Method for producing polyhydroxyalkanoate

Abstract

The present invention addresses the problem of providing a novel method for producing PHA with which it is possible to produce PHA having few impurities and a high degree of purification at a high yield without increasing the amount of wash water. The above-mentioned problem is solved by using a culture solution containing a thallus containing a PHB copolymer having a specific composition ratio, and by a production method comprising: (d) a step in which the culture solution is subjected to an enzyme treatment; (e) a step in which the thalli in the culture solution obtained in (d) are subjected to an enzyme treatment; (b) a step for adjusting the pH of the culture solution and adding a surfactant; and (f) a step for solid-liquid separation of the obtained aqueous suspension and recovery of a PHA-containing cake.

Description

Technical Field

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

[0002] It is known that polyhydroxyalkanoate (hereinafter sometimes referred to as "PHA") has biodegradability. PHA produced by microorganisms accumulates in the microbial cells. Therefore, in order to utilize PHA as a plastic, a step of separating / purifying PHA from the microbial cells is required. In the step of separating / purifying PHA, after dissolving the biological source components other than PHA, PHA is taken out from the obtained aqueous suspension. At this time, separation operations such as centrifugation, filtration, and drying are performed.

[0003] As a method for producing PHA, for example, the methods described in Patent Documents 1 to 3 are known. Patent Document 1 discloses a method for obtaining a polyhydroxybutyrate (PHB) copolymer with high purity by performing re-washing after spray drying. In addition, Patent Document 2 discloses a method for obtaining a PHB copolymer, which includes a step of subjecting the culture broth to disk centrifugation and then supplying it to a decanter. Patent Document 3 discloses a method for producing a PHA aqueous suspension with less impurities and excellent dispersibility from PHA with low crystallinity.

[0004] Prior Art Documents

[0005] Patent Documents

[0006] Patent Document 1: International Publication No. 2022 / 091685

[0007] Patent Document 2: Specification of Chinese Patent Application Publication No. 202111408282

[0008] Patent Document 3: Japanese Patent Application Laid-Open No. 2019-097518 Summary of the Invention

[0009] Problems to be Solved by the Invention

[0010] The methods described in Patent Documents 1 and 3 are excellent methods for obtaining a PHB copolymer with less impurities and high purity, but there is room for improvement from the viewpoint of reducing the amount of washing water (wastewater). In addition, in the method described in Patent Document 2, there is a problem of poor yield.

[0011] Therefore, the object of the present invention is to provide a new method for manufacturing PHA, which can manufacture PHA with few impurities and high purity in a high yield without increasing the amount of washing water (drainage). In particular, the object of the present invention is to provide a new method for manufacturing a polyhydroxybutyrate copolymer (hereinafter sometimes referred to as "PHB copolymer") in which the composition ratio of 3-hydroxybutyrate units (hereinafter sometimes referred to as "3HB") to hydroxyalkanoate units other than 3-hydroxybutyrate units is 80 / 20 to 88 / 12 (mol / mol).

[0012] Means for Solving the Problem

[0013] To solve the above problems, the present inventors conducted intensive studies and found that in the manufacturing process of PHA having a specific composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units, by including specific processes in a specific order and including a process of separation using a decanter centrifuge, it is possible to manufacture PHA with few impurities and high purity in a high yield without increasing the amount of washing water (drainage), thereby completing the present invention.

[0014] Therefore, one aspect of the present invention is a method for manufacturing PHA (hereinafter referred to as "the present manufacturing method"), which is a method for manufacturing PHA, wherein

[0015] the above-mentioned PHA is a PHB copolymer in which the composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units is 80 / 20 to 88 / 12 (mol / mol),

[0016] As the culture solution, a culture solution containing cells containing the above-mentioned polyhydroxyalkanoate is used,

[0017] including the following processes (d), (e), and (b);

[0018] (d) A process of subjecting the culture solution to enzymatic treatment by adding glycosidase,

[0019] (e) A process of subjecting the cells to enzymatic treatment by adding an alkaline protease to the culture solution obtained in the process (d),

[0020] (b) A process of adjusting the pH to 10.0 to 12.0 by adding an alkaline aqueous solution to the culture solution or the aqueous suspension obtained through the process (e), and adding a surfactant at any stage before, during, or after the adjustment; and

[0021] After the processes (d), (e), and (b), it includes:

[0022] (f) Step of subjecting the obtained aqueous suspension to solid-liquid separation using a decanter centrifuge and recovering the cake containing polyhydroxyalkanoate.

[0023] Effects of the Invention

[0024] According to one aspect of the present invention, there can be provided a new production method of PHA which can produce polyhydroxyalkanoate with few impurities and high purity at a high yield without increasing the amount of washing water (waste water). Detailed Description of the Invention

[0025] One embodiment of the present invention will be described in detail below. It should be noted that, in this specification, unless otherwise specified, the numerical range "A to B" means "A or more and B or less". In addition, all the documents described in this specification are incorporated herein by reference.

[0026] 〔1. Summary of the Present Invention〕

[0027] In the purification of a PHB copolymer having a specific composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units, it is difficult to obtain a PHB copolymer with high purity (i.e., low nitrogen content). In order to reduce the nitrogen content of the PHB copolymer, glycosidase treatment is required. However, if peptidoglycan is destroyed by a glycosidase such as lysozyme, the primary particles will irreversibly aggregate. Therefore, the operation of the PHB copolymer becomes difficult during the above enzyme treatment.

[0028] As described above, the method described in Patent Document 1 is an excellent technique for obtaining a PHB copolymer with few impurities and high purity, but since it is subjected to spray drying twice, the amount of washing water (waste water) increases, and there is room for improvement in this regard. In addition, there is room for improvement in the use of a dispersant. The method described in Patent Document 2 has a problem from an economic point of view because the yield is poor.

[0029] Therefore, in order to solve the above problems, the present inventors conducted intensive studies and as a result, for the first time, found that: in the production process of PHA having a specific composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units, by including a step of separating using a decanter centrifuge, it is possible to produce PHA with few impurities and high purity at a high yield without increasing the amount of washing water (waste water).

[0030] Therefore, according to this production method, it is possible to produce PHA (in particular, PHA having a specific composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units) with few impurities and high purity at a high yield without increasing the amount of washing water (waste water).

[0031] In addition, based on the above-described configuration, the amount of plastic waste generated can be reduced. Thus, for example, it can contribute to the achievement of sustainable development goals (SDGs) such as Goal 12, "Ensure sustainable consumption and production patterns," and Goal 14, "Conserve and sustainably use the oceans, seas and marine resources for sustainable development." Hereinafter, the configuration of this manufacturing method will be described in detail.

[0032] 〔2. Manufacturing method of PHA〕

[0033] This manufacturing method is a method that includes the following steps (d), (e), (b), and (f) as essential steps. In addition, the PHA in this manufacturing method is a PHB copolymer in which the composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units is 80 / 20 to 88 / 12 (mol / mol) (hereinafter, sometimes referred to as "specific PHB copolymer").

[0034] ･ Step (d): A step of performing enzymatic treatment by adding glycosidase to the culture solution

[0035] ･ Step (e): A step of adding an alkaline protease to the culture solution obtained in the above step (d) and performing enzymatic treatment on the cells

[0036] ･ Step (b): A step of adding an alkaline aqueous solution to the culture solution or the aqueous suspension obtained through step (e) to adjust the pH to 10.0 to 12.0, and adding a surfactant at any stage before, during, or after this adjustment

[0037] ･ Step (f): A step of solid-liquid separating the obtained aqueous suspension by a decanter centrifuge and recovering the PHA-containing cake.

[0038] In this specification, the "culture solution" refers to a solution containing cells containing PHA as a specific PHB copolymer. In addition, the "aqueous suspension" refers to a solution containing the above PHA after treating the cells in the above culture solution with an alkaline protease. In other words, the above culture solution becomes an aqueous suspension after passing through step (e).

[0039] In this manufacturing method, the order of steps (d) and (e) is different from that of step (b). In one embodiment of the present invention, the order of steps (d) and (e) and step (b) can be steps (d), (e), and (b) in sequence, or can be steps (b), (d), and (e) in sequence. That is, step (e) can be carried out immediately after step (d), and step (b) can be before step (d) or after step (e). In an alkaline solution or in the presence of a surfactant, the activity of glycosidase sometimes decreases, and the order of steps (d), (e), and (b) is preferred.

[0040] In this production method, step (f) is performed after steps (d), (e), and (b). Performing step (f) after step (e) disrupts the cell walls of the bacterial cells in the aqueous suspension, allowing the primary particles of PHA to aggregate. This improves the yield of PHA during solid-liquid separation using a decanter centrifuge. Furthermore, performing step (f) after step (d) disrupts the cell membranes of the bacterial cells in the aqueous suspension, allowing the primary particles of PHA to aggregate sufficiently, and also reduces the nitrogen content of PHA.

[0041] In addition, in one embodiment of the present invention, the production method may further include the following steps.

[0042] ･Step (c): The step of separating the obtained aqueous suspension into solid and liquid using a disc centrifuge to recover the PHA aqueous suspension

[0043] ･Step (a): Enzyme treatment by adding alkaline protease

[0044] Step (g): A step of adjusting the pH of the polyhydroxyalkanoate aqueous suspension obtained by adding an aqueous solution to the PHA-containing cake obtained in step (f) above to a value of 2.5 to 4.0

[0045] Step (h): After the above step (g), the aqueous PHA suspension is dehydrated and dried using a filter press.

[0046] Step (c) is preferably performed before steps (d) and (e) and after step (b). In addition, step (a) is preferably performed before steps (d) and (e) and the treatment of step (b), and before step (f).

[0047] Furthermore, in one embodiment of the present invention, each step may be performed multiple times for various purposes.

[0048] More specifically, the present manufacturing method can be carried out according to the following process sequence, but is not limited thereto:

[0049] (1) Step (d), Step (e), Step (b), Step (f)

[0050] (2) Step (b), Step (d), Step (e), Step (f)

[0051] (3) Step (d), Step (e), Step (b), Step (f), Step (g), Step (h)

[0052] (4) Step (b), Step (d), Step (e), Step (f), Step (g), Step (h)

[0053] (5) Step (b), Step (c), Step (d), Step (e), Step (f), Step (g), Step (h)

[0054] (6) Step (a), Step (b), Step (c), Step (d), Step (e), Step (f), Step (g), Step (h)

[0055] (7) Step (b), Step (a), Step (c), Step (d), Step (e), Step (f), Step (g), Step (h)

[0056] (8) Step (d), Step (e), Step (b), Step (f), Step (a), Step (f), Step (g), Step (h)

[0057] (9) Step (a), Step (b), Step (c), Step (d), Step (e), Step (f)

[0058] For example, in the case of (8) above, the content of impurities and the amount of water discharged in the PHB copolymer can be reduced, and the drying process is performed once, which has the advantage of low energy required for production. In addition, no dispersant is used. In addition, in the case of (9) above, the content of impurities in the PHB copolymer can be further reduced compared to the above (1), and the total process is short, and the drying process is performed once. In addition, in the case of (6) above, since a filter press filtration process (including air supply) is performed, the moisture content before drying is lower than that of the PHB copolymer in (2) above.

[0059] For the sake of convenience, the steps below will be described in the order of steps (d), (e), (b), (f), (c), (a), (g), and (h).

[0060] (2-1. Step (d))

[0061] Step (d) is a step of adding glycosidase to a culture solution containing bacterial cells containing PHA as a specific PHB copolymer to perform enzyme treatment.

[0062] <pha>

[0063] In this manufacturing method, the PHA is a PHB copolymer composed of 3HB and hydroxyalkanoate esters other than 3HB.

[0064] Examples of the hydroxyalkanoate esters other than 3HB include, for example, 3-hydroxyhexanoate (3HH), 3-hydroxypentanoate (3HV), 4-hydroxybutyrate (4HB), 3-hydroxyoctanoate (3HO), 3-hydroxyoctadecanoate (3HOD), 3-hydroxydecanoate (3HD), and the like.

[0065] As the PHB copolymer, for example, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (hereinafter sometimes referred to as "P3HB3HH") can be preferably exemplified, but it is not limited thereto. Hereinafter, for the sake of convenience of explanation, P3HB3HH will be mainly described as a representative example.

[0066] P3HB3HH changes the melting point and crystallinity by changing the composition ratio of the repeating units of 3HB and 3HH. As a result, physical properties such as Young's modulus and heat resistance can be changed, and physical properties between polypropylene and polyethylene can be imparted.

[0067] In this manufacturing method, the composition ratio of the 3HB unit / hydroxyalkanoate ester unit other than the 3HB unit of the PHB copolymer is 80 / 20 to 88 / 12 (mol / mol), preferably 81 / 19 to 87 / 13 (mol / mol), and more preferably 82 / 18 to 86 / 14 (mol / mol). When the composition ratio of the 3HB unit / hydroxyalkanoate ester unit other than the 3HB unit is 88 / 12 (mol / mol) or less, sufficient hardness can be obtained; when it is 80 / 20 (mol / mol) or more, sufficient softness can be obtained. The ratio of each repeating unit like this can be obtained by the method described in paragraph

[0047] of International Publication No. 2013 / 147139.

[0068] In one embodiment of the present invention, the weight-average molecular weight (hereinafter sometimes referred to as "Mw") of the PHB copolymer is not particularly limited, preferably 150,000 to 800,000, more preferably 200,000 to 700,000, and further preferably 250,000 to 600,000. When the weight-average molecular weight is 150,000 or more, sufficient mechanical properties and the like can be obtained; when it is 800,000 or less, sufficient crystallization rate can be obtained, and good moldability can be achieved. The weight-average molecular weight of the P3HB-based resin can be obtained by gel permeation chromatography (GPC) ("Shodex GPC-101" manufactured by Showa Denko), using a polystyrene gel ("Shodex K-804" manufactured by Showa Denko) as the chromatographic column, and chloroform as the mobile phase, as the molecular weight in terms of polystyrene.

[0069] <Bacterial cell>

[0070] The bacterial cell used in step (d) is not particularly limited as long as it is a microorganism capable of producing a PHB copolymer intracellularly. For example, microorganisms isolated from nature and those preserved in strain depositories (e.g., IFO, ATCC, etc.), or mutants and transformants prepared therefrom can be used. For example, the bacterial cell of P3HB, which is an example of a PHB copolymer, was first discovered as Bacillus megaterium in 1925. Other examples include natural microorganisms such as Cupriavidus necator (former classification: Alcaligenes eutrophus, Ralstonia eutropha), Alcaligenes latus, etc. It is known that PHB copolymers accumulate in these microorganisms.

[0071] In addition, as the bacterial cell of a copolymer of hydroxybutyrate and other hydroxyalkanoates, which is an example of a PHB copolymer, examples include Aeromonas caviae as a P3HB3HV and P3HB3HH producer, Alcaligenes eutrophus as a P3HB4HB producer, etc. In particular, regarding P3HB3HH, in order to improve the productivity of P3HB3HH, the Alcaligenes eutrophus AC32 strain (Alcaligenes eutrophus AC32, FERM BP-6038) (T. Fukui, Y. Doi, J. Bateriol., 179, p4821-4830 (1997)) into which the gene group of PHB copolymer synthase has been introduced is more preferably used. In addition, other than the above, the bacterial cell can also be a genetically engineered microorganism into which various genes related to the synthesis of PHB copolymers have been introduced according to the desired PHB copolymer to be produced.

[0072] In addition, the PHB copolymer can be produced, for example, by the method described in International Publication No. 2010 / 013483.

[0073] <Glycosidase>

[0074] In this specification, "glycosidase" refers to an enzyme having the activity of decomposing the cell wall (e.g., peptidoglycan) of a bacterial cell (lysing). As glycosidase, as long as it is within the above-defined scope, it is not particularly limited, and examples include, for example, lysozyme, Labiase, β-N-acetylglucosaminidase, endolysin, autolysin, etc. From an economic perspective, lysozyme, which is a general enzyme, is preferably used.

[0075] In step (d), water can be added to adjust the solid component concentration of the aqueous suspension before adding the glycosidase to the above-mentioned culture solution. The solid component concentration of the aqueous suspension at this time is preferably 10 to 40% by weight, more preferably 20 to 40% by weight. When the solid component concentration of the aqueous suspension is within the above range, the concentration of the aqueous suspension is not too high and is uniformly mixed with the glycosidase.

[0076] In addition, the pH of the culture solution can be adjusted before adding the glycosidase. The adjustment method at this time is not particularly limited, and examples thereof include: a method of adding an acid, etc. The acid is not particularly limited, and any of an organic acid and an inorganic acid can be used, 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.

[0077] It should be noted that before step (d), it is preferably not to perform the step of drying the aqueous suspension. That is, in the method for producing a polyhydroxybutyrate copolymer according to an embodiment of the present invention, before step (d), it is preferably not to perform the step of drying the aqueous suspension. Here, the drying can be the drying method described in the following description. Since the aqueous suspension is not dried before step (d), irreversible aggregation of the PHB copolymer particles can be suppressed.

[0078] (2-2. Step (e))

[0079] Step (e) is a step of adding an alkaline protease to the culture solution obtained in the above step (d) and performing an enzymatic treatment on the above-mentioned cells. By using an alkaline protease to perform an enzymatic treatment on the above-mentioned cells, the culture solution can be made into an aqueous suspension.

[0080] <Alkaline protease>

[0081] In the present specification, "alkaline protease" refers to a protease having the activity of decomposing proteins in an alkaline environment (for example, in a solution with a pH of 8.5).

[0082] In one embodiment of the present invention, the alkaline protease only needs to have the activity of decomposing proteins in an alkaline environment and is not particularly limited. For example, serine-specific proteases (for example, subtilisin, chymotrypsin); cysteine-specific proteases (for example, papain, bromelain), etc. can be cited. From the viewpoints of versatility / economy, serine-specific proteases are preferred, and alkaline proteases containing subtilisin are particularly preferred. These can be used alone or in combination of two or more.

[0083] As the alkaline proteolytic enzyme, commercially available products can be used, for example, "Alcalase" and "Esperase" 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.

[0084] In step (e), when the bacterial cells are enzymatically treated with an alkaline protease, the pH and temperature of the culture medium are preferably adjusted based on the optimal pH and temperature of the alkaline protease used. Furthermore, the pH in step (e) is preferably lower than the pH adjusted by adding the alkaline aqueous solution in step (b). The method for adjusting the pH and temperature of the culture medium is not particularly limited, and known methods can be used.

[0085] In one embodiment of the present invention, the optimal pH of the alkaline protease is not particularly limited as long as the alkaline protease is active in an alkaline environment, and is, for example, 8.0 to 12.0, preferably 8.0 to 11.0, 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 optimal temperature of the alkaline protease is not particularly limited, but is preferably 70°C or lower, more preferably 60°C or lower, from the perspective of avoiding excessive temperature increases and preventing thermal transition (thermal decomposition) of the PHB copolymer. The lower limit of the optimal temperature is not particularly limited, but is preferably room temperature (e.g., 25°C) or higher from the perspective of avoiding excessive cooling and economical considerations.

[0087] In one embodiment of the present invention, the amount of alkaline protease added is not particularly limited, and is, for example, 0.05 to 1.0 phr, preferably 0.1 to 0.5 phr, and more preferably 0.15 to 0.3 phr. When the amount of alkaline protease added is within the above range, bacterial cells can be appropriately degraded.

[0088] In step (e), it is preferable to substantially not add glycosidase while adding an alkaline protease. In this specification, "substantially not adding glycosidase" means that the addition amount of glycosidase is 0.0005 phr or less, and in one embodiment, the addition amount of glycosidase can also be 0 phr. The glycosidase is not particularly limited, and examples thereof include the glycosidases described in the items of step (d).

[0089] In step (e), it is preferable that the cells containing the PHB copolymer are inactivated. In other words, this production method may include a step of inactivating the cells after step (d) and before step (e). The method of inactivation is not particularly limited. For example, as described in the examples, a method of heating and stirring a culture solution containing cells containing the PHB copolymer at 60 to 70 °C for 7 hours can be cited. In addition, the culture solution after the heating and stirring treatment is preferably cooled to a temperature suitable for step (e).

[0090] (2-3. Step (b))

[0091] Step (b) is a step of adding an alkaline aqueous solution to a culture solution containing cells containing PHA as a specific PHB copolymer to adjust the pH to 10.0 to 12.0, and adding a surfactant at any stage before, during, or after the adjustment.

[0092] Step (b) includes the following steps (b1) and (b2).

[0093] ･ Step (b1): A step of adding an alkaline aqueous solution to a culture solution containing cells containing PHA as a specific PHB copolymer to adjust the pH to 10.0 to 12.0

[0094] ･ Step (b2): A step of adding a surfactant

[0095] (Step (b1))

[0096] As described above, step (b1) is a step of adding an alkaline aqueous solution to a culture solution containing cells containing PHA as a specific PHB copolymer to adjust the pH to 10.0 to 12.0. According to this step, by dispersing and dissolving impurities (such as nucleic acids and proteins) derived from the cells, a high-purity PHB copolymer can be separated from the cells.

[0097] In one embodiment of the present invention, the alkaline aqueous solution is an aqueous solution containing an alkaline compound. The alkaline compound contained in the alkaline aqueous solution is not particularly limited, and examples thereof include hydroxides of alkali metals or alkaline earth metals such as sodium hydroxide and potassium hydroxide; metal carbonates such as sodium carbonate and potassium carbonate; metal phosphates or metal hydrogen phosphates such as sodium phosphate, potassium phosphate, sodium hydrogen phosphate, and potassium hydrogen phosphate.

[0098] In one embodiment of the present invention, the basic compound contained in the aqueous basic solution is preferably an alkali metal hydroxide or an alkaline earth metal hydroxide, more preferably sodium hydroxide. The basic compound can be used alone or in combination of two or more kinds.

[0099] In step (b1), it is preferable to adjust the pH to 10.2 to 11.8, more preferably to 10.4 to 11.6, by adding an aqueous basic solution. By adjusting the pH to 10.0 or more, there is an advantage that the cell components can be decomposed and dissolved. In addition, by adjusting the pH to 12.0 or less, damage to unwanted cells can be prevented.

[0100] The temperature in step (b1) is preferably lower than 100 °C, more preferably lower than 80 °C. The lower limit is not particularly limited, and for example, it is preferably 40 °C or more.

[0101] (Step (b2))

[0102] Step (b2) is a step of adding a surfactant to the culture solution containing cells containing PHA as a specific PHB copolymer. Based on this step, the cell membrane can be treated efficiently in particular, and more impurities derived from the above cells can be removed, so that a higher purity PHB copolymer can be separated from the cells.

[0103] In one embodiment of the present invention, the surfactant is not particularly limited, and examples thereof include: anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, and the like. Among them, from the viewpoint of high cell membrane removal ability, an anionic surfactant is preferred. These can be used alone or in combination of two or more kinds.

[0104] Examples of the anionic surfactant include: alkyl sulfates, alkyl benzene sulfonates, alkyl sulfate esters, alkenyl sulfate esters, alkyl ether sulfate esters, alkenyl ether sulfate esters, α-olefin sulfonates, α-sulfo fatty acid salts, esters of α-sulfo fatty acid salts, alkyl ether carboxylates, alkenyl ether carboxylates, amino acid type surfactants, N-acyl amino acid type surfactants, and the like. Among them, alkyl sulfate esters are preferred, and sodium dodecyl sulfate (SDS) is particularly preferred from the viewpoints of high cell membrane removal ability and low cost. These can be used alone or in combination of two or more kinds.

[0105] In step (b2), the amount of the surfactant added is not particularly limited, and is, for example, 0.1 to 5.0% by weight, preferably 0.3 to 2.5% by weight, based on the above culture solution.

[0106] Step (b2) can be carried out before step (b1), simultaneously with step (b1), or after step (b1). Step (b2) is preferably carried out after step (b1).

[0107] (2-4. Step (f))

[0108] Step (f) is a step of solid-liquid separating the obtained aqueous suspension by a decanter centrifuge and recovering the PHA cake-containing material.

[0109] Since a decanter centrifuge is used in step (f), the aqueous suspension can be dehydrated without passing through a narrow passage, so that solid-liquid separation can be carried out without the risk of blockage. In addition, compared with a disc centrifuge or the like, the concentration of the solid component in the aqueous suspension can be increased, so that the efficiency of each solid-liquid separation is good, and thus the drainage volume can be reduced.

[0110] Examples of the decanter centrifuge include a horizontal type and a vertical type, and the horizontal type that can handle a large amount of liquid is preferred.

[0111] In step (f), the volume median diameter of the polyhydroxyalkanoate in the obtained aqueous suspension is preferably 13 to 500 μm, more preferably 15 to 400 μm, and still more preferably 19 to 300 μm. If the volume median diameter is within the above range, the yield in the washing step is excellent, the amount of the aqueous suspension that can be treated is increased, and the economy is good. The "volume median diameter of the polyhydroxyalkanoate" in step (f) refers to the volume median diameter of the polyhydroxyalkanoate in the aqueous suspension aggregated through step (d).

[0112] The centrifugal force when introducing the aqueous suspension into the decanter centrifuge is not particularly limited, and from the viewpoints of the yield and economy in the washing step, for example, it can be 2000 to 4000 G, 2500 to 3500 G, or 2800 to 3200 G.

[0113] (2-5. Step (c))

[0114] Step (c) is a step of solid-liquid separating the obtained aqueous suspension by a disc centrifuge and recovering the PHA aqueous suspension.

[0115] In step (c), the recovery of the PHA aqueous suspension is carried out 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 sedimentation centrifuge, a centrifugal dehydrator, or the like.

[0116] In step (c), after centrifuging the above aqueous suspension and removing the supernatant, it is preferable to repeat the steps of adding a solution to the sediment, centrifuging again, and removing the supernatant. By this operation, a more concentrated and purified PHA aqueous suspension can be obtained. Here, the solution added after removing the supernatant is preferably an alkaline aqueous solution whose pH is adjusted to the same as that of the above culture solution.

[0117] In step (c), the solvent constituting the PHA aqueous suspension (the "solvent" is also referred to as the "aqueous medium") is not particularly limited and may be water or a mixed solvent of water and an organic solvent. In addition, in this mixed solvent, the concentration of the organic solvent is not particularly limited as long as it is below the solubility of the organic solvent used in water. In addition, the organic solvent is not particularly limited, and examples 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, etc. Among them, from the perspective of easy removal, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, propionitrile, etc. are preferred. In addition, from the perspective of easy availability, methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone, etc. are more preferred. Further, methanol, ethanol, and acetone are particularly preferred.

[0118] The water content in the aqueous medium constituting the PHA aqueous suspension is preferably 5% by weight or more, more preferably 10% by weight or more, further preferably 30% by weight or more, and particularly preferably 50% by weight or more.

[0119] It should be noted that as long as the essence of the present invention is not damaged, the PHA aqueous suspension in step (c) may also contain other solvents, components derived from the bacterial cells, compounds generated during purification, etc.

[0120] In step (c), it is preferable to perform solid-liquid separation on the obtained aqueous suspension so that its conductivity reaches the range of 150 to 400 mS / m, more preferably 200 to 350 mS / m, and further preferably 250 to 300 mS / m, and further includes a step of recovering the aqueous suspension. The conductivity of the aqueous suspension is measured by the method described in the examples.

[0121] (2-6. Step (a))

[0122] Step (a) is a step of performing enzymatic treatment by adding an alkaline protease. Step (a) is preferably performed before step (c).

[0123] Regarding the alkaline protease in step (a), the description of <alkaline protease> in step (e) above is referred to.

[0124] (2-7. Step (g))

[0125] Step (g) involves adding an aqueous solution to the PHA-containing cake obtained in step (f) to adjust the pH to 2.5 to 4.0. Adjusting the pH to 2.5 to 4.0 facilitates maintaining the molecular weight of the PHA during processing and molding.

[0126] In step (g), the pH is preferably adjusted to 2.8 to 3.8, more preferably to 3.0 to 3.5.

[0127] In step (g), the aqueous solution used for adjusting the pH is not particularly limited, for example, ion exchange water, alkaline aqueous solution, acidic aqueous solution etc. can be used. Among these, ion exchange water and acidic aqueous solution are preferred, and acidic aqueous solution is more preferred. In acidic aqueous solution, it is particularly preferred to add sulfuric acid aqueous solution etc. and carry out. In one embodiment, the pH adjustment in step (g) can be carried out by dilution of an aqueous suspension.

[0128] (2-8. Step (h))

[0129] Step (h) is a step of dehydrating and drying the PHA aqueous suspension using a filter press after the above step (g).

[0130] In step (h), the PHA aqueous suspension can be squeezed and dehydrated using a filter press. The pressure during squeezing of the PHA aqueous suspension is not particularly limited, but 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. Squeezing may be performed only once or twice or more. When squeezing is performed twice or more, the pressure of the second and subsequent squeezing cycles is preferably higher than that of the first squeezing cycle.

[0131] In step (h), the PHA aqueous suspension can be further dehydrated by aeration. When drying by aeration, the pressure is not particularly limited, and for example, it can be 0.01 to 1.5 MPa, preferably 0.05 to 1.3 MPa, and more preferably 0.10 to 1.0 MPa. Furthermore, the aeration time is not particularly limited, as long as it allows the PHA aqueous suspension to be fully dried, and for example, it can be 10 minutes to 1 hour, preferably 15 minutes to 40 minutes, and more preferably 20 minutes to 30 minutes.

[0132] In step (h), the filter cake obtained after dehydration can be dried by a known method. Drying can be performed using, for example, a box dryer, a spray dryer, a fluidized bed dryer, or a drum dryer. For ease of operation, a box dryer is preferably used. The drying temperature is not particularly limited; for example, drying can be performed at 40 to 80°C.

[0133] 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.

[0134] That is, one embodiment of the present invention is as follows.

[0135] <1> A method for producing polyhydroxyalkanoate, wherein:

[0136] The polyhydroxyalkanoate is a polyhydroxybutyrate copolymer having a composition ratio of 3-hydroxybutyrate units / hydroxyalkanoate units other than 3-hydroxybutyrate units of 80 / 20 to 88 / 12 (mol / mol),

[0137] As the culture medium, a culture medium containing bacterial cells containing the above-mentioned polyhydroxyalkanoate is used.

[0138] The method for producing the polyhydroxyalkanoate comprises the following steps (d), (e), and (b):

[0139] (d) adding glycosidase to the culture solution to perform enzyme treatment;

[0140] (e) adding an alkaline protease to the culture solution obtained in the step (d) to perform an enzymatic treatment on the bacterial cells;

[0141] (b) adding an alkaline aqueous solution to the culture solution or the aqueous suspension obtained in step (e) to adjust the pH to 10.0 to 12.0, and adding a surfactant before, simultaneously with, or after the adjustment; and

[0142] After the steps (d), (e), and (b), the method comprises:

[0143] (f) A step of separating the obtained aqueous suspension into solid and liquid by a decanter centrifuge to recover a cake containing polyhydroxyalkanoate.

[0144] <2> according to <1> The method for producing polyhydroxyalkanoate, wherein:

[0145] In the step (f), the volume median diameter of the polyhydroxyalkanoate in the polyhydroxyalkanoate aqueous suspension is 13 to 500 μm.

[0146] <3> The method for producing a polyhydroxyalkanoate according to <1> or <2>, further comprising:

[0147] (g) A step of diluting the polyhydroxyalkanoate aqueous suspension obtained by adding an aqueous solution to the cake containing polyhydroxyalkanoate obtained in the step (f), thereby adjusting the pH to 2.5 to 4.0; and

[0148] (h) A step of dehydrating and drying the polyhydroxyalkanoate aqueous suspension with a filter press after the step (g).

[0149] <4> The method for producing a polyhydroxyalkanoate according to <3>, wherein

[0150] In the step (h), the dehydration by the filter press is carried out at a pressure of 0.2 to 1.0 MPa.

[0151] <5> The method for producing a polyhydroxyalkanoate according to <3> or <4>, wherein

[0152] In the step (h), the polyhydroxyalkanoate aqueous suspension is further dehydrated by blowing air.

[0153] <6> The method for producing a polyhydroxyalkanoate according to any one of <1> to <5>, wherein

[0154] The weight-average molecular weight of the polyhydroxybutyrate copolymer is 150,000 to 800,000.

[0155] <7> The method for producing a polyhydroxyalkanoate according to any one of <1> to <6>, wherein

[0156] The order of the steps of performing the steps (d), (e), and (b) is: performing steps (d) and (e) after performing step (b),

[0157] Before performing the step (d) after performing the step (b), the method for producing a polyhydroxyalkanoate further comprises: (c) a step of solid-liquid separating the obtained aqueous suspension with a disk centrifuge and recovering the polyhydroxyalkanoate aqueous suspension.

[0158] <8> The method for producing a polyhydroxyalkanoate according to <7>, further comprising:

[0159] In the step (c), solid-liquid separation is carried out using a disk centrifuge until the conductivity of the polyhydroxyalkanoate aqueous suspension reaches the range of 150 to 400 mS / m, and the step of recovering the polyhydroxyalkanoate aqueous suspension.

[0160] <9> The method for producing polyhydroxyalkanoate according to <7> or <8>, further comprising, before carrying out the step (c):

[0161] (a) A step of carrying out an enzymatic treatment by adding an alkaline protease.

[0162] <10> The method for producing polyhydroxyalkanoate according to any one of <1> to <9>, comprising, before carrying out the step (e):

[0163] A step of inactivating the bacterial cells containing the polyhydroxyalkanoate.

[0164] Examples

[0165] 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 "PHA" described in the examples may also be expressed as "P3HB3HH".

[0166] 〔Measurement method〕

[0167] The measurements in the examples and comparative examples were carried out by the following methods.

[0168] (Volume median diameter)

[0169] The volume median diameter of PHA was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA.

[0170] (pH of the PHA aqueous suspension)

[0171] The pH was measured using a pH meter (9652-10D manufactured by HORIBA).

[0172] (Conductivity of the PHA aqueous suspension)

[0173] The conductivity was measured using a conductivity meter (9382-10D manufactured by HORIBA).

[0174] (Solid component concentration)

[0175] The solid component concentration of the PHA aqueous suspension was measured using (manufactured by A&D Co., Ltd.). The PHA aqueous suspension was heated at 105 °C until the weight change rate was lower than 0.05% / min, and the solid component concentration was calculated from the weight change of the PHA aqueous suspension before and after heating.

[0176] (Determination of total nitrogen content)

[0177] The total nitrogen content of the PHA powder was measured using a trace total nitrogen analyzer TN-2100H (Analytech Co., Ltd., Nitto Seiko).

[0178] (Determination of PHA yield in the separation process)

[0179] The PHA yield in the separation process was calculated by the following formula:

[0180] 1 - ([Amount of PHA solid component in the PHA aqueous suspension discharged in the separation process] / [Amount of PHA solid component in the PHA aqueous suspension supplied to the separation process]).

[0181] (Total PHA yield in the separation process)

[0182] The total PHA yield in the separation process was calculated as the product of the overall yields in each separation step. For example, when the yields of the 1st, 2nd, 3rd, and 4th steps were all 98%, the result according to the following formula was 92.2%.

[0183] Total yield in the separation process = 98 / 100 × 98 / 100 × 98 / 100 × 98 / 100 = 0.922[-]

[0184] (Amount of PHA solid component in the PHA aqueous suspension)

[0185] After thoroughly stirring the aqueous suspension, 10.0 g was aliquoted into a 15 mL Falcon tube and separated using a tabletop centrifuge at 9000 G for 10 min. Subsequently, the supernatant was discarded, ethanol was added to the remaining precipitate to make 10 mL, and it was dispersed using a shaker. Then, it was put into a tabletop centrifuge and separated at 9000 G for 5 min. After discarding the supernatant, it was put into a vacuum dryer at 70 °C, and the weight of the remaining PHA solid component was measured after 24 hours. The obtained weight was taken as the concentration of the PHA aqueous suspension. The concentration of the PHA aqueous suspension was multiplied by the weight of the PHA aqueous suspension supplied to the separation process or the PHA aqueous suspension discharged in the separation process, and the product was taken as the amount of PHA solid component.

[0186] [Example 1]

[0187] (Preparation of bacterial cell culture solution)

[0188] The Alcaligenes eutrophus described in International Publication No. 2019 / 142717 was cultured according to the method described in paragraphs

[0041] to

[0048] of this document, and a cell culture solution containing cells containing PHA was obtained. It should be noted that Alcaligenes eutrophus is currently classified as Cupriavidus necator. The composition ratio of the repeating units of PHA (composition ratio of 3HB units / PHA units other than 3HB units) is 84 / 16 (mol / mol).

[0189] (Inactivation)

[0190] The cell culture solution obtained above was sterilized by heating at an internal temperature of 60 to 70 °C and stirring for 7 hours. Thereafter, ion-exchanged water at room temperature was added, and after dilution, it was cooled to 55 °C, thereby obtaining an inactivated culture solution.

[0191] (Alkali treatment)

[0192] To the inactivated culture solution obtained above, a 30% aqueous sodium hydroxide solution was added to adjust the pH to 9.5. While maintaining the solution at 70 °C, a 30% aqueous sodium hydroxide solution was continuously added and maintained for 6 hours to obtain a PHA aqueous suspension.

[0193] (Neutralization treatment and cell wall degrading enzyme treatment)

[0194] To the PHA aqueous suspension obtained above, 10% sulfuric acid was added to adjust the pH to 6.5. After the addition of sulfuric acid, 0.05 phr of lysozyme (manufactured by FUJIFILM Wako Pure Chemical Corporation), which is a cell wall degrading enzyme, was added and maintained at 50 °C for 2 hours.

[0195] (Alkaline enzyme treatment 1)

[0196] To the above PHA aqueous suspension, 30% sodium hydroxide was used to adjust the pH to 8.5 ± 0.2. Thereafter, 0.2 phr of Alcalase (manufactured by Novozymes), which is a proteolytic enzyme, was added, and while controlling the pH to 8.5 with 30% sodium hydroxide at 50 °C, it was maintained for more than 2 hours.

[0197] (Lysis)

[0198] To the enzyme-treated solution obtained above, an alkaline aqueous solution was added to adjust the pH to 11 or more. Thereafter, 1.0 wt% of sodium dodecyl sulfate (manufactured by Kao) relative to the weight of the PHA aqueous suspension was added, and it was maintained at 45 °C for 2 hours. When measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA, the volume median diameter of PHA in this PHA aqueous suspension was 19 μm. The solid component concentration of the PHA aqueous suspension at this time was 20%.

[0199] (Centrifugal separation process 1)

[0200] The above-mentioned PHA aqueous suspension was introduced into a decanter (PTM006 type, manufactured by B Industry) at a rate of 150 L / hr and centrifuged at 3100G to recover the water-containing cake of PHA. The yield of each centrifugal separation was 98%, and the drainage volume of each centrifugal separation was 3 times the amount of PHA in the PHA aqueous suspension after supply.

[0201] (Dilution after centrifugal separation)

[0202] An alkaline aqueous solution (pH 10 - 11) was added to the above-obtained water-containing cake of PHA to prepare a PHA aqueous suspension with a solid component concentration of 20%. The centrifugal separation process 1 and the dilution operation after centrifugal separation were carried out twice. In addition, the water content of the water-containing cake of PHA obtained in the second centrifugal separation was 46%.

[0203] (Alkaline enzyme treatment 2)

[0204] For the above-mentioned PHA aqueous suspension, the pH was adjusted to 11 ± 0.2 using 30% sodium hydroxide. Subsequently, 0.10 phr of Esperase (manufactured by Novozymes), a proteolytic enzyme, was added, and the pH was controlled at 11 with 30% sodium hydroxide at 50°C while maintaining for more than 2 hours.

[0205] (Centrifugal separation process 2)

[0206] The above enzyme-treated PHA aqueous suspension was introduced into a decanter (PTM006 type, manufactured by B Industry) at a rate of 150 L / hr and centrifuged at 3100G to recover the water-containing cake of PHA. An alkaline aqueous solution (pH 10 - 11) was added to the recovered cake to prepare a PHA aqueous suspension with a solid component concentration of 20%. The above operation was carried out three times. The yield of each centrifugal separation was 98%, and the drainage volume of each centrifugal separation was 3 times the amount of PHA in the supplied PHA aqueous suspension. In addition, the water content of the water-containing cake of PHA obtained in the third centrifugal separation was 43%. The total yield of the five centrifugal separations in total was 90.3%.

[0207] (pH adjustment)

[0208] Ion-exchanged water was added to the obtained water-containing cake of PHA to a solid component concentration of 20 wt% and heated to 40°C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0209] (Pressure filtration)

[0210] The PHA aqueous suspension was filtered using a filter press (ISD model 360, manufactured by Ishigaki Corporation). After pressing at a pressure of 0.4 MPa to obtain a filter cake, the filter was pressed again at a pressure of 0.7 MPa. The air supply pressure was adjusted to 0.4 MPa and air was supplied for 20 minutes to obtain a filter cake. The resulting filter cake had a moisture content of 13.5 wt% (WB).

[0211] (dry)

[0212] The obtained filter cake was dried for 24 hours in a box dryer (PV-211 manufactured by Espec) adjusted to 60° C. to obtain a dry PHA resin. The total nitrogen content of the obtained dry PHA resin was 450 ppm.

[0213] [Example 2]

[0214] The same operations as in Example 1 were performed up to the alkali treatment step.

[0215] (Alkaline enzyme treatment 1)

[0216] The pH of the PHA aqueous suspension was adjusted to 8.5±0.2 using 30% sodium hydroxide. Thereafter, 0.2 phr of Alcalase (Novozymes) was added as a proteolytic enzyme and the suspension was maintained at 50°C for at least 2 hours while controlling the pH to 8.5 using 30% sodium hydroxide.

[0217] (Bacteriolysis)

[0218] An alkaline aqueous solution was added to the enzyme-treated solution obtained above to adjust the pH to 11 or higher, and then 1.0 wt % of sodium lauryl sulfate (manufactured by Kao) was added based on the weight of the PHA aqueous suspension, and the mixture was maintained at 45° C. for 2 hours.

[0219] (Centrifugal separation step 1)

[0220] The PHA aqueous suspension was introduced into a disc separator at a rate of 150 L / hr and centrifuged at 3100 G for solid-liquid separation until the conductivity of the PHA aqueous suspension reached 290 mS / m. A PHA aqueous cake was recovered. The yield per centrifugation was 99.5%, and the amount of wastewater per centrifugation was 2.5 times the amount of PHA in the supplied PHA aqueous suspension.

[0221] (Centrifugation followed by dilution)

[0222] An alkaline aqueous solution (pH 10-11) was added to the PHA aqueous cake obtained above to prepare a PHA aqueous suspension with a solids concentration of 20%. The centrifugation step 1 and the dilution operation after centrifugation were performed three times. The moisture content of the PHA aqueous cake obtained in the third centrifugation was 64%.

[0223] (Neutralization and cell wall decomposing enzyme treatment)

[0224] The pH of the PHA aqueous suspension obtained above was adjusted to 6.5 by adding 10% sulfuric acid. After the addition of sulfuric acid, 0.05 phr of lysozyme (Fuji Film Wako Pure Chemical Industries, Ltd.), an enzyme that decomposes cell walls, was added, and the mixture was maintained at 50°C for 2 hours.

[0225] (Alkaline enzyme treatment 2)

[0226] The pH of the PHA aqueous suspension was adjusted to 11±0.2 using 30% sodium hydroxide. Subsequently, 0.10 phr of Esperase (Novozymes) was added as a proteolytic enzyme, and the pH was maintained at 11 using 30% sodium hydroxide at 50°C for at least 2 hours. The volume median particle size of the PHA in the PHA aqueous suspension was 25 μm, as measured using a HORIBA laser diffraction / scattering particle size distribution analyzer, LA-950.

[0227] (Centrifugal separation step 2)

[0228] The enzyme-treated PHA aqueous suspension was introduced into a decanter (PTM006, manufactured by Basilisk Industries) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA aqueous cake. An alkaline aqueous solution (pH 10-11) was added to the recovered cake to produce a PHA aqueous suspension with a solids concentration of 20%. This operation was repeated three times, with the yield per centrifugation being 99%, and the amount of water removed from each centrifugation being three times the amount of PHA in the supplied PHA aqueous suspension. The moisture content of the PHA aqueous cake obtained in the third centrifugation was 43%. The total yield of six centrifugal separations, including three in the disc separator and three in the decanter, was 95.6%.

[0229] (pH adjustment)

[0230] Ion-exchanged water was added to the obtained PHA aqueous cake so as to have a solid content concentration of 20% by weight, and the temperature was raised to 40° C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0231] (Pressure Filtration)

[0232] The PHA aqueous suspension was filtered using a filter press (ISD model 360, manufactured by Ishigaki Corporation). After pressing at a pressure of 0.4 MPa to obtain a filter cake, the filter was pressed again at a pressure of 0.7 MPa. The air flow pressure was adjusted to 0.4 MPa and air flow was continued for 20 minutes to obtain a filter cake. The resulting filter cake had a moisture content of 14 wt% (WB).

[0233] (dry)

[0234] The obtained filter cake was dried for 24 hours in a box dryer (PV-211 manufactured by Espec) adjusted to 60 degrees Celsius to obtain a dry PHA resin. The total nitrogen content of the obtained dry PHA resin was 350 ppm.

[0235] [Example 3]

[0236] The same operations as in Example 2 were carried out until the pH adjustment step.

[0237] (Dehydration process)

[0238] The pH-adjusted PHA aqueous suspension was introduced into a decanter (PTM006, manufactured by Basilisk Industries) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA water cake. The dehydration yield was 99.9%, and the water content of the PHA water cake was 35%.

[0239] (dry)

[0240] The obtained filter cake was dried for 24 hours in a box dryer (PV-211 manufactured by Espec) adjusted to 60 degrees Celsius to obtain a dry PHA resin. The total nitrogen content of the obtained dry PHA resin was 350 ppm.

[0241] [Comparative Example 1]

[0242] The same operations as in Example 1 were carried out up to the neutralization treatment and the cell wall decomposing enzyme treatment.

[0243] (Bacteriolysis)

[0244] An alkaline aqueous solution was added to the enzyme-treated solution obtained above to adjust the pH to 11 or higher. Subsequently, 1.0 wt% of sodium lauryl sulfate (manufactured by Kao) was added relative to the weight of the PHA aqueous suspension, and the mixture was maintained at 45°C for 2 hours. The volume median particle size of the PHA in the PHA aqueous suspension was 8 μm when measured using a HORIBA laser diffraction / scattering particle size distribution analyzer LA-950.

[0245] (Centrifugal separation step 1)

[0246] The above-mentioned PHA aqueous suspension was introduced into a decanter (PTM006 type, manufactured by BA Industry) at a rate of 150 L / hr and centrifuged at 3100G to recover the water-containing cake of PHA. The yield of each centrifugation was 90%, and the drainage volume of each centrifugation was 2.5 times the amount of PHA in the supplied PHA aqueous suspension.

[0247] (Dilution after centrifugation)

[0248] An alkaline aqueous solution (pH 10 - 11) was added to the obtained PHA water-containing cake above to prepare a PHA aqueous suspension with a solid component concentration of 20%. Two centrifugation processes 1 and the dilution operation after centrifugation were carried out. In addition, the water content of the PHA water-containing cake obtained in the second centrifugation was 43%.

[0249] (Alkaline enzyme treatment 2)

[0250] For the above-mentioned PHA aqueous suspension, the pH was adjusted to 11 ± 0.2 using 30% sodium hydroxide. Subsequently, 0.10 phr of Esperase (manufactured by Novozymes), a proteolytic enzyme, was added, and the pH was controlled at 11 using 30% sodium hydroxide at 50°C while maintaining for more than 2 hours.

[0251] (Centrifugation process 2)

[0252] The above-mentioned enzyme-treated PHA aqueous suspension was introduced into a decanter (PTM006 type, manufactured by BA Industry) at a rate of 150 L / hr and centrifuged at 3100G to recover the water-containing cake of PHA. An alkaline aqueous solution (pH 10 - 11) was added to the recovered cake to prepare a PHA aqueous suspension with a solid component concentration of 20%. The above operation was carried out 3 times. The yield of each centrifugation was 98%, and the drainage volume of each centrifugation was 3 times the amount of PHA in the supplied PHA aqueous suspension. In addition, the water content of the PHA water-containing cake obtained in the third centrifugation was 43%. The total yield of 5 centrifugations in total was 76.2%.

[0253] (pH adjustment)

[0254] Ion-exchanged water was added to the obtained PHA water-containing cake to a solid component concentration of 20 wt% and heated to 40°C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0255] (Pressure filtration)

[0256] The above-mentioned PHA aqueous suspension was filtered using a plate and frame filter press (Model ISD 360, manufactured by Ishigaki Corporation). After obtaining a filter cake by pressing at a pressure of 0.4 Mpa, it was pressed again at a pressure of 0.7 MPa, the air supply pressure was adjusted to 0.4 Mpa and air was supplied for 20 minutes to obtain a filter cake. The moisture content of the obtained filter cake was 13.5 wt% (W.B.).

[0257] (Drying)

[0258] The obtained filter cake was dried for 24 hours using a cabinet dryer (Model PV-211, manufactured by Espec) adjusted to 60 degrees to obtain a dried PHA resin. The total nitrogen content of the obtained dried PHA resin was 550 ppm.

[0259] [Comparative Example 2]

[0260] The same operations as in Example 2 were carried out until the neutralization treatment and the cell wall degrading enzyme treatment.

[0261] When measured using a laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA, the volume median diameter of PHA in the PHA aqueous suspension was 10 μm.

[0262] (Centrifugation step 2)

[0263] The above-mentioned PHA aqueous suspension after the enzyme treatment was introduced into a decanter (Model PTM006, manufactured by Ba Industries) at a rate of 150 L / hr and centrifuged at 3100 G to recover a water-containing cake of PHA. An alkaline aqueous solution (pH 10 - 11) was added to the recovered cake to prepare a PHA aqueous suspension with a solid component concentration of 20%. The above operation was carried out 3 times. The yield of each centrifugation was 90%, and the drainage volume of each centrifugation was 3 times the amount of PHA in the supplied PHA aqueous suspension. In addition, the moisture content of the PHA water-containing cake obtained in the third centrifugation was 43%. The total yield of 6 centrifugations in total, 3 times with a disc separator and 3 times with a decanter, was 71.8%.

[0264] (pH adjustment)

[0265] Ion-exchanged water was added to the obtained PHA water-containing cake to a solid component concentration of 20 wt% and the temperature was raised to 40 °C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0266] (Pressure filtration)

[0267] The PHA aqueous suspension was filtered using a filter press (ISD model 360, manufactured by Ishigaki Corporation). After pressing at a pressure of 0.4 MPa to obtain a filter cake, the filter was pressed again at a pressure of 0.7 MPa. The air flow pressure was adjusted to 0.4 MPa and air flow was continued for 20 minutes to obtain a filter cake. The resulting filter cake had a moisture content of 15 wt% (WB).

[0268] (dry)

[0269] The obtained filter cake was dried for 24 hours in a box dryer (PV-211 manufactured by Espec) adjusted to 60 degrees Celsius to obtain a dry PHA resin. The total nitrogen content of the obtained dry PHA resin was 500 ppm.

[0270] [Comparative Example 3]

[0271] The same operations as in Example 1 were performed up to the alkaline enzyme treatment 1.

[0272] The volume median particle size of the PHA in the PHA aqueous suspension was 10 μm when measured using a laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA.

[0273] (Centrifugal separation step 1)

[0274] The PHA aqueous suspension was introduced into a decanter (PTM006, manufactured by Basilisk Industries) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA aqueous cake. The yield per centrifugation was 90%, and the amount of wastewater per centrifugation was 2.5 times the amount of PHA in the supplied PHA aqueous suspension.

[0275] (Centrifugation followed by dilution)

[0276] An alkaline aqueous solution (pH 10-11) was added to the PHA aqueous cake obtained above to produce a PHA aqueous suspension with a solids concentration of 20%. The centrifugation step 1 and the dilution operation after centrifugation were performed twice. The water content of the PHA aqueous cake obtained in the second separation was 43%.

[0277] (Alkaline enzyme treatment 2)

[0278] The pH of the PHA aqueous suspension was adjusted to 11±0.2 using 30% sodium hydroxide. Thereafter, 0.10 phr of Esperase (Novozymes) was added as a proteolytic enzyme and maintained at 50°C for at least 2 hours while controlling the pH to 11 using 30% sodium hydroxide.

[0279] (Centrifugal separation step 2)

[0280] The enzyme-treated PHA aqueous suspension was introduced into a decanter (PTM006, manufactured by Basilisk Industries) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA aqueous cake. An alkaline aqueous solution (pH 10-11) was added to the recovered cake to produce a PHA aqueous suspension with a solids concentration of 20%. This operation was repeated three times, with the yield per centrifugation being 96%. The amount of water removed from each centrifugation was three times the amount of PHA in the supplied PHA aqueous suspension. The moisture content of the PHA aqueous cake obtained from the third separation was 43%. The total yield from the five centrifugal separations was 71.6%.

[0281] (pH adjustment)

[0282] Ion-exchanged water was added to the obtained PHA aqueous cake so as to have a solid content concentration of 20% by weight, and the temperature was raised to 40° C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0283] (Pressure Filtration)

[0284] The PHA aqueous suspension was filtered using a filter press (ISD model 360, manufactured by Ishigaki Corporation). After pressing at a pressure of 0.4 MPa to obtain a filter cake, the filter was pressed again at a pressure of 0.7 MPa. The air supply pressure was adjusted to 0.4 MPa and air was supplied for 20 minutes to obtain a filter cake. The resulting filter cake had a water content of 13.5 wt% (WB).

[0285] (dry)

[0286] The obtained filter cake was dried for 24 hours in a box dryer (PV-211 manufactured by Espec) adjusted to 60 degrees Celsius to obtain a dry PHA resin. The total nitrogen content of the obtained dry PHA resin was 600 ppm.

[0287] [Comparative Example 4]

[0288] The same operations as in Example 2 were performed until the dilution step after centrifugation.

[0289] (Alkaline enzyme treatment 2)

[0290] The pH of the PHA aqueous suspension was adjusted to 11±0.2 using 30% sodium hydroxide. Subsequently, 0.10 phr of Esperase (Novozymes) was added as a proteolytic enzyme, and the pH was maintained at 11 using 30% sodium hydroxide at 50°C for at least 2 hours. The volume median particle size of the PHA in the PHA aqueous suspension was 5 μm when measured using a HORIBA laser diffraction / scattering particle size distribution analyzer, LA-950.

[0291] (Centrifugal separation step 2)

[0292] The enzyme-treated PHA aqueous suspension was introduced into a decanter (PTM006, manufactured by Basilisk Industries) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA aqueous cake. An alkaline aqueous solution (pH 10-11) was added to the recovered cake to produce a PHA aqueous suspension with a solids concentration of 20%. This procedure was repeated three times, with the yield per centrifugation being 85%. The amount of wastewater per centrifugation was three times the amount of PHA in the supplied PHA aqueous suspension. The moisture content of the PHA aqueous cake obtained from the third centrifugation was 43%.

[0293] The total yield of the six centrifugal separations, three times using a disc separator and three times using a decanter, was 60.4%.

[0294] (pH adjustment)

[0295] Ion-exchanged water was added to the obtained PHA aqueous cake so as to have a solid content concentration of 20% by weight, and the temperature was raised to 40° C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0296] (Pressure Filtration)

[0297] The PHA aqueous suspension was filtered using a filter press (ISD model 360, manufactured by Ishigaki Corporation). After pressing at a pressure of 0.4 MPa to obtain a filter cake, the filter was pressed again at a pressure of 0.7 MPa. The air flow pressure was adjusted to 0.4 MPa and air flow was continued for 20 minutes to obtain a filter cake. The resulting filter cake had a moisture content of 14 wt% (WB).

[0298] (dry)

[0299] The obtained filter cake was dried for 24 hours in a box dryer (PV-211 manufactured by Espec) adjusted to 60 degrees Celsius to obtain a dry PHA resin. The total nitrogen content of the obtained dry PHA resin was 700 ppm.

[0300] 〔result〕

[0301] Table 1 shows the measurement results of Examples 1 to 3 and Comparative Examples 1 to 4.

[0302] [Table 1]

[0303]

[0304] As can be seen from Table 1, compared with Comparative Examples 1 to 4, the total yield in the washing process is higher for any one of Examples 1 to 3. In addition, there is basically no difference in the amount of washing wastewater between Examples 1 to 3 and Comparative Examples 1 to 4. Therefore, according to this manufacturing method, it is shown that the total yield of PHA in the washing process can be increased with less washing wastewater than in the past.

[0305] Industrial applicability

[0306] According to the present invention, it is possible to produce PHA with a high yield, less impurities, high purity, and a high composition ratio of hydroxyalkanoate units other than 3HB units without increasing the amount of washing water (wastewater). In addition, therefore, the present invention can be suitably used in agriculture, fishery, forestry, horticulture, medicine, hygiene products, clothing, non-clothing, packaging, automobiles, building materials, and other fields.< / pha>

Claims

1. A method for manufacturing a polyhydroxyalkanoate, wherein, the polyhydroxyalkanoate is a polyhydroxybutyrate copolymer with a composition ratio of 3-hydroxybutyrate units / hydroxyalkanoate units other than 3-hydroxybutyrate units of 80 / 20 to 88 / 12 (mol / mol), as the culture solution, a culture solution containing cells containing the polyhydroxyalkanoate is used, the method for manufacturing the polyhydroxyalkanoate includes the following steps (d), (e), and (b): (d) A step of performing an enzyme treatment by adding a glycosidase to the culture solution; (e) A step of adding an alkaline protease to the culture solution obtained in step (d) and performing an enzyme treatment on the cells; (b) A step of adding an alkaline aqueous solution to the culture solution or the aqueous suspension obtained through step (e) to adjust the pH to 10.0 to 12.0, and adding a surfactant at any stage before, during, or after the adjustment; and, after steps (d), (e), and (b), it includes: (f) A step of solid-liquid separating the obtained aqueous suspension by a decanter centrifuge and recovering the cake containing the polyhydroxyalkanoate.

2. The method for manufacturing a polyhydroxyalkanoate according to claim 1, wherein, in step (f), the volume median diameter of the polyhydroxyalkanoate in the polyhydroxyalkanoate aqueous suspension is 13 to 500 μm.

3. The method for manufacturing a polyhydroxyalkanoate according to claim 1 or 2, which further includes: (g) A step of adjusting the pH of the polyhydroxyalkanoate aqueous suspension obtained by adding an aqueous solution to the cake containing the polyhydroxyalkanoate obtained in step (f) to 2.5 to 4.0; and (h) A step of dehydrating and drying the polyhydroxyalkanoate aqueous suspension by a filter press after step (g).

4. The method for manufacturing a polyhydroxyalkanoate according to claim 3, wherein, in step (h), the dehydration by the filter press is performed at a pressure of 0.2 to 1.0 MPa.

5. The method for manufacturing a polyhydroxyalkanoate according to claim 3, wherein, in step (h), the polyhydroxyalkanoate aqueous suspension is further dehydrated by blowing air.

6. The method for manufacturing a polyhydroxyalkanoate according to claim 1 or 2, wherein, the weight average molecular weight of the polyhydroxybutyrate copolymer is 150,000 to 800,000.

7. The method for manufacturing a polyhydroxyalkanoate according to claim 1 or 2, wherein, the order of the steps of performing the treatments of steps (d), (e), and (b) is: performing steps (d) and (e) after performing step (b), after performing step (b) and before performing step (d), the method for manufacturing the polyhydroxyalkanoate further includes: (c) a step of solid-liquid separating the obtained aqueous suspension by a disk centrifuge and recovering the polyhydroxyalkanoate aqueous suspension.

8. The method for manufacturing a polyhydroxyalkanoate according to claim 7, which further includes: In the step (c), solid-liquid separation is carried out using a disk centrifuge until the conductivity of the polyhydroxyalkanoate aqueous suspension reaches the range of 150 to 400 mS / m, and the step of recovering the polyhydroxyalkanoate aqueous suspension.

9. The method for producing polyhydroxyalkanoate according to claim 7, further comprising, before carrying out the step (c): (a) A step of performing an enzymatic treatment by adding an alkaline proteolytic enzyme.

10. The method for producing polyhydroxyalkanoate according to claim 1 or 2, comprising, before carrying out the step (e): A step of inactivating the microbial cells containing the polyhydroxyalkanoate.

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