Method for producing polyhydroxyalkanoate and use thereof

Abstract

The present invention addresses the problem of providing a PHA having good heat stability even in a pH range where it is not necessary to use corrosion resistant devices. The aforesaid problem is solved by a method for producing a PHA that includes: a filter press filtration step including a compression step for compressing an aqueous PHA suspension having a pH of 2.5 or higher and lower than 4.0 with a filter press filtration machine; and a through-washing step for through-washing the filter cake obtained by the compression step until the pH becomes 4.0-5.5.

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 for separating and purifying the PHA, the cells of the PHA-containing microorganisms are crushed or biological 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. For the drying procedure, a spray dryer, a fluidized bed dryer, a drum dryer, etc. are used, but a spray dryer is preferably used because of its simple operation.

[0004] To date, the present inventors have developed a technique for preventing aggregation of PHA in an aqueous suspension having a pH of 7 or less and preventing an increase in viscosity by adding an alkylene oxide-based dispersant before adjusting the pH of the aqueous suspension to 7 or less, and then spray-drying the resulting aqueous suspension having a pH of 7 or less (see Patent Document 1).

[0005] Furthermore, Patent Document 2 discloses a method for producing a target product, which comprises mixing a polyhydroxyalkanoate with an acid having a pKa of 3 to 10.

[0006] International Publication No. 2021 / 085534 U.S. Patent No. 2013 / 0093119

[0007] However, there is room for further improvement in the techniques for producing PHA as described above.

[0008] Therefore, an object of the present invention is to provide a method for producing PHA with good thermal stability in a pH range that does not require the use of corrosion-resistant equipment.

[0009] As a result of intensive research to solve the above-mentioned problems, the present inventors have discovered a new finding that, in the production of PHA, a PHA having excellent thermal stability can be obtained by penetrating and washing a PHA filter cake obtained by a filter press filter until the pH reaches a specific range, and have completed the present invention.

[0010] Therefore, one aspect of the present invention is a method for producing a PHA (hereinafter referred to as "this production method"), which includes a filter press filtration step and a through-washing step, wherein the filter press filtration step includes a squeezing step of supplying an aqueous PHA suspension having a pH of 2.5 or more and less than 4.0 to a filter press filter and squeezing it, and the through-washing step is a step of through-washing the filter cake obtained by the squeezing step until the pH of the filter cake reaches 4.0 to 5.5.

[0011] Another aspect of the present invention is a PHA aggregate (hereinafter referred to as "the present PHA aggregate") containing water and having a pH of 4.0 to 5.5 and a water content of 5.0 to 25.0% (W.B.).

[0012] According to one aspect of the present invention, a method for producing a PHA having good thermal stability can be provided without using a corrosion-resistant device.

[0013] 1 is a graph showing the change in pH and electrical conductivity with respect to the ratio of the amount of washing water used to the amount of PHA aqueous suspension supplied in an example of the present invention. 2 is a graph showing the evaluation of the pH of the PHA aqueous suspension, and the thermal stability and YI of the obtained pH in an example of the present invention.

[0014] 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."

[0015] [1. Overview of the Invention] A method for drying an aqueous suspension of PHA is known as a method for producing PHA. In this method, in order to increase the thermal stability (molecular weight retention upon heating) of the resulting PHA (in other words, to suppress decomposition of the PHA upon heating), it is necessary to lower the pH of the PHA aqueous suspension using sulfuric acid or the like (usually pH 2.5 or higher and less than 4.0). However, when a PHA aqueous suspension with a low pH is used, a corrosion-resistant device is required for the drying operation in the PHA production process, which poses a problem of increased equipment costs. On the other hand, when a PHA aqueous suspension with a pH within a range that does not require a corrosion-resistant device is used, the thermal stability of the resulting PHA is reduced.

[0016] Therefore, the present inventors have conducted extensive research to provide a method for producing PHA that can produce PHA with good thermal stability without using corrosion-resistant equipment, and as a result, have discovered for the first time that PHA with high thermal stability can be produced without the need for corrosion-resistant equipment by compressing an aqueous PHA suspension using a filter press filter and performing through-washing.

[0017] The present inventors speculate that the mechanism of the present invention is as follows. Specifically, PHA aqueous suspensions contain metal ions and the like. When such PHA aqueous suspensions are dried at a high pH (e.g., 4.0 or higher) to avoid the need for corrosion-resistant equipment, the remaining metal ions and the like function as catalysts in the resulting PHA. Therefore, these metal ions and the like promote the thermal decomposition of the PHA, thereby reducing the thermal stability of the PHA. On the other hand, in the present invention, the low-pH PHA aqueous suspension is processed into a PHA filter cake by the squeezing and through-washing, and the solvent containing metal ions and the like in the PHA filter cake is replaced with wash water that does not contain impurities that can function as a catalyst for the thermal decomposition of metal ions and the like. Through these operations, impurities in the PHA filter cake are removed, and the pH of the PHA filter cake can be adjusted to 4.0 or higher, eliminating the need for corrosion-resistant equipment. Therefore, according to the present invention, a PHA with high thermal stability can be produced without the need for corrosion-resistant equipment.

[0018] As used herein, "PHA cake," "filter cake," or "PHA filter cake" refers to a solid composition containing PHA (formed by agglomeration of PHA in an aqueous PHA suspension) with a moisture content of more than 25.0% and not more than 50.0%, which is obtained, for example, by filtering or squeezing an aqueous PHA suspension. Furthermore, as used herein, "PHA agglomerates" refers to solid compositions containing PHA with a moisture content of 5.0 to 25.0%, which are obtained by dehydrating a PHA cake, for example, by air blowing. As used herein, "PHA powder" refers to a composition containing PHA with a moisture content of less than 5.0%, which is obtained, for example, by drying a PHA agglomerate.

[0019] This production method is extremely advantageous in PHA production because it can produce PHA with good thermal stability even in a pH range that does not require the use of corrosion-resistant equipment. Furthermore, the above-described configuration can reduce the amount of plastic waste generated, thereby contributing to the achievement of Sustainable Development Goals (SDGs), such as Goal 12 "Ensure sustainable consumption and production patterns" and Goal 14 "Conserve and sustainably use the oceans and marine resources for sustainable development." The following describes in detail this production method and the configuration of this aqueous suspension.

[0020] 2. PHA Production Method This production method comprises the following steps: a squeezing step of supplying an aqueous suspension of polyhydroxyalkanoate having a pH of 2.5 or more and less than 4.0 to a filter press filter and squeezing it, and a through-washing step of through-washing the filter cake obtained by the squeezing step (hereinafter referred to as step (d)).

[0021] 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 or higher and less than 4.0 (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").

[0022] 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."

[0023] (Step (d)) First, the step (d), which is a characteristic feature of the present invention, will be described.

[0024] Step (d) in this production method includes a filter press filtration step and a through-washing step. The filter press filtration step includes a squeezing step in which an aqueous PHA suspension having a pH of 2.5 or higher but lower than 4.0 is supplied to a filter press filter and squeezed. The through-washing step is a step in which the filter cake obtained in the squeezing step is through-washed until the pH of the filter cake reaches 4.0 to 5.5. Step (d) allows the production of a PHA with high thermal stability.

[0025] The pressure in the squeezing step in the step (d) 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. By setting the pressure in the squeezing step to 0.2 to 1.0 MPa, the PHA filter cake can be satisfactorily washed with a small amount of wash water.

[0026] In the through-washing step, the filter cake is washed until the pH of the filter cake reaches 4.0 to 5.5, preferably 4.1 to 5.4, and more preferably 4.2 to 5.3. If the pH of the filter cake is 4.0 or higher, a corrosion-resistant device is not required, particularly in the air blowing step described below. Furthermore, if the pH of the filter cake is 5.5 or lower, the thermal stability of the PHA is improved. Note that, since it is difficult to directly measure the pH of the filter cake in the through-washing step, the pH of the washing liquid (washing filtrate) discharged after washing the filter cake is measured and regarded as the pH of the filter cake.

[0027] In the through-washing step, the ratio (amount of washing water used) / (amount of polyhydroxyalkanoate aqueous suspension supplied to the filter chamber of the filter press filter) is 0.1 to 1.0, preferably 0.12 to 0.98, and more preferably 0.14 to 0.96. When this value is 0.1 or more, the pH of the PHA filter cake becomes 4.0 or higher. When this value is 1.0 or less, the amount of washing water in the PHA filter cake does not become too large, and PHA aggregates with a low water content can be obtained.

[0028] In one embodiment of the present invention, the filter press filtration step in step (d) includes an air blowing step in addition to the squeezing 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 subjecting the PHA cake to two dehydration steps, the squeezing step and the air blowing step, a PHA agglomerate with a low water content can be obtained.

[0029] 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.

[0030] 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.

[0031] In one embodiment of the present invention, the air blowing step can be a through air blowing step.

[0032] In one embodiment of the present invention, the number of times, order, etc. of the filter press filtration step and the through-washing step in step (d) are not particularly limited. For example, the through-washing step may be performed after the filter press filtration step, or the filter press filtration step may be performed before and / or after the through-washing step.

[0033] In the filter press filtration step and the through-washing step of step (d), it is preferable that the first squeezing step, the through-washing step, the second squeezing step, and the air blowing step are carried out in this order. By carrying out the first squeezing step and then the through-washing step, the washing water can easily pass through the PHA filter cake, improving the washing efficiency. Furthermore, by carrying out the second squeezing step after the through-washing step, it becomes possible to sufficiently discharge the washing water contained in the PHA filter cake.

[0034] In the above embodiment, the pressure in the first compressing step is preferably 0.2 to 0.6 MPa, more preferably 0.25 to 0.55 MPa, and even more preferably 0.3 to 0.5 MPa. The pressure in the second compressing step is preferably 0.4 to 1.0 MPa, more preferably 0.5 to 0.9 MPa, and even more preferably 0.6 to 0.8 MPa. From the viewpoint of sufficiently removing the wash water, the pressure in the second compressing step is preferably higher than the pressure in the first compressing step.

[0035] 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.

[0036] The apparatus for performing the filter press filtration is not particularly limited, and any known apparatus can be used.

[0037] 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 squeezing step of the present production method is measured by the method described in the Examples.

[0038] 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, and more preferably, a filter cloth having the above-mentioned air permeability is used.

[0039] In step (d), the liquid density of the PHA aqueous suspension is preferably 0.50 to 1.08 g / mL, more preferably 0.60 to 1.05 g / mL, even more preferably 0.70 to 1.03 g / mL, and particularly 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 resulting PHA aggregates. It is believed 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 interaction between the air and the PHA when the air is added. The liquid density of the PHA aqueous suspension can be adjusted, for example, by adding 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.

[0040] 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 that the filter cloth is less likely to clog. The lower limit is not particularly limited, and may be, for example, 0 mg / mL. The solids concentration of the filtrate in this production method is measured by the method described in the Examples.

[0041] In one embodiment of the present invention, the filter medium is preferably not pre-coated, which has the advantage that impurities are less likely to be mixed into the resulting PHA.

[0042] 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. Having the temperature of the PHA aqueous suspension within the above range has the advantage of increasing the filtrate permeation rate. The increase in the filtrate permeation rate is presumed to be due to the fact that the viscosity of the PHA aqueous suspension increases with increasing temperature, while the particle size of the PHA particles increases.

[0043] 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.

[0044] (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 impurities (cell walls, proteins, etc.) derived from the bacterial cells 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.

[0045] <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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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 1.2 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.

[0051] 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 in any of steps (a) to (d). Therefore, the volume median diameter may be measured in any of steps (a) to (d).

[0052] <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.

[0053] 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.

[0054] <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.

[0055] 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).

[0056] 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.

[0057] 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.

[0058] Commercially available lytic enzymes can also be used, such as "Lysozyme" and "Achromopeptidase" manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0059] 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.

[0060] 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.

[0061] 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).

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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, components derived from bacterial cells, compounds generated during purification, etc., as long as the essence of the present invention is not impaired.

[0069] 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.

[0070] (Other Steps) In one embodiment of the present invention, the production method may include the following steps before step (a).

[0071] <Step (a1)> Step (a1) is a step of culturing PHA-containing bacterial cells.

[0072] In step (a1), the bacterial cells used are, for example, the bacterial cells described in the above section <Bacterial cells (microorganisms)>.

[0073] 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.

[0074] <Step (a2)> Step (a2) is a step of inactivating the bacterial cells obtained in the step (a1) to obtain an inactivated culture solution.

[0075] 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.

[0076] <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 the solubilization of cell-derived components in step (a).

[0077] 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.

[0078] (Step (b)) In step (b) of the present production method, the PHA aqueous suspension is collected by centrifugation after step (a). By step (b), impurities (cell walls, proteins, etc.) derived from the bacterial cells in the PHA aqueous suspension can be removed.

[0079] 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.

[0080] 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.

[0081] 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 purification is the amount of protein attached to the PHA surface in the PHA aqueous suspension. This protein amount is 2000 ppm or less per PHA weight, 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 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.

[0082] (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 the present production method, the PHA aqueous suspension obtained in step (b) is adjusted to a pH of 2.5 or more and less than 4.0. By performing the pH adjustment in step (c'), the leakage rate during the filtration in step (d) is reduced.

[0083] In step (c'), the pH of the PHA aqueous suspension is at least 2.5 but less than 4.0, preferably 2.6 to 3.9, more preferably 2.7 to 3.8, even more preferably 2.8 to 3.7, and particularly preferably 2.9 to 3.6. 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 particle size not becoming too small and therefore easily agglomerating. Furthermore, the upper limit of the pH is preferably less than 4.0 from the viewpoints of reducing discoloration when the PHA is heated and melted, ensuring molecular weight stability during heating and / or drying, and obtaining a PHA with reduced discoloration during heating and melting and suppressed molecular weight reduction during heating and / or drying. The lower limit of the pH is preferably 2.5 or higher from the viewpoint of the acid resistance of the container.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] (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.

[0088] 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.

[0089] 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.

[0090] (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.

[0091] 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.

[0092] (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.

[0093] In step (f), the method of redispersion is not particularly limited, and can be any method used in the art.

[0094] 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 1.2 to 4.0 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.

[0095] 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.

[0096] 3. PHA Aggregates The PHA aggregates contain water and have a pH of 4.0 to 5.5, with a water content of 5.0 to 25.0% (W.B.).

[0097] The pH of the PHA aggregates is 4.0 to 5.5, preferably 4.1 to 5.4, and more preferably 4.2 to 5.3.

[0098] 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.

[0099] In one embodiment of the present invention, the PHA agglomerates are produced by the present production method.

[0100] 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.

[0101] 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.

[0102] The PHA agglomerates may be dried by known methods to form PHA powder. The thermal stability of the PHA powder obtained from the PHA agglomerates is preferably 70% or more, more preferably 75% or more, and even more preferably 80% or more. A thermal stability of 70% or more can suppress deterioration of the resin obtained by processing the PHA powder. The higher the thermal stability, the better, and there is no particular upper limit, but it may be, for example, 99% or less, or even 100%. The thermal stability is expressed by the following formula (2) based on the method described in the Examples below.

[0103] Thermal stability (%)=weight average molecular weight of a polyhydroxyalkanoate sheet obtained by pressing a polyhydroxyalkanoate powder at 160° C. and 5 MPa for 20 minutes / weight average molecular weight of polyhydroxyalkanoate powder×100 (2).

[0104] It is preferable that the PHA powder has reduced discoloration during heating. The degree of discoloration of the PHA powder can be evaluated by the YI (yellowness index) of a pressed sheet obtained by pressing the PHA powder. The lower the YI value of the pressed sheet, the more reduced the discoloration of the PHA powder can be evaluated. Note that a more specific method for evaluating the degree of discoloration of the PHA powder is as described in the Examples.

[0105] 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.

[0106] That is, one aspect of the present invention includes the following. <1> A method for producing a polyhydroxyalkanoate, comprising a filter press filtration step and a through-washing step, wherein the filter press filtration step comprises a squeezing step of supplying an aqueous suspension of polyhydroxyalkanoate having a pH of 2.5 or more and less than 4.0 to a filter press filter and squeezing it, and the through-washing step comprises a step of through-washing the filter cake obtained in the squeezing step until the pH of the filter cake reaches 4.0 to 5.5. <2> The production method according to <1>, wherein the filter press filtration step further comprises an air blowing step. <3> The production method according to <1> or <2>, wherein, in the filter press filtration step and the through-washing step, a first squeezing step, a through-washing step, a second squeezing step, and an air blowing step are performed in this order. <4> The method according to any one of <1> to <3>, wherein in the through-washing step, (amount of washing water used) / (amount of polyhydroxyalkanoate aqueous suspension supplied to the filtration chamber of the filter press filter) is 0.1 to 1.0. <5> In the filter press filtration step, ３ / cm ２ / min. <6> The production method according to any one of <1> to <5>, wherein the temperature of the aqueous polyhydroxyalkanoate suspension in the squeezing step is 20 to 95°C. <7> The production method according to any one of <1> to <6>, further comprising: (a) a step of disrupting and solubilizing cell-derived components other than polyhydroxyalkanoate from the polyhydroxyalkanoate-containing bacterial cells, wherein the volume median diameter of the polyhydroxyalkanoate in the step (a) is 0.5 to 5.0 μm. <8> The production method according to any one of <1> to <7>, further comprising: (b) a step of recovering the aqueous polyhydroxyalkanoate suspension by centrifugation after the step (a). <9> The production method according to any one of <1> to <8>, further comprising: a step of drying the polyhydroxyalkanoate obtained in the filtration step at 20 to 100°C. <10> The manufacturing method according to any one of <1> to <9>, wherein the pressure in the squeezing step is 0.2 to 1.0 MPa. <11> The manufacturing method according to any one of <1> to <10>, wherein the air blowing pressure in the air blowing step is 0.01 to 1.5 MPa. <12> A method for manufacturing an aqueous polyhydroxyalkanoate suspension, comprising a step of dispersing polyhydroxyalkanoate manufactured by the method according to any one of <1> to <11> in an aqueous solvent. <13> Polyhydroxyalkanoate agglomerates, containing water and having a pH of 4.0 to 5.5, and a water content of 5.0 to 25.0% (W.B.). <14> A polyhydroxyalkanoate powder obtained by drying the polyhydroxyalkanoate agglomerates according to <13>, wherein the polyhydroxyalkanoate powder has a thermal stability of 70% or more as represented by the following formula (2): thermal stability (%) = weight-average molecular weight of a polyhydroxyalkanoate sheet obtained by pressing the polyhydroxyalkanoate powder at 160°C and 5 MPa for 20 minutes / weight-average molecular weight of polyhydroxyalkanoate powder × 100 (2).

[0107] 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."

[0108] [Measurement Method] Measurements in the examples and comparative examples were carried out by the following methods.

[0109] (Thermal Stability of PHA Powder) PHA agglomerates obtained in the following Examples and Comparative Examples were used as evaluation samples. The PHA agglomerates were placed in a dryer (EYELA, WFO-700) and dried at 60°C for 24 hours to obtain PHA powder. The obtained PHA powder was preheated at 160°C for 7 minutes and then pressed at 5 MPa for 20 minutes to produce a PHA sheet. 10 mg of this PHA sheet was dissolved in 10 ml of chloroform, and insoluble matter was removed by filtration. This solution (filtrate) was subjected to molecular weight measurement using a Shimadzu GPC system equipped with a "Shodex K805L (300 x 8 mm, two connected)" (Showa Denko K.K.) using chloroform as the mobile phase. Commercially available standard polystyrene was used as a molecular weight standard sample. The molecular weight of the PHA powder was also measured using the same procedure as above, except that a PHA sheet was not produced.

[0110] The thermal stability was evaluated based on the following formula (2): Thermal stability (%) = weight average molecular weight of a PHA sheet obtained by pressing a PHA powder at 160°C and 5 MPa for 20 minutes / weight average molecular weight of PHA powder × 100 (2).

[0111] (YI (Yellowness Index)) A pressed sheet of PHA resin, which was a sample for measuring the YI value, was prepared by the following method. 3.0 g of PHA powder was sandwiched between 15 cm square metal plates, and 0.5 mm thick metal plates were inserted into the four corners of the metal plates. This was then set in a small laboratory press (H-15 model, manufactured by Takabayashi Rika Co., Ltd.). The sample was preheated at 160°C for 7 minutes, then pressed while heating at 5 MPa for 2 minutes, and left at room temperature after pressing to cure the PHA, thereby preparing a pressed sheet of PHA resin. The YI value was measured using a color difference meter "SE-2000" (manufactured by Nippon Denshoku Co., Ltd.) with a 30 mm measuring plate.

[0112] (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.

[0113] (Solid content concentration) The solid content concentration of the PHA aqueous suspension was measured using a heat-dry moisture meter ML-50 (manufactured by A&D Co., Ltd.) The PHA aqueous suspension was heated at 105°C until the weight change rate fell below 0.05% / min, and the solid content concentration was calculated from the weight change of the PHA aqueous suspension before and after heating.

[0114] (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.

[0115] (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.

[0116] [Example 1] (Preparation of fungal cell culture solution) Ralstonia eutropha described in International Publication No. WO2019 / 142717 was cultured by the method described in paragraphs

[0041] to

[0048] of the same document to obtain a fungal cell culture solution containing fungal cells containing PHA. Note that Ralstonia eutropha is currently classified as Capriavidus necator.

[0117] (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.

[0118] (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.

[0119] (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 the PHA aqueous suspension so that the liquid concentration was 10 ppm, and the suspension was maintained at 50 ° C. for 2 hours. Thereafter, Alcalase 2.5L (manufactured by Novozyme), a protease, was added so that the liquid concentration was 300 ppm, and then 30% sodium hydroxide was added at 50 ° C., and the suspension was maintained for 2 hours while adjusting the pH to 8.5.

[0120] (Alkali Treatment) Sodium dodecyl sulfate (SDS, manufactured by Kao) was added to the enzyme-treated solution to a concentration of 0.3 wt %. Then, the pH was 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 2x 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.

[0121] (pH Adjustment) The solid content 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 3.5. The liquid density was 1.00 g / mL.

[0122] (Filter press filtration and through-washing) 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. Compression was performed at a pressure of 0.4 MPa to obtain a filter cake, which was then thoroughly washed with wash water until the pH of the filter cake reached 5.07 and the ratio (amount of wash water used) / (amount of polyhydroxyalkanoate aqueous suspension supplied to the filter chamber of the filter press) reached 0.91. After washing, compression was performed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa, followed by air blowing for 20 minutes to obtain a filter cake. The pH and electrical conductivity of the washed filtrate are shown in Figure 1. The physical properties of the obtained filter cake are shown in Table 1. The water content of the filter cake was 13.5 wt% (W.B.).

[0123] Comparative Example 1 A filter cake was obtained in the same manner as in Example 1, except that through-cleaning was not performed. The physical properties of the obtained filter cake are shown in Table 1. The water content of the filter cake was 11.5 wt % (W.B.).

[0124] As shown in Table 1, Example 1, which was washed, had a lower YI and higher thermal stability than Comparative Example 1, which was not washed. FIG. 1 is a graph showing the changes in pH and electrical conductivity of the washing filtrate versus the ratio of the amount of washing water used to the amount of PHA aqueous suspension supplied. FIG. 1 shows that the pH of the washing filtrate increases and the electrical conductivity decreases as the amount of washing water used increases. Note that "stock solution" in FIG. 1 refers to the PHA aqueous suspension. The results shown in FIG. 1 show that the solvent derived from the PHA aqueous suspension contained in the PHA filter cake is replaced by washing water as washing continues.

[0125] From the above, it was demonstrated that the thermal stability of the resulting PHA was improved by this production method, and it was also found that this effect was due to the removal of the solvent in the PHA filter cake by washing.

[0126] [Example 2] A filter cake was obtained in the same manner as in Example 1, except that the through-washing step was carried out until the pH of the filter cake reached 4.3 and the ratio (amount of washing water used) / (amount of polyhydroxyalkanoate aqueous suspension supplied to the filter chamber of the filter press filter) reached 0.25, or until the pH of the filter cake reached 5.1 and the ratio (amount of washing water used) / (amount of polyhydroxyalkanoate aqueous suspension supplied to the filter chamber of the filter press filter) reached 0.91. The water content of the filter cake was 12.9 wt% (W.B.). Table 2 and Figure 2 show the YI and thermal stability of the obtained filter cake.

[0127] Comparative Examples 2-1 to 2-4: The same procedures as in Example 1 were carried out up to the pH adjustment, but the filter press filtration step and the through-washing step were not carried out, and all of the water contained in the PHA aqueous suspension was evaporated to obtain a PHA slurry. Table 2 and Figure 2 show the YI and thermal stability of the obtained PHA slurry.

[0128] 2, the PHAs of Examples 1 and 2 produced by this production method had higher thermal stability than those of Comparative Examples 2-3 to 2-4, which did not undergo the filter press filtration step and the through-washing step. Furthermore, the PHAs of Examples 1 and 2 had lower YIs than those of Comparative Examples 2-1 to 2-4. Comparative Example 2-1 has thermal stability comparable to that of Examples 1 and 2, but because Comparative Example 2-1 has a low pH of 3.8, corrosion-resistant equipment is required for production.

[0129] Example 3 The PHA agglomerates obtained in Example 1 were placed in a dryer (EYELA, WFO-700) and dried at 60°C for 24 hours. The dried PHA agglomerates were redispersed in water to adjust the solid content 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.

[0130] [Summary] From the above, it was found that according to the present production method, PHA with good thermal stability can be obtained even in a pH range of 4.0 or more, which does not require the use of a corrosion-resistant device.

[0131] The present method can be advantageously used in the production of PHAs because it can produce PHAs with good thermal stability in a pH range that does not require the use of corrosion-resistant equipment. Furthermore, the PHA agglomerates can be suitably used in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing, packaging, automobiles, building materials, and other fields.

Claims

1. A method for producing polyhydroxyalkanoate, comprising a filter press filtration step and a through-washing step, The filter press filtration step includes a squeezing step of supplying an aqueous suspension of polyhydroxyalkanoate having a pH of 2.5 or more and less than 4.0 to a filter press filter and squeezing the aqueous suspension, The production method, wherein the through-washing step includes a step of through-washing the filter cake obtained in the squeezing step until the pH of the filter cake becomes 4.0 to 5.

5.

2. The method according to claim 1 , wherein the filter press filtration step further comprises an air blowing step.

3. The production method according to claim 2, wherein in the filter press filtration step and the through-washing step, a first squeezing step, a through-washing step, a second squeezing step, and an air blowing step are carried out in this order.

4. 3. The method according to claim 1, wherein in the through-washing step, the ratio of (amount of washing water used) / (amount of aqueous suspension of polyhydroxyalkanoate supplied to a filtration chamber of the filter press filter) is 0.1 to 1.

0.

5. In the filter press filtration step, the air permeability is 0.1 to 2.5 cm ３ / cm ２ 3. The method according to claim 1, wherein a filter cloth having a flow rate of 100 / min is used.

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

7. 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.

8. The method according to claim 7, further comprising, after the step (a), a step (b) of recovering the aqueous suspension of polyhydroxyalkanoate by centrifugation.

9. The method according to claim 1 or 2, further comprising a step of drying the polyhydroxyalkanoate obtained by the filter press filtration step and the through-washing step at 20 to 100°C.

10. The method according to claim 1 or 2, wherein the pressure in the squeezing step is 0.2 to 1.0 MPa.

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

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