Process for producing polyhydroxybutyrate-based resin

Abstract

The present application relates to a method for producing a polyhydroxybutyric acid resin, including: a step (a) of disrupting or solubilizing microbial cells containing a polyhydroxybutyric acid resin; and a step (b) of separating the polyhydroxybutyric acid in the composition obtained in the step (a), in which steps (a) and (b), water having a concentration of calcium ions of 4.5 mg / L or less is used. The water used in steps (a) and (b) is preferably produced by, after subjecting drainage discharged from a production step of a polyhydroxybutyric acid resin to anaerobic treatment and aerobic treatment using microorganisms, performing a pretreatment filtration step based on a membrane separation activated sludge method, and a filtration step for removing a membrane using calcium ions. Thus, a method for producing a polyhydroxybutyric acid resin having a good color tone and high thermal stability is provided.

Description

Technical Field

[0001] This invention relates to a method for manufacturing polyhydroxybutyrate resins. Background Technology

[0002] Biodegradable plastics are completely biodegraded by microorganisms in soil and water and are incorporated into the natural carbon cycle. Therefore, they are expected to be actively used as environmentally friendly plastic materials that have virtually no adverse impact on ecosystems. Representative biodegradable plastics include plant-derived biodegradable plastics such as polyhydroxybutyrate (PHB). PHB is an aliphatic polyester (thermoplastic polyester) that is produced by microorganisms from natural organic acids and oils derived from plants, which serve as carbon sources and accumulate as energy within cells.

[0003] Polyhydroxybutyrate (PHB) produced by microorganisms is water-insoluble and typically accumulates within microbial cells in the form of particulate matter. Therefore, in order to use PHB as a plastic, a process is required to separate and remove PHB from the microbial cells. For example, Patent Document 1 reports a method combining alkali addition and high-pressure crushing as a method for the separation and purification of PHB. For example, Patent Document 2 reports a method combining physical cell disruption and chemical treatment using enzymes and surfactants as a method for the separation and purification of PHB.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 07-31489

[0007] Patent Document 2: Japanese Patent Application Publication No. 2008-193940 Summary of the Invention

[0008] The problem that the invention aims to solve

[0009] However, polyhydroxybutyrate separated by the methods described in Patent Documents 1 and 2 has problems such as coloring and low thermal stability, resulting in a decrease in molecular weight when heated at high temperatures.

[0010] In order to solve the above-mentioned problems, the present invention provides a method for manufacturing polyhydroxybutyrate resin that can produce polyhydroxybutyrate resin with good color and high thermal stability.

[0011] Problem Solving Methods

[0012] In one or more embodiments, the present invention relates to a method for manufacturing a polyhydroxybutyrate resin, comprising: a step (a) of breaking or soluble microbial cells containing the polyhydroxybutyrate resin; and a step (b) of separating the polyhydroxybutyrate resin from the composition obtained in step (a), wherein water with a calcium ion concentration of less than 4.5 mg / L is used in steps (a) and (b).

[0013] The effects of the invention

[0014] According to the manufacturing method of the present invention, a polyhydroxybutyrate resin with good color tone and high thermal stability can be provided. Detailed Implementation

[0015] The inventors of this invention conducted repeated research to solve the above-mentioned problems and found that by using water with a calcium ion concentration within a specific range in step (a) of breaking down or dissolving microbial cells containing polyhydroxybutyrate resin (hereinafter also simply referred to as "PHB") and step (b) of separating the polyhydroxybutyrate resin from the composition obtained in step (a), the resulting polyhydroxybutyrate resin has a better color tone, and even when heated at high temperatures, such as 160°C, the decrease in molecular weight can be suppressed. Furthermore, it is preferable to set the sodium ion concentration in the water used in steps (a) and (b) within a specific range.

[0016] Low-calcium ionized water

[0017] In one or more embodiments of the present invention, the concentration of calcium ions in the water used in steps (a) and (b) is 4.5 mg / L or less, preferably 3.0 mg / L or less, and more preferably 2.0 mg / L or less, from the viewpoint of improving color and thermal stability. In one or more embodiments of the present invention, ideally, the water used in steps (a) and (b) is free of calcium ions, but from a practical point of view, it can be 0.001 mg / L or more, or 0.005 mg / L or more.

[0018] In one or more embodiments of the present invention, from the viewpoint of improving color tone and thermal stability, the concentration of sodium ions in the water used in steps (a) and (b) is preferably 450 mg / L or less, more preferably 250 mg / L or less, and even more preferably 220 mg / L or less. In one or more embodiments of the present invention, it is ideal that the water used in steps (a) and (b) is free of sodium ions, but from a practical point of view, it can be 0.05 mg / L or more, or 0.1 mg / L or more.

[0019] Regarding the water used in steps (a) and (b), there are no particular limitations as long as the calcium ion concentration in the water is below 4.5 mg / L. From the viewpoint of reducing water consumption and mitigating environmental impact, it is preferable to use treated water obtained as described below: after treating the wastewater discharged from the polyhydroxybutyrate resin manufacturing process with anaerobic fermentation and aerobic fermentation using microorganisms, a pretreatment filtration process based on membrane separation activated sludge method and a filtration process using calcium ion removal membrane are performed, thereby obtaining treated water. The wastewater discharged from the polyhydroxybutyrate resin manufacturing process includes the wastewater discharged through any of the polyhydroxybutyrate resin manufacturing processes such as steps (a) and (b).

[0020] The use of microbial anaerobic and aerobic treatment, as well as pretreatment filtration based on membrane separation activated sludge processes, is not particularly limited and can be carried out using general methods employed in water treatment. Anaerobic treatment devices can, for example, consist of an acid-generating tank and a methane-generating reactor. The acid-generating tank uses acid-producing bacteria to decompose high-molecular-weight carbohydrates and lipids into organic acids and lower alcohols, while the methane-generating reactor uses granular methane-generating bacteria to decompose organic acids and lower alcohols into methane and carbon dioxide gases using an EGSB (Enhanced Electrolytic Cell) process. Aerobic treatment can, for example, be carried out using a device consisting of a denitrification tank (activated sludge treatment tank) and an aeration tank (activated sludge treatment tank), where undecomposed organic matter from anaerobic treatment is decomposed in the aeration tank by aerobic bacteria. An aerobic treatment device can consist of a denitrification tank (activated sludge treatment tank), an aeration tank (activated sludge treatment tank), a second denitrification tank (activated sludge treatment tank), and a re-aeration tank (activated sludge treatment tank). The pretreatment filtration process based on membrane separation activated sludge can be carried out, for example, by using an MBR (Membrane Bioreactor) with UF or MF membranes installed in an aeration tank (activated sludge treatment tank) or a re-aeration tank (activated sludge treatment tank).

[0021] In one or more embodiments of the present invention, as a calcium ion removal membrane, one or more selected from NF membranes and RO membranes are preferred because of their high calcium ion removal performance.

[0022] In one or more embodiments of the present invention, the MgSO4 rejection rate of the NF membrane or RO membrane when subjected to a pressure of 3000 kPa at 20°C is preferably 60% or more and 100% or less, more preferably 70% or more and 100% or less, and even more preferably 90% or more and 100% or less. When the MgSO4 rejection rate is 60% or more, the calcium ion permeability of the membrane does not increase. By using the treated water obtained in steps (a) and (b), the color tone of the polyhydroxybutyrate resin is easily improved, and the decrease in molecular weight at high temperatures is easily suppressed.

[0023] In one or more embodiments of the present invention, the inter-membrane differential pressure during filtration using an NF membrane or an RO membrane is not particularly limited. From the viewpoint of calcium ion removal rate and sodium ion removal rate, it is preferably 0.4 MPa or more and 4.14 MPa or less, more preferably 0.5 MPa or more and 2.5 MPa or less. When the inter-membrane differential pressure is 0.4 MPa or more, the permeate flow rate and ion removal rate will not decrease, and when it is 4.14 MPa or less, the membrane is less likely to break.

[0024] In one or more embodiments of the present invention, the permeation rate when using an NF membrane or an RO membrane for filtration is not particularly specified, but is preferably 0.01 L / min or more and 2000 L / min or less, more preferably 0.5 L / min or more and 1500 L / min or less. A permeation rate of 0.01 L / min or more results in good productivity.

[0025] In one or more embodiments of the present invention, the water temperature during filtration using an NF membrane or an RO membrane is not particularly specified, but the water temperature is preferably below 50°C, more preferably below 45°C. When the water temperature is below 50°C, the membrane is less prone to degradation.

[0026] [Microbial cells containing PHB]

[0027] In one or more embodiments of the present invention, microbial cells containing polyhydroxybutyrate resin can be obtained by culturing microorganisms capable of producing PHB.

[0028] In one or more embodiments of the present invention, polyhydroxybutyrate resins are a general term for polymers using 3-hydroxybutyric acid (hereinafter also referred to as 3HB) as a monomer unit. PHB can be poly(3-hydroxybutyric acid) as a homopolymer of 3-hydroxybutyric acid, or it can be a copolymer of 3-hydroxybutyric acid and other 3-hydroxyalkanoic acids. As other 3-hydroxyalkanoic acids, for example, one or more monomers selected from 3-hydroxyhexanoic acid (hereinafter also referred to as 3HH), 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytriadecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxypentadecanoic acid, and 3-hydroxyhexadecanoic acid can be used.

[0029] From the perspective of ease of industrial production, poly(3-hydroxybutyric acid), poly(3-hydroxybutyric acid-co-3-hydroxyhexanoic acid), and poly(3-hydroxybutyric acid-co-3-hydroxyoctanoic acid) are preferred as PHB, with poly(3-hydroxybutyric acid-co-3-hydroxyhexanoic acid) being particularly preferred. There is no particular limitation on the composition ratio of each monomer unit in the copolymer PHBH constituting the above-mentioned 3HB and 3HH components. When the total of all monomer units is set to 100 mol%, the 3HH unit can be 1 mol% or more and 50 mol% or less, or 1 mol% or more and 25 mol% or less, or 1 mol% or more and 15 mol% or less.

[0030] In one or more embodiments of the present invention, the microorganisms capable of producing PHB are not particularly limited, and microorganisms isolated from nature, microorganisms preserved in strain preservation institutions (e.g., IFO, ATCC, etc.), or mutants or transformants that can be prepared from these microorganisms can be used. Examples include bacteria of the genera *Cupriavidus*, *Alcaligenes*, *Ralstonia*, *Pseudomonas*, *Bacillus*, *Azotobacter*, *Nocardia*, and *Aeromonas*. *Alcaligenes lipolytica* (*A. lipolytica*), *A. latus*, *A. caviae*, *A. hydrophila*, and *C. necator* are particularly preferred strains. Alternatively, in cases where the microorganism does not inherently possess the ability to produce PHB, or where the production volume is low, a transformant obtained by introducing the target PHB synthase gene and / or its mutant into the microorganism can be used. The PHB synthase gene used for producing such a transformant is not particularly limited, but a PHB synthase gene from Aeromonas vaginalis is preferred.

[0031] By culturing the aforementioned microorganisms under appropriate conditions, microorganisms that accumulate PHB within their cells can be obtained. There are no particular limitations on the culturing method; for example, methods described in Japanese Patent Application Publication No. 05-93049 and International Publication No. 2008 / 010296 can be used. The PHB-containing microbial cells can be used directly from the culture medium after culturing, or sterilized culture medium can be used after the cells have been inactivated by heating. Sterilization can be performed, for example, by heat treatment at a temperature of 50°C or higher and 80°C for 5 minutes to 120 minutes.

[0032] [Process(a)]

[0033] In step (a), the microbial cells containing PHB are broken down or dissolved. Step (a) can be carried out, for example, by at least one treatment selected from chemical treatment and physical crushing treatment.

[0034] Chemical treatment can be carried out using at least one compound selected from alkaline compounds, proteolytic enzymes, and cell wall degrading enzymes.

[0035] There are no particular limitations on alkaline compounds, as long as they can break down the cell walls of PHB-containing microbial cells, causing PHB to leak out of the cells. Examples include: hydroxides of alkali metals such as sodium hydroxide, potassium hydroxide, and lithium hydroxide; carbonates of alkali metals such as sodium carbonate and potassium carbonate; bicarbonates of alkali metals such as sodium bicarbonate and potassium bicarbonate; alkali metal salts of organic acids such as sodium acetate and potassium acetate; borates of alkali metals such as borax; phosphates of alkali metals such as trisodium phosphate, disodium hydrogen phosphate, tripotassium phosphate, and dipotassium hydrogen phosphate; hydroxides of alkaline earth metals such as barium hydroxide; and ammonia. From the perspective of suitability for industrial production and cost reduction, it is preferable to select one or more of sodium hydroxide, sodium carbonate, potassium hydroxide, and lithium hydroxide.

[0036] There are no particular limitations on what constitutes a protein-degrading enzyme; examples include alkaline protease, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, and carboxypeptidase. Specific protein-degrading enzymes such as "Protease A," "Protease P," "Protease N" (manufactured by Amano Enzyme Inc.), "Alcalase," "Esperase," "Savinase," and "Everlase" (manufactured by Novozymes Japan Ltd.) can be used industrially and are suitable for use from the perspective of degradative activity.

[0037] There are no particular limitations on cell wall degrading enzymes; examples include lysozyme, amylase, cellulase, maltase, sucrase, α-glucosidase, and β-glucosidase. Among these cell wall degrading enzymes, lysozyme is preferred from the perspective of lysis efficiency. Specific cell wall degrading enzymes such as "Lysozyme" (manufactured by Shandong Huayuan Economic and Trade Co., Ltd.), "Biozyme A", "Cellulase A "Amano" 3", "Cellulase T "Amano" 4", "α-Glucosidase "Amano" (manufactured by Amano Enzyme Inc.), "Termamyl", and "Cellusoft" (manufactured by Novozymes Japan Ltd.) can be used industrially.

[0038] From the perspective of achieving high separation and purification efficiency, it is desirable that the above-described enzyme treatment be carried out in the presence of a surfactant. Furthermore, as the enzyme, for example, an enzyme composition containing an enzyme and one or more additives selected from enzyme stabilizers, surfactants, and anti-redeposition agents can be used.

[0039] Examples of surfactants include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. From the viewpoint of highly effective removal of residues resulting from cell membrane damage, anionic surfactants and / or nonionic surfactants are preferred. For the purpose of removing proteins, anionic surfactants are preferred; however, when the purpose is to remove fatty acids or oils, nonionic surfactants are preferred. Alternatively, both anionic and nonionic surfactants can be used. When using both, the weight ratio of anionic surfactant to nonionic surfactant is preferably 1 / 100 to 100 / 10, more preferably 5 / 100 to 100 / 20, further preferably 5 / 100 to 100 / 100, and particularly preferably 5 / 100 to 50 / 100.

[0040] Examples of anionic surfactants include: alkyl sulfates, alkylbenzene sulfonates, alkyl sulfate esters, alkenyl sulfate esters, alkyl ether sulfate esters, alkenyl ether sulfate esters, α-olefin sulfonates, α-sulfonyl fatty acid salts, esters of α-sulfonyl fatty acid salts, alkyl ether carboxylates, alkenyl ether carboxylates, amino acid-type surfactants, and N-acyl amino acid-type surfactants. Preferably, alkyl sulfates with 12 to 14 carbon atoms, straight-chain alkylbenzene sulfonates with 12 to 16 carbon atoms, alkyl sulfate esters with 10 to 18 carbon atoms, or alkyl ether sulfate esters with 10 to 18 carbon atoms are preferred. As counterions, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and alkanolamines such as monoethanolamine, diethanolamine, and triethanolamine are preferred.

[0041] Examples of nonionic surfactants include: polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene alkyl ethers, fatty acid sorbitan esters, alkyl polyglucosides, fatty acid diethanolamides, and alkyl monoglycidyl ethers. Considering high hydrophilicity and good biodegradability, polyoxyethylene alkyl ethers and polyoxyethylene alkyl ethers are preferred.

[0042] Examples of cationic surfactants include alkyltrimethylammonium salts and dialkyldimethylammonium salts.

[0043] Examples of amphoteric surfactants include carboxybetaine type and sulfobetaine type.

[0044] Among the surfactants mentioned above, considering cost, dosage, and additive effect, sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium cholate, sodium deoxycholate, and sodium oleate are preferred as anionic surfactants, while polyoxyethylene alkyl ethers and polyoxyethylene alkyl ethers are preferred as nonionic surfactants.

[0045] There is no particular limitation on the amount of surfactant added, but it is preferably 0.001 parts by weight or more and 10 parts by weight or less relative to 100 parts by weight of bacterial culture medium, and furthermore, from a cost perspective, it is preferably 5 parts by weight or less. A single surfactant may be used, or two or more surfactants may be used in combination.

[0046] Enzyme treatment is preferably carried out, for example, by adding an alkaline compound and / or a surfactant to the bacterial culture medium while stirring. Regarding enzyme treatment conditions, it is preferable to control the optimal values ​​for the enzyme used. The necessary amount of enzyme depends on the type and activity of the enzyme. There are no particular limitations, but 0.001 to 10 parts by weight relative to 100 parts by weight of PHB100 is preferred, and more preferably 0.001 to 5 parts by weight from a cost perspective.

[0047] From the perspective of improving crushing efficiency and facilitating PHB recovery, physical crushing is preferably performed after the addition of an alkaline compound, or an alkaline compound and a surfactant. The aforementioned alkaline compounds and surfactants can be appropriately used as alkaline compounds and surfactants. Among the aforementioned alkaline compounds, sodium hydroxide, sodium carbonate, potassium hydroxide, and lithium hydroxide are preferred from the perspective of suitability for industrial production and cost reduction. Among the aforementioned surfactants, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium cholate, sodium deoxycholate, and sodium oleate are preferred as anionic surfactants, and polyoxyethylene alkyl ethers and polyoxyethylene alkyl ethers are preferred as nonionic surfactants, considering cost, dosage, and additive effect.

[0048] Preferably, the pH of the bacterial culture medium is adjusted to a level above 8.0 and below 12.5 by adding an alkaline compound. This does not affect the pH and easily dissolves bacterial cell (microbial cell) residues, organic matter generated by the bacteria, and organic matter composed of the bacteria. After adding the alkaline compound to the bacterial culture medium, treatment can be carried out at a temperature above 20°C and below 80°C, preferably above 20°C and below 50°C, for a period of 30 minutes to 2 hours.

[0049] There is no particular limitation on the amount of surfactant added, but it is preferably 0.001 parts by weight or more and 10 parts by weight or less relative to 100 parts by weight of bacterial culture medium, and more preferably 5 parts by weight or less from a cost perspective. A single surfactant can be used, or two or more surfactants can be used in combination.

[0050] The apparatus used for physical crushing is not particularly limited, and examples include high-pressure homogenizers, ultrasonic crushers, emulsifying dispersers, and bead mills. Among these, a high-pressure homogenizer is preferred in terms of crushing efficiency, and more preferably a type that involves introducing the suspension into a pressure-resistant container with a small opening and applying high pressure, thereby extruding it from the opening. Examples of this type of crusher include the "PA2K type" high-pressure homogenizer manufactured by Niro Soavi SPA. When using a high-pressure homogenizer, the large shear force acts on the microbial cells, thus efficiently destroying them and improving the separability of PHB. Such a machine applies high pressure at the opening, instantly creating high temperatures; therefore, it is preferable to cool the bacterial culture medium as needed using a general low-temperature constant-temperature circulating tank to prevent temperature rise, performing crushing treatment at a temperature above 20°C and below 40°C. By treating at a temperature above 20°C and below 40°C, processing can be performed without significantly reducing the molecular weight of PHB. There is no particular limitation on the crushing pressure during high-pressure crushing, but from the perspective of crushing efficiency and cost, it is preferable to be above 30MPa and below 60MPa.

[0051] In step (a), chemical treatment and physical crushing treatment can be used in combination. In this case, from the viewpoint of improving the crushing effect, it is preferable to perform physical crushing treatment after chemical treatment. From the viewpoint of cost, step (a) can be performed by physical crushing treatment alone.

[0052] In step (a), water with a calcium ion concentration of 4.5 mg / L or less (preferably 3.0 mg / L or less, more preferably 2.0 mg / L or less) is used as the water. For example, water with a calcium ion concentration of 4.5 mg / L or less (preferably 3.0 mg / L or less, more preferably 2.0 mg / L or less) is used when adding surfactants, alkaline compounds, etc. The sodium ion concentration of this water is preferably 450 mg / L or less, more preferably 250 mg / L or less, and even more preferably 220 mg / L or less.

[0053] [Process (b)]

[0054] In step (b), the polyhydroxybutyrate resin in the composition obtained in step (a), such as the bacterial cell lysate, is separated. The separation method is not particularly limited; solid-liquid separation can be performed using methods such as filtration, sedimentation, or centrifugation, and the polyhydroxybutyrate resin is recovered as a water-insoluble component. From the viewpoint of being able to process on a large scale industrially and for continuous use, centrifugation is preferred.

[0055] There are no particular limitations on the centrifugal separator, but centrifugal sedimentation machines with non-porous rotating containers are preferred. Types include plate type, cylindrical type, and decanter type. Since PHB particles have a small specific gravity difference with water, plate type separators (intermittent discharge type, nozzle discharge type) are preferred as they offer a large separation and settling area and can achieve high acceleration. Nozzle discharge type separators are particularly preferred when the PHB concentration in the crushing solution is high. For decanter type separators, models with separation plates that increase the separation and settling area are preferred.

[0056] In step (b), prior to separation, 500 to 1000 parts by weight of an aqueous medium may be added to 100 parts by weight of the composition obtained in step (a), for example, the bacterial cell lysate.

[0057] In step (b), the aqueous medium may be water or a mixture of water and a water-miscible organic solvent. The water content in the aqueous medium is preferably 50% by weight or more, more preferably 70% by weight or more, further preferably 80% by weight or more, and particularly preferably 85% by weight or more. In step (b), water with a calcium ion concentration of 4.5 mg / L or less, preferably 3.0 mg / L or less, and more preferably 2.0 mg / L or less is used as the water. The sodium ion concentration in this water is preferably 450 mg / L or less, more preferably 250 mg / L or less, and further preferably 220 mg / L or less.

[0058] As a water-miscible organic solvent, there are no particular limitations, but examples include: alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, pentanol, hexanol, and heptanol; ketones such as acetone and methyl ethyl ketone; and tetrahydrofuran, dimethyl ethyl ketone, etc. Ethers such as alkanes; nitriles such as acetonitrile and propionitrile; amides such as dimethylformamide and acetamide; dimethyl sulfoxide, pyridine, piperidine, etc. Among these, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, acetone, methyl ethyl ketone, tetrahydrofuran, etc., are preferred for easy removal. Alkane, acetonitrile, propionitrile, etc. Furthermore, considering ease of availability, methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone, etc., are preferred. Moreover, methanol, ethanol, and acetone are particularly preferred.

[0059] In step (b), the separated PHB (containing the water-insoluble component of PHB) can be purified by washing it at least once with the aforementioned aqueous medium. For example, the washing with the aqueous medium can be performed by adding 500 to 1000 parts by weight of the aqueous medium to 100 parts by weight of the water-insoluble component containing the polyhydroxybutyrate resin. Furthermore, to efficiently remove impurities from microbial cells and improve the purification effect, alkaline compounds, surfactants, and proteolytic enzymes can be added to the aqueous medium.

[0060] [Process (c)]

[0061] The manufacturing method of one or more embodiments of the present invention may include a step (c) of drying the PHB separated in step (b). The PHB (dehydrated resin) that has been washed and dehydrated by an aqueous medium can be directly dried to obtain powdered PHB. The drying method can be appropriately selected and is not particularly limited; for example, general drying methods such as spray drying, airflow drying, flow drying, and belt drying are preferred.

[0062] Alternatively, a dispersant can be added to the concentrated PHB dispersion after cleaning with an aqueous medium to adjust the pH to below 7, followed by drying to obtain powdered PHB. Examples of dispersants include water-soluble polymers such as polyvinyl alcohol (PVA), methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, polyacrylic acid, sodium polyacrylate, potassium polyacrylate, polymethacrylic acid, and sodium polymethacrylate; and nonionic surfactants such as polyethylene glycol-polypropylene glycol-block ether type (polyoxyethylene-polyoxypropylene-block polymer type). A method for adjusting the pH to below 7 can be achieved by adding an acid. The acid is not particularly limited and can be any organic or inorganic acid, such as sulfuric acid, hydrochloric acid, phosphoric acid, or acetic acid.

[0063] Regarding the molecular weight of PHB, there is no limitation on its molecular weight, as long as it exhibits substantially sufficient physical properties for the intended use. For example, from the viewpoint of processability and the strength of the molded body, the weight-average molecular weight of PHB is preferably 50,000 or more and 3,000,000 or less, more preferably 60,000 or more and 1,500,000 or less. It should be noted that the weight-average molecular weight here refers to the molecular weight determined by gel permeation chromatography (GPC) using chloroform eluent and based on the molecular weight distribution converted from polystyrene. As the chromatographic column in this GPC, a chromatographic column suitable for determining the above-mentioned molecular weight can be used.

[0064] PHB has high thermal stability, and the weight-average molecular weight retention rate after heat treatment at 160°C for 20 minutes is preferably 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, and particularly preferably 90% or more.

[0065] PHB has a good hue, and the yellowness index (YI value) of a 5mm thick sheet after being pressed at 160°C is preferably 20 or less, more preferably 17 or less.

[0066] PHB can be molded into various shapes such as fibers, filaments, ropes, fabrics, woven fabrics, non-woven fabrics, paper, films, sheets, tubes, plates, rods, containers, bags, components, and foams. These molded bodies can be appropriately used in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing materials, packaging, and other fields.

[0067] Similarly, in the case of other biodegradable plastics produced by microorganisms, the microbial cells containing the biodegradable plastics are broken down or dissolved, and water with a calcium ion concentration of 4.5 mg / L or less can be used in the process of separating the biodegradable plastics.

[0068] Example

[0069] The present invention will be described in more detail below through embodiments, but the present invention is not limited to these embodiments.

[0070] [Measurement and Evaluation Methods]

[0071] The following illustrates the measurement / evaluation methods in the examples and comparative examples.

[0072] (chromaticity)

[0073] 200 mL of the prepared cleaning water was placed in a dedicated testing container, and the measuring instrument (TCR-5Z manufactured by Kasayasu Kagaku Kogyo Co., Ltd.) was gently immersed in it to prevent the formation of air bubbles, and the color of the cleaning water was measured. Based on the Ministry of Health, Labour and Welfare's "Tap Water Quality Standards," the color of tap water is 5 degrees or less.

[0074] (Thermal stability)

[0075] Regarding thermal stability, the weight-average molecular weight retention rate of polyhydroxybutyrate resins after heating at 160°C for 20 minutes is used. A weight-average molecular weight retention rate of 70% or higher is considered to indicate good thermal stability, while a weight-average molecular weight retention rate of less than 70% is considered to indicate poor thermal stability.

[0076] Weight-average molecular weight retention (%) = (weight-average molecular weight of PHB after heating / weight-average molecular weight of PHB before heating) × 100

[0077] <Weight-average molecular weight of PHB before heating>

[0078] 10 mg of polyhydroxybutyrate (PHB) resin powder was dissolved in 10 mL of chloroform, and insoluble matter was removed by filtration. The molecular weight of this solution (filtrate) was determined using a Shimadzu GPC system equipped with a Shodex K805L (300x8 mm, two in series) (manufactured by Showa Denko Corporation), with chloroform as the mobile phase. Commercially available standard polystyrene was used as the molecular weight standard.

[0079] <Weight-average molecular weight of PHB after heating>

[0080] Polyhydroxybutyrate (PHB) resin powder was preheated at 160°C for 7 minutes, then heated at 160°C for 20 minutes to prepare PHB resin tablets. Except for using 10 mg of the PHB resin tablets, the weight-average molecular weight of the heated PHB was determined using the same method as for determining the weight-average molecular weight of PHB before heating.

[0081] (Composition of PHB before heating)

[0082] Approximately 20 mg of polyhydroxybutyrate (PHB) resin powder was mixed with 2 mL of sulfuric acid-methanol (15:85) and 2 mL of chloroform, and the mixture was sealed tightly. The solution was heated at 100°C for 140 minutes to obtain methyl esters as a polyester decomposition product. After cooling, 1.5 g of sodium bicarbonate was added in small amounts each time for neutralization, and the mixture was allowed to stand until carbon dioxide production ceased. 4 mL of diisopropyl ether was added, and the mixture was thoroughly mixed and centrifuged. The composition of the methyl hydroxyalkanoate esters in the supernatant, as a polyester decomposition product, was analyzed by capillary gas chromatography to determine the composition (content) of the monomer units of the obtained polyester. A Shimadzu GC-17A gas chromatograph was used, and a NEUTRABOND-1 capillary column (25 m column length, 0.25 mm inner diameter, 0.4 μm film thickness) was used. The temperature conditions are as follows: the temperature is increased to the initial temperature of 100℃~200℃ at a rate of 8℃ / min, and then increased to 200℃~290℃ at a rate of 30℃ / min.

[0083] (Determination of Yellowness Index (YI value))

[0084] A pressed sheet of polyhydroxybutyrate (PHB) resin was prepared and used as the test sample. The PHB resin pressed sheet was prepared as follows: 3.0 g of PHB resin powder was held between four 15 cm square metal plates. 0.5 mm thick metal plates were inserted into the four corners of the metal plate. The sheet was placed in a small experimental press (Takabayashi Rika Co., Ltd., H-15 type), heated to 160°C for 7 minutes, and then pressed at approximately 5 MPa for 2 minutes while maintaining the temperature at 160°C. After pressing, the PHB resin was allowed to cure at room temperature. The YI value was measured as follows: using a colorimeter "SE-2000" (Nippon Denshoku Co., Ltd.), a 30 mm measuring plate was used. The pressed sheet was placed on the measuring plate, and a white standard plate was placed on top. A YI value below 20 was considered good hue, and a YI value above 20 was considered poor hue.

[0085] (Example 1 of the manufacture of cleaning water)

[0086] Wastewater discharged from the production of polyhydroxybutyrate resins underwent anaerobic and aerobic treatment using microorganisms, followed by pretreatment filtration using a UF membrane separation activated sludge process, and then filtration through an RO membrane. The anaerobic treatment was carried out as follows: in an acid-generating tank (pH around 7.1), acid-producing bacteria decomposed high molecular weight carbohydrates and lipids into organic acids and lower alcohols. Then, in an EGSB-based methane generation reactor (loading 15 kg-CODcr / m³), the methane was further processed. 3In the anaerobic treatment ( / d), organic acids and lower alcohols are decomposed into methane and carbon dioxide gases by the action of granular methanogenic bacteria. Aerobic treatment is carried out using a device consisting of a denitrification tank (activated sludge treatment tank), an aeration tank (activated sludge treatment tank), a second denitrification tank (activated sludge treatment tank), and a re-aeration tank (activated sludge treatment tank), and decomposes the organic matter that was not decomposed in the anaerobic treatment by the action of aerobic bacteria. Pretreatment filtration based on membrane separation activated sludge process is carried out by installing a UF membrane (hollow fiber membrane: PVDF, manufactured by Mitsubishi Chemical Corporation, nominal pore size: 0.05 μm, MgSO4 rejection rate of 0% at 20°C and 3000 kPa pressure) in the re-aeration tank (activated sludge treatment tank). Water permeates through the UF membrane at a filtration linear velocity of 0.86–1.15 m / day. UF membrane permeate was collected and fed to an RO membrane (material: composite polyamide, manufactured by Nitto Denko, with a MgSO4 rejection rate of 99.7% at 20°C and a permeation rate of 0.75–0.85 L / min) under conditions of 30°C, intermembrane differential pressure (permeation pressure) of 0.7–1.15 MPa, and permeation rate of 0.75–0.85 L / min). Water that did not permeate through the RO membrane was returned to the collection tank, while the permeated water was used as cleaning water. The UF membrane permeate was allowed to permeate through the RO membrane for 97 minutes, allowing 70% by weight of the UF membrane permeate to pass through. The RO membrane permeate was collected from the initial permeation until 13 minutes later to obtain cleaning water 1.

[0087] (Example 2 of manufacturing cleaning water)

[0088] The permeate water from 90 to 97 minutes was collected from the water passing through the RO membrane. In addition, cleaning water 2 was obtained in the same manner as in manufacturing example 1.

[0089] (Example 3 of the preparation of cleaning water)

[0090] Water that had permeated through the RO membrane under the same conditions as in Manufacturing Example 1 was further permeated through the RO membrane (material: composite polyamide, manufactured by Nitto Denko, with a MgSO4 rejection rate of 99.7% at 20°C and a pressure of 3000 kPa) at a water temperature of 30°C, a permeation pressure of 0.8 MPa, and a permeation rate of 1.25 L / min, thereby obtaining cleaning water 3. The permeate volume was 90% of the permeate volume of the UF membrane.

[0091] (Example 4 of the preparation of cleaning water)

[0092] Cleaning water 4 was obtained by taking the UF membrane permeate water after it had passed through the UF membrane under the same conditions as in manufacturing example 1.

[0093] (Example 5: Manufacturing of cleaning water)

[0094] Under the same conditions as in Manufacturing Example 1, the permeate water from the UF membrane was collected and allowed to pass through an NF membrane (material: composite polyamide, manufactured by Synder, with a MgSO4 rejection rate of 96.1% at 3000 kPa pressure applied at 20°C) at a water temperature of 20°C, a permeation pressure of 3 MPa, and a permeation rate of 30–300 g / h. The permeate water passing through the NF membrane was collected, thus obtaining cleaning water 5. The amount of permeate water was 75% of the amount of permeate water from the UF membrane.

[0095] (Example 6: Manufacturing of cleaning water)

[0096] Industrial water (made by KANEKA) was treated with ion exchange resins (manufactured by ORGANO, strong acid cation exchange resin and strong base anion exchange resin) to obtain cleaning water 6.

[0097] (Example 7: Manufacturing of cleaning water)

[0098] Under the same conditions as in Manufacturing Example 1, the permeate water from the UF membrane was collected and allowed to pass through an NF membrane (material: composite polyamide, manufactured by Synder, with a MgSO4 rejection rate of 6.7% when subjected to a pressure of 3000 kPa at 20°C) at a water temperature of 20°C, a permeation pressure of 3 MPa, and a permeation rate of 100–550 g / h. The permeate water passing through the NF membrane was collected, thus obtaining cleaning water 7. The amount of permeate water was 75% of the amount of permeate water from the UF membrane.

[0099] The concentrations of calcium ions, sodium ions, and color in the cleaning waters 1 through 7 are shown in Table 1 below.

[0100] [Table 1]

[0101]

[0102] [Example 1]

[0103] (Preparation of bacterial culture medium)

[0104] The method described in paragraphs

[0050] to

[0053] of International Publication No. 2008 / 010296 was used to culture the *Alcaligenes eeuoxygenobacter* strain KNK-005, as described in paragraph

[0049] of International Publication No. 2008 / 010296, to obtain a bacterial culture containing cells containing PHB (poly(3-hydroxybutyric acid-co-3-hydroxyhexanoic acid) with a 3HH content of 11.5 mol%). It should be noted that *Alcaligenes eeuoxygenobacter* is currently classified as a hookworm copper-loving bacterium.

[0105] (Sterilization treatment)

[0106] The bacterial culture broth obtained above was sterilized by heating / stirring at an internal temperature of 60-80℃ for 20 minutes.

[0107] (Processes (a) and (b))

[0108] Sodium dodecyl sulfate was added to the sterilized bacterial culture medium obtained above to reach 0.2% by weight. Further, washing water containing dissolved sodium hydroxide was added to bring the pH to 11.0, and then the mixture was kept at 50°C for 1 hour. Then, high-pressure crushing was performed using a high-pressure crusher (Niro Soavi SPA PA2K type high-pressure homogenizer) at a pressure of 44–54 MPa.

[0109] Add the same weight of washing water to the high-pressure crushed bacterial cell lysate obtained above. After centrifugation, remove the supernatant and concentrate the solution by 2 times. Add washing water with added sodium hydroxide (pH 11.0) to the concentrated PHB aqueous suspension until it reaches the same weight as the supernatant, and centrifuge again. Next, remove the supernatant, add washing water to suspend the solution, add 0.2% sodium dodecyl sulfate and 1 / 100 weight of PHB protease (Novozymes Esperase), and maintain the solution at 50°C at pH 10.0 for 2 hours with stirring. Then, remove the supernatant by centrifugation and concentrate the solution by 5 times. Add washing water with added sodium hydroxide (pH 11.0) to the concentrated PHB aqueous suspension until it reaches the same weight as the removed supernatant, and centrifuge again. Repeat the same operation 5 times, remove the supernatant, and adjust the polyhydroxybutyrate resin concentration to 52% by weight.

[0110] In processes (a) and (b), cleaning water 1 was used as cleaning water.

[0111] (Process (c))

[0112] A dispersant (polyethylene glycol-polypropylene glycol-block ether type nonionic surfactant, trade name "PLONON#208", manufactured by Nippon Oil Manufacturing Co., Ltd.) of 1 phr (1 part by weight relative to 100 parts by weight of polyhydroxybutyrate resin present in the aqueous suspension) was added to the above-obtained aqueous suspension (solid content concentration 52 wt%). Then, the solid content concentration was adjusted to 30 wt% with distilled water. After stirring the liquid for 30 minutes, sulfuric acid was added to adjust the pH to a stable value of 4. The resulting aqueous suspension of polyhydroxybutyrate resin was dried at 60°C for 12 hours to produce polyhydroxybutyrate resin powder.

[0113] [Example 2]

[0114] In steps (a) and (b), cleaning water 2 was used as the cleaning water, and otherwise, polyhydroxybutyrate resin powder was obtained in the same manner as in Example 1.

[0115] [Example 3]

[0116] In steps (a) and (b), cleaning water 3 was used as the cleaning water, and otherwise, polyhydroxybutyrate resin powder was obtained in the same manner as in Example 1.

[0117] [Comparative Example 1]

[0118] In steps (a) and (b), cleaning water 4 was used as the cleaning water, and otherwise, polyhydroxybutyrate resin powder was obtained in the same manner as in Example 1.

[0119] [Example 4]

[0120] In the preparation of the bacterial culture medium, a bacterial culture medium containing bacteria containing PHB (3HH content of 10.9 mol% poly(3-hydroxybutyric acid-co-3-hydroxyhexanoic acid)) was used, and in steps (a) and (b), washing water 2 was used as washing water. Otherwise, polyhydroxybutyrate resin powder was obtained by the same operation as in Example 1.

[0121] [Example 5]

[0122] In steps (a) and (b), cleaning water 5 was used as the cleaning water, and otherwise, polyhydroxybutyrate resin powder was obtained in the same manner as in Example 4.

[0123] [Example 6]

[0124] In steps (a) and (b), cleaning water 6 was used as the cleaning water, and otherwise, polyhydroxybutyrate resin powder was obtained in the same manner as in Example 4.

[0125] [Comparative Example 2]

[0126] In steps (a) and (b), cleaning water 7 was used as the cleaning water, and otherwise, polyhydroxybutyrate resin powder was obtained in the same manner as in Example 4.

[0127] [Comparative Example 3]

[0128] In steps (a) and (b), cleaning water 4 was used as the cleaning water, and otherwise, polyhydroxybutyrate resin powder was obtained in the same manner as in Example 4.

[0129] The thermal stability and YI value of the polyhydroxybutyrate resin powders obtained in the Examples and Comparative Examples were measured / evaluated as described above, and the results are shown in Table 2 below. In Table 2 below, the 3HH content before heating refers to the 3HH content in the polyhydroxybutyrate resin before heating.

[0130] [Table 2]

[0131]

[0132] In the embodiments, by using cleaning water with a calcium ion concentration of less than 4.5 mg / L in steps (a) and (b), the polyhydroxybutyrate (PHB) resin powder exhibits high thermal stability and a good color tone. Furthermore, the lower the calcium ion concentration in the cleaning water, the better the thermal stability and color tone of the PHB resin powder tend to be. In Comparative Example 2, although highly transparent water was used, the YI value exceeded the benchmark value, indicating that the calcium ion concentration has a strong influence on the YI value of the PHB resin powder. In particular, in Examples 1-5, where treated water obtained by treating the wastewater discharged from the PHB resin manufacturing process was used as cleaning water, the PHB resin powder exhibited high thermal stability and a good color tone. Therefore, water consumption can be reduced, and PHB resins with good color tone and thermal stability can be manufactured while reducing environmental impact.

[0133] This invention is not particularly limited, and may include one or more of the embodiments described below.

[0134] [1] A method for manufacturing a polyhydroxybutyrate resin, comprising:

[0135] The process of disrupting or solubleizing microbial cells containing polyhydroxybutyrate resin (a); and

[0136] Step (b) involves separating the polyhydroxybutyrate resin from the composition obtained in step (a).

[0137] In processes (a) and (b), water with a calcium ion concentration of less than 4.5 mg / L is used.

[0138] [2] According to the method for manufacturing polyhydroxybutyrate resin described in [1], wherein,

[0139] In the water used in processes (a) and (b), the concentration of sodium ions is below 450 mg / L.

[0140] [3] The method for manufacturing polyhydroxybutyrate resin according to [1] or [2], wherein,

[0141] The water used in processes (a) and (b) is obtained as follows: after anaerobic and aerobic treatment of the wastewater discharged from the manufacturing process of polyhydroxybutyrate resin using microorganisms, a pretreatment filtration process based on membrane separation activated sludge process and a filtration process using calcium ions to remove membrane ions are carried out.

[0142] [4] The method for manufacturing polyhydroxybutyrate resin according to any one of [1] to [3], wherein,

[0143] The calcium ion removal membrane mentioned above is selected from one or more of NF membranes and RO membranes.

[0144] [5] According to the method for manufacturing polyhydroxybutyrate resin described in [4], wherein,

[0145] The MgSO4 rejection rate of the above-mentioned NF membrane or RO membrane is above 60% and below 100% when a pressure of 3000 kPa is applied at 20°C.

[0146] [6] The method for manufacturing polyhydroxybutyrate resin according to any one of [1] to [5], wherein,

[0147] Process (a) includes at least one treatment selected from chemical treatment and physical crushing treatment.

[0148] [7] According to the method for manufacturing polyhydroxybutyrate resin described in [6], wherein,

[0149] The above chemical treatment utilizes at least one selected from alkaline compounds, protein-degrading enzymes, and cell wall-degrading enzymes.

[0150] [8] The method for manufacturing polyhydroxybutyrate resin according to any one of [1] to [7], wherein,

[0151] The above-mentioned polyhydroxybutyrate resin is poly(3-hydroxybutyric acid-co-3-hydroxyhexanoic acid).

[0152] [9] The method for manufacturing polyhydroxybutyrate resin according to any one of [1] to [8], wherein,

[0153] The weight-average molecular weight retention rate of the above-mentioned polyhydroxybutyrate resin is more than 70% when it is heat-treated at 160°C for 20 minutes.

[0154]

[10] The method for manufacturing polyhydroxybutyrate resin according to any one of [1] to [9], wherein,

[0155] The yellowness index (YI value) of a 5 mm thick sheet obtained by pressing the above-mentioned polyhydroxybutyrate resin at 160°C is below 20.

Claims

1. A method for producing a polyhydroxybutyric acid-based resin, comprising: a step (a) of disrupting or solubilizing microbial cells containing a polyhydroxybutyric acid-based resin; and a step (b) of separating the polyhydroxybutyric acid-based resin in the composition obtained in the step (a), wherein, in the step (a) and the step (b), water having a concentration of calcium ions of 4.5 mg / L or less is used, wherein the water used in the step (a) and the step (b) is obtained by, after subjecting drainage discharged from a production step of a polyhydroxybutyric acid-based resin to anaerobic treatment and aerobic treatment using microorganisms, carrying out a pretreatment filtration step based on a membrane separation activated sludge method, and a filtration step for removing a membrane using calcium ions, wherein the membrane for removing calcium ions is one or more selected from the group consisting of NF membranes and RO membranes, wherein the NF membrane or the RO membrane has a MgSO4 rejection rate of 60% or more and 100% or less when a pressure of 3000 kPa is applied at 20°C, wherein the polyhydroxybutyric acid-based resin is a copolymer of 3-hydroxybutyric acid and another 3-hydroxyalkanoic acid selected from one or more of the group consisting of 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytridecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxypentadecanoic acid, and 3-hydroxyhexadecanoic acid.

2. The method for producing a polyhydroxybutyric acid-based resin according to claim 1, wherein, in the water used in the step (a) and the step (b), the concentration of sodium ions is 450 mg / L or less.

3. The method for producing a polyhydroxybutyric acid-based resin according to claim 1 or 2, wherein, the step (a) comprises at least one treatment selected from the group consisting of chemical treatment and physical disruption treatment.

4. The method for producing a polyhydroxybutyric acid-based resin according to claim 3, wherein, the chemical treatment is a chemical treatment using at least one selected from the group consisting of an alkaline compound, a protein-decomposing enzyme, and a cell wall-decomposing enzyme.

5. The method for producing a polyhydroxybutyric acid-based resin according to claim 1 or 2, wherein, the polyhydroxybutyric acid-based resin is poly(3-hydroxybutyric acid-co-3-hydroxyhexanoic acid).

6. The method for producing a polyhydroxybutyric acid-based resin according to claim 1 or 2, wherein, the polyhydroxybutyric acid-based resin has a weight average molecular weight retention rate of 70% or more when subjected to heat treatment at 160°C for 20 minutes.

7. The method for producing a polyhydroxybutyric acid-based resin according to claim 1 or 2, wherein, a sheet having a thickness of 5 mm obtained by compression molding the polyhydroxybutyric acid-based resin at 160°C has a yellowness index (YI value) of 20 or less.

8. The method for producing a polyhydroxybutyric acid-based resin according to claim 3, wherein, the physical disruption treatment is carried out after adding an alkaline compound, or after adding an alkaline compound and a surfactant.

9. The method for producing a polyhydroxybutyric acid-based resin according to claim 1 or 2, further comprising: a step (c) of drying the polyhydroxybutyric acid-based resin separated in the step (b). ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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