Method for producing biomass-derived hydroxyalkanoic acid ester

Abstract

This method for producing a biomass-derived hydroxyalkanoic acid ester comprises: (1) causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in a system containing water; (2) dispersing the produced polyhydroxyalkanoate particles in an alcohol; (3) converting the polyhydroxyalkanoate that constitutes the polyhydroxyalkanoate particles into a hydroxyalkanoic acid ester in the alcohol dispersion to obtain a reaction product containing the hydroxyalkanoic acid ester; (4) adjusting the pH of the reaction product within the range of 3.5-13, inclusive; and (5-1) distilling the pH-adjusted reaction product to isolate the hydroxyalkanoic acid ester.

Description

Method for producing biomass-derived hydroxyalkanoate

[0006]

[0001] The present disclosure relates to a method for producing biomass-derived hydroxyalkanoate.

[0002] Propylene, butadiene, benzene and the like, which are important industrial raw materials, have conventionally been obtained by using a naphtha cracker and subjecting naphtha obtained by refining crude oil to thermal decomposition and then separating the reaction products by distillation. Among these, butadiene is obtained by extraction from the C4 fraction by-produced when producing ethylene using a naphtha cracker.

[0003] With the recent expansion of the shale gas market, the competitiveness of ethane crackers, which are devices for producing raw materials such as ethylene and propylene using ethane and propane contained in shale gas as raw materials, has been increasing. However, the production volume of propylene is small, and butadiene, benzene and the like cannot be directly produced using an ethane cracker. Therefore, there is concern that the expansion of ethane crackers in place of naphtha crackers will lead to a decline in the competitiveness of naphtha crackers and make it difficult to stably supply the above-mentioned raw materials that cannot be produced by ethane crackers.

[0004] By the way, in recent years, environmental problems, food problems, awareness of health and safety, and natural or nature orientation have been increasing. As an example, plastic waste has problems such as affecting the ecosystem, generating harmful gases during combustion, and causing global warming due to a large amount of combustion heat, imposing a large burden on the global environment. As a means to solve the above problems, the development of biodegradable plastics has been actively carried out.

[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 was originally in the air, and the carbon dioxide in the atmosphere does not increase. This is called carbon neutral, and is highly regarded under the Kyoto Protocol that sets a target value for carbon dioxide reduction, and active use is desired.

[0006] In line 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] Japanese Patent Publication No. 2012-236798, Japanese Patent Publication No. Hei 11-266891, International Publication No. 2004 / 029266, Japanese Patent Publication No. 2018-000032

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

[0009] While hydroxyalkanoate esters are obtained by the decomposition of PHA, for example, 3-hydroxybutyrate esters, which are hydroxyalkanoate esters, are obtained by the decomposition of poly(3-hydroxybutyrate) polymers (hereinafter, poly(3-hydroxybutyrate) will also be called PHB) among PHA. 3-hydroxybutyrate esters such as methyl 3-hydroxybutyrate are compounds that can serve as precursors for propylene and butadiene. Therefore, if 3-hydroxybutyrate esters can be produced efficiently, it will lead to the efficient production of propylene and butadiene.

[0010] Patent Document 1 discloses a method for obtaining 3-hydroxybutyrate ester using polyhydroxybutyrate derived from microorganisms, in which (3R)-3-hydroxybutyrate ester with a high enantiomeric excess was obtained using polyhydroxybutyrate derived from microorganisms obtained by the method described in the examples of Japanese Patent Application Publication No. 11-266891 (Patent Document 2). However, the yield of 3-hydroxybutyrate ester is not sufficient with the method described in Patent Document 1.

[0011] Patent Document 4 discloses a method for obtaining 3-hydroxybutyrate ester using microorganisms, which involves sequentially performing a fermentation step to obtain a fermented liquid by fermenting 3HB from 3-hydroxybutyrate (3HB) producing bacteria; an alcohol substitution step to obtain an alcohol solution containing 3HB by distilling off the water used as a solvent in the fermented liquid and replacing it with an alcohol solvent as an esterifying agent for 3HB; and a reaction step to obtain 3HB ester by adding a catalyst to the alcohol solution and heating it.

[0012] The method for producing 3HB ester described in document 4 uses 3HB released into the fermentation liquid by Halomonas bacteria after they decompose and consume PHB accumulated in their bodies as the raw material, resulting in poor raw material productivity (on a per-3HB monomer basis).

[0013] The purpose of this disclosure is to provide a method for producing biomass-derived hydroxyalkanoate esters that can yield hydroxyalkanoate esters in good yield.

[0014] In other words, this disclosure relates to a method for producing biomass-derived hydroxyalkanoate esters, comprising: (1) causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in a water-containing system; (2) dispersing the produced polyhydroxyalkanoate particles in alcohol; (3) converting the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles in the alcohol dispersion to a hydroxyalkanoate ester to obtain a reaction product containing a hydroxyalkanoate ester; (4) adjusting the pH of the reaction product to be within the range of 3.5 to 13; and (5-1) distilling the reaction product after the pH has been adjusted to separate the hydroxyalkanoate ester.

[0015] According to this disclosure, a method for producing biomass-derived hydroxyalkanoate esters can be provided that yields hydroxyalkanoate esters in good yield.

[0016] Figure 1 is a schematic diagram showing an example of an apparatus used to produce an alcohol dispersion of polyhydroxyalkanoate particles in a method for producing hydroxyalkanoate esters according to one embodiment of the present disclosure.

[0017] The embodiments will be described in detail below. [Method for producing biomass-derived hydroxyalkanoate esters - 1] This embodiment relates to a method for producing biomass-derived hydroxyalkanoate esters, comprising: (1) having a polyhydroxyalkanoate-producing microorganism produce polyhydroxyalkanoate particles in a water-containing system; (2) dispersing the produced polyhydroxyalkanoate particles in alcohol; (3) converting the polyhydroxyalkanoates constituting the polyhydroxyalkanoate particles in the alcohol dispersion to hydroxyalkanoate esters to obtain a reaction product containing hydroxyalkanoate esters; (4) adjusting the pH of the reaction product to be within the range of 3.5 to 13; and (5-1) distilling the reaction product after pH adjustment to separate the hydroxyalkanoate esters. The numbers in parentheses assigned to each action are symbols provided to facilitate understanding of the invention.

[0018] (Polyhydroxyalkanoate particles) The polyhydroxyalkanoate particles (hereinafter also referred to as PHA particles) are particles composed of PHA and are not particularly limited as long as they are PHA particles that can be produced by microorganisms. Furthermore, 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 acid. Specifically, examples include a homopolymer of one monomer selected from 3-hydroxyalkanoic acid having 4 to 16 carbon atoms, a copolymer of one monomer selected from 3-hydroxyalkanoic acid having 4 to 16 carbon atoms and other hydroxyalkanoic acids (for example, 2-hydroxyalkanoic acid, 4-hydroxyalkanoic acid, 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid, etc., having 4 to 16 carbon atoms), and a copolymer of two or more monomers selected from 3-hydroxyalkanoic acid having 4 to 16 carbon atoms.

[0019] The form in which the PHA is copolymerized is not particularly limited and may be random copolymerization, alternating copolymerization, block copolymerization, graft copolymerization, etc., but random copolymerization is preferred because it is readily available.

[0020] Among the PHAs mentioned above, poly(3-hydroxybutyrate) polymers are preferred from the viewpoint that they can be easily produced by microorganisms that exist in nature. A poly(3-hydroxybutyrate) polymer is a polymer in which 50% by weight or more of the constituent units of the polymer are 3-hydroxybutyrate, and includes homopolymers of 3-hydroxybutyrate units, copolymers containing 3-hydroxybutyrate units and other hydroxyalkanoate units, etc.

[0021] P(3HB), a homopolymer of 3-hydroxybutyrate units, refers to a polymer that substantially uses only 3-hydroxybutyrate units. A 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 a 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.

[0022] 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, but examples include 3-hydroxyhexanoate, 3-hydroxyvariate, 4-hydroxybutyrate, 3-hydroxyoctanoate, 3-hydroxyoctadecanoate, 3-hydroxydecanoate, and lactic acid, and one or more selected from the group consisting of these may be included as monomer units together with 3-hydroxybutyrate. Copolymers of 3HB and 3-hydroxyhexanoate (abbreviated as 3HH) are sometimes referred to as P(3HB-co-3HH) (abbreviated as PHBH), copolymers of 3HB and 3-hydroxyvalarate (abbreviated as 3HV) are sometimes referred to as P(3HB-co-3HV), copolymers of 3HB and 4-hydroxybutyrate (abbreviated as 4HB) are sometimes referred to as P(3HB-co-4HB), and PHA containing lactic acid (abbreviated as LA) as a component, for example, a copolymer of 3HB and LA is sometimes referred to as P(LA-co-3HB).

[0023] Poly(3-hydroxybutyrate) polymers containing 3-hydroxyhexanoate units are preferred because they yield C6 compounds (compounds with 6 carbon atoms).

[0024] The poly(3-hydroxybutyrate) polymer is preferably a homopolymer of 3-hydroxybutyrate units, as the microorganisms capable of producing it are readily available.

[0025] The shape of the PHA particles is not particularly limited and may be spherical or non-spherical, for example. The size of the PHA particles is also not particularly limited, but from the viewpoint of ease of washing and solvent replacement, and ease of aggregation during the preparation of alcohol dispersions, the average particle size of the primary particles is preferably 0.1 to 50 μm, more preferably 0.5 to 50 μm, and even more preferably 1 to 50 μm.

[0026] The average particle size of primary PHA particles can be determined by observation with an electron microscope or by a particle size distribution analyzer.

[0027] (PHA-producing microorganisms) The PHA-producing microorganisms can be any microorganisms that have the ability to produce PHA. The microorganisms can be microorganisms that have a PHA synthase gene. The microorganisms can be wild-type strains that inherently possess a PHA synthase gene, mutant strains obtained by artificially mutating such wild-type strains, or strains into which an exotic PHA synthase gene has been introduced by genetic engineering techniques.

[0028] The PHA-producing microorganism or its host is not particularly limited, but is preferably a rod-shaped bacillus, and more preferably a Gram-negative rod-shaped bacillus. Preferred examples of such rod-shaped bacilli include bacteria belonging to the Burkholderiaceae family, such as the genera Ralstonia, Cupriavidus, Woutersia, and Burkholderia, as well as bacteria belonging to the genera Pseudomonas, Halomonas, and Escherichia.

[0029] From the viewpoint of safety and PHA productivity, the bacteria more preferably belong to the genera Ralstonia, Capriavidus, or Escherichia, and even more preferably belong to the genera Capriavidus or Escherichia, and particularly preferably be Capriavidus necator or Escherichia coli.

[0030] (PHA synthase gene) The PHA synthase gene held by the PHA-producing microorganism is not particularly limited, but examples include PHA synthase genes derived from organisms belonging to the genera Ralstonia, Capriavidus, Woutersia, Alcaligenes, Aeromonas, Pseudomonas, Norcadia, and Chromobacterium, as well as modified versions thereof. As the modified version, a nucleotide sequence encoding PHA synthase in which one or more amino acid residues are deleted, added, inserted, or substituted can be used.

[0031] (Producing PHA particles (1)) In producing PHA particles in a water-containing system using PHA-producing microorganisms (1), the production of PHA particles can be achieved by culturing and growing the PHA-producing microorganisms under appropriate conditions, thereby accumulating PHA particles within the cells of the PHA-producing microorganisms.

[0032] The culture can be carried out by those skilled in the art based on common technical knowledge, and the culture method is not particularly limited. The culture medium composition, method of adding the carbon source, culture scale, aeration and stirring conditions, culture temperature, and culture time are also not particularly limited. For example, the method described in Japanese Patent Publication No. 05-93049 can be used. By performing the culture for an appropriate time, PHA particles can be accumulated within the cells of PHA-producing microorganisms.

[0033] (Dispersing PHA particles in alcohol (2)) Dispersing the produced PHA particles in alcohol will be described in detail below.

[0034] The alcohol used to disperse the PHA particles produced is preferably a water-soluble alcohol, and more preferably an alcohol capable of deriving the desired hydroxyalkanoate ester. Examples of water-soluble alcohols include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-butanol, and one or more selected from this group can be used. Among these, methanol or ethanol is more preferred in terms of reactivity with PHA, and methanol is even more preferred.

[0035] There are no particular limitations on the amount of alcohol used when dispersing PHA particles in alcohol to create an alcohol dispersion.

[0036] The alcohol dispersion obtained by dispersing the PHA particles produced in alcohol (2) is preferably produced by a method (which may be referred to as the "filter concentration method") comprising: agglomerating the PHA particles produced in a water-containing liquid (2-1); concentrating the agglomerated PHA particles in the liquid using a filter (2-2); and adding alcohol to the liquid being concentrated or the concentrated liquid obtained by the concentration (2-3). This is because an alcohol dispersion of microbially produced polyhydroxyalkanoate particles can be efficiently obtained. Furthermore, for the efficient progress of the esterification reaction, it is preferable to have a low water content in the reaction system. However, when removing water from the alcohol dispersion of PHA particles by distillation, heating is required, resulting in higher energy consumption and lower energy efficiency of solvent substitution compared to the filter concentration method. On the other hand, the filter concentration method allows for a reduction in the water content of the alcohol dispersion of PHA particles with low energy consumption and high energy efficiency.

[0037] (Agglutination of PHA particles (2-1)) The agglutination of the PHA particles produced above in a liquid containing water (2-1) will be described in detail. Here, agglutination of PHA particles refers to a state in which the primary PHA particles are not coalesced while maintaining their shape, but rather a state in which at least some of the primary PHA particles are bound together without maintaining their shape, forming a certain cohesive unit through interaction that does not cause separation of the PHA particles by normal stirring. If agglutination is incomplete, the entire dispersion of PHA particles may pass through the filter, or the filter may become clogged and concentration may not be possible.

[0038] In agglomerating the PHA particles (2-1), it is preferable to agglomerate more PHA particles from the viewpoint of concentration efficiency of the agglomerated PHA particles. In other words, it is preferable that the proportion of unagglomerated PHA particles be smaller. The weight ratio of unagglomerated PHA particles to the total PHA particles is preferably 50% by weight or less, 40% by weight or less, 30% by weight or less, 20% by weight or less, 10% by weight or less, 5% by weight or less, 3% by weight or less, and 1% by weight or less, in that order. The weight ratio of unagglomerated PHA particles to the total PHA particles is determined by image analysis of a microscopic image of the PHA particles.

[0039] The method for agglomerating the produced PHA particles in a water-containing liquid (2-1) is not particularly limited, as long as it can agglomerate the PHA particles in a water-containing liquid. For example, since the agglomerated PHA particles can be brought into a suitable agglomerated state by filter concentration, a method of agglomerating by adding 1.5 times the weight of alcohol relative to the water content in the water-containing liquid, and a method of agglomerating by lowering the pH of the water-containing liquid to 4 or less are preferred.

[0040] In the method of coagulation by adding 1.5 times or more by weight of alcohol relative to the water content in the aforementioned water-containing liquid, the amount of alcohol added is preferably in the following order: 1.75 times or more by weight, 2 times or more by weight, 2.25 times or more by weight, 2.5 times or more by weight, 2.75 times or more by weight, and 3 times or more by weight. Furthermore, there is no particular upper limit to the amount of alcohol added, but from the viewpoint of the concentration efficiency of the coagulated PHA particles, for example, it may be 20 times or less by weight relative to the water content in the liquid, or 10 times or less by weight.

[0041] In order to rapidly aggregate the PHA particles, it is preferable to add the entire amount of alcohol to the liquid at once.

[0042] As the type of alcohol, as long as PHA particles can be aggregated in a liquid containing water, it is not particularly limited. However, in particular, using the same alcohol as the alcohol constituting the target alcohol dispersion eliminates the trouble of removing unnecessary alcohol and is preferable because the target alcohol dispersion can be obtained more efficiently.

[0043] When converting PHA in the obtained alcohol dispersion of PHA particles into hydroxyalkanoate ester by an alcoholysis reaction, it is preferable to use the same alcohol as the alcohol used in the reaction. From the viewpoint of reactivity with PHA, methanol or ethanol is more preferable.

[0044] In the method of aggregating by adjusting the pH of the liquid containing water to 4 or less, it is more preferable that the pH is 3 or less, and further preferably 2 or less. Also, the pH may be 1 or more.

[0045] The method for adjusting the pH is not particularly limited, and examples include a method of adding an acid. The acid is also not particularly limited, and for example, at least one selected from the group consisting of organic acids such as acetic acid and inorganic acids such as hydrochloric acid, sulfuric acid, and nitric acid can be used. Sulfuric acid is preferable from the viewpoint of being able to lower the pH with a small amount.

[0046] After aggregating the PHA particles by adding alcohol, water can be added to the aggregated PHA particles. However, adding a large amount of water may cause the aggregated state to dissolve or change. Therefore, it is preferable to maintain an alcohol concentration of 1.5 times the weight or more with respect to the water content in the liquid containing water.

[0047] After aggregating the PHA particles by adjusting the pH of the liquid containing water to 4 or less, if the pH of the liquid containing water is increased to more than 4, the aggregated state may dissolve or change. Therefore, it is preferable to maintain the pH of the liquid containing water at 4 or less.

[0048] (Concentrating the aggregated PHA particles with a filter (2-2)) The method of concentrating the aggregated PHA particles in the liquid with a filter (2-2) will be described in detail. As long as it is a filter that can concentrate the aggregated PHA particles, its type, structure, and pore size are not particularly limited.

[0049] Examples of the types of filters include, for example, ceramic filters, metal filters, and zeolite filters. From the viewpoint of the efficiency of concentration, a ceramic filter is preferred.

[0050] From the viewpoint of efficiently concentrating the aggregated PHA particles, the aperture of the filter is preferably 1 / 10000 to 1 / 10 times the minor axis of the aggregated PHA particles. From the viewpoint of the efficiency of concentrating the aggregated PHA particles, the aperture of the filter is preferably 0.01 μm or more and 10 μm or less, more preferably 0.2 μm or more and 1.2 μm or less, and still more preferably 0.5 μm or more and 1.2 μm or less. The minor axis of the aggregated PHA particles refers to the average value of the diameters in the shortest direction of the aggregated PHA particles obtained from a microscopic image.

[0051] In the method for producing an alcohol dispersion of PHA particles by the filter concentration method, since the aggregated PHA particles are concentrated, a filter with a larger aperture can be selected compared to the case of concentrating non-aggregated PHA particles, and efficient concentration can be achieved. By efficiently concentrating the PHA particles, an alcohol dispersion can be obtained with high productivity and efficiency.

[0052] (Adding alcohol to the liquid during concentration or the concentrated liquid (2-3)) The addition of alcohol to the liquid during concentration or the concentrated liquid obtained by the concentration (2-3) will be described in detail. The alcohol is not particularly limited, but when the PHA in the obtained alcohol dispersion of PHA particles is converted to a hydroxyalkanoate ester by an alcoholysis reaction, it is preferable to use the same alcohol as that used in the reaction. From the viewpoint of reactivity with PHA, methanol or ethanol is more preferable.

[0053] The amount of alcohol added is not particularly limited, but from the viewpoint of the operability of the liquid during concentration or the concentrated liquid obtained by the concentration, it is preferable to adjust the amount of alcohol added so that the solid content concentration in the liquid during concentration or the concentrated liquid obtained by the concentration does not exceed 30% by weight. As the concentration of the aggregated PHA particles progresses, the viscosity in the liquid during concentration or the concentrated liquid may increase, resulting in poor operability.

[0054] The target of alcohol addition may be the liquid being concentrated or the concentrated liquid obtained by the concentration. Adding alcohol to the liquid being concentrated can be done by supplying alcohol at the same time that the dispersion of aggregated PHA particles containing water passes through the filter, and adding alcohol to the concentrated liquid can be done by supplying alcohol to the concentrated liquid after it has passed through the filter at least once.

[0055] Here, an embodiment of the apparatus used for concentrating the aggregated PHA particles in the liquid using a filter (2-2), and for adding alcohol to the liquid being concentrated or the concentrated liquid obtained by the concentration (2-3), will be described in detail with reference to Figure 1.

[0056] Figure 1 is a schematic diagram showing an example of an apparatus used to produce an alcohol dispersion of polyhydroxyalkanoate particles in a method for producing hydroxyalkanoate esters according to one embodiment of the present disclosure. The dispersion of aggregated PHA particles 2a in tank 1 is sent from tank 1 through flow path 11 by liquid transfer pump 3 to filter module 4. In filter module 4, a portion of the dispersion medium of the dispersion of aggregated PHA particles 2a (components other than aggregated PHA particles, mainly water; if PHA particles are aggregated by alcohol addition, mainly water and alcohol) is discharged as filtrate from outlet 12. The concentrated solution 2b of aggregated PHA particles, which has been concentrated by the removal of the filtrate, is returned to tank 1 through flow path 13.

[0057] Tank 1 is equipped with an alcohol inlet 14, and alcohol is supplied to the concentrated liquid 2b of aggregated PHA particles from the inlet 14, thereby obtaining an alcohol dispersion of PHA particles.

[0058] Furthermore, alcohol is continuously supplied to tank 1 from before the dispersion of agglomerated PHA particles 2a is sent to the filter module 4 until the concentrated solution of agglomerated PHA particles 2b is returned to tank 1, thereby adding alcohol to the liquid being concentrated and to the concentrated solution.

[0059] According to the method for producing an alcohol dispersion of PHA particles using the filter concentration method described above, an alcohol dispersion of microorganism-produced PHA particles can be efficiently obtained. Furthermore, an alcohol dispersion of PHA particles with a low water content and an alcohol dispersion of PHA particles with any desired PHA particle content can be obtained.

[0060] From the viewpoint of efficiency of conversion to hydroxyalkanoate esters, the water content in the alcohol dispersion of the PHA particles is preferably 10% by weight or less, 7.5% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, and 1% by weight or less, in that order.

[0061] The content of PHA particles in the alcohol dispersion of the PHA particles is preferably 10% by weight or more, more preferably 15% by weight or more, and even more preferably 20% by weight or more, from the viewpoint of concentration efficiency and purification efficiency after conversion to hydroxyalkanoate ester. Furthermore, from the viewpoint of ease of concentration, 25% by weight or less is preferred.

[0062] (Replacing water in the system with alcohol (6)) In the method for producing biomass-derived hydroxyalkanoate ester according to this embodiment, it is preferable to further include replacing water in the system with alcohol (6) after producing the PHA particles (1). This makes it possible to remove water that causes a hydrolysis reaction of PHA that competes with the conversion reaction from PHA to hydroxyalkanoate ester, and bacterial components that inhibit the conversion reaction, thereby enabling the conversion from PHA to hydroxyalkanoate ester in good yield. Here, "replacing water in the system with alcohol" means replacing the liquid portion containing water in the system (in other words, the part other than the solid portion) with alcohol, thereby removing water-soluble components derived from bacterial components and culture substrate residues along with the water.

[0063] The method for replacing water in a system with alcohol is not particularly limited and includes methods such as removing water from the system using filtration, centrifugation, sedimentation, or electrophoresis, and then adding alcohol.

[0064] The type of alcohol used to replace water in the system is not particularly limited, but water-soluble alcohols are preferred. Examples of water-soluble 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. These alcohols may be used individually or in combination of two or more.

[0065] When replacing water in the system, the alcohol used is preferably the same alcohol used in the reaction when converting PHA to a hydroxyalkanoate ester to obtain a reaction product containing the hydroxyalkanoate ester (3) is done by an alcohol decomposition reaction, and methanol or ethanol is more preferred.

[0066] Replacing the water in the system with alcohol (6) may involve separating the PHA particles from the cellular components of the PHA-producing microorganisms, that is, replacing the water in a system containing PHA-producing microorganisms while the PHA particles are still contained within the cells, by disrupting the cellular structure.

[0067] (Separation of PHA particles (7)) In the method for producing biomass-derived hydroxyalkanoate ester according to this embodiment, it is preferable to further include (7) separating the PHA particles from the microbial cell components in a water-containing system after producing the PHA particles (1). This is because the microbial cell components inhibit the alcohol decomposition reaction, which is the conversion reaction of PHA to hydroxyalkanoate ester, leading to the efficient removal and reduction of the microbial cell components. Furthermore, the efficient removal and reduction of the microbial cell components also leads to an increase in the flow rate of filter concentration when producing a dispersion of aggregated PHA particles.

[0068] Separating PHA particles from the cellular components of the aforementioned microorganism means destroying the cells of the microorganism and removing the PHA particles from inside the cell to outside. As a method for separating PHA particles, it is preferable to cause a PHA-producing microorganism to produce PHA particles (1), thereby accumulating PHA particles inside the cellular cells of the PHA-producing microorganism, and then crushing the cellular cells containing the PHA particles by physical, chemical, or biological treatment.

[0069] While there are no particular limitations on the crushing method, conventionally known methods utilizing fluid shear force, solid shear force, or grinding, such as French presses, homogenizers, X-presses, ball mills, colloid mills, DYNO mills, and ultrasonic homogenizers, can be used. Methods using drugs such as acids, alkalis, surfactants, organic solvents, and cell wall synthesis inhibitors, as well as enzymes such as lysozyme, amylase, pectinase, cellulase, thymolide, maltase, saccharase, α-glycosidase, β-glycosidase, and N-glycosidase, and supercritical fluids, as well as osmotic crushing, freezing, and dry grinding methods, can also be used. Furthermore, autolysis methods utilizing the action of proteases and esterases contained within the cells themselves are also possible. These crushing methods may be used individually or in combination, and the order of combination is not limited (for example, a method using enzymes may be followed by a method utilizing fluid shear force, solid shear force, or grinding). Batch processing or continuous processing may also be used.

[0070] In the method for producing biomass-derived hydroxyalkanoate esters according to this embodiment, it is preferable to further include, after producing the polyhydroxyalkanoate particles (1), separating the PHA particles from the microbial cell components in a water-containing system (7), and replacing the water in the system with alcohol (6). It is even more preferable to further include separating the PHA particles from the microbial cell components in a water-containing system (7), and replacing the water in the system with alcohol (6) in this order. This is because the microbial cell components and water can be removed more efficiently, and the conversion from PHA to hydroxyalkanoate esters can be performed with a higher yield.

[0071] (Washing of PHA particles (8)) The method for producing biomass-derived hydroxyalkanoate esters according to this embodiment may include washing the PHA particles (8) after producing the PHA particles (1). Here, "washing the PHA particles" means separating and removing nitrogen-containing components that adhere to or bind to the inside and outside of the PHA particles. In the process of converting the PHA constituting the PHA particles into hydroxyalkanoate esters to obtain a reaction product containing hydroxyalkanoate esters (3), the fewer impurities in the conversion reaction from PHA to hydroxyalkanoate esters, such as nitrogen-containing components other than PHA particles, culture substrate residues, and water, the easier it is to obtain hydroxyalkanoate esters in high yield. When converting the PHA in the obtained alcohol dispersion of PHA particles to hydroxyalkanoate esters by an alcohol decomposition reaction, bacterial components and the like can inhibit the alcohol decomposition reaction, which is the conversion reaction of PHA to hydroxyalkanoate esters. Therefore, efficient removal of bacterial components and the like is desirable, especially in order to obtain hydroxyalkanoate esters in high yield. Furthermore, including washing the PHA particles (8) enables efficient removal of bacterial components and the like, and also leads to an increase in the flow rate of the filter concentration of the dispersion of aggregated PHA particles.

[0072] Furthermore, the method for producing biomass-derived hydroxyalkanoate esters according to this embodiment may include, in this order, producing the PHA particles (1), separating the PHA particles from the cellular components of the microorganism (7), and washing the PHA particles (8). In particular, if washing the PHA particles (8) is performed using a detergent containing water, the method may include, in this order, separating the PHA particles from the cellular components of the microorganism (7), washing the PHA particles (8), and replacing the water in the system with alcohol (6).

[0073] The methods used to wash PHA particles are not particularly limited, but include filtration, centrifugation, sedimentation, and electrophoresis.

[0074] Examples of cleaning agents used for washing PHA particles include water, organic solvents, or mixed solutions of water and organic solvents. Surfactants and the like may be added to these cleaning agents. The pH of the water and mixed solutions of water and organic solvents used for washing PHA particles may be adjusted as appropriate.

[0075] Washing PHA particles with water is preferable because it does not involve the discharge of solvents. Washing PHA particles with an organic solvent is preferable because it easily reduces the nitrogen-containing components.

[0076] The type of organic solvent used for washing PHA particles is not particularly limited, but in the process of converting the PHA constituting the PHA particles into hydroxyalkanoate esters to obtain a reaction product containing hydroxyalkanoate esters (3), water-soluble organic solvents are preferred because they allow for an efficient reduction in the amount of water in the system, reduce the possibility of hydrolysis of PHA competing with the alcohol decomposition reaction of PHA, and make it easier to obtain hydroxyalkanoate esters in higher yield. 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. Examples of 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 individually or in combination of two or more.

[0077] As the organic solvent used for washing the PHA particles, alcohol is preferred, and methanol or ethanol is more preferred, because when the reaction is carried out by an alcohol decomposition reaction, it is efficient to use alcohol in the reaction to convert PHA to a hydroxyalkanoate ester and obtain a reaction product containing the hydroxyalkanoate ester (3).

[0078] Furthermore, in order to efficiently reduce the nitrogen-containing components, the PHA particles may be treated with enzymes, hypochlorous acid, or hydrogen peroxide before being washed with the detergent. In particular, further including treatment of the polyhydroxyalkanoate particles with an enzyme that decomposes the cellular components of the microorganisms (8-1) is preferable because it enables efficient removal of cellular components and the like, and increases the flow rate of the filter concentration of the dispersion of aggregated PHA particles.

[0079] In enzymatic treatment, the usable enzymes are not particularly limited, as long as they have the activity to decompose the components of the bacterial cell. Examples include proteolytic enzymes such as alcalase, lipid-degrading enzymes, cell wall-degrading enzymes, and nucleolytic enzymes. In addition, commercially available enzyme detergents for laundry, or enzyme compositions containing enzymes with enzyme stabilizers or anti-redeposition agents, can also be used. These may be used individually or in combination of two or more.

[0080] The time required for the enzyme treatment can be set appropriately considering the desired degree of purification, but for example, it may be 0.5 to 10 hours, 1 to 10 hours, or 1 to 2 hours.

[0081] The amount of enzyme used is not particularly limited and depends on the type and activity of the enzyme, but for example, it may be 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.

[0082] In the hypochlorous acid treatment, the pH of the PHA particle dispersion should be set to the alkaline range, and the treatment should be carried out under conditions that suppress heat, light, and contact with 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, and more preferably 20°C or lower.

[0083] In the hydrogen peroxide treatment, in order to enhance the effect of removing the nitrogen-containing components in a short time, it is preferable to add hydrogen peroxide to the dispersion of PHA particles and then heat the dispersion. The temperature is preferably 50°C or higher, and more preferably 70°C or higher. The upper limit of the temperature is preferably below the boiling point of the dispersion. In the hydrogen peroxide treatment, it is preferable to hold the dispersion under heating, and the holding time is preferably, for example, 10 minutes to 10 hours, more preferably 30 minutes to 5 hours, and even more preferably 1 to 3 hours.

[0084] In order to suppress the decrease in molecular weight of PHA caused by the treatment, it is preferable to carry out the hydrogen peroxide treatment while controlling the pH of the dispersion of PHA particles to 7 to 13 by continuously or intermittently adding an alkali to the dispersion. The alkali is not particularly limited, but examples include sodium hydroxide, sodium carbonate, and potassium hydroxide. Details of pH control can be found in Patent Document 3.

[0085] The various treatments for removing the nitrogen-containing components may be carried out individually or in combination of two or more.

[0086] (Drying of PHA particles (9)) In the method for producing biomass-derived hydroxyalkanoate ester according to this embodiment, the PHA particles may or may not be dried by removing the liquid component containing water in the system. In this disclosure, drying of PHA particles means removing the liquid component 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.

[0087] In the process of converting the PHA constituting the PHA particles into hydroxyalkanoate esters to obtain a reaction product containing hydroxyalkanoate esters (3), a larger contact area between the PHA and its reaction partner or solvent results in higher reactivity and easier acquisition of hydroxyalkanoate esters in high yield. By not drying the PHA particles (9) before converting the polyhydroxyalkanoate constituting the PHA particles into hydroxyalkanoate esters, it is possible to suppress the aggregation of PHA particles, the formation of secondary particles, and the resulting increase in particle size, thereby avoiding a decrease in the contact area between the PHA and its reaction partner or solvent, which is preferable.

[0088] (3) Obtaining a reaction product containing hydroxyalkanoate esters. The method of obtaining a reaction product containing hydroxyalkanoate esters by converting the PHA constituting the PHA particles in the alcohol dispersion to hydroxyalkanoate esters will be described in detail.

[0089] The conversion of PHA to a hydroxyalkanoate ester is not particularly limited as long as it can be converted to a hydroxyalkanoate ester, but it is preferably carried out by alcohol decomposition, and more preferably in the presence of a catalyst from the viewpoint of facilitating the alcohol decomposition of PHA. Examples of catalysts 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% by weight relative to the PHA, more preferably 0.01 to 5% by weight, and even more preferably 0.015 to 3% by weight.

[0090] The alcohol used in the alcoholic decomposition of PHA should be an alcohol capable of yielding the desired hydroxyalkanoate 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 this group can be used. Among these, methanol or ethanol is more preferred in terms of reactivity with PHA, and methanol is even more preferred. It is particularly preferable to use the same alcohol used in dispersing the PHA particles in alcohol (2).

[0091] In (3), where PHA is converted to a hydroxyalkanoate ester to obtain a reaction product containing the hydroxyalkanoate ester, the alcohol content in the alcohol dispersion is not particularly limited, but from the viewpoint of reactivity, 1 to 100 parts by weight is preferred, 1.5 to 100 parts by weight is more preferred, 1.5 to 50 parts by weight is even more preferred, 1.5 to 10 parts by weight is particularly preferred, and 1.5 to 5 parts by weight is most preferred per 1 part by weight of PHA.

[0092] When the conversion of PHA to hydroxyalkanoate esters is carried out by an alcohol decomposition reaction, the reaction proceeds at 60 to 200°C. However, from the viewpoint of easily carrying out the alcohol decomposition reaction and obtaining a sufficient yield, the temperature conditions are preferably 80 to 180°C, more preferably 90 to 160°C, and even more preferably 100 to 140°C. Furthermore, the time for maintaining the above temperature conditions is preferably 1 to 12 hours, more preferably 2 to 10 hours, and even more preferably 3 to 8 hours.

[0093] The hydroxyalkanoate ester obtained by the production method according to the above embodiment will be described using the case where the hydroxyalkanoate ester is 3-hydroxybutyrate ester as an example. The 3-hydroxybutyrate ester is a compound represented by the following formula (1). [In formula (1) above, R is a hydrocarbon group.]

[0094] In formula (1) above, R is not particularly limited as long as it is a hydrocarbon group, but from the viewpoint of ease of distillation, 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.

[0095] If the hydroxyalkanoate ester is a 3-hydroxybutyrate ester, PHA particles composed of a polyhydroxyalkanoate containing 3-hydroxybutyrate units, such as the poly(3-hydroxybutyrate) polymer, can be produced by a polyhydroxyalkanoate-producing microorganism.

[0096] Furthermore, the hydroxyalkanoate ester obtained by the manufacturing method according to the above embodiment will be described using the case where the hydroxyalkanoate ester is a 3-hydroxyhexanoate ester as an example. The 3-hydroxyhexanoate ester is a compound represented by the following formula (2). [In formula (2) above, R is a hydrocarbon group.]

[0097] In formula (2) above, R is not particularly limited as long as it is a hydrocarbon group, but from the viewpoint of ease of distillation, 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.

[0098] If the hydroxyalkanoate ester is a 3-hydroxyhexanoate ester, PHA particles composed of a polyhydroxyalkanoate containing 3-hydroxyhexanoate units, such as the poly(3-hydroxybutyrate) polymer, can be produced by a polyhydroxyalkanoate-producing microorganism.

[0099] (4) Adjusting the pH of the reaction product to be within the range of 3.5 to 13. The following is a detailed explanation of (4) adjusting the pH of the reaction product containing the hydroxyalkanoate ester to be within the range of 3.5 to 13.

[0100] The pH of the reaction product is preferably adjusted to a range of 3.5 to 13, more preferably to a range of 3.5 to 10, and even more preferably to a range of 5 to 9. If the pH is less than 3.5, the polymerization reaction of the hydroxyalkanoate ester converted from PHA will proceed, resulting in an insufficient yield of the hydroxyalkanoate ester. Adjusting the pH to a level above 13 may require a large amount of alkaline substance, and excess alkaline substance is likely to precipitate as residue after distillation. The adjustment of the pH of the reaction product may be an adjustment that changes the pH of the reaction product from outside the specified range to within the specified range, or an adjustment that changes the pH of the reaction product within the specified range.

[0101] Adjusting the pH of the reaction product (4) preferably involves adding an alkaline substance. The conversion of PHA to hydroxyalkanoate esters is preferably carried out by alcohol decomposition using an acid catalyst, and in that case, the pH of the resulting reaction product is also affected by the acid catalyst. In other words, it is preferable to raise the pH, which has decreased due to the effect of the acid catalyst, by adding an alkaline substance.

[0102] Examples of alkaline substances include hydroxides of alkali metals and alkaline earth metals, specifically sodium hydroxide (NaOH), potassium hydroxide (KOH), and calcium hydroxide, and one or more of these can be used in combination. Among these, sodium hydroxide (NaOH) is preferred because it is a strong alkali and can adjust the pH with a small amount.

[0103] (Distilling the reaction product to separate the hydroxyalkanoate ester (5-1)) Distilling the reaction product after adjusting the pH to separate the hydroxyalkanoate ester (5-1) will be described in detail.

[0104] The aforementioned distillation method is not particularly limited, but examples include single distillation, continuous distillation, atmospheric distillation, and reduced-pressure distillation. Furthermore, the temperature and pressure conditions during distillation, as well as the number of distillation cycles, are not particularly limited as long as they are within the range of general chemical manufacturing and can be selected according to the type of hydroxyalkanoate ester to be used.

[0105] The pressure conditions for distillation to separate the hydroxyalkanoate ester from the reaction product are preferably 300 torr or less, more preferably 200 torr or less, and even more preferably 150 torr or less. It may also be 0 torr or higher. This is because a higher degree of reduced pressure allows for a lower distillation temperature, thereby suppressing side reactions and enabling a higher yield of the hydroxyalkanoate ester.

[0106] The preferred temperature for distillation to separate the hydroxyalkanoate ester from the reaction product is 50 to 300°C, more preferably 50 to 200°C, and even more preferably 50 to 150°C. This is because lowering the distillation temperature suppresses side reactions and allows for efficient evaporation of the hydroxyalkanoate ester, resulting in a good yield of the hydroxyalkanoate ester.

[0107] For the distillation of the reaction product to separate the hydroxyalkanoate ester, it is particularly preferable to satisfy both the pressure of 300 torr or less and the temperature of 50 to 300°C.

[0108] When methanol is used as the alcohol to form the alcohol dispersion, and methyl 3-hydroxybutyrate is obtained as the hydroxyalkanoate ester, the boiling point of methanol is 65°C at 1 atmosphere, and the boiling point of methyl 3-hydroxybutyrate is 161°C at 1 atmosphere. Therefore, as the temperature of the reaction product is increased by starting distillation, methanol and then methyl 3-hydroxybutyrate will be distilled off in that order.

[0109] The pressure and temperature conditions during methanol distillation may be a pressure of 300 torr or less and a temperature of 50 to 300°C.

[0110] The pressure and temperature conditions during the distillation of methyl 3-hydroxybutyrate may be a pressure of 300 torr or less and a temperature of 50 to 300°C.

[0111] [Method 2 for Producing Biomass-Derived Hydroxyalkanoate Esters] Next, an embodiment of Method 2 for Producing Biomass-Derived Hydroxyalkanoate Esters will be described in detail. Method 2 is characterized by the conversion of PHA to hydroxyalkanoate esters compared to Method 1 for Producing Biomass-Derived Hydroxyalkanoate Esters. Except for obtaining a reaction product containing hydroxyalkanoate esters (3) and adjusting the pH of the reaction product to a range of 3.5 to 13 (4) in Method 1, the description of Method 1 for Producing Biomass-Derived Hydroxyalkanoate Esters can be applied mutatis mutandis, so the description will be omitted.

[0112] This embodiment relates to a method for producing biomass-derived hydroxyalkanoate esters, comprising: (1) causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in a water-containing system; (2) dispersing the produced polyhydroxyalkanoate particles in alcohol; and (3-2) converting the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles into a hydroxyalkanoate ester in a mixed solvent, wherein the mixed solvent contains alcohol and a solvent with a higher boiling point than the alcohol contained in the mixed solvent. According to this embodiment, it is possible to provide a method for producing biomass-derived hydroxyalkanoate esters that can convert polyhydroxyalkanoate to hydroxyalkanoate esters in good yield.

[0113] (Conversion of PHA to hydroxyalkanoate ester (3-2)) The conversion of PHA constituting PHA particles in an alcohol dispersion to a hydroxyalkanoate ester in a mixed solvent (3-2) will be described in detail.

[0114] The mixed solvent includes an alcohol and a solvent with a higher boiling point than the alcohol contained in the mixed solvent. The solvent with a higher boiling point than the alcohol contained in the mixed solvent refers to organic solvents other than alcohol that have a higher boiling point than the alcohol contained in the mixed solvent, and does not include water or solvents that chemically react with the alcohol contained in the mixed solvent.

[0115] By converting PHA to hydroxyalkanoate esters in a mixed solvent (3-2), the reaction can be carried out at higher temperatures even under the same pressure conditions compared to using alcohol alone as the solvent. This allows for a high yield of conversion from PHA to hydroxyalkanoate esters even under mild pressure conditions, such as atmospheric pressure or near atmospheric pressure. Here, atmospheric pressure refers to pressure equal to atmospheric pressure.

[0116] The alcohol contained in the mixed solvent can be any alcohol capable of yielding the desired hydroxyalkanoate 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 this group can be used. Among these, methanol or ethanol is more preferred in terms of reactivity with PHA, and methanol is even more preferred. It is particularly preferable to use the same alcohol used in dispersing the PHA particles in the alcohol (2).

[0117] In converting PHA to a hydroxyalkanoate ester (3-2), the alcohol content in the alcohol dispersion is not particularly limited, but from the viewpoint of reactivity, 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 per 1 part by weight of PHA.

[0118] As a solvent with a higher boiling point than the aforementioned alcohol, for example, if methanol is used as the alcohol, a solvent with a higher boiling point than methanol should be selected; and if ethanol is used as the alcohol, a solvent with a higher boiling point than ethanol should be selected.

[0119] Examples of solvents with higher boiling points than the aforementioned alcohols include 3-hydroxybutyrate esters such as methyl 3-hydroxybutyrate (161°C), ethyl 3-hydroxybutyrate (170°C), and butyl 3-hydroxybutyrate, as well as hydroxyalkanoates that are the target of this embodiment, including 3-hydroxyhexanoate esters such as methyl 3-hydroxyhexanoate, ethyl 3-hydroxyhexanoate, and methyl 3-hydroxyhexanoate; aromatic solvents such as toluene (110.6°C), xylene (139°C), benzene (80.1°C), dimethoxybenzene (212.6°C), chlorobenzene (132°C), dichlorobenzene (179°C), mesitylene (164.7°C), and ethylbenzene (136°C); and 1,4-dioxane (101°C), cyclopentyl Examples of solvents include ether-based solvents such as methyl ether (106°C), tetrahydrofuran (66°C), tetrahydropyran (88°C), and 1,3-dioxolane (78°C); chlorine-based solvents such as 1,2-dichloroethane (83.5°C), 1,1,2-trichloroethane (113.9°C), chlorobenzene (132°C), and o-dichlorobenzene (179°C); amide-based solvents such as N-methylpyrrolidone (202°C), N,N-dimethylformamide (153°C), N,N-dimethylacetamide (153°C), and hexamethylphosphate triamide (233°C); aliphatic hydrocarbon solvents such as n-hexane (68.7°C) and n-heptane (98.4°C); and sulfur-containing solvents such as dimethyl sulfoxide (189°C) and sulfolane (285°C). One or more solvents selected from this group can be used. The temperatures in parentheses indicate the boiling point of each solvent.

[0120] As a solvent with a higher boiling point than the aforementioned alcohol, it is preferable to select the same solvent as the target hydroxyalkanoate ester, as this allows for a higher yield of the target hydroxyalkanoate ester.

[0121] The content of the high-boiling point solvent in the mixed solvent is not particularly limited as long as it can raise the temperature of the ester conversion reaction system. However, in terms of achieving both mild pressure conditions and a good yield of conversion from PHA to hydroxyalkanoate ester, the content is preferably 3 to 100 parts by weight, more preferably 5 to 100 parts by weight, and even more preferably 5 to 50 parts by weight per 1 part by weight of alcohol in the mixed solvent.

[0122] The conversion of PHA to a hydroxyalkanoate ester is not particularly limited as long as it can be converted to a hydroxyalkanoate ester, but it is preferably carried out by alcohol decomposition, and more preferably in the presence of a catalyst from the viewpoint of facilitating the alcohol decomposition of PHA. Examples of catalysts 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% by weight relative to the PHA, more preferably 0.01 to 5% by weight, and even more preferably 0.015 to 3% by weight.

[0123] Furthermore, it is preferable that the mixed solvent contains a weak salt soluble in the mixed solvent. The conversion of PHA to hydroxyalkanoate esters is preferably carried out by alcohol decomposition using an acid catalyst. As a result, the reaction product obtained also contains the acid catalyst, and the polymerization reaction of the hydroxyalkanoate ester proceeds due to the presence of the acid catalyst, making it easy for oligomers to form. When a solvent with a higher boiling point than the alcohol is used, such as the same solvent as the target hydroxyalkanoate ester, or a compound that can polymerize with the generated hydroxyalkanoate ester, oligomer formation is particularly likely. On the other hand, by including a weak salt soluble in the mixed solvent, the formation of such oligomers can be avoided, and the conversion reaction of PHA to hydroxyalkanoate esters proceeds, thus increasing the yield of hydroxyalkanoate esters.

[0124] The weak salt may be added to the mixed solvent before the start of the conversion reaction of PHA to hydroxyalkanoate ester.

[0125] In the context of a weak salt soluble in the mixed solvent, "soluble" means that, when an acid catalyst is used for the conversion of the hydroxyalkanoate ester of PHA, it has enough solubility to accept protons generated by the ionization of the acid catalyst and form a weak acid. Examples of such weak salts include one or more selected from the group consisting of acetate salts such as ammonium acetate, sodium acetate, and potassium acetate, and organic salts such as sodium benzenesulfonate and sodium benzoate. When methanol or ethanol is used as the alcohol in the mixed solvent, acetate salts are preferred because they have high solubility in these alcohols and can more efficiently reduce the acidity in the reaction system.

[0126] The content of the weak salt in the mixed solvent is preferably 0.5 to 10% by weight, more preferably 1 to 10% by weight, and even more preferably 2 to 10% by weight, relative to the polyhydroxyalkanoate particles.

[0127] (Purification of the generated hydroxyalkanoate ester by distillation (5-2)) In the method for producing biomass-derived hydroxyalkanoate ester according to this embodiment, it is preferable to further include purification of the generated hydroxyalkanoate ester by distillation (5-2). The explanation of distillation in this embodiment is the same as the explanation of distillation in the separation of the hydroxyalkanoate ester by distillation of the reaction product in the above production method-1 (5-1), so it is omitted.

[0128] [Method for producing biomass-derived 1,3-butanediol] This embodiment relates to a method for producing biomass-derived 1,3-butanediol, which includes obtaining 3-hydroxybutyrate ester from the hydroxyalkanoate esters by the method for producing hydroxyalkanoate esters-1 or the method for producing hydroxyalkanoate esters-2, and reducing the 3-hydroxybutyrate ester to convert it into 1,3-butanediol.

[0129] (Obtaining 3-hydroxybutyrate ester) 3-hydroxybutyrate ester can be obtained by using the above-mentioned method for producing biomass-derived hydroxyalkanoate esters-1 or method for producing hydroxyalkanoate esters-2.

[0130] (Conversion of 3-hydroxybutyrate ester to 1,3-butanediol) The conversion of 3-hydroxybutyrate ester to 1,3-butanediol by reduction will be described in detail. 3-hydroxybutyrate ester is represented by the above formula (1). As a method for reducing the 3-hydroxybutyrate ester to 1,3-butanediol, known methods for hydrogen reduction of carboxylic acid esters can be used. Examples include hydrogen reduction reactions using catalysts containing noble metals such as ruthenium (Ru), palladium (Pd), rhenium (Re), and rhodium (Rh), and catalysts containing copper (Cu).

[0131] [Method for producing biomass-derived butadiene] This embodiment relates to a method for producing biomass-derived butadiene, which includes obtaining 1,3-butanediol by the above-described production method, and obtaining butadiene by a dehydration reaction of the 1,3-butanediol.

[0132] (Obtaining 1,3-butanediol) To obtain 1,3-butanediol, the above-mentioned method for producing biomass-derived 1,3-butanediol can be used.

[0133] (Obtaining Butadiene) The method for obtaining butadiene by the dehydration reaction of 1,3-butanediol will be described in detail. Known methods can be used to obtain butadiene by the dehydration reaction of 1,3-butanediol, such as intramolecular dehydration in the presence of a dehydration catalyst.

[0134] 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. Examples of metals constituting metal phosphates, metal sulfates, and metal hydrochlorides include 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.

[0135] [Method for producing biomass-derived polybutadiene rubber] This embodiment relates to a method for producing biomass-derived polybutadiene rubber, which includes obtaining butadiene by the above-described method and polymerizing the butadiene to obtain polybutadiene rubber.

[0136] (Obtaining butadiene) To obtain butadiene, the above-mentioned method for producing biomass-derived butadiene can be used.

[0137] (Obtaining Polybutadiene Rubber) The process of polymerizing butadiene to obtain polybutadiene rubber will be described in detail. Known methods can be used to polymerize butadiene to obtain polybutadiene rubber, including radical polymerization, coordination anionic polymerization, and anionic polymerization using organolithium compounds. The polymerization method should be appropriately selected according to the stereoregularity of the desired 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.

[0138] [Method for producing biomass-derived core-shell polymer particles] This embodiment relates to a method for producing biomass-derived core-shell polymer particles, comprising obtaining butadiene by the above-described method, and obtaining core-shell polymer particles containing polybutadiene rubber in the core layer using the butadiene.

[0139] (Obtaining butadiene) To obtain butadiene, the above-mentioned method for producing biomass-derived butadiene can be used.

[0140] (Obtaining core-shell polymer particles) The process of obtaining core-shell polymer particles containing polybutadiene rubber in the core layer using butadiene will be described in detail.

[0141] The method for producing the core-shell type polymer particles can be a conventional method and is not particularly limited. For example, bulk polymerization, solution polymerization, suspension polymerization, or emulsion polymerization can be employed.

[0142] In emulsion graft polymerization, specifically, a latex containing polymer particles of polybutadiene rubber, which forms the core layer, is first produced by emulsion polymerization. Then, monomer components for the shell layer and polymerization initiators are added to the latex to polymerize the monomer components.

[0143] 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.

[0144] Polymerization initiators include pyrolysis-type initiators and redox-type initiators. Examples of pyrolysis-type initiators include 2,2'-azobisisobutyronitrile, hydrogen peroxide, potassium persulfate, and ammonium persulfate. Examples of redox-type initiators include those that combine organic peroxides such as t-butyl peroxyisopropyl carbonate, paramenthane hydroperoxide, cumene hydroperoxide, dicumyl peroxide, t-butyl hydroperoxide, di-t-butyl peroxide, and t-hexyl peroxide; 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 ethylenediaminetetraacetate disodium; and phosphorus-containing compounds such as sodium pyrophosphate.

[0145] [Method for producing biomass-derived propylene] This embodiment relates to a method for producing biomass-derived propylene, which includes obtaining 3-hydroxybutyrate ester from the hydroxyalkanoate esters by the method for producing hydroxyalkanoate esters-1 or the method for producing hydroxyalkanoate esters-2, and converting the 3-hydroxybutyrate ester to propylene by a decarboxylation reaction and a dealcoholization reaction.

[0146] (Obtaining 3-hydroxybutyrate ester) 3-hydroxybutyrate ester can be obtained by using the above-mentioned method for producing biomass-derived hydroxyalkanoate esters-1 or method for producing hydroxyalkanoate esters-2.

[0147] (Conversion of 3-hydroxybutyrate ester to propylene) The conversion of 3-hydroxybutyrate ester to propylene by decarboxylation and dealcoholization reactions will be described in detail. Methods for carrying out decarboxylation and dealcoholization reactions of 3-hydroxybutyrate ester include reactions using catalysts such as acid catalysts and metal oxide catalysts, for example, reactions using solid acid catalysts such as amorphous silica-alumina and niobium phosphate.

[0148] [Method for producing biomass-derived 1,3-hexanediol] This embodiment relates to a method for producing biomass-derived 1,3-hexanediol, which includes obtaining 3-hydroxyhexanoic acid ester from the hydroxyalkanoic acid ester by the method for producing hydroxyalkanoic acid ester-1 or the method for producing hydroxyalkanoic acid ester-2, and reducing the 3-hydroxyhexanoic acid ester to convert it into 1,3-hexanediol.

[0149] (Obtaining 3-hydroxybutyrate ester) To obtain 3-hydroxyhexanoate ester, use the above-mentioned method for producing biomass-derived hydroxyalkanoate esters-1 or method for producing hydroxyalkanoate esters-2.

[0150] (Conversion of 3-hydroxyhexanoic acid ester to 1,3-hexanediol) The reduction of 3-hydroxyhexanoic acid ester to 1,3-hexanediol will be described in detail. 3-hydroxyhexanoic acid ester is represented by formula (2) above. As a method for reducing the 3-hydroxyhexanoic acid ester to 1,3-hexanediol, known methods for hydrogen reduction of carboxylic acid esters can be used. Examples include hydrogen reduction reactions using catalysts containing noble metals such as ruthenium (Ru), palladium (Pd), rhenium (Re), and rhodium (Rh), and catalysts containing copper (Cu).

[0151] The following items list preferred embodiments of the present disclosure, but the present invention is not limited to these items. [Item A-1] A method for producing biomass-derived hydroxyalkanoate esters, comprising: (1) causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in a water-containing system; (2) dispersing the produced polyhydroxyalkanoate particles in alcohol; (3) converting the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles in the alcohol dispersion to a hydroxyalkanoate ester to obtain a reaction product containing a hydroxyalkanoate ester; (4) adjusting the pH of the reaction product to be within the range of 3.5 to 13; and (5-1) distilling the reaction product after the pH has been adjusted to separate the hydroxyalkanoate ester. [Item A-2] The method according to item A-1, wherein the adjustment of the pH of the reaction product (4) includes adjustment by adding an alkaline substance. [Item A-3] The method according to item A-1 or A-2, wherein the distillation of the reaction product is carried out under a pressure of 300 torr or less. [Item A-4] The manufacturing method according to any one of items A-1 to A-3, wherein the distillation of the reaction product is carried out at a temperature of 50 to 300°C. [Item A-5] The manufacturing method according to any one of items A-1 to A-4, wherein the distillation of the reaction product is carried out under a pressure of 300 torr or less and at a temperature of 50 to 300°C. [Item A-6] The manufacturing method according to any one of items A-1 to A-5, further comprising (1) producing the polyhydroxyalkanoate particles and then replacing the water in the system with alcohol (6). [Item A-7] The manufacturing method according to any one of items A-1 to A-6, further comprising (1) producing the polyhydroxyalkanoate particles and then separating the polyhydroxyalkanoate particles from the cellular components of the microorganism in a water-containing system (7). [Item A-8] A manufacturing method according to any one of items A-1 to A-7, further comprising: (1) producing the polyhydroxyalkanoate particles; (7) separating the polyhydroxyalkanoate particles from the cellular components of the microorganism in a water-containing system; and (6) replacing the water in the system with alcohol.[Item A-9] The manufacturing method according to any one of items A-1 to A-8, wherein the conversion of the polyhydroxyalkanoate to a hydroxyalkanoate ester is carried out in the presence of an acid catalyst. [Item A-10] The manufacturing method according to any one of items A-1 to A-9, wherein the alcohol is methanol or ethanol. [Item A-11] The manufacturing method according to any one of items A-1 to A-10, wherein the polyhydroxyalkanoate particles constituting the polyhydroxyalkanoate particles are not dried (9) before being converted to a hydroxyalkanoate ester. [Item A-12] The method for producing an alcohol dispersion obtained by dispersing the produced polyhydroxyalkanoate particles in alcohol (2) is obtained by a method comprising: agglomerating the produced polyhydroxyalkanoate particles in a water-containing liquid (2-1); concentrating the agglomerated polyhydroxyalkanoate particles in the liquid with a filter (2-2); and adding alcohol to the liquid being concentrated or the concentrated liquid obtained by the concentration (2-3), as described in any one of Items A-1 to A-11. [Item A-13] The method for producing an alcohol dispersion obtained by agglomerating the polyhydroxyalkanoate particles (2-1) is obtained by adding alcohol at a weight of 1.5 times or more relative to the water content in the water-containing liquid. [Item A-14] The method for producing an alcohol dispersion obtained by agglomerating the polyhydroxyalkanoate particles (2-1) is methanol or ethanol, as described in Item A-13. [Item A-15] A manufacturing method according to any one of items A-12 to A-14, wherein the aggregation of the polyhydroxyalkanoate particles (2-1) is performed by making the pH of the water-containing liquid 4 or less. [Item A-16] A manufacturing method according to any one of items A-1 to A-15, further comprising producing the polyhydroxyalkanoate particles (1), and then treating the polyhydroxyalkanoate particles with an enzyme that decomposes the cellular components of the microorganism (8-1).[Item A-17] The manufacturing method according to any one of items A-12 to A-16, wherein the water content in the alcohol dispersion obtained by dispersing the produced polyhydroxyalkanoate particles in alcohol (2) is 10% by weight or less. [Item A-18] The manufacturing method according to any one of items A-1 to A-17, wherein the content of polyhydroxyalkanoate particles in the alcohol dispersion is 10% by weight or more. [Item A-19] The manufacturing method according to any one of items A-12 to A-18, wherein the mesh opening of the filter is 0.2 μm or more and 1.2 μm or less. [Item A-20] The manufacturing method according to any one of items A-12 to A-19, wherein the alcohol added in adding alcohol to the concentrate (2-3) is methanol or ethanol. [Item A-21] The manufacturing method according to any one of items A-1 to A-20, wherein the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles is a poly(3-hydroxybutyrate) polymer. [Item A-22] The manufacturing method according to any one of items A-1 to A-21, wherein the hydroxyalkanoate ester is 3-hydroxybutyrate ester. [Item A-23] A method for producing biomass-derived 1,3-butanediol, comprising obtaining 3-hydroxybutyrate ester from the hydroxyalkanoate ester by the manufacturing method of hydroxyalkanoate ester according to any one of items A-1 to A-22, and reducing the 3-hydroxybutyrate ester to 1,3-butanediol. [Item A-24] A method for producing biomass-derived butadiene, comprising obtaining 1,3-butanediol by the manufacturing method according to item A-23, and obtaining butadiene by a dehydration reaction of the 1,3-butanediol. [Item A-25] A method for producing biomass-derived polybutadiene rubber, comprising obtaining butadiene by the manufacturing method described in Item A-24, and polymerizing the butadiene to obtain polybutadiene rubber.[Item A-26] A method for producing biomass-derived core-shell polymer particles, comprising obtaining butadiene by the manufacturing method described in Item A-24, and using the butadiene to obtain core-shell polymer particles containing polybutadiene rubber in the core layer. [Item A-27] A method for producing biomass-derived propylene, comprising obtaining 3-hydroxybutyrate ester from the hydroxyalkanoate ester by the manufacturing method described in any one of Items A-1 to A-22, and converting the 3-hydroxybutyrate ester to propylene by a decarboxylation reaction and a de-alcoholization reaction. [Item A-28] The manufacturing method described in Item A-21, wherein the polyhydroxyalkanoate comprises a 3-hydroxyhexanoate unit. [Item A-29] The manufacturing method described in any one of Items A-1 to A-21 and A-28, wherein the hydroxyalkanoate ester is 3-hydroxyhexanoate ester. [Item A-30] A method for producing biomass-derived 1,3-hexanediol, comprising obtaining a 3-hydroxyhexanoate ester from the hydroxyalkanoate esters by the method for producing hydroxyalkanoate esters described in any one of items A-1 to A-21 or A-28 to A-29, and reducing the 3-hydroxyhexanoate ester to convert it to 1,3-hexanediol. [Item B-1] A method for producing biomass-derived hydroxyalkanoate esters, comprising: causing a polyhydroxyalkanoate-producing microorganism to produce polyhydroxyalkanoate particles in a water-containing system (1); dispersing the produced polyhydroxyalkanoate particles in alcohol (2); and converting the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles to a hydroxyalkanoate ester in a mixed solvent (3-2), wherein the mixed solvent includes alcohol and a solvent with a higher boiling point than the alcohol contained in the mixed solvent. [Item B-2] The manufacturing method according to Item B-1, wherein the amount of the high-boiling point solvent in the mixed solvent is 3 to 100 parts by weight per 1 part by weight of alcohol contained in the mixed solvent.[Item B-3] The method for producing polyhydroxyalkanoate in the presence of an acid catalyst, wherein the conversion of the polyhydroxyalkanoate to a hydroxyalkanoate ester is carried out in the presence of an acid catalyst, as described in item B-1 or B-2. [Item B-4] The method for producing polyhydroxyalkanoate in the presence of an acid catalyst, wherein the mixed solvent contains a weak salt soluble in the mixed solvent, as described in any one of items B-1 to B-3. [Item B-5] The method for producing polyhydroxyalkanoate in the mixed solvent in the presence of an acid catalyst, wherein the content of the weak salt in the mixed solvent is 0.5 to 10% by weight relative to the polyhydroxyalkanoate particles, as described in any one of items B-1 to B-4. [Item B-6] The method for producing polyhydroxyalkanoate in the presence of an acid catalyst, wherein the conversion reaction of polyhydroxyalkanoate to a hydroxyalkanoate ester is carried out under normal pressure, as described in any one of items B-1 to B-5. [Item B-7] The method for producing polyhydroxyalkanoate in the presence of an acid catalyst, wherein the water in the system is further replaced with an alcohol, as described in any one of items B-1 to B-6, as described in item further comprising the steps of producing polyhydroxyalkanoate particles (1) and then replacing the water in the system with an alcohol, as described in item B-1 to B-6. [Item B-8] A method for producing polyhydroxyalkanoate particles, further comprising (1) producing the polyhydroxyalkanoate particles, and then separating the polyhydroxyalkanoate particles from the cellular components of the microorganism in a water-containing system (7). [Item B-9] A method for producing polyhydroxyalkanoate particles, further comprising (1) producing the polyhydroxyalkanoate particles, and then separating the polyhydroxyalkanoate particles from the cellular components of the microorganism in a water-containing system (7), and then replacing the water in the system with alcohol (6). [Item B-10] A method for producing the generated hydroxyalkanoate ester, further comprising (5-2) purifying the generated hydroxyalkanoate ester by distillation. [Item B-11] A method for producing the polyhydroxyalkanoate particles, further comprising (2) dispersing the generated polyhydroxyalkanoate particles in alcohol, wherein the alcohol is methanol or ethanol. [Item B-12] The manufacturing method according to any one of items B-1 to B-11, wherein the alcohol contained in the mixed solvent is methanol or ethanol.[Item B-13] A method for producing polyhydroxyalkanoate particles according to any one of items B-1 to B-12, wherein the polyhydroxyalkanoate particles constituting the polyhydroxyalkanoate particles are not dried (9) before being converted to a hydroxyalkanoate ester. [Item B-14] A method for producing polyhydroxyalkanoate particles according to any one of items B-1 to B-13, wherein the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles is a poly(3-hydroxybutyrate) polymer. [Item B-15] A method for producing polyhydroxyalkanoate particles according to any one of items B-1 to B-14, wherein the hydroxyalkanoate ester is 3-hydroxybutyrate ester. [Item B-16] A method for producing biomass-derived 1,3-butanediol, comprising obtaining 3-hydroxybutyrate ester from the hydroxyalkanoate ester by the method for producing hydroxyalkanoate esters according to any one of items B-1 to B-15, and reducing the 3-hydroxybutyrate ester to 1,3-butanediol. [Item B-17] A method for producing biomass-derived butadiene, comprising obtaining 1,3-butanediol by the manufacturing method described in Item B-16, and obtaining butadiene by a dehydration reaction of the 1,3-butanediol. [Item B-18] A method for producing biomass-derived polybutadiene rubber, comprising obtaining butadiene by the manufacturing method described in Item B-17, and polymerizing the butadiene to obtain polybutadiene rubber. [Item B-19] A method for producing biomass-derived core-shell polymer particles, comprising obtaining butadiene by the manufacturing method described in Item B-17, and using the butadiene to obtain core-shell polymer particles containing polybutadiene rubber in the core layer. [Item B-20] A method for producing biomass-derived propylene, comprising obtaining 3-hydroxybutyrate ester from the hydroxyalkanoate esters by the manufacturing method described in any one of Items B-1 to B-15, and converting the 3-hydroxybutyrate ester to propylene by a decarboxylation reaction and a de-alcoholization reaction.[Item B-21] The method for producing a product according to Item B-14, wherein the polyhydroxyalkanoate contains a 3-hydroxyhexanoate unit. [Item B-22] The method for producing a product according to any one of Items B-1 to B-14 or B-21, wherein the hydroxyalkanoate ester is a 3-hydroxyhexanoate ester. [Item B-23] A method for producing a biomass-derived 1,3-hexanediol, comprising obtaining a 3-hydroxyhexanoate ester from the hydroxyalkanoate ester by the method for producing a hydroxyalkanoate ester according to any one of Items B-1 to B-14 or B-21 to B-22, and reducing the 3-hydroxyhexanoate ester to convert it to a 1,3-hexanediol.

[0152] The present disclosure will be explained in more detail below with reference to examples, but the present invention is not limited in any way to these examples.

[0153] (Yield of hydroxyalkanoate esters) The yield of hydroxyalkanoate esters was calculated from the GC analysis results according to the following formula: Yield (mol%) = (A / Molecular weight of hydroxyalkanoate ester) × 100 / (B / Average molecular weight of monomer units constituting polyhydroxyalkanoate) A: Weight of the hydroxyalkanoate ester finally obtained (g) B: Weight of polyhydroxyalkanoate particles (g)

[0154] (Comparative Example A1-1) 95% by weight sulfuric acid was added to a cell culture medium (inactivated culture medium) containing inactivated poly(3-hydroxybutyrate) particles [average particle size: 1 μm] to adjust the pH to 7.0 ± 0.2. Furthermore, industrial water (IW) was added to adjust the solid content concentration to 18% by weight. Then, lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme (lytic enzyme) that breaks down sugar chains (peptidoglycans) in the cell wall, was added, and the cells were disrupted by holding the mixture at 50°C for 2 hours.

[0155] Subsequently, 2.5 L of Alcalase (Novozymes), an alkaline proteolytic enzyme, was added, followed by the addition of 30% by weight sodium hydroxide at 50°C, and the mixture was maintained for 2 hours while adjusting the pH to 8.5. To the enzyme-treated culture medium obtained above, 30% by weight sodium hydroxide was added to adjust the pH to 10.5, and the mixture was maintained for 3 hours. Next, sodium dodecyl sulfate (SDS, Kao Corporation) was added to the enzyme-treated solution in an amount of 0.6–1.0% by weight (surfactant treatment).

[0156] Subsequently, the pH was adjusted to 11.0 ± 0.2 using 30% by weight sodium hydroxide, and a sodium hydroxide aqueous solution with a pH of 11.0 was added to dilute the enzyme-treated solution to twice its original weight, and the mixture was kept at 40°C for 1 hour.

[0157] The enzyme-treated solution was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed and the solution was concentrated. To this concentrated aqueous suspension of poly(3-hydroxybutyrate) particles, an aqueous sodium hydroxide solution (pH 11.0) was added and centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. This process was repeated three times.

[0158] Next, the supernatant was removed, methanol was added, and a methanol dispersion of poly(3-hydroxybutyrate) particles was obtained. The mixture was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. To the dispersion, 3% by weight of 95% sulfuric acid relative to the poly(3-hydroxybutyrate) particles was added, followed by the addition of methanol to a total weight of three times the poly(3-hydroxybutyrate) particles. The mixture was then heated to 120°C and held for 5 hours. 30% by weight of sodium hydroxide was added to the resulting reaction solution to adjust the pH to 3.04, and the methanol and water were distilled off under conditions of 60°C / 100 torr. Subsequently, the mixture was distilled off under conditions of 80°C / 0 torr, and the yield of the obtained 3-hydroxybutyrate ester was 81 mol%. A brownish, viscous liquid remained in the kettle.

[0159] (Example A1-1) The procedure was carried out in the same manner as in Comparative Example A1-1, except that 30% by weight of sodium hydroxide was added to the obtained reaction solution to adjust the pH to 4.06. The yield of the obtained hydroxyalkanoate ester, 3-hydroxybutyrate ester, was 94 mol%. A water-soluble solid remained in the vessel.

[0160] (Example A1-2) The procedure was carried out in the same manner as in Comparative Example A1-1, except that 30% by weight of sodium hydroxide was added to the obtained reaction solution to adjust the pH to 5.02. The yield of the obtained hydroxyalkanoate ester, 3-hydroxybutyrate ester, was 94 mol%. A water-soluble solid remained in the vessel.

[0161] (Example A1-3) The procedure was carried out in the same manner as in Comparative Example A1-1, except that 30% by weight of sodium hydroxide was added to the obtained reaction solution to adjust the pH to 7.02. The yield of the obtained hydroxyalkanoate ester, 3-hydroxybutyrate ester, was 96 mol%. A water-soluble solid remained in the vessel.

[0162] (Example A1-4) The procedure was carried out in the same manner as in Comparative Example A1-1, except that 30% by weight of sodium hydroxide was added to the obtained reaction solution to adjust the pH to 9.40. The yield of the obtained hydroxyalkanoate ester, 3-hydroxybutyrate ester, was 93 mol%. A water-soluble solid remained in the vessel.

[0163] (Example A1-5) The procedure was carried out in the same manner as in Comparative Example 1, except that 30% by weight of sodium hydroxide was added to the obtained reaction solution to adjust the pH to 12.05. The yield of the obtained hydroxyalkanoate ester, 3-hydroxybutyrate ester, was 91 mol%. A large amount of water-soluble solid remained in the vessel.

[0164] As described above, in Comparative Example A1-1, where the pH of the obtained reaction solution (reaction product) was adjusted to less than 3.5 and the hydroxyalkanoate ester was separated by distillation, the yield of 3-hydroxybutyrate ester, which is a hydroxyalkanoate ester, was 81 mol%, whereas in Examples A1-1 to A1-5, where the pH of the reaction product was adjusted to a range of 3.5 to 13 and the hydroxyalkanoate ester was separated by distillation, the yield of 3-hydroxybutyrate ester was 91 mol% or more. Therefore, it can be seen that the biomass-derived hydroxyalkanoate ester production method of this disclosure can yield hydroxyalkanoate esters in good yield.

[0165] (Comparative Example A1-1) 95% by weight sulfuric acid was added to a cell culture medium (inactivated culture medium) containing inactivated poly(3-hydroxybutyrate) particles [average particle size: 1 μm] to adjust the pH to 7.0 ± 0.2. Furthermore, industrial water (IW) was added to adjust the solid content concentration to 18% by weight. Then, lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme (lytic enzyme) that breaks down sugar chains (peptidoglycans) in the cell wall, was added, and the cells were disrupted by holding the mixture at 50°C for 2 hours.

[0166] Subsequently, 2.5 L of Alcalase (Novozymes), an alkaline proteolytic enzyme, was added, followed by the addition of 30% by weight sodium hydroxide at 50°C, and the mixture was maintained for 2 hours while adjusting the pH to 8.5. To the enzyme-treated culture medium obtained above, 30% by weight sodium hydroxide was added to adjust the pH to 10.5, and the mixture was maintained for 3 hours. Next, sodium dodecyl sulfate (SDS, Kao Corporation) was added to the enzyme-treated solution in an amount of 0.6–1.0% by weight (surfactant treatment).

[0167] Subsequently, the pH was adjusted to 11.0 ± 0.2 using 30% by weight sodium hydroxide, and the mixture was held at 40°C for 1 hour to obtain a poly(3-hydroxybutyrate) particle dispersion. When the obtained solution was then passed through a ceramic filter with a mesh size of 1.2 μm, the entire volume passed through the filter.

[0168] (Comparative Example A2-2) Instead of adjusting the pH to 11.0 ± 0.2 using 30% by weight sodium hydroxide and holding it at 40°C for 1 hour to obtain a poly(3-hydroxybutyrate) particle dispersion, and passing it through a ceramic filter with a mesh size of 1.2 μm, methanol was added to the dispersion at a ratio of 0.75 times its water content by weight, and the dispersion was passed through a ceramic filter with a mesh size of 1.2 μm, in the same manner as in Comparative Example A2-1. In this case, the entire amount passed through the filter.

[0169] (Comparative Example A2-3) The poly(3-hydroxybutyrate) particle dispersion obtained by adjusting the pH to 11.0 ± 0.2 using 30% by weight sodium hydroxide and holding it at 40°C for 1 hour was passed through a ceramic filter with a mesh size of 0.2 μm instead of a ceramic filter with a mesh size of 1.2 μm, but the procedure was the same as in Comparative Example A2-1. When the poly(3-hydroxybutyrate) particle dispersions of Comparative Examples A2-1 to A2-3 were observed under an optical microscope, no aggregation of PHA particles (the formation of any certain clumps) was observed at all.

[0170] (Example A2-1) Instead of adjusting the pH to 11.0 ± 0.2 using 30% by weight sodium hydroxide and holding it at 40°C for 1 hour, and passing the resulting poly(3-hydroxybutyrate) particle dispersion through a ceramic filter with a mesh size of 1.2 μm, methanol was added to the dispersion at a ratio of 2.26 times its water content by weight and passed through a ceramic filter with a mesh size of 1.2 μm. The process was carried out in the same manner as in Comparative Example A2-1, except that the water and methanol passed through the filter, while the poly(3-hydroxybutyrate) particles that agglomerated due to the addition of methanol did not pass through the filter, allowing the liquid to be concentrated. A poly(3-hydroxybutyrate) particle methanol dispersion with a solid content of 20% by weight and a water content of 3% by weight or less was obtained. When the obtained poly(3-hydroxybutyrate) particle methanol dispersion was observed under an optical microscope, a state in which certain clumps were formed (aggregation of PHA particles) was confirmed. Furthermore, image analysis of the optical microscope images showed that the weight ratio of unaggregated PHA particles to the total PHA particles was 50% by weight or less.

[0171] (Examples A2-2, A2-3, and A2-4) The procedure was carried out in the same manner as in Example A2-1, except that the 1.2 μm mesh ceramic filter used in Example A2-1 was replaced with ceramic filters with mesh sizes of 0.8 μm (Example A2-2), 0.4 μm (Example A2-3), and 0.2 μm (Example A2-4), respectively. In all cases, as in Example A2-1, water and methanol passed through the filter, but the poly(3-hydroxybutyrate) particles that agglomerated upon methanol addition did not pass through the filter, and the liquid could be concentrated to obtain a poly(3-hydroxybutyrate) particle methanol dispersion with a solid content of 20% by weight and a water content of 3% by weight or less. When the obtained poly(3-hydroxybutyrate) particle methanol dispersions were observed under an optical microscope, a state in which certain clumps were formed (aggregation of PHA particles) was confirmed. Furthermore, when the optical microscope images were analyzed, the weight ratio of unaglosed PHA particles to the total PHA particles was 50% by weight or less in all cases.

[0172] (Comparative Example B-1) 95% by weight sulfuric acid was added to a cell culture medium (inactivated culture medium) containing inactivated poly(3-hydroxybutyrate) particles [average particle size: 1 μm] to adjust the pH to 7.0 ± 0.2. Furthermore, industrial water (IW) was added to adjust the solid content concentration to 18% by weight. Then, lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme (lytic enzyme) that breaks down sugar chains (peptidoglycans) in the cell wall, was added, and the cells were disrupted by holding the mixture at 50°C for 2 hours.

[0173] Subsequently, 2.5 L of Alcalase (Novozymes), an alkaline proteolytic enzyme, was added, followed by the addition of 30% by weight sodium hydroxide at 50°C, and the mixture was maintained for 2 hours while adjusting the pH to 8.5. To the enzyme-treated culture medium obtained above, 30% by weight sodium hydroxide was added to adjust the pH to 10.5, and the mixture was maintained for 3 hours. Next, sodium dodecyl sulfate (SDS, Kao Corporation) was added to the enzyme-treated solution in an amount of 0.6–1.0% by weight (surfactant treatment).

[0174] Subsequently, the pH was adjusted to 11.0 ± 0.2 using 30% by weight sodium hydroxide, and a sodium hydroxide aqueous solution with a pH of 11.0 was added to dilute the enzyme-treated solution to twice its original weight, and the mixture was kept at 40°C for 1 hour.

[0175] The enzyme-treated solution was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed and the solution was concentrated. To this concentrated aqueous suspension of poly(3-hydroxybutyrate) particles, an aqueous sodium hydroxide solution (pH 11.0) was added and centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. This process was repeated three times.

[0176] Next, the supernatant was removed, methanol was added, and a methanol dispersion of poly(3-hydroxybutyrate) particles was obtained. The mixture was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed. To the dispersion, 1% by weight of 95% sulfuric acid relative to the poly(3-hydroxybutyrate) particles was added, followed by the addition of methanol in an amount three times the weight of the poly(3-hydroxybutyrate) particles. The mixture was heated under reflux, and the temperature of the system reached 65°C. After holding for 5 hours, the yield of the obtained hydroxyalkanoate ester, methyl 3-hydroxybutyrate, was 12 mol%.

[0177] (Example B-1) The same procedure as in Comparative Example B-1 was carried out, except that methanol was added to poly(3-hydroxybutyrate) particles in an amount three times their weight, and methyl 3-hydroxybutyrate was added to the amount of methanol in an amount 5.2 times its weight. The temperature of the system reached 100°C, and the yield of methyl 3-hydroxybutyrate, excluding the methyl 3-hydroxybutyrate added before the reaction, was 82 mol%.

[0178] (Example B-2) The same procedure as in Comparative Example B-1 was carried out, except that methanol was added to poly(3-hydroxybutyrate) particles in an amount three times their weight, and methyl 3-hydroxybutyrate was added to the amount of methanol in an amount 6.7 times its weight. The temperature of the system reached 110°C, and the yield of methyl 3-hydroxybutyrate, excluding the methyl 3-hydroxybutyrate added before the reaction, was 85 mol%.

[0179] (Example B-3) The same procedure as in Comparative Example B-1 was carried out, except that methanol was added to poly(3-hydroxybutyrate) particles in an amount three times their weight, and methyl 3-hydroxybutyrate was added to the amount of methanol in an amount 10.1 times its weight. The temperature of the system reached 120°C, and the yield of methyl 3-hydroxybutyrate, excluding the methyl 3-hydroxybutyrate added before the reaction, was 89 mol%.

[0180] (Example B-4) The procedure was carried out in the same manner as in Example B-3, except that ammonium acetate was further added at a concentration of 3% by weight relative to the poly(3-hydroxybutyrate) particles during the reaction. The temperature of the system reached 120°C, and the yield of methyl 3-hydroxybutyrate, excluding the methyl 3-hydroxybutyrate added before the reaction, was 92 mol%.

[0181] (Example B-5) The procedure was carried out in the same manner as in Example B-3, except that sodium acetate was added in an additional amount of 3% by weight relative to the poly(3-hydroxybutyrate) particles during the reaction. The temperature of the system reached 120°C, and the yield of methyl 3-hydroxybutyrate, excluding the methyl 3-hydroxybutyrate added before the reaction, was 92 mol%.

[0182] (Example B-6) The same procedure as in Example B-3 was carried out, except that potassium acetate was further added during the reaction in an amount of 3% by weight relative to the poly(3-hydroxybutyrate) particles. The temperature of the system reached 120°C, and the yield of methyl 3-hydroxybutyrate, excluding the methyl 3-hydroxybutyrate added before the reaction, was 92 mol%.

[0183] As described above, in the conversion to hydroxyalkanoate esters (3-2), Comparative Example B-1, which used a solvent that did not contain a solvent with a higher boiling point than methanol, yielded 3-hydroxybutyrate ester, a hydroxyalkanoate ester, at 12 mol%, whereas Examples B-1 to B-6, which used a mixed solvent containing methanol and methyl 3-hydroxybutyrate, which has a higher boiling point than methanol, all yielded a high yield of 82 mol% or more of 3-hydroxybutyrate ester. Therefore, it can be seen that the biomass-derived hydroxyalkanoate ester production method of this disclosure can convert PHA to hydroxyalkanoate esters with good yield. Among these, Examples B-4, B-5, and B-6, which added ammonium acetate as a weak salt, all yielded 3-hydroxybutyrate ester at 92 mol% or more, indicating particularly excellent yields.

Claims

1. A method for producing biomass-derived hydroxyalkanoate esters, comprising: (1) causing polyhydroxyalkanoate-producing microorganisms to produce polyhydroxyalkanoate particles in a water-containing system; (2) dispersing the produced polyhydroxyalkanoate particles in alcohol; (3) converting the polyhydroxyalkanoate constituting the polyhydroxyalkanoate particles in the alcohol dispersion to a hydroxyalkanoate ester to obtain a reaction product containing the hydroxyalkanoate ester; (4) adjusting the pH of the reaction product to be within the range of 3.5 to 13; and (5-1) distilling the reaction product after pH adjustment to separate the hydroxyalkanoate ester.

2. The manufacturing method according to claim 1, wherein the adjustment of the pH of the reaction product (4) includes adjustment by adding an alkaline substance.

3. The manufacturing method according to claim 1, further comprising (1) producing the polyhydroxyalkanoate particles, followed by (6) replacing the water in the system with alcohol.

4. The method for producing the polyhydroxyalkanoate particles (1), further comprising separating the polyhydroxyalkanoate particles from the cellular components of the microorganism in a water-containing system (7).

5. The method for producing the polyhydroxyalkanoate particles according to claim 1, further comprising: (1) producing the polyhydroxyalkanoate particles; (7) separating the polyhydroxyalkanoate particles from the cellular components of the microorganism in a water-containing system; and (6) replacing the water in the system with alcohol.

6. The manufacturing method according to claim 1, wherein the polyhydroxyalkanoate particles constituting the polyhydroxyalkanoate particles are not dried (9) before being converted to hydroxyalkanoate esters.

7. The manufacturing method according to claim 1, wherein the alcohol dispersion obtained by dispersing the produced polyhydroxyalkanoate particles in alcohol (2) is obtained by a method comprising: agglomerating the produced polyhydroxyalkanoate particles in a water-containing liquid (2-1); concentrating the agglomerated polyhydroxyalkanoate particles in the liquid with a filter (2-2); and adding alcohol to the liquid being concentrated or the concentrated liquid obtained by the concentration (2-3).

8. The manufacturing method according to claim 7, wherein the aggregation of the polyhydroxyalkanoate particles (2-1) is carried out by adding an alcohol of 1.5 times or more by weight relative to the water content in the liquid containing water.

9. The manufacturing method according to claim 7, wherein the aggregation of the polyhydroxyalkanoate particles (2-1) is carried out by lowering the pH of the water-containing liquid to 4 or less.

10. The manufacturing method according to claim 1, further comprising (1) producing the polyhydroxyalkanoate particles, and (8-1) treating the polyhydroxyalkanoate particles with an enzyme that decomposes the cellular components of the microorganism.

11. The manufacturing method according to claim 7, wherein the water content in the alcohol dispersion obtained by dispersing the produced polyhydroxyalkanoate particles in alcohol (2) is 10% by weight or less.

12. The manufacturing method according to claim 11, wherein the content of polyhydroxyalkanoate particles in the alcohol dispersion is 10% by weight or more.

13. The manufacturing method according to claim 7, wherein the mesh opening of the filter is 0.2 μm or more and 1.2 μm or less.

14. A method for producing biomass-derived 1,3-butanediol, comprising obtaining 3-hydroxybutyrate ester from the hydroxyalkanoate esters by the method for producing hydroxyalkanoate esters described in any one of claims 1 to 13, and reducing the 3-hydroxybutyrate ester to convert it to 1,3-butanediol.

15. A method for producing biomass-derived butadiene, comprising obtaining 1,3-butanediol by the manufacturing method described in claim 14, and obtaining butadiene by a dehydration reaction of the 1,3-butanediol.

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