PHA suspension is within the above range, it has the advantage that the leakage rate does not become too high. This effect is presumed to be because when the amount of protein attached to the surface of the PHA is low, the PHA molecules tend to stick together.

[0060] (Process (c')) In step (c'), the PHA aqueous suspension recovered by centrifugation typically has a pH greater than 7. Therefore, in step (c') of this manufacturing method, the PHA aqueous suspension obtained in step (b) is adjusted to a pH of 2.5 to 5.5. By adjusting the pH in step (c'), the leakage rate during filtration in step (d) is reduced.

[0061] In step (c'), the pH of the PHA aqueous suspension is 2.5 to 5.5, preferably 2.5 to 5.0, more preferably 2.5 to 4.5, even more preferably 2.5 to 4.0, and particularly preferably 2.5 to 3.5. When the pH of the PHA aqueous suspension is within the above range, it has the advantage that the filtrate permeation rate can be improved in the filtration step without increasing the leakage rate of PHA into the filtrate. This effect is presumed to be because the PHA does not become too small and is easily aggregated. Furthermore, regarding the upper limit of pH, a pH of 5.5 or lower is preferred from the viewpoint of reducing discoloration when PHA is heated and melted, ensuring molecular weight stability during heating and / or drying, and obtaining PHA with reduced discoloration during heating and melting and suppressed molecular weight reduction during heating and / or drying. Regarding the lower limit of pH, a pH of 2.5 or lower is preferred from the viewpoint of the acid resistance of the container.

[0062] In step (c'), the method for adjusting the pH is not particularly limited, and examples include adding an acid. The acid is not particularly limited and may be either an organic or inorganic acid, regardless of whether it is volatile or not. More specifically, examples of acids that can be used include sulfuric acid, hydrochloric acid, phosphoric acid, and acetic acid.

[0063] In one embodiment of the present invention, it is preferable not to perform any additional pH adjustment between the pH adjustment in step (c) and the performance of step (d).

[0064] (Process (c)) In step (c) of this manufacturing method, the PHA aqueous suspension is heated to a temperature of 60-120°C. Step (c) can increase the filtrate permeation rate during filtration.

[0065] In step (c), it is preferable to heat the PHA aqueous suspension to a temperature of 60 to 120°C, more preferably to 62 to 118°C, and even more preferably to 65 to 115°C. When the temperature of the PHA aqueous suspension is within the above range, the filtrate permeation rate during filtration can be further increased.

[0066] In step (c), the method of heat treatment is not particularly limited, but examples include (i) warming the container containing the PHA aqueous suspension using steam, (ii) warming the container containing the PHA aqueous suspension using oil, and (iii) directly introducing steam into the PHA aqueous suspension. The temperature of the steam in (i) and (iii) and the temperature of the oil in (ii) are not particularly limited as long as the temperature of the PHA aqueous suspension in step (c) is 60 to 120°C, for example, 95 to 150°C.

[0067] (Step (d)) In step (d) of this manufacturing method, a PHA aqueous suspension with a pH of 2.5 to 5.5 is subjected to an aeration rate of 0.01 to 5.0 cc / cm³. 2 Dead-end filtration is performed using a filter medium with a rate of / sec. In step (d), the amount of PHA surface-adhering protein in the PHA aqueous suspension is 2000 ppm or less, and the liquid density of the PHA aqueous suspension in the filtration step is 0.50 to 1.08 g / mL. Step (d) yields PHA with a constant ferret diameter and water content.

[0068] In this specification, the unit area (cm²) of the filter media per second is used. 2 The amount of air (cc) passing through the ) is called the permeability rate. In process (d), the permeability rate is 0.01 to 5.0 cc / cm³. 2 The rate is / sec, and the flow rate is 0.1~4.0cc / cm³. 2 / sec is preferred, and 0.2 to 3.5 cc / cm 2 / sec is more preferred, and 0.3 to 3.0 cc / cm 2 / sec is even more preferred, and 0.4 to 2.5 cc / cm 2 / sec is particularly preferred. When the ventilation volume is within the above range, it has the advantage that the leakage rate of PHA into the filtrate is low. The ventilation volume in the filtration step of this production method is measured by the method described in the examples.

[0069] In step (d), the liquid density of the PHA aqueous suspension is 0.50 to 1.08 g / mL, preferably 0.55 to 1.08 g / mL, more preferably 0.60 to 1.05 g / mL, and even more preferably 0.65 to 1.03 g / mL. When the liquid density of the PHA aqueous suspension is within the above range, it has the advantages that the filtrate permeation rate is high and the water content in the PHA agglomerates is low. When the liquid density is low, the decrease in the filtrate permeation rate is presumably due to the increase in the viscosity of the PHA aqueous suspension by containing air and the interaction between air and PHA resulting in an increase in viscosity. The liquid density of the PHA aqueous suspension can be adjusted, for example, by introducing air. Increasing the amount of air decreases the liquid density of the PHA aqueous suspension, and decreasing the amount of air increases the liquid density of the PHA aqueous suspension.

[0070] The filter medium used in step (d) is not particularly limited, and can be selected from various materials such as paper, filter cloth (woven fabric, non-woven fabric), screen, sintered plate, unglazed ware, polymer membrane, punched metal, wedge wire, etc. From the viewpoints of price and ease of cleaning, filter cloth is preferably used.

[0071] The filtration method in step (d) may be dead-end filtration and is not particularly limited. Examples include suction filtration, pressure filtration, centrifugal filtration, gravity filtration, etc. Among them, from the viewpoint of the size of the equipment, suction filtration, pressure filtration, and centrifugal filtration are preferably used. Further, from the viewpoint of the simplicity of the structure, suction filtration and pressure filtration are more preferably used.

[0072] In this specification, filtrate permeation rate means the rate at which the filtrate permeates through the PHA aggregates and filter media. In step (d) of this manufacturing method, the filtrate permeation rate is 200 L / m 2 Preferably, it is 300 L / m² or more. 2 It is more preferable that the rate is 400 L / m² or higher. 2 It is more preferable that the rate is 500 L / m² or higher. 2 It is more preferable that it be 600 L / m or more per hour. 2 It is more preferable that it be 800 L / m or more per hour. 2 It is even more preferable that the rate is 1000 L / m² or higher. 2 A rate of 1 / hr or higher is particularly preferable. A filtrate permeation rate within the above range has the advantage of reducing working time. A higher filtrate permeation rate is better, and there is no particular upper limit, but for example, 4000 L / m³ is preferable. 2 The rate is less than or equal to / hr. The filtrate permeation rate is measured by the method described in the examples.

[0073] In this specification, the leakage rate refers to the rate at which PHA leaks into the filtrate after the filtration step. In step (d) of this manufacturing method, the leakage rate is preferably 5% or less, more preferably 4% or less, more preferably 3% or less, more preferably 2% or less, more preferably 1% or less, even more preferably 0.5% or less, and particularly preferably 0.3% or less. When the leakage rate is within the above range, it has the advantage that PHA and water can be separated with high separation efficiency and PHA can be recovered in high yield. The leakage rate is measured by the method described in the examples.

[0074] The temperature of the PHA aqueous suspension in step (d) (filtration temperature) is preferably 20 to 95°C, more preferably 20 to 90°C, even more preferably 20 to 85°C, and particularly preferably 25 to 85°C. A temperature within the above range of the PHA aqueous suspension has the advantage of increasing the filtrate permeation rate. This increase in filtrate permeation rate is presumed to be due to the increase in viscosity as the temperature rises, while the particle size increases.

[0075] In this manufacturing method, if the heat treatment step (c) is included, the temperature of the PHA aqueous suspension during filtration is preferably 5°C or more lower than the temperature after the heat treatment step, more preferably 8°C or more lower, even more preferably 10°C or more lower, and particularly preferably 12°C or more lower. When the temperature of the PHA aqueous suspension during filtration is within the above range, it has the advantage that PHA can be filtered at a high filtrate permeation rate. Furthermore, the method for lowering the temperature after the heat treatment step is not particularly limited and includes, for example, cooling by a cooling device or air cooling.

[0076] Furthermore, in step (d), the "pH" is as described in (step (c')) above. Also, in step (d), the "amount of PHA surface-adhering protein in the PHA aqueous suspension" is as described in (step (b)) above.

[0077] (Step (e)) In step (e) of this manufacturing method, the PHA obtained in step (d) is dried at 20 to 80°C. Step (e) allows for the evaporation of water from the aqueous PHA suspension and adjustment of the water content.

[0078] In step (e), the method for drying the PHA is not particularly limited, but examples include heating, vacuum drying, and room temperature drying. Preferably, from the viewpoint of an appropriate drying rate, it is carried out by heating. The heat transfer medium during drying (e.g., hot air, jacket, etc.) is preferably 20 to 160°C, more preferably 40 to 160°C, even more preferably 40 to 150°C, and particularly preferably 50 to 150°C.

[0079] (Step (f)) In step (f) of this manufacturing method, the dried PHA is redispersed in an aqueous solvent to obtain an aqueous PHA suspension containing PHA with a volume median diameter of 0.5 to 5.0 μm. By performing step (f) following step (e), an aqueous PHA suspension containing PHA having a particle size substantially the same as the original particle size (primary particle size) is obtained.

[0080] In step (f), the method of redistribution is not particularly limited and can be any method used in the art.

[0081] In step (f), the volume median diameter of the PHA is not particularly limited as long as it is substantially the same as the volume median diameter of the PHA in step (a), but for example, 0.5 to 5.0 μm is preferred, 1.0 to 4.5 μm is more preferred, and 1.0 to 3.0 μm is even more preferred.

[0082] [3.PHA aggregate] These PHA aggregates have a ferret diameter of 1 to 100 mm and a water content of 25.0 to 50.0% (WB). These PHA aggregates are also sometimes referred to as "PHA cake," "filtration cake," or "PHA filtration cake."

[0083] The ferret diameter of the PHA aggregate is 1 to 100 mm, preferably 1 to 70 mm, more preferably 1 to 50 mm, even more preferably 1 to 30 mm, and particularly preferably 1 to 10 mm. Having the ferret diameter of the PHA aggregate within the above range has the advantage of being advantageous for transfer to the next process. It is also possible to sized the PHA aggregate obtained in step (d) to the above range by mechanical methods such as crushers or screws, or by breaking it due to the impact of dropping. In this PHA aggregate, images of 10 PHA aggregates (Figure 1) are taken and analyzed with ImageJ (ver1.50) to obtain the individual ferret diameter for each of the 10 PHA aggregates, and the simple average of the 10 individual ferret diameters is taken as the ferret diameter.

[0084] The moisture content of the PHA aggregate is 25.0 to 50.0% (WB), preferably 25.5 to 49.0% (WB), more preferably 26.0 to 48.0% (WB), even more preferably 26.5 to 47.0% (WB), and particularly preferably 27.0 to 46.0% (WB). When the moisture content of the PHA aggregate is within the above range, the PHA aggregate becomes solid rather than slurry, which has the advantage of being easy to put into a dryer. The moisture content of the PHA aggregate is measured by the method described in the examples.

[0085] In one embodiment of the present invention, the PHA aggregate is produced by the present manufacturing method.

[0086] The PHA aggregates may contain various components that were generated or not removed during the manufacturing process, as long as they achieve the effects of the present invention.

[0087] This PHA aggregate can be used in a variety of applications, including paper, film, sheets, tubes, plates, rods, containers (e.g., bottles), bags, parts, etc.

[0088] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0089] In other words, one aspect of the present invention includes the following: <1> A polyhydroxyalkanoate aqueous suspension with a pH of 2.5 to 5.5 is subjected to an aeration rate of 0.01 to 5.0 cc / cm³. 2 This process includes dead-end filtration using a filter media with a rate of / sec. The amount of polyhydroxyalkanoate surface-adhering protein in the aforementioned aqueous suspension of polyhydroxyalkanoate is 2000 ppm or less. A method for producing polyhydroxyalkanoate, wherein the liquid density of the aqueous suspension of polyhydroxyalkanoate in the filtration step is 0.50 to 1.08 g / mL. <2> In the filtration process described above, the filtrate permeation rate is 200 L / m 2 The data usage is above / hr, and the leakage rate is 5% or less. <1> The manufacturing method described above. <3> The temperature of the aqueous polyhydroxyalkanoate suspension in the filtration step is 20 to 95°C. <1> or <2> The manufacturing method described above. <4> Furthermore, the process includes (a) a step of destroying and solubilizing cell-derived components other than polyhydroxyalkanoates in bacterial cells containing polyhydroxyalkanoates, The volume median diameter of the polyhydroxyalkanoate in step (a) is 0.5 to 5.0 μm. <1> ~ <3> A manufacturing method described in any of the following. <5> Furthermore, after step (a), the process includes (b) recovering the aqueous suspension of polyhydroxyalkanoate by centrifugation. <4> The manufacturing method described above. <6> The process includes drying the polyhydroxyalkanoate obtained in the filtration step at 20 to 80°C. <1> ~ <5> A manufacturing method described in any of the following. <7> The process includes the step of redispersing the dried polyhydroxyalkanoate in an aqueous solvent to obtain an aqueous suspension of polyhydroxyalkanoate containing polyhydroxyalkanoate having a volume median diameter of 0.5 to 5.0 μm. <6> The manufacturing method described above. <8> Furthermore, prior to the filtration step, the process includes a step of heating (c) the aqueous suspension of polyhydroxyalkanoate to a temperature of 60-120°C. <1> ~ <7> A manufacturing method described in any of the following. <9> Furthermore, the temperature of the polyhydroxyalkanoate aqueous suspension during filtration is 5°C or more lower than the temperature of the polyhydroxyalkanoate aqueous suspension after the heat treatment step. <8> The manufacturing method described above. <10> The process includes a step of adjusting the aqueous suspension of polyhydroxyalkanoate obtained in step (b) above to a pH of 2.5 to 5.5. <5> The manufacturing method described above. <11> Polyhydroxyalkanoate aggregates having a feret diameter of 1 to 100 mm and a water content of 25.0 to 50.0% (WB). [Examples]

[0090] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples. In the examples, "P3HB3HH" is used as "PHA", and "PHA" in the examples can be read as "P3HB3HH".

[0091] [Measurement method] Measurements in the examples and comparative examples were performed using the following method.

[0092] (Airflow) The air permeability was measured according to the method described in JIS L 1096. Specifically, a Frazier type air permeability tester (permeometer P2, manufactured by Toyo Seiki Seisakusho Co., Ltd.) was used to adjust the air intake so that the inclined barometer showed 125 Pa, and the airflow rate at that time was measured.

[0093] (Amount of PHA surface-attached protein) The amount of PHA surface-adhering protein was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific). Specifically, 20-50 mg of the PHA aqueous suspension immediately before step (c') (containing approximately 10 mg of P3HB3HH particles in the solution) was placed in a 15 mL Falcon tube, 2 mL of the reagent from the kit was added, and the mixture was shaken at 60°C for 30 minutes. After 30 minutes from the end of shaking, the mixture was cooled to 25°C, and the absorbance at a wavelength of 562 nm was measured.

[0094] (solid content concentration) The solid content concentration of the inactivated PHA-containing culture medium and the pH-adjusted PHA aqueous suspension was measured using a heat-drying type moisture meter ML-50 (manufactured by A&D Corporation). The culture medium was heated at 130°C until the weight change rate fell below 0.05% / min, and the solid content concentration was determined from the weight change before and after heating.

[0095] (liquid density) The PHA aqueous suspension was heated to the filtration temperature immediately before the filtration process, and 20 mL was drawn into a 20 mL plastic syringe (manufactured by Terumo) whose weight had been measured in advance. Next, the weight of the plastic syringe and the 20 mL aqueous suspension were measured, and the liquid density at the filtration temperature was calculated by subtracting the weight of the 20 mL plastic syringe from the suspension weight (g) and dividing the result by the volume of the liquid (20 mL).

[0096] (heat treatment temperature) The heat treatment temperature was measured at the temperature of the PHA aqueous suspension furthest from the heat source while the PHA aqueous suspension was in a fluid state using a stirring blade or similar device. For example, when heating from the outside of the container, the temperature at the center of the container was measured, and when steam was directly introduced into the center of the container, the temperature of the container wall was measured. The temperature was measured using a K-type thermocouple (AD5601A, manufactured by AND Corporation).

[0097] (Temperature during filtration) The temperature during filtration was measured using a K-type thermocouple (AD5601A, manufactured by AND Corporation) just before the PHA aqueous suspension was added to the filter.

[0098] (pH of the PHA aqueous suspension in step (c')) The pH was measured using a pH meter (9652-10D, manufactured by HORIBA). The pH measurement point was the furthest point from the acid addition point in the PHA aqueous suspension, while the suspension was in a flowing state using a stirring blade or similar device. For example, when the acid was added from the side of the container, the pH at the center of the container was measured.

[0099] (Permeation rate of filtrate in suction filtration) A filter cloth was placed in a 47mm inner diameter filter (KST-47, Advantec), and with the filter's top lid removed, it was attached to a suction bell (2L, SHIBATA). A 50mL glass graduated cylinder was placed inside the suction bell so that all the filtrate would enter the graduated cylinder. Filtration was performed by adding a PHA aqueous suspension to the filter while suctioning to -76kPa with a vacuum pump. The flow of the filtrate was filmed with a video camera. The time (hr) required from when 5mL of filtrate was discharged until 25mL of filtrate was discharged, and the cross-sectional area (m²) of the filter were recorded. 2 By dividing the difference (25-5=20mL) in the volume of filtrate (L) by this, the filtrate permeation rate (L / m) can be calculated. 2 The calculation was performed using the suction filtration method ( / hr). In this specification, the cross-sectional area of ​​the filter during suction filtration was calculated as the area of ​​a circle with a diameter of 47 mm.

[0100] (Permeation rate of filtrate in pressurized filtration) A 2.0 kg aqueous PHA suspension is pumped using a diaphragm pump to a pressure of 0.2 MPa, with a filtration area of ​​0.05 m². 2 Filtration was performed using a pressurized filter (YTO type filter press, manufactured by Yabuta Machinery). The filtrate permeation rate (L / m³) was calculated from the time taken from when 100 mL of filtrate was discharged to when 1000 mL of filtrate was discharged. 2 The calculation ( / hr) was performed.

[0101] (Leakage rate) The weight of the filtrate obtained by the filtration process was measured. The absorbance of the filtrate was measured using a spectrophotometer (Jasco V-770, manufactured by JASCO), and the absorbance at a wavelength of 600 nm was measured. The PHA concentration in the filtrate was calculated using a calibration curve prepared from an aqueous PHA suspension with a known solid content concentration. The leakage rate (%) was calculated by dividing the weight of solid content (g) from the PHA concentration in the filtrate and the weight of the filtrate (g) by the weight of solid content (g) of the aqueous PHA suspension before the filtration process (g).

[0102] (Moisture content of PHA aggregates) The PHA aggregates obtained after filtration were measured using a heat-drying type moisture meter ML-50 (manufactured by A&D Corporation). The PHA aggregates were heated at 105°C until the weight change rate fell below 0.05% (WB) / min, and the moisture content of the PHA aggregates was determined from the weight change before and after heating.

[0103] (Volume median diameter) The volume median diameter of PHA was measured using a HORIBA LA-950 laser diffraction / scattering particle size distribution analyzer.

[0104] (Ferret diameter) Images of 10 PHA aggregates (Figure 1) were taken and analyzed using ImageJ (ver1.50). Individual ferret diameters were determined for each of the 10 PHA aggregates, and the simple average of these ferret diameters was used as the ferret diameter.

[0105] [Example 1] (Preparation of bacterial culture medium) Ralstonia eutropha, as described in International Publication No. WO2019 / 142717, was cultured using the method described in paragraphs

[0041] to

[0048] of the same document to obtain a bacterial culture medium containing PHA-containing cells. Note that Ralstonia eutropha is now classified as Capriavidus necatol.

[0106] (inactivation) The bacterial culture solution obtained above was sterilized by heating and stirring at an internal temperature of 60-70°C for 7 hours to obtain an inactivated culture solution.

[0107] (Viscosity reduction treatment) To the inactivated culture medium obtained above, 35% by weight hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries) was added to a concentration of 1% by weight. Next, a 30% sodium hydroxide aqueous solution was added to adjust the pH to 11.0. While maintaining the solution at 60°C, the 30% sodium hydroxide aqueous solution was continuously added to maintain the pH at 11.0 for 180 minutes, thereby obtaining an aqueous PHA suspension.

[0108] (Enzyme treatment) To the PHA aqueous suspension obtained above, 95% sulfuric acid was added to adjust the pH to 7.0 ± 0.2. The solid content concentration of the PHA aqueous suspension with added sulfuric acid was measured to be 30% by weight. After adding sulfuric acid, lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries), an enzyme that degrades sugar chains (peptidoglycans) in the cell wall, was added to a concentration of 10 ppm in the solution and maintained at 50°C for 2 hours. Subsequently, 2.5 L of alcalase (manufactured by Novozyme), a proteolytic enzyme, was added to a concentration of 300 ppm in the solution, and then 30% sodium hydroxide was added at 50°C to adjust the pH to 8.5 while maintaining the solution for 2 hours.

[0109] (Alkaline treatment) Sodium dodecyl sulfate (SDS, manufactured by Kao Corporation) was added to the enzyme-treated solution to a concentration of 0.3% by weight. The pH was then adjusted to 11.0 ± 0.2 using an aqueous sodium hydroxide solution. Next, the enzyme-treated solution was centrifuged (4000 G, 10 minutes), and the supernatant was removed to obtain a 2-fold concentrated aqueous PHA suspension. The same amount of sodium hydroxide as the removed supernatant was added to the concentrated aqueous PHA suspension, and the mixture was centrifuged again (4000 G, 10 minutes), with the supernatant removed. This process was repeated four times. The amount of PHA surface-adhering protein in the obtained aqueous PHA suspension was 1000 ppm. The volume median diameter of the PHA was 2.2 μm.

[0110] (pH adjustment) The solid content concentration of the PHA aqueous suspension obtained above was adjusted to 25% by weight and maintained at 60°C. Then, 10% sulfuric acid was added to adjust the pH to 3.0. The liquid density was 1.00 g / mL.

[0111] (filtration) The PHA aqueous suspension was placed in a 63°C water bath, heated to a temperature of 60°C, and then subjected to suction filtration. Aeration rate: 0.5 cc / cm² 2 When using a filter cloth (T7104C, manufactured by Yabuta Machinery) with a filtration rate of / sec, the filtrate permeation rate was 1010 L / m 2 The leakage rate was 0.2% (per hour). The water content of the obtained PHA agglutinations was 40.3% (WB). The ferret diameter of the PHA agglutinations was 18 mm.

[0112] [Example 2] An aqueous suspension of PHA at pH 3.0 was obtained using the same method as in Example 1, except that 2.5 L of alcalase (Novozyme), a proteolytic enzyme, was added to the solution at a concentration of 200 ppm during enzymatic treatment. The amount of protein adhering to the PHA surface was 1500 ppm, and the liquid density was 0.95 g / mL. The volume median diameter of the PHA was 2.3 μm. Filtration was performed using the same method as in Example 1, and the filtrate permeation rate was 580 L / m². 2 The leakage rate was 1.0% (per hour). The water content of the obtained PHA agglutinations was 38.9% (WB). The ferret diameter of the PHA agglutinations was 25 mm.

[0113] [Comparative Example 1] An aqueous suspension of PHA at pH 3.0 was obtained using the same method as in Example 1, except that 2.5 L of alcalase (Novozyme), a proteolytic enzyme, was added to the solution at a concentration of 100 ppm during enzymatic treatment. The amount of protein adhering to the PHA surface was 2500 ppm, and the liquid density was 0.95 g / mL. The volume median diameter of the PHA was 2.5 μm. Filtration was performed using the same method as in Example 1, and the filtrate permeation rate was 2500 L / m². 2 The leakage rate was 10.2% per hour. The water content of the obtained PHA agglutinates was 40.8% (WB). The ferret diameter of the PHA agglutinates was 38 mm.

[0114] [Table 1] Table 1 shows that the higher the amount of PHA surface-adhered protein, the higher the particle leakage rate.

[0115] [Example 3] A pH 3.0 aqueous PHA suspension was obtained using the same method as in Example 1, except that after adjusting the pH to 3.0 in the pH adjustment step, the PHA aqueous suspension was placed in a container and manually shaken to incorporate air. The amount of protein attached to the PHA surface was 1000 ppm, and the liquid density was 0.7 g / mL. The volume median diameter of the PHA was 2.2 μm. When filtration was performed using the same method as in Example 1, the filtrate permeation rate was 450 L / m 2 The leakage rate was 0.2% (per hour). The water content of the obtained PHA agglutinations was 36.8% (WB). The ferret diameter of the PHA agglutinations was 25 mm.

[0116] [Comparative Example 2] Filtration was performed using the same method as in Example 1, except that vigorous stirring was performed during the pH adjustment step to achieve a liquid density of 0.40 g / mL. The filtrate permeation rate was 150 L / m 2 The leakage rate was 3.2% (per hour). The water content of the obtained PHA agglutinates was 35.4% (WB). The ferret diameter of the PHA agglutinates was 34 mm.

[0117] [Table 2] Table 2 shows that as liquid density increases, the filtrate permeation rate increases, but the water content also increases.

[0118] [Comparative Example 3] Filtration was performed using the same method as in Example 1, except that the pH was adjusted to 6.0 in the pH adjustment step. The filtration rate was 11800 L / m³. 2 The result was / h, the leakage rate was 99%, and no PHA aggregates were obtained.

[0119] [Comparative Example 4] Filtration was performed using the same method as in Example 1, except that the pH was adjusted to 2.0 in the pH adjustment step. The filtrate permeation rate was 9800 L / m 2 The result was / hr, the leakage rate was 95%, and no PHA aggregates were obtained.

[0120] [Table 3] Table 3 shows that the leakage rate changes with pH, ​​and that lower pH is more suitable for recovering PHA aggregates.

[0121] [Example 4] In the filtration process, the air permeability is 2.0 cc / cm³. 2 A pH 3.0 aqueous suspension of PHA was obtained using the same method as in Example 1, except that a filter cloth (T7302C, manufactured by Yabuta Machinery) with a filtration rate of / sec was used. The amount of protein attached to the PHA surface was 1000 ppm, and the liquid density was 1.00 g / mL. The volume median diameter of the PHA was 2.2 μm. Filtration was performed using the same method as in Example 1, and the filtration transmission rate was 1210 L / m². 2 The leakage rate was 0.2% (per hour). The water content of the obtained PHA agglutinates was 42.3% (WB). The ferret diameter of the PHA agglutinates was 17 mm.

[0122] [Comparative Example 5] Air permeability during filtration: 12.0 cc / cm³ 2 Filtration was performed using the same method as in Example 1, except that a filter cloth (PP26F, manufactured by Nakao Filter Co., Ltd.) with a density of / sec was used. The filtration transmission rate was 9440 L / m³. 2 The leakage rate was 73.0%. The water content of the obtained PHA agglutinations was 47.0% (WB). The ferret diameter of the PHA agglutinations was 11 mm.

[0123] [Table 4] Table 4 shows that using a filter media with high air permeability increases the filtrate permeation rate, but also increases the leakage rate.

[0124] [Example 5] An aqueous suspension of PHA at pH 3.0 was obtained using the same method as in Example 1, except that the filtration process involved heating the water bath at 78°C until the internal temperature reached 75°C. The amount of protein adhering to the PHA surface was 1000 ppm, and the liquid density was 1.00 g / mL. The volume median diameter of the PHA was 2.2 μm. When filtration was performed using the same method as in Example 1, the filtrate permeation rate was 1580 L / m³. 2 The leakage rate was 0.3% (per hour). The water content of the obtained PHA agglutinates was 42.5% (WB). The ferret diameter of the PHA agglutinates was 18 mm.

[0125] [Example 6] An aqueous suspension of PHA at pH 3.0 was obtained using the same method as in Example 1, except that the filtration step involved cooling the water bath at 28°C until the internal temperature reached 25°C. The amount of protein adhering to the PHA surface was 1000 ppm, and the liquid density was 1.00 g / mL. The volume median diameter of the PHA was 2.2 μm. When filtration was performed using the same method as in Example 1, the filtrate permeation rate was 450 L / m 2 The leakage rate was 0.1% (per hour). The water content of the obtained PHA agglutinates was 44.8% (WB). The ferret diameter of the PHA agglutinates was 26 mm.

[0126] [Table 5] Table 5 shows that the temperature of the PHA aqueous suspension affects the filtrate permeation rate, with the rate increasing as the temperature rises.

[0127] [Example 7] A pH 3.0 aqueous suspension of PHA was obtained using the same method as in Example 1, except that before the filtration step, the solution after the pH adjustment step was heated in a 125°C oil bath until the internal temperature reached 75°C, and then cooled in a 63°C water bath until the internal temperature reached 60°C. The amount of protein attached to the PHA surface was 1000 ppm, and the liquid density was 1.00 g / mL. The volume median diameter of the PHA was 2.2 μm. When filtration was performed using the same method as in Example 1, the filtrate permeation rate was 1220 L / m 2 The leakage rate was 0.2% ( / hr). The water content of the obtained PHA agglutinates was 42.2% (WB). The ferret diameter of the PHA agglutinates was 28 mm.

[0128] [Example 8] A pH 3.0 aqueous suspension of PHA was obtained by the same method as in Example 1, except that the pH-adjusted solution was heated in a 125°C oil bath until the internal temperature reached 90°C before the filtration step, and then cooled in a 63°C water bath until the internal temperature reached 60°C. The amount of protein attached to the PHA surface was 1000 ppm, and the liquid density was 1.00 g / mL. The volume median diameter of the PHA was 2.2 μm. When filtration was performed by the same method as in Example 1, the filtrate permeation rate was 1320 L / m 2 The leakage rate was 0.1% (per hour). The water content of the obtained PHA agglutinates was 44.0% (WB). The ferret diameter of the PHA agglutinates was 21 mm.

[0129] [Example 9] In the pH adjustment step, the solid content concentration in the PHA aqueous suspension was adjusted to 28% by weight. Before the filtration step, the pH-adjusted liquid was placed in a pressure vessel, 0.3 MPaG of steam was added, and the temperature was raised to 110°C. Then, it was cooled in a 63°C water bath until the internal temperature reached 60°C. Distilled water was added to adjust the concentration in the PHA aqueous suspension to 25% by weight. Except for these steps, a pH 3.0 PHA aqueous suspension was obtained using the same method as in Example 1. The amount of protein attached to the PHA surface was 1000 ppm, and the liquid density was 1.00 g / mL. The volume median diameter of the PHA was 2.2 μm. Filtration was performed using the same method as in Example 1, and the filtrate permeation rate was 2840 L / m². 2 The leakage rate was 0.0% ( / hr). The water content of the obtained PHA agglutinates was 44.9% (WB). The ferret diameter of the PHA agglutinates was 15 mm.

[0130] [Table 6] Table 6 shows that when the PHA aqueous suspension is heated to a high temperature, the filtrate permeation rate increases, and this rate increases with increasing heating temperature.

[0131] [Example 10] The same procedure as in Example 1 was followed up to the pH adjustment step. Air permeability: 0.5 cc / cm 2 A filter cloth (T7104C, manufactured by Yabuta Machinery) with a flow rate of / second was used, and pressure filtration was performed using a pressure filter (YTO type filter press, manufactured by Yabuta Machinery). Using the method described above, the filtrate permeation rate was calculated to be 1500 L / m³. 2 The ratio was / hr. Furthermore, when dewatering was performed by compression at a pressure of 0.5 MPa, the water content of the obtained PHA aggregates was 31.9% (WB). The leakage rate of all obtained filtrates was 2.6%. Filtration could be performed regardless of the filtration method. The ferret diameter of the PHA aggregates was 20 mm.

[0132] [Example 11] (Drying) The PHA aggregates obtained in Example 1 were placed in a dryer (EYELA, WFO-700) and dried at 60°C for 24 hours. The dried PHA aggregates were redispersed in water to adjust the solid content concentration to 15% by weight. The pH was adjusted to between 7 and 9 using 1% NaOH aqueous solution and 1% H2SO4 aqueous solution, and the mixture was stirred to prepare an aqueous PHA suspension. After stirring for 30 minutes, the particle size of the PHA particles in the aqueous PHA suspension was measured, and the volume median diameter was found to be 2.8 μm.

[0133] 〔summary〕 Based on the above, it was found that the amount of protein attached to the PHA surface, the liquid density, the pH of the PHA aqueous suspension, the air permeability of the filter media, and the temperature of the PHA aqueous suspension all affect the filtrate permeation rate and leakage rate during filtration using this manufacturing method. In other words, it was found that PHA can be efficiently filtered by controlling at least the amount of protein attached to the PHA surface, the liquid density, the pH of the PHA aqueous suspension, and the air permeability of the filter media. Furthermore, it was revealed that PHA can be filtered using this manufacturing method regardless of the filtration method. [Industrial applicability]

[0134] This manufacturing method allows for the production of PHA with simple operations, making it advantageous for PHA production. Furthermore, the PHA obtained by this manufacturing method can be suitably used in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing products, packaging, automobiles, building materials, and other fields.< / pha>

Claims

1. A polyhydroxyalkanoate aqueous suspension with a pH of 2.5 to 5.5 is subjected to an aeration rate of 0.01 to 5.0 cc / cm². ２ This process includes dead-end filtration using a filter media that is / sec. The amount of polyhydroxyalkanoate surface-adhering protein in the aforementioned aqueous suspension of polyhydroxyalkanoate is 2000 ppm or less. A method for producing polyhydroxyalkanoate, wherein the liquid density of the aqueous suspension of polyhydroxyalkanoate in the filtration step is 0.50 to 1.08 g / mL.

2. In the filtration process described above, the filtrate permeation rate is 200 L / min. ２ The manufacturing method according to claim 1, wherein the rate is 1 / hr or more and the leakage rate is 5% or less.

3. The manufacturing method according to claim 1 or 2, wherein the temperature of the aqueous suspension of polyhydroxyalkanoate in the filtration step is 20 to 95°C.

4. Furthermore, the process includes (a) a step of destroying and solubilizing cell-derived components other than polyhydroxyalkanoates in bacterial cells containing polyhydroxyalkanoates, The manufacturing method according to claim 1 or 2, wherein the volume median diameter of the polyhydroxyalkanoate in step (a) is 0.5 to 5.0 μm.

5. Furthermore, the manufacturing method according to claim 4, further comprising the step of recovering the aqueous suspension of polyhydroxyalkanoate by centrifugation after step (a).

6. The manufacturing method according to claim 1 or 2, further comprising the step of drying the polyhydroxyalkanoate obtained in the filtration step at 20 to 80°C.

7. The manufacturing method according to claim 6, comprising the step of redispersing the dried polyhydroxyalkanoate in an aqueous solvent to obtain an aqueous suspension of polyhydroxyalkanoate containing a polyhydroxyalkanoate having a volume median diameter of 0.5 to 5.0 μm.

8. Furthermore, the manufacturing method according to claim 1 or 2, further comprising the step of heating (c) the aqueous suspension of polyhydroxyalkanoate to 60 to 120°C before the filtration step.

9. Furthermore, the manufacturing method according to claim 8, wherein the temperature of the aqueous polyhydroxyalkanoate suspension during filtration is 5°C or more lower than the temperature of the aqueous polyhydroxyalkanoate suspension after the heat treatment step.

10. The manufacturing method according to claim 5, further comprising the step of adjusting the pH of the aqueous suspension of polyhydroxyalkanoate obtained in step (b) to 2.5 to 5.5.

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