Method for producing polyhydroxyalkanoate

Abstract

The present invention addresses the problem of providing a new method for producing PHA by which PHA having a small amount of impurities and a high degree of purity can be produced at high yield without increasing the amount of washing water. The problem is resolve by a method for producing polyhydroxyalkanoate that uses a culture solution containing bacterial cells containing a PHB copolymer having a specific composition ratio, said method comprising: (d) a step for enzymatically treating the culture solution; (e) a step for enzymatically treating the bacterial cells in the culture solution obtained in step (d); (b) a step for adjusting the pH of the culture solution and adding a surfactant; and (f) a step for subjecting the obtained aqueous suspension to solid-liquid separation to recover PHA-containing cake.

Description

Method for producing polyhydroxyalkanoic acid

[0001] The present invention relates to a method for producing polyhydroxyalkanoic acid.

[0002] Polyhydroxyalkanoic acid (hereinafter sometimes referred to as "PHA") is known to be biodegradable. PHA produced by microorganisms accumulates within the microbial cells. Therefore, in order to use PHA as a plastic, a process of separating and purifying PHA from the microbial cells is required. In the process of separating and purifying PHA, biologically derived components other than PHA are solubilized, and then PHA is extracted from the resulting aqueous suspension. At this time, separation procedures such as centrifugation, filtration, and drying are performed.

[0003] Known methods for producing PHA include those described in, for example, Patent Documents 1 to 3. Patent Document 1 discloses a method for obtaining a highly purified polyhydroxybutyric acid (PHB) copolymer by spray drying followed by re-washing. Patent Document 2 discloses a method for obtaining a PHB copolymer, which includes a step of disc-centrifuging a culture broth and then subjecting it to a decanter. Patent Document 3 discloses a method for producing an aqueous PHA suspension with a low impurity content and excellent dispersibility from a PHA with a low degree of crystallinity.

[0004] International Publication No. 2022 / 091685 Chinese Patent Application Publication No. 202111408282 Japanese Patent Application Publication No. 2019-097518

[0005] The methods described in Patent Documents 1 and 3 are excellent in that they yield a highly purified PHB copolymer with few impurities, but there is room for improvement in terms of reducing the amount of washing water (wastewater).Furthermore, the method described in Patent Document 2 has the problem of low yield.

[0006] Therefore, an object of the present invention is to provide a new method for producing PHA, which can produce highly purified PHA with few impurities in a high yield without increasing the amount of wash water (wastewater).In particular, an object of the present invention is to provide a new method for producing, among the above-mentioned PHAs, a polyhydroxybutyric acid copolymer (hereinafter sometimes referred to as a "PHB copolymer") having a composition ratio of 3-hydroxybutyrate (hereinafter sometimes referred to as "3HB") units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80 / 20 to 88 / 12 (mol / mol).

[0007] As a result of intensive research to solve the above-mentioned problems, the present inventors have discovered that a highly purified PHA with few impurities can be produced in a high yield without increasing the amount of wash water (wastewater) by including specific steps in a specific order in a production process for PHA having a composition ratio of a specific 3HB unit / hydroxyalkanoate unit other than 3HB unit, and by including a step of separation using a decanter-type centrifuge, and have thus completed the present invention.

[0008] Therefore, one aspect of the present invention is a method for producing a PHA, wherein the PHA is a PHB copolymer having a composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units of 80 / 20 to 88 / 12 (mol / mol), and a culture solution containing bacterial cells containing the PHA is used as a culture solution, and the method comprises the following steps (d) and (e), and (b): (d) adding glucosidase to the culture solution to perform an enzymatic treatment; (e) adding an alkaline protease to the culture solution obtained in step (d) to perform an enzymatic treatment of the bacterial cells; (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 either before, simultaneously with, or after the adjustment; and after steps (d) and (e), and (b), (f) subjecting the obtained aqueous suspension to solid-liquid separation using a decanter-type centrifuge to recover a PHA-containing cake (hereinafter referred to as "this production method").

[0009] According to one aspect of the present invention, a new method for producing PHA can be provided, which can produce highly purified polyhydroxyalkanoic acid with few impurities in good yield without increasing the amount of wash water (wastewater).

[0010] An embodiment of the present invention will be described in detail below. In this specification, unless otherwise specified, "A to B" representing a numerical range means "A or more, B or less." In addition, all documents described in this specification are incorporated herein by reference.

[0011] [1. Overview of the Invention] In purifying a PHB copolymer having a specific 3HB unit / hydroxyalkanoate unit other than 3HB unit composition ratio, it has been difficult to obtain a PHB copolymer with high purity (i.e., a low nitrogen content). In order to reduce the nitrogen content of the PHB copolymer, glucosidase enzyme treatment is required. However, if peptidoglycan is destroyed by a glucosidase enzyme such as lysozyme, the primary particles will irreversibly aggregate. Therefore, the enzyme treatment makes it difficult to manipulate the PHB copolymer.

[0012] As described above, the method described in Patent Document 1 is an excellent technique for obtaining a highly purified PHB copolymer with few impurities, but there is room for improvement in that the amount of washing water (wastewater) increases because spray drying is performed twice. There is also room for improvement in the use of a dispersant. The method described in Patent Document 2 has a problem from an economic viewpoint due to its low yield.

[0013] Therefore, the present inventors have conducted extensive research to solve the above problems, and as a result have discovered for the first time that by including a separation step using a decanter centrifuge in the process for producing PHA having a specific composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units, it is possible to produce highly purified PHA with few impurities in a high yield without increasing the amount of wash water (wastewater).

[0014] Therefore, according to this production method, highly purified PHA with few impurities (especially PHA having a specific composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units) can be produced with high yield without increasing the amount of wash water (wastewater).

[0015] Furthermore, the above-described configuration can reduce the amount of plastic waste generated, thereby contributing to the achievement of Sustainable Development Goals (SDGs) such as Goal 12 "Ensure sustainable consumption and production patterns" and Goal 14 "Conserve and sustainably use the oceans and marine resources for sustainable development." The configuration of this manufacturing method will be described in detail below.

[0016] 2. PHA Production Method This production method includes the following steps (d), (e), (b), and (f) as essential steps. The PHA used in this production method is a PHB copolymer (hereinafter sometimes referred to as a "specific PHB copolymer") having a composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units of 80 / 20 to 88 / 12 (mol / mol). - Step (d): A step of adding glucosidase to the culture liquid to perform enzymatic treatment. - Step (e): A step of adding an alkaline protease to the culture liquid obtained in step (d) to perform enzymatic treatment of the bacterial cells. - Step (b): A step of adding an alkaline aqueous solution to the culture liquid or the aqueous suspension obtained in step (e) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment. - Step (f): A step of performing solid-liquid separation of the obtained aqueous suspension using a decanter-type centrifuge to recover a PHA-containing cake.

[0017] In this specification, the term "culture solution" refers to a solution containing bacterial cells containing PHA, which is a specific PHB copolymer. The term "aqueous suspension" refers to a solution containing the PHA after the bacterial cells in the culture solution have been treated with an alkaline protease. In other words, the culture solution becomes an aqueous suspension by undergoing step (e).

[0018] In this production method, steps (d) and (e) and step (b) can be performed in any order. In one embodiment of the present invention, the order of steps (d) and (e) and step (b) may be steps (d), (e), and (b), or steps (b), (d), and (e). That is, step (e) may be performed immediately after step (d), and step (b) may be performed before step (d) or after step (e). Since the activity of some glucosidases decreases in alkaline solutions or in the presence of surfactants, the order of steps (d), (e), and (b) is preferred.

[0019] In this production method, step (f) is performed after steps (d) and (e), and (b). By performing step (f) after step (e), the cell walls of the bacterial cells contained in the aqueous suspension are destroyed, and the primary particles of the PHA are aggregated, thereby improving the yield of PHA during solid-liquid separation using a decanter centrifuge. Furthermore, by performing step (f) after step (d), the cell membranes of the bacterial cells contained in the aqueous suspension are destroyed, and the primary particles of the PHA are sufficiently aggregated, and the nitrogen content of the PHA can also be reduced.

[0020] In one embodiment of the present invention, the production method may further include the following steps: Step (c): A step of subjecting the obtained aqueous suspension to solid-liquid separation using a disc centrifuge and recovering an aqueous PHA suspension; Step (a): A step of adding an alkaline protease to perform an enzymatic treatment; Step (g): A step of adjusting the pH of the aqueous polyhydroxyalkanoic acid suspension obtained by adding an aqueous solution to the PHA-containing cake obtained in step (f) to 2.5 to 4.0; Step (h): A step of dehydrating and drying the aqueous PHA suspension using a filter press after step (g).

[0021] It is preferable that step (c) is carried out before steps (d) and (e) and after step (b), and it is preferable that step (a) is carried out before steps (d), (e), and (b) and before step (f).

[0022] In one embodiment of the present invention, each step may be performed multiple times for various purposes.

[0023] More specifically, the present production method can be carried out in the following order of steps, for example, but is not limited to this: (1) step (d), step (e), step (b), step (f) (2) step (b), step (d), step (e), step (f) (3) step (d), step (e), step (b), step (f), step (g), step (h) (4) step (b), step (d), step (e), step (f), step (g), step (h) (5) step (b), step (c), step (d), step (e), step (f), step (g), step (h) (6) step (a), step (b), step (c), step (d), step (e), step (f), step (g), step (h) (7) step (b), step (a), step (c), step (d), step (e), step (f), step (g), step (h) (8) Step (d), Step (e), Step (b), Step (f), Step (a), Step (f), Step (g), Step (h) (9) Step (a), Step (b), Step (c), Step (d), Step (e), Step (f)

[0024] For example, in the case of (8) above, the content of impurities contained in the PHB copolymer and the amount of wastewater can be reduced, and since the drying step is performed only once, the energy required for production is low. Furthermore, no dispersant is used. Furthermore, in the case of (9) above, the content of impurities contained in the PHB copolymer can be further reduced compared to the case of (1) above, and the overall process is shorter, and the drying step is performed only once. Furthermore, in the case of (6) above, since a filter press filtration step (including air blowing) is performed, the water content before drying is lower than that of the PHB copolymer of (2) above.

[0025] For convenience of explanation, the steps will be described below in the order of steps (d), (e), (b), (f), (c), (a), (g), and (h).

[0026] (2-1. Step (d)) Step (d) is a step of adding glucosidase to a culture solution containing bacterial cells containing PHA, which is a specific PHB copolymer, to perform an enzymatic treatment.

[0027] <PHA> The PHA in this production method is a PHB copolymer composed of 3HB and a hydroxyalkanoate other than 3HB.

[0028] Examples of hydroxyalkanoates other than 3HB include 3-hydroxyhexanoate (3HH), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 3-hydroxyoctanoate (3HO), 3-hydroxyoctadecanoate (3HOD), and 3-hydroxydecanoate (3HD).

[0029] A preferred example of a PHB copolymer is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (hereinafter, sometimes referred to as "P3HB3HH"). However, the present invention is not limited to this. For convenience of explanation, the following description will be mainly based on P3HB3HH as a representative example.

[0030] By changing the composition ratio of the repeating units 3HB and 3HH, the melting point and degree of crystallinity of P3HB3HH can be changed, and as a result, physical properties such as Young's modulus and heat resistance can be changed, making it possible to impart physical properties between those of polypropylene and polyethylene.

[0031] The PHB copolymer used in this production method has a composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units of 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 3HB units to hydroxyalkanoate units other than 3HB units is 88 / 12 (mol / mol) or less, sufficient hardness is obtained, and when it is 80 / 20 (mol / mol) or more, sufficient flexibility is obtained. The ratio of each repeating unit can be determined by the method described in paragraph

[0047] of WO 2013 / 147139.

[0032] 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, but is preferably 150,000 to 800,000, more preferably 200,000 to 700,000, and even more preferably 250,000 to 600,000. A weight-average molecular weight of 150,000 or more provides sufficient mechanical properties, while a weight-average molecular weight of 800,000 or less provides a sufficient crystallization rate and achieves good moldability. The weight-average molecular weight of the P3HB resin can be determined by gel permeation chromatography (GPC) (Shodex GPC-101 manufactured by Showa Denko K.K.) using a polystyrene gel (Shodex K-804 manufactured by Showa Denko K.K.) as a column and chloroform as the mobile phase, and expressed as the molecular weight in terms of polystyrene.

[0033] <Bacterial Cells> The bacterial cells used in step (d) are not particularly limited as long as they are microorganisms capable of producing PHB copolymers intracellularly. For example, microorganisms isolated from nature and deposited in a depository institution for strains (e.g., IFO, ATCC, etc.), or mutants and transformants prepared from them, can be used. For example, the first bacterial cell to produce P3HB, an example of a PHB copolymer, was Bacillus megaterium, discovered in 1925. Other examples include naturally occurring microorganisms such as Cupriavidus necator (formerly classified as Alcaligenes eutrophus and Ralstonia eutropha) and Alcaligenes latus. It is known that PHB copolymers accumulate intracellularly in these microorganisms.

[0034] Examples of bacteria that produce copolymers of hydroxybutyrate and other hydroxyalkanoates, which are examples of PHB copolymers, include Aeromonas caviae, which produces P3HB3HV and P3HB3HH, and Alcaligenes eutrophus, which produces P3HB4HB. In particular, with regard to P3HB3HH, Alcaligenes eutrophus AC32 strain (FERM BP-6038) (T. Fukui, Y. Doi, J. Bateriol., 179, pp. 4821-4830 (1997)) into which genes encoding PHB copolymer synthases have been introduced is more preferred. In addition to the above, the bacterial cells may be genetically modified microorganisms into which various PHB copolymer synthesis-related genes have been introduced according to the PHB copolymer to be produced.

[0035] The PHB copolymer can also be produced by the method described in, for example, WO 2010 / 013483.

[0036] <Glucosidase> As used herein, the term "glucosidase" refers to an enzyme that has the activity of degrading (lysing) the cell wall (e.g., peptidoglycan) of a fungus. Glucosidase is not particularly limited as long as it falls within the scope of the above definition, and examples thereof include lysozyme, labia, β-N-acetylglucosaminidase, endolysin, autolysin, and the like. From an economical viewpoint, lysozyme, a general-purpose enzyme, is preferred.

[0037] In step (d), water may be added to the culture solution before adding the glucosidase to adjust the solids concentration of the aqueous suspension. The solids concentration of the aqueous suspension at this time is preferably 10 to 40 wt %, more preferably 20 to 40 wt %. When the solids concentration of the aqueous suspension is within the above range, the concentration of the aqueous suspension is not too high and the glucosidase is uniformly mixed.

[0038] The pH of the culture medium may be adjusted before adding the glucosidase. In this case, the adjustment method is not particularly limited, and examples thereof include adding an acid. The acid is not particularly limited, and may be either an organic acid or an inorganic acid, regardless of whether it is volatile. More specifically, examples of acids that can be used include sulfuric acid, hydrochloric acid, phosphoric acid, and acetic acid.

[0039] It is preferable that a step of drying the aqueous suspension is not carried out before step (d). That is, in the method for producing a polyhydroxybutyric acid copolymer according to one embodiment of the present invention, it is preferable that a step of drying the aqueous suspension is not carried out before step (d). Here, the drying may be a drying method described later. By not drying the aqueous suspension before step (d), irreversible aggregation of the PHB copolymer particles can be suppressed.

[0040] (2-2. Step (e)) Step (e) is a step of adding an alkaline protease to the culture solution obtained in step (d) to enzymatically treat the bacterial cells. By enzymatically treating the bacterial cells with the alkaline protease, the culture solution can be converted into an aqueous suspension.

[0041] <Alkaline protease> As used herein, the term "alkaline protease" refers to a protease that has the activity of decomposing proteins in an alkaline environment (for example, in a solution of pH 8.5).

[0042] In one embodiment of the present invention, the alkaline protease is not particularly limited as long as it has the activity of degrading proteins in an alkaline environment, and examples thereof include serine-specific proteases (e.g., subtilisin, chymotrypsin), cysteine-specific proteases (e.g., papain, bromelain), etc. From the viewpoints of versatility and economy, serine-specific proteases, particularly alcalase including subtilisin, are preferred. These may be used alone or in combination of two or more.

[0043] Commercially available alkaline protease enzymes can also be used, including, for example, "Alcalase" and "Esperase" manufactured by Novozyme; "Protin SD-AY10" and "Protease P 'Amano' 3SD" manufactured by Amano Enzyme Inc.; "Multifect PR6L" and "Optimase PR89L" manufactured by Danisco Japan Co., Ltd.; "Sumiteam MP" manufactured by Shin Nippon Chemical Industry Co., Ltd.; "Delvolase" manufactured by DSM Japan Co., Ltd.; "Bioprase OP," "Bioprase SP-20FG," and "Bioprase SP-4FG" manufactured by Nagase ChemteX Corporation; "Orientase 22BF" manufactured by HBI Corporation; and "Aroase XA-10" manufactured by Yakult Pharmaceutical Co., Ltd.

[0044] In step (e), when the bacterial cells are treated with an alkaline protease, the pH and temperature of the culture medium are preferably adjusted to match the optimum pH and temperature of the alkaline protease used. Furthermore, the pH in step (e) is preferably lower than the pH adjusted by adding the aqueous alkaline 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.

[0045] In one embodiment of the present invention, the optimal pH of the alkaline protease is not particularly limited as long as the alkaline protease has activity in an alkaline environment, but is, for example, 8.0 to 12.0, preferably 8.0 to 11.0, more preferably 8.0 to 10.0, even more preferably 8.0 to 9.0, and most preferably 8.5.

[0046] In one embodiment of the present invention, the optimum temperature of the alkaline protease is not particularly limited, but is preferably 70° C. or lower, more preferably 60° C. or lower, from the viewpoint that excessive heating is not required and thermal changes (thermal decomposition) of the PHB copolymer can be prevented. The lower limit of the optimum temperature is not particularly limited, but is preferably room temperature (e.g., 25° C.) or higher, from the viewpoint that excessive cooling is not required and it is economical.

[0047] In one embodiment of the present invention, the amount of alkaline protease added is not particularly limited, but 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 decomposed.

[0048] In step (e), it is preferable that substantially no glucosidase is added simultaneously with the alkaline protease. As used herein, "substantially no glucosidase is added" means that the amount of glucosidase added is 0.0005 phr or less, and in one embodiment, the amount of glucosidase added may be 0 phr. The glucosidase is not particularly limited, but examples include those described in the section on step (d).

[0049] In step (e), the bacterial cells containing the PHB copolymer are preferably inactivated. In other words, the present production method may include a step of inactivating the bacterial cells after step (d) and before step (e). The inactivation method is not particularly limited, but an example thereof includes a method in which a culture solution containing bacterial cells containing the PHB copolymer is heated and stirred at 60 to 70°C for 7 hours, as described in the Examples. After the heating and stirring treatment, the culture solution is preferably further cooled to a temperature suitable for step (e).

[0050] (2-3. Step (b)) Step (b) is a step of adding an alkaline aqueous solution to a culture solution containing bacterial cells containing PHA, which is a specific PHB copolymer, to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment.

[0051] Step (b) includes the following steps (b1) and (b2): Step (b1): Adding an alkaline aqueous solution to a culture solution containing bacterial cells containing PHA, which is a specific PHB copolymer, to adjust the pH to 10.0 to 12.0; and Step (b2): Adding a surfactant.

[0052] (Step (b1)) As described above, step (b1) is a step of adding an alkaline aqueous solution to a culture solution containing bacterial cells containing PHA, which is a specific PHB copolymer, to adjust the pH to 10.0 to 12.0. This step disperses and dissolves bacterial cell-derived impurities (nucleic acids, proteins, etc.), thereby allowing a highly pure PHB copolymer to be separated from the bacterial cells.

[0053] In one embodiment of the present invention, the alkaline aqueous solution is an aqueous solution containing a basic compound. The basic compound contained in the alkaline aqueous solution is not particularly limited, but examples thereof include alkali metal or alkaline earth metal hydroxides 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, and the like.

[0054] In one embodiment of the present invention, the basic compound contained in the alkaline aqueous solution is preferably an alkali metal hydroxide or an alkaline earth metal hydroxide, more preferably sodium hydroxide. The basic compounds may be used alone or in combination of two or more.

[0055] In step (b1), the pH is preferably adjusted to 10.2 to 11.8, more preferably 10.4 to 11.6, by adding an alkaline aqueous solution. Adjusting the pH to 10.0 or higher has the advantage of enabling decomposition and dissolution of bacterial components. Adjusting the pH to 12.0 or lower can prevent unintended damage to the bacterial cells.

[0056] The temperature in step (b1) is preferably less than 100° C., more preferably less than 80° C. There is no particular lower limit to the temperature, but it is preferably, for example, 40° C. or higher.

[0057] (Step (b2)) Step (b2) is a step of adding a surfactant to a culture solution containing bacterial cells containing PHA, which is a specific PHB copolymer. This step can efficiently treat cell membranes in particular and remove a larger amount of bacterial cell-derived impurities, allowing a more highly pure PHB copolymer to be separated from the bacterial cells.

[0058] In one embodiment of the present invention, the surfactant is not particularly limited, but examples thereof include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. Among these, anionic surfactants are preferred from the viewpoint of their high cell membrane removal ability. These surfactants may be used alone or in combination of two or more.

[0059] Examples of anionic surfactants include alkyl sulfates, alkylbenzene sulfonates, alkyl sulfate ester salts, alkenyl sulfate ester salts, alkyl ether sulfate ester salts, alkenyl ether sulfate ester salts, α-olefin sulfonates, α-sulfofatty acid salts, esters of α-sulfofatty acid salts, alkyl ether carboxylates, alkenyl ether carboxylates, amino acid surfactants, and N-acylamino acid surfactants. Among these, alkyl sulfate ester salts are preferred, and sodium dodecyl sulfate (SDS) is particularly preferred from the viewpoints of its high cell membrane removal ability and low cost. These surfactants may be used alone or in combination of two or more.

[0060] In step (b2), the amount of surfactant to be added is not particularly limited, and is, for example, 0.1 to 5.0% by weight, preferably 0.3 to 2.5% by weight, relative to the culture medium.

[0061] Step (b2) may be carried out before, simultaneously with, or after step (b1), and preferably after step (b1).

[0062] (2-4. Step (f)) Step (f) is a step of subjecting the obtained aqueous suspension to solid-liquid separation using a decanter-type centrifuge to recover a PHA-containing cake.

[0063] By using a decanter centrifuge in step (f), it is possible to dehydrate the aqueous suspension without passing through a narrow flow path, and therefore solid-liquid separation can be carried out without the risk of clogging. Furthermore, compared to a disc stack centrifuge or the like, the solid concentration of the aqueous suspension can be increased, and therefore the efficiency of each solid-liquid separation is high, and the amount of wastewater can be reduced.

[0064] Decanter centrifuges include, for example, horizontal and vertical types, but horizontal types that can process large amounts of liquid are preferred.

[0065] In step (f), the volume median diameter of the polyhydroxyalkanoic acid in the aqueous suspension obtained is preferably 13 to 500 μm, more preferably 15 to 400 μm, and even more preferably 19 to 300 μm. If the volume median diameter is within the above range, the yield in the washing step is excellent, and the amount of aqueous suspension that can be treated increases, making it economical. In step (f), the "volume median diameter of the polyhydroxyalkanoic acid" means the volume median diameter of the polyhydroxyalkanoic acid in the aqueous suspension aggregated in step (d).

[0066] The centrifugal force when introducing the aqueous suspension into the decanter centrifuge is not particularly limited, but may be, for example, 2000 to 4000 G, 2500 to 3500 G, or 2800 to 3200 G from the viewpoint of yield in the washing step and economic efficiency.

[0067] (2-5. Step (c)) Step (c) is a step of subjecting the obtained aqueous suspension to solid-liquid separation using a disc-type centrifuge to recover an aqueous PHA suspension.

[0068] In step (c), the PHA aqueous suspension is collected by any centrifugation method known in the art. The centrifugation method is not particularly limited, but examples thereof include centrifugation using a centrifugal settler, a centrifugal dehydrator, etc.

[0069] In step (c), it is preferable to repeat the steps of centrifuging the aqueous suspension, removing the supernatant, adding a solution to the precipitate, and then centrifuging again and removing the supernatant. This operation allows for a more concentrated and purified PHA aqueous suspension to be obtained. Here, the solution added after removing the supernatant is preferably an alkaline aqueous solution adjusted to the same pH as the culture solution.

[0070] In step (c), the solvent constituting the PHA aqueous suspension ("solvent" is also referred to as "aqueous medium") is not particularly limited, and may be water or a mixed solvent of water and an organic solvent. Furthermore, in the mixed solvent, the concentration of the organic solvent is not particularly limited as long as it is equal to or lower than the solubility of the organic solvent used in water. Furthermore, the organic solvent is not particularly limited, and examples thereof include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, pentanol, hexanol, and heptanol; ketones such as acetone and methyl ethyl ketone; ethers such as tetrahydrofuran and dioxane; nitriles such as acetonitrile and propionitrile; amides such as dimethylformamide and acetamide; dimethyl sulfoxide, pyridine, and piperidine. Among these, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, propionitrile, etc. are preferred because they are easily removed. Furthermore, methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone, etc. are more preferred because they are easily available. Furthermore, methanol, ethanol, and acetone are particularly preferred.

[0071] 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, even more preferably 30% by weight or more, and particularly preferably 50% by weight or more.

[0072] The PHA aqueous suspension in step (c) may contain other solvents, components derived from bacterial cells, compounds generated during purification, etc., as long as the essence of the present invention is not impaired.

[0073] In step (c), it is preferable to further include a step of performing solid-liquid separation until the electrical conductivity of the obtained aqueous suspension falls within the range of preferably 150 to 400 mS / m, more preferably 200 to 350 mS / m, and even more preferably 250 to 300 mS / m, and recovering the aqueous suspension. The electrical conductivity of the aqueous suspension is measured by the method described in the Examples.

[0074] (2-6. Step (a)) Step (a) is a step of adding an alkaline protease to perform an enzymatic treatment. Step (a) is preferably carried out before step (c).

[0075] For the alkaline protease in step (a), the description of <Alkaline protease> in step (e) above is applicable.

[0076] (2-7. Step (g)) Step (g) is a step of adding an aqueous solution to the PHA-containing cake obtained in step (f) and adjusting the pH to 2.5 to 4.0. Adjusting the pH to 2.5 or more and 4.0 or less has the advantage of making it easier to maintain the molecular weight of the PHA during processing and molding.

[0077] In step (g), the pH is preferably adjusted to 2.8 to 3.8, more preferably 3.0 to 3.5.

[0078] In step (g), the aqueous solution used to adjust the pH is not particularly limited, and for example, ion-exchanged water, an alkaline aqueous solution, an acidic aqueous solution, etc. can be used. Among these, ion-exchanged water and an acidic aqueous solution are preferred, and an acidic aqueous solution is more preferred. Among the acidic aqueous solutions, it is particularly preferred to add an aqueous sulfuric acid solution, etc. In one embodiment, the pH adjustment in step (g) may be performed by diluting the aqueous suspension.

[0079] (2-8. Step (h)) Step (h) is a step following step (g) in which the aqueous PHA suspension is dehydrated and dried using a filter press.

[0080] In step (h), the PHA aqueous suspension may be dehydrated by squeezing it with a filter press. When squeezing the PHA aqueous suspension, the pressure 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. The squeezing may be carried out only once, or may be carried out two or more times. When squeezing is carried out two or more times, the pressure from the second time onwards is preferably higher than that of the first time.

[0081] In step (h), the aqueous PHA suspension may be further dehydrated by air blowing. When drying is performed by air blowing, the pressure is not particularly limited, but is, for example, 0.01 to 1.5 MPa, preferably 0.05 to 1.3 MPa, and more preferably 0.10 to 1.0 MPa. The duration of air blowing is also not particularly limited as long as the aqueous PHA suspension can be sufficiently dried, but is, for example, 10 minutes to 1 hour, preferably 15 to 40 minutes, and more preferably 20 to 30 minutes.

[0082] In step (h), the filter cake obtained after dehydration may be dried by a known method. Drying can be carried out using, for example, a tray dryer, a spray dryer, a fluidized bed dryer, a drum dryer, etc., but it is preferable to use a tray dryer from the viewpoint of ease of operation. The temperature during drying is not particularly limited, and drying may be carried out, for example, at 40 to 80°C.

[0083] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0084] That is, one embodiment of the present invention is as follows: <1> A method for producing a polyhydroxyalkanoic acid, wherein the polyhydroxyalkanoic acid 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), and a culture solution containing bacterial cells containing the polyhydroxyalkanoic acid is used as a culture solution, and the method comprises the following steps (d) and (e), and (b): (d) a step of adding glucosidase to the culture solution to perform an enzymatic treatment, (e) a step of adding an alkaline protease to the culture solution obtained in step (d) to perform an enzymatic treatment of the bacterial cells, and (b) a step of 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 either before, simultaneously with, or after the adjustment; A method for producing polyhydroxyalkanoic acid, comprising, after steps (d) and (e) and (b), (f) subjecting the obtained aqueous suspension to solid-liquid separation using a decanter centrifuge to recover a polyhydroxyalkanoic acid-containing cake.<2> The method for producing polyhydroxyalkanoic acid according to <1>, wherein in step (f), the volume median diameter of polyhydroxyalkanoic acid in the polyhydroxyalkanoic acid aqueous suspension is 13 to 500 μm.<3> The method for producing polyhydroxyalkanoic acid according to <1> or <2>, further comprising: (g) a step of adding an aqueous solution to the polyhydroxyalkanoic acid-containing cake obtained in step (f) to dilute the polyhydroxyalkanoic acid aqueous suspension and adjusting the pH of the polyhydroxyalkanoic acid aqueous suspension to 2.5 to 4.0, and (h) a step of dehydrating and drying the polyhydroxyalkanoic acid suspension using a filter press after step (g). <4> The method for producing a polyhydroxyalkanoic acid according to <3>, wherein in the step (h), the dehydration using a filter press is carried out at a pressure of 0.2 to 1.0 MPa. <5> The method for producing a polyhydroxyalkanoic acid according to <3> or <4>, wherein in the step (h), the aqueous suspension of polyhydroxyalkanoic acid is further dehydrated by air blowing.<6> The method for producing polyhydroxyalkanoic acid according to any one of <1> to <5>, wherein the weight-average molecular weight of the polyhydroxybutyric acid copolymer is 150,000 to 800,000. <7> The method for producing polyhydroxyalkanoic acid according to any one of <1> to <6>, wherein the step of carrying out the treatments of steps (d) and (e) and (b) is carried out after step (b), and further comprises, after step (b) and before step (d), a step (c) of performing solid-liquid separation of the obtained aqueous suspension using a disc centrifuge and recovering the aqueous polyhydroxyalkanoic acid suspension. <8> The method for producing polyhydroxyalkanoic acid according to <7>, further comprising, in step (c), a step of performing solid-liquid separation using a disc centrifuge until the electrical conductivity of the aqueous polyhydroxyalkanoic acid suspension falls within the range of 150 to 400 mS / m and recovering the aqueous polyhydroxyalkanoic acid suspension. <9> The method for producing a polyhydroxyalkanoic acid according to <7> or <8>, further comprising, before carrying out the step (c), (a) a step of adding an alkaline protease to perform an enzymatic treatment. <10> The method for producing a polyhydroxyalkanoic acid according to any one of <1> to <9>, further comprising, before carrying out the step (e), a step of inactivating the bacterial cells containing the polyhydroxyalkanoic acid.

[0085] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples. In the examples, "P3HB3HH" is used as "PHA," and "PHA" in the examples can also be read as "P3HB3HH."

[0086] [Measurement Methods] Measurements in the Examples and Comparative Examples were carried out by the following methods: (Volume Median Diameter) The volume median diameter of PHA was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA.

[0087] (pH of PHA aqueous suspension) The pH was measured using a pH meter (9652-10D, manufactured by HORIBA) (Electrical conductivity of PHA aqueous suspension) The electrical conductivity was measured using an electrical conductivity meter (9382-10D, manufactured by HORIBA).

[0088] (Solid Content Concentration) The solid content concentration of the PHA aqueous suspension was measured using a meter (manufactured by A&D Co., Ltd.) The PHA aqueous suspension was heated at 105°C until the weight change rate fell below 0.05% / min, and the solid content concentration was calculated from the weight change of the PHA aqueous suspension before and after heating.

[0089] (Measurement of Total Nitrogen Amount) The total nitrogen amount of the PHA powder was measured using a trace total nitrogen analyzer TN-2100H (Nitto Seiko Analytech Co., Ltd.).

[0090] (Measurement of PHA Yield in Separation Step) The PHA yield in the separation step was calculated using the following formula: 1 - ([Amount of PHA solids in the PHA aqueous suspension discharged in the separation step] / [Amount of PHA solids in the PHA aqueous suspension supplied to the separation step]). (Total PHA Yield in Separation Step) The total PHA yield in the separation step was calculated as the product of all the yields in the separations in each separation step. For example, if the yields in the first, second, third, and fourth runs are all 98%, the result is 92.2% according to the formula below. Total Yield in Separation Step = 98 / 100 x 98 / 100 x 98 / 100 x 98 / 100 = 0.922 [-] (Amount of PHA solids in the PHA aqueous suspension) After thorough stirring, the aqueous PHA suspension was dispensed in an amount of 10.0 g into a 15 mL Falcon tube and separated in a tabletop centrifuge at 9000 G for 10 minutes. The supernatant was then discarded, and ethanol was added to the remaining precipitate until it reached 10 mL, and the precipitate was dispersed using a shaker. The mixture was then placed in a tabletop centrifuge and separated at 9000 G for 5 minutes. After discarding the supernatant, the mixture was placed in a vacuum dryer at 70 ° C., and after 24 hours, the weight of the remaining PHA solids was measured. The resulting weight was used as the concentration of the PHA aqueous suspension. The product of the concentration of the PHA aqueous suspension and the weight of the PHA aqueous suspension supplied to the separation step or the PHA aqueous suspension discharged in the separation step was used as the PHA solids content.

[0091] Example 1 (Preparation of fungal cell culture solution) Ralstonia eutropha described in International Publication No. 2019 / 142717 was cultured by the method described in paragraphs

[0041] to

[0048] of the same document to obtain a fungal cell culture solution containing PHA-containing fungal cells. Ralstonia eutropha is currently classified as Capriavidus necator. The composition ratio of the repeating units of PHA (composition ratio of 3HB units / PHA units other than 3HB units) was 84 / 16 (mol / mol).

[0092] (Inactivation) The bacterial cell culture solution obtained above was sterilized by heating and stirring at an internal temperature of 60 to 70°C for 7 hours, and then ion-exchanged water at room temperature was added to dilute the solution, followed by cooling to 55°C to obtain an inactivated culture solution.

[0093] (Alkali Treatment) A 30% aqueous solution of sodium hydroxide was added to the inactivated culture solution obtained above to adjust the pH to 9.5. The solution was maintained at 70°C for 6 hours by continuously adding the 30% aqueous solution of sodium hydroxide, thereby obtaining an aqueous PHA suspension.

[0094] (Neutralization Treatment and Cell Wall-Degrading Enzyme Treatment) 10% sulfuric acid was added to the PHA aqueous suspension obtained above to adjust the pH to 6.5. After the addition of sulfuric acid, 0.05 phr of lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), a cell wall-degrading enzyme, was added, and the mixture was maintained at 50°C for 2 hours.

[0095] (Alkaline Enzyme Treatment 1) The pH of the aqueous PHA suspension was adjusted to 8.5±0.2 using 30% sodium hydroxide, followed by adding 0.2 phr of Alcalase (Novozymes), a protease, and maintaining the pH at 8.5 with 30% sodium hydroxide at 50° C. for at least 2 hours.

[0096] (Bacteriolysis) 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 dodecyl sulfate (manufactured by Kao) relative to the weight of the PHA aqueous suspension was added, followed by maintaining at 45°C for 2 hours. The volume median diameter of PHA in this PHA aqueous suspension was measured using a laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA and found to be 19 µm. The solids concentration of the PHA aqueous suspension at this time was 20%.

[0097] (Centrifugation step 1) The above PHA aqueous suspension was introduced into a decanter (PTM006 type, manufactured by Tomoe Kogyo) at a rate of 150 L / hr and centrifuged at 3100 G to recover a wet cake of PHA. The yield per centrifugation was 98%, and the amount of wastewater per centrifugation was three times the amount of PHA in the supplied PHA aqueous suspension.

[0098] (Dilution after centrifugation) An alkaline aqueous solution (pH 10 to 11) was added to the PHA hydrous 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 twice. The water content of the PHA hydrous cake obtained after the second centrifugation was 46%.

[0099] (Alkaline Enzyme Treatment 2) The pH of the aqueous PHA suspension was adjusted to 11±0.2 using 30% sodium hydroxide, followed by the addition of 0.10 phr of Esperase (Novozymes), a protease, and the mixture was kept at 50° C. for 2 hours or more while being controlled at pH 11 with 30% sodium hydroxide.

[0100] (Centrifugation Step 2) The PHA aqueous suspension after the enzyme treatment was introduced into a decanter (PTM006 type, manufactured by Tomoe Kogyo) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA wet cake. An alkaline aqueous solution (pH 10-11) was added to the recovered cake to prepare a PHA aqueous suspension with a solids concentration of 20%. The above operation was performed three times, with a yield per centrifugation of 98% and a drainage volume per centrifugation of three times the amount of PHA in the supplied PHA aqueous suspension. The water content of the PHA wet cake obtained in the third centrifugation was 43%. The overall yield for a total of five centrifugations was 90.3%.

[0101] (pH Adjustment) Ion-exchanged water was added to the obtained PHA water cake so that the solid content concentration was 20% by weight, and the temperature was raised to 40° C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0102] (Filter press filtration) The PHA aqueous suspension was filtered using a filter press (ISD type 360, manufactured by Ishigaki Co., Ltd.). After pressing at a pressure of 0.4 MPa to obtain a filter cake, the filter cake was pressed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa, followed by air blowing for 20 minutes to obtain a filter cake. The water content of the obtained filter cake was 13.5 wt% (W.B.).

[0103] (Drying) The obtained filter cake was dried for 24 hours in a shelf dryer (PV-211, manufactured by Espec) adjusted to 60° C. to obtain a dried PHA resin. The total nitrogen content of the obtained dried PHA resin was 450 ppm.

[0104] Example 2 The same operations as in Example 1 were carried out up to the alkali treatment step.

[0105] (Alkaline Enzyme Treatment 1) The pH of the aqueous PHA suspension was adjusted to 8.5±0.2 using 30% sodium hydroxide, followed by adding 0.2 phr of Alcalase (Novozymes), a protease, and maintaining the pH at 8.5 with 30% sodium hydroxide at 50° C. for at least 2 hours.

[0106] (Bacteriolysis) 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 dodecyl sulfate (manufactured by Kao) was added relative to the weight of the PHA aqueous suspension, and the mixture was kept at 45°C for 2 hours.

[0107] (Centrifugation step 1) The above PHA aqueous suspension was introduced into a disk separator at a rate of 150 L / hr and centrifuged at 3100 G to perform solid-liquid separation until the electrical conductivity of the PHA aqueous suspension reached 290 mS / m, and a PHA wet 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.

[0108] (Dilution after centrifugation) An alkaline aqueous solution (pH 10 to 11) was added to the PHA hydrous 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 repeated three times. The water content of the PHA hydrous cake obtained after the third centrifugation was 64%.

[0109] (Neutralization Treatment and Cell Wall-Degrading Enzyme Treatment) 10% sulfuric acid was added to the PHA aqueous suspension obtained above to adjust the pH to 6.5. After adding sulfuric acid, 0.05 phr of lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme that degrades cell walls, was added, and the mixture was maintained at 50°C for 2 hours.

[0110] (Alkaline Enzyme Treatment 2) The pH of the above PHA aqueous suspension was adjusted to 11±0.2 using 30% sodium hydroxide. Thereafter, 0.10 phr of Esperase (manufactured by Novozymes), a protease, was added, and the mixture was held at 50° C. for 2 hours or more while controlling the pH to 11 with 30% sodium hydroxide. The volume median diameter of the PHA in this PHA aqueous suspension was measured using a laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA, and was found to be 25 μm.

[0111] (Centrifugation Step 2) The above-described enzyme-treated PHA aqueous suspension was introduced into a decanter (PTM006, manufactured by Tomoe Kogyo) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA wet cake. An alkaline aqueous solution (pH 10-11) was added to the recovered cake to prepare a PHA aqueous suspension with a solids concentration of 20%. The above procedure was performed three times, with a yield of 99% per centrifugation, and 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 wet cake obtained in the third centrifugation was 43%. The overall yield of a total of six centrifugations, three times using the disc separator and three times using the decanter, was 95.6%.

[0112] (pH Adjustment) Ion-exchanged water was added to the obtained PHA water cake so that the solid content concentration was 20% by weight, and the temperature was raised to 40° C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0113] (Filter press filtration) The PHA aqueous suspension was filtered using a filter press (ISD type 360, manufactured by Ishigaki Co., Ltd.). After pressing at a pressure of 0.4 MPa to obtain a filter cake, the filter cake was pressed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa, followed by air blowing for 20 minutes to obtain a filter cake. The water content of the obtained filter cake was 14 wt% (W.B.).

[0114] (Drying) The obtained filter cake was dried for 24 hours in a shelf dryer (PV-211, manufactured by Espec) adjusted to 60° C. to obtain a dried PHA resin. The total nitrogen content of the obtained dried PHA resin was 350 ppm.

[0115] Example 3 The same operations as in Example 2 were carried out up to the pH adjustment step.

[0116] (Dehydration step) The pH-adjusted PHA aqueous suspension was introduced into a decanter (PTM006 type, manufactured by Tomoe Kogyo) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA wet cake. The yield in the dehydration step was 99.9%, and the water content of the PHA wet cake was 35%.

[0117] (Drying) The obtained filter cake was dried for 24 hours in a shelf dryer (PV-211, manufactured by Espec) adjusted to 60° C. to obtain a dried PHA resin. The total nitrogen content of the obtained dried PHA resin was 350 ppm.

[0118] Comparative Example 1 The same procedures as in Example 1 were carried out up to the neutralization treatment and cell wall-decomposing enzyme treatment.

[0119] (Bacteriolysis) 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 dodecyl sulfate (manufactured by Kao) relative to the weight of the PHA aqueous suspension was added, followed by maintaining the suspension at 45° C. for 2 hours. The volume median diameter of the PHA in this PHA aqueous suspension was measured using a laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA, and was found to be 8 μm.

[0120] (Centrifugation step 1) The above PHA aqueous suspension was introduced into a decanter (PTM006 type, manufactured by Tomoe Kogyo) at a rate of 150 L / hr and centrifuged at 3100 G to recover a wet cake of PHA. The yield per centrifugation was 90%, and the amount of wastewater per centrifugation was 2.5 times the amount of PHA in the supplied aqueous PHA suspension.

[0121] (Dilution after centrifugation) An alkaline aqueous solution (pH 10 to 11) was added to the PHA hydrous 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 twice. The water content of the PHA hydrous cake obtained after the second centrifugation was 43%.

[0122] (Alkaline Enzyme Treatment 2) The pH of the aqueous PHA suspension was adjusted to 11±0.2 using 30% sodium hydroxide, followed by the addition of 0.10 phr of Esperase (Novozymes), a protease, and the mixture was kept at 50° C. for 2 hours or more while being controlled at pH 11 with 30% sodium hydroxide.

[0123] (Centrifugation Step 2) The PHA aqueous suspension after the enzyme treatment was introduced into a decanter (PTM006, manufactured by Tomoe Kogyo) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA wet cake. An alkaline aqueous solution (pH 10-11) was added to the recovered cake to prepare a PHA aqueous suspension with a solids concentration of 20%. The above procedure was carried out three times, with a yield of 98% per centrifugation, and 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 wet cake obtained in the third centrifugation was 43%. The overall yield from a total of five centrifugations was 76.2%.

[0124] (pH Adjustment) Ion-exchanged water was added to the obtained PHA water cake so that the solid content concentration was 20% by weight, and the temperature was raised to 40° C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0125] (Filter press filtration) The PHA aqueous suspension was filtered using a filter press (ISD type 360, manufactured by Ishigaki Co., Ltd.). After pressing at a pressure of 0.4 MPa to obtain a filter cake, the filter cake was pressed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa, followed by air blowing for 20 minutes to obtain a filter cake. The water content of the obtained filter cake was 13.5 wt% (W.B.).

[0126] (Drying) The obtained filter cake was dried for 24 hours in a shelf dryer (PV-211, manufactured by Espec) adjusted to 60° C. to obtain a dried PHA resin. The total nitrogen content of the obtained dried PHA resin was 550 ppm.

[0127] Comparative Example 2 The same procedures as in Example 2 were carried out up to the neutralization treatment and cell wall-degrading enzyme treatment. The volume median diameter of the PHA in this PHA aqueous suspension was measured using a laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA, and was found to be 10 μm.

[0128] (Centrifugation Step 2) The PHA aqueous suspension after the enzyme treatment was introduced into a decanter (PTM006, manufactured by Tomoe Kogyo) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA wet cake. An alkaline aqueous solution (pH 10-11) was added to the recovered cake to prepare a PHA aqueous suspension with a solids concentration of 20%. The above procedure was carried out three times, with a yield of 90% per centrifugation, and 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 wet cake obtained in the third centrifugation was 43%. The overall yield of a total of six centrifugations, three times using the disc separator and three times using the decanter, was 71.8%.

[0129] (pH Adjustment) Ion-exchanged water was added to the obtained PHA water cake so that the solid content concentration was 20% by weight, and the temperature was raised to 40° C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0130] (Filter press filtration) The PHA aqueous suspension was filtered using a filter press (ISD type 360, manufactured by Ishigaki Co., Ltd.). After compressing at a pressure of 0.4 MPa to obtain a filter cake, the filter cake was compressed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa, followed by air blowing for 20 minutes to obtain a filter cake. The water content of the obtained filter cake was 15 wt% (W.B.).

[0131] (Drying) The obtained filter cake was dried for 24 hours in a shelf dryer (PV-211, manufactured by Espec) adjusted to 60°C to obtain a dried PHA resin. The total nitrogen content of the obtained dried PHA resin was 500 ppm.

[0132] Comparative Example 3 The same procedures as in Example 1 were carried out up to alkaline enzyme treatment 1.

[0133] The volume median diameter of the PHA in this PHA aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA, and was found to be 10 μm.

[0134] (Centrifugation step 1) The above PHA aqueous suspension was introduced into a decanter (PTM006 type, manufactured by Tomoe Kogyo) at a rate of 150 L / hr and centrifuged at 3100 G to recover a wet cake of PHA. 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.

[0135] (Dilution after centrifugation) An alkaline aqueous solution (pH 10 to 11) was added to the PHA hydrous 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 twice. The water content of the PHA hydrous cake obtained in the second separation was 43%.

[0136] (Alkaline Enzyme Treatment 2) The pH of the aqueous PHA suspension was adjusted to 11±0.2 using 30% sodium hydroxide, followed by the addition of 0.10 phr of Esperase (Novozymes), a protease, and the mixture was kept at 50° C. for 2 hours or more while being controlled at pH 11 with 30% sodium hydroxide.

[0137] (Centrifugation Step 2) The PHA aqueous suspension after the enzyme treatment was introduced into a decanter (PTM006, manufactured by Tomoe Kogyo) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA wet cake. An alkaline aqueous solution (pH 10-11) was added to the recovered cake to prepare a PHA aqueous suspension with a solids concentration of 20%. The above operation was performed three times, with a yield per centrifugation of 96% and a drainage volume per centrifugation of three times the amount of PHA in the supplied PHA aqueous suspension. The moisture content of the PHA wet cake obtained in the third separation was 43%. The overall yield for a total of five centrifugations was 71.6%.

[0138] (pH Adjustment) Ion-exchanged water was added to the obtained PHA water cake so that the solid content concentration was 20% by weight, and the temperature was raised to 40° C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0139] (Filter press filtration) The PHA aqueous suspension was filtered using a filter press (ISD type 360, manufactured by Ishigaki Co., Ltd.). After pressing at a pressure of 0.4 MPa to obtain a filter cake, the filter cake was pressed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa, followed by air blowing for 20 minutes to obtain a filter cake. The water content of the obtained filter cake was 13.5 wt% (W.B.).

[0140] (Drying) The obtained filter cake was dried for 24 hours in a shelf dryer (PV-211, manufactured by Espec) adjusted to 60° C. to obtain a dried PHA resin. The total nitrogen content of the obtained dried PHA resin was 600 ppm.

[0141] Comparative Example 4 After centrifugation, the same procedures as in Example 2 were carried out up to the dilution step.

[0142] (Alkaline Enzyme Treatment 2) The pH of the above PHA aqueous suspension was adjusted to 11±0.2 using 30% sodium hydroxide. Thereafter, 0.10 phr of Esperase (manufactured by Novozymes), a protease, was added, and the mixture was held at 50° C. for 2 hours or more while controlling the pH to 11 with 30% sodium hydroxide. The volume median diameter of the PHA in this PHA aqueous suspension was measured using a laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA, and was found to be 5 μm.

[0143] (Centrifugation Step 2) The PHA aqueous suspension after the enzyme treatment was introduced into a decanter (PTM006 type, manufactured by Tomoe Kogyo) at a rate of 150 L / hr and centrifuged at 3100 G to recover a PHA wet cake. An alkaline aqueous solution (pH 10-11) was added to the recovered cake to prepare a PHA aqueous suspension with a solids concentration of 20%. The above operation was carried out three times, with a yield per centrifugation of 85% and a drainage volume per centrifugation of three times the amount of PHA in the supplied PHA aqueous suspension. The water content of the PHA wet cake obtained in the third centrifugation was 43%.

[0144] The overall yield of six centrifugations, three with the disc separator and three with the decanter, was 60.4%.

[0145] (pH Adjustment) Ion-exchanged water was added to the obtained PHA water cake so that the solid content concentration was 20% by weight, and the temperature was raised to 40° C. Then, 10% sulfuric acid was added to adjust the pH to 3.5.

[0146] (Filter press filtration) The PHA aqueous suspension was filtered using a filter press (ISD type 360, manufactured by Ishigaki Co., Ltd.). After pressing at a pressure of 0.4 MPa to obtain a filter cake, the filter cake was pressed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa, followed by air blowing for 20 minutes to obtain a filter cake. The water content of the obtained filter cake was 14 wt% (W.B.).

[0147] (Drying) The obtained filter cake was dried for 24 hours in a shelf dryer (PV-211, manufactured by Espec) adjusted to 60° C. to obtain a dried PHA resin. The total nitrogen content of the obtained dried PHA resin was 700 ppm.

[0148] [Results] The measurement results for Examples 1 to 3 and Comparative Examples 1 to 4 are shown in Table 1.

[0149]

[0150] As can be seen from Table 1, Examples 1 to 3 all had higher overall yields in the washing step compared to Comparative Examples 1 to 4. Furthermore, there was almost no difference in the amount of washing wastewater between Examples 1 to 3 and Comparative Examples 1 to 4. Therefore, it was demonstrated that the present production method can improve the overall yield of PHA in the washing step with less washing wastewater than conventional methods.

[0151] According to the present invention, it is possible to produce a highly purified PHA with few impurities and a high composition ratio of hydroxyalkanoate units other than 3HB units with a high yield without increasing the amount of wash water (wastewater). Therefore, the present invention can be suitably used in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing, packaging, automobiles, building materials, and other fields.

Claims

1. 1. A method for producing a polyhydroxyalkanoic acid, comprising: the polyhydroxyalkanoic acid is a polyhydroxybutyric acid copolymer having a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80 / 20 to 88 / 12 (mol / mol); a culture medium containing the polyhydroxyalkanoic acid-containing fungus cells is used as the culture medium; The method comprises the following steps (d) and (e), and (b); (d) adding glucosidase to the culture solution to perform an enzyme treatment; (e) adding an alkaline protease to the culture solution obtained in the step (d) to enzymatically treat the bacterial cells; (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 either before, simultaneously with, or after the adjustment; After steps (d) and (e), and (b), (f) subjecting the obtained aqueous suspension to solid-liquid separation using a decanter-type centrifuge to recover a polyhydroxyalkanoic acid-containing cake, The step of carrying out the treatments of steps (d) and (e) and (b) is to carry out steps (d) and (e) after carrying out step (b), The method for producing polyhydroxyalkanoic acid further comprises, after carrying out step (b) and before carrying out step (d), a step (c) of subjecting the obtained aqueous suspension to solid-liquid separation using a disc centrifuge to recover an aqueous suspension of polyhydroxyalkanoic acid.

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

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

4. The method for producing a polyhydroxyalkanoic acid according to claim 3, wherein in the step (h), the dehydration by a filter press is carried out at a pressure of 0.2 to 1.0 MPa.

5. 4. The method for producing a polyhydroxyalkanoic acid according to claim 3, wherein in the step (h), the aqueous suspension of polyhydroxyalkanoic acid is further dehydrated by air blowing.

6. The method for producing a polyhydroxyalkanoic acid according to claim 1 or 2, wherein the weight average molecular weight of the polyhydroxybutyric acid copolymer is 150,000 to 800,000.

7. 3. The method for producing a polyhydroxyalkanoic acid according to claim 1 or 2, further comprising the step of performing solid-liquid separation using a disk centrifuge in the step (c) until the electrical conductivity of the aqueous polyhydroxyalkanoic acid suspension falls within the range of 150 to 400 mS / m, and recovering the aqueous polyhydroxyalkanoic acid suspension.

8. 3. The method for producing a polyhydroxyalkanoic acid according to claim 1, further comprising the step (a) of adding an alkaline protease to perform an enzymatic treatment before carrying out the step (c).

9. 3. The method for producing a polyhydroxyalkanoic acid according to claim 1, further comprising a step of inactivating the polyhydroxyalkanoic acid-containing bacterial cells before carrying out the step (e).