Method for producing polyhydroxyalkanoate and use thereof

Abstract

The purpose of the present invention is to provide a method for producing PHA having a low water content. The problem is solved by providing a method for producing PHA, the method comprising a step in which an aqueous PHA suspension having a pH of 2.5-5.5 is press-filtered using a filter material having an air permeability of 0.1-2.5 cm < 3 > / cm < 2 > / min, the liquid density of the aqueous PHA suspension in the filtration step being 0.50-1.08 g / mL, and the press-filtering step comprising a pressing step and an air supply step.

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 cells of microorganisms. Therefore, in order to use PHA as plastic, a process of separating and purifying PHA from the cells of microorganisms is required. In the process of separating and purifying PHA, the cells of microorganisms containing PHA are destroyed or microorganism-derived 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.

[0004] As a method for producing PHA using filtration, for example, Patent Document 1 discloses a method for producing PHA, which includes: a step of inoculating a PHA fermentation strain on a fermentation medium to ferment it; a step of separating the fermentation liquid into solid and liquid to obtain a fermentation supernatant and a bacterial precipitate; and a step of destroying the cell wall of the bacterial precipitate and performing plate and frame filtration using a filter pre-coated with a cell wall destroying agent to obtain PHA.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Chinese Patent No. 111500650 Summary of the invention

[0008] Problems to be solved by the invention

[0009] However, the PHA obtained by the above-mentioned technique has a high water content, and energy is required to volatilize the water thereafter, so there is room for further improvement.

[0010] Therefore, an object of the present invention is to provide a method for producing PHA having a low water content, and an aggregate of PHA having a low water content.

[0011] Solutions to the problem

[0012] In order to solve the above-mentioned problems, the inventors have conducted intensive research and found a new insight: in the production of PHA, by controlling the pH of the PHA aqueous suspension, the air permeability of the filter material, and the liquid density of the PHA aqueous suspension and performing a specific filtration process, PHA with a low water content can be obtained, thereby completing the present invention.

[0013] Therefore, one embodiment of the present invention is a method for producing PHA (hereinafter referred to as "the present production method"), which comprises: treating a PHA aqueous suspension having a pH of 2.5 to 5.5 with an aeration rate of 0.1 to 2.5 cm 3 / cm 2 / min of filter material for filter pressure filtration, in which the liquid density of the PHA aqueous suspension is 0.50-1.08 g / mL, and the filter pressure filtration step includes a pressing step and an air supply step.

[0014] 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").

[0015] Effects of the Invention

[0016] According to one embodiment of the present invention, a method for producing PHA having a low water content and a PHA aggregate having a low water content can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing photographs of filter cloth filtrates of Example 1-1 and Comparative Example 2 according to one embodiment of the present invention. DETAILED DESCRIPTION

[0018] 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.

[0019] [1. Summary of the Invention]

[0020] The particle size of PHA produced in the fungus is about 1 to 2 μm, so there is a problem of difficulty in filtering. In addition, centrifugal separation is mainly used for the recovery of PHA, but the separation form is an aqueous suspension, and PHA is recovered in a state containing a large amount of water. In order to separate and recover PHA from water, the water in the aqueous suspension must be evaporated, which has the problem of requiring a large amount of energy. Therefore, as a method for producing PHA using filtration, for example, the above-mentioned patent document 1 is known. It is known that in the technology of patent document 1, the filter material is pre-coated, and filtration is performed before purification (in a state where a large amount of biological-derived residue remains), so there are problems such as complicated operation and a large number of impurities in the obtained PHA agglomerates. In addition, it is known that in the technology of patent document 1, there is also a problem that the water content is not sufficiently reduced.

[0021] Therefore, in the production of PHA, the present inventors have conducted intensive research from the perspective of reducing the water content of PHA. By controlling the pH of the PHA aqueous suspension, the air permeability of the filter material, and the liquid density of the PHA aqueous suspension, and performing a specific filtration process, they finally discovered for the first time that PHA with a low water content can be obtained. Specifically, a technology has been developed to adjust the pH of the PHA aqueous suspension to a pH range where PHA agglomerates, and to recover the agglomerated PHA by filtration. This production method can provide PHA with a low water content. In addition, this production method can produce PHA with a low water content without adding impurities such as filter aids, so impurities can be reduced. Therefore, this production method is very advantageous in the industrial production of PHA.

[0022] In addition, according to the above scheme, the required heat, time and cost, i.e., energy, can be reduced in the drying process after filtration. Therefore, it is possible to contribute to the realization of the Sustainable Development Goals (SDGs), such as Goal 7 "Ensure access to affordable, reliable and sustainable modern energy for all." The present invention is described in detail below.

[0023] [2. Method for producing PHA]

[0024] This manufacturing method comprises the following steps:

[0025] ■Contains a PHA aqueous suspension with a pH of 2.5 to 5.5 and an aeration of 0.1 to 2.5 cm 3 / cm 2 / min, the filter press filtration step (hereinafter referred to as step (d)) comprises a pressing step and an air supply step, and the liquid density of the polyhydroxyalkanoate aqueous suspension in the filtration step is 0.50 to 1.08 g / mL.

[0026] 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).

[0027] ■ 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.

[0028] ■Step (b): After the above-mentioned step (a), a step of recovering the PHA aqueous suspension by centrifugal separation (also referred to as "recovery step").

[0029] ■Step (c'): A step of adjusting the pH of the PHA aqueous suspension obtained in the above step (b) to 2.5 to 5.5 (also referred to as "adjustment step").

[0030] ■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”).

[0031] ■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”).

[0032] ■Step (f): A step of redispersing the dried PHA in an aqueous solvent (also referred to as a "redispersion step").

[0033] 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".

[0034] (Step (d))

[0035] First, the step (d) which is a characteristic feature of the present invention will be described.

[0036] In step (d) of the present production method, the PHA aqueous suspension having a pH of 2.5 to 5.5 is treated with an aeration rate of 0.1 to 2.5 cm 3 / cm 2 / min filter material for filter press filtration. In step (d), the liquid density of the PHA aqueous suspension in the above filtration step is 0.50 to 1.08 g / mL. In addition, in step (d), the above filter press filtration step includes a pressing step and an air supply step. PHA with a low water content can be obtained by step (d).

[0037] The filter press filtration step of step (d) includes a pressing step and 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 above-mentioned PHA cake by air supply. Due to the two-stage dehydration process of the pressing step and the air supply step, a PHA agglomerate with a low moisture content can be obtained.

[0038] In one embodiment of the present invention, the gas supply process may be a through-gas supply process.

[0039] 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.

[0040] The pressure in the pressing step is preferably 0.2 to 1.0 MPa, more preferably 0.25 to 0.9 MPa, and even more preferably 0.3 to 0.8 MPa. The pressure in the pressing step is 0.2 to 1.0 MPa, which has the advantage of reducing the water content of the PHA aggregate.

[0041] The gas supply pressure in the gas supply 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 gas supply pressure is within the above range, there is an advantage in that the water content of the PHA aggregate is reduced.

[0042] The gas supply time can be appropriately set according to the gas supply pressure, and is, for example, 1 to 50 minutes, preferably 5 to 40 minutes.

[0043] The apparatus for performing filter press filtration is not particularly limited, and any known apparatus can be used.

[0044] 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.

[0045] 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 filtration step of the present production method is measured by the method described in the Examples.

[0046] In step (d), the liquid density of the PHA aqueous suspension is 0.50 to 1.08 g / mL, preferably 0.60 to 1.05 g / mL, more preferably 0.70 to 1.03 g / mL, and further 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 it has the advantage of lowering the water content in the PHA agglomerates. 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 inclusion of air, and the viscosity increases due to the interaction between air and 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.

[0047] The solid content concentration of the filtrate in step (d) is preferably 1000 mg / L or less, more preferably 500 mg / L or less, further preferably 200 mg / L or less, and particularly preferably 100 mg / L or less. When the solid content concentration of the filtrate is within the above range, it has the advantage of high recovery rate of PHA agglomerates. In addition, there is no particular limitation on the lower limit, for example, it can also be 0 mg / mL. It should be noted that the solid content concentration of the filtrate in the filtration step of the present manufacturing method is measured by the method described in the examples.

[0048] 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 advantages of eliminating the complexity of operation and reducing the amount of impurities in PHA.

[0049] 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 viscosity and the increase in particle size caused by the temperature increase.

[0050] 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.

[0051] In addition, in step (d), the description of the later-mentioned step (c') is used with respect to "pH".

[0052] (Step (a))

[0053] In step (a) of the present production method, cell-derived components other than PHA in the PHA-containing bacterial cells are disrupted and dissolved. By disrupting and removing the cell-derived impurities (cell wall, protein, etc.) 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.

[0054] <pha>

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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 after step (a). Therefore, the volume median particle size can be measured in any step of steps (a) to (d).

[0062] <Bacteria (microorganisms)>

[0063] 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.

[0064] 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.

[0065] <Destruction and dissolution of cell-derived components>

[0066] 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.

[0067] 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).

[0068] In the present specification, “lysozyme” refers to an enzyme having an activity of decomposing (lysing) the cell wall (eg, peptidoglycan) of bacteria.

[0069] 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.

[0070] As lysozyme, commercially available products can be used, and examples thereof include "Lysozyme" and "Achromopeptidase" manufactured by Fuji Film & Wako Pure Chemical Industries, Ltd.

[0071] 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.

[0072] 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.

[0073] 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).

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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, cell-derived components, compounds produced during purification, etc., as long as the essence of the present invention is not impaired.

[0081] 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.

[0082] (Other processes)

[0083] In one embodiment of the present invention, the production method may include the following steps before step (a).

[0084] <Step (a1)>

[0085] Step (a1) is a step of culturing bacterial cells containing PHA.

[0086] In step (a1), the bacterial cells that can be used include, for example, those described in the above-mentioned <Bacterial cells (microorganisms)>.

[0087] 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.

[0088] <Step (a2)>

[0089] 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.

[0090] 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.

[0091] <Step (a3)>

[0092] 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).

[0093] In step (a3), the method for adjusting the concentration and pH of the inactivated culture solution is 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.

[0094] (Step (b))

[0095] After the above step (a), the step (b) of the present production method is to recover the PHA aqueous suspension by centrifugal separation. The above cell-derived impurities (cell wall, protein, etc.) in the PHA aqueous suspension can be removed by the step (b).

[0096] 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.

[0097] 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.

[0098] 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 PHA surface-attached protein in the PHA aqueous suspension can be cited. For each unit weight of PHA, the protein amount is preferably less than 2000ppm, more preferably less than 1900ppm, further preferably less than 1800ppm, and most preferably less than 1700ppm. When the amount of PHA surface-attached protein 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 PHA surface is small, PHAs tend to aggregate with each other.

[0099] (Process (c'))

[0100] 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 to 5.5. By performing the pH adjustment in step (c'), the leakage rate in the filtration in step (d) is reduced.

[0101] In step (c'), the pH of the PHA aqueous suspension is 2.5 to 5.5, preferably 2.6 to 5.0, more preferably 2.7 to 4.5, further preferably 2.8 to 4.0, and particularly preferably 3.0 to 3.8. 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 PHA does not become too small and is easy to coagulate. In addition, with regard to the upper limit of pH, from the viewpoint of reducing coloration when heating and molten PHA, and from the viewpoint of ensuring the stability of the molecular weight of PHA during heating and / or drying, it is preferably pH 5.5 or less. 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.

[0102] 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.

[0103] 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.

[0104] 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).

[0105] (Step (c))

[0106] In step (c) of the present production method, the PHA aqueous suspension is heat-treated to a temperature of 60 to 120° C. The filtrate permeation rate during filtration can be increased by step (c).

[0107] 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.

[0108] 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.

[0109] (Step (e))

[0110] 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).

[0111] 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.

[0112] (Process (f))

[0113] 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.

[0114] In step (f), the redispersion method is not particularly limited and may be carried out by any method used in the relevant technical field.

[0115] 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 1.0 to 3.0 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.

[0116] 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.

[0117] [3. PHA aggregate]

[0118] The moisture content of the PHA agglomerate is 5.0 to 25.0% (WB). The PHA agglomerate is sometimes referred to as "PHA cake", "filter cake" or "PHA filter cake".

[0119] 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.

[0120] In one embodiment of the present invention, the present PHA aggregate is produced by the present production method.

[0121] 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.

[0122] 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.

[0123] The present invention is not limited to the above-mentioned embodiments, and various modifications can be made within the scope of the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments are also included in the technical scope of the present invention. That is, one embodiment of the present invention includes the following.

[0124] <1> A method for producing PHA, the method comprising:

[0125] The PHA aqueous suspension with a pH of 2.5 to 5.5 was aerated with a flow rate of 0.1 to 2.5 cm 3 / cm 2 / min filter material for filter press filtration,

[0126] Wherein, the liquid density of the polyhydroxyalkanoate aqueous suspension in the filtering step is 0.50 to 1.08 g / mL,

[0127] The filter pressing and filtering process includes a pressing process and an air supply process.

[0128] <2> according to <1> The manufacturing method, wherein

[0129] In the filtration step, the solid content concentration of the filtrate is 1000 mg / L or less.

[0130] <3> according to <1> or <2> The manufacturing method, wherein

[0131] The filter material is not pre-coated.

[0132] <4> according to <1> ~ <3> The manufacturing method described in any one of the above, wherein

[0133] In the filtering step, the temperature of the PHA aqueous suspension is 20 to 95°C.

[0134] <5> according to <1> ~ <4> The manufacturing method described in any one of the above further comprises:

[0135] (a) a step of disrupting and solubilizing cell-derived components other than PHA in PHA-containing bacterial cells,

[0136] In the step (a), the volume median particle size of the polyhydroxyalkanoate is 0.5 to 5.0 μm.

[0137] <6> according to <5> The manufacturing method further comprises, after step (a),

[0138] (b) A step of recovering the PHA aqueous suspension by centrifugal separation.

[0139] <7> according to <1> ~ <6> The manufacturing method described in any one of the above comprises:

[0140] A step of drying the polyhydroxyalkanoate obtained in the filtration step at 20 to 100°C.

[0141] <8> according to <1> ~ <7> The manufacturing method described in any one of the above, wherein

[0142] In the gas supply step, the gas supply pressure is 0.01 to 1.5 MPa.

[0143] <9> according to <1> ~ <8> The manufacturing method described in any one of the above, wherein

[0144] In the gas supply step, the gas supply time is 1 to 50 minutes.

[0145] <10> A method for preparing a polyhydroxyalkanoate aqueous suspension, the method comprising:

[0146] Will pass <1> ~ <9> A step of dispersing the polyhydroxyalkanoate produced by any one of the methods described in any one of the above in an aqueous solvent.

[0147] <11> A PHA agglomerate having a water content of 5.0 to 25.0% (WB).

[0148] Example

[0149] 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".

[0150] [Measurement method]

[0151] The measurements in Examples and Comparative Examples were performed by the following methods.

[0152] (Filtrate solid content concentration)

[0153] An appropriate amount of the filtrate was evacuated using a glass filter through glass filter paper GS-25 (manufactured by ADVANTEC Co., Ltd.) Then, the solid content concentration was calculated from the weight after the filter paper was dried.

[0154] (Water content of PHA agglomerate)

[0155] 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.

[0156] (Ventilation)

[0157] The air permeability was measured by the method described in JIS L 1096. Specifically, the air intake amount was adjusted using a Frazier air permeability tester (Permeameter P2, manufactured by Toyo Seiki Seisaku-sho, Ltd.) so that the inclined barometer indicated 125 Pa, and the air flow rate at that time was measured.

[0158] (Liquid Density)

[0159] The PHA aqueous suspension immediately before the filtration step was heated to the filtration temperature, and 20 mL of the suspension was drawn into a 20 mL plastic syringe (manufactured by Terumo Co., Ltd.) whose weight was measured in advance. Next, the weight of the plastic syringe and the 20 mL aqueous suspension was measured, the weight of the 20 mL plastic syringe was subtracted, and the obtained weight (g) was divided by the volume of the liquid (20 mL), thereby calculating the liquid density at the filtration temperature.

[0160] (Volume median particle size)

[0161] The volume median particle size of PHA was measured using a laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA.

[0162] (Filtration temperature)

[0163] The temperature during filtration was measured using a K thermocouple (AD5601A manufactured by AND Co., Ltd.) at a temperature immediately before the PHA aqueous suspension was placed in the filter.

[0164] (pH of PHA aqueous suspension in step (c'))

[0165] 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.

[0166] [Example 1]

[0167] (Preparation of bacterial culture fluid)

[0168] 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 Cupriavidus hookworms. The composition ratio of the repeating units of PHA (P3HB3HH) in the bacterial culture solution (composition ratio of 3HB unit / 3HH unit) is 94 / 6 (mol / mol).

[0169] (Inactivated)

[0170] 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.

[0171] (Viscosity reduction treatment)

[0172] 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 weight percentage 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 the 30% sodium hydroxide aqueous solution, thereby obtaining a PHA aqueous suspension.

[0173] (Enzyme treatment)

[0174] 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) was added as an enzyme for decomposing sugar chains (peptidoglycan) in the cell wall to a concentration of 10 ppm in the liquid, and the mixture was kept at 50°C for 2 hours. Thereafter, 2.5 L of alkaline protease (manufactured by Novozyme) was added as a proteolytic enzyme to a concentration of 300 ppm in the liquid, and then 30% sodium hydroxide was added at 50°C to adjust the pH to 8.5, and the mixture was kept at the same time for 2 hours.

[0175] (Alkali treatment)

[0176] Sodium dodecyl sulfate (SDS, Kao) was added to the enzyme treatment solution to make its weight percentage 0.3% by weight. 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: To the concentrated PHA aqueous suspension, an amount of sodium hydroxide equal to the removed supernatant was added, and the supernatant was removed by centrifugation again (4000G, 10 minutes). The volume median particle size of PHA is 2.2μm. The protein content in the obtained PHA aqueous suspension is 1000ppm.

[0177] (pH adjustment)

[0178] 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.

[0179] (filter)

[0180] 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.). It should be noted that the filter cloth used was not pre-coated. Filtration was performed at a pressing pressure of 0.7 MPa, and air was supplied at an air supply pressure of 0.2 to 0.6 MPa. Table 1 records the solid content concentration of the obtained filtrate and the water content of the PHA agglomerates. In addition, a photograph of the filter cloth filtrate of Example 1-1 is shown in Figure 1 .

[0181] [Comparative Example 1]

[0182] In the filter press filtration, a PHA agglomerate was obtained by the same method as in Example 1 except that air was not supplied. Table 1 shows the solid content concentration of the obtained filtrate and the water content of the PHA agglomerate.

[0183] [Table 1]

[0184]

[0185] [Example 2]

[0186] The same method as in Example 1 was used to perform the enzyme treatment until the enzyme treatment, and an enzyme treatment solution was obtained. SDS was added to the enzyme treatment solution until it reached 0.2% by weight. 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 twice: to the concentrated PHA aqueous suspension, an amount of sodium hydroxide equal to the removed supernatant was added, and the supernatant was removed again by centrifugation (4000G, 10 minutes). The protein content in the obtained PHA aqueous suspension was 3000ppm. The volume median particle size of PHA was 2.2μm. The pH adjustment process and the filtration process were carried out under the same conditions as in Example 1-2 to obtain a filter cake. The liquid density after the pH adjustment process was 1.00g / mL. The moisture content (WB) of the obtained cake was 13.1%, and the solid content concentration of the filter cloth filtrate was 10 mg / L or less.

[0187] [Example 3]

[0188] (Preparation of bacterial culture fluid)

[0189] The Bacillus eutrophus described in International Publication No. WO2019 / 142717 was cultured by the method described in paragraphs

[0041] to

[0048] of the document to obtain a bacterial culture solution containing PHA. It should be noted that the composition ratio of the repeating units of PHA in the bacterial culture solution (the composition ratio of 3HB unit / 3HH unit) is 80 / 20 to 88 / 12 (mol / mol), more precisely 85 / 15 (mol / mol). The obtained bacterial culture solution was filtered in the same manner as in Example 1, except that lysozyme was not added in the enzyme treatment step. It should be noted that the volume median particle size of PHA after the alkali treatment step is 2.3 μm. The amount of protein in the obtained PHA aqueous suspension is 1800 ppm. The liquid density after the pH adjustment step is 1.00 g / mL.

[0190] [Table 2]

[0191]

[0192] [Comparative Example 2]

[0193] In addition to using a ventilation rate of 3.0 cm 3 / cm 2 / min filter cloth (IP126C, manufactured by Ishigaki Co., Ltd.), filter press filtration was performed in the same manner as in Comparative Example 1. Figure 1 The photo of the filter cloth filtrate is shown in Figure 2. It can be seen that the air permeability is 1.0 cm 3 / cm 2 / min filter cloth does not allow particles to pass through, and the air permeability is 3.0cm 3 / cm 2 The filter cloth with a flow rate of 1000 rpm allowed particles to pass through. The solid content concentration of the filtrate was 2060 mg / L.

[0194] [Example 4]

[0195] The PHA agglomerates obtained in Example 1-3 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.

[0196] 〔Summarize〕

[0197] As described above, it is clear that the water content of the PHA aggregate can be greatly reduced by the present production method.

[0198] Industrial Applicability

[0199] The present production method can produce PHA with low water content by simple operation, and thus can be advantageously used in the production of PHA. In addition, the present PHA agglomerate can be suitably used in agriculture, fishery, forestry, gardening, 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: The polyhydroxyalkanoate aqueous suspension at pH 2.5 to 5.5 is prepared by using an aeration system of 0.1 to 2.5 cm 3 / cm 2 / min filter material for filter press filtration, Wherein, in the filtering step, the liquid density of the polyhydroxyalkanoate aqueous suspension is 0.50 to 1.08 g / mL, The filter pressing and filtering process includes a pressing process and an air supply process.

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

3. The manufacturing method according to claim 1 or 2, wherein: The filter material is not pre-coated.

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

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

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

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

8. The manufacturing method according to claim 1 or 2, wherein: In the gas supply step, the gas supply pressure is 0.01 to 1.5 MPa.

9. The manufacturing method according to claim 1 or 2, wherein: In the gas supply step, the gas supply time is 1 to 50 minutes.

10. 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.

11. A polyhydroxyalkanoate agglomerate having a water content of 5.0 to 25.0% (WB).

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