Method for producing biomass-derived hydroxyalkanoic acid ester

Abstract

The purpose of the present disclosure is to provide a method which is for producing a biomass-derived hydroxyalkanoic acid ester and by which the conversion of polyhydroxyalkanoates into hydroxyalkanoic acid esters can be performed in good yield.　This method for producing a biomass-derived hydroxyalkanoic acid ester comprises: a step for causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in a water-containing system; a step for dispersing the produced polyhydroxyalkanoate particles in an alcohol; and a step for converting, into hydroxyalkanoic acid esters, polyhydroxyalkanoates constituting the polyhydroxyalkanoate particles in the alcohol dispersion, wherein said method has one feature among features i) to iv).

Description

Method for producing biomass-derived hydroxyalkanoic acid esters

[0001] The present invention relates to a method for producing a biomass-derived hydroxyalkanoic acid ester.

[0002] Important industrial raw materials such as propylene, butadiene, and benzene have conventionally been obtained by distillation separation of reaction products from the thermal cracking of naphtha obtained by refining crude oil using a naphtha cracker. Of these, butadiene can be obtained by extraction from the C4 fraction produced as a by-product when ethylene is produced using a naphtha cracker.

[0003] With the expansion of the shale gas market in recent years, the competitiveness of ethane crackers, which are devices that produce raw materials such as ethylene and propylene using the ethane and propane contained in shale gas as raw materials, has increased, but the amount of propylene produced is small, and butadiene, benzene, etc. cannot be produced directly using ethane crackers. Therefore, there are concerns that the expansion of ethane crackers as a substitute for naphtha crackers will lead to a decline in the competitiveness of naphtha crackers, making it difficult to ensure a stable supply of the above-mentioned raw materials that cannot be produced by ethane crackers.

[0004] In recent years, there has been growing awareness of environmental issues, food issues, health and safety, and a preference for natural or natural products. As an example of this, there is the problem that plastic waste places a heavy burden on the global environment, such as its impact on the ecosystem, the generation of harmful gases when burned, and global warming due to the large amount of heat generated by combustion. There has been active development of biodegradable plastics as a solution to these problems.

[0005] In particular, when biodegradable plastics are produced from biomass-derived raw materials such as plants, the carbon dioxide emitted when the biodegradable plastics are burned is the same carbon dioxide that was originally present in the air, and does not increase the amount of carbon dioxide in the atmosphere. This is called carbon neutrality, and is considered important under the Kyoto Protocol, which imposed carbon dioxide reduction targets, and active use of biodegradable plastics is desired.

[0006] Along with this trend toward carbon neutrality, the use of biomass is being considered to ensure a stable supply of important industrial raw materials, including butadiene.

[0007] JP 2012-236798 A JP 11-266891 A International Publication No. 2004 / 029266

[0008] Against the backdrop of growing awareness of the above-mentioned environmental issues and a growing preference for natural or natural products, the significance and importance of material production using microorganisms (fermentation production, bioconversion, etc.), which offers significant benefits in terms of energy efficiency and environmental impact, is also increasing, and microorganisms are being used to produce degradable plastics. One biodegradable plastic that is expected to be used industrially is polyhydroxyalkanoate (hereinafter also referred to as PHA). PHA is a thermoplastic polyester that is produced and accumulated as an energy storage substance in the cells of many microbial species.

[0009] Hydroxyalkanoic acid esters are obtained by decomposition of PHA, and 3-hydroxybutyric acid esters, which are hydroxyalkanoic acid esters, are obtained by decomposition of, for example, poly(3-hydroxybutyrate)-based polymers (hereinafter, poly(3-hydroxybutyrate) may also be referred to as PHB) among PHAs. 3-Hydroxybutyric acid esters, such as methyl 3-hydroxybutyrate, are compounds that can serve as precursors for propylene, butadiene, and the like. Therefore, efficient production of 3-hydroxybutyric acid esters leads to efficient production of propylene, butadiene, and the like.

[0010] As a method for obtaining a 3-hydroxybutyrate ester using a microbially-derived polyhydroxybutyrate, Patent Document 1 discloses that a (3R)-3-hydroxybutyrate ester with a high enantiomeric excess was obtained using a microbially-derived polyhydroxybutyrate obtained by the method described in the examples of JP-A-11-266891 (Patent Document 2). However, the method described in Patent Document 1 does not provide a sufficient yield of the 3-hydroxybutyrate ester.

[0011] An object of the present invention is to provide a method for producing a biomass-derived hydroxyalkanoate, which can convert a polyhydroxyalkanoate into a hydroxyalkanoate in a high yield.

[0012] The present invention relates to a method for producing a biomass-derived hydroxyalkanoate ester, which comprises the steps of causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in a system containing water, dispersing the produced polyhydroxyalkanoate particles in alcohol, and converting the polyhydroxyalkanoate that constitutes the polyhydroxyalkanoate particles in the alcohol dispersion into a hydroxyalkanoate ester, and which has any one of the following configurations i) to iv): Structure i) In the step of converting the polyhydroxyalkanoate particles into the hydroxyalkanoate ester, the total nitrogen content in the polyhydroxyalkanoate particles is 2% by weight or less. Structure ii) In the step of converting the polyhydroxyalkanoate particles into the hydroxyalkanoate ester, the water content in the alcohol dispersion is 10% by weight or less relative to 100% by weight of the polyhydroxyalkanoate particles. Structure iii) The weight-average molecular weight of the polyhydroxyalkanoate is 2,000,000 or less. Structure iv) No step of drying the polyhydroxyalkanoate particles is performed before converting the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles into the hydroxyalkanoate ester.

[0013] According to the present invention, it is possible to provide a method for producing a biomass-derived hydroxyalkanoate, which is capable of converting a polyhydroxyalkanoate into a hydroxyalkanoate with good yield.

[0014]

[0023] The first embodiment of the present invention relates to a method for producing a biomass-derived hydroxyalkanoate ester having the above-mentioned configuration i), that is, a method for producing a biomass-derived hydroxyalkanoate ester, comprising the steps of causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in an aqueous system, dispersing the produced polyhydroxyalkanoate particles in alcohol, and converting the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles into a hydroxyalkanoate ester in the alcohol dispersion, wherein the total nitrogen content in the polyhydroxyalkanoate particles in the step of converting into the hydroxyalkanoate ester is 2% by weight or less. The first embodiment of the present invention will be described in detail below.

[0015] (Polyhydroxyalkanoate Particles) The polyhydroxyalkanoate particles (hereinafter also referred to as PHA particles) refer to particles composed of PHA, and are not particularly limited as long as they are PHA particles that can be produced by microorganisms. The type of PHA may be a homopolymer composed of one type of hydroxyalkanoic acid, or a copolymer composed of two or more types of hydroxyalkanoic acids. Specific examples include homopolymers of one type of monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, copolymers of one type of monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and other hydroxyalkanoic acids (e.g., 2-hydroxyalkanoic acids, 4-hydroxyalkanoic acids, 5-hydroxyalkanoic acids, 6-hydroxyalkanoic acids, etc. having 4 to 16 carbon atoms), and copolymers of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms.

[0016] When the PHA is a copolymer, the type is not particularly limited, and may be a random copolymer, an alternating copolymer, a block copolymer, a graft copolymer, or the like, but a random copolymer is preferred because it is easily available.

[0017] Among the PHAs, poly(3-hydroxybutyrate) polymers are preferred from the viewpoint that they can be easily produced even by microorganisms present in nature. Poly(3-hydroxybutyrate) polymers refer to polymers in which 50% by weight or more of the structural units of the polymer are 3-hydroxybutyrate, and include homopolymers of 3-hydroxybutyrate units, copolymers containing 3-hydroxybutyrate units and other hydroxyalkanoate units, etc.

[0018] P(3HB), a homopolymer of 3-hydroxybutyrate units, refers to a polymer essentially containing only 3-hydroxybutyrate units. The homopolymer of 3-hydroxybutyrate units may contain trace amounts of monomer units other than 3-hydroxybutyrate, but the content of monomer units other than 3-hydroxybutyrate in the homopolymer of 3-hydroxybutyrate units is preferably 5% by weight or less, more preferably 3% by weight or less, even more preferably 1% by weight or less, and particularly preferably 0.5% by weight or less.

[0019] When the poly(3-hydroxybutyrate) polymer is a copolymer containing 3-hydroxybutyrate units and other hydroxyalkanoate units, the hydroxyalkanoate units other than 3-hydroxybutyrate are not particularly limited, and examples thereof include 3-hydroxyhexanoate, 3-hydroxyvalerate, 4-hydroxybutyrate, 3-hydroxyoctanoate, 3-hydroxyoctadecanoate, 3-hydroxydecanoate, and lactic acid, and one or more selected from the group consisting of these may be contained as monomer units together with 3-hydroxybutyrate. A copolymer of 3HB and 3-hydroxyhexanoate (abbreviation: 3HH) may be referred to as P(3HB-co-3HH) (abbreviation: PHBH), a copolymer of 3HB and 3-hydroxyvalerate (abbreviation: 3HV) as P(3HB-co-3HV), a copolymer of 3HB and 4-hydroxybutyrate (abbreviation: 4HB) as P(3HB-co-4HB), and a PHA containing lactic acid (abbreviation: LA) as a constituent component, for example, a copolymer of 3HB and LA as P(LA-co-3HB).

[0020] A poly(3-hydroxybutyrate) polymer containing a 3-hydroxyhexanoate unit is suitable in that a C6 compound (a compound having 6 carbon atoms) can be obtained.

[0021] The poly(3-hydroxybutyrate) polymer is preferably a homopolymer of 3-hydroxybutyrate units, since microorganisms capable of producing it are readily available.

[0022] The shape of the PHA particles is not particularly limited and may be, for example, spherical or non-spherical. The size of the PHA particles is also not particularly limited, but from the viewpoint of ease of washing and solvent substitution, the average particle size is preferably 0.1 to 50 μm, more preferably 0.5 to 50 μm, and even more preferably 1 to 50 μm.

[0023] The average particle size of the PHA particles can be determined by observation with an electron microscope or by a particle size distribution measuring device.

[0024] (PHA-producing microorganism) The PHA-producing microorganism may be a microorganism capable of producing PHA. The microorganism may be a microorganism having a PHA synthase gene. The microorganism may be a wild-type strain that inherently has the PHA synthase gene, a mutant strain obtained by artificially mutating such a wild-type strain, or a strain into which an exogenous PHA synthase gene has been introduced by genetic engineering techniques.

[0025] The PHA-producing microorganism or the host of the microorganism is not particularly limited, but is preferably a bacillus, more preferably a gram-negative bacillus. Preferred examples of the bacillus include bacteria belonging to the family Burkholderiaceae, such as the genera Ralstonia, Cupriavidus, Wautersia, and Burkholderia, as well as bacteria belonging to the genus Pseudomonas, Halomonas, and Escherichia.

[0026] From the viewpoints of safety and PHA productivity, bacteria belonging to the genus Ralstonia, Cupriavidus, or Escherichia are more preferred, bacteria belonging to the genus Cupriavidus or Escherichia are even more preferred, and Cupriavidus necator or Escherichia coli are particularly preferred.

[0027] (PHA synthase gene) The PHA synthase gene carried by the PHA-producing microorganism is not particularly limited, and examples thereof include PHA synthase genes derived from organisms similar to the genera Ralstonia, Capriavidus, Wautersia, Alcaligenes, Aeromonas, Pseudomonas, Norcadia, and Chromobacterium, as well as modified versions thereof. Examples of the modified versions include a base sequence encoding a PHA synthase in which one or more amino acid residues have been deleted, added, inserted, or substituted.

[0028] (Step of producing PHA particles) In the step of causing a PHA-producing microorganism to produce PHA particles in a system containing water, the PHA particles can be produced by culturing and growing the PHA-producing microorganism under appropriate conditions, thereby accumulating PHA particles within the cells of the PHA-producing microorganism.

[0029] The culture can be carried out by a person skilled in the art based on common technical knowledge, and the culture method is not particularly limited. The medium composition, the method of adding the carbon source, the culture scale, the aeration and agitation conditions, the culture temperature, and the culture time are also not particularly limited. For example, the method described in JP-A-05-93049 can be used. By culturing for an appropriate period of time, PHA particles can be accumulated within the cells of the PHA-producing microorganism.

[0030] (Step of Dispersing PHA Particles in Alcohol) The step of dispersing the produced PHA particles in alcohol will be described in detail below.

[0031] The alcohol in which the produced PHA particles are dispersed is preferably a water-soluble alcohol, more preferably an alcohol capable of deriving the desired hydroxyalkanoic acid ester. Examples of water-soluble alcohols include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-butanol, and one or more selected from the group consisting of these may be used. Among these, methanol or ethanol are more preferred, and methanol is even more preferred, in terms of reactivity with PHA.

[0032] When dispersing PHA particles in alcohol to prepare an alcohol dispersion, the amount of alcohol used is not particularly limited.

[0033] (Step of reducing the total nitrogen content in the PHA particles) After the step of producing the PHA particles, it is preferable to further include a step of reducing the total nitrogen content in the PHA particles. In the step of converting into a hydroxyalkanoic acid ester, the total nitrogen content in the PHA particles can be reduced, and the yield of the hydroxyalkanoic acid ester can be further increased.

[0034] The total nitrogen content in the PHA particles can be reduced by removing nitrogen-containing components such as bacterial cell constituents and culture substrate residues from the system. The method for removing nitrogen-containing components such as bacterial cell constituents and culture substrate residues is not particularly limited, and examples include a method for washing PHA particles, a method for treating PHA particles with an enzyme that decomposes bacterial cell constituents of microorganisms, and a method for replacing water in the system with alcohol, each of which can be used alone or in combination of two or more, and a method for combining two or more can be used, as the total nitrogen content in the PHA particles can be efficiently reduced.

[0035] (Method for washing PHA particles) A ​​method for washing PHA particles will be described in detail. In the step of converting the PHA constituting the PHA particles into a hydroxyalkanoic acid ester, the lower the total nitrogen content in the PHA particles, the easier it is to obtain a high yield of hydroxyalkanoic acid ester. However, by including a step of reducing the total nitrogen content in the PHA particles by washing the PHA particles after the step of producing the PHA particles, the yield of hydroxyalkanoic acid ester can be increased. In the present disclosure, "washing PHA particles" refers to separating and removing nitrogen-containing components attached to or bonded to the inside or outside of the PHA particles.

[0036] The method used to wash the PHA particles is not particularly limited, but examples thereof include filtration, centrifugation, sedimentation, and electrophoresis.

[0037] Examples of cleaning agents used for washing PHA particles include water, organic solvents, and mixed solutions of water and organic solvents. These cleaning agents may contain a surfactant or the like. The pH of water and mixed solutions of water and organic solvents used for washing PHA particles may be appropriately adjusted.

[0038] The method of washing PHA particles with water is preferable because no solvent is discharged, whereas the method of washing PHA particles with an organic solvent is preferable because the total nitrogen content of the PHA particles is likely to decrease.

[0039] The type of organic solvent used to wash the PHA particles is not particularly limited. However, water-soluble organic solvents are preferred because they enable efficient reduction of the water content in the system during the process of converting the PHA that constitutes the PHA particles into a hydroxyalkanoic acid ester, reduce the possibility of PHA hydrolysis reactions competing with the alcoholysis reaction of the PHA, and facilitate the production of hydroxyalkanoic acid esters in higher yields. Examples of water-soluble organic solvents include alcohols and other aprotic polar solvents. Examples of alcohols include monohydric alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, and t-butyl alcohol; and dihydric alcohols such as ethylene glycol and diethylene glycol. Other aprotic polar solvents include acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, acetone, tetrahydrofuran, methyl acetate, and diethyl ether, as well as ketones, amines, esters, and ethers. These organic solvents may be used alone or in combination of two or more.

[0040] When the step of converting PHA into a hydroxyalkanoic acid ester is carried out by alcoholysis reaction, the organic solvent is preferably an alcohol, more preferably methanol or ethanol, since it can be used in the reaction and is efficient.

[0041] (Method of treating PHA particles with an enzyme) The method of treating with an enzyme that decomposes the bacterial cell components of the microorganism will be described in detail. In the step of converting the PHA that constitutes the PHA particles into a hydroxyalkanoic acid ester, the lower the total nitrogen content in the PHA particles, the easier it is to obtain a high yield of hydroxyalkanoic acid ester. However, by including a step of treating the PHA particles with an enzyme that decomposes the bacterial cell components of the microorganism after the step of producing the PHA particles, thereby reducing the total nitrogen content in the PHA particles, the yield of the hydroxyalkanoic acid ester can be increased.

[0042] Examples of enzymes used in the method of treating with an enzyme that decomposes the components of the microorganism's body include cell wall-decomposing enzymes; and proteolytic enzymes, lipolytic enzymes, and nucleolytic enzymes that decompose proteins, lipids, nucleic acids, and the like that constitute the microorganism. Also, commercially available enzyme laundry detergents and enzyme compositions containing enzymes and enzyme stabilizers, anti-redeposition agents, etc. can be used. These may be used alone, or two or more types may be used in combination. After the enzyme treatment, the PHA particles may be washed or subjected to solvent substitution, etc.

[0043] Examples of the cell wall-degrading enzyme include lysozyme, amylase, pectinase, cellulase, zymolyase, maltase, saccharase, α-glycosidase, β-glycosidase, and N-glycosidase. Lysozyme is preferred because it has an excellent bacteriolytic effect and can decompose peptidoglycan in the cell wall of the microorganism to lyse the microorganism even when the microorganism contains PHA particles, thereby enabling efficient removal of the components constituting the microorganism's body and further reducing the total nitrogen content in the PHA particles.

[0044] Examples of the proteolytic enzyme include esperase, alcalase, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, and carboxypeptidase, with alcalase being preferred in terms of its ability to efficiently decompose proteins.

[0045] Examples of the lipolytic enzyme include lipase.

[0046] Examples of the nucleolytic enzyme include nucleopolymerase.

[0047] The time required for the enzyme treatment can be appropriately set taking into consideration the desired degree of purification, and may be, for example, 0.5 to 10 hours, 1 to 10 hours, or 1 to 2 hours.

[0048] The amount of enzyme used depends on the type and activity of the enzyme and is not particularly limited, but may be, for example, about 0.0001 to 10 parts by weight per 100 parts by weight of PHA particles, and from the viewpoint of cost, 0.01 to 5 parts by weight is preferred.

[0049] Other examples of the method for reducing the total nitrogen content in the PHA particles include a method of treating with hypochlorous acid or a method of treating with hydrogen peroxide. After the treatment with hypochlorous acid or hydrogen peroxide, the PHA particles may be washed or subjected to solvent substitution.

[0050] In the method of treating with hypochlorous acid, the pH of the dispersion of PHA particles is set in the alkaline region, and the treatment is carried out under conditions that suppress contact with heat, light, and metals. The pH is preferably 8 or higher, more preferably 10 or higher, and even more preferably 12 or higher. The temperature during the treatment is preferably 40° C. or lower, more preferably 20° C. or lower.

[0051] In the method of treating with hydrogen peroxide, in order to enhance the effect of reducing the total nitrogen content in the PHA particles in a short time, it is preferable to add hydrogen peroxide to a dispersion of PHA particles and then heat the dispersion. The temperature is preferably 50°C or higher, more preferably 70°C or higher. The upper limit of the temperature is preferably the boiling point of the dispersion or lower. In the hydrogen peroxide treatment, it is preferable to hold the dispersion under heating, and the holding time is, for example, preferably about 10 minutes to 10 hours, more preferably 30 minutes to 5 hours, and even more preferably 1 to 3 hours.

[0052] In order to suppress a decrease in the molecular weight of the PHA due to the treatment using hydrogen peroxide, it is preferable to carry out the treatment while controlling the pH of the dispersion to 7 to 13 by continuously or intermittently adding an alkali to the dispersion of PHA particles. The alkali is not particularly limited, but examples thereof include sodium hydroxide, sodium carbonate, and potassium hydroxide. For details of pH control, see Patent Document 3.

[0053] (Method of Replacing Water in the System with Alcohol) In the method for producing a biomass-derived hydroxyalkanoic acid ester according to the first embodiment of the present invention, it is preferable to further include a step of replacing the water in the system with an alcohol after the step of producing the PHA particles. This is because the total nitrogen content in the PHA particles can be reduced by alcohol replacement. In the present disclosure, "replacing the water in the system with an alcohol" refers to removing water-soluble components derived from bacterial cell components, culture substrate residues, etc. together with water by replacing the liquid portion containing water in the system (in other words, other than the solid portion) with alcohol.

[0054] The type of alcohol used for alcohol substitution is not particularly limited, but water-soluble alcohols are preferred, and examples thereof include monohydric alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, and t-butyl alcohol; and dihydric alcohols such as ethylene glycol and diethylene glycol. These alcohols may be used alone or in combination of two or more.

[0055] When the step of converting PHA into a hydroxyalkanoic acid ester is carried out by alcoholysis reaction, it is preferable to use the same alcohol as that used in the reaction, and methanol or ethanol is more preferable, since it can be used in the reaction and is efficient.

[0056] (Step of Separating PHA Particles) In the method for producing a biomass-derived hydroxyalkanoic acid ester according to the first embodiment of the present invention, it is preferable to further include a step of separating the PHA particles from the bacterial body components of the microorganism in a system containing water after the step of producing the PHA particles, because this leads to an efficient reduction in the total nitrogen content in the PHA particles.

[0057] Separating PHA particles from the constituent components of the microorganism's cells means destroying the cells of the microorganism and removing the PHA particles from inside the microorganism to outside the microorganism. A preferred method for separating PHA particles is to accumulate PHA particles within the microorganism's cells by causing the PHA-producing microorganism to produce PHA particles, and then to disrupt the microorganism (cells) containing the PHA particles by physical, chemical, or biological treatment.

[0058] The disruption method is not particularly limited, but can be any of the conventional methods utilizing fluid shear force, solid shear force, or grinding, such as those using a French press, homogenizer, X-press, ball mill, colloid mill, DYNO mill, or ultrasonic homogenizer. Other methods that can be used include those using chemicals such as acids, alkalis, surfactants, organic solvents, and cell wall synthesis inhibitors; those using enzymes such as lysozyme, amylase, pectinase, cellulase, zymolyase, maltase, saccharase, α-glycosidase, β-glycosidase, and N-glycosidase; those using supercritical fluids; osmotic disruption; freezing; and dry grinding. Another example is autolysis, which utilizes the activity of proteases and esterases contained in the cells themselves. These disruption methods can be used alone or in combination, and the order in which they are combined is not limited (for example, a method using an enzyme can be followed by a method utilizing fluid shear force, solid shear force, or grinding). Batch processing or continuous processing can also be used.

[0059] In the first embodiment of the method for producing a biomass-derived hydroxyalkanoic acid ester of the present invention, it is more preferable to include, after the step of producing the polyhydroxyalkanoate particles, a step of separating PHA particles from the bacterial cell components of the microorganism in a system containing water, and a step of reducing the total nitrogen content in the PHA particles, in this order, since this leads to an efficient reduction in the total nitrogen content in the PHA particles.

[0060] (Drying of PHA Particles) In the method for producing a biomass-derived hydroxyalkanoic acid ester according to the first embodiment of the present invention, after the step of producing the PHA particles, the PHA particles may or may not be dried by removing liquid components containing water from the system. In the first embodiment, drying the PHA particles refers to removing liquid components to the extent that the PHA particles as a whole become powdery or granular. For example, the amount of liquid component relative to the PHA particles may be 5% by weight or less, 3% by weight or less, 1% by weight or less, or 0% by weight. The amount of liquid component relative to the PHA particles can be measured using a heated moisture meter such as the MOC63u manufactured by Shimadzu Corporation, or a Karl Fischer moisture meter. The method for measuring the amount of liquid component is the same as in the other embodiments.

[0061] In the process of converting the PHA that constitutes the PHA particles into a hydroxyalkanoic acid ester, the larger the contact area between the PHA and its reaction partners and the solvent, the higher the reactivity and the easier it is to obtain a hydroxyalkanoic acid ester in high yield. In addition, it is preferable not to go through a process of drying the PHA particles before converting the polyhydroxyalkanoate that constitutes the PHA particles into a hydroxyalkanoic acid ester, as this can prevent the PHA particles from agglomerating and forming secondary particles, which would result in an increase in particle size, and it can avoid the contact area between the PHA and its reaction partners and the solvent from becoming smaller.

[0062] (Step of Converting PHA to Hydroxyalkanoic Acid Ester) The step of converting the PHA constituting the PHA particles in the alcohol dispersion into a hydroxyalkanoic acid ester will be described in detail.

[0063] In this step, the total nitrogen content in the PHA particles is preferably 2% by weight or less, more preferably 1% by weight or less, more preferably 0.5% by weight or less, more preferably 0.2% by weight or less, more preferably 0.1% by weight or less, more preferably 0.09% by weight or less, more preferably 0.08% by weight or less, more preferably 0.07% by weight or less, more preferably 0.06% by weight or less, more preferably 0.05% by weight or less, and most preferably 0% by weight. When the conversion to a hydroxyalkanoic acid ester is by alcoholysis, nitrogen derived from the PHA-producing microorganism in the alcohol dispersion (e.g., nitrogen constituting proteins) inhibits the alcoholysis of PHA, and if the total nitrogen content in the PHA particles is more than 2% by weight, the yield of the hydroxyalkanoic acid ester will be insufficient.

[0064] The total nitrogen content in the PHA particles can be measured by a dry combustion method using an NC analyzer.

[0065] The method for converting PHA to a hydroxyalkanoic acid ester is not particularly limited as long as it can convert PHA to a hydroxyalkanoic acid ester, but alcoholysis is preferred, and from the viewpoint of facilitating the alcoholysis of PHA, it is more preferred to carry out the conversion in the presence of a catalyst. Examples of the catalyst include acid catalysts, base catalysts, and metal oxide catalysts. Among these, acid catalysts are preferred, and sulfuric acid is more preferred. The amount of catalyst used is preferably 0.005 to 10 wt % relative to the PHA, more preferably 0.01 to 5 wt %, and even more preferably 0.015 to 3 wt %.

[0066] The alcohol used in the alcoholysis of PHA may be any alcohol capable of deriving the desired hydroxyalkanoic acid ester, and a water-soluble alcohol is preferred. Examples of water-soluble alcohols include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-butanol, and one or more selected from the group consisting of these. Among these, methanol or ethanol are more preferred, and methanol is even more preferred, in terms of reactivity with PHA. It is particularly preferred to use the same alcohol as that used in the step of dispersing PHA particles in alcohol.

[0067] In the step of converting PHA into a hydroxyalkanoic acid ester, the content of alcohol in the alcohol dispersion is not particularly limited, but from the viewpoint of reactivity, the content is preferably 1 to 100 parts by weight, more preferably 1.5 to 100 parts by weight, even more preferably 1.5 to 50 parts by weight, particularly preferably 1.5 to 10 parts by weight, and most preferably 1.5 to 5 parts by weight, relative to 1 part by weight of PHA.

[0068] According to the method for producing a biomass-derived hydroxyalkanoic acid ester according to the first embodiment of the present invention, the total nitrogen content in the PHA particles during the conversion to a hydroxyalkanoic acid ester is low, which is less likely to inhibit the alcoholysis reaction. Therefore, the conversion of PHA to a hydroxyalkanoic acid ester can be carried out with high yield. Because the total nitrogen content in the PHA particles is below a specific amount, the reaction conditions for converting PHA to a hydroxyalkanoic acid ester can be relaxed. Specifically, for example, as described above, the amount of catalyst used can be reduced, and the temperature conditions can be relaxed as described below. Reducing the amount of catalyst used reduces the effort and cost required for catalyst removal, and relaxing the temperature conditions improves safety and efficiency.

[0069] When the conversion of PHA to a hydroxyalkanoic acid ester is carried out by alcoholysis, the reaction proceeds at 60 to 200° C., but from the viewpoint of facilitating the alcoholysis reaction, the temperature condition is preferably 80 to 180° C., more preferably 90 to 160° C., and even more preferably 100 to 140° C. The time for maintaining the temperature condition is preferably 1 to 12 hours, more preferably 2 to 10 hours, and even more preferably 3 to 8 hours.

[0070] (3-Hydroxybutyrate Ester) The hydroxyalkanoate ester obtained by the production method according to the first embodiment of the present invention will be described below by taking as an example a case where the hydroxyalkanoate ester is a 3-hydroxybutyrate ester. The 3-hydroxybutyrate ester is a compound represented by the following formula (1): [In the above formula (1), R is a hydrocarbon group]

[0071] R in the above formula (1) is not particularly limited as long as it is a hydrocarbon group. From the viewpoint of ease of distillation, however, a linear or branched lower alkyl group having 1 to 4 carbon atoms is preferred, and a methyl group or an ethyl group is more preferred.

[0072] When the hydroxyalkanoate ester is a 3-hydroxybutyrate ester, PHA particles composed of a polyhydroxyalkanoate containing a 3-hydroxybutyrate unit, such as the poly(3-hydroxybutyrate) polymer, may be produced by a polyhydroxyalkanoate-producing microorganism.

[0073] (3-Hydroxyhexanoic acid ester) The hydroxyalkanoic acid ester obtained by the production method according to the first embodiment of the present invention will be described below by taking as an example a case where the hydroxyalkanoic acid ester is a 3-hydroxyhexanoic acid ester. The 3-hydroxyhexanoic acid ester is a compound represented by the following formula (2): [In the above formula (2), R is a hydrocarbon group]

[0074] R in the above formula (2) is not particularly limited as long as it is a hydrocarbon group. From the viewpoint of ease of distillation, however, a linear or branched lower alkyl group having 1 to 4 carbon atoms is preferred, and a methyl group or an ethyl group is more preferred.

[0075] When the hydroxyalkanoate ester is a 3-hydroxyhexanoate ester, PHA particles composed of a polyhydroxyalkanoate containing a 3-hydroxyhexanoate unit, such as the poly(3-hydroxybutyrate) polymer, may be produced by a polyhydroxyalkanoate-producing microorganism.

[0076] (Step of purifying the produced hydroxyalkanoic acid ester by distillation) Preferably, the method for producing a biomass-derived hydroxyalkanoic acid ester according to the first embodiment of the present invention further comprises a step of purifying the produced hydroxyalkanoic acid ester by distillation.

[0077] The distillation method is not particularly limited, but examples thereof include single distillation, continuous distillation, atmospheric distillation, reduced pressure distillation, etc. In addition, the temperature and pressure conditions during distillation and the number of distillations are also not particularly limited as long as they are within the range of general chemical production.

[0078] [Method for producing biomass-derived hydroxyalkanoate ester ii)] A second embodiment of the present invention relates to a method for producing a biomass-derived hydroxyalkanoate ester having the above-mentioned configuration ii), that is, a method for producing a biomass-derived hydroxyalkanoate ester, comprising the steps of causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in a system containing water, dispersing the produced polyhydroxyalkanoate particles in alcohol, and converting the polyhydroxyalkanoate that constitutes the polyhydroxyalkanoate particles in the alcohol dispersion into a hydroxyalkanoate ester, wherein the water content in the alcohol dispersion in the step of converting into the hydroxyalkanoate ester is 10% by weight or less relative to 100% by weight of the polyhydroxyalkanoate particles. The second embodiment of the present invention will be described in detail below.

[0079] The explanations of the first embodiment regarding (polyhydroxyalkanoate particles), (PHA-producing microorganisms), (PHA synthase gene), (process for producing PHA particles), and (process for dispersing PHA particles in alcohol) are common to the second embodiment.

[0080] (Step of Removing Water from the System) In the second embodiment, it is preferable to further include a step of removing water from the system after the step of producing the PHA particles, which makes it possible to easily adjust the water content in the alcohol dispersion within the above range in the step of converting the PHA particles into the hydroxyalkanoic acid ester.

[0081] The PHA particles may or may not be dried by removing the liquid components including water in the system. In the second embodiment, drying the PHA particles refers to removing the liquid components to the extent that the PHA particles as a whole become powdery or granular, and for example, the amount of the liquid component relative to the PHA particles may be 5 wt% or less, 3 wt% or less, 1 wt% or less, or 0 wt%.

[0082] In the process of converting the PHA that constitutes the PHA particles of the second embodiment into a hydroxyalkanoic acid ester, the lower the water content in the alcohol dispersion, the easier it is to obtain a high yield of hydroxyalkanoic acid ester. Therefore, it is advantageous to include a process of removing water from the system and drying the PHA particles after the process of producing the PHA particles, as this enables efficient reduction of the water content in the alcohol dispersion.

[0083] On the other hand, it is preferable not to carry out a step of drying the PHA particles until the polyhydroxyalkanoate constituting the PHA particles is converted into a hydroxyalkanoic acid ester, for the reasons described in the first embodiment.

[0084] Specific examples of the method for removing water from the system include a method for evaporating or sublimating water in the system by adjusting the humidity, temperature, and pressure in the system, and a method for replacing water in the system with an organic solvent. These methods may be used either alone or in combination of two or more.

[0085] A method for replacing water in the system with an organic solvent will be described in detail. The method for producing a biomass-derived hydroxyalkanoic acid ester according to the second embodiment of the present invention may include a step of replacing water in the system with an organic solvent to remove the water after the step of producing the PHA particles. Here, the "water in the system" in "replacing water in the system with an organic solvent" refers to water as a component in the system, as well as liquid portions containing water (in other words, portions other than solid portions) such as aqueous solutions and aqueous dispersions.

[0086] The method for replacing water in the system with an organic solvent is not particularly limited, and examples thereof include a method in which water is removed from the system by filtration, centrifugation, sedimentation, electrophoresis, or the like, and then an organic solvent is added.

[0087] The type of organic solvent is not particularly limited as long as it can remove water from the system. However, water-soluble organic solvents are preferred because they enable efficient reduction of the water content in the alcohol dispersion during the process of converting the PHA constituting the PHA particles into a hydroxyalkanoic acid ester. Examples of water-soluble organic solvents include alcohols and other aprotic polar solvents. Examples of alcohols include monohydric alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, and t-butyl alcohol; and dihydric alcohols such as ethylene glycol and diethylene glycol. Other aprotic polar solvents include acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, acetone, tetrahydrofuran, methyl acetate, and diethyl ether, as well as ketones, amines, esters, and ethers. These organic solvents may be used alone or in combination of two or more. Surfactants and the like may also be added to the organic solvent.

[0088] When the step of converting PHA into a hydroxyalkanoic acid ester is carried out by alcoholysis reaction, the organic solvent is preferably an alcohol, more preferably methanol or ethanol, since it can be used in the reaction and is efficient.

[0089] When the step of replacing the water in the system with an organic solvent and removing the water is performed before separating the PHA particles from the bacterial cell components of the PHA microorganism, that is, when replacing the water in a system containing PHA microorganisms in which the PHA particles are contained within the bacterial cells with an organic solvent, depending on the type of organic solvent, separation of the PHA particles may be accompanied by disruption of the bacterial cells.

[0090] (Step of Separating PHA Particles) The method for producing a biomass-derived hydroxyalkanoic acid ester according to the second embodiment of the present invention preferably further comprises a step of separating the PHA particles from the bacterial cell components of the microorganism in a system containing water after the step of producing the PHA particles. This is because the bacterial cell components inhibit the alcoholysis reaction, which is a reaction for converting PHA to a hydroxyalkanoic acid ester, and this leads to an efficient reduction of the bacterial cell components.

[0091] The description of the section (Step of separating PHA particles) in the first embodiment is common to the second embodiment.

[0092] In the second embodiment of the present invention, the method for producing a biomass-derived hydroxyalkanoic acid ester preferably includes both a step of separating the PHA particles from the microbial cell components in a water-containing system and a step of removing the water from the system after the step of producing PHA particles. It is more preferable to perform a step of removing the water from the system after the separation step. The method for removing the water from the system after the separation step preferably includes a method of replacing the water in the system with an organic solvent, since this increases the contact area between the PHA and its reaction partner or the solvent. Furthermore, in order to enable efficient reduction of the water content in the system, it is preferable to remove the water from the system and dry the PHA particles after the separation step.

[0093] In addition, from the following viewpoints, a method of replacing the water in the system with an organic solvent is preferred as a method of removing water after the step of separating PHA particles from the bacterial cell components of the microorganism in an aqueous system. The cell lysate obtained by disrupting bacterial cells (cells) containing PHA particles contains, in addition to the PHA particles, bacterial cell components such as proteins, nucleic acids, lipids, and sugar components in the cells, as well as culture substrate residues. By replacing the water in the system with an organic solvent after disrupting the cells, impurities other than water in the conversion reaction from PHA to hydroxyalkanoic acid ester, such as bacterial cell components other than PHA particles, can also be replaced and removed by the organic solvent together with water, allowing for a more efficient conversion of PHA to hydroxyalkanoic acid ester.

[0094] Another example of a treatment for removing impurities in the conversion reaction from PHA to hydroxyalkanoic acid ester after the step of separating PHA particles from the bacterial cell components of the microorganism in a system containing water is a method in which the PHA particles are washed or solvent-substituted after treatment such as enzyme treatment, hypochlorous acid treatment, or hydrogen peroxide treatment to remove the impurities.

[0095] The descriptions regarding the enzyme treatment, hypochlorous acid treatment, and hydrogen peroxide treatment in the first embodiment are common to the second embodiment.

[0096] The various treatments for removing impurities may be carried out either alone or in combination of two or more.

[0097] (Step of Converting PHA to Hydroxyalkanoic Acid Ester) The step of converting the PHA constituting the PHA particles in the alcohol dispersion of the second embodiment to a hydroxyalkanoic acid ester will be described in detail.

[0098] In this step, the water content in the alcohol dispersion is preferably 10% by weight or less, more preferably 6% by weight or less, even more preferably 4% by weight or less, particularly preferably 1% by weight or less, and most preferably 0% by weight, relative to 100% by weight of the PHA particles. If the water content exceeds 10% by weight, when the conversion to a hydroxyalkanoic acid ester is by alcoholysis, the hydrolysis reaction of PHA due to the water in the alcohol dispersion competes with the alcoholysis reaction of PHA, resulting in an insufficient yield of the hydroxyalkanoic acid ester.

[0099] The water content in the alcohol dispersion refers to the water content before the start of the conversion reaction from PHA to a hydroxyalkanoic acid ester, and in the case of an alcoholysis reaction, it is the water content in the alcohol dispersion before the addition of a catalyst. Also, it can be measured by the Karl Fischer method (coulometric titration or volumetric titration).

[0100] The explanation in the section (Step of converting PHA into hydroxyalkanoic acid ester) of the first embodiment of the present invention is common to the second embodiment and the third and fourth embodiments described below.

[0101] According to the method for producing a biomass-derived hydroxyalkanoic acid ester according to the second embodiment of the present invention, the water content in the alcohol dispersion in the step of converting PHA to a hydroxyalkanoic acid ester is low, making it difficult for a hydrolysis reaction competing with the alcoholysis reaction to occur. Therefore, the conversion of PHA to a hydroxyalkanoic acid ester can be carried out with high yield. Furthermore, because the water content in the alcohol dispersion is below a specific amount, the reaction conditions for converting PHA to a hydroxyalkanoic acid ester can be relaxed. Specifically, for example, as described above, the amount of catalyst used can be reduced, and the temperature conditions can be relaxed as described below. Reducing the amount of catalyst used reduces the effort and cost required for catalyst removal, and relaxing the temperature conditions improves safety and efficiency.

[0102] The descriptions in the sections (3-hydroxybutyrate ester) and (3-hydroxyhexanoate ester) of the first embodiment of the present invention are common to the second embodiment of the present invention and the third and fourth embodiments described below.

[0103] The explanation in the section (step of purifying the produced hydroxyalkanoic acid ester by distillation) of the first embodiment of the present invention is common to the second embodiment, and the third and fourth embodiments described below.

[0104] [Method for Producing Biomass-Derived Hydroxyalkanoate Esters (iii)] The third embodiment of the present invention relates to a method for producing a biomass-derived hydroxyalkanoate ester having the above-mentioned configuration (iii), i.e., the method includes the steps of (1) causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in an aqueous system, (2) dispersing the produced polyhydroxyalkanoate particles in alcohol, and (3) converting the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles in the alcohol dispersion into a hydroxyalkanoate ester, wherein the weight-average molecular weight of the polyhydroxyalkanoate is 2,000,000 or less. Note that in the third embodiment, numbers ((1), (2), (3), etc.) may be used to denote each step, but these numbers are used to concisely represent each step and facilitate understanding of the third embodiment of the present invention, and do not restrict the presence or absence or order of each step. The third embodiment of the present invention will be described in detail below.

[0105] The explanations of the first embodiment regarding (polyhydroxyalkanoate particles), (PHA-producing microorganisms), (PHA synthase gene), (process for producing PHA particles), and (process for dispersing PHA particles in alcohol) are common to the third embodiment.

[0106] (Step (4) of Reducing the Weight-Average Molecular Weight of PHA) In the third embodiment, it is preferable to include step (4) of reducing the weight-average molecular weight of the PHA after step (1) of producing the PHA particles and before step (3) of converting the PHA to a hydroxyalkanoic acid ester. This can increase the reaction rate of the conversion reaction from PHA to a hydroxyalkanoic acid ester, allowing the conversion of PHA to a hydroxyalkanoic acid ester to be carried out in a higher yield.

[0107] It is more preferable that the step (4) of reducing the weight-average molecular weight of the PHA after the step (1) of producing the PHA particles is included before the step (2) of dispersing the PHA particles in alcohol. When the conversion of PHA to a hydroxyalkanoic acid ester is by alcoholysis, the hydrolysis reaction of PHA due to water in the alcohol dispersion competes with the alcoholysis reaction of PHA, so the lower the water content in the alcohol dispersion, the more efficiently the conversion of PHA to a hydroxyalkanoic acid ester can be carried out, and the less water content there is, the less water can be brought into the alcohol dispersion and the lower the water content can be.

[0108] The method for reducing the weight-average molecular weight of PHA is not particularly limited, but includes a method of carrying out a hydrolysis reaction. The hydrolysis reaction is preferably carried out in the presence of a hydrolysis catalyst. Conventionally known catalysts can be used as the hydrolysis catalyst, and acid catalysts or base catalysts can be used. Examples of acid catalysts include inorganic acids and organic acids such as sulfuric acid, hydrochloric acid, acetic acid, and phosphoric acid. Examples of base catalysts include alkali metal salts such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, and sodium bicarbonate; amines such as triethylamine, diethylamine, and aniline; and aqueous ammonia. Among these, base catalysts are preferred, and sodium hydroxide is more preferred.

[0109] When an acid catalyst is used as a hydrolysis reaction catalyst, the amount used is preferably adjusted so that the pH becomes 1 to 6 or 1 to 5, and when a base catalyst is used, the amount used is preferably adjusted so that the pH becomes 8 to 14 or 9 to 14. If necessary, a neutralizing agent may be added to neutralize the acid catalyst or base catalyst to complete the hydrolysis reaction.

[0110] The hydrolysis reaction time is not particularly limited as long as it is within a range in which the weight average molecular weight of the PHA is 2,000,000 or less, and may be, for example, 15 to 600 minutes, or 60 to 400 minutes.

[0111] The hydrolysis reaction temperature is not particularly limited, but may be 40 to 100°C, or 60 to 90°C.

[0112] (Step (5) of substituting water in the system with alcohol) In the method for producing a biomass-derived hydroxyalkanoate ester according to the third embodiment of the present invention, it is preferable to further include step (5) of substituting water in the system with alcohol after step (1) of producing PHA particles. This is because water that competes with the conversion reaction of PHA to hydroxyalkanoate ester and causes a hydrolysis reaction of PHA, as well as bacterial cell components that inhibit the conversion reaction, can be removed, allowing for a higher yield of conversion of PHA to hydroxyalkanoate ester.

[0113] The method for replacing water in the system with alcohol is not particularly limited, and examples thereof include a method in which water is removed from the system by filtration, centrifugation, sedimentation, electrophoresis, or the like, and then alcohol is added.

[0114] It is more preferable that after the step (1) of producing the PHA particles, the step (5) of substituting water in the system with alcohol is included before the step (2) of dispersing the PHA particles in alcohol. In other words, it is preferable to perform the step (5) of substituting water in the system with alcohol and the step (2) of dispersing the PHA particles in alcohol in that order. Furthermore, when the step (4) of reducing the weight-average molecular weight of the PHA is included, it is preferable to perform the steps (4), (5), and (2) in that order. When the conversion to a hydroxyalkanoic acid ester is by alcoholysis, the hydrolysis reaction of PHA due to the water in the alcohol dispersion competes with the alcoholysis reaction of PHA, so a lower water content in the alcohol dispersion allows for a higher conversion yield from PHA to a hydroxyalkanoic acid ester, and also allows for efficient removal of water from the alcohol dispersion.

[0115] The type of alcohol used to replace water in the system is not particularly limited, and the alcohols listed in the first embodiment can be used, and the description of suitable alcohols is also the same. In addition, a surfactant or the like may be added to the alcohol.

[0116] When the step (5) of replacing the water in the system with alcohol is carried out before separating the PHA particles from the bacterial cell components of the PHA microorganism, that is, when replacing the water in the system containing the PHA microorganism with alcohol while the PHA particles are contained within the bacterial cell, the separation of the PHA particles may be accompanied by disruption of the bacterial cell.

[0117] (Step (6) of Separating PHA Particles) The description in the section (Step (6) of Separating PHA Particles) of the first embodiment is common to (Step (6) of Separating PHA Particles) of the third embodiment.

[0118] (Washing of PHA particles) In the method for producing a biomass-derived hydroxyalkanoic acid ester according to the third embodiment of the present invention, in the step (3) of converting the PHA constituting the PHA particles into a hydroxyalkanoic acid ester, the fewer impurities present in the conversion reaction from PHA to a hydroxyalkanoic acid ester, such as nitrogen-containing components such as bacterial cell components other than the PHA particles, culture substrate residues, and water, the easier it is to obtain a high yield of the hydroxyalkanoic acid ester. However, by including a step of washing the PHA particles after the step (1) of producing the PHA particles to reduce the nitrogen-containing components, the yield of the hydroxyalkanoic acid ester can be increased. The explanation in the section (Method for washing PHA particles) of the first embodiment is common to the third embodiment.

[0119] In order to efficiently reduce the nitrogen-containing components, the PHA particles may be washed after being subjected to a treatment such as an enzyme treatment, a hypochlorous acid treatment, or a hydrogen peroxide treatment.

[0120] The descriptions regarding the enzyme treatment, hypochlorous acid treatment, and hydrogen peroxide treatment in the first embodiment are common to the third embodiment.

[0121] The various treatments for removing the nitrogen-containing components may be carried out either alone or in combination of two or more.

[0122] (Procedure) A third embodiment of the method for producing a hydroxyalkanoic acid ester, including the steps described above, will be further described below. The method for producing a biomass-derived hydroxyalkanoic acid ester according to the third embodiment of the present invention may include, after step (1) of producing the PHA particles, at least one of step (4) of reducing the weight-average molecular weight of the PHA, step (5) of substituting water in the system with an alcohol, and step (6) of separating the PHA particles from the microbial cell components in an aqueous system. Even if the method includes two or more steps, the order of these steps is not particularly limited. Step (2) of dispersing the PHA particles in an alcohol and step (3) of converting the PHA into a hydroxyalkanoic acid ester may be performed in this order after steps (1), (4), (5), and (6).

[0123] It is preferable that the method includes a step (6) of separating PHA particles from the bacterial cell components of the microorganism in a system containing water, and a step (5) of replacing the water in the system with alcohol, and it is particularly preferable that the step (6) of separating PHA particles from the bacterial cell components of the microorganism in a system containing water, and the step (5) of replacing the water in the system with alcohol are performed in this order.

[0124] The cell lysate obtained by disrupting bacterial cells (cells) containing PHA particles contains, in addition to the PHA particles, bacterial cell components such as proteins, nucleic acids, lipids, and sugar components in the cells, as well as culture substrate residues, etc. Therefore, by substituting water in the system with alcohol after disrupting the cells, impurities in the conversion reaction from PHA to hydroxyalkanoic acid ester, such as bacterial cell components other than PHA particles and water, can be removed, and the conversion from PHA to hydroxyalkanoic acid ester can be carried out in a higher yield.

[0125] The inclusion of step (4) of reducing the weight-average molecular weight of the PHA and step (6) of separating the PHA particles from the bacterial cell components of the microorganism in a system containing water in this order after step (1) of producing the PHA particles is advantageous in that it allows for efficient reduction of the content of impurities such as bacterial cell components other than PHA particles.

[0126] After the step (1) of producing the PHA particles, it is preferable to include the steps (6) of separating the PHA particles from the bacterial cell components of the microorganism in a system containing water, the step (4) of reducing the weight-average molecular weight of the PHA, and the step (5) of replacing the water in the system with alcohol in this order, since this makes it easier to reduce the molecular weight to the desired molecular weight.

[0127] A more specific third embodiment of the present invention will be described below. After the step (1) of producing PHA particles, the method may include a step (6) of separating the PHA particles from the bacterial cell components of the microorganism in a system containing water, a step (2) of dispersing the PHA particles in alcohol, and a step (3) of converting the PHA into a hydroxyalkanoic acid ester, in this order.

[0128] After the step (1) of producing the PHA particles, the method may include the steps of: separating the PHA particles from the bacterial cell components of the microorganism in a system containing water (6); replacing the water in the system with alcohol (5); dispersing the PHA particles in alcohol (2); and converting the PHA into a hydroxyalkanoic acid ester (3), in this order.

[0129] After the step (1) of producing the PHA particles, the method may include the steps of: separating the PHA particles from the bacterial cell components of the microorganism in a system containing water (6); reducing the weight-average molecular weight of the PHA (4); dispersing the PHA particles in alcohol (2); and converting the PHA into a hydroxyalkanoic acid ester (3), in this order.

[0130] After the step (1) of producing the PHA particles, the method may include the steps of: separating the PHA particles from the bacterial cell components of the microorganism in a system containing water (6); reducing the weight-average molecular weight of the PHA (4); replacing the water in the system with alcohol (5); dispersing the PHA particles in alcohol (2); and converting the PHA into a hydroxyalkanoic acid ester (3), in this order.

[0131] After the step (1) of producing the PHA particles, the method may include the steps of: (4) reducing the weight average molecular weight of the PHA particles from the bacterial cell components of the microorganism; (6) separating the PHA particles in a system containing water; (2) dispersing the PHA particles in alcohol; and (3) converting the PHA into a hydroxyalkanoic acid ester, in this order.

[0132] After the step (1) of producing the PHA particles, the method may include the steps of: (4) reducing the weight-average molecular weight of the PHA particles from the bacterial cell components of the microorganism; (6) separating the PHA particles in a system containing water; (5) replacing the water in the system with alcohol; (2) dispersing the PHA particles in alcohol; and (3) converting the PHA into a hydroxyalkanoic acid ester, in this order.

[0133] The explanation in the section (Drying of PHA particles) of the first embodiment is common to the third embodiment.

[0134] (Step (3) of Converting PHA to Hydroxyalkanoic Acid Ester) The step (3) of converting the PHA constituting the PHA particles in the alcohol dispersion into a hydroxyalkanoic acid ester will be described in detail.

[0135] In step (3), the weight-average molecular weight of the PHA constituting the PHA particles is preferably 2,000,000 or less, more preferably 1,000,000 or less, more preferably 500,000 or less, more preferably 300,000 or less, and particularly preferably 250,000 or less. If it is greater than 2,000,000, the reaction rate of conversion to hydroxyalkanoic acid ester is low, and the yield of hydroxyalkanoic acid ester becomes insufficient. In addition, the weight-average molecular weight of the PHA may be 10,000 or more, or may be 50,000 or more.

[0136] The weight-average molecular weight of the PHA can be determined as a molecular weight converted into polystyrene using gel permeation chromatography (GPC) (Shimadzu Corporation's "High Performance Liquid Chromatograph 20A System"), using a polystyrene gel (Showa Denko K.K.'s "K-G 4A" or "K-806M" or the like) as a column, and chloroform as a mobile phase. In this case, a calibration curve can be prepared using polystyrenes with weight-average molecular weights of 31,400, 197,000, 668,000, and 1,920,000. As the column for the GPC, a column appropriate for measuring the molecular weight may be used.

[0137] In the step (3) of converting PHA into a hydroxyalkanoic acid ester, the content of alcohol in the alcohol dispersion is not particularly limited, but from the viewpoint of reactivity, the content is preferably 0.5 to 100 parts by weight, more preferably 0.5 to 100 parts by weight, more preferably 0.5 to 50 parts by weight, more preferably 0.5 to 10 parts by weight, more preferably 0.5 to 5 parts by weight, and most preferably 0.5 to 3 parts by weight, relative to 1 part by weight of PHA.

[0138] According to the method for producing a biomass-derived hydroxyalkanoic acid ester according to the third embodiment of the present invention, the weight-average molecular weight of the PHA is equal to or less than a specific amount, and the reaction rate of the conversion reaction from the PHA to the hydroxyalkanoic acid ester is high, allowing the conversion of the PHA to the hydroxyalkanoic acid ester to be carried out with high yield. Furthermore, because the weight-average molecular weight of the PHA is equal to or less than a specific amount, the reaction conditions for the conversion of the PHA to the hydroxyalkanoic acid ester can be relaxed. Specifically, for example, as described above, the amount of catalyst used can be reduced, and the temperature conditions can be relaxed as described below. Reducing the amount of catalyst used reduces the effort and cost required for catalyst removal, and relaxing the temperature conditions improves safety and efficiency.

[0139] [Method for producing biomass-derived hydroxyalkanoate ester iv)] A fourth embodiment of the present invention relates to a method for producing a biomass-derived hydroxyalkanoate ester having the above-mentioned configuration iv), that is, the method includes the steps of causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in an aqueous system, dispersing the produced polyhydroxyalkanoate particles in alcohol, and converting the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles into a hydroxyalkanoate ester in the alcohol dispersion, without a step of drying the polyhydroxyalkanoate particles until the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles is converted into a hydroxyalkanoate ester. The fourth embodiment of the present invention will be described in detail below.

[0140] The explanations of the first embodiment regarding (polyhydroxyalkanoate particles), (PHA-producing microorganisms), (PHA synthase gene), and (step of producing PHA particles) are common to the fourth embodiment.

[0141] (Step of Dispersing PHA Particles in Alcohol) In the fourth embodiment, the step of dispersing the produced PHA particles in alcohol will be described in detail.

[0142] In this step, the PHA particles are dispersed in alcohol without drying to obtain an alcohol dispersion of PHA particles. In the fourth embodiment, drying of the PHA particles refers to removing the liquid component to the extent that the PHA particles aggregate to form secondary particles, but does not include removing the liquid component to the extent that secondary particles of the PHA particles are not formed.

[0143] The alcohol is preferably a water-soluble alcohol. Examples of the water-soluble alcohol include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-butanol, and one or more selected from the group consisting of these may be used. Among these, methanol or ethanol is more preferred, and methanol is even more preferred, in terms of reactivity with PHA.

[0144] When dispersing PHA particles in alcohol to prepare an alcohol dispersion, the amount of alcohol used is not particularly limited.

[0145] Furthermore, the method for producing a biomass-derived hydroxyalkanoate ester according to the fourth embodiment does not involve a step of drying the polyhydroxyalkanoate particles until the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles is converted into a hydroxyalkanoate ester.

[0146] The fourth embodiment of the present invention provides a method for producing a biomass-derived hydroxyalkanoate ester, which includes the above-described configuration iv) and thus can suppress the formation of secondary particles due to aggregation of PHA particles and the increase in particle size. This allows the PHA to have a large contact area with the reaction partner and the solvent in the step of converting the PHA into a hydroxyalkanoate ester, thereby achieving good reactivity. As a result, the hydroxyalkanoate ester can be produced in high yield.

[0147] (Step of replacing water in the system with alcohol) In the method for producing a biomass-derived hydroxyalkanoic acid ester according to the fourth embodiment of the present invention, it is preferable that the method further includes a step of replacing water in the system with alcohol after the step of producing the PHA particles.

[0148] The explanation in the section (Method for substituting water in a system with an alcohol) of the first embodiment is common to the section (Step for substituting water in a system with an alcohol) of the fourth embodiment.

[0149] Furthermore, in the method for producing a biomass-derived hydroxyalkanoic acid ester according to the fourth embodiment of the present invention, specific examples of the step of replacing water in the system with alcohol after the step of producing polyhydroxyalkanoate particles include: (1) a case in which water in the system is replaced with alcohol immediately after the step of producing polyhydroxyalkanoate particles, while the PHA particles remain accumulated and contained within the cells of the PHA-producing microorganism; and (2) a case in which water in the system is replaced with alcohol after the step of producing polyhydroxyalkanoate particles and the step of separating the PHA particles described below.

[0150] (Step of separating PHA particles) In the fourth embodiment of the method for producing biomass-derived hydroxyalkanoic acid esters of the present invention, it is preferable that after the step of producing the PHA particles, the method further includes a step of separating the PHA particles from the bacterial body components of the microorganism in a system containing water.

[0151] It is also preferable to include both the separation step and the step of substituting the water in the system with an alcohol, and the order of these steps is not particularly limited.

[0152] The description of the section (Step of separating PHA particles) in the first embodiment is common to the fourth embodiment.

[0153] (Purification of PHA particles) In addition to PHA particles, the cell lysate obtained by disrupting cells contains contaminants such as bacterial cell components in the cells, such as proteins, nucleic acids, lipids, and sugar components, as well as culture substrate residues, etc. Therefore, after the step of separating PHA particles by disrupting the cells, etc., it is preferable to carry out a purification treatment to decompose and / or remove impurities such as bacterial cell components other than the PHA particles.

[0154] For example, after cell disruption, the content of impurities can be reduced by separating water containing water-soluble components such as bacterial cell constituents and culture substrate residues. Methods for separating water containing water-soluble components such as bacterial cell constituents and culture substrate residues are not particularly limited, but include filtration, centrifugation, sedimentation, electrophoresis, etc. The separation of water containing water-soluble components refers to the separation of water to the extent that secondary particles of PHA particles are not formed.

[0155] Examples of purification treatments include a method using an enzyme, a method using hypochlorous acid, and a method using hydrogen peroxide, and the explanations of each method are common to the explanations of the enzyme treatment, hypochlorous acid treatment, and hydrogen peroxide treatment in the first embodiment.

[0156] The above-mentioned various purification treatments may be carried out either alone or in combination of two or more.

[0157] After the purification treatment described above, dehydration can be performed as necessary, and the resulting PHA particles can be washed with water or the like as necessary to further increase the degree of purification of the PHA particles. For washing, an organic solvent may be used, or a mixture of water and an organic solvent may be used. The pH of the water may also be adjusted. The organic solvent used as the washing solvent is preferably a hydrophilic solvent, and specific examples include methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, ketones, amines, etc. Two or more of these organic solvents may be mixed and used. A surfactant or the like may also be added to the water. Note that dehydration performed to increase the degree of purification of the PHA particles refers to the removal of water to an extent that secondary particles of the PHA particles are not formed.

[0158] In the fourth embodiment, the PHA particles can be purified by applying the methods described in the sections (Method for washing PHA particles) and (Method for treating PHA particles with an enzyme) listed as steps for reducing the total nitrogen content in the PHA particles in the first embodiment.

[0159] According to the method for producing a biomass-derived hydroxyalkanoic acid ester according to the fourth embodiment of the present invention, since a step of drying the PHA particles is not performed, the PHA particles do not aggregate to form secondary particles, and therefore the reaction conditions for converting the PHA to a hydroxyalkanoic acid ester can be relaxed. Specifically, for example, as described above, the amount of catalyst used can be reduced, and the temperature conditions can be relaxed as described below. Reducing the amount of catalyst used reduces the effort and cost required for catalyst removal, and relaxing the temperature conditions improves safety and efficiency.

[0160] The above description of the methods for producing a hydroxyalkanoic acid ester according to the first, second, third, and fourth embodiments has been divided into Structure i), Structure ii), Structure iii), and Structure iv), respectively, but this does not preclude the inclusion of other structures in each embodiment. The method for producing a hydroxyalkanoic acid ester according to the first embodiment may have at least one of Structure ii), Structure iii), and Structure iv), the method for producing a hydroxyalkanoic acid ester according to the second embodiment may have at least one of Structure i), Structure iii), and Structure iv), the method for producing a hydroxyalkanoic acid ester according to the third embodiment may have at least one of Structure i), Structure ii), and Structure iv), and the method for producing a hydroxyalkanoic acid ester according to the fourth embodiment may have at least one of Structure i), Structure ii), and Structure iii).

[0161] [Method for Producing Biomass-Derived 1,3-Butanediol] One embodiment of the present invention relates to a method for producing biomass-derived 1,3-butanediol, comprising: obtaining a 3-hydroxybutyrate ester from among the hydroxyalkanoate esters by the method for producing a hydroxyalkanoate ester according to any one of the first, second, third, and fourth embodiments; and reducing the 3-hydroxybutyrate ester to convert it into 1,3-butanediol.

[0162] (Step of Obtaining 3-Hydroxybutyric Acid Ester) The step of obtaining 3-hydroxybutyric acid ester may be performed using the above-described method for producing a biomass-derived hydroxyalkanoic acid ester.

[0163] (Step of converting 3-hydroxybutyrate ester into 1,3-butanediol) The step of converting 3-hydroxybutyrate ester into 1,3-butanediol by reducing it will be described in detail. 3-Hydroxybutyrate ester is represented by the above formula (1), and as a method of reducing 3-hydroxybutyrate ester into 1,3-butanediol by reducing it, a known method for reducing a carboxylic acid ester with hydrogen can be used, and examples thereof include a hydrogen reduction reaction using a catalyst containing a noble metal such as ruthenium (Ru), palladium (Pd), rhenium (Re), or rhodium (Rh), or a catalyst containing copper (Cu).

[0164] [Method for Producing Biomass-Derived Butadiene] One embodiment of the present invention relates to a method for producing biomass-derived butadiene, comprising the steps of obtaining 1,3-butanediol by the above-described production method and obtaining butadiene by a dehydration reaction of the 1,3-butanediol.

[0165] (Step of Obtaining 1,3-Butanediol) The step of obtaining 1,3-butanediol may be performed using the above-described method for producing biomass-derived 1,3-butanediol.

[0166] (Step of Obtaining Butadiene) The step of obtaining butadiene by dehydration of 1,3-butanediol will be described in detail. As a method of obtaining butadiene by dehydration of 1,3-butanediol, a known method can be used, for example, an intramolecular dehydration reaction in the presence of a dehydration catalyst.

[0167] Examples of dehydration catalysts include metal phosphates, metal sulfates, metal hydrochlorides, metal oxides, and inorganic acids. These catalysts may be used alone or supported on a carrier. Metals constituting metal phosphates, metal sulfates, and metal hydrochlorides include, for example, alkali metals, alkaline earth metals, Ti, Zr, Hf, Nb, Ta, Al, B, and Sn. Examples of metal oxides include silica, alumina, magnesia, titania, zirconia, niobia, silica-alumina, silica-magnesia, and zeolites. Examples of carriers include silica, alumina, titania, zirconia, silica-alumina, zeolites, activated carbon, and graphite. Examples of inorganic acids include sulfuric acid, nitric acid, hydrochloric acid, and phosphoric acid.

[0168] [Method for producing biomass-derived polybutadiene rubber] One embodiment of the present invention relates to a method for producing biomass-derived polybutadiene rubber, including a step of obtaining butadiene by the above-described production method, and a step of polymerizing the butadiene to obtain polybutadiene rubber.

[0169] (Step of Obtaining Butadiene) As the step of obtaining butadiene, the above-described method for producing biomass-derived butadiene may be used.

[0170] (Step of Obtaining Polybutadiene Rubber) The step of polymerizing butadiene to obtain polybutadiene rubber will be described in detail. Known methods can be used to obtain polybutadiene rubber by polymerizing butadiene, including radical polymerization, coordination anionic polymerization, and anionic polymerization using an organolithium compound. The polymerization method may be appropriately selected depending on the stereoregularity of the target polybutadiene rubber. Examples of catalysts used in coordination anionic polymerization include transition metal compounds such as titanium (Ti), cobalt (Co), or nickel (Ni), or catalysts combining rare earth metals such as neodymium (Nd) with organoaluminum.

[0171] [Method for producing biomass-derived core-shell polymer particles] One embodiment of the present invention relates to a method for producing biomass-derived core-shell polymer particles, comprising the steps of obtaining butadiene by the above-described production method, and using the butadiene to obtain core-shell polymer particles containing polybutadiene rubber in a core layer.

[0172] (Step of Obtaining Butadiene) As the step of obtaining butadiene, the above-described method for producing biomass-derived butadiene may be used.

[0173] (Step of Obtaining Core-Shell Polymer Particles) The step of obtaining core-shell polymer particles containing polybutadiene rubber in the core layer using butadiene will be described in detail.

[0174] The core-shell polymer particles can be produced by a conventional method, and is not particularly limited. For example, any of bulk polymerization, solution polymerization, suspension polymerization, and emulsion polymerization can be used.

[0175] Specifically, in emulsion graft polymerization, first, a latex of polymer particles containing polybutadiene rubber corresponding to the core layer is produced by emulsion polymerization, and then monomer components for the shell layer, a polymerization initiator, and the like are added to the latex to polymerize the monomer components.

[0176] Examples of monomer components for the shell layer include acrylic acid ester monomers, methacrylic acid ester monomers, and other monomers copolymerizable with acrylic acid ester monomers or methacrylic acid ester monomers.

[0177] Examples of the polymerization initiator include thermal decomposition initiators and redox initiators. Examples of the thermal decomposition initiator include 2,2'-azobisisobutyronitrile, hydrogen peroxide, potassium persulfate, and ammonium persulfate. Examples of the redox initiator include redox initiators that combine at least one selected from the group consisting of organic peroxides such as t-butylperoxyisopropyl carbonate, paramenthane hydroperoxide, cumene hydroperoxide, dicumyl peroxide, t-butyl hydroperoxide, di-t-butyl peroxide, and t-hexyl peroxide; and inorganic peroxides such as hydrogen peroxide, potassium persulfate, and ammonium persulfate, with at least one selected from the group consisting of reducing agents such as sodium formaldehyde sulfoxylate and glucose; transition metal salts such as iron (II) sulfate; chelating agents such as disodium ethylenediaminetetraacetate; and phosphorus-containing compounds such as sodium pyrophosphate.

[0178] [Method for Producing Biomass-Derived Propylene] One embodiment of the present invention relates to a method for producing biomass-derived propylene, comprising the steps of obtaining a 3-hydroxybutyrate ester from among the hydroxyalkanoate esters by the method for producing a hydroxyalkanoate ester, and converting the 3-hydroxybutyrate ester into propylene by a decarboxylation reaction and a dealcoholization reaction.

[0179] (Step of Obtaining 3-Hydroxybutyric Acid Ester) The step of obtaining 3-hydroxybutyric acid ester may be performed using the above-described method for producing a biomass-derived hydroxyalkanoic acid ester.

[0180] (Step of converting 3-hydroxybutyrate ester into propylene) The step of converting 3-hydroxybutyrate ester into propylene by decarboxylation and dealcoholization will be described in detail. Methods for carrying out the decarboxylation and dealcoholization of 3-hydroxybutyrate ester include reactions using a catalyst such as an acid catalyst or a metal oxide catalyst, for example, reactions using a solid acid catalyst such as amorphous silica-alumina and niobium phosphate.

[0181] [Method for Producing Biomass-Derived 1,3-Hexanediol] One embodiment of the present invention relates to a method for producing biomass-derived 1,3-hexanediol, comprising the steps of obtaining a 3-hydroxyhexanoate ester from among the hydroxyalkanoate esters by the method for producing a hydroxyalkanoate ester, and reducing the 3-hydroxyhexanoate ester to convert it into 1,3-hexanediol.

[0182] (Step of Obtaining 3-Hydroxybutyrate Ester) The step of obtaining 3-hydroxyhexanoate ester may be performed using the above-described method for producing a biomass-derived hydroxyalkanoate ester.

[0183] (Step of converting 3-hydroxyhexanoate ester into 1,3-hexanediol) The step of converting 3-hydroxyhexanoate ester into 1,3-hexanediol by reducing it will be described in detail. 3-Hydroxyhexanoate ester is represented by the above formula (2), and as a method of reducing 3-hydroxyhexanoate ester to convert it into 1,3-hexanediol, a known method for reducing a carboxylic acid ester with hydrogen can be used, and examples thereof include a hydrogen reduction reaction using a catalyst containing a noble metal such as ruthenium (Ru), palladium (Pd), rhenium (Re), or rhodium (Rh), or a catalyst containing copper (Cu).

[0184] The following items list preferred embodiments of the present disclosure, but the present invention is not limited to the following items. [Item 1] A method for producing a biomass-derived hydroxyalkanoate ester, comprising the steps of: causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in a system containing water; dispersing the produced polyhydroxyalkanoate particles in alcohol; and converting the polyhydroxyalkanoate that constitutes the polyhydroxyalkanoate particles in the alcohol dispersion into a hydroxyalkanoate ester, the method having any one of the following configurations i) to iv): Item 2. The method according to item 1, comprising: i) the polyhydroxyalkanoate particles have a total nitrogen content of 2% by weight or less in the step of converting to a hydroxyalkanoate ester; ii) the water content in the alcohol dispersion is 10% by weight or less relative to 100% by weight of the polyhydroxyalkanoate particles in the step of converting to a hydroxyalkanoate ester; iii) the polyhydroxyalkanoate has a weight-average molecular weight of 2,000,000 or less; and iv) no step of drying the polyhydroxyalkanoate particles is performed before the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles is converted to a hydroxyalkanoate ester. [Item 3] The production method according to Item 1 or 2, which has the configuration i), and further comprises, after the step of producing the polyhydroxyalkanoate particles, a step of washing the polyhydroxyalkanoate particles with an organic solvent to reduce the total nitrogen content in the polyhydroxyalkanoate particles.[Item 4] The production method according to any one of Items 1 to 3, which has the feature i), and further comprises, after the step of producing the polyhydroxyalkanoate particles, a step of treating the polyhydroxyalkanoate particles with an enzyme that decomposes bacterial body components of the microorganism, thereby reducing the total nitrogen content in the polyhydroxyalkanoate particles. [Item 5] The production method according to any one of Items 1 to 4, which has the feature ii) but does not have the feature iv), and further comprises, after the step of producing the polyhydroxyalkanoate particles, a step of removing water from the system and drying the polyhydroxyalkanoate particles. [Item 6] The production method according to any one of Items 1 to 5, which has the feature ii), and further comprises, after the step of producing the polyhydroxyalkanoate particles, a step of replacing the water in the system with an organic solvent to remove the water. [Item 7] The production method according to any one of Items 1 to 6, which has the feature iii) and further comprises a step of reducing the weight-average molecular weight of the polyhydroxyalkanoate before the step of converting the polyhydroxyalkanoate into a hydroxyalkanoic acid ester. [Item 8] The production method according to Item 7, which has the feature iii), and wherein the reduction in the weight-average molecular weight of the polyhydroxyalkanoate is achieved by a reaction in the presence of a base catalyst. [Item 9] The production method according to any one of Items 1 to 8, which further comprises a step of substituting water in the system with an alcohol after the step of producing polyhydroxyalkanoate particles. [Item 10] The production method according to any one of Items 1 to 9, which further comprises a step of separating the polyhydroxyalkanoate particles from bacterial body components of the microorganism in a system containing water after the step of producing polyhydroxyalkanoate particles. [Item 11] The production method according to any one of Items 1 to 10, further comprising, after the step of producing polyhydroxyalkanoate particles, a step of separating the polyhydroxyalkanoate particles from the bacterial body components of the microorganism in a system containing water, and a step of substituting water in the system with an alcohol.[Item 12] The production method according to any one of Items 1 to 11, which has the configuration ii), and further comprises, after the step of producing the polyhydroxyalkanoate particles, a step of separating the polyhydroxyalkanoate particles from the bacterial cell components of the microorganism in a system containing water, and a step of replacing the water in the system with an organic solvent to remove the water. [Item 13] The production method according to any one of Items 1 to 12, which has the configuration iii), and further comprises, after the step of producing the polyhydroxyalkanoate particles, a step of reducing the weight-average molecular weight of the polyhydroxyalkanoate, and a step of separating the polyhydroxyalkanoate particles from the bacterial cell components of the microorganism in a system containing water, in this order. [Item 14] The production method according to any one of Items 1 to 12, which has the configuration iii), and further comprises, after the step of producing polyhydroxyalkanoate particles, a step of separating the polyhydroxyalkanoate particles from bacterial cell components of the microorganism in a system containing water, a step of reducing the weight-average molecular weight of the polyhydroxyalkanoate, and a step of substituting water in the system with an alcohol, in this order. [Item 15] The production method according to any one of Items 1 to 14, wherein the conversion of the polyhydroxyalkanoate to a hydroxyalkanoate ester is carried out in the presence of an acid catalyst. [Item 16] The production method according to any one of Items 1 to 15, further comprising a step of purifying the produced hydroxyalkanoate ester by distillation. [Item 17] The production method according to any one of Items 1 to 16, wherein the alcohol is methanol or ethanol. [Item 18] The method according to any one of items 1 to 17, wherein the polyhydroxyalkanoate particles have any one of the structures i) to iii), and the polyhydroxyalkanoate particles are not dried before the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles is converted into a hydroxyalkanoic acid ester. [Item 19] The method according to any one of items 1 to 18, wherein the polyhydroxyalkanoate is a poly(3-hydroxybutyrate)-based polymer.[Item 20] The production method according to any one of Items 1 to 19, wherein the hydroxyalkanoate ester is a 3-hydroxybutyrate ester. [Item 21] A method for producing biomass-derived 1,3-butanediol, comprising the steps of obtaining a 3-hydroxybutyrate ester from among the hydroxyalkanoate esters by the production method for a hydroxyalkanoate ester according to any one of Items 1 to 20, and reducing the 3-hydroxybutyrate ester to convert it into 1,3-butanediol. [Item 22] A method for producing biomass-derived butadiene, comprising the steps of obtaining 1,3-butanediol by the production method according to Item 21, and obtaining butadiene by a dehydration reaction of the 1,3-butanediol. [Item 23] A method for producing biomass-derived polybutadiene rubber, comprising the steps of obtaining butadiene by the production method according to Item 22, and polymerizing the butadiene to obtain polybutadiene rubber. [Item 24] A method for producing biomass-derived core-shell polymer particles, comprising the steps of obtaining butadiene by the production method according to item 22, and using the butadiene to obtain core-shell polymer particles having a core layer containing polybutadiene rubber. [Item 25] A method for producing biomass-derived propylene, comprising the steps of obtaining a 3-hydroxybutyrate ester from among the hydroxyalkanoate esters by the production method according to any one of items 1 to 20, and converting the 3-hydroxybutyrate ester into propylene by decarboxylation and dealcoholization. [Item 26] The production method according to item 19, wherein the polyhydroxyalkanoate contains a 3-hydroxyhexanoate unit. [Item 27] ​​The production method according to any one of items 1 to 19 and 26, wherein the hydroxyalkanoate ester is a 3-hydroxyhexanoate ester. [Item 28] A method for producing biomass-derived 1,3-hexanediol, comprising the steps of obtaining a 3-hydroxyhexanoate ester from among the hydroxyalkanoate esters by the method for producing a hydroxyalkanoate ester according to any one of Items 1 to 19 and 26 to 27, and reducing the 3-hydroxyhexanoate ester to convert it into 1,3-hexanediol.

[0185] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0186] (Yield of Hydroxyalkanoic Acid Ester) The yield of hydroxyalkanoic acid ester was calculated from the GC analysis results according to the following formula: Yield (mol %) = (A / Molecular Weight of Hydroxyalkanoic Acid Ester) x 100 / (B / Average Molecular Weight of Monomer Units Constituting Polyhydroxyalkanoate), A: Weight (g) of the finally obtained hydroxyalkanoic acid ester, B: Weight (g) of the polyhydroxyalkanoate particles.

[0187] Example i-1: 95% by weight sulfuric acid was added to a cell culture solution (inactivated culture solution) containing inactivated poly(3-hydroxybutyrate) particles [average particle size: 1 μm] to adjust the pH to 7.0±0.2. Furthermore, IW (industrial water) was added to adjust the solids concentration to 18% by weight, and then lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme (lytic enzyme) that degrades sugar chains (peptidoglycans) in cell walls, was added, and the mixture was kept at 50°C for 2 hours to disrupt the cells.

[0188] Thereafter, 2.5 L of Alcalase (Novozymes), an alkaline protease, was added, followed by the addition of 30 wt. % sodium hydroxide at 50°C to adjust the pH to 8.5 and maintaining the mixture for 2 hours. 30 wt. % sodium hydroxide was added to the enzyme-treated culture solution obtained above to adjust the pH to 10.5 and maintaining the mixture for 3 hours. Sodium dodecyl sulfate (SDS, Kao) was then added to the enzyme-treated solution to a concentration of 0.6 to 1.0 wt. % (surfactant treatment).

[0189] The pH was then adjusted to 11.0±0.2 using 30% by weight sodium hydroxide, and an aqueous solution of sodium hydroxide at pH 11.0 was added to dilute the solution to twice the weight of the enzyme-treated solution, and the solution was kept at 40°C for 1 hour.

[0190] The enzyme-treated solution was centrifuged (4500 rpm, 10 minutes), the supernatant was removed, and the solution was concentrated. An aqueous solution of sodium hydroxide (pH 11.0) was added to the concentrated aqueous suspension of poly(3-hydroxybutyrate) particles, and the mixture was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. This process was repeated three times.

[0191] Next, the supernatant was removed, and methanol was added to obtain a poly(3-hydroxybutyrate) particle methanol dispersion. The mixture was centrifuged (4500 rpm, 10 minutes) and the supernatant was removed. After drying the dispersion, 2.0 wt % 95 wt % sulfuric acid was added relative to the poly(3-hydroxybutyrate) particles, followed by the addition of methanol in an amount 2.2 times the weight of the poly(3-hydroxybutyrate) particles. The mixture was heated to 120°C and maintained for 5 hours. The yield of the resulting hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 84.5 mol %. Furthermore, the total nitrogen content of the dried poly(3-hydroxybutyrate) particles was analyzed using an NC analyzer (Sumigraph NC-220F, Sumika Chemical Analysis Center), and was found to be 0.137 wt %.

[0192] Example i-2 The same procedure as in Example i-1 was carried out, except that the total nitrogen content in the dry poly(3-hydroxybutyrate) particles obtained in Example i-1 was adjusted to 0.094 wt % by repeating the addition of methanol and centrifugation twice. The yield of the obtained hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 93.8 mol %.

[0193] Example i-3 The same procedure as in Example i-1 was carried out, except that the total nitrogen content in the dry poly(3-hydroxybutyrate) particles obtained in Example i-1 was adjusted to 0.085 wt % by repeating the addition of methanol and centrifugation three times. The yield of the obtained hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 96.6 mol %.

[0194] Comparative Example i-1: A cell culture solution (inactivated culture solution) containing inactivated poly(3-hydroxybutyrate) particles [average particle size: 1 μm] was centrifuged (4,500 rpm, 10 minutes), and the supernatant was removed. After drying, 2.0 wt % 95 wt % sulfuric acid was added relative to the poly(3-hydroxybutyrate) particles, followed by addition of methanol in an amount 2.2 times the weight of the poly(3-hydroxybutyrate) particles. The mixture was heated to 120°C and maintained for 5 hours. No 3-hydroxybutyrate ester, a hydroxyalkanoate ester, was obtained. Furthermore, the total nitrogen content of the dried poly(3-hydroxybutyrate) particles was analyzed using an NC analyzer (Sumigraph NC-220F, Sumika Chemical Analysis Center), and the total nitrogen content was found to be 2.242 wt %.

[0195] As described above, in Comparative Example i-1, in which the total nitrogen content in the PHA particles was greater than 2 wt. % in the step of converting to a hydroxyalkanoate ester, the hydroxyalkanoate ester, 3-hydroxybutyrate ester, was not obtained, whereas in Examples i-1, i-2, and i-3, in which the total nitrogen content was 2 wt. % or less, the yield of 3-hydroxybutyrate ester was 84 mol. Thus, it can be seen that the method for producing a biomass-derived hydroxyalkanoate ester according to the first embodiment of the present invention can convert polyhydroxyalkanoate to a hydroxyalkanoate ester with good yield. Furthermore, the results of Examples i-1, i-2, and i-3 show that the lower the total nitrogen content in the PHA particles, the higher the yield of 3-hydroxybutyrate ester.

[0196] Comparative Example ii-1: 95% by weight sulfuric acid was added to a cell culture solution (inactivated culture solution) containing inactivated poly(3-hydroxybutyrate) particles [average particle size: 1 μm] to adjust the pH to 7.0±0.2. Furthermore, IW (industrial water) was added to adjust the solids concentration to 18% by weight, and then lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme (lytic enzyme) that degrades sugar chains (peptidoglycans) in cell walls, was added, and the mixture was kept at 50°C for 2 hours to disrupt the cells.

[0197] Thereafter, 2.5 L of Alcalase (Novozymes), an alkaline protease, was added, followed by the addition of 30 wt. % sodium hydroxide at 50°C to adjust the pH to 8.5 and maintaining the mixture for 2 hours. 30 wt. % sodium hydroxide was added to the enzyme-treated culture solution obtained above to adjust the pH to 10.5 and maintaining the mixture for 3 hours. Sodium dodecyl sulfate (SDS, Kao) was then added to the enzyme-treated solution to a concentration of 0.6 to 1.0 wt. % (surfactant treatment).

[0198] The pH was then adjusted to 11.0±0.2 using 30% by weight sodium hydroxide, and an aqueous solution of sodium hydroxide at pH 11.0 was added to dilute the solution to twice the weight of the enzyme-treated solution, and the solution was kept at 40°C for 1 hour.

[0199] The enzyme-treated solution was centrifuged (4500 rpm, 10 minutes), the supernatant was removed, and the solution was concentrated. An aqueous solution of sodium hydroxide (pH 11.0) was added to the concentrated aqueous suspension of poly(3-hydroxybutyrate) particles, and the mixture was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. This process was repeated three times.

[0200] Next, the supernatant was removed, and methanol was added to obtain a poly(3-hydroxybutyrate) particle methanol dispersion. The mixture was centrifuged (4500 rpm, 10 minutes) and the supernatant was removed. After the addition of methanol, the water content in the dispersion was analyzed using a Karl Fischer moisture meter and found to be 11.3 wt % relative to the poly(3-hydroxybutyrate) particles.

[0201] To the dispersion, 2.0% by weight of 95% by weight sulfuric acid relative to the poly(3-hydroxybutyrate) particles was added, and then methanol was added in an amount twice the weight of the poly(3-hydroxybutyrate) particles, and the mixture was heated to 120°C and maintained for 5 hours to carry out an exchange reaction of PHA to an ester. The yield of the resulting hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 79 mol%.

[0202] Example ii-1 The same procedure as in Comparative Example ii-1 was carried out, except that the amount of methanol added was increased and the number of cycles of centrifugation, supernatant removal, and methanol addition was further increased by one, so that the water content in the dispersion before the reaction obtained in Comparative Example ii-1 was 8.8% by weight relative to the poly(3-hydroxybutyrate) particles. The yield of the resulting hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 85 mol%.

[0203] Example ii-2 The same procedure as in Comparative Example ii-1 was carried out, except that the amount of methanol added in Comparative Example ii-1 was increased and the number of cycles of centrifugation, supernatant removal, and methanol addition was increased by two, thereby making the water content in the dispersion before the reaction obtained in Comparative Example ii-1 5.3% by weight relative to the poly(3-hydroxybutyrate) particles. The yield of the resulting hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 89 mol%.

[0204] Example ii-3 The same procedure as in Comparative Example ii-1 was carried out, except that the amount of methanol added in Comparative Example ii-1 was increased and the number of cycles of the steps of centrifugation, supernatant removal, and methanol addition was increased by three, so that the water content in the dispersion before the reaction obtained in Comparative Example ii-1 was 3.8% by weight relative to the poly(3-hydroxybutyrate) particles. The yield of the obtained hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 91 mol%.

[0205] Example ii-4 The same procedure as in Comparative Example ii-1 was carried out, except that the amount of methanol added was increased and the number of cycles of centrifugation, supernatant removal, and methanol addition was increased by four, so that the water content in the dispersion before the reaction obtained in Comparative Example ii-1 was 0.5% by weight relative to the poly(3-hydroxybutyrate) particles. The yield of the resulting hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 94 mol%.

[0206] As described above, in Comparative Example ii-1, in which the water content in the alcohol dispersion in the step of converting to a hydroxyalkanoate ester was more than 10 wt. % relative to 100 wt. % of the PHA particles, the yield of 3-hydroxybutyrate ester was less than 80 mol. In contrast, in Examples ii-1 to ii-4, in which the water content was 10 wt. % or less, the yield of 3-hydroxybutyrate ester was 85 mol. Thus, it can be seen that the method for producing a biomass-derived hydroxyalkanoate ester according to the second embodiment of the present invention can convert PHA to a hydroxyalkanoate ester with good yield. Furthermore, as is clear from the results of Examples ii-1 to ii-4, the lower the water content in the alcohol dispersion, the higher the yield of 3-hydroxybutyrate ester.

[0207] (Weight-average molecular weight) The resin to be measured was dissolved in chloroform and heated in a hot water bath at 60°C for 30 minutes. The soluble matter was filtered through a disposable PTFE filter with a 0.45 μm pore size. The filtrate was then subjected to GPC measurement under the following conditions to determine the weight-average molecular weight. GPC measurement device: High-performance liquid chromatograph 20A system manufactured by Shimadzu Corporation Columns: K-G 4A (1 column), K-806M (2 columns) manufactured by Showa Denko K.K. Sample concentration: 1 mg / ml Free liquid: chloroform solution Free liquid flow rate: 1.0 ml / min Sample injection amount: 100 μL Analysis time: 30 minutes Standard sample: standard polystyrene

[0208] Comparative Example iii-1: 95% by weight sulfuric acid was added to a cell culture solution (inactivated culture solution) containing inactivated poly(3-hydroxybutyrate) particles [average particle size: 1 μm] to adjust the pH to 7.0±0.2. Furthermore, IW (industrial water) was added to adjust the solids concentration to 18% by weight, and then lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme (lytic enzyme) that degrades sugar chains (peptidoglycans) in cell walls, was added, and the mixture was kept at 50°C for 2 hours to disrupt the cells.

[0209] Thereafter, 2.5 L of Alcalase (Novozymes), an alkaline protease, was added, followed by the addition of 30 wt. % sodium hydroxide at 50°C to adjust the pH to 8.5 and maintaining the mixture for 2 hours. 30 wt. % sodium hydroxide was added to the enzyme-treated culture solution obtained above to adjust the pH to 10.5 and maintaining the mixture for 3 hours. Sodium dodecyl sulfate (SDS, Kao) was then added to the enzyme-treated solution to a concentration of 0.6 to 1.0 wt. % (surfactant treatment).

[0210] The pH was then adjusted to 11.0±0.2 using 30% by weight sodium hydroxide, and an aqueous solution of sodium hydroxide at pH 11.0 was added to dilute the solution to twice the weight of the enzyme-treated solution, and the solution was kept at 40°C for 1 hour.

[0211] The enzyme-treated solution was centrifuged (4500 rpm, 10 minutes), the supernatant was removed, and the solution was concentrated. An aqueous solution of sodium hydroxide (pH 11.0) was added to the concentrated aqueous suspension of poly(3-hydroxybutyrate) particles, and the mixture was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. This process was repeated three times.

[0212] Next, the supernatant was removed, and methanol was added to obtain a poly(3-hydroxybutyrate) particle methanol dispersion. The mixture was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. To the dispersion was added 95% by weight sulfuric acid in an amount of 0.5% by weight relative to the poly(3-hydroxybutyrate) particles, and then methanol was added to the dispersion in an amount equal to 1 weight relative to the poly(3-hydroxybutyrate) particles, and the mixture was heated to 120°C and maintained for 5 hours. The yield of the resulting hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 62.7 mol%.

[0213] The poly(3-hydroxybutyrate) particle methanol dispersion obtained above was dried, and the weight-average molecular weight of the (3-hydroxybutyrate) constituting the poly(3-hydroxybutyrate) particles obtained was 2,600,000.

[0214] Example iii-1: To a cell culture solution (inactivated culture solution) containing inactivated poly(3-hydroxybutyrate) particles [average particle size: 1 μm], 30 wt % sodium hydroxide was added to adjust the pH to 10, and the solution was maintained at 70°C for 1 hour. Then, 95 wt % sulfuric acid was added to adjust the pH to 7.0±0.2. Furthermore, IW (industrial water) was added to adjust the solids concentration to 18 wt %, and then lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme (lytic enzyme) that degrades sugar chains (peptidoglycans) in cell walls, was added, and the solution was maintained at 50°C for 2 hours to disrupt the cells.

[0215] Thereafter, 2.5 L of Alcalase (Novozymes), an alkaline protease, was added, followed by the addition of 30 wt. % sodium hydroxide at 50°C to adjust the pH to 8.5 and maintaining the mixture for 2 hours. 30 wt. % sodium hydroxide was added to the enzyme-treated culture solution obtained above to adjust the pH to 10.5 and maintaining the mixture for 3 hours. Sodium dodecyl sulfate (SDS, Kao) was then added to the enzyme-treated solution to a concentration of 0.6 to 1.0 wt. % (surfactant treatment).

[0216] The pH was then adjusted to 11.0±0.2 using 30% by weight sodium hydroxide, and an aqueous solution of sodium hydroxide at pH 11.0 was added to dilute the solution to twice the weight of the enzyme-treated solution, and the solution was kept at 40°C for 1 hour.

[0217] The enzyme-treated solution was centrifuged (4500 rpm, 10 minutes), the supernatant was removed, and the solution was concentrated. An aqueous solution of sodium hydroxide (pH 11.0) was added to the concentrated aqueous suspension of poly(3-hydroxybutyrate) particles, and the mixture was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. This process was repeated three times.

[0218] Next, the supernatant was removed, and methanol was added to obtain a methanol dispersion of poly(3-hydroxybutyrate) particles. The mixture was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. To the dispersion, 0.5% by weight of 95% by weight sulfuric acid relative to the poly(3-hydroxybutyrate) particles was added, and then methanol was added to make a 1x weight ratio relative to the poly(3-hydroxybutyrate) particles, and the mixture was heated to 120°C and maintained for 5 hours. The yield of the resulting hydroxyalkanoate ester, 3-hydroxybutyrate ester, was 70.2 mol%.

[0219] The poly(3-hydroxybutyrate) particle methanol dispersion obtained above was dried, and the weight-average molecular weight of the (3-hydroxybutyrate) constituting the poly(3-hydroxybutyrate) particles obtained was 1,220,000.

[0220] Example iii-2 The same procedure as in Example iii-1 was carried out, except that the weight-average molecular weight of the (3-hydroxybutyrate) constituting the obtained poly(3-hydroxybutyrate) particles was set to 770,000 by adding 30 wt % sodium hydroxide to adjust the pH to 10 and maintaining the mixture at 70°C for 2 hours. As a result, the yield of the obtained hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 72.4 mol %.

[0221] Example iii-3 The same procedure as in Example iii-1 was carried out, except that the weight-average molecular weight of the (3-hydroxybutyrate) constituting the obtained poly(3-hydroxybutyrate) particles was set to 460,000 by adding 30 wt % sodium hydroxide to adjust the pH to 10 and maintaining the mixture at 70°C for 3 hours. As a result, the yield of the obtained hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 75.5 mol %.

[0222] Example iii-4 The same procedure as in Example iii-1 was carried out, except that the weight-average molecular weight of the (3-hydroxybutyrate) constituting the obtained poly(3-hydroxybutyrate) particles was set to 280,000 by adding 30 wt % sodium hydroxide to adjust the pH to 10 and maintaining the mixture at 70°C for 4 hours. As a result, the yield of the obtained hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 78.9 mol %.

[0223] Example iii-5 The same procedure as in Example iii-1 was carried out, except that the weight-average molecular weight of the (3-hydroxybutyrate) constituting the obtained poly(3-hydroxybutyrate) particles was set to 180,000 by adding 30 wt % sodium hydroxide to adjust the pH to 10 and maintaining the mixture at 70°C for 5 hours. As a result, the yield of the obtained hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 80.8 mol %.

[0224] As described above, Comparative Example iii-1, in which PHA with a weight-average molecular weight of more than 2 million was converted into a hydroxyalkanoate ester, had a yield of 3-hydroxybutyrate ester of 62.7 mol%, whereas Examples iii-1 to iii-5, in which PHA with a weight-average molecular weight of 2 million or less, all had a yield of 3-hydroxybutyrate ester of 70 mol% or more. Therefore, it can be seen that the method for producing a biomass-derived hydroxyalkanoate ester according to the third embodiment of the present invention can convert PHA into a hydroxyalkanoate ester with good yield. Furthermore, as is clear from the results of Examples iii-1 to iii-5, the smaller the weight-average molecular weight of the PHA, below 2 million, the higher the yield of 3-hydroxybutyrate ester.

[0225] (Comparative Example iv-1) 95% by weight sulfuric acid was added to a cell culture solution (inactivated culture solution) containing inactivated poly(3-hydroxybutyrate) particles [average particle size: 1 μm] to adjust the pH to 7.0±0.2. Furthermore, IW (industrial water) was added to adjust the solids concentration to 18% by weight, and then lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme (lytic enzyme) that degrades sugar chains (peptidoglycans) in cell walls, was added, and the mixture was kept at 50°C for 2 hours to disrupt the cells.

[0226] Thereafter, 2.5 L of Alcalase (Novozymes), an alkaline protease, was added, followed by the addition of 30 wt. % sodium hydroxide at 50°C to adjust the pH to 8.5 and maintaining the mixture for 2 hours. 30 wt. % sodium hydroxide was added to the enzyme-treated culture solution obtained above to adjust the pH to 10.5 and maintaining the mixture for 3 hours. Sodium dodecyl sulfate (SDS, Kao) was then added to the enzyme-treated solution to a concentration of 0.6 to 1.0 wt. % (surfactant treatment).

[0227] Thereafter, the pH was adjusted to 11.0±0.2 using 30% by weight sodium hydroxide, and an aqueous solution of sodium hydroxide at pH 11.0 was added to dilute the solution to twice the volume of the enzyme-treated solution, and the solution was kept for 1 hour.

[0228] The enzyme-treated solution was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed and concentrated. A sodium hydroxide solution (pH 11.0) was added to the concentrated aqueous suspension of poly(3-hydroxybutyrate) particles, and the mixture was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. This process was repeated three times. Finally, the supernatant was removed and the mixture was dried to obtain poly(3-hydroxybutyrate) particle aggregates. The poly(3-hydroxybutyrate) particles were separated from the bacterial cell components by the above-mentioned disruption and purification treatments. To the dried poly(3-hydroxybutyrate) particle aggregates, 1.2 wt % of 95 wt % sulfuric acid and three times the amount of methanol were added, and the mixture was heated to 120°C and maintained for 5 hours. The yield of the obtained hydroxyalkanoic acid ester, 3-hydroxybutyric acid ester, was 80 mol %.

[0229] Example iv-1 Methanol was added to the poly(3-hydroxybutyrate) particles obtained in Comparative Example iv-1 before drying to prepare a poly(3-hydroxybutyrate) particle methanol dispersion. The dispersion was centrifuged (4,500 rpm, 10 minutes), the supernatant was removed, and the dispersion was concentrated. To the concentrated dispersion, 1.2% by weight of 95% by weight sulfuric acid relative to the poly(3-hydroxybutyrate) particles was added, followed by adding methanol in an amount three times the amount relative to the poly(3-hydroxybutyrate) particles, and the mixture was heated to 120°C and maintained for 5 hours. The yield of the resulting hydroxyalkanoate ester, 3-hydroxybutyrate ester, was 98 mol%.

[0230] Comparative Example iv-2: A cell culture solution (inactivated culture solution) containing inactivated poly(3-hydroxybutyrate) particles [average particle size: 1 μm] was dried to obtain poly(3-hydroxybutyrate) particle aggregates. 5% by weight of 95% by weight sulfuric acid and a five-fold amount of methanol were added to the dried poly(3-hydroxybutyrate) particle aggregates, and the mixture was heated to 120°C and maintained for 5 hours. The yield of the resulting hydroxyalkanoate ester, 3-hydroxybutyrate ester, was 67 mol%.

[0231] Example iv-2: Methanol was added to a cell culture medium (inactivated culture medium) containing inactivated poly(3-hydroxybutyrate) particles [average particle size: 1 μm] to prepare a poly(3-hydroxybutyrate) particle methanol dispersion. The dispersion was centrifuged (4500 rpm, 10 minutes), the supernatant was removed, and the dispersion was concentrated. To the concentrated dispersion, 5% by weight of 95% sulfuric acid relative to the poly(3-hydroxybutyrate) particles was added, followed by addition of methanol in an amount five times the amount of the poly(3-hydroxybutyrate) particles, and the mixture was heated to 120°C and maintained for 5 hours. The yield of the resulting hydroxyalkanoate ester, 3-hydroxybutyrate ester, was 87 mol%.

[0232] Example iv-3: A cell culture solution (inactivated culture solution) containing inactivated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate polymer) particles [90% by weight of 3-hydroxybutyrate, 10% by weight of 3-hydroxyhexanoate, average particle size: 1 μm] was added with 95% by weight sulfuric acid to adjust the pH to 7.0±0.2. Furthermore, IW (industrial water) was added to adjust the solids concentration to 18% by weight, and then lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme (lytic enzyme) that degrades sugar chains (peptidoglycans) in cell walls, was added, and the mixture was kept at 50°C for 2 hours to disrupt the cells.

[0233] Thereafter, 2.5 L of Alcalase (Novozymes), an alkaline protease, was added, followed by the addition of 30 wt. % sodium hydroxide at 50°C to adjust the pH to 8.5 and maintaining the mixture for 2 hours. 30 wt. % sodium hydroxide was added to the enzyme-treated culture solution obtained above to adjust the pH to 10.5 and maintaining the mixture for 3 hours. Sodium dodecyl sulfate (SDS, Kao) was then added to the enzyme-treated solution to a concentration of 0.6 to 1.0 wt. % (surfactant treatment).

[0234] Thereafter, the pH was adjusted to 11.0±0.2 using 30% by weight sodium hydroxide, and an aqueous solution of sodium hydroxide at pH 11.0 was added to dilute the solution to twice the volume of the enzyme-treated solution, and the solution was kept for 1 hour.

[0235] The enzyme-treated solution was centrifuged (4500 rpm, 10 minutes), the supernatant was removed, and the solution was concentrated. An aqueous solution of sodium hydroxide (pH 11.0) was added to the concentrated aqueous suspension of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate polymer) particles, and the mixture was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. This process was repeated three times. By the above disruption and purification treatments, the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate polymer) particles were separated from the bacterial cell components.

[0236] Furthermore, methanol was added to the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate polymer) particles to prepare a poly(3-hydroxybutyrate-co-3-hydroxyhexanoate polymer) particle methanol dispersion. The dispersion was centrifuged (4,500 rpm, 10 minutes), the supernatant was removed, and the mixture was concentrated. To the concentrated dispersion, 1.2% by weight of 95% by weight sulfuric acid relative to the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate polymer) particles was added, and then methanol was added in an amount three times the amount of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate polymer) particles. The mixture was heated to 120°C and maintained for 5 hours. The yield of the resulting mixture of hydroxyalkanoate esters, 3-hydroxybutyrate ester and 3-hydroxyhexanoate ester, was 97 mol%.

[0237] As described above, in Comparative Examples iv-1 and iv-2, in which poly(3-hydroxybutyrate) particles were dispersed in alcohol after a drying step, the yield of 3-hydroxybutyrate ester in Examples iv-1 and iv-2, in which the particles were dispersed in alcohol without a drying step, was 1.2 times or more higher. Example iv-3, in which poly(3-hydroxybutyrate-co-3-hydroxyhexanoate polymer) particles were dispersed in alcohol without a drying step, also achieved a high yield similar to that of Example iv-1. Therefore, it can be seen that the method for producing a biomass-derived hydroxyalkanoate ester according to the fourth embodiment of the present invention can convert polyhydroxyalkanoate to a hydroxyalkanoate ester with high yield.

[0238] (Production of 1,3-butanediol) 50 g of the 3-hydroxybutyric acid ester obtained in each Example and 2.5 g of a chromium-free copper oxide catalyst (ACA-1, manufactured by Sakai Chemical Industry Co., Ltd.) were reacted (hydrogen reduction reaction) under a hydrogen atmosphere at 15 MPa for 8 hours. It was confirmed by gas chromatography (GC) that 1,3-butanediol was produced by this reaction.

Claims

1. A method for producing a biomass-derived hydroxyalkanoate ester, comprising: a step of producing polyhydroxyalkanoate particles in a system containing water using a polyhydroxyalkanoate-producing microorganism; a step of dispersing the produced polyhydroxyalkanoate particles in an alcohol; and a step of converting the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles in the alcohol dispersion into a hydroxyalkanoate ester, and having any one of the following configurations i) to iv). Configuration i): The total nitrogen content in the polyhydroxyalkanoate particles in the step of converting to the hydroxyalkanoate ester is 2% by weight or less. Configuration ii): In the step of converting to the hydroxyalkanoate ester, the water content in the alcohol dispersion is 10% by weight or less based on 100% by weight of the polyhydroxyalkanoate particles. Configuration iii): The weight average molecular weight of the polyhydroxyalkanoate is 2 million or less. Configuration iv): Before converting the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles into a hydroxyalkanoate ester, the step of drying the polyhydroxyalkanoate particles is not performed 2. The production method according to claim 1, further comprising, after the step of producing the polyhydroxyalkanoate particles, a step of reducing the total nitrogen content in the polyhydroxyalkanoate particles by washing the polyhydroxyalkanoate particles with water, having the above configuration i).

3. The production method according to claim 1, further comprising, after the step of producing the polyhydroxyalkanoate particles, a step of reducing the total nitrogen content in the polyhydroxyalkanoate particles by washing the polyhydroxyalkanoate particles with an organic solvent, having the above configuration i).

4. The production method according to claim 1, further comprising, after the step of producing the polyhydroxyalkanoate particles, a step of reducing the total nitrogen content in the polyhydroxyalkanoate particles by treating the polyhydroxyalkanoate particles with an enzyme that decomposes the cell components of the microorganism, having the above configuration i).

5. The manufacturing method according to claim 1, further comprising, after the step of producing the polyhydroxyalkanoate particles, which has the configuration ii) and does not have the configuration iv), a step of removing water in the system to dry the polyhydroxyalkanoate particles.

6. The manufacturing method according to claim 1, further comprising, after the step of producing the polyhydroxyalkanoate particles, which has the configuration ii), a step of replacing water in the system with an organic solvent to remove the water.

7. The manufacturing method according to claim 1, further comprising, which has the configuration iii), a step of reducing the weight average molecular weight of the polyhydroxyalkanoate before the step of converting the polyhydroxyalkanoate to a hydroxyalkanoate ester.

8. The manufacturing method according to claim 1, further comprising, after the step of producing the polyhydroxyalkanoate particles, a step of replacing water in the system with alcohol.

9. The manufacturing method according to claim 1, further comprising a step of purifying the produced hydroxyalkanoate ester by distillation.

10. The manufacturing method according to claim 1, which has any one of the configurations i) to iii) and does not go through a step of drying the polyhydroxyalkanoate particles until the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles is converted to a hydroxyalkanoate ester.

11. A method for producing 1,3 - butanediol derived from biomass, comprising a step of obtaining 3 - hydroxybutyrate ester among the hydroxyalkanoate esters by the manufacturing method of the hydroxyalkanoate ester according to any one of claims 1 to 10, and a step of reducing the 3 - hydroxybutyrate ester to convert it to 1,3 - butanediol.

12. A method for producing butadiene derived from biomass, comprising a step of obtaining 1,3 - butanediol by the manufacturing method according to claim 11, and a step of obtaining butadiene by a dehydration reaction of the 1,3 - butanediol.

13. A method for producing polybutadiene rubber derived from biomass, comprising a step of obtaining butadiene by the manufacturing method according to claim 12, and a step of polymerizing the butadiene to obtain polybutadiene rubber.

14. A method for producing biomass-derived core-shell type polymer particles, comprising a step of obtaining butadiene by the production method according to claim 12, and a step of obtaining core-shell type polymer particles containing polybutadiene rubber in the core layer using the butadiene.

15. A method for producing biomass-derived propylene, comprising a step of obtaining 3-hydroxybutyric acid ester among the hydroxyalkanoic acid esters by the production method of the hydroxyalkanoic acid ester according to any one of claims 1 to 10, and a step of converting the 3-hydroxybutyric acid ester into propylene by a decarboxylation reaction and a dealcoholization reaction.

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