Method for producing polyhydroxyalkanoates

Abstract

The present invention provides a method for producing PHA that can suppress the thickening and deterioration of the liquid transfer properties of the PHA aqueous suspension. [Solution] A method for producing PHA, comprising the steps of (a) adding modified cellulose to an aqueous PHA suspension having a pH of 7.0 or higher, and (b) adding a phosphate compound and / or a phosphate compound to the aqueous suspension obtained in step (b).

Description

Technical Field

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

Background Art

[0002] Polyhydroxyalkanoate (hereinafter sometimes referred to as "PHA") is known to have biodegradability and has been utilized in recent years from the perspective of environmental consideration.

[0003] One of the advantages of PHA is that it can be produced by microorganisms using renewable plant-based raw materials. When utilizing the PHA produced by these microorganisms, first, the microbial cells and PHA are separated to obtain an aqueous suspension containing PHA (hereinafter sometimes referred to as "PHA aqueous suspension" or simply "aqueous suspension"), and an operation to recover PHA from this PHA aqueous suspension is performed.

[0004] In order to improve the thermal stability of the recovered PHA (particularly, the molecular weight retention rate during heating), a method is known in which sulfuric acid or the like is used to make the PHA aqueous suspension acidic to about pH 4 and spray-dried (Patent Documents and ).

[0005] Patent Document discloses a method for improving the thermal stability of PHA without making the PHA aqueous suspension acidic to a low pH that requires a corrosion-resistant device by using a specific compound.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

[0007] While the above-mentioned conventional technology is excellent, the PHA aqueous suspension can become thicker, leading to poor liquid delivery. From this perspective, there was room for improvement.

[0008] In light of the circumstances described above, the objective of one embodiment of the present invention is to provide a method for producing PHA with excellent thermal stability that can suppress the thickening and deterioration of the liquid transfer properties of the PHA aqueous suspension. [Means for solving the problem]

[0009] The inventors of the present invention have conducted extensive research to solve the above problems and have for the first time discovered that by (a) adding modified cellulose to a PHA aqueous suspension with a pH of 7.0 or higher, and then (b) adding a phosphate compound or a phosphate compound, it is possible to improve the thermal stability of PHA and suppress the thickening of the PHA aqueous suspension and the deterioration of its liquid transferability, thus completing the present invention.

[0010] In other words, one aspect of the present invention includes the following configuration. [1] A method for producing a polyhydroxyalkanoate, comprising the steps of (a) adding modified cellulose to an aqueous suspension of a polyhydroxyalkanoate having a pH of 7.0 or higher, and (b) adding a phosphate compound and / or a phosphate compound to the aqueous suspension obtained in step (a). [2] The method for producing a polyhydroxyalkanoate according to [1], wherein the phosphate compound comprises one or more compounds selected from the group consisting of tetrapotassium pyrophosphate, disodium dihydrogen pyrophosphate, tetrasodium pyrophosphate, potassium polyphosphate, sodium polyphosphate, potassium metaphosphate, sodium metaphosphate, tripotassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, diammonium hydrogen phosphate, and ammonium dihydrogen phosphate. [3] A method for producing polyhydroxyalkanoate according to [1] or [2], wherein the modified cellulose comprises one or more compounds selected from the group consisting of methylcellulose, ethylcellulose, propylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, carboxyethylcellulose, carboxypropylcellulose, carboxymethylhydroxyethylcellulose, acetylcellulose, cyanoethylcellulose, and sodium cellulose sulfate. [4] A method for producing a polyhydroxyalkanoate according to any one of [1] to [3], wherein in step (b), the pH of the aqueous suspension to which the phosphoric acid compound and / or phosphate compound have been added is adjusted to 5.0 or more and less than 7.0. [5] A method for producing a polyhydroxyalkanoate according to any one of [1] to [4], wherein in step (a), an alkylene oxide compound and / or polyvinyl alcohol are further added to the aqueous suspension of the polyhydroxyalkanoate. [6] A method for producing a polyhydroxyalkanoate according to any one of [1] to [5], wherein in step (a), the pH of the aqueous suspension to which the modified cellulose is added is 7.0 or higher, and before step (b), the method further includes (a') adjusting the pH of the aqueous suspension obtained in step (a) to less than 7.0. [7] A method for producing a polyhydroxyalkanoate according to any one of [1] to [6], wherein the polyhydroxyalkanoate is a copolymer comprising a 3-hydroxybutyrate unit and other hydroxyalkanoate units, and the molar ratio (3-hydroxybutyrate unit / other hydroxyalkanoate unit) of the 3-hydroxybutyrate unit to the other hydroxyalkanoate unit in the copolymer is 90 / 10 to 70 / 30. [8] The method for producing a polyhydroxyalkanoate according to any one of [1] to [7], wherein the polyhydroxyalkanoate is a copolymer comprising a 3-hydroxybutyrate unit and a 3-hydroxyhexanoate unit. [Effects of the Invention]

[0011] According to one aspect of the present invention, a method for producing PHA can be provided that can suppress the thickening and deterioration of the liquid transfer properties of the PHA aqueous suspension. [Modes for carrying out the invention]

[0012] One embodiment of the present invention will be described in detail below. Unless otherwise specified in this specification, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B".

[0013] [1. Method for producing PHA] A method for producing PHA according to one embodiment of the present invention (hereinafter sometimes referred to as "this production method") includes the steps of (a) adding modified cellulose to an aqueous suspension of PHA having a pH of 7.0 or higher, and (b) adding a phosphate compound and / or a phosphate compound to the aqueous suspension obtained in step (a).

[0014] As described in Patent Document 2, conventional methods for producing PHA sometimes result in increased viscosity of the PHA aqueous suspension, leading to poor liquid transferability. From this perspective, there was room for improvement. However, this production method makes it possible to suppress the increased viscosity of the PHA aqueous suspension and improve liquid transferability.

[0015] The following provides a detailed explanation of each step that may be included in this manufacturing method.

[0016] (Step (a)) This manufacturing method includes the step of adding modified cellulose to a PHA aqueous suspension with a pH of 7.0 or higher (which may be simply referred to as "step (a)"). Step (a) can also be said to be the step of preparing a PHA aqueous suspension containing modified cellulose, or the step of preparing a PHA aqueous suspension to which modified cellulose has been added.

[0017] (PHA aqueous suspension) In this specification, the PHA aqueous suspension is intended to be a solution in which PHA is suspended (dispersed) in water (an aqueous medium).

[0018] ·PHA "PHA" is a general term for polymers containing hydroxyalkanoate as monomer units (monomer units), and generally has biodegradability. In particular, in this specification, "PHA" refers to (co)polymers containing hydroxyalkanoate repeating units at 50 mol% or more of all monomer units (100 mol%) and resins composed of such (co)polymers. Specific examples of the hydroxyalkanoate units constituting PHA include 3-hydroxybutyrate (3HB) units, 4-hydroxybutyrate units, 3-hydroxypropionate units, 3-hydroxypentanoate units, 3-hydroxyhexanoate units, 3-hydroxyheptanoate units, 3-hydroxyoctanoate units, 2-hydroxypropionate units, etc. In this specification, the (co)polymer is a concept that includes both homopolymers composed of only one type of monomer unit and copolymers composed of two or more types of monomer units.

[0019] Examples of the PHA provided by this manufacturing method include poly(3-hydroxyalkanoate) (hereinafter, may also be referred to as "P3HA"), poly(4-hydroxyalkanoate), etc. Among them, P3HA is preferred because it is suitable for use in molded articles.

[0020] P3HA has a 3-hydroxyalkanoate repeating unit represented by the formula: [-CHR-CH2-CO-O-] (wherein R is C H2n+1 It is a PHA that contains an alkyl group represented by , where n is an integer between 1 and 15, and ) as an essential repeating unit.

[0021] Specific examples of P3HA include the homopolymers of 3HB: poly(3-hydroxybutyrate) (sometimes referred to as "P3HB"), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (sometimes referred to as "P3HB3HH"), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (sometimes referred to as "P3HB4HB"), poly(3-hydroxybutyrate-co-3-hydroxyvariate), and poly(3-hydroxy Examples include poly(3-hydroxybutyrate-co-3-hydroxyoctanoate), poly(3-hydroxybutyrate-co-3-hydroxyoctadecanoate), poly(3-hydroxybutyrate-co-3-hydroxydecanoate), poly(3-hydroxybutyrate-co-3-hydroxyvalate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-2-hydroxypropionate), and poly(3-hydroxybutyrate-co-3-hydroxypropionate). Among these, P3HB, P3HB3HH, and P3HB4HB are preferred, and P3HB3HH and P3HB4HB are more preferred, due to their ease of industrial production using microorganisms. In other words, the PHA related to this production method is preferably a copolymer containing 3HB units and 3-HH units, or a copolymer containing 3HB units and 4-HB units.

[0022] In this specification, "poly(X-co-Y)" refers to a copolymer containing X repeating units and Y repeating units, and is intended to be a copolymer formed by copolymerizing the monomer from which the X units originate with the monomer from which the Y units originate. As described above, the name of P3HA is determined by the monomer units contained in the P3HA. However, trace amounts (approximately 1 mol% or less) of monomers contained in P3HA may not be reflected in the name of the P3HA, provided that they do not significantly affect the physical properties of the P3HA. In other words, P3HA may contain trace amounts of other monomer units in addition to the monomer units corresponding to its name.

[0023] When P3HA is a copolymer containing 3HB units and hydroxyalkanoate units other than 3HB units (other hydroxyalkanoate units), the molar ratio of 3HB units to other hydroxyalkanoate units (3-hydroxybutyrate units / other hydroxyalkanoate units) in the total monomer units (100 mol%) of the P3HA is preferably 90 / 10 (mol% / mol%) to 70 / 30 (mol% / mol%), more preferably 87 / 13 (mol% / mol%) to 75 / 25 (mol% / mol%), and even more preferably 85 / 15 (mol% / mol%) to 80 / 20 (mol% / mol%). The monomer composition ratio of P3HA can be measured by gas chromatography (see, for example, International Publication No. 2014 / 020838).

[0024] In copolymers containing 3HB units and other hydroxyalkanoate units, the lower the proportion of 3HB units, the more likely the copolymer is to be a P3HA with low crystallinity. Aqueous suspensions containing such low-crystallinity P3HA have problems such as being prone to thickening and having poor liquid transfer properties.

[0025] As described above, this manufacturing method can suppress thickening and deterioration of liquid transfer properties even in aqueous PHA suspensions containing P3HA with a low proportion of 3HB units and low crystallinity. Therefore, this manufacturing method can be particularly suitably used as a method for producing P3HA with a high proportion of other hydroxyalkanoate units (for example, a molar ratio of 3HB units to other hydroxyalkanoate units of 10 / 90 or higher).

[0026] Microorganisms capable of producing PHA PHA can be produced using microorganisms capable of producing PHA, with plant-derived materials (e.g., oils and fats) as raw materials. P3HA-producing microorganisms are preferably used as such PHA-producing microorganisms. Examples of such P3HA-producing microorganisms include P3HB-producing microorganisms, with Bacillus megaterium, discovered in 1925, being the first. Other known natural microorganisms include Cupriavidus necator (formerly classified as Alcaligenes eutrophus, Ralstonia eutropha), and Alcaligenes latus. In these microorganisms, P3HB accumulates within the bacterial cells.

[0027] Furthermore, known microorganisms that produce P3HA, a copolymer of 3HB and other hydroxyalkanoates, include Aeromonas caviae, which produces P3HB3HH, and Alcaligenes eutrophus, which produces poly(3-hydroxybutyrate-co-4-hydroxybutyrate). In particular, to increase the productivity of P3HB3HH, Alcaligenes eutrophus AC32 strain (FERM BP-6038) (T.Fukui, Y.Doi, J.Bacteriol., 179, p4821-4830 (1997)) into which genes for P3HA synthases have been introduced is preferred. In addition to the above, genetically modified microorganisms into which various P3HA synthesis-related genes have been introduced can also be used to suit the desired physical properties and composition of P3HA.

[0028] • Method for producing PHA aqueous suspension As the PHA aqueous suspension used in step (a), a PHA aqueous suspension derived from the culture medium of a microorganism capable of producing PHA can be suitably used. Such a PHA aqueous suspension derived from the culture medium of a microorganism capable of producing PHA can be prepared, for example, by the following method: (1) Culturing a microorganism capable of producing PHA; (2) Inactivating the culture medium of the microorganism by heating to obtain an inactivated culture medium; (3) Adding alkali to the inactivated culture medium and performing alkali treatment; (4) Enzymatically treating the alkali-treated inactivated culture medium to treat the PHA and impurities derived from the microbial cells (proteins, etc.); (5) Adding an oxidizing agent to the enzymatically treated culture medium; (6) Further adjusting the pH of the culture medium and adding a surfactant to decompose and / or remove (lyse) substances derived from the microbial cells (especially cell membranes); (7) Further removing components other than PHA from the treated solution by centrifugation and / or filtration. As specific conditions for each of these operations, for example, the conditions described in the examples of International Publication 2023 / 120310 can be adopted.

[0029] This manufacturing method may include, prior to step (a), a step of culturing a microorganism capable of producing PHA, which includes one or more of the operations (1) to (7) above, and preparing a PHA aqueous suspension from the culture medium (PHA aqueous suspension preparation step). Furthermore, the order of the above processes is not particularly limited, and the order of processes can be arbitrarily changed.

[0030] In the inactivation step described in (2) above, the temperature at which the culture medium is heated (inactivation temperature) is not particularly limited as long as it is a temperature that can kill the microorganisms in the culture medium, for example, 40°C to 80°C, preferably 50°C to 80°C, and more preferably 60°C to 70°C. The heating time at the inactivation temperature is not particularly limited as long as it can kill the microorganisms in the culture medium, but for example, it may be about 30 minutes to 12 hours.

[0031] The alkali added in the alkali treatment step described in (3) above can be any alkali that is conventionally known, such as alkali metal hydroxides including sodium hydroxide, potassium hydroxide, lithium hydroxide, etc.; alkali metal carbonates such as sodium carbonate and potassium carbonate; alkali metal bicarbonates such as sodium bicarbonate and potassium bicarbonate; alkali metal salts of organic acids such as sodium acetate and potassium acetate; alkali metal borates such as borax; alkali metal phosphates such as trisodium phosphate, disodium hydrogen phosphate, tripotassium phosphate, and dipotassium hydrogen phosphate; alkali earth metal hydroxides such as barium hydroxide; and ammonia water. The amount of alkali added in the alkali treatment step is not particularly limited, but it is preferable to add an amount of alkali such that the pH of the aqueous suspension after alkali treatment becomes 8.0 to 12.0.

[0032] The enzyme used in the enzyme treatment step described in (4) above is not particularly limited, but lytic enzymes and / or proteolytic enzymes are preferred.

[0033] In this specification, "lytic enzyme" refers to an enzyme that has the activity to break down (lyse) sugar chains (e.g., peptidoglycans) that constitute the cell wall of a microbial cell. Such lytic enzymes are not particularly limited as long as they have the above activity, but examples include lysozyme, labiases, β-N-acetylglucosaminidase, endolysin, and autolysin.

[0034] In this specification, "proteinase" refers to an enzyme that has the activity to break down proteins. Such proteinases are not particularly limited as long as they have the above activity, but examples include serine-specific proteinases (e.g., subtilisin, chymotrypsin, trypsin), cysteine-specific proteinases (e.g., papain, bromelain, cathepsin), and aspartate-specific proteinases (e.g., pepsin, cathepsin D, HIV protease).

[0035] In the enzyme treatment process, one of the above-mentioned enzymes (and other enzymes) may be used alone, or two or more may be used.

[0036] In the enzyme treatment process, it is preferable to adjust the pH and temperature of the culture medium to which the enzyme has been added to match the optimal pH and temperature of the added enzyme. Furthermore, the reaction time for the added enzyme is not particularly limited and may be, for example, 30 minutes to 6 hours, or 1 hour to 3 hours.

[0037] The oxidizing agent used in the oxidizing agent addition step described in (5) above is not particularly limited and includes, for example, hydrogen peroxide (H2O2), ozone; other inorganic peroxides such as sodium peroxide (Na2O2), sodium perborate (Na2H4B2O8), sodium percarbonate (Na2H3CO6), and sodium persulfate (Na2S2O8); similar halogen compounds such as chlorites, chlorates, metachloroperbenzoic acid (C7H5ClO3) perchlorate, perchloric acid (ClO4), and chlorine dioxide (ClO2); peracids such as performic acid (CH2O3) and peracetic acid (CH3CO3H); permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate (KNO3); sodium bismuthate; and cerium(IV) compounds such as cerium ammonium nitrate and cerium sulfate. Among these, hydrogen peroxide is preferred as the oxidizing agent because it can provide PHA with superior thermal stability.

[0038] In the oxidizing agent addition step, from the viewpoint of efficiently carrying out the oxidation treatment, it is preferable to adjust the pH of the culture medium to which the oxidizing agent has been added to 10.0 to 13.0, and more preferably to 10.5 to 13.0. The pH adjustment in the oxidation treatment step can be carried out, for example, by adding the above-mentioned alkali to the culture medium.

[0039] Furthermore, in the oxidizing agent addition step, from the viewpoint of efficiently carrying out the oxidation treatment, it is preferable to adjust the temperature of the culture medium to which the oxidizing agent has been added to 30°C to 75°C, more preferably to 35°C to 70°C, and even more preferably to 40°C to 60°C.

[0040] The surfactant used in the lysis step described in (6) above is not particularly limited, and anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, etc., can be used. Among these, anionic surfactants such as sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium cholate, sodium deoxycholate, and sodium oleate are preferred, and nonionic surfactants such as polyoxyethylene alkyl ethers and polyoxyalkylene alkyl ethers are preferred, and two or more of these may be used in combination. From the viewpoint of price, amount used, and additive effect, sodium dodecyl sulfate (SDS) is more preferably used.

[0041] In the lysis process, it is preferable to adjust the pH of the culture medium to be alkaline, more specifically, to adjust the pH of the culture medium to 10.0 to 13.0, and more preferably to 10.5 to 12.0. The pH adjustment in the lysis process can be carried out, for example, by adding the above-mentioned alkali to the culture medium.

[0042] In the separation step described in (7) above, the method for separating impurities from the culture medium is not particularly limited, but centrifugation and / or filtration are preferred methods.

[0043] The form of centrifugal separation that can be performed in the separation process is not particularly limited, and known centrifugal separation methods such as centrifugal sedimentation or centrifugal dehydration can be employed.

[0044] The filtration methods that can be performed in the separation process are not particularly limited, and known filtration methods such as membrane filtration, filter press filtration, suction filtration, pressure filtration, centrifugal filtration, and gravity filtration can be employed.

[0045] The conditions for each separation operation in the separation process (e.g., the number of operations) are not particularly limited and can be set as appropriate by those skilled in the art. Furthermore, in the separation process, either filtration or centrifugation may be performed alone, or a combination of both may be performed. In addition, each process may be performed only once, or two or more times.

[0046] pH of PHA aqueous suspension If the pH of the PHA aqueous suspension used in step (a), for example, the pH of the PHA aqueous suspension obtained by the above method, is less than 7.0, it is preferable in this manufacturing method to adjust the pH of the PHA aqueous suspension to 7.0 or higher before step (a). Such pH adjustment can be performed, for example, by adding the above-mentioned alkali to the PHA aqueous suspension.

[0047] In step (a), the pH of the PHA aqueous suspension to which the modified cellulose is added is not particularly limited as long as it is 6.5 or higher, but it is preferably 7.0 or higher, more preferably 8.0 or higher, more preferably 9.0 or higher, and even more preferably 10.0 or higher, as this has the advantage of making the PHA easier to disperse.

[0048] • Solid content concentration of PHA aqueous suspension The solid content concentration of the PHA aqueous suspension in step (a), in other words, the concentration of PHA, is preferably 30% by weight or more, more preferably 40% by weight or more, and even more preferably 50% by weight or more, from the viewpoint of improving economic efficiency in terms of dry utility and improving productivity. Furthermore, the solid content concentration of the PHA aqueous suspension is preferably 70% by weight or less, and more preferably 60% by weight or less, from the viewpoint of ensuring sufficient fluidity of the aqueous suspension.

[0049] In step (a), the solid content concentration of the PHA aqueous suspension may be adjusted to fall within the above range. The method for adjusting the solid content concentration of the PHA aqueous suspension is not particularly limited and includes methods such as adding water or removing some of the water (for example, by removing the supernatant after centrifugation).

[0050] (Modified Cellulose) In this specification, "modified cellulose" means cellulose in which at least one of the hydroxyl groups present in the cellulose is replaced with any substituent. Note that "modified cellulose" is sometimes also referred to as "cellulose derivative."

[0051] The substituents that may be included in the modified cellulose added in step (a) are not particularly limited, but examples include alkyl groups, alkoxy groups, alkoxycarbonyl groups, carboxyl groups, cyano groups, acyl groups, sulfate groups, halogen atoms, and the like.

[0052] More specifically, the modified cellulose added in step (a) can be one or more compounds selected from the group consisting of methylcellulose, ethylcellulose, propylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxyethylethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, carboxyethylcellulose, carboxypropylcellulose, carboxymethylhydroxyethylcellulose, acetylcellulose, cyanoethylcellulose, and sodium cellulose sulfate. Among these, methylcellulose is preferred because it exhibits excellent aggregation suppression effects on PHA particles.

[0053] The amount of modified cellulose added in step (a) is not particularly limited, but is preferably 0.01 to 10 parts by weight, more preferably 0.1 to 1 part by weight, and even more preferably 0.1 to 0.5 parts by weight per 100 parts by weight of solids (i.e., PHA) in the aqueous PHA suspension. By adding 0.01 parts by weight or more of modified cellulose, the effect of suppressing aggregation of PHA particles can be sufficiently exerted, and by adding 10 parts by weight or less, excessive leakage of modified cellulose during molding of the manufactured PHA can be suppressed, and the water resistance of the molded article can be improved.

[0054] In this manufacturing method, by adding modified cellulose and a phosphate compound and / or a phosphate compound to a PHA aqueous suspension under predetermined pH conditions, aggregation of PHA particles in the aqueous suspension is suppressed, and as a result, thickening of the aqueous suspension is suppressed. Conventionally in this art, modified cellulose has been used as a binder to promote aggregation of PHA particles during granulation (see Patent Document 1). In this context, the effect discovered by the present inventors—that using modified cellulose in combination with a phosphate compound and / or a phosphate compound can actually inhibit aggregation of PHA particles in an aqueous suspension—is a unique and remarkable effect that could not be predicted from the prior art.

[0055] (Alkylene oxide compounds, and / or polyvinyl alcohol) In step (a), in addition to modified cellulose, alkylene oxide compounds and / or polyvinyl alcohol may be added to the aqueous suspension. Adding these components has the advantage of further suppressing the aggregation of PHA particles.

[0056] • Alkylene oxide compounds In this specification, alkylene oxide compounds refer to single compounds or copolymers containing structural units derived from one or more alkylene oxides such as ethylene oxide, propylene oxide, and butylene oxide (preferably formed by the (co)polymerization of such structural units).

[0057] The alkylene oxide compound used in step (a) is not particularly limited, but it is preferable that it contains structural units derived from ethylene oxide and / or propylene oxide, as it can improve the water resistance of the resulting molded article and has an effect of improving thermal stability. It is more preferable that it contains polyethylene oxide structures formed by the polymerization of multiple structural units derived from ethylene oxide and / or polypropylene oxide structures formed by the polymerization of multiple structural units derived from propylene oxide. It is particularly preferable that it is a copolymer composed of a block made of polyethylene oxide structures (EO block) and a block made of polypropylene oxide structures (PO block).

[0058] The amount of alkylene oxide compound added in step (a) is preferably 0.01 to 10 parts by weight, more preferably 0.1 to 5 parts by weight, and even more preferably 0.5 to 1 part by weight, per 100 parts by weight of solids (i.e., PHA) in the aqueous PHA suspension. By adding 0.01 parts by weight or more of the alkylene oxide compound, the aggregation suppression effect of PHA particles can be sufficiently exhibited, and by adding 10 parts by weight or less, excessive outflow of the alkylene oxide compound during molding of the manufactured PHA can be suppressed, thereby improving the water resistance of the molded article.

[0059] • Polyvinyl alcohol The polyvinyl alcohol used in step (a) is not particularly limited, and in addition to ordinary polyvinyl alcohol, modified polyvinyl alcohols such as ethylene-modified polyvinyl alcohol can also be used.

[0060] The amount of polyvinyl alcohol added in step (a) is not particularly limited, but is preferably 0.01 to 10 parts by weight, more preferably 0.1 to 5 parts by weight, and even more preferably 0.5 to 1 part by weight per 100 parts by weight of solids (i.e., PHA) in the aqueous PHA suspension. By adding 0.01 parts by weight or more of polyvinyl alcohol, the effect of suppressing aggregation of PHA particles can be sufficiently exhibited, and by adding 10 parts by weight or less, excessive leakage of polyvinyl alcohol during molding of the manufactured PHA can be suppressed, and the water resistance of the molded article can be improved.

[0061] (Process (a')) If the pH of the aqueous suspension to which modified cellulose is added in step (a) is 7.0 or higher, it is preferable that the present manufacturing method includes, before step (b), a step (a') in which the pH of the aqueous suspension obtained in step (a) is adjusted to less than 7.0 (sometimes simply referred to as "step (a')"). By adjusting the pH of the PHA aqueous suspension to the above range in step (a'), aggregation of PHA particles in the final PHA aqueous suspension can be further suppressed, and as a result, thickening of the aqueous suspension and deterioration of its liquid transferability can be further suppressed. Step (a') can also be described as a step in which a PHA aqueous suspension containing modified cellulose and having a pH of less than 7.0 is obtained.

[0062] In step (a'), the pH of the aqueous suspension should be adjusted to less than 7.0. The lower limit is not particularly limited, but it is preferable that it be 5.0 or higher so that the aqueous suspension can be handled without using corrosion-resistant equipment. In other words, in step (a'), it is preferable to adjust the pH of the aqueous suspension to 5.0 or higher and less than 7.0.

[0063] In step (a'), the method for adjusting the pH of the PHA aqueous suspension is not particularly limited, but a preferred method is to add an acid.

[0064] The acid that can be used in step (a') is not particularly limited and may be either an organic or inorganic acid, regardless of whether it is volatile or not. For example, sulfuric acid, hydrochloric acid, acetic acid, etc., can be used in step (a').

[0065] (Step (b)) This manufacturing method includes (b) a step of adding a phosphate compound and / or a phosphate compound to the aqueous suspension obtained in step (a) (sometimes simply referred to as "step (b)"). Step (b) can also be described as a step of obtaining a PHA aqueous suspension containing modified cellulose and a phosphate compound and / or a phosphate compound, with a pH of less than 7.0. The PHA aqueous suspension obtained through steps (a) and (b) of this manufacturing method has suppressed aggregation of PHA particles in the suspension, and as a result, the viscosity increase and deterioration of the liquid transferability of the aqueous suspension are suppressed, resulting in a PHA aqueous suspension. If this manufacturing method includes step (a'), the phosphate compound and / or phosphate compound are added to the aqueous suspension obtained in such step (a').

[0066] Examples of phosphoric acid compounds that can be used in step (b) include one or more compounds selected from the group consisting of orthophosphoric acid, pyrophosphoric acid, metaphosphoric acid, and phosphorus pentoxide.

[0067] Examples of phosphate compounds that can be used in step (b) include one or more compounds selected from the group consisting of tetrapotassium pyrophosphate, disodium dihydrogen pyrophosphate, tetrasodium pyrophosphate, potassium polyphosphate, sodium polyphosphate, potassium metaphosphate, sodium metaphosphate, tripotassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, lithium dihydrogen phosphate, diammonium hydrogen phosphate, and ammonium dihydrogen phosphate.

[0068] Among phosphoric acid compounds and phosphate compounds, it is preferable to add a phosphate compound in step (b) because it has the advantage of having a high viscosity-inhibiting effect.

[0069] The amount of phosphate compounds and / or phosphate compounds added in step (b) (the total amount if multiple types are combined) is not particularly limited, but is preferably 0.01 to 10 parts by weight, more preferably 0.1 to 1 part by weight, and even more preferably 0.02 to 0.3 parts by weight per 100 parts by weight of solids (i.e., PHA) in the aqueous PHA suspension. By adding 0.01 parts by weight or more of phosphate compounds and / or phosphate compounds, the aggregation suppression effect of PHA particles can be sufficiently exhibited, and by adding 10 parts by weight or less, excessive outflow of phosphate compounds and / or phosphate compounds during molding of the manufactured PHA can be suppressed, and the water resistance of the molded article can be improved.

[0070] The pH of the PHA aqueous suspension obtained through step (b) is not particularly limited, but from the viewpoint of suppressing PHA aggregation, it is preferably less than 7.0, and from the viewpoint of obtaining an aqueous suspension that can be handled without using corrosion-resistant equipment, it is preferably 5.0 or higher. That is, in step (b), the pH of the aqueous suspension to which the phosphate compound and / or phosphate compound has been added is preferably 5.0 or more and less than 7.0, more preferably 5.0 or more and 6.5 or less, and even more preferably 5.0 or more and 6.0 or less. In step (b), it is preferable to adjust the amount of phosphate compound and / or phosphate compound added so that the pH of the aqueous suspension falls within the above range. Alternatively, the pH can be adjusted by adding the above-mentioned acid or alkali.

[0071] In conventional techniques, acidic conditions were necessary to suppress the hydrolysis of PHA in aqueous PHA suspensions. However, under acidic conditions, PHA tends to aggregate, leading to increased viscosity of the aqueous suspension and potentially poor liquid transfer. This tendency was particularly pronounced with PHAs with a low 3HB ratio (e.g., P3HB3HH). In this context, the aqueous suspension obtained through steps (a) and (b) of the present manufacturing method can suppress the hydrolysis of PHA in the suspension even under relatively weak acidic conditions of 5.0 or higher, and further suppresses the thickening of the aqueous suspension and deterioration of liquid transfer. Moreover, this manufacturing method is particularly suitable for PHAs with a low 3HB ratio that tend to aggregate under low pH conditions.

[0072] (drying process) This manufacturing method may also include a drying step in which the aqueous suspension of PHA obtained through steps (a) and (b) above is dried (in other words, the water is removed) to obtain PHA.

[0073] In the drying process, the method for drying the PHA aqueous suspension is not particularly limited, and any drying method of the process, such as using a dryer, oven, spray dryer, fluidized bed dryer, drum dryer, etc., can be employed.

[0074] (Other processes) This manufacturing method may include, in addition to the above-mentioned steps, steps (other steps) that perform various operations that can be carried out in the PHA manufacturing process (such as granulation and addition of additives). [Examples]

[0075] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0076] [Measurement and evaluation methods] Measurements and evaluations in the examples and comparative examples were performed using the following methods.

[0077] (Shear viscosity of PHA aqueous suspension) The shear viscosity of the PHA aqueous suspension was measured using a rheometer MCR302 (Anton Paar) in a coaxial double cylinder. More specifically, using the above apparatus, the target PHA aqueous suspension was placed in a 17 mL cylinder, and the shear viscosity was measured under conditions of a liquid temperature of 40°C and a shear rate of 100 1 / s.

[0078] A lower shear viscosity indicates a PHA aqueous suspension with suppressed thickening. In particular, a viscosity of 150 mPa·s or less indicates a PHA aqueous suspension with sufficiently suppressed thickening and excellent liquid transfer properties, while a viscosity of 100 mPa·s indicates a PHA aqueous suspension with particularly suppressed thickening and particularly excellent liquid transfer properties.

[0079] (Thermal stability of PHA) For evaluation purposes, the water content of the PHA aqueous suspensions obtained in the following examples and comparative examples was evaporated using a dryer (EYELA NDO-600ND) to obtain PHA powder. The PHA powder was preheated at 160°C for 3 minutes, and then pressed at 13 MPa for 20 minutes to produce a PHA sheet. 5 mg of this PHA sheet was dissolved in 5 ml of chloroform, and unwanted materials were removed by filtration. This solution (filtrate) was subjected to molecular weight measurement using a Shimadzu GPC system equipped with a "TOSOH TSKgel GMHxl 7.8 mm ID × 30 cm, 9 μm × 2 tubes," with chloroform as the mobile phase. A commercially available standard polystyrene was used as the molecular weight standard sample. The molecular weight of the PHA powder was measured using the same procedure as above, except that a PHA sheet was not prepared. The thermal stability of PHA was evaluated based on the following formula (1): Thermal stability (%) = Weight-average molecular weight of PHA sheet pressed at 160°C and 13 MPa for 20 minutes / Weight-average molecular weight of PHA powder × 100 ... (1) A higher thermal stability value indicates a more thermally stable PHA. In particular, a PHA with a thermal stability of 50% or higher can be said to have sufficient thermal stability, while a PHA with a thermal stability of 70% or higher can be said to have excellent thermal stability.

[0080] [Example 1] (Preparation of PHA aqueous suspension) ·culture Ralstonia eutropha, as described in International Publication No. WO2021 / 206155, was cultured using the method described in Example 2 of the same publication to obtain a bacterial culture medium containing PHA. Note that Ralstonia eutropha is now classified as Capriavidus nekator. The composition ratio of the repeating units of this PHA (3HB units / 3HH units) was 84.3 / 15.7 (mol / mol).

[0081] ·Inactivation The bacterial culture medium obtained above was sterilized by heating and stirring at an internal temperature of 70°C for 8 hours to obtain an inactivated culture medium. The solid content concentration of the inactivated culture medium was 30% by weight.

[0082] • Alkaline treatment The pH of the inactivated culture medium obtained above was adjusted to 9.0 ± 1.0 by adding 30% sodium hydroxide. The pH was maintained for 16 hours by continuously adding a 30% by weight sodium hydroxide aqueous solution at an internal temperature of 70 ± 2°C. The weight-average molecular weight of PHA in the resulting treated solution was 700,000.

[0083] • Enzyme treatment To the alkaline treatment solution obtained above, 10% sulfuric acid was added to adjust the pH to 7.0 ± 0.2. Industrial water (IW) was added to adjust the solid content to 18%, then 2.5 L of Alcalase (Novozyme), an alkaline proteolytic enzyme, was added, followed by the addition of 30% sodium hydroxide at 50°C, and the mixture was maintained for 2 hours while adjusting the pH to 9.0.

[0084] • Hydrogen peroxide treatment Hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries) was added to the enzyme-treated solution obtained above. Next, a 30% sodium hydroxide aqueous solution was added to adjust the pH to 10.5. While maintaining the solution at 50°C, the 30% sodium hydroxide aqueous solution was continuously added to maintain the pH at 10.5 for 4 hours, thereby obtaining a hydrogen peroxide-treated solution.

[0085] ·Bacterial lysis Sodium dodecyl sulfate (SDS, manufactured by Kao Corporation) was added to the hydrogen peroxide treatment solution to a concentration of 0.4 wt%. Subsequently, the pH was adjusted to 11.0 ± 0.2 using an aqueous sodium hydroxide solution.

[0086] Centrifugal separation The lysing solution was prepared by removing the supernatant, then resuspending it in water. Protease (Novozyme, Esperase) was added, and the mixture was stirred for 2 hours at pH 11 and 45°C. The mixture was then centrifuged (4500 rpm, 10 minutes), the supernatant was removed, and the solution was concentrated twice. An equal volume of sodium hydroxide aqueous solution (pH 11.0) was added to the removed supernatant, and the mixture was centrifuged again (4500 rpm, 10 minutes). Finally, the supernatant was removed to obtain an aqueous PHA suspension with a solid content of 30% by weight (PHA particle content: 300 g / L).

[0087] • Membrane filtration A filtration system using a tubular membrane (MEMBRALOX® 1T1-70, manufactured by PALL, material: alumina ceramic) was used to circulate the PHA aqueous suspension through the tubular membrane, and filtration was carried out until the solid content concentration reached 52% by weight. The linear velocity during filtration (linear velocity of liquid passing through the tubular membrane) was 3-4 m / s, and the intermembrane pressure differential was 80 kPa.

[0088] (Step (a)) To the PHA aqueous suspension obtained above (pH 11.0, solids concentration 52% by weight), methylcellulose (product name: Metroze SM-15 (manufactured by Shin-Etsu Chemical Co., Ltd.)), a modified cellulose, was added at a rate of 0.3 parts by weight per 100 parts by weight of solids (PHA present in the aqueous suspension), and polyethylene glycol / polypropylene glycol / block ether type polymer (product name: Pronon #208), an alkylene oxide compound, was added at a rate of 0.95 parts by weight per 100 parts by weight of solids. Subsequently, the solids concentration of the aqueous suspension was adjusted to 50% by weight, and the mixture was stirred for 30 minutes.

[0089] (Process (a')) Sulfuric acid was added to the PHA aqueous suspension containing the modified cellulose obtained in step (a) until the pH stabilized at 6.7.

[0090] (Step (b)) To the aqueous suspension obtained in step (a'), 0.2 parts by weight of sodium dihydrogen phosphate, a phosphate compound, was added and the pH was adjusted to a stable 5.8 to obtain an aqueous PHA suspension containing modified cellulose and the phosphate compound. The shear viscosity and thermal stability of the obtained aqueous PHA suspension were measured. The results are shown in Table 1.

[0091] [Examples 2-3] A PHA aqueous suspension containing modified cellulose and a phosphate compound was obtained using the same procedure as in Example 1, except that P3HB3HH with the 3HH ratio shown in Table 1 was used as the PHA. The shear viscosity and thermal stability of the obtained PHA aqueous suspension were measured. The results are shown in Table 1.

[0092] [Example 4] In step (b), a phosphoric acid compound was used instead of a phosphate compound to obtain an aqueous PHA suspension containing modified cellulose and a phosphate compound, following the same procedure as in Example 1. The shear viscosity and thermal stability of the obtained aqueous PHA suspension were measured. The results are shown in Table 1.

[0093] [Comparative Example 1] Except for the absence of modified cellulose in step (a), an aqueous PHA suspension containing a phosphate compound and free of modified cellulose was obtained using the same procedure as in Example 1. The shear viscosity and thermal stability of the obtained aqueous PHA suspension were measured. The results are shown in Table 1.

[0094] [Comparative Example 2] Except for the absence of modified cellulose in step (a), an aqueous PHA suspension containing a phosphate compound and free of modified cellulose was obtained using the same procedure as in Example 4. The shear viscosity and thermal stability of the obtained aqueous PHA suspension were measured. The results are shown in Table 1.

[0095] [Comparative Example 3] Except for the absence of a phosphate compound in step (b), an aqueous PHA suspension containing modified cellulose and free of phosphate compounds was obtained using the same procedure as in Example 1. The shear viscosity and thermal stability of the obtained aqueous PHA suspension were measured. The results are shown in Table 1.

[0096] [Table 1]

[0097] 〔result〕 Table 1 shows that the PHA aqueous suspensions obtained through steps (a) and (b) of this manufacturing method, which contain a phosphate compound or phosphate compound and modified cellulose, have low shear viscosity, suppressed thickening of the aqueous suspension, and excellent liquid transfer properties. Furthermore, the thermal stability of the obtained PHAs is all 70% or higher, indicating excellent thermal stability. On the other hand, the results of Comparative Examples 1 to 3 show that the desired thickening suppression effect cannot be obtained when either a phosphate compound or phosphate compound, or modified cellulose, is not added. These results indicate that the synergistic effect of modified cellulose and a phosphate compound and / or phosphate compound is important in order to obtain a PHA aqueous suspension with excellent liquid transfer properties, suppressed thickening, and improved thermal stability of PHA. [Industrial applicability]

[0098] This manufacturing method can be advantageously used in the production of PHA because it can improve the thermal stability of PHA while suppressing the thickening and deterioration of liquid transfer properties of the PHA aqueous suspension. Furthermore, the PHA obtained by this manufacturing method can be suitably used in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing products, packaging, automobiles, building materials, and other fields.

Claims

1. (a) A step of adding modified cellulose to an aqueous suspension of polyhydroxyalkanoate with a pH of 6.5 or higher, and (b) A method for producing a polyhydroxyalkanoate, comprising the step of adding a phosphoric acid compound and / or a phosphate compound to the aqueous suspension obtained in step (a).

2. A method for producing a polyhydroxyalkanoate according to claim 1, wherein the phosphate compound comprises one or more compounds selected from the group consisting of tetrapotassium pyrophosphate, disodium dihydrogen pyrophosphate, tetrasodium pyrophosphate, potassium polyphosphate, sodium polyphosphate, potassium metaphosphate, sodium metaphosphate, tripotassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, diammonium hydrogen phosphate, and ammonium dihydrogen phosphate.

3. A method for producing a polyhydroxyalkanoate according to claim 1 or 2, wherein the modified cellulose comprises one or more compounds selected from the group consisting of methylcellulose, ethylcellulose, propylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, carboxyethylcellulose, carboxypropylcellulose, carboxymethylhydroxyethylcellulose, acetylcellulose, cyanoethylcellulose, and sodium cellulose sulfate.

4. The method for producing a polyhydroxyalkanoate according to claim 1 or 2, wherein in step (b), the pH of the aqueous suspension to which the phosphoric acid compound and / or phosphate compound has been added is adjusted to 5.0 or more and less than 7.

0.

5. A method for producing a polyhydroxyalkanoate according to claim 1 or 2, wherein in step (a), an alkylene oxide compound and / or polyvinyl alcohol is further added to the aqueous suspension of the polyhydroxyalkanoate.

6. In step (a) above, the pH of the aqueous suspension to which the modified cellulose is added is 7.0 or higher, A method for producing a polyhydroxyalkanoate according to claim 1 or 2, further comprising (a') a step of adjusting the pH of the aqueous suspension obtained in step (a) to less than 7.0, prior to step (b).

7. The polyhydroxyalkanoate is a copolymer comprising a 3-hydroxybutyrate unit and other hydroxyalkanoate units. A method for producing a polyhydroxyalkanoate according to claim 1 or 2, wherein the molar ratio (3-hydroxybutyrate units / other hydroxyalkanoate units) of the copolymer to the 3-hydroxybutyrate units to the other hydroxyalkanoate units is 90 / 10 to 70 / 30.

8. The method for producing a polyhydroxyalkanoate according to claim 1 or 2, wherein the polyhydroxyalkanoate is a copolymer comprising a 3-hydroxybutyrate unit and a 3-hydroxyhexanoate unit.

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