WO2026014323A1 - Method for producing polyhydroxyalkanoate - Google Patents

Abstract

The problem addressed is to provide a method for producing PHA that can sufficiently remove impurities while reducing the amount of wastewater in a purification process and can realize a practical filtration rate. The problem is solved by a method for producing PHA, the method comprising: a centrifugal separation step in which a PHA aqueous suspension (1) is centrifuged to obtain a PHA aqueous suspension (2) having a predetermined protein content; and a filtration step in which the PHA aqueous suspension (2), which has been adjusted to a pH of from more than 5.5 to 11.0, is subjected to dead end filtration.

Description

Method for producing polyhydroxyalkanoate

The present invention relates to a method for producing polyhydroxyalkanoates.

Polyhydroxyalkanoates (hereinafter sometimes referred to as "PHA") are known to be biodegradable, and in recent years have been increasingly utilized from an environmentally friendly perspective.

One of the advantages of PHA is that it can be produced by microorganisms using renewable plant-based raw materials. To use PHA produced by microorganisms, it is first necessary to destroy the PHA-containing microorganism cells or solubilize biological components other than PHA, disperse the PHA in the cells in water to obtain an aqueous PHA suspension, and then purify the PHA by further removing impurities other than PHA from this aqueous PHA suspension.

Known techniques for purifying such PHA include centrifuging an aqueous PHA suspension (Patent Documents 1 and 2) and filtering an aqueous PHA suspension (Patent Documents 3 and 4).

International Publication No. WO2023/120193 Japanese Patent Publication No. 2023-86317 Japanese Patent Publication No. 2016-524926 Chinese Patent No. 111500650

However, among conventional PHA purification techniques, the method of centrifuging an aqueous PHA suspension requires multiple centrifugation steps (generally four or more) during purification, which can result in excessively large amounts of wastewater being discharged during the purification process. In other words, there is room for improvement in terms of the amount of wastewater being discharged during the purification process. On the other hand, when the number of centrifugation steps was limited in order to reduce the amount of wastewater, it was not possible to sufficiently remove impurities.

Furthermore, the method of filtering the PHA aqueous suspension had an extremely low filtration rate, making it difficult to use (practically) for industrial purposes.

In light of the above situation, the present invention aims to provide a PHA production method that can sufficiently remove impurities while reducing the amount of wastewater discharged during the purification process, and achieve a filtration rate sufficient for practical use.

As a result of extensive research to solve the above-mentioned problems, the inventors discovered a new method for purifying PHA 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 with the pH adjusted to a predetermined range. This method reduces the amount of wastewater discharged during the purification process, while sufficiently removing impurities and achieving a filtration speed sufficient for practical use, leading to the completion of the present invention.

In other words, one aspect of the present invention is a method for producing 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 greater than 5.5 and equal to or less than 11.0.

One aspect of the present invention provides a PHA production method that can reduce the amount of wastewater discharged during the purification process, while sufficiently removing impurities and achieving a filtration rate suitable for practical use.

One embodiment of the present invention is described below, but the present invention is not limited to this. 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. Furthermore, new technical features can be created 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 A and greater than A) and less than or equal to B (including B and less than B)."

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.

<Technical Concept of the Present Invention>
PHA produced by a microorganism may naturally contain impurities (particularly proteins) derived from the microorganism. The impurities contained in such PHA produced by a microorganism may cause quality degradation, such as contamination with foreign matter or deterioration in color tone, in products made by processing the PHA. For this reason, when using PHA produced by a microorganism in a product, it is first necessary to remove the impurities contained in the PHA and purify it.

However, 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 through four centrifugation steps (which are then dehydrated by filtration), resulting in the generation of a large amount of wastewater.

In investigating a method for purifying PHA that would produce less wastewater, with a view to reducing environmental impact, the inventors focused on a method for purifying PHA by filtration. However, as described above, it was difficult to achieve a filtration rate sufficient for practical use when purifying PHA by filtration.

In researching the causes of reduced filtration speed during PHA purification by filtration, the inventors discovered that one of the causes of reduced filtration speed is blockage of the filter media used for filtration due to the large amount of impurities contained in the unpurified PHA aqueous suspension.

Further research by the inventors, who gained the above knowledge, led them to discover that by reducing the amount of impurities in the PHA aqueous suspension to a certain level beforehand and then subjecting it to filtration, clogging of the filter media during the filtration process can be suppressed, making it possible to purify PHA through filtration at a practical filtration speed. They also discovered 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 clog the filter media during filtration, can be carried out with a relatively small number of centrifugation cycles (i.e., with a small amount of wastewater). Additionally, they discovered that the ability to remove impurities can be further improved by controlling the pH of the PHA aqueous suspension within a specified range during filtration.

With these findings in mind, the inventors discovered that by combining a centrifugation process that reduces the amount of impurities in the aqueous PHA suspension to the above-mentioned level with a filtration process that filters the PHA aqueous 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, achieve a filtration speed sufficient for practical use that is difficult to achieve with filtration by filtration alone, and further discover that this process also has excellent impurity removal capabilities.In other words, it is possible to reduce the amount of wastewater in the purification process while sufficiently removing impurities, and achieve a filtration speed sufficient for practical use, which led to the completion of the present invention.

This production method can be said to be a method for purifying PHA (PHA purification method) that can sufficiently remove impurities while reducing the amount of wastewater generated during the purification process, and can be carried out at a filtration speed sufficient for practical use.

The following provides a detailed explanation of each step involved in this manufacturing method.

<Centrifugal separation step>
This production method includes a centrifugation step in which an aqueous PHA suspension (1) is centrifuged to obtain an aqueous PHA suspension (2) having a protein content of 6,000 to 30,000 ppm.

(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 is intended to mean a solution in which PHA is suspended (dispersed) in water (aqueous medium) and has fluid properties. Furthermore, 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. That is, the PHA aqueous suspension (1) according to this production method may contain these components in addition to PHA and water.

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, "unpurified PHA aqueous suspension" refers to an aqueous PHA 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 cells in water, preferably by disrupting the PHA-containing microorganism cells and solubilizing biological components other than PHA, and which has not undergone any purification treatment, specifically, centrifugation or filtration. The method for destroying the cells of PHA-containing microorganisms 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 or other lytic enzymes and/or alcalase or other protease enzymes.

If the purification history of a certain PHA aqueous suspension is unknown, whether the aqueous suspension is unpurified can be determined, for example, based on the amount of impurities, particularly the amount of protein, in the aqueous suspension. Specifically, in this specification, if the protein content of a certain PHA aqueous suspension is 50,000 ppm or more, the aqueous suspension is considered to be an unpurified PHA aqueous 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 PHA aqueous suspension." It goes without saying that 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) used in this production method if it is unpurified. The protein content of a PHA aqueous suspension is measured using the method described in the Examples.

The shear viscosity of the PHA aqueous suspension (1) is not particularly limited, but from the perspective of improving the filtration rate in the subsequent filtration process, the shear viscosity at 40°C and a shear rate of 10 1/s is preferably 4 to 15 mPa·s, and more preferably 5 to 10 mPa·s. In this specification, unless otherwise specified, "shear viscosity of the PHA aqueous suspension" refers to the shear viscosity at 40°C and a shear rate of 10 1/s. The shear viscosity of the PHA aqueous suspension is measured by the method described in the Examples.

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

Next, the PHA contained in PHA aqueous suspension (1) will be described in detail. Note that the basic physical properties of the PHA contained in PHA aqueous suspension (1) do not change during the centrifugation and filtration steps of this production method, and therefore the following description also applies to the specific aspects of the PHA contained in PHA aqueous suspension (2) made from PHA aqueous suspension (1).

・PHA
"PHA" is a general term for polymers containing hydroxyalkanoate as a monomer unit (monomer repeating unit) and is generally biodegradable. In particular, in this specification, "PHA" refers to a (co)polymer containing 50 mol% or more of hydroxyalkanoate repeating units out of all monomer repeating units (100 mol%), and a resin composed of such a (co)polymer. Specific examples of hydroxyalkanoate repeating units that constitute 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" is used to refer to both a homopolymer composed of only one type of monomer and a copolymer composed of two or more types of monomers.

PHAs provided by this manufacturing method include, for example, poly(3-hydroxyalkanoate) (hereinafter sometimes referred to as "P3HA") and poly(4-hydroxyalkanoate). Of these, P3HA is preferred because it is suitable for use in molded articles.

P3HA is a PHA that contains, as an essential repeating unit, a 3-hydroxyalkanoate repeating unit represented by the formula: [-CHR-CH ₂ -CO-O-] (wherein R is an alkyl group represented by C _n H _2n+1 , and n is an integer of 1 or more and 15 or less).

Specific examples of P3HA include poly(3-hydroxybutyrate) (hereinafter sometimes referred to as "P3HB"), which is a homopolymer of 3HB, 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), poly(3-hydroxy Examples of suitable polyhydroxybutyrates include 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), and poly(3-hydroxybutyrate-co-3-hydroxypropionate). Among these, P3HB, P3HB3HH, and P3HB4HB are preferred, with P3HB3HH and P3HB4HB being more preferred, due to the ease of industrial production using microorganisms.

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 units are derived and a monomer from which the Y repeating units are derived. As mentioned above, the name of P3HA is determined by the repeating units contained in the P3HA. However, very small 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 very small amounts of other repeating units in addition to the repeating units corresponding to its name.

When P3HA contains 3HB repeat units, from the viewpoint of the balance between flexibility and strength, the composition ratio (3HB repeat units/other repeat units) of 3HB repeat units to repeat units other than 3HB repeat units (other repeat units) among all monomer repeat units (100 mol%) in 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 repeat units in P3HA is 60 mol% or more, it has the advantage of being able to provide resin products with superior rigidity. On the other hand, when the composition ratio of 3HB repeat units in P3HA is 99 mol% or less, it has the advantage of being able to provide resin products with superior flexibility. The monomer composition ratio of P3HA can be measured by gas chromatography or the like (see, for example, WO 2014/020838).

P3HA can be produced by microorganisms. Examples of microorganisms capable of producing P3HA include P3HB-producing bacteria, such as Bacillus megaterium, which was discovered in 1925, and other microorganisms such as Cupriavidus necator.
Known natural microorganisms include Alcaligenes eutrophus (formerly classified as Alcaligenes eutrophus and Ralstonia eutropha), Alcaligenes latus, and others. In these microorganisms, P3HB accumulates intracellularly.

Furthermore, known bacteria 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). In particular, Alcaligenes eutrophus AC32 (FERM BP-6038) (T. Fukui, Y. Doi, J. Bacteriol., 179, pp. 4821-4830 (1997)), which has been introduced with genes encoding P3HA synthases, is preferred for increasing P3HB3HH productivity. 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 P3HA.

- Method for Producing PHA Aqueous Suspension As the PHA aqueous suspension (1) to be subjected to the centrifugation step, a PHA aqueous suspension derived from a culture broth of a PHA-producing microorganism can be suitably used. Such an aqueous PHA suspension derived from the culture solution of a PHA-producing microorganism can be prepared, for example, by the following methods: (1) culturing a microorganism capable of producing PHA; (2) inactivating the culture solution of the microorganism by heating to obtain an inactivated culture solution; (3) treating the inactivated culture solution with hydrogen peroxide to reduce the viscosity of the culture solution; (4) treating the inactivated culture solution treated with hydrogen peroxide with alkali; (5) adding a lytic enzyme (a cell wall-degrading enzyme) to the alkali-treated inactivated culture solution to lyse the microbial cells and disperse the substances within the cells, including PHA, in the culture solution; (6) adding a protease to the culture solution to decompose substances derived from the microbial cells other than PHA (particularly proteins); (7) further adjusting the pH of the culture solution and adding a surfactant to decompose substances derived from the microbial cells other than PHA (particularly cell membranes).

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 (PHA aqueous suspension preparation step), which includes one or more of the above-mentioned operations. Furthermore, when the present production method includes two or more of the above-mentioned operations, the order in which the operations are performed is not limited to the above-mentioned order.

(Centrifugation operation)
The centrifugation operation in the centrifugation step is described in detail below. 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.

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

Centrifugal settlers that can be used in the centrifugation process include, for example, separator 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 and continuous type centrifugal settlers can be used. Similarly, either palindrome or continuous type centrifugal dehydrators can be used.

The number of centrifugation steps in the centrifugation step significantly affects the amount of wastewater generated in this production method. Therefore, from the perspective of reducing the amount of wastewater generated by this production method, the number of centrifugation steps in this production method is preferably two or fewer, and may be one. In this specification, one centrifugation step refers to a series of operations in which the target liquid (e.g., PHA aqueous suspension (1)) is centrifuged under any conditions, followed by partial or complete removal of the supernatant (e.g., an amount equivalent to 40% or more by volume 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 from the above-mentioned 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.

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, this production method combines a centrifugation step and a filtration step to perform the PHA purification process, making it possible to obtain PHA (PHA cake) with sufficiently reduced impurities despite performing only two or fewer centrifugations. Therefore, it is possible to reduce the amount of wastewater generated throughout the entire purification process compared to conventional PHA purification processes using centrifugation.

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

(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, since the PHA aqueous suspension (2) is a PHA aqueous suspension derived from the PHA aqueous suspension (1), the PHA contained in the PHA aqueous suspension (2) has the same composition as that of the PHA aqueous suspension (1).

The protein content of PHA aqueous suspension (2) is 6,000 to 30,000 ppm. A protein content of 30,000 ppm or less in PHA aqueous suspension (2) enables a sufficient filtration rate to be achieved in the subsequent filtration step. From the perspective of improving the filtration rate in the filtration step, the lower the protein content of PHA aqueous suspension (2), the better. Specifically, the protein content of 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. In other words, 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) is within the above range.

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

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 greater than 5.5 and less than 11.0. By adjusting the pH of the PHA aqueous suspension (2) to be subjected to the filtration step to greater than 5.5 and less than 11.0, it is possible to improve the impurity removal efficiency while maintaining a sufficient filtration rate in the filtration step, and as a result, it is possible to efficiently obtain PHA from which impurities have been sufficiently removed.

The higher the pH of the PHA aqueous suspension (2) subjected to the filtration step, the more likely it is that the impurity removal efficiency in the filtration step will be improved. Therefore, from the perspective of further improving the impurity removal efficiency, the pH of the PHA aqueous suspension (2) subjected to the filtration step is preferably 6.2 or higher, more preferably 6.7 or higher, more preferably 7.2 or higher, even more preferably 7.7 or higher, even more preferably 8.2 or higher, even more preferably 8.7 or higher, even more preferably 9.0 or higher, even more preferably 9.2 or higher, even more preferably 9.7 or higher, and even more preferably 10.2 or higher.

pH Adjustment Step: In one embodiment of the present production method, the pH of the PHA aqueous suspension (2) immediately after centrifugation may be 5.5 or less, or 11.0 or more. In such cases, it is preferable to adjust the pH of the PHA aqueous suspension (2) to the above-mentioned range, for example, greater than 5.5 and less than 11.0, 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 greater than 5.5 and less than 11.0 prior to the filtration step.

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, but it is preferable to adjust the pH of the PHA aqueous suspension (2) by adding, for example, an acid or alkali.

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

The alkali used in the pH adjustment step is also not particularly limited, and examples that can be used 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.

The amount of these acids or alkalis used in the pH adjustment step can be appropriately determined by those skilled in the art, depending on 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.

In another aspect of the present invention, by adjusting the pH of PHA aqueous suspension (1) to greater than 5.5 and equal to or less than 11.0 prior to the centrifugation step, it is possible to directly obtain PHA aqueous suspension (2) having a pH greater than 5.5 and equal to or less than 11.0. In other words, the pH adjustment step is performed before the filtration step, and as long as it is possible to provide PHA aqueous suspension (2) having the desired pH, the timing of the pH adjustment step is not particularly limited. It may be performed before or after the centrifugation step, but it is preferable to perform it after the centrifugation step, as this enables the production of PHA with a lower impurity content.

<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."

Specific modes of dead-end filtration operations in the filtration process are not particularly limited, and include suction filtration, pressure filtration, centrifugal filtration, gravity filtration, etc.

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

The air permeability of the filter medium used in the filtration step is not particularly limited, and may be, for example, 0.50 cc/cm ² /sec or less, 0.40 cc/cm ² /sec or less, or 0.30 cc/cm ² /sec or less, but is preferably 0.25 cc/cm ² /sec or less, and more preferably 0.20 cc/cm ² /sec or less. By performing filtration using a filter medium ^with the above air permeability, particularly a filter medium with an air permeability of 0.25 cc/cm 2 /sec or less, leakage of PHA into the filtrate can be suppressed and the recovery rate (yield) of PHA can be improved. From the above perspective, the filtration step in this production method is preferably a step of performing dead-end filtration using a filter medium with an air permeability of 0.25 cc/cm ² /sec or less. From the above viewpoint, the lower the air permeability of the filter medium used in the filtration step, the better. There is no particular lower limit to the air permeability, but it can be, for example, 0.01 cc/cm ² /sec or more.

In this specification, the air permeability of a filter medium refers to the amount of air (cc) that passes through a unit area (cm ² ) of the target filter medium per second. The air permeability of a filter medium can be measured by the method described in the Examples.

In this manufacturing method, the protein content of the PHA aqueous suspension is reduced to 30,000 ppm or less during the centrifugation process, making it possible to achieve a practical filtration speed during the filtration process.As a result, it is now possible to purify PHA (remove impurities) by filtration, which was previously difficult to implement due to the filtration speed limitations.

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. A higher filtrate permeation rate in the filtration step means a faster filtration rate in the filtration step. More specifically, when the filtrate permeation rate in the filtration step is 100 L/m ² /hr or more, it can be evaluated that a filtration rate sufficient for practical use has been achieved. From the above viewpoint, the filtrate permeation rate in the filtration step of the present production method is preferably 100 L/m ² /hr or more, more preferably 150 L/m ² /hr or more, and may be 200 L/m ² /hr or more, or may be 250 L/m ² /hr or more.

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.

The amount of impurities in the filter cake obtained by this production method can be evaluated based on the protein content of the filter cake. Specifically, if the protein content of the filter cake obtained through the centrifugation and filtration steps is 5,500 ppm or less, the filter cake can be said to be a filter cake from which impurities have been sufficiently removed. The lower the protein content of the filter cake, the more impurities have been removed from the filter cake. From the above perspective, the protein content of the filter cake obtained by this production method is 5,500 ppm or less, preferably 5,000 ppm or less, and more preferably 4,500 ppm or less.

Furthermore, in the filtration step, if a filter cake satisfying the above conditions is not obtained through 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, albeit in a smaller amount than in the centrifugation operation, from the viewpoint of reducing the amount of wastewater generated throughout the production method, the fewer the number of filtration operations performed in the filtration step, the better. For example, seven or fewer filtration operations are preferred, six or fewer filtration operations are more preferred, five or fewer filtration operations are more preferred, four or fewer filtration operations are more preferred, three or fewer filtration operations are even more preferred, two or fewer filtration operations are even more preferred, and one filtration operation is particularly preferred. On the other hand, from the viewpoint of removing as many impurities as possible, the more the number of filtration operations is preferred, one or more filtration operations are preferred, two or more filtration operations are more preferred, and three or more filtration operations are even more preferred. From the viewpoint of achieving both a reduction in the amount of wastewater and the removal of impurities, the number of filtration operations performed in the filtration step is preferably one to three filtration operations.

By drying the filter cake obtained from the filtration process using a known method, it is possible to obtain PHA particles or PHA powder with a sufficiently reduced amount of impurities. Such PHA particles or PHA powder can be used as a molded product by molding using a known molding method, such as injection molding, extrusion molding, blow molding, or compression molding. They can also 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 a variety of purposes, including paper, film, sheets, tubes, plates, rods, containers (e.g., bottle containers), tableware (e.g., straws, cutlery), bags, and parts.

〔others〕
One aspect of the present invention may include the following configuration.

[1] A method for producing 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 greater than 5.5 and less than or equal to 11.0.

[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 greater than 5.5 and not greater than 11.0 prior to the filtration step.

[3] The method for producing a polyhydroxyalkanoate according to [2], wherein the pH is adjusted by adding an acid or alkali to the aqueous polyhydroxyalkanoate suspension (2) in the pH adjustment step.

[4] The method for producing polyhydroxyalkanoate according to any one of [1] to [3], wherein in the filtration step, dead-end filtration is carried out using a filter medium having an air permeability of 0.50 cc/cm ² /sec or less.

[5] The method for producing polyhydroxyalkanoate according to any one of [1] to [4], wherein in the filtration step, dead-end filtration is carried out using a filter medium having an air permeability of 0.25 cc/cm ² /sec or less.

[6] The method for producing polyhydroxyalkanoate described in any one of [1] to [5], wherein the number of centrifugation steps in the centrifugation step is two or less.

[7] The method for producing a polyhydroxyalkanoate according to any one of [1] to [6], wherein the protein content of the polyhydroxyalkanoate aqueous suspension (1) is 50,000 ppm or more.

[8] The method for producing a polyhydroxyalkanoate according to any one of [1] to [7], wherein the shear viscosity of the aqueous polyhydroxyalkanoate suspension (1) at 40°C and 10 1/s is 4 to 15 mPa·s.

[9] The method for producing polyhydroxyalkanoate according to any one of [1] to [8], wherein the protein content of the filter cake obtained in the filtration step is 5,500 ppm or less.

[10] The method for producing a polyhydroxyalkanoate according to any one of [1] to [9], wherein the pH of the aqueous polyhydroxyalkanoate suspension (2) subjected to the filtration step is 9.0 to 11.0.

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

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

(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).

(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 MCR302 manufactured by Anton Paar. 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%.

(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, a PHA aqueous suspension in an amount equivalent to 10 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.

(Measurement of filter media air permeability)
The air permeability of the filter material (filter cloth) used in the filtration step was measured using a FX3345 Flex Air manufactured by Textest Instruments.

(Filtrate permeation rate in 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).

(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 mixture was shaken at 60 ° C for 30 minutes. 30 minutes after the end of shaking, the mixture 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.

(PHA recovery rate)
The PHA recovery rate was measured using the following procedure. The total weight of the filtrate obtained by the filtration process was measured. The filtrate was then thoroughly mixed, and 1 g of the filtrate was dispensed using a dropper. The dispensed filtrate was heated at 105°C using a heat-dry moisture meter ML-50 (manufactured by A&D Co., Ltd.) until the weight change rate fell below 0.05% (W.B.)/min, thereby measuring the solids concentration in the filtrate. The solids content (g) in the total filtrate was calculated by multiplying the obtained solids concentration by the total weight of the filtrate. The leakage rate (%) was calculated by dividing the solids weight (g) in the filtrate by the solids weight (g) of the PHA aqueous suspension before the filtration process. The PHA recovery rate (%) was then calculated by subtracting the leakage rate (%) from 100%.

Example 1
(Preparation of PHA aqueous suspension (1))
Preparation of Bacterial Cell Culture Solution Ralstonia eutropha described in International Publication No. WO 2019/142717 was cultured by the method described in paragraphs [0041] to [0048] of the same document to obtain a bacterial cell culture solution containing PHA-containing bacterial cells. The obtained PHA was a copolymer composed 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 (composition ratio of 3HB units/3HH units) was 99/1 to 92/8 (mol/mol).

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, thereby obtaining an inactivated culture solution. The weight-average molecular weight of PHA in the obtained inactivated culture solution was 1,800,000. The solids concentration of the inactivated culture solution was 30% by weight.

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

Alkali Treatment: A 30% aqueous solution of sodium hydroxide was added to the inactivated culture solution with reduced viscosity obtained above to adjust the pH to 11.0. The solution was maintained at 60°C, and the 30% aqueous solution of sodium hydroxide was added continuously to maintain the pH at 11.0 for 1.5 hours, thereby obtaining an aqueous PHA suspension.

Neutralization and enzyme treatment: The pH of the resulting aqueous PHA suspension was adjusted to 7.0±0.2 by adding 95% sulfuric acid. The solids concentration of this aqueous PHA 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 decomposes sugar chains (peptidoglycans) in cell walls, was added to a liquid concentration of 10 ppm and maintained at 50 ° C. for 2 hours. Then, Alcalase 2.5L (manufactured by Novozyme), a protease, was added to a liquid concentration of 300 ppm, and then 30% sodium hydroxide was added at 50 ° C., and the mixture was maintained for 2 hours while adjusting the pH to 8.5.

Surfactant Addition Treatment Sodium dodecyl sulfate (SDS, manufactured by Kao Corporation) 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.

(Centrifugation step)
The resulting PHA aqueous suspension (1) was centrifuged (4500 rpm, 10 minutes), and then an amount of supernatant equivalent to 50% by volume of the aqueous suspension centrifuged was removed to obtain a 2-fold concentrated PHA aqueous suspension. To this PHA aqueous suspension, an amount of sodium hydroxide solution equal to the amount of the removed supernatant was added, and the mixture was centrifuged again (4500 rpm, 10 minutes), and an amount of supernatant equivalent to 50% by volume of the aqueous suspension centrifuged was removed (centrifugation step). A sodium hydroxide solution was added again to the PHA aqueous suspension obtained after two centrifugation operations, and a PHA aqueous suspension (2) with a pH of 11.0 and a solids concentration of 30 wt% was obtained (pH adjustment step). The protein content of the resulting PHA aqueous suspension (2) is shown in Table 1.

(Filtration process)
18.2 g of the obtained pH-adjusted aqueous PHA suspension (2) was subjected to dead-end filtration using a filter cloth with an air permeability of 0.16 cc/cm ² /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 (2 L, manufactured by SHIBATA). Then, while sucking to -90 kPa with a vacuum pump, the aqueous PHA suspension (2) was introduced into the filter, whereby suction filtration was performed. The protein content of the obtained filter cake and the filtration rate in the filtration step (the filtration rate 1 minute after the start of filtration) are shown in Table 1.

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 7.7 using sulfuric acid. Table 1 shows the viscosity of the resulting PHA aqueous suspension (1), the air permeability of the filter cloth used, the protein content of each PHA aqueous suspension and filter cake, and the filtration rate in the filtration step.

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 7.0 using sulfuric acid. Table 1 shows the viscosity of the resulting PHA aqueous suspension (1), the air permeability of the filter cloth used, the protein content of each PHA aqueous suspension and filter cake, and the filtration rate in the filtration step.

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 6.4 using sulfuric acid. Table 1 shows the viscosity of the resulting PHA aqueous suspension (1), the air permeability of the filter cloth used, the protein content of each PHA aqueous suspension and filter cake, and the filtration rate in the filtration step.

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 9.0 using sulfuric acid. The viscosity of the resulting PHA aqueous suspension (1), the air permeability of the filter cloth used, the protein content of each PHA aqueous suspension and filter cake, and the filtration rate in the filtration step are shown in Table 1. The PHA recovery rate was measured and found to be 99.1%.

Example 6
PHA purification was carried out in the same manner as in Example 1, except that a filter cloth with an air permeability of 0.30 cc/cm ² /sec was used in the filtration step. The viscosity of the resulting PHA aqueous suspension (1), the air permeability of the filter cloth used, the protein content of each PHA aqueous suspension and filter cake, and the filtration rate in the filtration step are shown in Table 1. The PHA recovery rate was measured and found to be 91.5%.

(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. The viscosity and protein content of the obtained PHA aqueous suspension (1), the air permeability of the filter cloth used, and the filtration rate in the filtration step are shown in Table 1.

(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 adjusted to 4.8 using sulfuric acid. Table 1 shows the viscosity of the resulting PHA aqueous suspension (1), the air permeability of the filter cloth used, the protein content of each PHA aqueous suspension and filter cake, and the filtration rate in the filtration step.

(Comparative Example 3)
A 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 viscosity of the obtained PHA aqueous suspension (1), the protein content of the filter cake, and the filtration rate in the filtration step are shown in Table 1.

Example 7
The filter cake obtained in Example 1 was dispersed in an aqueous sodium hydroxide solution to obtain an aqueous suspension with a solids concentration of 30 wt % and a pH of 11.0. This aqueous suspension was then filtered again under the same conditions as in Example 1, thereby obtaining a filter cake that had undergone a total of two filtration operations. This filter cake was further dispersed in an aqueous sodium hydroxide solution to obtain an aqueous suspension with a solids concentration of 30 wt % and a pH of 11.0. This aqueous suspension was then filtered again under the same conditions as in Example 1, thereby obtaining a filter cake that had undergone a total of three filtration operations. The protein contents of the obtained filter cakes and the amount of wastewater generated before obtaining the filter cakes (amount per kg of resin (PHA)) are shown in Table 2.

(Example 8)
The filter cake obtained in Example 3 was dispersed in an aqueous sodium hydroxide solution to obtain an aqueous suspension with a solids concentration of 30 wt % and a pH of 7.0. This aqueous suspension was then filtered again under the same conditions as in Example 1, yielding a filter cake that had undergone a total of two filtration operations. This filter cake was then dispersed in an aqueous sodium hydroxide solution to obtain an aqueous suspension with a solids concentration of 30 wt % and a pH of 7.0. This aqueous suspension was then filtered again under the same conditions as in Example 1, yielding a filter cake that had undergone a total of three filtration operations. The protein content of the obtained filter cake and the amount of wastewater (amount per kg of resin (PHA)) generated before obtaining the filter cake were measured. The results are shown in Table 3.

(Comparative Example 4)
The PHA aqueous suspension (1) obtained in Example 1 was centrifuged a total of six times under the same conditions as in Example 1. After each centrifugation, the protein content of the precipitate and the cumulative amount of wastewater (amount per kg of resin (PHA)) were measured. The results are shown in Table 4. The protein content of the precipitate was measured by the method described above in the section (Protein content of filter cake), except that precipitate was used instead of filter cake.

〔summary〕
As is clear from Table 1, the protein content of the PHA aqueous suspension (1) obtained by the centrifugation step was 9885 ppm, which indicated 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 to 6 indicated that by performing a filtration step in addition to two centrifugations, the amount of wastewater could be reduced by minimizing the number of centrifugations, while still sufficiently removing impurities from the PHA aqueous suspension.

Furthermore, a comparison of Examples 1 to 6 with Comparative Example 1 showed that if a centrifugation step was not performed before the filtration step, a filtration speed sufficient for practical use would not be achieved, but that by performing a centrifugation step before the filtration step and controlling the protein content in the aqueous suspension, it was possible to purify PHA by filtration at a filtration speed sufficient for practical use. Furthermore, a comparison of Examples 1 to 6 with Comparative Examples 2 and 3 showed that by adjusting the pH of the PHA aqueous suspension (2) subjected to the filtration step to greater than 5.5 and equal to or less than 11.0, a greater amount of impurities could be removed in the filtration step, making it possible to provide PHA with a sufficiently reduced amount of impurities.

Furthermore, a comparison of Examples 7 and 8 with Comparative Example 4 showed that, while a total of four centrifugation operations are required to produce PHA with a sufficiently reduced amount of impurities using centrifugation alone, this production method, which combines centrifugation and filtration, makes it possible to produce PHA with a sufficiently reduced amount of impurities using a smaller amount of wastewater. Furthermore, the results of Examples 7 and 8 also showed that by increasing the number of filtration operations, it is possible to remove a greater amount of impurities while reducing the amount of wastewater compared to centrifugation alone.

This production method is suitable for use in the production of PHA, as it can sufficiently remove impurities while reducing the amount of wastewater discharged during the refining process and achieves a filtration rate suitable for practical use. PHA produced by this production method can be used, for example, as a molded product in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing, packaging, automobiles, building materials, and other fields.

Claims (10)

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 greater than 5.5 and not greater than 11.0. The method for producing polyhydroxyalkanoate according to claim 1, further comprising a pH adjustment step of adjusting the pH of the polyhydroxyalkanoate aqueous suspension (2) to greater than 5.5 and not greater than 11.0 prior to the filtration step. The method for producing polyhydroxyalkanoate according to claim 2, wherein the pH is adjusted by adding an acid or alkali to the polyhydroxyalkanoate aqueous suspension (2) in the pH adjustment step. 2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the filtration step is performed by dead-end filtration using a filter medium having an air permeability of 0.50 cc/cm ^<2> /sec or less. 2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the filtration step is performed by dead-end filtration using a filter medium having an air permeability of 0.25 cc/cm ^<2> /sec or less. The method for producing polyhydroxyalkanoate according to claim 1, wherein the number of centrifugation steps in the centrifugation step is two or less. The method for producing polyhydroxyalkanoate according to claim 1, wherein the protein content of the polyhydroxyalkanoate aqueous suspension (1) is 50,000 ppm or more. The method for producing 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. The method for producing polyhydroxyalkanoate according to claim 1, wherein the protein content of the filter cake obtained in the filtration step is 5,500 ppm or less. The method for producing polyhydroxyalkanoate according to claim 1, wherein the pH of the aqueous polyhydroxyalkanoate suspension (2) subjected to the filtration step is 9.0 to 11.0.