Method for producing polyhydroxyalkanoate

Abstract

To provide a method for producing PHA (polyhydroxyalkanoate) by which the amount of drainage in a purification process can be reduced and which is excellent in filtration rate.SOLUTION: A method for producing PHA, comprising a centrifugation step of centrifuging a PHA aqueous dispersion (1) to obtain a PHA aqueous dispersion (2) containing proteins of 6000 to 30 000ppm, and a filtration step of dead-end filtering the PHA aqueous dispersion (2) adjusted to pH3. 0 to 5.5.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing polyhydroxyalkanoates. [Background technology]

[0002] Polyhydroxyalkanoates (hereinafter sometimes referred to as "PHAs") are known to be biodegradable, and in recent years, their use has been promoted from the viewpoint of environmental considerations.

[0003] One of the advantages of PHA is that it can be produced by microorganisms using renewable plant materials. To utilize PHA produced by microorganisms, first, the PHA-containing microorganism cells are disrupted or biological components other than PHA are solubilized, and the PHA in the cells is dispersed in water to obtain an aqueous PHA suspension. Then, impurities other than PHA are further removed from this PHA aqueous suspension to purify the PHA.

[0004] Known techniques for purifying such PHA include a method of centrifuging an aqueous PHA suspension (Patent Documents 1 and 2) and a method of filtering an aqueous PHA suspension (Patent Documents 3 and 4). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2023 / 120193 [Patent Document 2] Patent Publication No. 2023-86317 [Patent Document 3] Special table number 2016-524926 [Patent Document 4] Chinese Patent No. 111500650 Details Summary of the Invention [Problem to be solved by the invention]

[0006] However, among the conventional PHA purification techniques, the method of industrially centrifuging an aqueous PHA suspension requires multiple (generally four or more) centrifugation steps for purification, and the amount of wastewater discharged during the purification process may be excessively large. In other words, there is room for improvement in terms of the amount of wastewater discharged during the purification process.

[0007] Furthermore, the method of filtering an aqueous PHA suspension may not achieve a filtration rate that is sufficient for industrial use, meaning that there is room for improvement in terms of filtration rate.

[0008] An object of the present invention is to provide a method for producing PHA that can reduce the amount of wastewater discharged during the purification process and also has an excellent filtration rate. [Means for solving the problem]

[0009] As a result of intensive research to solve the above-mentioned problems, the present inventors have discovered a new finding that, in purifying PHA, a sufficient filtration rate can be achieved while reducing the amount of wastewater discharged during the purification process by first centrifuging an aqueous PHA suspension until a predetermined protein concentration is reached, and then subjecting the centrifuged aqueous PHA suspension to dead-end filtration, which has led to the completion of the present invention.

[0010] That is, one aspect of the present invention includes the following configuration. [1] A method for producing a polyhydroxyalkanoate, comprising: a centrifugation step of centrifuging a polyhydroxyalkanoate aqueous suspension (1) to obtain a polyhydroxyalkanoate aqueous suspension (2) having a protein content of 6,000 to 30,000 ppm; and a filtration step of subjecting the obtained polyhydroxyalkanoate aqueous suspension (2) to dead-end filtration, wherein the pH of the polyhydroxyalkanoate aqueous suspension (2) subjected to the filtration step is 3.0 to 5.0. [2] The method for producing a polyhydroxyalkanoate according to [1], further comprising a pH adjustment step of adjusting the pH of the aqueous polyhydroxyalkanoate suspension (2) to 3.0 to 5.0 before the filtration step. [3] In the filtration step, the air permeability is 0.25 cc / cm 2 The method for producing a polyhydroxyalkanoate according to [1] or [2], wherein dead-end filtration is performed using a filter medium having a flow rate of 1 / sec or less. [4] The method for producing a polyhydroxyalkanoate according to any one of [1] to [3], wherein the number of times of centrifugation in the centrifugation step is two or less. [5] The method for producing a polyhydroxyalkanoate according to any one of [1] to [4], wherein the protein content of the polyhydroxyalkanoate aqueous suspension (1) is 50,000 ppm or more. [6] The method for producing a polyhydroxyalkanoate according to any one of [1] to [5], wherein the shear viscosity of the aqueous polyhydroxyalkanoate suspension (1) at 40°C and 10 1 / s is 4 to 15 mPa·s. [7] The method for producing a polyhydroxyalkanoate according to any one of [1] to [6], wherein the protein content of the filter cake obtained by the filtration step is lower by 2,000 ppm or more than the protein content of the polyhydroxyalkanoate aqueous suspension (2) that is the raw material for the filter cake. [Effects of the Invention]

[0011] According to one aspect of the present invention, it is possible to provide a method for producing PHA that can reduce the amount of wastewater discharged during the purification process and also has an excellent filtration rate. DETAILED DESCRIPTION OF THE INVENTION

[0012] One embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the respective 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. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic literature and patent documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, the term "A to B" representing a numerical range means "greater than or equal to A (including and greater than A) and less than or equal to B (including and less than B)."

[0013] 1. PHA manufacturing method A method for producing a PHA according to one embodiment of the present invention (hereinafter, "a method for producing a PHA according to one embodiment of the present invention" may be referred to as "this production method") is a method for producing a PHA, comprising a centrifugation step of centrifuging an aqueous PHA suspension (1) to obtain an aqueous PHA suspension (2) having a protein content of 6,000 to 30,000 ppm, and a filtration step of subjecting the obtained aqueous PHA suspension (2) to dead-end filtration.

[0014] <Technical Concept of the Present Invention> As described above, conventional purification of PHA by centrifuging an aqueous PHA suspension requires multiple centrifugation steps to sufficiently remove impurities, resulting in the generation of a large amount of wastewater during the purification process. For example, in the technology of Patent Document 1, PHA is purified by four centrifugation steps (which are then dehydrated by filtration), resulting in the generation of a large amount of wastewater.

[0015] The present inventors have been studying a method for purifying PHA with a smaller amount of wastewater from the viewpoint of reducing environmental load, etc., and have focused on a method for purifying PHA by filtration. However, as described above, the purification of PHA by filtration is not sufficient in terms of filtration rate.

[0016] When the inventors investigated the cause of the decrease in filtration rate during the purification of PHA by filtration, they found that the decrease in filtration rate was caused by the clogging of the filter media used for filtration due to the large amount of impurities contained in the unpurified PHA aqueous suspension.

[0017] After gaining the above knowledge, the present inventors conducted further research and found that by reducing the amount of impurities in the PHA aqueous suspension to a certain level in advance and adjusting the pH to a predetermined range before filtering, clogging of the filter material during the filtration process can be suppressed, thereby enabling PHA purification by filtration at an excellent filtration rate.The present inventors also found that the operation of reducing the amount of impurities in the PHA aqueous suspension to the above level, i.e., to a level that does not cause clogging of the filter material during filtration, can be carried out with a relatively small number of centrifugation cycles (i.e., with a small amount of wastewater).

[0018] Having gained these findings, the present inventors have discovered that by combining a centrifugation step for reducing the amount of impurities in the aqueous PHA suspension to the above-mentioned level with a filtration step for filtering the aqueous PHA suspension with the impurity amount reduced while adjusting the pH to a predetermined range, it is possible to reduce the amount of wastewater compared to filtration by centrifugation alone and to purify PHA at a superior filtration rate compared to filtration by filtration alone, i.e., it is possible to achieve both a reduced amount of wastewater and a superior filtration rate, thereby completing the present invention.

[0019] This production method can also be said to be a method for purifying PHA (a method for purifying PHA) while reducing the amount of wastewater discharged during the purification process and at an excellent filtration rate.

[0020] Each step included in this manufacturing method will be described in detail below.

[0021] <Centrifugal separation process> The present production method includes a centrifugation step of centrifuging a PHA aqueous suspension (1) to obtain a PHA aqueous suspension (2) having a protein content of 6,000 to 30,000 ppm.

[0022] (PHA aqueous suspension (1)) First, the PHA aqueous suspension (1) subjected to the centrifugation step will be described. In this specification, the PHA aqueous suspension refers to a solution in which PHA is suspended (dispersed) in water (aqueous medium) and has fluid properties. The PHA aqueous suspension may contain, in addition to water, other solvents (e.g., organic solvents compatible with water), components derived from PHA-producing microorganisms (e.g., cell walls, proteins, etc.), and / or other compounds generated during purification. In other words, the PHA aqueous suspension (1) used in this production method may contain these components in addition to PHA and water.

[0023] The PHA aqueous suspension (1) in this production method refers to an unpurified PHA aqueous suspension and a substantially unpurified PHA aqueous suspension. In this specification, the term "unpurified PHA aqueous suspension" refers to a PHA aqueous suspension produced by using a culture medium of a PHA-producing microorganism or an aqueous solution containing a PHA-producing microorganism as a raw material, disrupting the PHA-containing microorganism cells in the raw material and / or solubilizing biological components other than PHA, and dispersing the PHA in the microorganism cells in water, preferably by disrupting the PHA-containing microorganism cells and solubilizing biological components other than PHA, without any purification treatment, specifically, centrifugation or filtration. The method for disrupting the PHA-containing microorganism cells and / or solubilizing biological components other than PHA is not particularly limited, and known methods can be used, such as methods using enzymes such as lysozyme and / or alcalase.

[0024] When the purification history of a certain aqueous PHA suspension is unknown, whether the suspension is unpurified can be determined, for example, based on the amount of impurities, particularly the amount of protein, in the suspension. Specifically, in this specification, if the protein content in a certain aqueous PHA suspension is 50,000 ppm or more, the suspension is considered to be an unpurified aqueous PHA suspension. In addition, a PHA aqueous suspension that has undergone some processing but still has a protein content of 50,000 ppm or more is also considered to be a "substantially unpurified aqueous PHA suspension." Needless to say, even if the protein content of a PHA aqueous suspension is less than 50,000 ppm, the aqueous suspension can be used as the PHA aqueous suspension (1) according to the present production method if it is unpurified. The protein content of the aqueous PHA suspension is measured by the method described in the Examples.

[0025] The shear viscosity of the aqueous PHA suspension (1) is not particularly limited, but from the viewpoint of improving the filtration rate in the subsequent filtration step, it is preferably 4 to 15 mPa·s, more preferably 5 to 10 mPa·s, at a shear rate of 10 1 / s at 40° C. The shear viscosity of the aqueous PHA suspension is measured by the method described in the Examples.

[0026] The solids concentration (i.e., PHA concentration) of the PHA aqueous suspension (1) is not particularly limited, but from the viewpoint of improving the fluidity of the PHA aqueous suspension, it is preferably 5 to 30% by weight, more preferably 10 to 20% by weight.

[0027] Next, the PHA contained in the PHA aqueous suspension (1) will be described in detail. Note that, since the basic physical properties of the PHA contained in the PHA aqueous suspension (1) do not change in the centrifugation step and filtration step of the present production method, the following description also applies to the specific aspects of the PHA contained in the PHA aqueous suspension (2) made from the PHA aqueous suspension (1) as a raw material.

[0028] PHA "PHA" is a general term for polymers containing hydroxyalkanoate as a monomer unit (monomer repeat unit) and is generally biodegradable. In particular, "PHA" herein refers to a (co)polymer containing hydroxyalkanoate repeat units at 50 mol% or more of the total monomer repeat units (100 mol%), and a resin composed of such a (co)polymer. Specific examples of hydroxyalkanoate repeat units constituting PHA include 3-hydroxybutanoic acid unit, 4-hydroxybutanoic acid unit, 3-hydroxypropionic acid unit, 3-hydroxypentanoic acid unit, 3-hydroxyhexanoic acid unit, 3-hydroxyheptanoic acid unit, 3-hydroxyoctanoic acid unit, and 2-hydroxypropionic acid unit. In this specification, the term "(co)polymer" encompasses both a homopolymer composed of only one type of monomer and a copolymer composed of two or more types of monomers.

[0029] Examples of PHAs provided by this production method include poly(3-hydroxyalkanoate) (hereinafter sometimes referred to as "P3HA"), poly(4-hydroxyalkanoate), etc. Among these, P3HA is preferred because it is suitable for use in molded articles.

[0030] P3HA is a 3-hydroxyalkanoate repeating unit of the formula: [—CHR—CH—CO—O—] (wherein R is C n H 2n+1 and n is an integer of 1 or more and 15 or less.) as an essential repeating unit.

[0031] Specific examples of P3HA include: 3HB homopolymers 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-hydroxyvalerate), and poly(3-hydroxybutyrate -co-3-hydroxyoctanoate), poly(3-hydroxybutyrate-co-3-hydroxyoctadecanoate), poly(3-hydroxybutyrate-co-3-hydroxydecanoate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-2-hydroxypropionate), poly(3-hydroxybutyrate-co-3-hydroxypropionate), etc. Among these, P3HB, P3HB3HH, and P3HB4HB are preferred, with P3HB3HH and P3HB4HB being more preferred, in view of ease of industrial production using microorganisms.

[0032] In this specification, "poly(X-co-Y)" refers to a copolymer containing X repeating units and Y repeating units, and is intended to mean a copolymer obtained by copolymerizing a monomer from which the X repeating unit is derived and a monomer from which the Y repeating unit is derived. As described above, the name of a P3HA is determined by the repeating units contained in the P3HA. However, a very small amount (about 1 mol % or less) of a monomer contained in a P3HA may not be reflected in the name of the P3HA, provided that such a monomer does not significantly affect the physical properties of the P3HA. In other words, a P3HA may contain, in addition to the repeating units corresponding to its name, very small amounts of other repeating units.

[0033] When P3HA contains 3HB repeating units, from the viewpoint of the balance between flexibility and strength, the composition ratio of 3HB repeating units to repeating units other than 3HB repeating units (other repeating units) in the total monomer repeating units (100 mol%) in the P3HA (3HB repeating units / other repeating units) 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, it is advantageous 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, it is advantageous in that a resin product with superior flexibility can be provided. The monomer composition ratio of P3HA can be measured by gas chromatography or the like (see, for example, WO 2014 / 020838).

[0034] P3HA can be produced by microorganisms. Examples of microorganisms capable of producing P3HA include Bacillus megaterium, a P3HB-producing bacterium discovered in 1925, as well as other naturally occurring microorganisms such as Cupriavidus necator (formerly Alcaligenes eutrophus and Ralstonia eutropha) and Alcaligenes latus. These microorganisms accumulate P3HB within their cells.

[0035] Known microorganisms that produce P3HA, a copolymer of 3HB and other hydroxyalkanoic acids, include Aeromonas caviae, which produces P3HB3HH, and Alcaligenes eutrophus, which produces poly(3-hydroxybutyrate-co-4-hydroxybutyrate). To increase P3HB3HH productivity, Alcaligenes eutrophus AC32 (FERM BP-6038) (T. Fukui, Y. Doi, J. Bacteriol., 179, pp. 4821-4830 (1997)) is particularly preferred. In addition to the above, genetically modified microorganisms containing various P3HA synthesis-related genes can also be used depending on the desired physical properties of P3HA.

[0036] ·Method of manufacturing PHA aqueous suspension The PHA aqueous suspension (1) to be subjected to the centrifugation step is preferably a PHA aqueous suspension derived from a culture medium of a PHA-producing microorganism. Such a PHA aqueous suspension derived from a culture medium of a PHA-producing microorganism can be prepared, for example, by the following method: (1) culturing a microorganism capable of producing PHA; (2) inactivating the culture medium by heating to obtain an inactivated culture medium; (3) treating the inactivated culture medium with hydrogen peroxide to reduce the viscosity of the culture medium; (4) treating the hydrogen peroxide-treated inactivated culture medium with alkali; (5) adding a lytic enzyme (a cell wall-degrading enzyme) to the alkali-treated inactivated culture medium to lyse the microbial cells and disperse the intracellular substances, including PHA, in the culture medium; (6) adding a protease to the culture medium to degrade substances derived from the microbial cells other than PHA (particularly proteins); and (7) further adjusting the pH of the culture medium and adding a surfactant to degrade substances derived from the microbial cells other than PHA (particularly cell membranes).

[0037] The present production method may include, prior to the centrifugation step, a step of culturing a PHA-producing microorganism and preparing a PHA aqueous suspension (1) from the culture solution, which includes one or more of the above-mentioned steps (a PHA aqueous suspension preparation step). When the present production method includes two or more of the above-mentioned steps, the order in which the steps are performed is not limited to the above-mentioned order.

[0038] (Centrifugation) Next, the centrifugation operation performed in the centrifugation step will be described in detail. The centrifugation operation in the centrifugation step is not particularly limited as long as it can remove impurities (particularly proteins) derived from the cells of the PHA-containing microorganism in the PHA aqueous suspension (1) by centrifugation and obtain a PHA aqueous suspension (2) having a protein content of 6,000 to 30,000 ppm, and can be performed by any centrifugation method known in the technical field of the present invention.

[0039] Such known centrifugation methods include, for example, centrifugation using a centrifugal settler or a centrifugal dehydrator.

[0040] Examples of centrifugal settlers that can be used in the centrifugation step include separation plate type (e.g., disk type, self-cleaning type, nozzle type, screw decanter type, skimming type, etc.) and cylindrical type centrifugal settlers. Furthermore, both palindrome type and continuous type centrifugal settlers can be used. Similarly, either palindrome type or continuous type centrifugal dehydrators can be used.

[0041] The number of centrifugation steps in the centrifugation step significantly affects the amount of wastewater generated in the present production method. Therefore, from the viewpoint of reducing the amount of wastewater generated by the present production method, the number of centrifugation steps in the present production method is preferably two or less, and may be one. In this specification, one centrifugation step refers to a series of operations from centrifuging a target liquid (e.g., PHA aqueous suspension (1)) under any conditions to removing a part or all of the supernatant (e.g., an amount equivalent to 40% by volume or more of the liquid subjected to centrifugation). When two or more centrifugations are performed, the liquid to be centrifuged may be a concentrated liquid (concentrated PHA aqueous suspension) obtained by removing the supernatant obtained by the above centrifugation step, or may be a liquid obtained by adding an aqueous solvent to the concentrated liquid and adjusting the concentration to any desired level.

[0042] In conventional PHA purification processes using centrifugation, four or more centrifugation steps are required to sufficiently remove impurities from a PHA aqueous suspension, resulting in the generation of a large amount of wastewater (equivalent to at least four steps). In contrast, the present production method combines a centrifugation step and a filtration step to perform PHA purification, thereby enabling the production of PHA (PHA cake) with sufficiently reduced impurities despite requiring only two or fewer centrifugations. Therefore, compared to conventional PHA purification processes using centrifugation, it is possible to reduce the amount of wastewater generated throughout the entire purification process.

[0043] The centrifugation conditions (rotation speed, rotation time) in the centrifugation step are not particularly limited as long as a PHA aqueous suspension (2) with a desired protein content can be obtained with the desired number of centrifugations. For example, the rotation speed per centrifugation may be 3,000 to 6,000 rpm, and the rotation time may be 1 to 30 minutes.

[0044] (PHA aqueous suspension (2)) The PHA aqueous suspension (2) obtained by the centrifugation step is a PHA aqueous suspension derived from the PHA aqueous suspension (1) and has a protein content of 6,000 to 30,000 ppm. As described above, the PHA aqueous suspension (2) is derived from the PHA aqueous suspension (1), and therefore the PHA contained in the PHA aqueous suspension (2) has the same composition as that of the PHA aqueous suspension (1).

[0045] The protein content of the PHA aqueous suspension (2) is 6,000 to 30,000 ppm. When the protein content of the PHA aqueous suspension (2) is 30,000 ppm or less, a sufficient filtration rate can be achieved in the subsequent filtration step. From the viewpoint of improving the filtration rate in the filtration step, the lower the protein content of the PHA aqueous suspension (2), the more preferable. Specifically, the protein content of the PHA aqueous suspension (2) is preferably 25,000 ppm or less, more preferably 15,000 ppm or less, and even more preferably 10,000 ppm or less. That is, in the centrifugation step, it is preferable to carry out the centrifugation operation so that the protein content of the resulting PHA aqueous suspension (2) falls within the above range.

[0046] The solids concentration (i.e., PHA concentration) of the PHA aqueous suspension (2) is not particularly limited, but from the viewpoint of improving the fluidity of the PHA aqueous suspension, it is preferably 5 to 30% by weight, more preferably 10 to 20% by weight.

[0047] The pH of the PHA aqueous suspension (2) immediately after centrifugation is not particularly limited, but the pH of the PHA aqueous suspension (2) immediately before being subjected to the subsequent filtration step is 3.0 to 5.5. By adjusting the pH of the PHA aqueous suspension (2) to be subjected to the filtration step to 3.0 to 5.5, it is possible to achieve an excellent filtration rate in the filtration step, even though the PHA aqueous suspension (2) contains a relatively large amount of protein, at 6,000 ppm or more.

[0048] ·pH adjustment process In one embodiment of the present production method, the pH of the PHA aqueous suspension (2) immediately after centrifugation may be outside the range of 3.0 to 5.5. In such cases, it is preferable to adjust the pH of the PHA aqueous suspension (2) to 3.0 to 5.5 prior to the filtration step. That is, the present production method preferably includes a pH adjustment step of adjusting the pH of the PHA aqueous suspension (2) to 3.0 to 5.5 prior to the filtration step.

[0049] In the pH adjustment step, the method for adjusting the pH of the PHA aqueous suspension (2) to the above range is not particularly limited. For example, it is preferable to adjust the pH of the PHA aqueous suspension (2) by adding an acid or alkali.

[0050] The acid used in the pH adjustment step is not particularly limited and may be either an organic acid or an inorganic acid, regardless of whether it is volatile. More specifically, examples of the acid used in the pH adjustment step include sulfuric acid, hydrochloric acid, phosphoric acid, and acetic acid.

[0051] The alkali used in the pH adjustment step is also not particularly limited, and examples thereof include alkali metal or alkaline earth metal hydroxides 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.

[0052] The amount of these acids or alkalis used in the pH adjustment step can be appropriately determined by those skilled in the art according to the pH of the PHA aqueous suspension (2) before pH adjustment and the pH of the PHA aqueous suspension (2) after the desired pH adjustment.

[0053] In another embodiment of the present invention, the pH of the PHA aqueous suspension (1) can be adjusted to 3.0 to 5.5 prior to the centrifugation step, thereby directly obtaining an aqueous PHA suspension (2) having a pH of 3.0 to 5.5. That is, the timing of the pH adjustment step is not particularly limited as long as it is performed before the filtration step and provides an aqueous PHA suspension (2) having the desired pH. The pH adjustment step may be performed before or after the centrifugation step, but is preferably performed after the centrifugation step because this enables the production of a PHA with a lower impurity content.

[0054] <Filtration process> This production method includes a filtration step of subjecting the aqueous polyhydroxyalkanoate suspension (2) obtained in the above-mentioned centrifugation step to dead-end filtration. In this specification, the term "dead-end filtration" means "filtration by the dead-end filtration method."

[0055] The specific mode of the dead-end filtration operation in the filtration step is not particularly limited, and examples thereof include suction filtration, pressure filtration, centrifugal filtration, and gravity filtration.

[0056] The material of the filter medium used in the filtration step is not particularly limited and can be selected from various materials, such as paper, filter cloth (woven or nonwoven), screen, sintered plate, bisque, polymer membrane, punched metal, wedge wire, etc. From the viewpoints of cost and ease of cleaning, filter cloth is preferably used.

[0057] The air permeability of the filter medium used in the filtration step is not particularly limited, but is preferably 0.25 cc / cm 2 / sec or less, and 0.20cc / cm 2 / sec or less. 2 By carrying out filtration using a filter medium with an air permeability of 0.25 cc / cm or less, leakage of PHA into the filtrate can be suppressed, and the yield of PHA can be improved. 2From the above viewpoint, the lower the air permeability of the filter medium used in the filtration step, the more preferable it is. The lower limit of the air permeability is not particularly limited, but for example, it is 0.01 cc / cm 2 / sec or more.

[0058] In this specification, the air permeability of a filter medium is the air permeability of a unit area (cm ) of the filter medium per second. 2 The air permeability of a filter medium can be measured by the method described in the Examples.

[0059] This manufacturing method reduces the protein content of the PHA aqueous suspension to 30,000 ppm or less in the centrifugation process, thereby enabling a sufficient filtration speed in the filtration process.As a result, it is now possible to purify PHA by filtration (remove impurities), which was previously difficult to put into practical use due to the filtration speed.

[0060] In this specification, the filtration rate in the filtration step can be evaluated by the filtrate permeation rate measured under the conditions described in the Examples. The higher the filtrate permeation rate in the filtration step, the faster the filtration rate in the filtration step. More specifically, if the filtrate permeation rate in the filtration step is 350 L / m 2 From the above viewpoint, it can be evaluated that an excellent filtration rate has been achieved when the filtrate permeation rate in the filtration step of the present production method is 350 L / m 2 / hr or more is preferable, and 400 L / m 2 / hr or more is more preferable, and 450 L / m 2 / hr or more is more preferable.

[0061] Filter cake In the filtration step, the PHA aqueous suspension (2) is filtered to obtain a filter cake as a residue. The filter cake undergoes a two-stage purification process consisting of a centrifugation step and a filtration step, thereby becoming a PHA aggregate from which impurities have been sufficiently removed.

[0062] From the viewpoint of obtaining a filter cake from which impurities have been sufficiently removed, the protein content of the filter cake obtained by the filtration step is preferably at least 2,000 ppm lower than the protein content of the PHA aqueous suspension (2) that is the raw material for the filter cake. In other words, the filtration step is preferably performed under conditions such that the protein content of the filter cake obtained by the filtration step is less than 6,000 ppm, which is at least 2,000 ppm lower than the protein content of the PHA aqueous suspension (2) that is the raw material for the filter cake.

[0063] The protein content of the filter cake is not particularly limited as long as it is 2,000 ppm or more lower than the protein content of the PHA aqueous suspension (2) that is the raw material of the filter cake, but is preferably less than 6,000 ppm.If the protein content of the filter cake is less than 6,000 ppm, it can be said that the filter cake is a filter cake from which impurities have been sufficiently removed.The protein content of the filter cake can be measured by the method described in Examples.

[0064] Furthermore, in the filtration step, if a filter cake satisfying the above conditions is not obtained by a single filtration operation, the obtained filter cake may be suspended in water or the like and subjected to another filtration operation. That is, the filtration step may include two or more filtration operations. However, since wastewater is generated during the filtration operation, although the amount is smaller than that of the centrifugation operation, from the viewpoint of reducing the amount of wastewater generated throughout the present production method, the fewer the number of filtration operations performed in the filtration step, the more preferable. For example, it is preferably seven or fewer times, more preferably six or fewer times, more preferably five or fewer times, more preferably four or fewer times, more preferably three or fewer times, even more preferably two or fewer times, and particularly preferably one time.

[0065] By drying the filter cake obtained by the filtration step using a known method, PHA particles or PHA powder with a sufficiently reduced amount of impurities can be obtained. Such PHA particles or PHA powder can be used as a molded product by a known molding method, such as injection molding, extrusion molding, blow molding, or compression molding. In addition, they can be used as a foamed molded product by foaming using a known method and then molding. These molded products and foamed molded products made from PHA particles or PHA powder can be used for various purposes, such as paper, film, sheet, tube, plate, rod, container (e.g., bottle container), bag, parts, etc. [Example]

[0066] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0067] [Measurement method] Measurements in the examples and comparative examples were carried out by the following methods.

[0068] (pH of PHA aqueous suspension) The pH of the PHA aqueous suspensions (PHA aqueous suspensions (1) and (2)) was measured using a pH meter (9652-10D, manufactured by HORIBA).

[0069] (Shear viscosity of PHA aqueous suspension) The shear viscosity of the PHA aqueous suspension was measured by the following method. Specifically, the shear viscosity was measured using a coaxial double cylinder with an Anton Paar MCR302. The PHA aqueous suspension was poured into a 20 mL cylinder, and the liquid temperature was adjusted to 40°C or 50°C. After reaching the target shear rate (10 1 / s), the viscosity was measured when the change in torque with time became less than 1%.

[0070] (Protein content of PHA aqueous suspension) The protein content of the PHA aqueous suspension was measured using a BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Specifically, an amount of PHA aqueous suspension equivalent to 2 mg of PHA in solid content was placed in a 15 mL Falcon tube, 2 mL of the kit's reagent was added, and the suspension was shaken at 60°C for 30 minutes. 30 minutes after the end of shaking, the suspension was cooled to 25°C, and the absorbance at a wavelength of 562 nm was measured. The protein content of the PHA aqueous suspension was calculated based on the measured absorbance.

[0071] (Measurement of filter media air permeability) The air permeability of the filter material (filter cloth) used in the filtration process was measured using a TEXTEST INSTRUMENTS FX3345 Flex Air.

[0072] (Filtrate permeation rate during filtration process) Filtration was carried out under the conditions described in the filtration step of each Example and Comparative Example, and the amount of filtrate was measured for 1 minute from the start of the filtration. The filtrate permeation rate (LMH) was calculated based on the following formula: Filtrate permeation rate (LMH) = Filtrate volume (L) / Filtration area (m 2 ) / filtration time (h).

[0073] (Protein content of filter cake) The protein content of the filter cake was measured using a BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Specifically, the filter cake was dried to obtain PHA dried particles, and 2 mg of the dried particles was placed in a 15 mL Falcon tube, 2 mL of the kit's reagent was added, and the tube was shaken at 60 ° C for 30 minutes. 30 minutes after the end of shaking, the tube was cooled to 25 ° C, and the absorbance at a wavelength of 562 nm was measured. The protein content of the filter cake was calculated based on the measured absorbance.

[0074] Example 1 (Preparation of PHA aqueous suspension (1)) Preparation of bacterial culture medium Ralstonia eutropha, as described in International Publication No. WO 2019 / 142717, was cultured using the method described in paragraphs

[0041] to

[0048] of the same document to obtain a bacterial cell culture broth containing PHA. The obtained PHA was a copolymer consisting of 3HB repeating units and 3HH repeating units (i.e., poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)), and the composition ratio of the repeating units in the PHA (3HB unit / 3HH unit composition ratio) was 99 / 1 to 92 / 8 (mol / mol).

[0075] Inactivation treatment The bacterial cell culture solution obtained above was sterilized by heating and stirring at an internal temperature of 60 to 70°C for 7 hours to obtain an inactivated culture solution. The weight-average molecular weight of PHA in the obtained inactivated culture solution was 1.8 million. The solids concentration of the inactivated culture solution was 30% by weight.

[0076] Hydrogen peroxide treatment Hydrogen peroxide (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the inactivated culture medium obtained above to a concentration of 0.66%, and the inactivated culture medium was treated with hydrogen peroxide. After treatment, the shear viscosity of the inactivated culture medium at a temperature of 50°C and a shear rate of 10 1 / s was 5.01 mPa s.

[0077] Alkali treatment To the inactivated culture solution with reduced viscosity obtained above, a 30% aqueous solution of sodium hydroxide was added to adjust the pH to 11.0. While maintaining the solution at 60°C, the pH was maintained at 11.0 for 1.5 hours by continuing to add the 30% aqueous solution of sodium hydroxide, thereby obtaining an aqueous PHA suspension.

[0078] Neutralization and enzyme treatment The pH of the resulting PHA aqueous suspension was adjusted to 7.0±0.2 by adding 95% sulfuric acid. The solids concentration of this PHA aqueous suspension was measured and found to be 30% by weight. After adding sulfuric acid, lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme that degrades sugar chains (peptidoglycans) in cell walls, was added to a liquid concentration of 10 ppm, and the mixture was maintained at 50°C for 2 hours. Subsequently, 2.5 L of Alcalase (manufactured by Novozyme), a protease, was added to a liquid concentration of 300 ppm, and then 30% sodium hydroxide was added at 50°C to adjust the pH to 8.5, and the mixture was maintained for 2 hours.

[0079] Surfactant addition treatment Sodium dodecyl sulfate (SDS, manufactured by Kao) was added to the resulting enzyme-treated PHA aqueous suspension to a concentration of 0.6 to 1.0 wt %. The pH was then adjusted to 11.0 ± 0.2 using aqueous sodium hydroxide. After holding the suspension at 40°C for 1 hour, the PHA aqueous suspension was diluted with aqueous sodium hydroxide to obtain PHA aqueous suspension (1) with a solids concentration of 15 wt % and a pH of 11. The protein content of the resulting PHA aqueous suspension (1) is shown in Table 1. The shear viscosity of the resulting PHA aqueous suspension (1) at a liquid temperature of 40°C and a shear rate of 10 1 / s was 8.58 mPa s.

[0080] (Centrifugation process) The resulting PHA aqueous suspension (1) was centrifuged (4500 rpm, 10 minutes), and then a volume of supernatant equivalent to 50% by volume of the aqueous suspension centrifuged was removed, yielding a 2x concentrated PHA aqueous suspension. To this PHA aqueous suspension, an amount of sodium hydroxide solution equal to the removed supernatant was added, and the suspension was centrifuged again (4500 rpm, 10 minutes), and a volume of supernatant equivalent to 50% by volume of the aqueous suspension centrifuged was removed. To the PHA aqueous suspension obtained after these two centrifugation procedures, sodium hydroxide solution was added again, yielding PHA aqueous suspension (2) with a pH of 11.0 and a solids concentration of 30 wt%. The protein content of the resulting PHA aqueous suspension (2) is shown in Table 1.

[0081] (filtration process) The pH of the resulting PHA aqueous suspension (2) was adjusted to 4.8 using sulfuric acid. The air permeability of 18.2 g of the pH-adjusted PHA aqueous suspension (2) was 0.16 cc / cm. 2 Dead-end filtration was performed using a filter cloth of 1000 kJ / sec to obtain a filter cake. Specifically, the filter cloth was placed in a filter with an inner diameter of 40 mm and attached to a suction bottle (2L, manufactured by SHIBATA). Suction filtration was then performed by adding the PHA aqueous suspension (2) to the filter while applying suction to -90 kPa with a vacuum pump. The protein content of the obtained filter cake, the filtration rate in the filtration process (the filtration rate at 1 minute after the start of filtration), and the amount of wastewater (amount per 1 kg of resin (PHA)) generated during the centrifugation step to the filtration step (until the filter cake was obtained) are shown in Table 1.

[0082] Example 2 PHA purification treatment was carried out in the same manner as in Example 1, except that the pH of the PHA aqueous suspension (2) to be subjected to the filtration step was adjusted to 3.4 using sulfuric acid. The protein contents of the PHA aqueous suspension and filter cake obtained in each operation, the filtration rate in the filtration step, and the amount of wastewater are shown in Table 1.

[0083] (Comparative Example 1) A PHA filter cake was obtained in the same manner as in Example 1, except that the centrifugation step was not performed, i.e., the PHA aqueous suspension (1) was directly subjected to the filtration step. Table 1 shows the protein contents of the PHA aqueous suspensions and filter cakes obtained in each operation, the filtration rates in the filtration step, and the wastewater volumes.

[0084] (Comparative Example 2) PHA purification treatment was carried out in the same manner as in Example 1, except that the pH of the PHA aqueous suspension (2) to be subjected to the filtration step was not adjusted (i.e., PHA aqueous suspension (2) at pH 11 was subjected to the filtration step). The protein contents of the PHA aqueous suspensions and filter cakes obtained in each operation, the filtration rates in the filtration step, and the wastewater volumes are shown in Table 1.

[0085] (Comparative Example 3) PHA purification treatment was carried out in the same manner as in Example 1, except that the pH of the PHA aqueous suspension (2) to be subjected to the filtration step was adjusted to 7.7 using sulfuric acid. The protein contents of the PHA aqueous suspension and filter cake obtained in each operation, the filtration rate in the filtration step, and the amount of wastewater are shown in Table 1.

[0086] Comparative Example 4 PHA purification treatment was carried out in the same manner as in Example 1, except that the pH of the PHA aqueous suspension (2) to be subjected to the filtration step was adjusted to 7.0 using sulfuric acid. The protein contents of the PHA aqueous suspension and filter cake obtained in each operation, the filtration rate in the filtration step, and the amount of wastewater are shown in Table 1.

[0087] (Comparative Example 5) PHA purification treatment was carried out in the same manner as in Example 1, except that the pH of the PHA aqueous suspension (2) to be subjected to the filtration step was adjusted to 6.4 using sulfuric acid. The protein contents of the PHA aqueous suspension and filter cake obtained in each operation, the filtration rate in the filtration step, and the amount of wastewater are shown in Table 1.

[0088] (Comparative Example 6) The PHA aqueous suspension (1) obtained in Example 1 was centrifuged a total of three times under the same conditions as in Example 1 to obtain an aqueous suspension. The protein content of the obtained aqueous suspension was 6,533 ppm, and the amount of wastewater generated (amount per kg of resin (PHA)) until the aqueous suspension was obtained was 9.99 kg / kg of resin.

[0089] [Table 1]

[0090] 〔summary〕 As is clear from Table 1, the protein content of the PHA aqueous suspension (1) obtained by the centrifugation step was 9885 ppm, indicating that impurities in the PHA aqueous suspension could not be sufficiently treated by only two centrifugations that took into consideration the reduction of the amount of wastewater. On the other hand, the results of Examples 1 and 2 indicated that impurities in the PHA aqueous suspension could be sufficiently treated by performing a filtration step in addition to two centrifugations, while minimizing the number of centrifugations and reducing the amount of wastewater.

[0091] Furthermore, a comparison of Examples 1 and 2 with Comparative Example 1 showed that PHA can be purified by filtration at a high filtration rate by controlling the protein content in the aqueous suspension through a centrifugation step prior to the filtration step. Also, a comparison of Examples 1 and 2 with Comparative Examples 2 to 5 showed that PHA can be purified by filtration at a high filtration rate by adjusting the pH of the PHA aqueous suspension (2) to be subjected to the filtration step to 3.0 to 5.5.

[0092] Furthermore, a comparison of Examples 1 and 2 with Comparative Example 6 showed that the present production method, which combines a centrifugation step and a filtration step, can produce a higher purity PHA with a smaller amount of wastewater than when PHA is purified by centrifugation alone. [Industrial Applicability]

[0093] This production method can reduce the amount of wastewater discharged during the purification process and has an excellent filtration rate, making it suitable for use in the production of PHA. PHA produced by this production method can be used, for example, as molded articles in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing, packaging, automobiles, building materials, and other fields.

Claims

1. a centrifugation step of centrifuging the polyhydroxyalkanoate aqueous suspension (1) to obtain a polyhydroxyalkanoate aqueous suspension (2) having a protein content of 6,000 to 30,000 ppm; a filtration step of subjecting the obtained polyhydroxyalkanoate aqueous suspension (2) to dead-end filtration, The method for producing a polyhydroxyalkanoate, wherein the pH of the aqueous polyhydroxyalkanoate suspension (2) subjected to the filtration step is 3.0 to 5.

5.

2. 2. The method for producing a polyhydroxyalkanoate according to claim 1, further comprising a pH adjustment step of adjusting the pH of the polyhydroxyalkanoate aqueous suspension (2) to 3.0 to 5.5 before the filtration step.

3. In the filtration step, the air permeability is 0.25 cc / cm ２ 2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein dead-end filtration is carried out using a filter medium having a flow rate of 1 / sec or less.

4. 2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the number of times of centrifugation in the centrifugation step is two or less.

5. 2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the protein content of the aqueous polyhydroxyalkanoate suspension (1) is 50,000 ppm or more.

6. 2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the shear viscosity of the aqueous polyhydroxyalkanoate suspension (1) at 40° C. and 10 1 / s is 4 to 15 mPa·s.

7. 2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the protein content of the filter cake obtained in the filtration step is lower by 2,000 ppm or more than the protein content of the polyhydroxyalkanoate aqueous suspension (2) that is the raw material for the filter cake.

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