Method for producing polyhydroxyalkanoate and use thereof

Abstract

The present invention addresses the problem of providing a method with which PHA having good thermal stability can be achieved even in a pH range in which no corrosion-resistant device is used. The above-described problem is solved by a PHA production method comprising: a press filtration step including a press filtration step in which an aqueous suspension of a polyhydroxyalkanoate having a pH of 2.5 or more and less than 4.0 is supplied to a press filter and pressed, and a through-washing step in which the aqueous suspension of a polyhydroxyalkanoate having a pH of 2.5 or more and less than 4.0 is washed by washing the aqueous suspension of a polyhydroxyalkanoate having a pH of 2.5 or more and less than 4.0; the through-cleaning step includes a step for carrying out through-cleaning on the filter cake obtained in the pressing step until the pH of the filter cake reaches 4.0-5.5.

Description

Technical Field

[0001] The present invention relates to a method for producing polyhydroxyalkanoate and utilization thereof. Background Art

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

[0003] PHA produced by microorganisms accumulates in the microbial cells of the microorganisms. Therefore, in order to use PHA as a plastic, a process of separating / purifying PHA from the microbial cells is required. In the process of separating / purifying PHA, the microbial cells containing PHA are destroyed, or the biological components other than PHA are dissolved, and then PHA is taken out from the obtained aqueous suspension. At this time, separation operations such as centrifugation, filtration, and drying are performed. For the drying operation, a spray dryer, a fluidized bed dryer, a drum dryer, etc. can be used. From the perspective of ease of operation, a spray dryer is preferably used.

[0004] To date, in order to prevent PHA aggregation and viscosity increase in an aqueous suspension at a pH of less than 7, the present inventors have developed a technology in which an alkylene oxide-based dispersant is added before the pH of the aqueous suspension is adjusted to 7 or less, and then the obtained aqueous suspension at a pH of less than 7 is spray-dried (see Patent Document 1).

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

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2021 / 085534

[0009] Patent Document 2: U.S. Patent No. 2013 / 0093119 Summary of the invention

[0010] Problems to be solved by the invention

[0011] However, the above-mentioned techniques related to the method for producing PHA still have room for improvement.

[0012] Therefore, an object of the present invention is to provide a method for producing PHA having excellent thermal stability even in a pH range that does not require the use of a corrosion-resistant device.

[0013] Solutions to the problem

[0014] The present inventors have conducted intensive studies to solve the above problems and have found a new finding: in the production of PHA, by through-washing a PHA cake obtained by a filter press until the pH of the cake reaches a specific range, PHA with excellent thermal stability can be obtained.

[0015] Therefore, one embodiment of the present invention is a method for producing PHA (hereinafter referred to as "the present production method"), which includes a filter press filtration step and a through-cleaning step, wherein the filter press filtration step includes a pressing step of supplying a PHA aqueous suspension having a pH of greater than 2.5 and less than 4.0 to a filter press filter for pressing, and the through-cleaning step includes a step of through-cleaning a filter cake obtained by the pressing step until the pH of the filter cake reaches 4.0 to 5.5.

[0016] Furthermore, one embodiment of the present invention is a PHA aggregate having a water content of 5.0 to 25.0% (WB) (hereinafter referred to as "the present PHA aggregate"), which contains water at a pH of 4.0 to 5.5.

[0017] Effects of the Invention

[0018] According to one embodiment of the present invention, a method for producing PHA having good thermal stability can be provided, which method does not require the use of a corrosion-resistant device. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Graphs showing changes in pH and conductivity with respect to the ratio of the amount of wash water used and the amount of PHA aqueous suspension supplied in Examples of the present invention.

[0020] Figure 2 The graph shows the pH of the PHA aqueous suspension in the examples of the present invention and the evaluation of the thermal stability and YI of the obtained pH. DETAILED DESCRIPTION

[0021] Hereinafter, one embodiment of the present invention will be described in detail. It should be noted that in the present specification, "A to B" indicating a numerical range means "A or more and B or less" unless otherwise specified.

[0022] [1. Summary of the Invention]

[0023] As a method for producing PHA, a method of drying an aqueous suspension of PHA is known. In the method, in order to improve the thermal stability (molecular weight retention rate when heated) of PHA (in other words, to suppress the decomposition of PHA when heated), sulfuric acid or the like is used, and the pH of the aqueous suspension of PHA must be lowered (usually, pH 2.5 or more and less than 4.0). However, when using an aqueous suspension of PHA with a low pH, a corrosion-resistant device is required in the drying operation of the manufacturing process of PHA, and therefore, there is a problem of high device cost. On the other hand, when using an aqueous suspension of PHA with a pH in a range that does not require a corrosion-resistant device, there is a problem of reduced thermal stability of the resulting PHA.

[0024] Therefore, in order to provide a PHA production method that can produce PHA with good thermal stability even without using a corrosion-resistant device, the present inventors conducted intensive research and found for the first time that PHA with high thermal stability can be produced by pressing the PHA aqueous suspension using a filter press and through-washing without the need for a corrosion-resistant device.

[0025] As the mechanism of the present invention, the present inventors presume the following. That is, when a PHA aqueous suspension contains metal ions and the like, and such a PHA aqueous suspension is dried at a high pH (for example, above 4.0) without using a corrosion resistant device, the above-mentioned metal ions and the like remaining in the obtained PHA act as a catalyst. As a result, since these metal ions and the like promote the thermal decomposition of PHA, the thermal stability of PHA decreases. In contrast, in the present invention, by the above-mentioned pressing and through-cleaning, while processing the PHA aqueous suspension with a low pH into a PHA filter cake, the solvent containing metal ions and the like in the PHA filter cake is replaced with washing water that does not contain impurities such as metal ions that act as a thermal decomposition catalyst. Through this operation, the impurities in the PHA filter cake can be removed while the pH of the PHA filter cake can be adjusted to above 4.0 without the need for a corrosion resistant device. Therefore, the present invention can produce PHA with high thermal stability without the need for a corrosion resistant device.

[0026] In addition, in this specification, "PHA cake", "filter cake" or "PHA filter cake" refers to a solid composition containing PHA (PHA in the PHA aqueous suspension is agglomerated) with a moisture content of more than 25.0% and less than 50.0%, obtained by filtering or squeezing a PHA aqueous suspension. In addition, in this specification, "PHA agglomerate" refers to a solid composition containing PHA with a moisture content of 5.0 to 25.0% obtained by dehydrating a PHA cake by, for example, blowing air. In this specification, "PHA powder" refers to a composition containing PHA with a moisture content of less than 5.0%, obtained by, for example, drying a PHA agglomerate.

[0027] This manufacturing method can produce PHA with good thermal stability even in a pH range that does not require the use of a corrosion-resistant device, which is extremely beneficial for the production of PHA. In addition, based on the above invention, the amount of plastic waste generated can be reduced, which can be beneficial to the realization of sustainable development goals (SDGs) such as Goal 12 "Ensure sustainable consumption and production patterns" and Goal 14 "Protect and sustainably use the oceans and marine resources and promote sustainable development". The following describes the composition of this manufacturing method and this aqueous suspension.

[0028] [2. Method for producing PHA]

[0029] This manufacturing method comprises the following steps:

[0030] A squeezing step of supplying the aqueous suspension of polyhydroxyalkanoate having a pH of 2.5 or higher and lower than 4.0 to a filter press to perform squeezing; and a through-cleaning step of through-cleaning the filter cake obtained in the squeezing step (hereinafter referred to as step (d)).

[0031] Furthermore, in one embodiment of the present invention, the production method preferably includes at least one of the following steps in addition to the above step (d).

[0032] Step (a): A step of disrupting and solubilizing cell-derived components other than PHA in bacterial cells containing PHA (also referred to as a "solubilization step"), wherein the volume median particle size of the PHA is 0.5 to 5.0 μm.

[0033] Step (b): After the step (a), a step of recovering the PHA aqueous suspension by centrifugal separation (also referred to as a "recovery step").

[0034] Step (c'): A step of adjusting the pH of the PHA aqueous suspension obtained in the above step (b) to 2.5 or more and less than 4.0 (also referred to as a "preparation step").

[0035] Step (c): a step of heating the PHA aqueous suspension to a temperature of 60 to 120° C. (also referred to as a “heating step”).

[0036] Step (e): A step of drying the PHA obtained in the above step (d) at 20 to 100° C. (also referred to as a “drying step”).

[0037] Step (f): a step of redispersing the dried PHA in an aqueous solvent (also referred to as a "redispersion step").

[0038] In the present manufacturing method, the above-mentioned steps are preferably carried out in the order of steps (a), (b), (c'), (c), (d), (e), and (f), and the order may be appropriately changed according to the purpose. For example, the order of steps (a) and (b) may be changed to carry out in the order of steps (b) and (a); and the order of steps (c') and (c) may be changed to carry out in the order of steps (c) and (c'). In addition, depending on the purpose, steps (a), (b), (c'), and (c) may be carried out more than two times. That is, for example, the steps may be carried out in the order of steps (b), (a), (b), or (c'), (c), (c'). It should be noted that in the present specification, an aqueous suspension containing at least PHA is sometimes simply described as "PHA aqueous suspension".

[0039] (Step (d))

[0040] First, the step (d) which is a characteristic feature of the present invention will be described.

[0041] Step (d) of the present production method includes a filter press filtration step and a through-cleaning step. The filter press filtration step includes a squeezing step of supplying an aqueous suspension of polyhydroxyalkanoate having a pH of greater than 2.5 and less than 4.0 to a filter press filter for squeezing. In addition, the through-cleaning step includes a step of through-cleaning the filter cake obtained by the squeezing step until the pH of the filter cake reaches 4.0 to 5.5. PHA with high thermal stability can be obtained by step (d).

[0042] The pressure in the pressing 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. When the pressure in the pressing step is 0.2 to 1.0 MPa, the PHA cake can be washed well even with a small amount of washing water.

[0043] In the through-cleaning step, the filter cake is cleaned 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 more, no corrosion resistance device is required, especially in the air supply step described later. In addition, if the pH of the filter cake is 5.5 or less, the thermal stability of PHA is improved. In addition, since it is difficult to directly measure the pH of the filter cake in the through-cleaning step, the pH of the cleaning liquid (cleaning filtrate) discharged after cleaning the filter cake can be measured and regarded as the pH of the filter cake.

[0044] In the through-cleaning step, (the amount of washing water used) / (the amount of the polyhydroxyalkanoate aqueous suspension supplied to the filter chamber of the filter press) is 0.1 to 1.0, preferably 0.12 to 0.98, and more preferably 0.14 to 0.96. When the value is 0.1 or more, the pH of the PHA filter cake is 4.0 or more. In addition, when the value is 1.0 or less, the amount of washing water in the PHA filter cake is not too much, and a PHA agglomerate with a low water content can be obtained.

[0045] In one embodiment of the present invention, in addition to the pressing step, the filter pressing and filtering step of step (d) further includes an air supply step. In the pressing step, the PHA cake is squeezed out by pressing to squeeze out water from the PHA cake. In the air supply step, water is squeezed out of the PHA cake by air supply. Since the PHA cake undergoes two stages of dehydration steps, namely, the pressing step and the air supply step, a PHA agglomerate with a low moisture content can be obtained.

[0046] The air blowing pressure in the air blowing step is not particularly limited, and 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 aggregates is reduced.

[0047] The air blowing time can be appropriately set according to the air blowing pressure, and is, for example, 1 to 50 minutes, preferably 5 to 40 minutes.

[0048] In one embodiment of the present invention, the air supply process may be a through-air supply process.

[0049] In one embodiment of the present invention, the number of times and order of implementation of the filter press filtration step and the penetration cleaning step of step (d) are not particularly limited. For example, the penetration cleaning step can be performed after the filter press filtration step, or the filter press filtration step can be performed before and / or after the penetration cleaning step.

[0050] The filter press filtration step and the through-cleaning step of step (d) are preferably performed in the following order: the first pressing step, the through-cleaning step, the second pressing step, and the air supply step. By performing the through-cleaning step after the first pressing step, the washing water can easily pass through the PHA filter cake, and the washing efficiency is improved. In addition, by performing the second pressing step after the through-cleaning step, the washing water contained in the PHA filter cake can be fully discharged.

[0051] In the above embodiment, the pressure in the first pressing step is preferably 0.2 to 0.6 MPa, more preferably 0.25 to 0.55 MPa, and further preferably 0.3 to 0.5 MPa. In addition, the pressure in the second pressing step is preferably 0.4 to 1.0 MPa, more preferably 0.5 to 0.9 MPa, and further preferably 0.6 to 0.8 MPa. From the viewpoint of sufficiently removing the washing water, the pressure in the second pressing step is preferably higher than the pressure in the first pressing step.

[0052] In one embodiment of the present invention, the filter press filtration step may include a step of supplying a stock solution (PHA aqueous suspension) into a filtration chamber before the pressing step.

[0053] The apparatus for performing filter press filtration is not particularly limited, and any known apparatus can be used.

[0054] In this specification, the amount of water passing through the filter medium per unit area (cm 2 ) air volume (cm 3 ) is called air permeability. In step (d), the air permeability is 0.1 to 2.5 cm 3 / cm 2 / min, preferably 0.2 to 2.0 cm 3 / cm 2 / min, more preferably 0.3 to 1.8 cm 3 / cm 2 / min, more preferably 0.5 to 1.5 cm 3 / cm 2 / min, particularly preferably 0.8 to 1.2 cm 3 / cm 2 When the air permeability is within the above range, there is an advantage that the leakage rate of PHA to the filtrate is low. It should be noted that the air permeability in the pressing step of the present production method is measured by the method described in the Examples.

[0055] The filter material used in step (d) is not particularly limited, and can be selected from various raw materials such as paper, filter cloth (woven fabric, non-woven fabric), screen, sintered plate, bisque, polymer film, punched metal, wedge wire, etc. From the viewpoint of price and easy cleaning, filter cloth is preferably used, and filter cloth having the above-mentioned air permeability is more preferably used.

[0056] 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, further preferably 0.70 to 1.03 g / mL, and particularly preferably 0.80 to 1.02 g / mL. When the liquid density of the PHA aqueous suspension is within the above range, the filtrate permeation rate is high, and there is an advantage that the water content in the obtained PHA agglomerate is low. When the liquid density is low, the filtrate permeation rate decreases. It is speculated that this is because the viscosity of the PHA aqueous suspension increases due to the interaction between the air and the PHA. The liquid density of the PHA aqueous suspension can be adjusted, for example, by containing air. When the amount of air is increased, the liquid density of the PHA aqueous suspension decreases; when the amount of air is reduced, the liquid density of the PHA aqueous suspension increases.

[0057] The solid content concentration of the filtrate in step (d) is preferably below 1000mg / L, more preferably below 500mg / L, further preferably below 200mg / L, and particularly preferably below 100mg / L. When the solid content concentration of the filtrate is within the above range, there is an advantage that the filter cloth is not easily blocked. In addition, there is no particular limitation on the lower limit, for example, 0mg / mL is also possible. It should be noted that the solid content concentration of the filtrate is measured by the method described in the examples in this manufacture method.

[0058] In one embodiment of the present invention, the filter material is preferably not pre-coated. Since the filter material is not pre-coated, it has the advantage that impurities are not easily mixed into the obtained PHA.

[0059] In step (d), the temperature of the PHA aqueous suspension (temperature during filtration) is preferably 20 to 95° C., more preferably 25 to 90° C., further 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 is increased. The increase in the filtrate permeation rate is presumably due to the increase in the viscosity of the PHA aqueous suspension due to the increase in temperature and the increase in the particle size of the PHA particles.

[0060] In the present production method, when the heat treatment step (c) is included, the temperature of the PHA aqueous suspension during filtration is preferably 5°C or more lower, more preferably 8°C or more lower, further preferably 10°C or more lower, and particularly preferably 12°C or more lower than the temperature after the heat treatment step. When the temperature of the PHA aqueous suspension during filtration is within the above range, there is an advantage that 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, natural cooling, and the like.

[0061] (Step (a))

[0062] In step (a) of the present production method, cell-derived components other than PHA in PHA-containing bacterial cells are disrupted and dissolved. By disrupting and removing the impurities (cell wall, protein, etc.) derived from the bacterial cells in step (a), PHA having a volume median particle size of 0.5 to 5.0 μm can be efficiently recovered from the bacterial cells.

[0063] <pha>

[0064] In this specification, "PHA" is a general term for polymers having hydroxyalkanoic acid as a monomer unit. The hydroxyalkanoic acid constituting PHA is not particularly limited, and examples thereof include 3-hydroxybutyric acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, 3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, and 3-hydroxyoctanoic acid. These polymers may be homopolymers or copolymers containing two or more monomer units.

[0065] 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), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (P3HB3HV3HH). Among them, P3HB, P3HB3HH, P3HB3HV, and P3HB4HB are preferred from the viewpoint of easy industrial production.

[0066] In addition, by changing the composition ratio of the repeating units, the melting point and crystallinity can be changed, and as a result, the physical properties such as Young's modulus and heat resistance can be changed. Moreover, from the viewpoint that physical properties between those of polypropylene and polyethylene can be imparted and that it is easy to produce industrially and is a plastic useful in terms of physical properties as described above, P3HB3HH, which is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, is more preferred.

[0067] In one embodiment of the present invention, regarding the composition ratio of the repeating units of P3HB3HH, from the viewpoint of the balance between flexibility and strength, the composition ratio of 3-hydroxybutyrate unit / 3-hydroxyhexanoate unit is preferably 80 / 20 to 99.9 / 0.1 (mol / mol), and more preferably 85 / 15 to 97 / 3 (mol / mol). When the composition ratio of 3-hydroxybutyrate unit / 3-hydroxyhexanoate unit is 99.9 / 0.01 (mol / mol) or less, sufficient flexibility can be obtained; when it is 80 / 20 (mol / mol) or more, sufficient hardness can be obtained.

[0068] In step (a), the volume median particle size of PHA is preferably 50 times or less, more preferably 20 times or less, and even more preferably 10 times or less of the volume median particle size of the primary particles of the PHA (hereinafter referred to as "primary particle size"). By setting the volume median particle size of PHA to be 50 times or less of the primary particle size, the PHA aqueous suspension exhibits more excellent fluidity, and thus the productivity of PHA tends to be further improved.

[0069] In one embodiment of the present invention, for example, from the viewpoint of achieving excellent fluidity, the volume median particle size of 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. The volume median particle size of PHA was measured using a laser diffraction / scattering particle size distribution measuring apparatus LA-950 manufactured by HORIBA.

[0070] It should be noted that, for the sake of convenience, although the volume median particle size of PHA is specified in step (a), the volume median particle size is usually the same value in steps (a) to (d). Therefore, the volume median particle size can be measured in any step (a) to (d).

[0071] <Bacteria (microorganisms)>

[0072] The microorganism used in step (a) is not particularly limited, as long as it is a microorganism that can produce PHA in the cell. For example, microorganisms isolated from nature and microorganisms preserved in strain preservation institutions (e.g., IFO, ATCC, etc.), or mutants and transformants that can be prepared from them can be used. For example, the bacterial body of P3HB, which is an example of producing PHA, was first discovered in 1925 as Bacillus megaterium. Other natural microorganisms include: Cupriavidus necator (old classification: Alcaligenes eutrophus, Ralstonia eutropha), Alcaligenes latus, etc. It is known that these microorganisms accumulate PHA in the bacteria.

[0073] In addition, as an example of a bacterial cell that produces a copolymer of hydroxybutyrate and other hydroxyalkanoates, there can be mentioned Aeromonas caviae as a P3HB3HV and P3HB3HH producing bacteria, Alcaligenes eutrophus as a P3HB4HB producing bacteria, etc. In particular, regarding P3HB3HH, in order to improve the productivity of P3HB3HH, Alcaligenes eutrophus AC32 strain (Alcaligenes eutrophus AC32, FERM BP-6038) (T. Fukui, Y. Doi, J. Bateriol., 179, p4821-4830 (1997)) into which genes of the PHA synthase group are introduced is preferred. In addition to the above, the bacterial cell may also be a genetically modified microorganism into which various PHA synthesis-related genes are introduced according to the PHA to be produced.

[0074] <Destruction and dissolution of cell-derived components>

[0075] 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.

[0076] In one embodiment of the present invention, the above-mentioned destruction and solubilization are performed by using, for example, lysozyme or a protease (for example, alkaline protease).

[0077] In the present specification, “lysozyme” refers to an enzyme having an activity of decomposing (lysing) the cell wall (eg, peptidoglycan) of bacteria.

[0078] In one embodiment of the present invention, lysozyme is not particularly limited, for example, lysozyme, Labiase, β-N-acetylglucosaminidase, endolysin, autolysin, etc. can be mentioned. From the economically advantageous viewpoint, lysozyme is preferably used. These can be used alone or in combination of two or more.

[0079] As lysozyme, commercially available products can be used, and examples thereof include "Lysozyme" and "Achromopeptidase" manufactured by Fuji Film & Wako Pure Chemical Industries, Ltd.

[0080] In one embodiment of the present invention, the optimum pH of lysozyme is not particularly limited as long as the lysozyme has cell wall decomposing activity, and is, for example, 5.0 to 11.0, preferably 6.0 to 9.0, and more preferably 6.0 to 8.0.

[0081] In one embodiment of the present invention, the optimum temperature of lysozyme is not particularly limited, but is preferably 60° C. or less, and more preferably 50° C. or less, from the viewpoint of preventing thermal transition (thermal decomposition) of PHA without excessive temperature increase. The lower limit of the optimum temperature is not particularly limited, but is preferably room temperature (e.g., 25° C.) or more from the viewpoint of not requiring excessive cooling operations and economic efficiency.

[0082] In the present specification, “alkaline protease” refers to a protease having an activity of degrading protein in an alkaline environment (for example, in a solution of pH 8.5).

[0083] In one embodiment of the present invention, alkaline protease is not particularly limited, as long as it has the activity of decomposing proteins under alkaline environment, for example, can be cited: serine-specific protease (for example, subtilisin, chymotrypsin, trypsin); cysteine-specific protease (for example, papain, bromelain, cathepsin); aspartate-specific protease (for example, pepsin, cathepsin D, HIV protease) etc. Consider from the economically advantageous viewpoint, preferably serine-specific protease, especially preferably subtilisin (for example, alkaline protease). These can be used alone or in combination of two or more.

[0084] As the alkaline protease, commercially available products can be used, for example, "Alkaline Protease 2.5L" manufactured by Novozyme; "Protin SD-AY10" and "Protease P" Amano "3SD" manufactured by Amano Enzyme Co., Ltd.; "Multifect PR6L" and "Optimase PR89L" manufactured by Danisco Japan Co., Ltd.; "Sumizyme MP" manufactured by Shin Nippon Chemical Co., Ltd.; "Delvolase" manufactured by DSM Japan Co., Ltd.; "Bioplase OP", "Bioplase SP-20FG" and "Bioplase SP-4FG" manufactured by Nagase ChemteX Co., Ltd.; "Orientase 22BF" manufactured by HBI Co., Ltd.; "Aroase XA-10" manufactured by Yakult Pharmaceutical Co., Ltd., etc.

[0085] In one embodiment of the present invention, the optimum pH of the alkaline protease is not particularly limited as long as the alkaline protease is active in an alkaline environment, and is, for example, 8.0 to 14.0, preferably 8.0 to 12.0, more preferably 8.0 to 10.0, further preferably 8.0 to 9.0, and most preferably 8.5.

[0086] In one embodiment of the present invention, the optimum temperature of the alkaline protease is not particularly limited, and there is no need to increase the temperature excessively. From the viewpoint of being able to prevent thermal changes (thermal decomposition) of PHA, it is preferably 60° C. or less, and more preferably 50° C. or less. The lower limit of the optimum temperature is not particularly limited, but from the viewpoint of not requiring excessive cooling operations and being economical, it is preferably room temperature (e.g., 25° C.) or more.

[0087] In one embodiment of the present invention, in step (a), lysozyme and alkaline protease can be used in combination to disrupt and solubilize cell-derived components.

[0088] In step (a), the enzyme treatment time can be changed depending on the type of enzyme, pH, temperature and other conditions, and is, for example, 1 to 8 hours, preferably 2 to 6 hours.

[0089] It should be noted that the solvent ("solvent" is also referred to as "aqueous medium") constituting the aqueous suspension of PHA in the present production method may be water or a mixed solvent of water and an organic solvent. In addition, in the mixed solvent, the concentration of the organic solvent miscible with water is not particularly limited, as long as it is less than the solubility of the organic solvent used in water. In addition, the organic solvent miscible with water 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, piperidine, and the like. Among them, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, propionitrile, etc. are preferred from the perspective of easy removal. In addition, methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone, etc. are more preferred from the perspective of easy availability. Furthermore, methanol, ethanol, and acetone are particularly preferred. It should be noted that the aqueous medium constituting the PHA aqueous suspension may also contain other solvents, bacterial cell-derived components, compounds produced during purification, etc., as long as the essence of the present invention is not impaired.

[0090] In the present production method, the aqueous medium constituting the PHA aqueous suspension preferably contains water. The content of water in the aqueous medium is preferably 5 wt % or more, more preferably 10 wt % or more, further preferably 30 wt % or more, and particularly preferably 50 wt % or more.

[0091] (Other processes)

[0092] In one embodiment of the present invention, the production method may include the following steps before step (a).

[0093] <Step (a1)>

[0094] Step (a1) is a step of culturing bacterial cells containing PHA.

[0095] In step (a1), the bacterial cells that can be used include, for example, those described in the above-mentioned <Bacterial cells (microorganisms)>.

[0096] In step (a1), the method for culturing the bacteria is not particularly limited, and examples thereof include the methods described in paragraphs

[0041] to

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

[0097] <Step (a2)>

[0098] Step (a2) is a step of inactivating the bacterial cells obtained in the above step (a1). In this step, the bacterial cells obtained in the above step (a1) are inactivated to obtain an inactivated culture solution.

[0099] In step (a2), the inactivation method is not particularly limited, and an example thereof includes 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.

[0100] <Step (a3)>

[0101] Step (a3) ​​is a step of adjusting the concentration and pH of the inactivated culture solution obtained in the above step (a2). Step (a3) ​​is mainly performed when the viscosity of the inactivated culture solution obtained in the above step (a2) is high, and the viscosity of the inactivated culture solution is reduced by adjusting the concentration and pH of the inactivated culture solution. Step (a3) ​​facilitates the dissolution in step (a).

[0102] In step (a3), the method for adjusting the concentration and pH of the inactivated culture solution can be carried out by any method that can be used in the technical field, and is not particularly limited. For example, the concentration of the inactivated culture solution can be adjusted by adding hydrogen peroxide or the like to the inactivated culture solution. In addition, as a method for adjusting the pH, for example, a method of adding an alkaline compound to the inactivated culture solution can be cited. The alkaline compound is not particularly limited, but preferably an alkali metal hydroxide or an alkaline earth metal hydroxide, and more preferably sodium hydroxide. The alkaline compound can be used alone or in combination of two or more.

[0103] (Step (b))

[0104] After the above step (a), the step (b) of the present production method is to recover the PHA aqueous suspension by centrifugation. The above-mentioned bacterial cell-derived impurities (cell wall, protein, etc.) in the PHA aqueous suspension can be removed by the step (b).

[0105] In step (b), the PHA aqueous suspension can be recovered by any centrifugal separation method known in the art. The centrifugal separation method is not particularly limited, and examples thereof include centrifugal separation using a centrifugal sedimentation machine, a centrifugal dehydrator, and the like.

[0106] Examples of centrifugal sedimentation machines include separation plate type (e.g., disc type, self-cleaning type, nozzle type, spiral decanter type, scraping type, etc.), cylinder type, and decanter type centrifugal sedimentation machines. Depending on the method of discharging each precipitated component, there are batch type and continuous type. In addition, regarding centrifugal dehydrators, there are also batch type and continuous type. By using these devices, it is possible to separate the precipitate containing PHA and the culture solution component according to the difference in specific gravity.

[0107] The amount of impurities remaining in the final product can be roughly determined by steps (a) and (b), so it is preferred to minimize these impurities. Of course, depending on the application, impurities may be mixed in as long as the physical properties of the final product are not damaged, but in the case of medical applications requiring high-purity PHA, it is preferred to minimize these impurities. As an indicator of the degree of purification at this time, for example, the amount of protein attached to the surface of PHA in the PHA aqueous suspension can be cited. For each unit weight of PHA, the protein amount is less than 2000ppm, preferably less than 1900ppm, more preferably less than 1800ppm, and most preferably less than 1700ppm. When the amount of protein attached to the surface of PHA in the PHA aqueous suspension is within the above range, it has the advantage of preventing the leakage rate from becoming too high. It is speculated that this effect is due to the fact that when the amount of protein attached to the surface of PHA is small, PHAs tend to aggregate with each other.

[0108] (Process (c'))

[0109] In step (c'), the PHA aqueous suspension recovered by centrifugal separation generally has a pH exceeding 7. Therefore, in step (c') of the present production method, the pH of the PHA aqueous suspension obtained in step (b) is adjusted to 2.5 or more and less than 4.0. By performing the pH adjustment in step (c'), the leakage rate in the filtration in step (d) is reduced.

[0110] In step (c'), the pH of the PHA aqueous suspension is pH 2.5 or more and less than 4.0, preferably pH 2.6 to 3.9, more preferably 2.7 to 3.8, further preferably 2.8 to 3.7, and particularly preferably 2.9 to 3.6. When the pH of the PHA aqueous suspension is within the above range, it has the advantage of being able to increase the filtrate permeation rate of the filtration step without increasing the leakage rate of PHA into the filtrate. It is speculated that this effect is caused by the fact that the PHA particle size does not become too small and is easy to aggregate. In addition, with regard to the upper limit of pH, from the viewpoint of reducing coloration when heating and melting PHA, ensuring the stability of the molecular weight during heating and / or drying, and obtaining PHA with reduced coloration during heating and melting and suppressed molecular weight reduction during heating and / or drying, the pH is preferably lower than 4.0. With regard to the lower limit of pH, from the viewpoint of the acid resistance of the container, it is preferably pH 2.5 or more.

[0111] In step (c'), the pH adjustment method is not particularly limited, and for example, a method of adding an acid can be cited. The acid is not particularly limited, and can be any organic acid or inorganic acid, regardless of whether it is volatile. More specifically, as the acid, for example, sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, etc. can be used.

[0112] In step (c'), the PHA aqueous suspension is preferably heated. The heating temperature is not particularly limited, and is, for example, 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'), pH adjustment becomes easier, and thus PHA with high thermal stability can be obtained.

[0113] In one embodiment of the present invention, it is preferred that no additional pH adjustment is performed during the period from after the pH adjustment in step (c) to the step (d).

[0114] (Step (c))

[0115] In step (c) of the present production method, the PHA aqueous suspension is heat-treated to a temperature of 60 to 120° C. The step (c) can increase the permeation rate of the filtrate during filtration.

[0116] 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. When the temperature of the PHA aqueous suspension is within the above range, the filtrate permeation rate during filtration can be further increased.

[0117] In step (c), the method of heat treatment is not particularly limited, and examples thereof include: (i) a method of heating the container containing the PHA aqueous suspension with steam, (ii) a method of heating the container containing the PHA aqueous suspension with oil, (iii) a method of directly injecting steam into the PHA aqueous suspension, etc. The temperature of the steam in (i) and (iii) and the temperature of the oil in (ii) are not particularly limited, as long as the temperature of the PHA aqueous suspension in step (c) can be adjusted to 60 to 120°C, for example, 95 to 150°C.

[0118] (Step (e))

[0119] In step (e) of the present production method, the PHA obtained in step (d) is dried at 20 to 100° C. The water content can be adjusted by evaporating the water in the PHA aqueous suspension in step (e).

[0120] In step (e), the method of drying PHA is not particularly limited, and examples thereof include heating, vacuum drying, and room temperature drying. From the viewpoint of an appropriate drying speed, it is preferably dried by heating. 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.

[0121] (Process (f))

[0122] In step (f) of the present production method, the dried PHA is redispersed in an aqueous solvent to obtain a PHA aqueous suspension. By performing step (f) after 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.

[0123] In step (f), the redispersion method is not particularly limited and may be carried out by any method used in the relevant technical field.

[0124] In step (f), the volume median particle size of PHA is not particularly limited as long as it is substantially the same as the volume median particle size of PHA in step (a), and 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 the present specification, "substantially the same as the volume median particle size" means that the difference between the volume median particle size and the volume median particle size of PHA in step (a) is 1.0 μm or less.

[0125] The above steps (e) and (f) can be performed continuously. That is, the PHA dried in step (e) can be redispersed in step (f) to obtain an aqueous suspension of PHA. In one embodiment of the present invention, the production method includes: a step of drying the polyhydroxyalkanoate obtained by the above filtration step at 20 to 100°C, and a step of redispersing the dried polyhydroxyalkanoate in an aqueous solvent to obtain an aqueous suspension of polyhydroxyalkanoate containing polyhydroxyalkanoate having a volume median particle size of 0.5 to 5.0 μm.

[0126] [3. PHA aggregate]

[0127] The PHA aggregate contains water at a pH of 4.0 to 5.5 and a water content of 5.0 to 25.0% (WB).

[0128] The pH of the present PHA aggregate is 4.0 to 5.5, preferably 4.1 to 5.4, and more preferably 4.2 to 5.3.

[0129] The moisture content of the PHA agglomerate is 5.0 to 25.0% (WB), preferably 5.5 to 23.0% (WB), more preferably 6.0 to 21.0% (WB), further preferably 6.5 to 20.0% (WB), and particularly preferably 7.0 to 19.0% (WB). When the moisture content of the PHA agglomerate is within the above range, the PHA agglomerate is solid rather than slurry, and has the advantage of being easy to put into a dryer. It should be noted that the moisture content of the PHA agglomerate can be measured by the method described in the Examples.

[0130] In one embodiment of the present invention, the present PHA aggregate is produced by the present production method.

[0131] As long as the effects of the present invention can be achieved, the PHA aggregates may contain various components generated during the production process or not removed.

[0132] The PHA aggregate can be used for various purposes such as paper, film, sheet, tube, plate, rod, container (for example, bottle container), bag, and member.

[0133] The PHA agglomerate can be dried into a PHA powder by a known method. The thermal stability of the PHA powder obtained from the PHA agglomerate is preferably 70% or more, more preferably 75% or more, and further preferably 80% or more. When the thermal stability is 70% or more, the degradation of the resin obtained by processing the PHA powder can be suppressed. The higher the thermal stability, the better, and there is no particular upper limit, for example, it is 99% or less, and it can also be 100%. The above thermal stability is expressed by the following formula (2) based on the method described in the examples described later.

[0134] Thermal stability (%) = weight average molecular weight of polyhydroxyalkanoate sheet obtained by pressing polyhydroxyalkanoate powder at 160°C and 5 MPa for 20 minutes / weight average molecular weight of polyhydroxyalkanoate powder × 100 (2)

[0135] It is preferred that the coloration of the PHA powder is reduced when heated. The coloration degree of the PHA powder can be evaluated by the YI (yellowness) of the pressed tablets obtained by pressing the PHA powder. The lower the YI value of the pressed tablet, the lower the coloration of the PHA powder. In addition, a more specific method for evaluating the coloration degree of the PHA powder is described in the Examples.

[0136] The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims. Embodiments obtained by appropriately combining technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0137] That is, one embodiment of the present invention includes the following.

[0138] <1> A method for producing polyhydroxyalkanoate, comprising a filter press filtration step and a through-cleaning step.

[0139] The filter press filtration step includes a pressing step of supplying the polyhydroxyalkanoate aqueous suspension having a pH of 2.5 or more and less than 4.0 to a filter press for pressing.

[0140] 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 reaches 4.0 to 5.5.

[0141] <2> according to <1> The manufacturing method, wherein

[0142] The filter pressing and filtering process further includes an air supply process.

[0143] <3> according to <1> or <2> The manufacturing method, wherein

[0144] In the filter press filtration step and the penetration cleaning step, the first pressing step, the penetration cleaning step, the second pressing step, and the air supply step are performed in this order.

[0145] <4> according to <1> ~ <3> The manufacturing method described in any one of the above, wherein

[0146] In the penetration cleaning step, (the amount of cleaning water) / (the amount of the polyhydroxyalkanoate aqueous suspension supplied to the filter chamber of the filter press) is 0.1 to 1.0.

[0147] <5> according to <1> ~ <4> The manufacturing method described in any one of the above, wherein

[0148] In the filter press filtration process, the air permeability is 0.1 to 2.5 cm 3 / cm 2 / min filter cloth.

[0149] <6> according to <1> ~ <5> The manufacturing method described in any one of the above, wherein

[0150] In the pressing step, the temperature of the polyhydroxyalkanoate aqueous suspension is 20 to 95°C.

[0151] <7> according to <1> ~ <6> The manufacturing method described in any one of the above further comprises:

[0152] (a) a step of disrupting and solubilizing cell-derived components other than polyhydroxyalkanoate in bacterial cells containing polyhydroxyalkanoate,

[0153] In the step (a), the volume median particle size of the polyhydroxyalkanoate is 0.5 to 5.0 μm.

[0154] <8> according to <1> ~ <7> The manufacturing method described in any one of the above, further comprising, after the step (a):

[0155] (b) A step of recovering the polyhydroxyalkanoate aqueous suspension by centrifugal separation.

[0156] <9> according to <1> ~ <8> The manufacturing method described in any one of the above comprises:

[0157] A step of drying the polyhydroxyalkanoate obtained by the filter press filtration step at 20 to 100°C.

[0158] <10> according to <1> ~ <9> The manufacturing method described in any one of the above, wherein

[0159] The pressure of the pressing process is 0.2 to 1.0 MPa.

[0160] <11> according to <1> ~ <10> The manufacturing method described in any one of the above, wherein

[0161] In the air supply process, the air supply pressure is 0.01 to 1.5 MPa.

[0162] <12> A method for preparing a polyhydroxyalkanoate aqueous suspension, the method comprising:

[0163] Will pass <1> ~ <11> A step of redispersing the polyhydroxyalkanoate produced by any one of the methods described in any one of the above in an aqueous solvent.

[0164] <13> A polyhydroxyalkanoate aggregate containing water at a pH of 4.0 to 5.5 and a water content of 5.0 to 25.0% (WB).

[0165] <14> A polyhydroxyalkanoate powder, which is <13> The polyhydroxyalkanoate agglomerate is dried, and the polyhydroxyalkanoate powder has a thermal stability represented by the following formula (2) of 70% or more.

[0166] Thermal stability (%) = weight average molecular weight of polyhydroxyalkanoate sheet obtained by pressing polyhydroxyalkanoate powder at 160° C. and 5 MPa for 20 minutes / weight average molecular weight of polyhydroxyalkanoate powder × 100 (2).

[0167] Example

[0168] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples. It should be noted that in the examples, "P3HB3HH" is used as "PHA", and the "PHA" described in the examples can also be expressed as "P3HB3HH".

[0169] [Measurement method]

[0170] The measurements in Examples and Comparative Examples were performed by the following methods.

[0171] (Thermal stability of PHA powder)

[0172] As the evaluation sample, the PHA agglomerates obtained in the following examples and comparative examples were used. The PHA agglomerates were placed in a dryer (manufactured by 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 5MPa for 20 minutes to prepare PHA sheets. After dissolving 10 mg of the PHA sheet in 10 ml of chloroform, the insoluble matter was removed by filtration. The solution (filtrate) was subjected to molecular weight measurement using a GPC system manufactured by Shimadzu Corporation equipped with "Shodex K805L (300x8mm, 2 in series)" (manufactured by Showa Denko Co., Ltd.) using chloroform as the mobile phase. Commercially available standard polystyrene was used as the molecular weight standard sample. The molecular weight of the PHA powder was measured in the same order as above, except that no PHA sheet was prepared.

[0173] The thermal stability was evaluated based on the following formula (2):

[0174] Thermal stability (%) = weight average molecular weight of polyhydroxyalkanoate sheet obtained by pressing PHA powder at 160° C. and 5 MPa for 20 minutes / weight average molecular weight of polyhydroxyalkanoate powder × 100 (2).

[0175] (YI (Yellowness Index))

[0176] The pressed PHA resin tablets used as samples for the determination of the YI value were prepared according to the following method. 3.0 g of PHA powder was clamped between 15 cm square metal plates, and metal plates with a thickness of 0.5 mm were further inserted into the four corners of the metal plates. The tablets were placed in a small experimental press (H-15 model manufactured by Kobayashi Rika Co., Ltd.). After preheating at 160°C for 7 minutes, the tablets were pressed while being heated at 5 MPa for 2 minutes. After pressing, the tablets were left at room temperature to harden the PHA, and pressed PHA resin tablets were prepared. The YI value was measured using a colorimeter "SE-2000" (manufactured by Nippon Denshoku Co., Ltd.) using a 30 mm measuring plate.

[0177] (Water content of PHA agglomerate)

[0178] The dehydrated cake produced in the test was divided into 3 or 9 equal parts, and appropriate amounts were taken from each part. Then, it was dried in a constant temperature dryer at 105°C for about 15 hours, and the moisture content of the PHA aggregate was calculated from the difference in mass before and after drying.

[0179] (Solid content concentration)

[0180] The solid content concentration of the PHA aqueous suspension was measured using a heat-drying moisture meter ML-50 (manufactured by A&D Co., Ltd.) The PHA aqueous suspension was heated at 105°C until the weight change rate dropped to 0.05% / min, and the solid content concentration was calculated from the weight change of the PHA aqueous suspension before and after heating.

[0181] (Volume median particle size)

[0182] The volume median particle size of PHA was measured using a laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA.

[0183] (pH of PHA aqueous suspension in step (c'))

[0184] The measurement was performed using a pH meter (9652-10D manufactured by HORIBA). The pH was measured at the position of the PHA aqueous suspension farthest from the acid addition position while the PHA aqueous suspension was in a flowing state by a stirring blade, etc. For example, when the acid was added from the wall surface of the container, the pH at the center of the container was measured.

[0185] [Example 1]

[0186] (Preparation of bacterial culture fluid)

[0187] Alcaligenes eutrophus described in International Publication No. WO2019 / 142717 was cultured according to the method described in paragraphs

[0041] to

[0048] of the document to obtain a bacterial culture solution containing PHA. It should be noted that Alcaligenes eutrophus is currently classified as Copperworm.

[0188] (Inactivated)

[0189] The bacterial cell culture solution obtained above was sterilized by heating at an internal temperature of 60 to 70° C. and stirring for 7 hours to obtain an inactivated culture solution.

[0190] (Viscosity reduction treatment)

[0191] To the inactivated culture solution obtained above, 35 wt% hydrogen peroxide (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added to make the concentration 1 wt%. Then, a 30% sodium hydroxide aqueous solution was added to adjust the pH to 11.0. While the solution was kept at 60°C, the pH was kept at 11.0 for 180 minutes by continuously adding a 30% sodium hydroxide aqueous solution, thereby obtaining a PHA aqueous suspension.

[0192] (Enzyme treatment)

[0193] 95% sulfuric acid was added to the PHA aqueous suspension obtained above to adjust the pH to 7.0±0.2. The solid content concentration of the PHA aqueous suspension to which sulfuric acid was added was measured and found to be 30% by weight. After the addition of sulfuric acid, lysozyme (manufactured by Fuji Film and Wako Pure Chemical Industries, Ltd.) as an enzyme for decomposing sugar chains (peptidoglycan) in the cell wall was added to the PHA aqueous suspension to make its liquid concentration 10ppm, and it was kept at 50°C for 2 hours. Thereafter, 2.5L of alkaline protease (manufactured by Novozyme) as a proteolytic enzyme was added to make its liquid concentration 300ppm, and then 30% sodium hydroxide was added at 50°C to adjust the pH to 8.5, and it was kept at the same time for 2 hours.

[0194] (Alkali treatment)

[0195] Sodium dodecyl sulfate (SDS, Kao) was added to the enzyme treatment solution to make its weight percentage 0.3 wt%. Thereafter, the pH was adjusted to 11.0±0.2 using an aqueous sodium hydroxide solution. Next, the enzyme treatment solution was centrifuged (4000G, 10 minutes), and the supernatant was removed to obtain a 2-fold concentrated PHA aqueous suspension. The following operation was repeated 4 times: an amount of sodium hydroxide equal to the removed supernatant was added to the concentrated PHA aqueous suspension, and the solution was centrifuged again (4000G, 10 minutes) to remove the supernatant. The volume median particle size of PHA is 2.2 μm.

[0196] (pH adjustment)

[0197] The solid content concentration in the PHA aqueous suspension obtained above was adjusted to 25% by weight, and the suspension was 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.

[0198] (Pressure filtration and through cleaning)

[0199] The PHA aqueous suspension was added to a 63°C water bath, heated to a liquid temperature of 60°C, and filtered using a filter press (ISD model 360, manufactured by Ishigaki Corporation). A filter cloth with an air permeability of 1.0 cm 3 / cm 2 / min filter cloth (IP196C-1, manufactured by Ishigaki Co., Ltd.). After squeezing at a pressure of 0.4 MPa, a filter cake was obtained, which was then washed with washing water until the pH of the filter cake was 5.07 and (amount of washing water used) / (amount of polyhydroxyalkanoate aqueous suspension supplied to the filter chamber of the filter press) was 0.91. After washing, squeezing was performed again at a pressure of 0.7 MPa, and the air supply pressure was adjusted to 0.4 MPa, and air was supplied for 20 minutes to obtain a filter cake. In addition, the pH and conductivity of the washing filtrate were as follows Figure 1 The solid content concentration of the obtained filtrate and the physical properties of the PHA agglomerate filter cake are shown in Table 1. The water content of the filter cake was 13.5 wt% (WB).

[0200] [Comparative Example 1]

[0201] A filter cake was obtained in the same manner as in Example 1 except that the 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% (WB).

[0202] [Table 1]

[0203]

[0204] According to the description in Table 1, the YI of Example 1 in which cleaning was performed was lower and the thermal stability was higher than that of Comparative Example 1 in which cleaning was not performed. Figure 1 The graph shows the changes in pH and conductivity of the wash filtrate relative to the ratio of the wash water usage to the PHA aqueous suspension supply. Figure 1 As shown in the figure, as the amount of washing water used increases, the pH of the washing filtrate increases and the conductivity decreases. Figure 1 The "stock solution" here refers to the PHA aqueous suspension. Figure 1 As shown in the results, the solvent contained in the PHA filter cake from the PHA aqueous suspension was replaced with wash water by continuous washing.

[0205] As described above, the thermal stability of PHA obtained by the present production method is improved. This effect is due to the removal of the solvent in the PHA cake by washing.

[0206] [Example 2]

[0207] A filter cake was obtained in the same manner as in Example 1 except that the through-cleaning step was performed until the pH of the filter cake was 4.3 and the ratio of (the amount of washing water used) / (the amount of the aqueous suspension of polyhydroxyalkanoate supplied to the filter chamber of the filter press) was 0.25, or the pH of the filter cake was 5.1 and the ratio of (the amount of washing water used) / (the amount of the aqueous suspension of polyhydroxyalkanoate supplied to the filter chamber of the filter press) was 0.91. The water content of the filter cake was 12.9 wt% (WB). Table 2 and Figure 2 The YI and thermal stability of the resulting filter cake are shown.

[0208] [Comparative Examples 2-1 to 2-4]

[0209] The same operation as in Example 1 was performed until the pH was adjusted, without performing the filter press filtration step and the through-cleaning step, and all the water contained in the PHA aqueous suspension was volatilized, thereby obtaining a PHA slurry. Figure 2 The YI and thermal stability of the resulting PHA slurries are shown.

[0210] [Table 2]

[0211] pH[-] Thermal stability [%] YI[-] Example 1 5.1 82.1 14.3 Example 2 4.3 81.9 14.0 Comparative Example 2-1 3.8 82.2 22.2 Comparative Example 2-2 4.3 77.7 17.8 Comparative Example 2-3 4.7 48.9 18.1 Comparative Examples 2-4 5.0 32.1 16.9

[0212] According to Table 2 and Figure 2 As shown, the PHAs of Examples 1 and 2 produced by the present production method have improved thermal stability compared to Comparative Examples 2-3 to 2-4 in which the filter press filtration step and the through-cleaning step are not performed. In addition, the PHAs of Examples 1 and 2 have reduced YI compared to Comparative Examples 2-1 to 2-4. In addition, although Comparative Example 2-1 has the same degree of thermal stability as Examples 1 and 2, since the pH of Comparative Example 2-1 is as low as 3.8, a corrosion-resistant device is required during production.

[0213] [Example 3]

[0214] The PHA agglomerates obtained in Example 1 were added to a dryer (manufactured by EYELA, WFO-700) and dried at 60°C for 24 hours. The dried PHA agglomerates were redispersed in water and the solid content concentration was adjusted to 15% by weight. The pH was adjusted to between 7 and 9 using a 1% NaOH aqueous solution and a 1% H2SO4 aqueous solution, and stirred to prepare a PHA aqueous suspension. When the particle size of the PHA particles in the PHA aqueous suspension was measured after stirring for 30 minutes, the volume median particle size was 2.8 μm.

[0215] 〔Summarize〕

[0216] As described above, it is understood that the present production method can obtain PHA with excellent thermal stability even in a pH range of pH 4.0 or higher that does not require the use of a corrosion-resistant device.

[0217] Industrial Applicability

[0218] The present production method can produce PHA with good thermal stability even in a pH range that does not require the use of a corrosion-resistant device, and thus can be advantageously used in the production of PHA. In addition, the present PHA agglomerate can be suitably used in agriculture, fisheries, forestry, horticulture, medicine, sanitary products, clothing, non-clothing materials, packaging, automobiles, building materials, and other fields.< / pha>

Claims

1. A method for producing a polyhydroxyalkanoate, the method comprising a filter press filtration step and a through-cleaning step, The filter press filtration step includes a pressing step of supplying the polyhydroxyalkanoate aqueous suspension having a pH of 2.5 or more and less than 4.0 to a filter press for pressing. 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 reaches 4.0 to 5.

5.

2. The manufacturing method according to claim 1, wherein: The filter pressing and filtering process further includes an air supply process.

3. The manufacturing method according to claim 2, wherein: In the filter press filtration step and the penetration cleaning step, the first pressing step, the penetration cleaning step, the second pressing step, and the air supply step are performed in this order.

4. The manufacturing method according to claim 1 or 2, wherein: In the penetration cleaning step, (the amount of cleaning water) / (the amount of the polyhydroxyalkanoate aqueous suspension supplied to the filter chamber of the filter press) is 0.1 to 1.

0.

5. The manufacturing method according to claim 1 or 2, wherein: In the filter press filtration process, the air permeability is 0.1 to 2.5 cm. 3 / cm 2 / min filter cloth.

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

7. The manufacturing method according to claim 1 or 2, further comprising: (a) a step of disrupting and solubilizing cell-derived components other than polyhydroxyalkanoate in bacterial cells containing polyhydroxyalkanoate, In the step (a), the volume median particle size of the polyhydroxyalkanoate is 0.5 to 5.0 μm.

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

9. The manufacturing method according to claim 1 or 2, comprising: A step of drying the polyhydroxyalkanoate obtained in the filter press filtration step and the penetration washing step at 20 to 100°C.

10. The manufacturing method according to claim 1 or 2, wherein: The pressure of the pressing process is 0.2 to 1.0 MPa.

11. The manufacturing method according to claim 2, wherein: In the air supply process, the air supply pressure is 0.01 to 1.5 MPa.

12. A method for preparing an aqueous suspension of polyhydroxyalkanoate, the method comprising: A step of redispersing the polyhydroxyalkanoate produced by the method of claim 1 or 2 in an aqueous solvent.

13. A polyhydroxyalkanoate aggregate containing water at a pH of 4.0 to 5.5 and a water content of 5.0 to 25.0% (WB).

14. A polyhydroxyalkanoate powder obtained by drying the polyhydroxyalkanoate agglomerate according to claim 13, wherein the polyhydroxyalkanoate powder has a thermal stability represented by the following formula (2) of 70% or more. Thermal stability (%) = weight average molecular weight of polyhydroxyalkanoate sheet obtained by pressing polyhydroxyalkanoate powder at 160° C. and 5 MPa for 20 minutes / weight average molecular weight of polyhydroxyalkanoate powder × 100 (2).

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