Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
  • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
  • C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
  • C08G63/88—Post-polymerisation treatment
  • C08G63/89—Recovery of the polymer
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L101/00—Compositions of unspecified macromolecular compounds
  • C08L101/16—Compositions of unspecified macromolecular compounds the macromolecular compounds being biodegradable
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/62—Carboxylic acid esters

Definitions

• the present invention relates to a method for producing polyhydroxyalkanoic acid.
• PHA Polyhydroxyalkanoic acid
• PHA Polyhydroxyalkanoic acid
• 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 subjecting a culture broth to disk centrifugation and then to a decanter.
• Patent Document 3 discloses a method for producing an aqueous PHA suspension with low impurity levels and excellent dispersibility from a PHA with a low degree of crystallinity.
• Patent Documents 1 and 3 are excellent in that they produce PHB copolymers with few impurities and a high degree of purification, but there is room for improvement in terms of reducing the amount of washing water (wastewater). Also, the method described in Patent Document 2 has the problem of low yield.
• the object of the present invention is to provide a new method for producing PHA that can produce highly refined PHA with few impurities at a high yield without increasing the amount of wash water (wastewater).
• the object of the present invention is to provide a new method for producing polyhydroxybutyric acid copolymer (hereinafter sometimes referred to as "PHB copolymer”), among the above-mentioned PHAs, in which the composition ratio of 3-hydroxybutyrate (hereinafter sometimes referred to as "3HB”) units to hydroxyalkanoate units other than 3-hydroxybutyrate units is 80/20 to 88/12 (mol/mol).
• PHA copolymer polyhydroxybutyric acid copolymer
• a manufacturing process for PHA having a specific composition ratio of 3HB units/hydroxyalkanoate units other than 3HB units includes specific steps in a specific order, and also includes a step of separation using a decanter-type centrifuge, which allows for the production of highly refined PHA with few impurities in a good yield without increasing the amount of wash water (wastewater), thus completing the present invention.
• one aspect of the present invention is a method for producing a PHA, comprising the steps of:
• 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);
• a culture solution containing the PHA-containing bacterial cells is used as the culture solution,
• 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 aqueous alkaline solution to the culture solution or the aqueous suspension obtained by 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-
• a new method for producing PHA can be provided that can produce highly purified polyhydroxyalkanoic acid with few impurities with good yield without increasing the amount of washing water (wastewater).
• Patent Document 1 is an excellent technology that can obtain 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 poor yield, which is problematic from an economic standpoint.
• the inventors therefore conducted extensive research to solve the above problems, and as a result discovered for the first time that by including a separation step using a decanter-type 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 refined PHA with few impurities and in good yield without increasing the amount of wash water (wastewater).
• this manufacturing method makes it possible to produce highly purified PHA with few impurities (especially PHA with a specific composition ratio of 3HB units/hydroxyalkanoate units other than 3HB units) with good yield without increasing the amount of wash water (wastewater).
• the amount of plastic waste generated can be reduced, which can contribute to the achievement of the 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.”
• SDGs Sustainable Development Goals
• the PHA in this production method 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) (hereinafter, sometimes referred to as a "specific PHB copolymer").
• culture solution refers to a solution containing bacterial cells that contain PHA, which is a specific PHB copolymer.
• 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).
• steps (d) and (e) and step (b) can be performed in any order.
• the order of steps (d) and (e) and step (b) may be steps (d), (e) and (b), or steps (b), (d) and (e).
• step (e) may be performed immediately after step (d)
• step (b) may be performed before step (d) or after step (e). Since the activity of some glucosidases decreases in an alkaline solution or in the presence of a surfactant, the order of steps (d), (e) and (b) is preferred.
• step (f) is performed after steps (d) and (e), and after (b).
• step (f) is performed after steps (d) and (e), and after (b).
• step (f) is performed after step (e)
• the cell walls of the bacteria 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.
• step (f) after step (d) the cell membranes of the bacteria 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.
• the manufacturing method may further include the following steps.
• step (c) is performed before steps (d) and (e) and after step (b). It is also preferable that step (a) is performed before steps (d) and (e) and the step of carrying out the treatment of (b) and before step (f).
• each step may be performed multiple times for various purposes.
• the present production method can be carried out, for example, in the following order of steps, but is not limited thereto: (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
• Step (d) is a step of adding glucosidase to a culture solution containing the bacterial cells containing PHA, which is a specific PHB copolymer, to perform an enzyme treatment.
• the PHA in this production method is a PHB copolymer composed of 3HB and a hydroxyalkanoate other than 3HB.
• Hydroxyalkanoates other than 3HB include, for example, 3-hydroxyhexanoate (3HH), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 3-hydroxyoctanoate (3HO), 3-hydroxyoctadecanoate (3HOD), 3-hydroxydecanoate (3HD), etc.
• P3HB3HH poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
• P3HB3HH poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
• composition ratio of the repeating units 3HB and 3HH By changing the composition ratio of the repeating units 3HB and 3HH, it is possible to change the melting point and crystallinity of P3HB3HH, which in turn changes physical properties such as Young's modulus and heat resistance, giving it physical properties between those of polypropylene and polyethylene.
• the PHB copolymer in this manufacturing method has a composition ratio of 3HB units/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).
• the ratio of each repeating unit can be determined by the method described in paragraph [0047] of WO 2013/147139.
• 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. If the weight average molecular weight is 150,000 or more, sufficient mechanical properties are obtained, and if it is 800,000 or less, a sufficient crystallization rate is obtained and good moldability is achieved.
• the weight average molecular weight of the P3HB resin can be determined as the molecular weight in polystyrene equivalent terms by gel permeation chromatography (GPC) (Shodex GPC-101 manufactured by Showa Denko) using a polystyrene gel (Shodex K-804 manufactured by Showa Denko) in a column and chloroform as the mobile phase.
• GPC gel permeation chromatography
• the bacterial cells used in step (d) are not particularly limited as long as they are microorganisms capable of producing PHB copolymers within the cells.
• microorganisms isolated from nature and deposited in a depository institution for strains (e.g., IFO, ATCC, etc.), or mutants and transformants that can be prepared from them can be used.
• the first bacterial cell to produce P3HB an example of a PHB copolymer, was Bacillus megaterium, discovered in 1925, and other natural microorganisms include Cupriavidus necator (old classification: Alcaligenes eutrophus, Ralstonia eutropha), Alcaligenes latus, etc. It is known that PHB copolymers accumulate within the bacterial cells of these microorganisms.
• examples of bacteria that produce copolymers of hydroxybutyrate and other hydroxyalkanoates which are examples of PHB copolymers
• examples of bacteria that produce copolymers of hydroxybutyrate and other hydroxyalkanoates include Aeromonas caviae, which produces P3HB3HV and P3HB3HH, and Alcaligenes eutrophus, which produces P3HB4HB.
• Alcaligenes eutrophus AC32 (FERM BP-6038) (T. Fukui, Y. Doi, J. Bateriol., 179, p4821-4830 (1997)) into which genes of the PHB copolymer synthase group have been introduced is more preferred.
• 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.
• PHB copolymers can also be produced, for example, by the method described in WO 2010/013483.
• glucosidase refers to an enzyme that has the activity of decomposing (lysing) the cell wall (e.g., peptidoglycan) of a fungus.
• the glucosidase is not particularly limited as long as it falls within the scope of the above definition, and examples thereof include lysozyme, labia, ⁇ -N-acetylglucosaminidase, endolysin, autolysin, etc. From an economical viewpoint, lysozyme, which is a versatile enzyme, is preferred.
• step (d) water may be added to the culture solution before adding 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% by weight, and more preferably 20 to 40% by weight. When the solids concentration of the aqueous suspension is within the above range, the concentration of the aqueous suspension is not too high, and is mixed uniformly with the glucosidase.
• the pH of the culture medium may be adjusted before the addition of glucosidase.
• the method of adjustment is not particularly limited, and examples include a method of adding an acid.
• the acid is not particularly limited, and may be either an organic acid or an inorganic acid, whether or not it is volatile. More specifically, examples of acids that can be used include sulfuric acid, hydrochloric acid, phosphoric acid, and acetic acid.
• a step of drying the aqueous suspension before step (d) it is preferable not to carry out a step of drying the aqueous suspension 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 not to carry out a step of drying the aqueous suspension before step (d).
• the drying may be a drying method described in the explanation below.
• Step (e) is a step of adding an alkaline protease to the culture solution obtained in step (d) to enzymatically treat the bacterial cells.
• the culture medium By treating the culture medium with an enzyme, the culture medium can be made into an aqueous suspension.
• 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).
• the alkaline protease is not particularly limited as long as it has the activity of decomposing proteins in an alkaline environment, and examples thereof include serine-specific protease (e.g., subtilisin, chymotrypsin), cysteine-specific protease (e.g., papain, bromelain), etc. From the viewpoint of versatility and economy, serine-specific protease, particularly alcalase including subtilisin, is preferred. One of these may be used alone, or two or more may be used in combination.
• serine-specific protease e.g., subtilisin, chymotrypsin
• cysteine-specific protease e.g., papain, bromelain
• serine-specific protease particularly alcalase including subtilisin
• One of these may be used alone, or two or more may be used in combination.
• Alkaline protease may be commercially available, such as “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.; “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.
• step (e) when performing enzymatic treatment of the bacterial cells with an alkaline protease, it is preferable to adjust the pH and temperature of the culture solution to the optimum pH and temperature of the alkaline protease used. In addition, it is preferable that the pH in step (e) is lower than the pH adjusted by adding an alkaline aqueous solution in step (b).
• the method for adjusting the pH and temperature of the culture solution there are no particular limitations on the method for adjusting the pH and temperature of the culture solution, and any known method can be used.
• 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.
• the optimum temperature of the alkaline protease is not particularly limited, but is preferably 70°C or less, and more preferably 60°C or less, from the viewpoint of not requiring excessive heating and being able to prevent thermal changes (thermal decomposition) of the PHB copolymer.
• 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 of not requiring excessive cooling operations and being economical.
• 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. If the amount of alkaline protease added is within the above range, the bacterial cells can be appropriately decomposed.
• step (e) it is preferable that substantially no glucosidase is added simultaneously with the alkaline protease.
• 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).
• the bacterial cells containing the PHB copolymer are preferably inactivated.
• 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 of the method is heating and stirring a culture solution containing bacterial cells containing the PHB copolymer 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).
• Step (b) In the step (b), an alkaline aqueous solution is added to a culture solution containing a bacterial cell containing a specific PHA copolymer, thereby adjusting the pH to 10.0 to 12.0. Either simultaneously with the preparation or after the preparation, a surfactant is added.
• the step (b) includes the following steps (b1) and (b2).
• Step (b1) A step of adding an alkaline aqueous solution to a culture solution containing bacteria containing a specific PHA copolymer, thereby adjusting the pH to 10.0 to 12.0.
• Step (b2) A step of adding a surfactant.
• 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.
• PHA which is a specific PHB copolymer
• impurities derived from the bacterial cells are dispersed and dissolved, and a high-purity PHB copolymer can be separated from the bacterial cells.
• 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 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.
• the basic compound contained in the alkaline aqueous solution is preferably an alkali metal hydroxide or an alkaline earth metal hydroxide, and more preferably sodium hydroxide.
• the basic compounds may be used alone or in combination of two or more kinds.
• 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 being able to decompose and dissolve the bacterial components. In addition, adjusting the pH to 12.0 or lower can prevent unintended damage to the bacterial cells.
• the temperature in step (b1) is preferably less than 100°C, and more preferably less than 80°C. There is no particular lower limit, but it is preferably, for example, 40°C or higher.
• 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 effectively treat cell membranes and remove a larger amount of impurities derived from the bacterial cells, so that a higher purity PHB copolymer can be separated from the bacterial cells.
• the surfactant is not particularly limited, but examples thereof include anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, etc.
• anionic surfactants are preferred from the viewpoint of their high cell membrane removal ability.
• One of these surfactants may be used alone, or two or more of them may be used in combination.
• 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, ⁇ -olefin sulfonates, ⁇ -sulfofatty acid salts, esters of ⁇ -sulfofatty acid salts, alkyl ether carboxylates, alkenyl ether carboxylates, amino acid surfactants, and N-acyl amino acid surfactants.
• 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.
• SDS sodium dodecyl sulfate
• These surfactants may be used alone or in combination of two or more.
• the amount of 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, relative to the culture medium.
• Step (b2) may be performed before, simultaneously with, or after step (b1).
• step (b2) is performed after step (b1).
• 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.
• step (f) By using a decanter centrifuge in step (f), it is possible to dehydrate the aqueous suspension without passing through a narrow flow path, so solid-liquid separation can be performed without the risk of clogging.
• the solid concentration of the aqueous suspension can be increased, so the efficiency of each solid-liquid separation is high, and the amount of wastewater can be reduced.
• Decanter centrifuges can be of horizontal or vertical type, but horizontal type is preferred as it can process large amounts of liquid.
• the volume median diameter of the polyhydroxyalkanoic acid in the aqueous suspension obtained is preferably 13 to 500 ⁇ m, more preferably 15 to 400 ⁇ m, and even more preferably 19 to 300 ⁇ m. If the volume median diameter is within the above-mentioned range, the yield in the washing step is excellent, the amount of aqueous suspension that can be treated is increased, and it is economical.
• the "volume median diameter of polyhydroxyalkanoic acid" means the volume median diameter of the polyhydroxyalkanoic acid in the aqueous suspension aggregated in step (d).
• 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 standpoint of yield and economy in the washing process.
• Step (c) is a step of subjecting the obtained aqueous suspension to solid-liquid separation using a disk-type centrifuge to recover an aqueous PHA suspension.
• step (c) the PHA aqueous suspension is recovered by any centrifugation method known in the art.
• the centrifugation method is not particularly limited, but examples include centrifugation using a centrifugal settler, a centrifugal dehydrator, etc.
• step (c) it is preferable to centrifuge the aqueous suspension, remove the supernatant, add a solution to the sediment, and repeat the steps of centrifuging again and removing the supernatant.
• This operation makes it possible to obtain a more concentrated and purified aqueous PHA suspension.
• the solution added after removing the supernatant is an alkaline aqueous solution adjusted to the same pH as the culture solution.
• 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.
• aqueous medium 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.
• the organic solvent is not particularly limited, and examples thereof include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, 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.
• alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, pentanol, hexanol, and heptanol
• ketones such as acetone and methyl ethyl ketone
• methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, propionitrile, etc. are preferred because they are easy to remove.
• methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone, etc. are more preferred because they are easily available.
• methanol, ethanol, and acetone are particularly preferred.
• 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.
• the PHA aqueous suspension in step (c) may contain other solvents, components derived from bacteria, compounds generated during purification, etc., as long as this does not impair the essence of the present invention.
• 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.
• Step (a) is a step of adding an alkaline protease to carry out an enzymatic treatment. Step (a) is preferably carried out before step (c).
• step (e) For the alkaline protease in step (a), the description of the alkaline protease in step (e) above is applicable.
• Step (g) is a step of 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 molecular weight to 0.0 or less has the advantage that the molecular weight of the PHA can be easily maintained during processing and molding.
• step (g) it is preferable to adjust the pH to 2.8 to 3.8, and more preferably to 3.0 to 3.5.
• 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 or the like. In one embodiment, the pH adjustment in step (g) may be performed by diluting the aqueous suspension.
• Step (h) is a step following step (g) of dehydrating and drying the PHA aqueous suspension using a filter press.
• the PHA aqueous suspension may be dehydrated by squeezing it with a filter press.
• 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 performed only once, or may be performed two or more times. When squeezing is performed two or more times, it is preferable that the pressure from the second time onwards is higher than that of the first time.
• the aqueous PHA suspension may be dehydrated by further blowing air.
• 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 the blowing air 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 minutes to 40 minutes, and more preferably 20 minutes to 30 minutes.
• the filter cake obtained after dehydration may be dried by a known method.
• the drying may be performed using, for example, a shelf dryer, a spray dryer, a fluidized bed dryer, a drum dryer, etc., but it is preferable to use a shelf dryer from the viewpoint of ease of operation.
• the temperature during drying is not particularly limited, but drying may be performed at, for example, 40 to 80°C.
• a method for producing 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 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 aqueous alkaline solution to the culture solution or the aqueous suspension obtained by step (e) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment;
• ⁇ 2> The method for producing a polyhydroxyalkanoic acid according to ⁇ 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 ⁇ m.
• the method for producing polyhydroxyalkanoic acid according to ⁇ 1> or ⁇ 2> further comprises: (g) a step of adding an aqueous solution to the polyhydroxyalkanoic acid-containing cake obtained in the step (f) to dilute the polyhydroxyalkanoic acid aqueous suspension obtained by the step (f) 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 the step (g).
• ⁇ 4> The method for producing a polyhydroxyalkanoic acid according to ⁇ 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> 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 a polyhydroxyalkanoic acid according to any one of ⁇ 1> to ⁇ 5>, wherein the polyhydroxybutyric acid copolymer has a weight average molecular weight of 150,000 to 800,000.
• the step of carrying out the treatments of the steps (d) and (e) and (b) is to carry out the steps (d) and (e) after carrying out the step (b),
• the method for producing a polyhydroxyalkanoic acid according to any one of ⁇ 1> to ⁇ 6> further comprising, after carrying out the step (b) and before carrying out the step (d), a step (c) of subjecting the obtained aqueous suspension to solid-liquid separation using a disk-type centrifuge to recover an aqueous suspension of polyhydroxyalkanoic acid.
• ⁇ 8> The method for producing polyhydroxyalkanoic acid according to ⁇ 7>, further comprising a step of performing solid-liquid separation using a disk-type centrifuge until the electrical conductivity of the aqueous suspension of polyhydroxyalkanoic acid falls within the range of 150 to 400 mS/m in the step (c), and recovering the aqueous suspension of polyhydroxyalkanoic acid.
• ⁇ 9> The method for producing a polyhydroxyalkanoic acid according to ⁇ 7> or ⁇ 8>, further comprising the step of (a) adding an alkaline protease to perform an enzymatic treatment before carrying out the step (c).
• ⁇ 10> The method for producing a polyhydroxyalkanoic acid according to any one of ⁇ 1> to ⁇ 9>, further comprising a step of inactivating a bacterial cell containing the polyhydroxyalkanoic acid before carrying out the step (e).
• P3HB3HH is used as “PHA”
• PHA in the examples can also be read as “P3HB3HH”.
• ⁇ Measuring method ⁇ The 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 type particle size distribution measuring device LA-950 manufactured by HORIBA.
• 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).
• Solid content concentration The solid content of the aqueous PHA suspension was measured using a centrifuge (manufactured by A&D Co., Ltd.) The aqueous PHA suspension was heated at 105° C. until the weight change rate fell below 0.05%/min, and the solid content was calculated from the weight change of the aqueous PHA suspension before and after heating.
• the total nitrogen content of the PHA powder was measured using a total nitrogen trace analyzer TN-2100H (Nitto Seiko Analytech Co., Ltd.).
• the PHA yield in the separation process was calculated using the following formula: 1-([PHA solid content in the PHA aqueous suspension discharged in the separation step]/[PHA solid content in the PHA aqueous suspension supplied to the separation step]). (Total PHA yield in separation process)
• the overall PHA yield in the separation steps was calculated as the product of all the yields in each separation step. For example, if the yields in the first, second, third, and fourth steps are all 98%, the overall yield is 92.2% according to the following formula.
• 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 obtained weight was taken 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 process or the PHA aqueous suspension discharged in the separation process was taken as the PHA solids content.
• Example 1 (Preparation of bacterial 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 cell culture solution containing cells containing PHA. Ralstonia eutropha is currently classified as Capriavidus necator.
• the composition ratio of the repeating units of PHA (3HB unit/composition ratio of PHA units other than 3HB units) was 84/16 (mol/mol).
• Alkaline enzyme treatment 1 The pH of the aqueous suspension of PHA was adjusted to 8.5 ⁇ 0.2 with 30% sodium hydroxide, after which 0.2 phr of Alcalase (Novozymes), a protease, was added and the pH was controlled to 8.5 with 30% sodium hydroxide at 50° C. and held at that temperature for 2 hours or more.
• Alcalase Novozymes
• Alkaline enzyme treatment 2 The pH of the aqueous suspension of PHA was adjusted to 11 ⁇ 0.2 with 30% sodium hydroxide, after which 0.10 phr of Esperase (Novozymes) was added, and the pH was controlled to 11 with 30% sodium hydroxide at 50° C. and held for 2 hours or more.
• the PHA aqueous suspension was filtered using a filter press (ISD type 360, Ishigaki Co., Ltd.). After squeezing at a pressure of 0.4 MPa to obtain a filter cake, squeezing was performed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa to perform air blow for 20 minutes to obtain a filter cake.
• the water content of the obtained filter cake was 13.5 wt% (W.B.).
• 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.
• Example 2 The same operations as in Example 1 were carried out up to the alkali treatment step.
• Alkaline enzyme treatment 1 The pH of the aqueous suspension of PHA was adjusted to 8.5 ⁇ 0.2 with 30% sodium hydroxide, after which 0.2 phr of Alcalase (Novozymes), a protease, was added and the pH was controlled to 8.5 with 30% sodium hydroxide at 50° C. and held at that temperature for 2 hours or more.
• Alcalase Novozymes
• the PHA aqueous suspension was filtered using a filter press (ISD type 360, Ishigaki Co., Ltd.). After squeezing at a pressure of 0.4 MPa to obtain a filter cake, squeezing was performed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa to perform air blow for 20 minutes to obtain a filter cake.
• the water content of the obtained filter cake was 14 wt% (W.B.).
• 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.
• Example 3 The same operations as in Example 2 were carried out up to the pH adjustment step.
• the pH-adjusted PHA aqueous suspension was introduced into a decanter (PTM006, manufactured by Tomoe Kogyo Co., Ltd.) 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%.
• 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.
• Comparative Example 1 The same procedures as in Example 1 were carried out up to the neutralization treatment and cell wall-decomposing enzyme treatment.
• Alkaline enzyme treatment 2 The pH of the aqueous suspension of PHA was adjusted to 11 ⁇ 0.2 with 30% sodium hydroxide, after which 0.10 phr of Esperase (Novozymes) was added, and the pH was controlled to 11 with 30% sodium hydroxide at 50° C. and held for 2 hours or more.
• the PHA aqueous suspension was filtered using a filter press (ISD type 360, Ishigaki Co., Ltd.). After squeezing at a pressure of 0.4 MPa to obtain a filter cake, squeezing was performed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa to perform air blow for 20 minutes to obtain a filter cake.
• the water content of the obtained filter cake was 13.5 wt% (W.B.).
• 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.
• Comparative Example 2 The same procedures as in Example 2 were carried out up to the neutralization treatment and cell wall-decomposing enzyme treatment.
• the volume median diameter of the PHA in this PHA aqueous suspension was measured using a HORIBA laser diffraction/scattering type particle size distribution measuring device LA-950 and was found to be 10 ⁇ m.
• the PHA aqueous suspension was filtered using a filter press (ISD type 360, Ishigaki Co., Ltd.). After squeezing at a pressure of 0.4 MPa to obtain a filter cake, squeezing was performed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa to perform air blow for 20 minutes to obtain a filter cake.
• the water content of the obtained filter cake was 15 wt% (W.B.).
• 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.
• Comparative Example 3 The same procedures as in Example 1 were carried out up to the alkaline enzyme treatment 1.
• the volume median diameter of the PHA in this aqueous PHA suspension was measured using a HORIBA laser diffraction/scattering particle size distribution analyzer LA-950 and found to be 10 ⁇ m.
• Alkaline enzyme treatment 2 The pH of the aqueous suspension of PHA was adjusted to 11 ⁇ 0.2 with 30% sodium hydroxide, after which 0.10 phr of Esperase (Novozymes) was added, and the pH was controlled to 11 with 30% sodium hydroxide at 50° C. and held for 2 hours or more.
• the PHA aqueous suspension was filtered using a filter press (ISD type 360, Ishigaki Co., Ltd.). After squeezing at a pressure of 0.4 MPa to obtain a filter cake, squeezing was performed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa to perform air blow for 20 minutes to obtain a filter cake.
• the water content of the obtained filter cake was 13.5 wt% (W.B.).
• 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.
• the PHA aqueous suspension was filtered using a filter press (ISD type 360, Ishigaki Co., Ltd.). After squeezing at a pressure of 0.4 MPa to obtain a filter cake, squeezing was performed again at a pressure of 0.7 MPa, and the air blow pressure was adjusted to 0.4 MPa to perform air blow for 20 minutes to obtain a filter cake.
• the water content of the obtained filter cake was 14 wt% (W.B.).
• 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.
• PHA with a high composition ratio of hydroxyalkanoate units other than 3HB units, with few impurities and a high degree of purification can be produced with good yield without increasing the amount of washing water (wastewater).
• the present invention can be suitably used in agriculture, fisheries, forestry, horticulture, medicine, sanitary products, clothing, non-clothing, packaging, automobiles, building materials, and other fields.

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