Method for producing polyhydroxyalkanoate

Abstract

The present invention addresses the problem of providing a method for producing PHA, the method being capable of suppressing the generation of fine powder. The problem is solved by a method for producing PHA, the method comprising: (a) a step for enzymatically treating a culture solution containing PHA-containing bacterial cells to obtain an aqueous PHA suspension; (b) a step for heating the aqueous suspension obtained in step (a) at a heating rate of 50 °C / sec to 180 °C / sec to a temperature ranging from (Tm-32) °C to (Tm-2) °C (where, Tm is the melting point of the PHA); and (c) a step for cooling the obtained aqueous suspension, wherein the pH of the aqueous suspension during heating is 2.0-5.0.

Description

Method for producing polyhydroxyalkanoate

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

[0002] Polyhydroxyalkanoate (hereinafter sometimes referred to as "PHA") is known to have biodegradability, and in recent years, utilization has been attempted 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 using PHA produced by microorganisms, it is necessary to separate and recover PHA from the microbial cells. In the process of separating and recovering this PHA, a dehydration operation for removing moisture from an aqueous suspension containing PHA (PHA aqueous suspension, sometimes simply referred to as "aqueous suspension") is performed.

[0004] As a method for dehydrating a PHA aqueous suspension, a technique of directly spray-drying the aqueous suspension is known. However, such a method has a large amount of moisture to be dried, and there is room for improvement from an economic perspective. Therefore, dehydration methods for PHA that do not rely on spray drying have been studied, and as such dehydration methods, for example, the techniques described in Patent Documents 1 and 2 are known.

[0005] International Publication Gazette "WO2023 / 120310" Japanese Published Patent Gazette "2019-041606"

[0006] Although the conventional PHA recovery techniques described in Patent Documents 1 and 2 are excellent, fine powder may be generated in the process of recovering PHA separated from the microbial cells, and there is room for improvement from this perspective.

[0007] In such a situation, an object of the present invention is to provide a method for producing PHA that can suppress the generation of fine powder in the process of separating and recovering PHA from microbial cells.

[0008] The present inventors, after diligently studying to solve the aforementioned problems, have found that by enzymatically treating bacterial cells containing PHA, and then heating the resulting aqueous suspension of PHA under acidic conditions to a range of (Tm-32)°C or higher and (Tm-2)°C or lower (where Tm is the melting point of the polyhydroxyalkanoate) at a heating rate of 50°C / sec or higher and 180°C / sec or lower, the decrease in molecular weight of the obtained PHA can be suppressed, and the generation of fine powder can be suppressed, thus completing the present invention.

[0009] In other words, one aspect of the present invention is a method for producing polyhydroxyalkanoate, comprising the steps of (a) enzymatically treating a culture medium containing bacterial cells containing polyhydroxyalkanoate to obtain an aqueous suspension of polyhydroxyalkanoate, (b) heating the aqueous suspension obtained in step (a) to a range of (Tm-32)°C or higher and (Tm-2)°C or lower (where Tm is the melting point of the polyhydroxyalkanoate) at a heating rate of 50°C / sec or higher and 180°C / sec or lower, and (c) cooling the aqueous suspension obtained in step (b), wherein the pH of the aqueous suspension heated in step (b) is 2.0 or higher and 5.0 or lower, and the temperature is (Tm-75)°C or lower.

[0010] According to one aspect of the present invention, it is possible to provide a method for producing PHA that can suppress the generation of fine powder during the process of separating and recovering PHA from bacterial cells.

[0011] This figure shows the schematic configuration of the heating apparatus used to carry out steps (b) and (c) in the examples and comparative examples.

[0012] One embodiment of the present invention is described below, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims. Furthermore, embodiments or examples obtained by combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Moreover, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic and patent documents mentioned herein are incorporated herein by reference. [1. [Method for Producing PHA] A method for producing PHA according to one embodiment of the present invention (hereinafter, "the method for producing PHA according to one embodiment of the present invention" may be referred to as "this production method") comprises the steps of: (a) enzymatically treating a culture medium containing a polyhydroxyalkanoate to obtain an aqueous suspension of polyhydroxyalkanoate; (b) heating the aqueous suspension obtained in step (a) to a range of (Tm-32)°C or higher and (Tm-2)°C or lower (where Tm is the melting point of the polyhydroxyalkanoate) at a heating rate of 50°C / sec or higher and 180°C / sec or lower; and (c) cooling the aqueous suspension obtained in step (b), wherein the pH of the aqueous suspension heated in step (b) is 2.0 or higher and 5.0 or lower, and the temperature is (Tm-75)°C or lower.

[0013] While researching PHA recovery technology, the inventors discovered that the fine particle size of PHA in aqueous suspensions, approximately 1 μm to 3 μm, is the cause of the generation of fine powder during the PHA recovery process from the aqueous suspension, particularly after dehydration and drying. Based on this finding, the inventors hypothesized that if these fine particles of PHA could be agglomerated, the generation of fine powder after dehydration could be suppressed. Based on this hypothesis, the inventors investigated means for agglomerating PHA and found that PHA in an aqueous suspension can be agglomerated by rapidly heating the PHA-containing aqueous suspension from a temperature of (Tm-75)°C or lower, specifically at a heating rate of 50°C / sec to 180°C / sec. Furthermore, we found that if the temperature of the aqueous suspension after heating is excessively low (specifically, below (Tm-32)°C), aggregation is insufficient and the generation of fine powder cannot be adequately suppressed. On the other hand, if the heating temperature is excessively high (specifically, above (Tm-2)°C), the PHA aggregates excessively, resulting in the generation of coarse particles with poor handling properties. In other words, we found that by heating the aqueous suspension containing PHA to a range of (Tm-32)°C or higher and (Tm-2)°C or lower, at a heating rate of 50°C / sec or higher and 180°C / sec or lower, the PHA in the aqueous suspension can be appropriately aggregated. Furthermore, we found that by setting the pH of the aqueous suspension to acidic conditions (specifically, pH 2.0 or higher and 5.0 or lower) during the above heating process, the decrease in the molecular weight of PHA during heating can also be suppressed, thus completing the present invention.

[0014] The structure of this manufacturing method is based on novel findings discovered by the inventors described above. This manufacturing method, having such a structure, provides a method for producing PHA that can suppress the generation of fine powder during the PHA recovery process. Furthermore, it can also suppress the reduction of PHA's molecular weight during the above process, and can also suppress the generation of coarse particles due to excessive aggregation of PHA. This allows for efficient dehydration and provides excellent handling properties for the resulting PHA.

[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 (a) a step of enzymatically treating a culture medium containing PHA-containing bacterial cells to obtain a PHA aqueous suspension (sometimes simply referred to as "step (a)"). Step (a) can be described as an enzymatic treatment step or as a PHA aqueous suspension preparation step.

[0017] (Culture medium containing PHA-containing microorganisms) The culture medium containing PHA-containing microorganisms used in step (a) is a culture medium obtained by culturing microorganisms capable of producing PHA, and includes the microorganisms (and the PHA produced in the microorganisms) of the cultured microorganisms.

[0018] PHA "PHA" is a general term for polymers that contain hydroxyalkanoates as monomer units (monomer repeating units) and are generally biodegradable. In particular, in this specification, "PHA" refers to a (co)polymer containing hydroxyalkanoate repeating units in an amount of 50 mol% or more of the total monomer repeating units (100 mol%) and a resin made from such (co)polymer. Specific examples of hydroxyalkanoate repeating units that constitute PHA include 3-hydroxybutanoic acid units, 4-hydroxybutanoic acid units, 3-hydroxypropionic acid units, 3-hydroxypentanoic acid units, 3-hydroxyhexanoic acid units, 3-hydroxyheptanoic acid units, 3-hydroxyoctanoic acid units, and 2-hydroxypropionic acid units. In this specification, the term (co)polymer includes both homopolymers consisting of only one type of monomer and copolymers consisting of two or more types of monomers.

[0019] Examples of PHAs provided by this manufacturing method include poly(3-hydroxyalkanoate) (hereinafter sometimes referred to as "P3HA") and poly(4-hydroxyalkanoate). Among these, P3HA is preferred because it is suitable for molded article applications.

[0020] P3HA is given by the formula: [-CHR-CH ２ 3-hydroxyalkanoate repeating unit represented by -CO-O- (where R is C ｎ H ２ｎ＋１It is an alkyl group represented by , where n is an integer between 1 and 15. It is a PHA that contains ) as an essential repeating unit.

[0021] Specific examples of P3HA include poly(3-hydroxybutyrate) (hereinafter sometimes referred to as "P3HB"), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (hereinafter sometimes referred to as "P3HB3HH"), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (hereinafter sometimes referred to as "P3HB4HB"), poly(3-hydroxybutyrate-co-3-hydroxyvariate), and poly(3-hydroxybutyrate-co-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-hydroxyvariate-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.

[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 repeating units originate with the monomer from which the Y repeating units originate. As described above, the name of P3HA is determined by the repeating 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 repeating units in addition to the repeating units corresponding to its name.

[0023] When P3HA contains 3HB repeating units, from the viewpoint of balancing flexibility and strength, the composition ratio (3HB repeating units / other repeating units) of 3HB repeating units to other repeating units (100 mol%) in the total monomer repeating units (100 mol%) of the P3HA is preferably 99 / 1 (mol% / mol%) to 60 / 40 (mol% / mol%), more preferably 97 / 3 (mol% / mol%) to 70 / 30 (mol% / mol%), and even more preferably 95 / 5 (mol% / mol%) to 80 / 20 (mol% / mol%). When the composition ratio of 3HB repeating units in P3HA is 60 mol% or more, there is an advantage in that a resin product with superior rigidity can be provided. On the other hand, when the composition ratio of 3HB repeating units in P3HA is 99 mol% or less, there is an advantage in that a resin product with superior flexibility can be provided. The monomer composition ratio of P3HA can be measured by gas chromatography or other methods (see, for example, International Publication No. 2014 / 020838).

[0024] The melting point of PHA produced by this manufacturing method is not particularly limited, but is preferably 120°C or higher and 160°C or lower, more preferably 130°C or higher and 150°C or lower, and even more preferably 135°C or higher and 145°C or lower. The melting point of PHA in this specification is the value measured by the method described in the examples.

[0025] Microorganisms capable of producing PHA As microorganisms capable of producing PHA, P3HA-producing bacteria can be suitably used. Examples of such P3HA-producing bacteria include P3HB-producing bacteria, the first of which was Bacillus megaterium discovered in 1925, and other known natural microorganisms such as Cupriavidus necator (formerly classified as Alcaligenes eutrophus, Ralstonia eutropha) and Alcaligenes latus. In these microorganisms, P3HB accumulates within the bacterial cells.

[0026] Furthermore, known microorganisms that produce P3HA, a copolymer of 3HB and other hydroxyalkanoates, include Aeromonas caviae, a P3HB3HH-producing microorganism, and Alcaligenes eutrophus, a poly(3-hydroxybutyrate-co-4-hydroxybutyrate)-producing microorganism. In particular, Alcaligenes eutrophus AC32 strain (FERM BP-6038) (T. Fukui, Y. Doi, J. Bacteriol., 179, pp. 4821-4830 (1997)), into which genes for the P3HA synthase group have been introduced to increase the productivity of P3HB3HH, is preferred. In addition to the above, genetically modified microorganisms into which various P3HA synthesis-related genes have been introduced can also be used, depending on the desired physical properties of the P3HA.

[0027] This manufacturing method may include a step of culturing the above-mentioned various PHA-producing bacteria using a known culture method to obtain a culture medium containing PHA-containing bacterial cells.

[0028] - The culture medium containing PHA-containing microorganisms to be used in the inactivation step (a) may be a culture medium in which microorganisms capable of producing PHA have been cultured, or an inactivated culture medium in which the microorganisms in the culture medium have been inactivated may be used. In other words, it is preferable that this manufacturing method includes an inactivation step in which the culture medium containing PHA-containing microorganisms to be used in step (a) is inactivated.

[0029] In the inactivation step of this manufacturing method, the method for inactivating microorganisms in the culture medium is not particularly limited, but one example is heating the culture medium at an inactivation temperature.

[0030] Here, "inactivation temperature" refers to the temperature at which microorganisms in the culture medium can be killed. Specifically, the inactivation temperature is, 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 is sufficient to kill the microorganisms in the culture medium, but it may be, for example, 30 minutes to 12 hours.

[0031] (Enzymes) The enzyme used in step (a) is not particularly limited, but lytic enzymes and / or proteolytic enzymes are preferred. That is, in one embodiment of the present invention, step (a) may be a step of adding a lytic enzyme and / or proteolytic enzyme to a culture medium containing PHA, or a step of treating a culture medium containing PHA with a lytic enzyme and / or proteolytic enzyme.

[0032] - Lysolytic enzymes: In this specification, "lysolytic enzymes" refers to enzymes that have the activity to break down (lyse) sugar chains (e.g., peptidoglycans) that make up the cell wall of a microbial cell.

[0033] The lytic enzymes that can be used in step (a) are not particularly limited as long as they have the above-mentioned activity, but examples include lysozyme, labiases, β-N-acetylglucosaminidase, endolysin, autolysin, etc.

[0034] • Proteolytic enzyme treatment In this specification, "proteolytic enzyme" refers to an enzyme that has the activity to break down proteins.

[0035] The proteolytic enzymes that can be used in step (a) are not particularly limited as long as they have the above-mentioned activity, but examples include serine-specific proteolytic enzymes (e.g., subtilisin, chymotrypsin, trypsin), cysteine-specific proteolytic enzymes (e.g., papain, bromelain, cathepsin), and aspartate-specific proteolytic enzymes (e.g., pepsin, cathepsin D, HIV protease).

[0036] In step (a), one of the above-mentioned enzymes (and other enzymes) may be used alone, or two or more may be used, but in step (a), it is preferable to use at least a lytic enzyme and a proteolytic enzyme.

[0037] In step (a), when using a lytic enzyme and a proteolytic enzyme, the order in which the two enzymes are added is not particularly limited. However, from the viewpoint of improving the efficiency of enzyme treatment, it is preferable to add the lytic enzyme first, allow it to react sufficiently, and then add the proteolytic enzyme. Alternatively, after allowing the proteolytic enzyme to react sufficiently, if necessary, a lytic enzyme and / or a proteolytic enzyme (or other enzymes) may be added and reacted further.

[0038] In step (a), when adding and reacting the enzyme, 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 or more and 6 hours or less, or 1 hour or more and 3 hours or less.

[0039] (PHA aqueous suspension) In this specification, a PHA aqueous suspension refers to a solution in which PHA is suspended (dispersed) in water (an aqueous medium). That is, the PHA aqueous suspension obtained by step (a) is a solution in which the PHA contained in the aforementioned bacterial cells is suspended in water. In addition to PHA and water, the PHA aqueous suspension obtained by step (a) may also contain other solvents (e.g., organic solvents compatible with water), components derived from the PHA-producing microorganism (e.g., cell walls, proteins, etc.), and / or other compounds generated during purification.

[0040] <Separation Step> As described above, the PHA aqueous suspension obtained by step (a) may contain impurities in addition to PHA, particularly components derived from PHA-producing microorganisms (e.g., cell walls, proteins, etc.). These impurities may cause product defects in products made using the obtained PHA. Therefore, it is preferable that this manufacturing method includes a separation step to separate these impurities from the PHA aqueous suspension.

[0041] In the separation process, the method for separating impurities from the PHA aqueous suspension is not particularly limited, but a method including a solubilization treatment to solubilize the impurities and a separation treatment to separate the solubilized impurities can be suitably used.

[0042] ・Solubilization treatment As the solubilization treatment of the PHA aqueous suspension, chemical treatments such as alkali treatment and surfactant treatment can be preferably used. These solubilization treatments may be carried out by implementing only one kind or in combination of two or more kinds.

[0043] The alkali treatment can be carried out, for example, by adding an alkali to the PHA aqueous suspension. As the alkali added in the alkali treatment, conventionally known ones can be used. For example, hydroxides of alkali metals including sodium hydroxide, potassium hydroxide, lithium hydroxide, etc.; carbonates of alkali metals such as sodium carbonate, potassium carbonate, etc.; bicarbonates of alkali metals such as sodium bicarbonate, potassium bicarbonate, etc.; alkali metal salts of organic acids such as sodium acetate, potassium acetate, etc.; borates of alkali metals such as borax; phosphates of alkali metals such as trisodium phosphate, disodium hydrogen phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, etc.; hydroxides of alkaline earth metals such as barium hydroxide; aqueous ammonia, etc. can be mentioned. The addition amount of the alkali in the alkali treatment is not particularly limited, but it is preferable to add an amount of alkali such that the pH of the aqueous suspension after the alkali treatment is 8.0 or more and 12.0 or less.

[0044] The surfactant treatment can be carried out according to a conventionally known method, and the method is not particularly limited. For example, the method described in JP-A-2012-115145 (a method of adding a surfactant to the PHA aqueous suspension) etc. can be used. As the surfactant added in the surfactant treatment, conventionally known ones can be used. For example, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants can be mentioned.

[0045] More specifically, as the surfactant used in the surfactant treatment, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium cholate, sodium deoxycholate, and sodium oleate, which are anionic surfactants, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, etc., which are nonionic surfactants, are preferable, and two or more of these may be used in combination. Among them, sodium dodecyl sulfate (SDS) is preferably used from the viewpoints of price, usage amount, and addition effect. The surfactant treatment is preferably carried out under alkaline conditions, that is, preferably carried out together with the alkali treatment.

[0046] - Separation treatment As a method for separating impurities from the PHA aqueous suspension, preferably the PHA aqueous suspension that has undergone the solubilization treatment, conventionally known methods such as centrifugation and membrane separation can be used. Among them, centrifugation is preferable because it enables industrial large-scale treatment and can be used continuously.

[0047] The separation step may be carried out prior to step (a) or after step (a). Also, a part of the treatment in the separation step (for example, the alkali treatment) may be carried out prior to step (a), and the remaining treatment (for example, the surfactant treatment and the molecular treatment) may be carried out after step (a). Each treatment may be carried out only once or may be carried out two or more times.

[0048] <Process (b)> This production method includes a step of heating the aqueous suspension obtained in step (a) above (Tm - 32)°C and below (Tm - 2)°C at a heating rate of 50°C / sec or more and 180°C / sec or less (which may be simply referred to as "step (b)"). Step (b) can also be said to be a rapid heating step that rapidly heats the aqueous suspension.

[0049] In step (b), the aqueous suspension is heated to a temperature range of (Tm-32)°C or higher and (Tm-2)°C or lower (this temperature at which the aqueous suspension is heated is sometimes referred to as the heating temperature). Here, Tm means the melting point of the PHA contained in the aqueous suspension used in step (b). For example, in step (b), if the melting point of the PHA contained in the aqueous suspension used in step (b) is 139°C, the aqueous suspension is heated to a temperature of 107°C or higher and 137°C or lower; if the melting point of the PHA contained in the aqueous suspension used in step (b) is 151°C, the aqueous suspension is heated to a temperature of 119°C or higher and 149°C or lower; and if the melting point of the PHA contained in the aqueous suspension used in step (b) is 170°C, the aqueous suspension is heated to a temperature of 138°C or higher and 168°C or lower. In step (b), by rapidly heating the aqueous suspension to a temperature range of (Tm-32)°C or higher and (Tm-2)°C or lower, the PHA can be appropriately aggregated, suppressing the generation of fine powder, while also suppressing excessive aggregation of PHA and thus suppressing the generation of coarse particles.

[0050] The heating temperature in step (b) is not particularly limited as long as it is between (Tm-32)°C and (Tm-2)°C, but is preferably between (Tm-32)°C and (Tm-5)°C, more preferably between (Tm-32)°C and (Tm-10)°C, even more preferably between (Tm-32)°C and (Tm-17)°C, and may be between (Tm-30)°C and (Tm-20)°C.

[0051] In step (b), the aqueous suspension is heated to the above temperature range at a heating rate of 50°C / sec or more and 180°C / sec or less. By rapidly heating the aqueous suspension to the above temperature range at such a relatively fast heating rate, the PHA can be appropriately aggregated as described above, suppressing the generation of fine powder, and also suppressing excessive aggregation of PHA, thereby suppressing the generation of coarse particles. The heating rate of the aqueous suspension in step (b) is calculated using the method described in the examples.

[0052] The heating rate in step (b) is not particularly limited as long as it is within the above range, but is preferably 55°C / sec or more and 120°C / sec, more preferably 60°C / sec or more and 100°C / sec or less, and even more preferably 60°C / sec or more and 80°C / sec or less.

[0053] The pH of the aqueous suspension heated in step (b) is acidic, more specifically, between 2.0 and 5.0. By heating the aqueous suspension with a pH within the above range, the decomposition of PHA due to heating can be suppressed, and the decrease in molecular weight of the resulting (recovered) PHA can be suppressed.

[0054] The pH of the aqueous suspension heated in step (b) is not particularly limited as long as it is any pH within the above range, but it is preferably 2.5 or higher and 4.5 or lower, and more preferably 2.8 or higher and 3.8 or lower.

[0055] In step (b), the time for heating the PHA aqueous suspension at the above temperature (heating time) is not particularly limited, but is preferably 0.1 minutes or more and 30 minutes or less, more preferably 0.2 minutes or more and 20 minutes or less, and even more preferably 0.3 minutes or more and 15 minutes or less. By setting the heating time within this range, the cost of heating equipment can be reduced, which has the advantage of improving economic efficiency. It also has the advantage of suppressing the decomposition of PHA due to heating, and providing PHA with a uniform molecular weight (less variation in intermolecular molecular weight).

[0056] In step (b), the temperature of the aqueous suspension to be heated immediately before heating is (Tm-75)°C or lower. By rapidly heating the aqueous suspension at this temperature as described above, the PHA can be appropriately aggregated, suppressing the generation of fine powder, while also suppressing excessive aggregation of PHA and thus suppressing the generation of coarse particles.

[0057] In step (b), the temperature of the aqueous suspension to be heated immediately before heating is not particularly limited as long as it is within the above range. The lower limit is also not particularly limited, but it is preferably 0°C or higher, may be 25°C (room temperature) or higher, or 50°C or higher. That is, in one embodiment, in particular, when heating an aqueous suspension heated to a specific temperature or higher (for example, 25°C (room temperature) or higher, or 50°C or higher, etc.) in step (b), step (b) may further include a step of preheating the aqueous suspension to be heated to the above specific temperature.

[0058] In step (b), the method for heating (including preheating) the aqueous suspension is not particularly limited, and known methods such as heating using a heat exchanger or heating using a heating tank (heat treatment tank) can be employed.

[0059] Furthermore, if preheating is performed in step (b), the preheating and the main heating (i.e., the operation of heating the aqueous suspension to a range of (Tm-32)°C or higher and (Tm-2)°C or lower at a heating rate of 50°C / sec or higher and 180°C / sec or lower) may be performed continuously or intermittently (in a so-called batch manner).

[0060] <Step (b')> The present manufacturing method preferably includes a step (b') before step (b) in which the pH of the aqueous suspension obtained in step (a) is adjusted to 2.0 or higher and 5.0 or lower (sometimes simply referred to as "step (b')"). In one embodiment of the present manufacturing method, the pH of the aqueous suspension obtained in step (a) may be less than 2.0 or greater than 5.0, but by performing step (b'), it becomes possible to carry out heating under appropriate pH conditions in the subsequent step (b).

[0061] If the manufacturing method includes a separation step, step (b') may be a step of adjusting the pH of the aqueous suspension obtained in the separation step to between 2.0 and 5.0. In particular, if the separation step includes an alkaline treatment, the pH of the aqueous suspension obtained in the separation step will be alkaline, so it is preferable to perform step (b') to adjust its pH to between 2.0 and 5.0.

[0062] In step (b'), the method for adjusting the pH of the PHA aqueous suspension to the above range is not particularly limited, but for example, it could be done by adding an acid or alkali.

[0063] The acid that can be used in step (b') is not particularly limited and may be either an organic or inorganic acid, regardless of whether it is volatile or not. More specifically, examples of acids that can be used in the pH adjustment step include sulfuric acid, hydrochloric acid, phosphoric acid, and acetic acid.

[0064] The alkalis that can be used in step (b') are also not particularly limited, and for example, hydroxides of alkali metals or alkaline earth metals such as sodium oxide 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 can be used.

[0065] The amounts of these acids or alkalis used in step (b') can be appropriately set by those skilled in the art in accordance with the pH of the PHA aqueous suspension before pH adjustment and the desired pH conditions.

[0066] <Step (c)> This manufacturing method includes (c) a step of cooling the heated aqueous suspension obtained in step (b).

[0067] In step (c), the degree to which the aqueous suspension is cooled is not particularly limited, but it is preferable to cool it until the temperature of the aqueous suspension after cooling is 98°C or lower, more preferably to 85°C or lower, and even more preferably to 70°C or lower. Also, in step (c), the lower limit of the temperature of the aqueous suspension after cooling is not particularly limited, but from the viewpoint of eliminating adverse effects on the dehydration operation, it is preferable to be 0°C or higher.

[0068] In step (c), the method for cooling the aqueous suspension is not particularly limited, and known methods such as cooling by a cooling tank or cooling device, or air cooling can be employed.

[0069] <Step (d)> Preferably, this manufacturing method further includes a step of dehydrating the aqueous suspension obtained in step (c), i.e., the aqueous suspension obtained after the heating and cooling operation in step (b). Because this manufacturing method includes step (b) as described above, the PHA in the aqueous suspension is in the form of aggregates, and efficient dehydration is possible. In addition, the PHA aggregates obtained after the dehydration operation have the advantage of being aggregates in which the generation of fine powder is suppressed when dried.

[0070] In step (d), the method for dehydrating the aqueous suspension is not particularly limited, but filtration or centrifugation are preferred methods. That is, step (d) is preferably a step in which the aqueous suspension obtained in step (c) is dehydrated by filtration or centrifugation.

[0071] (Filtration Operation) The filtration method that can be carried out in step (d) is not particularly limited as long as the aqueous suspension obtained in step (c) is dehydrated and PHA aggregates can be obtained, and known filtration methods can be used, but dead-end filtration is preferred because it can be carried out with simple equipment and operation, and can be carried out in a more space-saving and low-cost manner. The specific form of dead-end filtration is not particularly limited, and any method such as filter press filtration, suction filtration, pressure filtration, centrifugal filtration, and gravity filtration can be used.

[0072] (Centrifugal Separation) The manner of centrifugal separation that can be carried out in step (d) is not particularly limited as long as the aqueous suspension obtained in step (c) is dehydrated and PHA aggregates can be obtained, and known centrifugal separation methods can be employed. Such known centrifugal separation methods include, for example, centrifugal sedimentation or centrifugal dehydration.

[0073] Examples of centrifugal sedimentation machines that can be used in the centrifugal separation process include separation plate type (e.g., disk type, self-cleaning type, nozzle type, screw decanter type, skimming type, etc.) or cylindrical type centrifugal sedimentation machines. Both batch and continuous type centrifugal sedimentation machines can be used. Similarly, both batch and continuous type centrifugal dehydrators can be used.

[0074] The conditions for each dewatering operation in step (d) (e.g., the number of operations) are not particularly limited and can be set as appropriate by those skilled in the art. Furthermore, in step (d), either filtration or centrifugal separation may be performed alone, or a combination of both may be performed.

[0075] <Drying Process> This manufacturing method may include a drying process in which the PHA aggregates obtained in process (d) are dried. That is, in one embodiment of the present invention, a dried product obtained by drying the aggregates is provided. In the drying process, the method for drying the PHA aggregates is not particularly limited, and examples include using a shelf dryer, band dryer, conveyor dryer, spray dryer, fluidized bed dryer, airflow dryer, vibrating dryer, plate dryer, or rotary dryer.

[0076] <PHA Aggregates> In one embodiment of the present invention, a PHA aggregate (hereinafter sometimes referred to as "the aggregate") is provided, which is obtained by dehydrating the aqueous suspension obtained through steps (a) to (c) above (i.e., by further step (d)). The aggregate is a PHA aggregate produced by the present manufacturing method, and therefore, the generation of fine powder during drying is suppressed, furthermore, the decrease in the molecular weight of PHA is suppressed, it has an appropriate particle size, and does not contain coarse particles.

[0077] (Molecular Weight Retention Rate) The molecular weight retention rate of this aggregate is calculated as the ratio of the weight-average molecular weight of PHA contained in this aggregate (i.e., after heating) to the weight-average molecular weight of PHA in the aqueous suspension before heating (i.e., before carrying out step (b)), and more specifically, it is the value measured by the method described in the examples. The higher the molecular weight retention rate of this aggregate, the more it means that the decrease in molecular weight of PHA is suppressed during the manufacturing process of this aggregate, i.e., during the PHA recovery process in this manufacturing method. In particular, a molecular weight retention rate of 60% or more means that the decrease in molecular weight of PHA is sufficiently suppressed, and a molecular weight retention rate of 80% or more means that it is particularly excellent in suppressing the decrease in molecular weight of PHA.

[0078] From the viewpoint of suppressing a decrease in molecular weight during the recovery process, the molecular weight retention rate of the aggregate is preferably 60% or more, and more preferably 70% or more. Furthermore, from the viewpoint of improving the processability of the obtained PHA aggregate, it is preferably 80% or more, and particularly preferably 90% or more.

[0079] (Powdering Rate) The powdering rate of this aggregate is the percentage of fine powder (particles with a particle size of less than 10 μm) after irradiating the aggregate with ultrasound under specific conditions, and more specifically, it is a value measured by the method described in the examples. The lower the powdering rate of this aggregate, the more the generation of fine powder during drying is suppressed. In particular, a powdering rate of 5.0% or less means that the generation of fine powder is sufficiently suppressed, and a powdering rate of 0% means that the generation of fine powder is almost completely suppressed.

[0080] From the viewpoint of suppressing the generation of fine particles, the pulverization rate of the aggregate is preferably 5.0% or less, more preferably 3.0% or less, even more preferably 1.0% or less, and particularly preferably 0%.

[0081] (Volume median diameter) The volume median diameter of this aggregate is measured by laser diffraction and scattering, and more specifically, by the method described in the examples.

[0082] The volume median diameter of the aggregate is preferably 50 μm or more and 3000 μm or less. Having a volume median diameter of 50 μm or more and 3000 μm or less means that the PHA in the aqueous suspension, which is the raw material for the aggregate, has a volume median diameter within this range. Furthermore, having a volume median diameter of 50 μm or more and 3000 μm or less for the PHA in the aqueous suspension means that the dehydration process of the aqueous suspension is easy and efficient.

[0083] From the above viewpoint, the volume median diameter of the aggregate is preferably 50 μm or more and 3000 μm or less, more preferably 100 μm or more and 2000 μm or less, and even more preferably 200 μm or more and 1000 μm or less.

[0084] In this specification, PHA aggregates with a volume median diameter exceeding 10 mm are referred to as coarse particles. Coarse particles cause deterioration in the handling properties of PHA aggregates during processes such as dehydration and drying. In other words, PHA aggregates that do not contain coarse particles may have superior handling properties. From the above viewpoint, it is preferable that the aggregates do not contain coarse particles. Whether or not the PHA aggregates contain coarse particles can be evaluated by the method described in the examples.

[0085] (Uses of the aggregate) This aggregate, and its dried form, can be used as a molded article by molding using known molding methods, such as injection molding, extrusion molding, hollow molding, or compression molding. Furthermore, this aggregate can be foamed using known methods and then molded to produce a foamed molded article. These molded articles and foamed molded articles made from this aggregate can be used in a variety of applications, such as paper, film, sheets, tubes, plates, rods, containers (e.g., bottles), bags, parts, etc.

[0086] One aspect of the present invention may include the following configuration.

[0087] [1] A method for producing polyhydroxyalkanoate, comprising the steps of: (a) enzymatically treating a culture medium containing bacterial cells containing polyhydroxyalkanoate to obtain an aqueous suspension of polyhydroxyalkanoate; (b) heating the aqueous suspension obtained in step (a) to a range of (Tm-32)°C or higher and (Tm-2)°C or lower (where Tm is the melting point of the polyhydroxyalkanoate) at a heating rate of 50°C / sec or higher and 180°C / sec or lower; and (c) cooling the aqueous suspension obtained in step (b), wherein the pH of the aqueous suspension heated in step (b) is 2.0 or higher and 5.0 or lower, and the temperature is (Tm-75)°C or lower.

[0088] [2] The method for producing a polyhydroxyalkanoate according to [1], wherein in step (b), the aqueous suspension obtained in step (a) is heated at a temperature of (Tm-32)°C or higher and (Tm-17)°C or lower.

[0089] [3] A method for producing a polyhydroxyalkanoate according to [1] or [2], comprising (b') adjusting the pH of the aqueous suspension obtained in step (a) to 2.0 or higher and 5.0 or lower, prior to step (b).

[0090] [4] A method for producing a polyhydroxyalkanoate according to any one of [1] to [3], wherein the heating time in step (b) is 0.1 minutes or more and 30 minutes or less.

[0091] [5] A method for producing a polyhydroxyalkanoate according to any one of [1] to [4], wherein in step (c), the aqueous suspension obtained in step (b) is cooled to 98°C or below.

[0092] [6] A method for producing a polyhydroxyalkanoate according to any one of [1] to [5], wherein the temperature of the aqueous suspension heated in step (b) is 0°C or higher.

[0093] [7] A method for producing a polyhydroxyalkanoate according to any one of [1] to [6], further comprising (d) a step of dehydrating the aqueous suspension obtained in step (c) above.

[0094] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0095] [Measurement Method] Measurements in the examples and comparative examples were performed using the following method.

[0096] (Molecular weight retention of PHA) In the examples and comparative examples, PHA aggregates were placed in a dryer (EYELA, WFO-700) and dried at 60°C for 24 hours to obtain PHA powder. 10 mg of this PHA powder was dissolved in 10 ml of chloroform, and insoluble matter was removed by filtration. This solution (filtrate) was subjected to molecular weight measurement using a Shimadzu GPC system equipped with a "Shodex K805L (300 x 8 mm, 2 connected)" (Showa Denko Corporation) with chloroform as the mobile phase. A commercially available standard polystyrene was used as the molecular weight standard sample. The obtained weight-average molecular weight was taken as the PHA molecular weight after heating. The weight-average molecular weight of the PHA powder obtained by dehydrating the PHA aqueous suspension without heating (before being subjected to step (b)) was also measured in the same manner as above, and the obtained weight-average molecular weight was taken as the PHA weight-average molecular weight before heating.

[0097] Using the measurement results, the molecular weight retention rate of PHA in the PHA aggregates was calculated based on the following formula (1): Molecular weight retention rate (%) = Weight-average molecular weight of PHA after heating / Weight-average molecular weight of PHA before heating × 100 ... (1).

[0098] (Melting point of PHA) The melting point of PHA was measured using a differential scanning calorimetry system (PerkinElmer, DSC8500). For the measurement, the powder obtained by drying the aqueous PHA suspension obtained in step (b') at 60°C was used. Specifically, the measurement was performed using the following temperature profile: The target PHA powder was heated at 10°C / min to a temperature 20°C higher than the melting point peak termination temperature and held for 10 minutes. Then, it was cooled at 5°C / min to a temperature at least 50°C lower than the melting point peak start temperature. After that, the temperature at the peak top of the DSC curve when heated at 50°C / min was taken as the melting point (Tm) of the target PHA. If the DSC curve obtained above has two peaks, Tm was calculated based on the following equation (2), where n is the number of peaks, X1, X2...Xn is the proportion of enthalpy of melting of each peak, and Tm1, Tm2...Tmn is the temperature of each peak top: Tm = (Tm1 × X1 + Tm2 × X2... + Tmn × Xn) / n ... (2) The proportion of enthalpy of melting of each peak (Xn) in equation (2) was calculated based on the following equation (3): Proportion of enthalpy of melting of the target peak = enthalpy of melting of the target peak / enthalpy of melting of the entire DSC curve × 100 ... (3) Also, if the proportion of enthalpy of melting was less than 5%, it was not counted as a melting point peak.

[0099] (Volume median diameter and pulverization rate of PHA aggregates) The volume median diameter of PHA aggregates was measured using a laser diffraction particle analyzer (MASTERSIZER3000, Malvern). The pulverization rate of PHA aggregates was determined by irradiating the target PHA aggregates with ultrasound for 2 minutes, then measuring the particle size distribution using the method described above, and defining the pulverization rate as the percentage of particles smaller than 10 μm in the obtained volume-based integrated distribution.

[0100] (Presence or absence of coarse particles in PHA aggregates) The target PHA aggregates were placed into a metal mesh sieve with a mesh size of 9.5 mm. If aggregates remained on the sieve, it was evaluated as containing coarse particles. If no aggregates remained on the sieve, it was evaluated as not containing coarse particles.

[0101] (pH of PHA aqueous suspension) The pH of the PHA aqueous suspension was measured using a pH meter (9652-10D, manufactured by HORIBA). The pH was measured at the point furthest from the acid addition point while the PHA aqueous suspension was in a flowing state using a stirring blade or similar device. For example, when the acid was added from the side of the container, the pH at the center of the container was measured.

[0102] (Measurement of heating rate) The heating rate in process (b) was calculated using the following method: (1) When the aqueous suspension is continuously supplied and heated as shown in Figure 1, it was assumed that the aqueous suspension was heated to the temperature inside the heating tank 1 second after it was supplied to the heating tank. That is, the temperature of the aqueous suspension after heating was treated as the temperature inside the heating tank, and the time taken to heat up was assumed to be 1 second. (2) When the aqueous suspension is heated by batch processing as in Comparative Example 2, the heating rate was calculated based on the following formula (4): [Heating rate (°C / sec)] = [(Aqueous suspension temperature after heating (°C)) - (Aqueous suspension temperature before heating (°C)] ÷ [(Time when the temperature of the aqueous suspension first reached the target temperature (sec)) - (Time when heating started (sec))] ... (4).

[0103] [Example 1] (Preparation of bacterial culture medium) Ralstonia eutropha, as described in International Publication No. WO2019 / 142717, was cultured using the method described in paragraphs

[0041] to

[0048] of the same document to obtain a culture medium (bacterial culture medium) containing PHA-containing bacterial cells. The monomer unit composition (mol%) of the obtained PHA, such as 3HH units, can be analyzed by gas chromatography, nuclear magnetic resonance spectroscopy, etc. The 3HH composition of the obtained PHA was 6 mol%. Note that Ralstonia eutropha is now classified as Capriavidus necatol.

[0104] (Inactivation) The bacterial culture solution obtained above was sterilized by heating and stirring at an internal temperature of 60 to 70°C for 7 hours to obtain an inactivated bacterial culture solution.

[0105] (Separation Process - 1) Alkali Treatment: A 30% by weight sodium hydroxide aqueous solution was added to the inactivated bacterial culture solution obtained above to adjust the pH to 9.5. While maintaining this bacterial culture solution at 70°C, the pH was maintained at 9.5 for 6 hours by continuously adding the 30% by weight sodium hydroxide aqueous solution.

[0106] (Step (a)) Deionized water was added to the alkali-treated bacterial culture solution obtained above, and the solution was diluted to a solid content concentration of 20% by weight. Then, 95% sulfuric acid was added to adjust the pH to 7.0 ± 0.2. After adding sulfuric acid, lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries), a lytic enzyme, was added to a concentration of 10 ppm in the solution, and the solution was maintained at 50°C for 2 hours. Then, 2.5 L of alcalase (manufactured by Novozyme), a proteolytic enzyme, was added to a concentration of 300 ppm in the solution, and then 30% sodium hydroxide was added at 50°C to adjust the pH to 8.5 while maintaining the solution for 2 hours. The bacterial culture solution was treated with enzymes by this procedure.

[0107] (Separation process - 2) Surfactant treatment and alkali treatment Sodium dodecyl sulfate (SDS, manufactured by Kao Corporation) was added to the enzyme treatment solution obtained above to a concentration of 0.3% by weight (surfactant treatment). Then, the pH was adjusted to 11.0 ± 0.2 using an aqueous sodium hydroxide solution (alkali treatment).

[0108] - Separation Treatment Next, the enzyme-treated solution was centrifuged (4000 G, 10 minutes), and the supernatant was removed to obtain a PHA aqueous suspension concentrated 2 times. An equal amount of sodium hydroxide aqueous solution was added to the concentrated PHA aqueous suspension, and the mixture was centrifuged again (4000 G, 10 minutes), and the supernatant was removed. This process was repeated four times. A PHA aqueous suspension was obtained by this procedure.

[0109] (Step (b')) The solid content concentration of the PHA aqueous suspension obtained above was adjusted to 10% by weight and maintained at 60°C. Next, 10% sulfuric acid was added to adjust the pH to 3.5. The melting point of PHA in this aqueous suspension was measured to be 139°C.

[0110] (Step (b)) Rapid heating of the PHA aqueous suspension (i.e., step (b)) was performed using the heating device 100 shown in Figure 1. Specifically, the PHA aqueous suspension supplied to the raw material tank 1 was supplied to the coagulation tank 4 via the heat exchanger 3 using the Mono pump 2 (manufactured by Hyoshin Equipment Co., Ltd.). In the middle of the process, the PHA aqueous suspension was preheated by the heat exchanger 3. At this time, the heat exchanger 3 was heated by steam, and its temperature was 60°C (i.e., the aqueous suspension was preheated to 60°C). Subsequently, the preheated aqueous suspension was heated in the coagulation tank 4 equipped with a jacket (step (b)). The coagulation tank 4 was heated by supplying steam to the jacket, and its temperature was 120°C (i.e., in step (b), the aqueous suspension was heated to 120°C). The heating rate of the aqueous suspension in step (b) was 60°C / sec, and the heating time was 11 minutes. Thermocouples were installed at various points in the heating device 100, and these thermocouples were used to measure the temperature of the aqueous suspension at each stage of the process.

[0111] (Step (c)) The heated aqueous suspension was supplied to a cooling tank equipped with a jacket, and cooled to 65°C by supplying cold water to the jacket.

[0112] (Step (d)) The cooled aqueous suspension was centrifuged using a basket dehydrator (Kokusan H-122) to dehydrate it and obtain PHA aggregates. The presence or absence of coarse particles was checked in the obtained aggregates, and their various physical properties were measured. The results are shown in Table 1.

[0113] [Examples 2 to 10, Comparative Examples 1, 3 to 6] Except for changing the type of PHA used, the pH of the aqueous suspension, and / or the heating conditions in step (b) (preheating temperature (temperature of the aqueous suspension before heating), heating rate, heating temperature, etc.) as shown in Table 1, the PHA aqueous suspension was heated, cooled, and dehydrated in the same manner as in Example 1 to obtain PHA aggregates. The presence or absence of coarse particles was checked in the obtained aggregates, and each physical property was measured. The results are shown in Table 1.

[0114] [Comparative Example 2] Up to step (b'), the same procedure as in Example 1 was followed to obtain a pH-adjusted aqueous PHA suspension. 2.5 L of the aqueous PHA suspension was directly added to the coagulation tank 4, and 143°C steam was supplied to the jacket of the coagulation tank 4 to heat-treat the aqueous suspension until its temperature reached 120°C. Immediately after the aqueous suspension reached 120°C, the temperature of the supplied steam was changed to 120°C, and the aqueous suspension was maintained at 120°C for 20 minutes. After that, the supply of steam was stopped, and 20°C cold water was supplied to the jacket to cool the heated aqueous suspension to 60°C, and it was discharged from the coagulation tank 4. The cooled aqueous suspension was dewatered using the same procedure as in Example 1 to obtain PHA aggregates. The presence or absence of coarse particles was checked in the obtained aggregates, and each physical property was measured. The results are shown in Table 1.

[0115] [Summary] From Table 1, it can be seen that in Examples 1 to 10, where heating was carried out in step (b) at the temperature, heating rate, and pH specified in this manufacturing method, the pulverization rate of the obtained PHA aggregates was 5% or less, indicating that the generation of fine powder was suppressed. Furthermore, the molecular weight retention rate was 80% or more, indicating that the demolecularization of PHA was also suppressed. In addition, the volume median diameter of the PHA aggregates was neither excessively low nor excessively large, and no coarse particles were generated, indicating that PHA aggregates with excellent dewatering efficiency were obtained.

[0116] On the other hand, the results from Comparative Examples 1, 3 to 5 show that if the temperature of the aqueous suspension before heating in step (b) is higher than specified, and the heating rate is lower than the specified rate, the aggregation of PHA is insufficient, resulting in the generation of fine powder. The results from Comparative Example 2 also show that if the heating rate is lower than the specified rate, the aggregation of PHA is insufficient, resulting in the generation of fine powder. Furthermore, the results from Comparative Example 6 show that if the pH of the aqueous suspension is outside the specified range, the suppression of fine powder generation and the suppression of low molecular weight are insufficient. From the results of these comparative examples, it is clear that rapid heating under the heating conditions specified in this manufacturing method is necessary to suppress the generation of fine powder during the PHA recovery process.

[0117] This manufacturing method can be suitably used in the production of PHA. PHA produced by this method can be suitably used, for example, as molded articles in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing products, packaging, automobiles, building materials, and other fields.

Claims

1. A method for producing polyhydroxyalkanoate, comprising: (a) enzymatically treating a culture medium containing bacterial cells containing polyhydroxyalkanoate to obtain an aqueous suspension of polyhydroxyalkanoate; (b) heating the aqueous suspension obtained in step (a) to a range of (Tm-32)°C or higher and (Tm-2)°C or lower (where Tm is the melting point of the polyhydroxyalkanoate) at a heating rate of 50°C / sec or higher and 180°C / sec or lower; and (c) cooling the aqueous suspension obtained in step (b), wherein the pH of the aqueous suspension heated in step (b) is 2.0 or higher and 5.0 or lower, and the temperature is (Tm-75)°C or lower.

2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein in step (b), the aqueous suspension obtained in step (a) is heated to a temperature of (Tm-32)°C or higher and (Tm-17)°C or lower.

3. The method for producing a polyhydroxyalkanoate according to claim 1, further comprising (b') a step of adjusting the pH of the aqueous suspension obtained in step (a) to 2.0 or higher and 5.0 or lower, prior to step (b).

4. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the heating time in step (b) is 0.1 minutes or more and 30 minutes or less.

5. The method for producing a polyhydroxyalkanoate according to claim 1, wherein in step (c), the aqueous suspension obtained in step (b) is cooled to 98°C or below.

6. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the temperature of the aqueous suspension heated in step (b) is 0°C or higher.

7. The method for producing a polyhydroxyalkanoate according to claim 1, further comprising (d) a step of dehydrating the aqueous suspension obtained in step (c).

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