Method for producing polyhydroxyalkanoate and use thereof

Abstract

The purpose of the present invention is to provide a method for producing a PHA having a low moisture content. The above is addressed by providing a method for producing a PHA that comprises a filter press filtration step for filtering an aqueous PHA suspension having a pH of 2.5-5.5 using a filtering material having an air permeability of 0.1-2.5 cm3 / cm2 / min, wherein the liquid density of the aqueous PHA suspension in the filtration step is 0.50-1.08 g / mL, and the filter press filtration step includes a compression step and an air blow step.

Description

Method for producing polyhydroxyalkanoate and its use

[0001] The present invention relates to a method for producing polyhydroxyalkanoates and their use.

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

[0003] Since PHA produced by microorganisms accumulates within the cells of the microorganisms, a process for separating and purifying the PHA from the cells of the microorganisms is required to use the PHA as a plastic. In the process of separating and purifying the PHA, the cells of the PHA-containing microorganisms are disrupted or microbial components other than the PHA are solubilized, and then the PHA is extracted from the resulting aqueous suspension. At this time, separation procedures 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, which includes the steps of inoculating a PHA-fermenting bacterial species into a fermentation medium and fermenting it, separating the fermented liquid into solids and liquids to obtain a fermentation supernatant and a bacterial cell precipitate, and precipitating the bacterial cells to crush the cell walls, and filtering the crushed cell walls through a plate and frame using a precoated filter to obtain PHA.

[0005] Chinese Patent No. 111500650

[0006] However, the PHA obtained by the above-mentioned technique has a high water content, and energy is required to subsequently volatilize the water, leaving room for further improvement.

[0007] Therefore, an object of the present invention is to provide a method for producing PHA with a low moisture content, and PHA agglomerates with a low moisture content.

[0008] The present inventors have conducted extensive research to solve the above-mentioned problems, and as a result have discovered a new finding that in the production of PHA, PHA with a low water content can be obtained by controlling the pH of the PHA aqueous suspension, the aeration rate of the filter medium, and the liquid density of the PHA aqueous suspension and performing a specific filtration step, and have completed the present invention.

[0009] Therefore, one aspect of the present invention is to prepare a PHA aqueous suspension having a pH of 2.5 to 5.5 by subjecting the suspension to an aeration rate of 0.1 to 2.5 cm ３ / cm ２ / min, wherein the liquid density of the PHA aqueous suspension in the filtration step is 0.50 to 1.08 g / mL, and the filter press filtration step includes a squeezing step and an air blowing step (hereinafter referred to as "this production method").

[0010] Another aspect of the present invention is a PHA agglomerate (hereinafter referred to as "the present PHA agglomerate") having a water content of 5.0 to 25.0% (W.B.).

[0011] According to one aspect of the present invention, a method for producing a PHA having a low water content and a PHA agglomerate having a low water content can be provided.

[0012] FIG. 1 is a view showing photographs of the filter cloth permeate of Example 1-1 according to one embodiment of the present invention and Comparative Example 2.

[0013] An embodiment of the present invention will be described in detail below. In this specification, unless otherwise specified, the numerical range "A to B" means "A or more and B or less."

[0014] [1. Overview of the Invention] PHA produced within bacterial cells has a particle diameter of approximately 1 to 2 μm, making filtration difficult. Furthermore, centrifugation is primarily used to recover PHA, but the separated form is an aqueous suspension, and the PHA is recovered in a state containing a large amount of water. In order to recover PHA by separating it from water, the water in the aqueous suspension must be evaporated, which poses problems such as the need for a large amount of energy. Here, for example, Patent Document 1, mentioned above, is known as a method for producing PHA using filtration. The technology of Patent Document 1 involves pre-coating a filter medium and performing filtration before purification (when a large amount of biological residue remains), which is found to be cumbersome and to result in problems such as a high level of impurities in the resulting PHA aggregates. Furthermore, it has been found that the technology of Patent Document 1 does not achieve a sufficiently low water content.

[0015] Therefore, the present inventors conducted extensive research from the perspective of reducing the water content of PHA in the production of PHA, and as a result, they discovered for the first time that PHA with a low water content can be obtained by controlling the pH of the PHA aqueous suspension, the air permeability of the filter medium, and the liquid density of the PHA aqueous suspension and performing a specific filtration process. Specifically, they developed a technology for adjusting the pH of the PHA aqueous suspension to a pH range in which PHA aggregates and recovering the aggregated PHA by filtration. This production method can provide PHA with a low water content. Furthermore, this production method can produce PHA with a low water content without adding impurities such as filter aids, thereby reducing impurities. Therefore, this production method is extremely advantageous in the industrial production of PHA.

[0016] Furthermore, the above-described configuration can reduce the amount of heat, time, and cost required in the drying process after filtration, i.e., energy. This can contribute to the achievement of the 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.

[0017] 2. PHA Production Method This production method includes the following steps: A PHA aqueous suspension having a pH of 2.5 to 5.5 is immersed in a filtrate having an aeration rate of 0.1 to 2.5 cm ３ / cm ２ / min, wherein the liquid density of the aqueous suspension of polyhydroxyalkanoate in the filtration step is 0.50 to 1.08 g / mL, and the filter press filtration step includes a squeezing step and an air blowing step (hereinafter referred to as step (d)).

[0018] In one embodiment of the present invention, the production method preferably includes at least one of the following steps in addition to step (d): step (a): disrupting and solubilizing cell-derived components other than PHA in PHA-containing bacterial cells, whereby the volume median diameter of the PHA is 0.5 to 5.0 μm (also referred to as the "solubilization step"); step (b): recovering the PHA aqueous suspension by centrifugation after step (a) (also referred to as the "recovery step"); step (c'): adjusting the pH of the PHA aqueous suspension obtained in step (b) to 2.5 to 5.5 (also referred to as the "preparation step"); step (c): heat-treating the PHA aqueous suspension to a temperature of 60 to 120°C (also referred to as the "heat treatment step"); step (e): drying the PHA obtained in step (d) at 20 to 100°C (also referred to as the "drying step"). Step (f): A step of redispersing the dried PHA in an aqueous solvent (also referred to as the "redispersion step").

[0019] In this production method, the above steps are preferably performed in the order of steps (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 perform steps (b) and (a), and the order of steps (c') and (c) can be changed to perform steps (c) and (c'). Furthermore, depending on the purpose, steps (a), (b), (c'), and (c) can be performed two or more times. That is, for example, steps can be performed in the order of steps (b), (a), (b), or (c'), (c), and (c'). In this specification, an aqueous suspension containing at least PHA may be abbreviated as "PHA aqueous suspension."

[0020] (Step (d)) First, the step (d), which is a characteristic feature of the present invention, will be described.

[0021] In step (d) of this production method, the PHA aqueous suspension having a pH of 2.5 to 5.5 is mixed with a sieve having an air permeability of 0.1 to 2.5 cm ３ / cm ２The PHA is filtered through a filter press using a filter medium having a flow rate of 0.50 to 1.08 g / min. In step (d), the liquid density of the PHA aqueous suspension in the filtration step is 0.50 to 1.08 g / mL. In step (d), the filter press filtration step includes a squeezing step and an air blowing step. A PHA having a low water content is obtained by step (d).

[0022] The filter press filtration step (d) includes a squeezing step and an air blowing step. In the squeezing step, the PHA cake is squeezed by squeezing to squeeze out water from the PHA cake. In the air blowing step, water is forced out of the PHA cake by air blown. By undergoing the two-stage dehydration step of the squeezing step and the air blowing step, a PHA agglomerate with a low water content can be obtained.

[0023] In one embodiment of the present invention, the air blowing step can be a through air blowing step.

[0024] In one embodiment of the present invention, the filter press filtration step may include a step of supplying a raw liquid (aqueous PHA suspension) to a filter chamber before the squeezing step.

[0025] The pressure in the squeezing step is preferably 0.2 to 1.0 MPa, more preferably 0.25 to 0.9 MPa, and even more preferably 0.3 to 0.8 MPa. The pressure in the squeezing step of 0.2 to 1.0 MPa has the advantage of reducing the water content of the PHA aggregates.

[0026] The air blowing pressure in the air blowing step is not particularly limited, but is, for example, 0.01 to 1.5 MPa, preferably 0.05 to 1.3 MPa, and more preferably 0.10 to 1.0 MPa. When the air blowing pressure is within the above range, there is an advantage that the water content of the PHA agglomerates is reduced.

[0027] The air blowing time can be set appropriately depending on the air blowing pressure, but is, for example, 1 to 50 minutes, preferably 5 to 40 minutes.

[0028] The apparatus for performing the filter press filtration is not particularly limited, and any known apparatus can be used.

[0029] The filter material used in step (d) is not particularly limited, and can be selected from various materials, such as paper, filter cloth (woven or nonwoven), screen, sintered plate, bisque, polymer membrane, punched metal, wedge wire, etc. From the viewpoints of cost and ease of cleaning, filter cloth is preferably used.

[0030] In this specification, the unit area (cm ) of the filter medium per minute is used. ２ ) the amount of air passing through (cm ３ In step (d), the air permeability is 0.1 to 2.5 cm ３ / cm ２ / min, and 0.2 to 2.0 cm ３ / cm ２ / min is preferable, and 0.3 to 1.8 cm ３ / cm ２ / min is more preferable, and 0.5 to 1.5 cm ３ / cm ２ / min is more preferable, and 0.8 to 1.2 cm ３ / cm ２ / min is particularly preferred. When the air permeability is within the above range, there is an advantage that the leakage rate of PHA into the filtrate is low. The air permeability in the filtration step of this production method is measured by the method described in the Examples.

[0031] In step (d), the liquid density of the PHA aqueous suspension is 0.50 to 1.08 g / mL, preferably 0.60 to 1.05 g / mL, more preferably 0.70 to 1.03 g / mL, and even more preferably 0.80 to 1.02 g / mL. Having a liquid density of the PHA aqueous suspension within the above range has the advantages of a high filtrate permeation rate and a low water content in the PHA aggregates. It is presumed that the reason for the decrease in filtrate permeation rate when the liquid density is low is that the viscosity of the PHA aqueous suspension increases due to the inclusion of air, which in turn increases the viscosity through interaction between the air and the PHA. The liquid density of the PHA aqueous suspension can be adjusted, for example, by incorporating air. Increasing the amount of air decreases the liquid density of the PHA aqueous suspension, while decreasing the amount of air increases the liquid density of the PHA aqueous suspension.

[0032] The solids concentration of the filtrate in step (d) is preferably 1000 mg / L or less, more preferably 500 mg / L or less, even more preferably 200 mg / L or less, and particularly preferably 100 mg / L or less. When the solids concentration of the filtrate is within the above range, it has the advantage of a high recovery rate of PHA aggregates. There is no particular restriction on the lower limit, and it may be, for example, 0 mg / mL. The solids concentration of the filtrate in the filtration step of this production method is measured by the method described in the Examples.

[0033] In one embodiment of the present invention, the filter medium is preferably not pre-coated, which has the advantages of eliminating the complexity of the operation and reducing the amount of impurities in the PHA.

[0034] The temperature of the PHA aqueous suspension in step (d) (temperature during filtration) is preferably 20 to 95°C, more preferably 25 to 90°C, even more preferably 30 to 85°C, and particularly preferably 35 to 70°C. When the temperature of the PHA aqueous suspension is within the above range, there is an advantage that the filtrate permeation rate increases. The increase in the filtrate permeation rate is presumed to be due to an increase in viscosity caused by an increase in temperature, while the particle size also increases.

[0035] When the present production method includes the heat treatment step of step (c), the temperature of the PHA aqueous suspension during filtration is preferably at least 5°C lower than the temperature after the heat treatment step, more preferably at least 8°C lower, even more preferably at least 10°C lower, and particularly preferably at least 12°C lower. When the temperature of the PHA aqueous suspension during filtration is within the above range, there is an advantage that the PHA can be filtered at a high filtrate permeation rate. In addition, the method for lowering the temperature after the heat treatment step is not particularly limited, and examples thereof include cooling with a cooling device and leaving it to cool.

[0036] In step (d), the "pH" is the same as that described in step (c') below.

[0037] (Step (a)) In the step (a) of this production method, cell-derived components other than PHA in the PHA-containing bacterial cells are disrupted and solubilized. By disrupting and removing the cell-derived impurities (cell walls, proteins, etc.) in the step (a), PHA having a volume median diameter of 0.5 to 5.0 μm can be efficiently recovered from the bacterial cells.

[0038] <PHA> In this specification, "PHA" is a general term for polymers containing hydroxyalkanoic acid as a monomer unit. The hydroxyalkanoic acid constituting PHA is not particularly limited, but examples thereof 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 types of monomer units.

[0039] More specifically, examples of PHA include poly(3-hydroxybutyrate) (P3HB), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P3HB3HH), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (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), poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (P3HB3HV3HH), and the like. Among these, P3HB, P3HB3HH, P3HB3HV, and P3HB4HB are preferred because they are easy to produce industrially.

[0040] Furthermore, by changing the composition ratio of the repeating units, it is possible to change the melting point and degree of crystallinity, and as a result, it is possible to change physical properties such as Young's modulus and heat resistance, and it is possible to impart physical properties between those of polypropylene and polyethylene.In addition, from the viewpoint that it is easy to produce industrially as described above and is a physically useful plastic, P3HB3HH, which is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, is more preferred.

[0041] In one embodiment of the present invention, from the viewpoint of a balance between flexibility and strength, the composition ratio of the repeating units of P3HB3HH is preferably 80 / 20 to 99.9 / 0.1 (mol / mol), and more preferably 85 / 15 to 97 / 3 (mol / mol), as the composition ratio of 3-hydroxybutyrate units to 3-hydroxyhexanoate units. When 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 when it is 80 / 20 (mol / mol) or more, sufficient hardness is obtained.

[0042] The volume median diameter of the PHA in step (a) 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 the primary particle diameter, the aqueous PHA suspension exhibits superior fluidity, which tends to further improve the productivity of the PHA.

[0043] 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 laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA.

[0044] For convenience, the volume median diameter of the PHA is specified in step (a), but the volume median diameter of the PHA usually has the same value after step (a). Therefore, the volume median diameter may be measured in any of steps (a) to (d).

[0045] <Bacteria (Microorganisms)> The microorganisms used in step (a) are not particularly limited as long as they are capable of producing PHA intracellularly. For example, microorganisms isolated from nature and deposited in a depository institution for strains (e.g., IFO, ATCC, etc.), or mutants and transformants prepared from them, can be used. For example, the first bacteria to produce P3HB, an example of PHA, was Bacillus megaterium, discovered in 1925. Other examples include naturally occurring microorganisms such as Cupriavidus necator (formerly classified as Alcaligenes eutrophus and Ralstonia eutropha) and Alcaligenes latus. It is known that PHA accumulates intracellularly in these microorganisms.

[0046] Examples of bacteria that produce copolymers of hydroxybutyrate and other hydroxyalkanoates, which are examples of PHA, include Aeromonas caviae, which produces P3HB3HV and P3HB3HH, and Alcaligenes eutrophus, which produces P3HB4HB. In particular, with regard to P3HB3HH, more preferred is Alcaligenes eutrophus AC32 (FERM BP-6038) (T. Fukui, Y. Doi, J. Bateriol., 179, pp. 4821-4830 (1997)), into which genes encoding PHA synthases have been introduced, in order to increase the productivity of P3HB3HH. In addition to the above, the bacterial cells may be genetically modified microorganisms into which various PHA synthesis-related genes have been introduced depending on the PHA to be produced.

[0047] <Disruption and solubilization of cell-derived components> In step (a), the method for disrupting and solubilizing cell-derived components other than PHA in the PHA-containing bacterial cells is not particularly limited.

[0048] In one embodiment of the present invention, the disruption and solubilization is carried out using, for example, a lytic enzyme, a proteolytic enzyme (eg, alkaline protease).

[0049] As used herein, the term "lytic enzyme" refers to an enzyme that has the activity of degrading (lysing) the cell wall (for example, peptidoglycan) of a bacterial cell.

[0050] In one embodiment of the present invention, the lytic enzyme is not particularly limited, and examples thereof include lysozyme, raviol, β-N-acetylglucosaminidase, endolysin, and autolysin. From the viewpoint of economical advantages, lysozyme is preferred. These enzymes may be used alone or in combination of two or more.

[0051] Commercially available lytic enzymes can also be used, such as "Lysozyme" and "Achromopeptidase" manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0052] 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 decomposition activity, but is, for example, 5.0 to 11.0, preferably 6.0 to 9.0, and more preferably 6.0 to 8.0.

[0053] In one embodiment of the present invention, the optimum temperature of the lytic enzyme is not particularly limited, but is preferably 60° C. or lower, more preferably 50° C. or lower, from the viewpoint that excessive heating is not required and thermal changes (thermal decomposition) of the PHA can be prevented. The lower limit of the optimum temperature is not particularly limited, but is preferably room temperature (e.g., 25° C.) or higher, from the viewpoint that excessive cooling is not required and it is economical.

[0054] As used herein, the term "alkaline protease" refers to a protease that has the activity of decomposing proteins in an alkaline environment (for example, in a solution of pH 8.5).

[0055] In one embodiment of the present invention, the alkaline protease is not particularly limited as long as it has the activity of degrading proteins in an alkaline environment, and examples thereof include serine-specific proteases (e.g., subtilisin, chymotrypsin, trypsin), cysteine-specific proteases (e.g., papain, bromelain, cathepsin), aspartic acid-specific proteases (e.g., pepsin, cathepsin D, HIV protease), etc. From the viewpoint of economical advantage, serine-specific proteases, particularly subtilisins (e.g., alcalase), are preferred. These may be used alone or in combination of two or more.

[0056] Commercially available alkaline protease products can also be used, such as "Alcalase 2.5L" manufactured by Novozyme; "Protin SD-AY10" and "Protease P "Amano" 3SD" manufactured by Amano Enzyme Inc.; "Multifect PR6L" and "Optimase PR89L" manufactured by Danisco Japan Co., Ltd.; "Sumiteam MP" manufactured by Shin Nippon Chemical Industry Co., Ltd.; "Delvolase" manufactured by DSM Japan Co., Ltd.; "Bioprase OP," "Bioprase SP-20FG," and "Bioprase SP-4FG" manufactured by Nagase ChemteX Corporation; "Orientase 22BF" manufactured by HBI Corporation; and "Aroase XA-10" manufactured by Yakult Pharmaceutical Co., Ltd.

[0057] In one embodiment of the present invention, the optimal pH of the alkaline protease is not particularly limited as long as the alkaline protease has activity 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.

[0058] In one embodiment of the present invention, the optimum temperature of the alkaline protease is not particularly limited, but is preferably 60° C. or lower, more preferably 50° C. or lower, from the viewpoint that excessive heating is not required and thermal changes (thermal decomposition) of the PHA can be prevented. The lower limit of the optimum temperature is not particularly limited, but is preferably room temperature (e.g., 25° C.) or higher, from the viewpoint that excessive cooling is not required and it is economical.

[0059] In one embodiment of the present invention, the disruption and solubilization of cell-derived components in step (a) may be carried out using a combination of lysozyme and alcalase.

[0060] The enzyme treatment time in step (a) may vary depending on conditions such as the type of enzyme, pH, and temperature, but is, for example, 1 to 8 hours, preferably 2 to 6 hours.

[0061] The solvent ("solvent" is also referred to as "aqueous medium") constituting the PHA aqueous suspension in this production method may be water or a mixed solvent of water and an organic solvent. In addition, the concentration of the water-compatible organic solvent in the mixed solvent is not particularly limited as long as it is equal to or lower than the solubility in water of the organic solvent used. In addition, the water-compatible organic solvent is not particularly limited, and examples thereof include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, 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; dimethyl sulfoxide, pyridine, and piperidine. Among these, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, propionitrile, etc. are preferred because they are easily removed. Furthermore, methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone, etc. are more preferred because they are easily available. Furthermore, methanol, ethanol, and acetone are particularly preferred. Note that the aqueous medium constituting the PHA aqueous suspension may contain other solvents, cell-derived components, compounds generated during purification, etc., as long as the essence of the present invention is not impaired.

[0062] The aqueous medium constituting the PHA aqueous suspension in this production method preferably contains water, and the water content in the aqueous medium is preferably 5 wt % or more, more preferably 10 wt % or more, even more preferably 30 wt % or more, and particularly preferably 50 wt % or more.

[0063] (Other Steps) In one embodiment of the present invention, the production method may include the following steps before step (a).

[0064] <Step (a1)> Step (a1) is a step of culturing PHA-containing bacterial cells.

[0065] In step (a1), the bacterial cells used are, for example, the bacterial cells described above in the section <Bacterial cells (microorganisms)>.

[0066] In step (a1), the method for culturing the bacterial cells is not particularly limited, and examples thereof include the methods described in paragraphs

[0041] to

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

[0067] <Step (a2)> Step (a2) is a step of inactivating the bacterial cells obtained in the step (a1) to obtain an inactivated culture solution.

[0068] In step (a2), the inactivation method is not particularly limited, but examples thereof include a method in which a culture solution containing P3HA-containing bacterial cells is heated and stirred at an internal temperature of 60 to 70°C for 7 hours.

[0069] <Step (a3)> Step (a3) ​​is a step of adjusting the concentration and pH of the inactivated culture solution obtained in step (a2). Step (a3) ​​is mainly performed when the viscosity of the inactivated culture solution obtained in step (a2) is high, and the concentration and pH of the inactivated culture solution are adjusted to reduce the viscosity of the inactivated culture solution. Step (a3) ​​facilitates solubilization in step (a).

[0070] In step (a3), the method for adjusting the concentration and pH of the inactivated culture solution is not particularly limited, and any method used in the art can be used. For example, the concentration of the inactivated culture solution can be adjusted by adding hydrogen peroxide or the like to the inactivated culture solution. Another method for adjusting the pH is, for example, adding a basic compound to the inactivated culture solution. The basic compound is not particularly limited, but is preferably an alkali metal hydroxide or alkaline earth metal hydroxide, and more preferably sodium hydroxide. One basic compound may be used alone, or two or more basic compounds may be used in combination.

[0071] (Step (b)) In step (b) of the present production method, the PHA aqueous suspension is recovered by centrifugation after step (a). By step (b), impurities derived from the cells (cell walls, proteins, etc.) in the PHA aqueous suspension can be removed.

[0072] In step (b), the PHA aqueous suspension is collected by any centrifugation method known in the art. The centrifugation method is not particularly limited, but examples thereof include centrifugation using a centrifugal settler, a centrifugal dehydrator, etc.

[0073] Examples of centrifugal settlers include separator plate type (e.g., disk type, self-cleaning type, nozzle type, screw decanter type, skimming type, etc.), cylindrical type, and decanter type centrifugal settlers. Each type is classified into batch type and continuous type depending on the method of discharging the sediment components. Centrifugal dehydrators are also classified into batch type and continuous type. By using these devices, it is possible to separate the PHA-containing sediment from the culture solution components due to the difference in specific gravity.

[0074] Since the amount of impurities remaining in the final product is largely determined by steps (a) and (b), it is preferable to reduce these impurities as much as possible. Naturally, depending on the application, impurities may be present as long as they do not impair the physical properties of the final product. However, when a highly pure PHA is required, such as for medical applications, it is preferable to reduce the impurities as much as possible. An example of an indicator of the degree of purity in this case is the amount of protein attached to the PHA surface in the PHA aqueous suspension. This protein amount is preferably 2000 ppm or less per PHA weight, more preferably 1900 ppm or less, even more preferably 1800 ppm or less, and most preferably 1700 ppm or less. When the amount of protein attached to the PHA surface of the PHA aqueous suspension is within the above range, there is an advantage that the leakage rate does not become too high. It is presumed that this effect is due to the fact that when the amount of protein attached to the PHA surface is low, PHA particles tend to clump together.

[0075] (Step (c')) In step (c'), the PHA aqueous suspension recovered by centrifugation usually has a pH of more than 7. Therefore, in step (c') of this production method, the PHA aqueous suspension obtained in step (b) is adjusted to a pH of 2.5 to 5.5. By performing the pH adjustment in step (c'), the leakage rate during the filtration in step (d) is reduced.

[0076] In step (c'), the pH of the PHA aqueous suspension is 2.5 to 5.5, preferably 2.6 to 5.0, more preferably 2.7 to 4.5, even more preferably 2.8 to 4.0, and particularly preferably 3.0 to 3.8. Having a pH within the above range of the PHA aqueous suspension has the advantage of improving the filtrate permeation rate in the filtration step without increasing the PHA leakage rate into the filtrate. This effect is presumably due to the PHA not becoming too small and being more likely to aggregate. Furthermore, the upper limit of the pH is preferably 5.5 or less from the viewpoints of reducing coloration when the PHA is heated and melted, ensuring stability of the molecular weight of the PHA during heating and / or drying, and suppressing molecular weight reduction. The lower limit of the pH is preferably 2.5 or more from the viewpoint of the acid resistance of the container.

[0077] In step (c'), the method for adjusting the pH is not particularly limited, and examples thereof include a method of adding an acid. The acid is not particularly limited, and may be either an organic acid or an inorganic acid, regardless of whether it is volatile. More specifically, examples of acids that can be used include sulfuric acid, hydrochloric acid, phosphoric acid, and acetic acid.

[0078] In step (c'), it is preferable to heat the PHA aqueous suspension. The heating temperature is not particularly limited, but is preferably 40 to 90°C, more preferably 50 to 80°C, and even more preferably 60 to 70°C. By heating the PHA aqueous suspension in step (c'), it becomes easier to adjust the pH, and therefore it is possible to obtain a PHA with high thermal stability.

[0079] In one embodiment of the present invention, it is preferred that no additional pH adjustment is carried out after the pH adjustment in step (c) and before step (d) is carried out.

[0080] (Step (c)) In step (c) of the present production method, the PHA aqueous suspension is heated to a temperature of 60 to 120° C. Step (c) can increase the filtrate permeation rate during filtration.

[0081] In step (c), the PHA aqueous suspension is preferably heated to a temperature of 60 to 120° C., more preferably 62 to 118° C., and even more preferably 65 to 115° C. If the temperature of the PHA aqueous suspension is within the above range, the filtrate permeation rate during filtration can be further increased.

[0082] The method of heat treatment in step (c) is not particularly limited, and examples thereof include (i) a method of using steam to warm a container containing the PHA aqueous suspension, (ii) a method of using oil to warm a container containing the PHA aqueous suspension, and (iii) a method of directly introducing steam into the PHA aqueous suspension. The temperatures of the steam in (i) and (iii) and the oil in (ii) are not particularly limited as long as they bring the temperature of the PHA aqueous suspension in step (c) to 60 to 120°C, and are, for example, 95 to 150°C.

[0083] (Step (e)) In step (e) of the present production method, the PHA obtained in step (d) is dried at 20 to 100° C. In step (e), the water content in the PHA aqueous suspension is evaporated, and the water content can be adjusted.

[0084] In step (e), the method for drying the PHA is not particularly limited, but examples thereof include heating, vacuum drying, and room temperature drying. Preferably, the drying is performed by heating from the viewpoint of an appropriate drying rate. The temperature of the heat medium (e.g., hot air, jacket, etc.) during drying is preferably 30 to 90°C, more preferably 40 to 80°C, and even more preferably 50 to 70°C.

[0085] (Step (f)) In step (f) of this production method, the dried PHA is redispersed in an aqueous solvent to obtain a PHA aqueous suspension. By performing step (f) subsequent to step (e), an aqueous PHA suspension containing PHA having a particle size substantially the same as the original particle size (primary particle size) can be obtained.

[0086] In step (f), the method of redispersion is not particularly limited, and can be any method used in the art.

[0087] 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 is, for example, preferably 0.5 to 5.0 μm, more preferably 1.0 to 4.5 μm, and even more preferably 1.0 to 3.0 μm. In this specification, "substantially the same volume median diameter" means that the difference from the volume median diameter of the PHA in step (a) is 1.0 μm or less.

[0088] The steps (e) and (f) may be carried out consecutively. That is, the PHA dried in the step (e) may be redispersed in the step (f) to obtain an aqueous PHA suspension. In one embodiment of the present invention, this production method includes a step of drying the polyhydroxyalkanoate obtained in the filtration step at 20 to 100°C, and a step of redispersing the dried polyhydroxyalkanoate in an aqueous solvent to obtain an aqueous polyhydroxyalkanoate suspension containing a polyhydroxyalkanoate having a volume median diameter of 0.5 to 5.0 μm.

[0089] 3. PHA Agglomerates The PHA agglomerates have a water content of 5.0 to 25.0% (W.B.) The PHA agglomerates are also referred to as "PHA cake," "filter cake," or "PHA filter cake."

[0090] The water content of the PHA agglomerates is 5.0 to 25.0% (W.B.), preferably 5.5 to 23.0% (W.B.), more preferably 6.0 to 21.0% (W.B.), even more preferably 6.5 to 20.0% (W.B.), and particularly preferably 7.0 to 19.0% (W.B.). When the water content of the PHA agglomerates is within the above range, the PHA agglomerates are in a solid state rather than a slurry state, which has the advantage of making them easier to put into a dryer. The water content of the PHA agglomerates is measured by the method described in the Examples.

[0091] In one embodiment of the present invention, the PHA agglomerates are produced by the present production method.

[0092] The PHA aggregates may contain various components that have been generated or not removed during the production process, as long as the effects of the present invention are achieved.

[0093] The PHA agglomerates can be used in a variety of applications, including paper, films, sheets, tubes, plates, rods, containers (e.g., bottles), bags, and parts.

[0094] The present invention is not limited to the above-described embodiments, 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.

[0095] That is, one aspect of the present invention includes the following: <1> A PHA aqueous suspension having a pH of 2.5 to 5.5 is mixed with an aqueous suspension having an air permeability of 0.1 to 2.5 cm ３ / cm ２A method for producing a PHA, comprising a step of filter press filtration using a filter medium having a flow rate of 0.50 to 1.08 g / min, wherein the liquid density of the PHA aqueous suspension in the filtration step is 0.50 to 1.08 g / mL, and the filter press filtration step comprises a squeezing step and an air blowing step. <2> The method according to <1>, wherein the solids concentration of the filtrate in the filtration step is 1000 mg / L or less. <3> The method according to <1> or <2>, wherein the filter medium is not pre-coated. <4> The method according to any one of <1> to <3>, wherein the temperature of the PHA aqueous suspension in the filtration step is 20 to 95°C. <5> The method according to any one of <1> to <4>, further comprising a step (a) of disrupting and solubilizing cell-derived components other than PHA from PHA-containing bacterial cells, wherein the volume median diameter of the PHA in the step (a) is 0.5 to 5.0 μm. <6> The method for producing a polyhydroxyalkanoate according to <5>, further comprising, after step (a), (b) a step of recovering the aqueous PHA suspension by centrifugation. <7> The method for producing a polyhydroxyalkanoate according to any one of <1> to <6>, further comprising a step of drying the polyhydroxyalkanoate obtained in the filtration step at 20 to 100°C. <8> The method for producing a polyhydroxyalkanoate according to any one of <1> to <7>, wherein the air blowing step uses an air blowing pressure of 0.01 to 1.5 MPa. <9> The method for producing a polyhydroxyalkanoate according to any one of <1> to <8>, wherein the air blowing step uses an air blowing time of 1 to 50 minutes. <10> A method for producing a polyhydroxyalkanoate aqueous suspension, comprising a step of dispersing the polyhydroxyalkanoate produced by the method according to any one of <1> to <9> in an aqueous solvent. <11> A PHA aggregate having a water content of 5.0 to 25.0% (W.B.).

[0096] 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 also be read as "P3HB3HH."

[0097] [Measurement Method] Measurements in the examples and comparative examples were carried out by the following methods.

[0098] (Solid content concentration of filtrate) A suitable amount of filtrate was vacuumed using a glass filter, glass filter paper GS-25 (manufactured by ADVANTEC Co., Ltd.) The solid content concentration was then calculated from the mass of the dried filter paper.

[0099] (Water content of PHA agglomerates) The dehydrated cake produced in the test was divided into 3 or 9 equal parts, and an appropriate amount was collected from each part. Then, the cake was dried in a thermostatic oven at 105°C for about 15 hours, and the water content of the PHA agglomerates was calculated from the difference in mass before and after drying.

[0100] (Air permeability) The air permeability was measured by the method described in JIS L 1096. Specifically, using a Frazier-type air permeability tester (Permeameter P2, manufactured by Toyo Seiki Seisakusho, Ltd.), the amount of air intake was adjusted so that the inclined barometer indicated 125 Pa, and the air flow rate at that time was measured.

[0101] (Liquid Density) The PHA aqueous suspension immediately before the filtration step was heated to the filtration temperature, and 20 mL of the suspension was drawn into a 20 mL plastic syringe (manufactured by Terumo) whose weight had been measured in advance. The weight of the plastic syringe and the 20 mL of the aqueous suspension was then measured, and the weight (g) obtained by subtracting the weight of the 20 mL plastic syringe was divided by the liquid volume (20 mL) to calculate the liquid density at the filtration temperature.

[0102] (Volume Median Diameter) The volume median diameter of PHA was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA.

[0103] (Temperature during filtration) The temperature during filtration was measured using a K thermocouple (AD5601A, manufactured by AND) immediately before the PHA aqueous suspension was introduced into the filter.

[0104] (pH of PHA aqueous suspension in step (c')) The pH was measured using a pH meter (9652-10D, manufactured by HORIBA). The pH was measured at the position of the PHA aqueous suspension farthest from the position where the acid was added, while the PHA aqueous suspension was kept in a fluidized state using a stirring blade or the like. For example, when the acid was added from the wall of the container, the pH was measured at the center of the container.

[0105] Example 1 Preparation of Fungal Cell Culture Solution Ralstonia eutropha, as described in International Publication No. WO 2019 / 142717, was cultured by the method described in paragraphs

[0041] to

[0048] of the same document to obtain a fungal cell culture solution containing PHA-containing fungal cells. Ralstonia eutropha is currently classified as Capriavidus necator. The composition ratio of repeating units of PHA (P3HB3HH) in the fungal cell culture solution (composition ratio of 3HB unit / 3HH unit) was 94 / 6 (mol / mol).

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

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

[0108] (Enzyme Treatment) 95% sulfuric acid was added to the PHA aqueous suspension obtained above to adjust the pH to 7.0±0.2. The solids concentration of the PHA aqueous suspension to which sulfuric acid had been added was measured and found to be 30% by weight. After adding sulfuric acid, lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme that decomposes sugar chains (peptidoglycans) in cell walls, was added to a liquid concentration of 10 ppm and maintained at 50 ° C. for 2 hours. Thereafter, Alcalase 2.5L (manufactured by Novozyme), a protease, was added to a liquid concentration of 300 ppm, and then 30% sodium hydroxide was added at 50 ° C., and the mixture was maintained for 2 hours while adjusting the pH to 8.5.

[0109] (Alkali Treatment) Sodium dodecyl sulfate (SDS, manufactured by Kao) was added to the enzyme-treated solution to a concentration of 0.3 wt %. The pH was then adjusted to 11.0±0.2 using aqueous sodium hydroxide. The enzyme-treated solution was then centrifuged (4000 G, 10 minutes), and the supernatant was removed to obtain a 2-fold concentrated PHA aqueous suspension. The concentrated PHA aqueous suspension was added with an equal amount of sodium hydroxide to the removed supernatant, and the suspension was centrifuged again (4000 G, 10 minutes), followed by removal of the supernatant. This process was repeated four times. The volume median diameter of the PHA was 2.2 μm. The protein content of the resulting PHA aqueous suspension was 1000 ppm.

[0110] (pH Adjustment) The solid content of the PHA aqueous suspension obtained above was adjusted to 25 wt % and maintained at 60° C. Then, 10% sulfuric acid was added to adjust the pH to 3.5. The liquid density was 1.00 g / mL.

[0111] (Filtration) The PHA aqueous suspension was placed in a water bath at 63°C, heated to a liquid temperature of 60°C, and filtered using a filter press (ISD type 360, manufactured by Ishigaki Co., Ltd.). ３ / cm ２ A filter cloth (IP196C-1, manufactured by Ishigaki Co., Ltd.) with a flow rate of 1 / min was used. The filter cloth used was not pre-coated. Filtration was carried out at a compression pressure of 0.7 MPa, and air blowing was carried out by adjusting the air blow pressure to 0.2 to 0.6 MPa. Table 1 shows the solid concentration of the obtained filtrate and the water content of the PHA aggregates. A photograph of the filter cloth permeate of Example 1-1 is shown in Figure 1.

[0112] Comparative Example 1 PHA aggregates were obtained in the same manner as in Example 1, except that air blowing was not performed during filter press filtration. Table 1 shows the solid concentration of the obtained filtrate and the water content of the PHA aggregates.

[0113]

[0114] [Example 2] An enzyme-treated solution was obtained using the same method as in Example 1 up to the enzyme treatment. SDS was added to the enzyme-treated solution to a concentration of 0.2 wt %. The pH was then adjusted to 11.0±0.2 using aqueous sodium hydroxide. The enzyme-treated solution was then centrifuged (4000 G, 10 minutes), and the supernatant was removed to obtain a 2-fold concentrated PHA aqueous suspension. The concentrated PHA aqueous suspension was added with an equal amount of sodium hydroxide to the removed supernatant, and the suspension was centrifuged again (4000 G, 10 minutes), followed by removal of the supernatant. This process was repeated twice. The protein content in the resulting PHA aqueous suspension was 3000 ppm. The volume median diameter of the PHA was 2.2 μm. The pH adjustment step and filtration step were performed under the same conditions as in Examples 1-2, and a filter cake was obtained. The liquid density after the pH adjustment step was 1.00 g / mL. The water content (W.B.) of the obtained cake was 13.1%, and the solid concentration of the liquid permeating through the filter cloth was 10 mg / L or less.

[0115] Example 3 (Preparation of Bacterial Cell Culture Solution) Ralstonia eutropha, as described in International Publication No. WO 2019 / 142717, was cultured using the method described in paragraphs

[0041] to

[0048] of the same document to obtain a bacterial cell culture solution containing PHA-containing bacterial cells. The composition ratio of the repeating units of PHA in the bacterial cell culture solution (composition ratio of 3HB units / 3HH units) was 80 / 20 to 88 / 12 (mol / mol), more precisely, 85 / 15 (mol / mol). Filtration was performed using the obtained bacterial cell culture solution in the same manner as in Example 1, except that lysozyme was not added in the enzyme treatment step. The volume median diameter of the PHA after the alkali treatment step was 2.3 μm. The protein content of the obtained PHA aqueous suspension was 1,800 ppm. The liquid density after the pH adjustment step was 1.00 g / mL.

[0116]

[0117] [Comparative Example 2] Air permeability 3.0 cm ３ / cm ２ Filter press filtration was carried out in the same manner as in Comparative Example 1, except that a filter cloth (IP126C, manufactured by Ishigaki Co., Ltd.) with a permeability of 1.0 cm / min was used.３ / cm ２ / min filter cloth does not allow particles to pass through, but the air permeability is 3.0 cm ３ / cm ２ It can be seen that the particles pass through the filter cloth with a flow rate of 1 / min. The solid concentration of the filtrate was 2060 mg / L.

[0118] [Example 4] The PHA agglomerates obtained in Examples 1-3 were placed in a dryer (EYELA, WFO-700) and dried at 60°C for 24 hours. The dried PHA agglomerates were redispersed in water, and the solid concentration was adjusted to 15% by weight. ２ SO ４ The pH was adjusted to between 7 and 9 using an aqueous solution, and the mixture was stirred to prepare a PHA aqueous suspension. After stirring for 30 minutes, the particle size of the PHA particles in the PHA aqueous suspension was measured, and the volume median size was found to be 2.8 μm.

[0119] [Summary] From the above, it was found that the present production method can significantly reduce the water content of PHA aggregates.

[0120] The present production method can be advantageously used in the production of PHA because it can produce PHA with a low moisture content through a simple operation. Furthermore, the PHA agglomerates of the present invention can be suitably used in the fields of agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing, packaging, automobiles, building materials, and other fields.

Claims

1. Aqueous suspension of polyhydroxyalkanoate having a pH of 2.5 to 5.5 is subjected to aeration of 0.1 to 2.5 cm ３ / cm ２ / min, The liquid density of the aqueous suspension of polyhydroxyalkanoate in the filtration step is 0.50 to 1.08 g / mL; The method for producing polyhydroxyalkanoate, wherein the filter press filtration step includes a squeezing step and an air blowing step.

2. The method according to claim 1 , wherein the solid content of the filtrate in the filtration step is 1000 mg / L or less.

3. The method of claim 1 or 2, wherein the filter medium is not precoated.

4. The 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.

5. The method further comprises the step of (a) disrupting and solubilizing cellular components other than polyhydroxyalkanoate from the polyhydroxyalkanoate-containing bacterial cells, The method according to claim 1 or 2, wherein the polyhydroxyalkanoate in the step (a) has a volume median diameter of 0.5 to 5.0 μm.

6. The method according to claim 5, further comprising, after the step (a), a step (b) of recovering the aqueous suspension of polyhydroxyalkanoate by centrifugation.

7. The method according to claim 1 or 2, further comprising a step of drying the polyhydroxyalkanoate obtained in the filtration step at 20 to 100°C.

8. The method according to claim 1 or 2, wherein the air blowing pressure in the air blowing step is 0.01 to 1.5 MPa.

9. The method according to claim 1 or 2, wherein the air blowing time in the air blowing step is 1 to 50 minutes.

10. A method for producing an aqueous suspension of a polyhydroxyalkanoate, comprising a step of redispersing the polyhydroxyalkanoate produced by the method according to claim 1 or 2 in an aqueous solvent.