Method for producing polyhydroxybutyric acid resin

Abstract

The present invention relates, in one or more embodiments, to a method for producing a polyhydroxybutyric acid resin that comprises a step (a) for crushing or solubilizing microbial cells that contain polyhydroxybutyric acid resin, and a step (b) for separating the polyhydroxybutyric acid in the composition obtained in step (a). Water having a calcium ion concentration of 4.5 mg / L or less is used in step (a) and step (b). The water used in step (a) and step (b) is preferably produced by subjecting water discharged during the polyhydroxybutyric acid resin production step to a microorganism-assisted anaerobic treatment and aerobic treatment, and subsequently carrying out a pretreatment filtration step performed via a membrane-separation activated sludge process and a filtration step performed via a calcium-ion-removing membrane. What is thereby provided is method for producing a polyhydroxybutyric acid resin having excellent color tone and high thermal stability.

Description

[Technical field]

[0001] The present invention relates to a method for producing a polyhydroxybutyric acid-based resin. [Background technology]

[0002] Biodegradable plastics are completely biodegraded by microorganisms in soil and water and are incorporated into the natural carbon cycle process, so there is a demand for their active use as environmentally friendly plastic materials that have almost no adverse effects on ecosystems. Representative biodegradable plastics that have attracted attention include plant-derived biodegradable plastics such as polyhydroxybutyrate. Polyhydroxybutyrate is an aliphatic polyester (thermoplastic polyester) that is produced by microorganisms using natural plant-derived organic acids and oils and fats as carbon sources and is accumulated within cells as an energy storage substance.

[0003] Since polyhydroxybutyric acid produced by microorganisms is water-insoluble and usually accumulates as granules within microbial cells, a process of separating and extracting polyhydroxybutyric acid from microbial cells is required to use polyhydroxybutyric acid as a plastic. For example, Patent Document 1 reports a method of separating and purifying polyhydroxybutyric acid by combining alkali addition and high-pressure crushing. For example, Patent Document 2 reports a method of separating and purifying polyhydroxybutyric acid by combining physical cell crushing and chemical treatment with enzymes and surfactants. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 07-31489 [Patent Document 2] JP 2008-193940 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, polyhydroxybutyric acid separated by the methods described in Patent Documents 1 and 2 has problems such as coloration and low thermal stability, resulting in a decrease in molecular weight when heated at high temperatures.

[0006] In order to solve the above problems, the present invention provides a method for producing a polyhydroxybutyric acid-based resin that can produce a polyhydroxybutyric acid-based resin having a good color tone and high thermal stability. [Means for solving the problem]

[0007] In one or more embodiments, the present invention relates to a method for producing a polyhydroxybutyric acid-based resin, comprising step (a) of disrupting or solubilizing microbial cells containing a polyhydroxybutyric acid-based resin, and step (b) of separating the polyhydroxybutyric acid-based resin from the composition obtained in step (a), wherein steps (a) and (b) use water having a calcium ion concentration of 4.5 mg / L or less. Effect of the Invention

[0008] According to the production method of the present invention, it is possible to provide a polyhydroxybutyric acid-based resin that has a good color tone and high thermal stability. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] As a result of extensive investigations to solve the above-mentioned problems, the inventors of the present invention have found that by using water having a specific calcium ion concentration in step (a) of disrupting or solubilizing microbial cells containing a polyhydroxybutyric acid resin (hereinafter also referred to simply as "PHB") and in step (b) of separating the polyhydroxybutyric acid resin from the composition obtained in step (a), the color tone of the obtained polyhydroxybutyric acid resin becomes good and a decrease in molecular weight is suppressed even when heated at a high temperature, for example, at 160°C. Furthermore, it is preferable to set the sodium ion concentration in the water used in steps (a) and (b) to a specific range.

[0010] [Low calcium ionized water] 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, and from the viewpoint of improving color tone and thermal stability, it is preferably 3.0 mg / L or less, and more preferably 2.0 mg / L or less. In one or more embodiments of the present invention, the water used in steps (a) and (b) ideally does not contain calcium ions, but from the viewpoint of practicality, it may be 0.001 mg / L or more, or may be 0.005 mg / L or more.

[0011] 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, the water used in steps (a) and (b) ideally does not contain sodium ions, but from the viewpoint of practicality, the concentration may be 0.05 mg / L or more, or may be 0.1 mg / L or more.

[0012] The water used in steps (a) and (b) is not particularly limited as long as the calcium ion concentration in the water is 4.5 mg / L or less, but from the viewpoint of reducing the amount of water used and reducing the environmental load, it is preferable to use treated water obtained by subjecting wastewater discharged from the production process of polyhydroxybutyric acid-based resin to anaerobic fermentation treatment and aerobic fermentation treatment using microorganisms, followed by a pretreatment filtration step using a membrane separation activated sludge method and a filtration step using a calcium ion removal membrane. The wastewater discharged from the production process of polyhydroxybutyric acid-based resin includes wastewater discharged from any production process of polyhydroxybutyric acid-based resin, such as steps (a) and (b).

[0013] The anaerobic and aerobic treatments using microorganisms, and the pretreatment filtration using the membrane separation activated sludge method are not particularly limited and can be carried out by a general method used in water treatment. The anaerobic treatment device can be composed of, for example, an acid generation tank that decomposes high molecular weight carbohydrates and lipids into organic acids and lower alcohols by the action of acid-producing bacteria, and an EGSB type methane generation reaction tank that decomposes organic acids and lower alcohols into methane gas and carbon dioxide gas by the action of granular methanogens. The aerobic treatment can be carried out by, for example, using a device composed of a denitrification tank (activated sludge treatment tank) and an aeration tank (activated sludge treatment tank), and by decomposing organic matter that has not been decomposed by the anaerobic treatment by the action of aerobic bacteria in the aeration tank. The aerobic treatment device may be composed 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 reaeration tank (activated sludge treatment tank). The pretreatment filtration process using the membrane separation activated sludge method can be carried out, for example, by installing a UF membrane or MF membrane MBR (membrane bioreactor membrane separation activated sludge method permeation membrane) in an aeration tank (activated sludge treatment tank) or a reaeration tank (activated sludge treatment tank).

[0014] In one or more embodiments of the present invention, it is preferable to use, as the calcium ion removal membrane, one or more selected from the group consisting of an NF membrane and an RO membrane, since these membranes have high calcium ion removal performance.

[0015] In one or more embodiments of the present invention, the NF membrane or RO membrane preferably has an MgSO4 rejection of 60% to 100%, more preferably 70% to 100%, and even more preferably 90% to 100% when a pressure of 3000 kPa is applied at 20° C. If the MgSO4 rejection is 60% or more, the calcium ion permeability of the membrane does not increase, and by using the obtained treated water in steps (a) and (b), the color tone of the polyhydroxybutyric acid-based resin tends to be good, and a decrease in molecular weight at high temperatures is also easily suppressed.

[0016] In one or more embodiments of the present invention, the transmembrane pressure during filtration through an NF membrane or an RO membrane is not particularly specified, but 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. If the transmembrane pressure is 0.4 MPa or more, the amount of permeate and the ion removal rate do not decrease, and if it is 4.14 MPa or less, the membrane is unlikely to be damaged.

[0017] In one or more embodiments of the present invention, the permeation rate during filtration through an NF membrane or an RO membrane is not particularly specified, but is preferably 0.01 L / min to 2000 L / min, more preferably 0.5 L / min to 1500 L / min. A permeation rate of 0.01 L / min or more provides good productivity.

[0018] In one or more embodiments of the present invention, the water temperature during filtration through the NF membrane or RO membrane is not particularly specified, but the water temperature is preferably 50° C. or less, and more preferably 45° C. or less. If the water temperature is 50° C. or less, the membrane is less likely to deteriorate.

[0019] [Microbial cells containing PHB] In one or more embodiments of the present invention, the microbial cells containing the polyhydroxybutyrate resin can be obtained by culturing a microorganism capable of producing PHB.

[0020] In one or more embodiments of the present invention, the polyhydroxybutyric acid resin is a general term for polymers having 3-hydroxybutyric acid (hereinafter also referred to as 3HB) as a monomer unit. PHB may be poly(3-hydroxybutyric acid), which is a homopolymer of 3-hydroxybutyric acid, or may be a copolymer of 3-hydroxybutyric acid and another 3-hydroxyalkanoic acid. As the other 3-hydroxyalkanoic acid, for example, one or more monomers selected from the group consisting of 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-hydroxytridecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxypentadecanoic acid, and 3-hydroxyhexadecanoic acid can be used.

[0021] As PHB, poly(3-hydroxybutyric acid), poly(3-hydroxybutyric acid-co-3-hydroxyhexanoic acid), poly(3-hydroxybutyric acid-co-3-hydroxyoctanoic acid), etc. are preferred from the viewpoint of ease of industrial production, and poly(3-hydroxybutyric acid-co-3-hydroxyhexanoic acid) is particularly preferred. The composition ratio of each monomer unit constituting the two-component copolymer PHBH of 3HB and 3HH is not particularly limited, but when the total of all monomer units is taken as 100 mol%, the 3HH unit may be 1 mol% or more and 50 mol% or less, 1 mol% or more and 25 mol% or less, or 1 mol% or more and 15 mol% or less.

[0022] In one or more embodiments of the present invention, the microorganism capable of producing PHB is not particularly limited, and may be a microorganism isolated from nature or deposited in a depository institution for strains (e.g., IFO, ATCC, etc.), or a mutant or transformant that can be prepared from such a microorganism. Examples of such a microorganism include bacteria of the genera Cupriavidus, Alcaligenes, Ralstonia, Pseudomonas, Bacillus, Azotobacter, Nocardia, and Aeromonas. In particular, strains such as A. lipolytica, A. latus, A. caviae, A. hydrophila, and C. necator are preferred. In addition, when a microorganism does not inherently have the ability to produce PHB or produces only a low amount of PHB, a transformant obtained by introducing a target PHB synthase gene and / or a mutant thereof into the microorganism can be used. There is no particular limitation on the PHB synthase gene used to prepare such a transformant, but a PHB synthase gene derived from Aeromonas caviae is preferred.

[0023] By culturing the above-mentioned microorganism under appropriate conditions, a microorganism having PHB accumulated in the cells can be obtained. The culturing method is not particularly limited, and for example, the methods described in JP-A-05-93049 and WO 2008 / 010296 can be used. As the microbial cells containing PHB, a bacterial cell culture solution containing PHB-containing microbial cells after the completion of the culture can be used as it is, or a sterilized bacterial cell culture solution after the bacterial cells are killed by heating the bacterial cell culture solution can be used. The sterilization can be performed, for example, by heat treatment at a temperature of 50° C. to 80° C. for 5 minutes to 120 minutes.

[0024] [Process (a)] In step (a), microbial cells containing PHB are disrupted or solubilized. Step (a) can be carried out, for example, by at least one treatment selected from the group consisting of a chemical treatment and a physical disruption treatment.

[0025] The chemical treatment can be carried out with at least one compound selected from the group consisting of alkaline compounds, proteolytic enzymes and cell wall decomposing enzymes.

[0026] The alkaline compound is not particularly limited as long as it can destroy the cell wall of the PHB-containing microbial cell and release the PHB from the cell to the outside, and examples thereof include alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, and lithium hydroxide, alkali metal carbonates such as sodium carbonate and potassium carbonate, alkali metal bicarbonates such as sodium bicarbonate and potassium bicarbonate, alkali metal salts of organic acids such as sodium acetate and potassium acetate, alkali metal borates such as borax, alkali metal phosphates such as trisodium phosphate, disodium hydrogen phosphate, tripotassium phosphate, and dipotassium hydrogen phosphate, hydroxides of alkaline earth metals such as barium hydroxide, and aqueous ammonia. Among these, from the viewpoint of suitability for industrial production and cost reduction, one or more selected from the group consisting of sodium hydroxide, sodium carbonate, potassium hydroxide, and lithium hydroxide are preferred.

[0027] The proteolytic enzyme is not particularly limited, but examples thereof include Alcalase, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, carboxypeptidase, etc. Specific proteolytic enzymes that can be used industrially include, for example, "Protease A," "Protease P," and "Protease N" (all manufactured by Amano Enzyme Co., Ltd.), "Alcalase," "Esperase," "Zavinase," and "Evalase" (all manufactured by Novozymes Co., Ltd.), and are preferably used in terms of decomposition activity.

[0028] Examples of cell wall decomposition enzymes include, but are not limited to, lysozyme, amylase, cellulase, maltase, saccharase, α-glycosinase, and β-glycosinase. Among the above cell wall decomposition enzymes, lysozyme is preferred in terms of its bacteriolytic effect. Specific examples of cell wall decomposition enzymes that can be used industrially include "Lysozyme" (manufactured by Huayuan Jingmao Co., Ltd., Shandong Province), "Biozyme A", "Cellulase A "Amano" 3", "Cellulase T "Amano" 4", "α-Glucosidase "Amano"" (all manufactured by Amano Enzyme Co., Ltd.), "Termamyl", and "Cellsoft" (all manufactured by Novozymes Co., Ltd.).

[0029] The above-mentioned enzyme treatment is desirably carried out in the presence of a surfactant, since this provides a high separation and purification effect. As the enzyme, for example, an enzyme composition containing an enzyme and one or more additives selected from the group consisting of an enzyme stabilizer, a surfactant, and a redeposition inhibitor may be used.

[0030] Examples of surfactants include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. From the viewpoint of high effect of removing residues due to destruction of cell membranes, anionic surfactants and / or nonionic surfactants are preferred. For the purpose of removing proteins and the like, it is preferred to use anionic surfactants, and for the purpose of removing fatty acids and oils and fats, it is preferred to use nonionic surfactants. In addition, both anionic surfactants and nonionic surfactants can be used. When both are used, the weight ratio of anionic surfactant / 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.

[0031] 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-sulfofatty acid salts, esters of α-sulfofatty acid salts, alkyl ether carboxylates, alkenyl ether carboxylates, amino acid surfactants, and N-acylamino acid surfactants. Among them, alkyl sulfates having an alkyl group with 12 to 14 carbon atoms, linear alkylbenzene sulfonates having an alkyl group with 12 to 16 carbon atoms, alkyl sulfates having an alkyl group with 10 to 18 carbon atoms, and alkyl ether sulfates having an alkyl group with 10 to 18 carbon atoms. As counter ions, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and alkanolamines such as monoethanolamine, diethanolamine, and triethanolamine are preferred.

[0032] Examples of nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyalkylene alkyl ethers, fatty acid sorbitan esters, alkyl polyglucosides, fatty acid diethanolamides, alkyl monoglyceryl ethers, etc. Polyoxyethylene alkyl ethers and polyoxyalkylene alkyl ethers are preferred because of their high hydrophilicity and relatively good biodegradability.

[0033] Examples of the cationic surfactant include alkyltrimethylammonium salts and dialkyldimethylammonium salts.

[0034] Examples of amphoteric surfactants include carbobetaine type and sulfobetaine type.

[0035] Of the above-mentioned surfactants, anionic surfactants such as sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium cholate, sodium deoxycholate, and sodium oleate, and nonionic surfactants such as polyoxyethylene alkyl ethers and polyoxyalkylene alkyl ethers are preferred in terms of cost, amount used, and addition effect.

[0036] The amount of the surfactant to be added is not particularly limited, but is preferably 0.001 to 10 parts by weight, and more preferably 5 parts by weight or less, based on 100 parts by weight of the bacterial culture solution, from the viewpoint of cost. One type of surfactant may be used alone, or two or more types may be used in combination.

[0037] The enzyme treatment is preferably carried out, for example, by adding an alkaline compound and / or a surfactant to the bacterial cell culture solution and stirring the mixture. The enzyme treatment conditions are preferably controlled to the optimum value of the enzyme used. The required amount of the enzyme depends on the type and activity of the enzyme. Although not particularly limited, the amount is preferably 0.001 to 10 parts by weight per 100 parts by weight of PHB100, and more preferably 0.001 to 5 parts by weight from the viewpoint of cost.

[0038] From the viewpoint of enhancing the crushing effect and facilitating the recovery of PHB, the physical crushing treatment is preferably carried out after adding an alkaline compound, or an alkaline compound and a surfactant. As the alkaline compound and the surfactant, the above-mentioned ones can be appropriately used. Among the above-mentioned alkaline compounds, sodium hydroxide, sodium carbonate, potassium hydroxide, and lithium hydroxide are preferable from the viewpoint of suitability for industrial production and reducing costs. Among the above-mentioned surfactants, anionic surfactants such as sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium cholate, sodium deoxycholate, and sodium oleate, and nonionic surfactants such as polyoxyethylene alkyl ether and polyoxyalkylene alkyl ether are preferable in terms of cost, usage amount, and addition effect.

[0039] It is preferable to adjust the pH of the bacterial cell culture solution to 8.0 or more and 12.5 or less by adding an alkaline compound. It is easy to solubilize bacterial cell (microbial cell) residues, organic matter produced by the bacterial cell, organic matter constituting the bacterial cell, etc. without affecting the PHB. After adding the alkaline compound to the bacterial cell culture solution, it may be treated at a temperature of 20°C to 80°C, preferably 20°C to 50°C, for 30 minutes to 2 hours.

[0040] The amount of the surfactant to be added is not particularly limited, but is preferably 0.001 to 10 parts by weight, and more preferably 5 parts by weight or less, based on 100 parts by weight of the bacterial culture solution, from the viewpoint of cost. One type of surfactant may be used alone, or two or more types may be used in combination.

[0041] The device for performing the physical disruption treatment is not particularly limited, but examples thereof include a high-pressure homogenizer, an ultrasonic disrupter, an emulsifying disperser, a bead mill, and the like. Among them, the use of a high-pressure homogenizer is preferred from the viewpoint of the disruption efficiency, and a type in which the suspension is introduced into a pressure-resistant container having a minute opening and is pushed out from the opening by applying high pressure is more preferred. Examples of this type of disrupter include the high-pressure homogenizer model "PA2K type" manufactured by Nilosoavi. When a high-pressure homogenizer is used, a large shear force acts on the microbial cells, so that the microbial cells are efficiently destroyed and the separability of PHB is improved. Since such equipment is subjected to high pressure at the opening and becomes hot instantaneously, it is preferable to cool the bacterial culture liquid using a general low-temperature constant temperature circulation tank as necessary to prevent the temperature from increasing, and to perform the disruption treatment at 20°C to 40°C. By performing the treatment at 20°C to 40°C, the molecular weight of PHB can be treated with almost no decrease. The crushing pressure when performing high-pressure crushing is not particularly limited, but from the viewpoints of crushing efficiency and cost, it is preferably 30 MPa or more and 60 MPa or less.

[0042] In step (a), chemical treatment and physical crushing treatment may be used in combination, and in this case, from the viewpoint of enhancing the crushing effect, it is preferable to perform physical crushing treatment after chemical treatment. From the viewpoint of cost, step (a) may be performed by physical crushing treatment alone.

[0043] In step (a), water having 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. For example, water having 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 a surfactant, an alkaline compound, etc. The water preferably has a sodium ion concentration of 450 mg / L or less, more preferably 250 mg / L or less, and even more preferably 220 mg / L or less.

[0044] [Step (b)] In step (b), the composition obtained in step (a), for example, the polyhydroxybutyric acid resin in the cell disruption liquid, is separated. The method of separation is not particularly limited, and the polyhydroxybutyric acid resin can be recovered together with the water-insoluble component by solid-liquid separation using a method such as filtration, sedimentation, or centrifugation. Centrifugation is preferred from the viewpoint of industrial mass processing and continuous use.

[0045] The centrifuge is not particularly limited, but a centrifugal sedimentation machine with a rotating container without holes is preferred, and the types include separation plate type, cylindrical type, decanter type, etc. Since the specific gravity difference between PHB particles and water is small, a separation plate type (intermittent discharge type, nozzle discharge type) that has a large separation and settling area and can obtain high acceleration is preferred, and the nozzle discharge type is particularly preferred when the PHB concentration in the crushing treatment liquid is high. As for the decanter type, a model with a separation plate and a large separation and settling area is preferred.

[0046] In step (b), prior to separation, 500 parts by weight to 1,000 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 cell disruption liquid.

[0047] In step (b), the aqueous medium may be water or a mixed solvent of water and a water-miscible organic solvent. The content of water in the aqueous medium is preferably 50% by weight or more, more preferably 70% by weight or more, even more preferably 80% by weight or more, and particularly preferably 85% by weight or more. In step (b), water having 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. The sodium ion concentration of the water is preferably 450 mg / L or less, more preferably 250 mg / L or less, and even more preferably 220 mg / L or less.

[0048] The water-miscible organic solvent is not particularly limited, but examples thereof include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, pentanol, hexanol, and heptanol; ketones such as acetone and methyl ethyl ketone; ethers such as tetrahydrofuran and dioxane; nitriles such as acetonitrile and propionitrile; amides such as dimethylformamide and acetamide; dimethyl sulfoxide, pyridine, and piperidine. Among these, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, and propionitrile are preferred because they are easy to remove. In addition, methanol, ethanol, 1-propanol, 2-propanol, butanol, and acetone are more preferred because they are easily available. Furthermore, methanol, ethanol, and acetone are particularly preferred.

[0049] In step (b), the separated PHB (water-insoluble components including PHB) may be purified by washing with the above-mentioned aqueous medium at least once. Washing with an aqueous medium may be performed, for example, by adding 500 parts by weight to 1000 parts by weight of the aqueous medium to 100 parts by weight of the water-insoluble components including the polyhydroxybutyric acid resin. In order to effectively remove impurities derived from microbial cells and to enhance the purification effect, an alkaline compound, a surfactant, a protease, etc. may be added to the aqueous medium.

[0050] [Process (c)] The manufacturing method according to one or more embodiments of the present invention may include a step (c) of drying the PHB separated in the step (b). The PHB (dehydrated resin) washed with an aqueous medium and dehydrated can be dried as it is to obtain powdered PHB. The drying method can be appropriately selected and is not particularly limited, but for example, a general drying method such as spray drying, air flow drying, fluidized drying, band drying, etc. can be preferably used.

[0051] Alternatively, a dispersant can be added to the PHB dispersion liquid that has been washed with an aqueous medium and concentrated, and the pH can be adjusted to 7 or less, and then the liquid can be dried to obtain powdered PHB. Examples of dispersants include water-soluble polymers such as polyvinyl alcohol (PVA), methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, 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). Examples of a method for adjusting the pH to 7 or less include a method of adding an acid. The acid is not particularly limited, and may be either an organic acid or an inorganic acid. For example, sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, and the like can be appropriately used.

[0052] The molecular weight of PHB is not particularly limited as long as it shows substantially sufficient physical properties for the intended use. For example, from the viewpoint of moldability and strength of molded bodies, the weight average molecular weight of PHB is preferably 50,000 to 3,000,000, more preferably 60,000 to 1,500,000. The weight average molecular weight here refers to a molecular weight distribution measured in terms of polystyrene using gel permeation chromatography (GPC) with a chloroform eluent. As a column for the GPC, a column suitable for measuring the molecular weight may be used.

[0053] PHB has high thermal stability, and when heat-treated at 160°C for 20 minutes, the weight-average molecular weight retention 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.

[0054] PHB has a good color tone, and a yellowness index (YI value) of a 5 mm-thick sheet press-molded at 160° C. is preferably 20 or less, and more preferably 17 or less.

[0055] PHB can be molded into various molded articles such as fibers, threads, ropes, woven fabrics, knitted fabrics, nonwoven fabrics, paper, films, sheets, tubes, plates, rods, containers, bags, parts, foams, etc. The molded articles can be suitably used in agriculture, fisheries, forestry, horticulture, medicine, sanitary products, clothing, non-clothing, packaging, and other fields.

[0056] In the case of other biodegradable plastics produced by microorganisms, water with the above-mentioned calcium ion concentration of 4.5 mg / L or less can be used in the process of crushing or solubilizing the microbial cells containing the biodegradable plastic and separating the biodegradable plastic. EXAMPLES

[0057] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0058] [Measurement and evaluation methods] The measurement and evaluation methods used in the examples and comparative examples are shown below.

[0059] (chromaticity) 200mL of the prepared cleaning water was placed in a special measuring container, and a measuring device (Kasahara Chemical Industries Co., Ltd. TCR-5Z) was gently immersed in the container while being careful not to create air bubbles, and the color of the cleaning water was measured. According to the Ministry of Health, Labor and Welfare's "Tap Water Quality Standards," tap water has a color of 5 or less.

[0060] (thermal stability) Thermal stability was evaluated based on the weight-average molecular weight retention of the polyhydroxybutyric acid resin after heating at 160°C for 20 minutes. If the weight-average molecular weight retention was 70% or more, the thermal stability was judged to be good, and if the weight-average molecular weight retention was less than 70%, the thermal stability was judged to be poor. Weight average molecular weight retention rate (%) = (weight average molecular weight of PHB after heating / weight average molecular weight of PHB before heating) × 100 <Weight average molecular weight of PHB before heating> 10 mg of polyhydroxybutyric acid resin powder was dissolved in 10 mL of chloroform, and insoluble matter was removed by filtration. The solution (filtrate) was subjected to molecular weight measurement using a Shimadzu GPC system equipped with "Shodex K805L (300x8mm, 2 connected)" (Showa Denko) and chloroform as the mobile phase. Commercially available standard polystyrene was used as the molecular weight standard sample. <Weight average molecular weight of PHB after heating> A polyhydroxybutyric acid-based resin sheet was produced by preheating the polyhydroxybutyric acid-based resin powder at 160° C. for 7 minutes and then heating at 160° C. for 20 minutes. The weight average molecular weight of the PHB after heating was measured in the same manner as in the measurement of the weight average molecular weight of the PHB before heating, except that 10 mg of the polyhydroxybutyric acid-based resin sheet was used.

[0061] (Composition of PHB before heating) Approximately 20 mg of polyhydroxybutyric acid-based resin powder was added with 2 mL of sulfuric acid-methanol mixture (15:85) and 2 mL of chloroform, sealed, and heated at 100 ° C for 140 minutes to obtain methyl ester of polyester decomposition product. After cooling, 1.5 g of sodium bicarbonate was gradually added to neutralize it, and the mixture was left until the generation of carbon dioxide gas stopped. 4 mL of diisopropyl ether was added and mixed well, and then centrifuged. The composition of hydroxyalkanoic acid methyl ester of polyester decomposition product in the supernatant was analyzed by capillary gas chromatography to obtain the composition (content) of monomer unit of the obtained polyester. The gas chromatograph used was GC-17A manufactured by Shimadzu Corporation, and the capillary column used was NEUTRA BOND-1 manufactured by GL Science Co., Ltd. (column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 μm). The temperature conditions were as follows: the initial temperature was raised from 100°C to 200°C at a rate of 8°C / min, and then the temperature was raised further from 200°C to 290°C at a rate of 30°C / min.

[0062] (Yellowness Index (YI) Measurement) A press sheet of polyhydroxybutyric acid resin was prepared, and the press sheet was used as a measurement sample. The press sheet of polyhydroxybutyric acid resin was prepared by sandwiching 3.0 g of polyhydroxybutyric acid resin powder between 15 cm square metal plates, inserting metal plates of 0.5 mm thickness into the four corners of the metal plates, setting this in a small experimental press machine (H-15 type manufactured by Takabayashi Rika Co., Ltd.), heating at 160 ° C for 7 minutes, pressing at about 5 MPa for 2 minutes while heating at 160 ° C, and leaving it at room temperature after pressing to harden the polyhydroxybutyric acid resin. The YI value was measured using a color difference meter "SE-2000" (manufactured by Nippon Denshoku Co., Ltd.) by placing the press sheet on a 30 mm measuring plate and covering it with a white standard plate. If the YI value was 20 or less, it was judged to have good color tone, and if the YI value was more than 20, it was judged to have poor color tone.

[0063] (Cleaning water production example 1) Wastewater discharged from the production of polyhydroxybutyrate resin was treated by microorganisms for anaerobic and aerobic processes, then pretreated and filtered using a membrane separation activated sludge method with UF membranes, and then filtered using an RO membrane. In the anaerobic process, the acid generating bacteria in the acid generating tank (pH 7.1) decompose high molecular weight carbohydrates and lipids into organic acids and lower alcohols, and then the wastewater is passed through an EGSB type methane generation reaction tank (load capacity 15 kg-CODcr / m 3 The aerobic treatment was carried out by decomposing organic acids and lower alcohols into methane gas and carbon dioxide gas by the action of granular methanogens in a 2000-methane tank (1000-methanol tank / day). The aerobic treatment was carried out by decomposing the organic matter that was not decomposed in the anaerobic treatment by the action of aerobic bacteria in 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 reaeration tank (activated sludge treatment tank). The pretreatment filtration using the membrane separation activated sludge method was 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 0% when a pressure of 3000 kPa was applied at 20°C) in the reaeration tank (activated sludge treatment tank). Water was passed through the UF membrane at a filtration linear velocity of 0.86 to 1.15 m / day. The UF membrane permeate was collected and sent to an RO membrane (material: composite polyamide, manufactured by Nitto Denko, MgSO4 rejection rate 99.7% when pressure of 3000 kPa was applied at 20°C) with a water temperature of 30°C, transmembrane pressure difference (permeation pressure) of 0.7 to 1.15 MPa, and a permeation rate of 0.75 to 0.85 L / min. The water that did not permeate the RO membrane was returned to the water collection tank, and the permeated water was used as washing water. The UF membrane permeate was permeated through the RO membrane for 97 minutes, and 70% by weight of the UF membrane permeate was permeated. The RO membrane permeate from the beginning of the permeation to 13 minutes was collected to obtain washing water 1.

[0064] (Cleaning water production example 2) Washing water 2 was obtained in the same manner as in Production Example 1, except that the permeated water from 90 minutes to 97 minutes was collected from the water that had permeated the RO membrane.

[0065] (Cleaning water production example 3) The water permeated through the RO membrane under the same conditions as in Production Example 1 was further permeated through an RO membrane (material: composite polyamide, manufactured by Nitto Denko Corporation, MgSO4 rejection rate 99.7% when a pressure of 3000 kPa was applied at 20°C) at a water temperature of 30°C, a permeation pressure of 0.8 MPa, and a permeation rate of 1.25 L / min to obtain wash water 3. The amount of permeated water was 90% of the amount of the UF membrane permeated water.

[0066] (Cleaning Water Production Example 4) The UF membrane permeate was passed through the UF membrane under the same conditions as in Production Example 1, and collected to obtain wash water 4.

[0067] (Cleaning Water Production Example 5) The UF membrane permeate was collected under the same conditions as in Production Example 1, and permeated through an NF membrane (material: composite polyamide, manufactured by Synder, MgSO4 rejection rate 96.1% when a pressure of 3000 kPa was applied at 20°C) at a water temperature of 20°C, a permeation pressure of 3 MPa, and a permeation rate of 30 to 300 g / hour, to obtain wash water 5. The amount of permeated water was 75% of the amount of the UF membrane permeate.

[0068] (Cleaning Water Production Example 6) Wash water 6 was obtained by treating industrial water (manufactured by Kaneka) with ion exchange resins (manufactured by Organo, strong acid cation resin and strong base anion resin).

[0069] (Washing Water Production Example 7) The UF membrane permeate was collected under the same conditions as in Production Example 1, and permeated through an NF membrane (material: composite polyamide, manufactured by Synder, MgSO4 rejection rate 6.7% when a pressure of 3000 kPa was applied at 20°C) at a water temperature of 20°C, a permeation pressure of 3 MPa, and a permeation rate of 100 to 550 g / hour, to obtain wash water 7. The amount of permeated water was 75% of the amount of the UF membrane permeate.

[0070] The calcium ion concentrations, sodium ion concentrations and color in the wash waters 1 to 7 are shown in Table 1 below.

[0071] [Table 1]

[0072] [Example 1] (Preparation of bacterial culture solution) The Ralstonia eutropha KNK-005 strain described in paragraph

[0049] of International Publication No. 2008 / 010296 was cultured by the method described in paragraphs

[0050] to

[0053] of the same document to obtain a bacterial culture solution containing bacterial cells containing PHB (poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with a 3HH content of 11.5 mol %). Ralstonia eutropha is currently classified as Capriavidus necator.

[0073] (Sterilization) The bacterial culture solution obtained above was sterilized by heating and stirring at an internal temperature of 60 to 80° C. for 20 minutes.

[0074] (Steps (a) and (b)) Sodium dodecyl sulfate was added to the sterilized bacterial culture solution obtained above to a concentration of 0.2% by weight. Furthermore, washing water in which sodium hydroxide was dissolved was added to adjust the pH to 11.0, and the mixture was then kept at 50°C for 1 hour. After that, high-pressure homogenization was performed at a pressure of 44 to 54 MPa using a high-pressure homogenizer (Nilosoavi High-Pressure Homogenizer Model PA2K). The same weight of washing water was added to the bacterial cell lysate obtained after high-pressure lysis. After centrifuging, the supernatant was removed and the mixture was concentrated twice. To this concentrated aqueous suspension of PHB, washing water (pH 11.0) containing sodium hydroxide was added to the same weight as the removed supernatant, and centrifuged. Next, the supernatant was removed, and washing water was added to suspend the mixture, and 0.2% by weight of sodium dodecyl sulfate and 1 / 100 weight of protease (Novozymes, Esperase) of PHB were added, and the mixture was stirred for 2 hours while maintaining pH 10.0 and 50°C. Thereafter, the supernatant was removed by centrifugation, and the mixture was concentrated 5 times. To this concentrated aqueous suspension of PHB, washing water (pH 11.0) containing sodium hydroxide was added to the same weight as the removed supernatant, and centrifuged. The same operation was repeated 5 times, and the supernatant was removed, and the polyhydroxybutyric acid resin concentration was adjusted to 52% by weight. In steps (a) and (b), wash water 1 was used as the wash water.

[0075] (Process (c)) To the aqueous suspension of polyhydroxybutyric acid resin obtained above (solid concentration 52% by weight), 1 phr (1 part by weight per 100 parts by weight of polyhydroxybutyric acid resin present in the aqueous suspension) of a dispersant (polyethylene glycol-polypropylene glycol-block ether type nonionic surfactant, product name "Pronon #208", NOF Corp.) was added, and then the solid concentration was adjusted to 30% by weight with distilled water. After stirring this liquid for 30 minutes, sulfuric acid was added to adjust the pH until it stabilized at 4. The aqueous suspension of polyhydroxybutyric acid resin obtained was dried at 60°C for 12 hours to produce polyhydroxybutyric acid resin powder.

[0076] [Example 2] A polyhydroxybutyric acid-based resin powder was obtained in the same manner as in Example 1, except that in steps (a) and (b), washing water 2 was used as the washing water.

[0077] [Example 3] A polyhydroxybutyric acid-based resin powder was obtained in the same manner as in Example 1, except that in steps (a) and (b), washing water 3 was used as the washing water.

[0078] [Comparative Example 1] A polyhydroxybutyric acid-based resin powder was obtained in the same manner as in Example 1, except that in steps (a) and (b), washing water 4 was used as the washing water.

[0079] [Example 4] A polyhydroxybutyrate-based resin powder was obtained by the same procedure as in Example 1, except that a bacterial cell culture solution containing bacterial cells containing PHB (poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with a 3HH content of 10.9 mol %) was used in the preparation of the bacterial cell culture solution, and washing water 2 was used as the washing water in steps (a) and (b).

[0080] [Example 5] A polyhydroxybutyric acid-based resin powder was obtained in the same manner as in Example 4, except that in steps (a) and (b), washing water 5 was used as the washing water.

[0081] [Example 6] A polyhydroxybutyric acid-based resin powder was obtained in the same manner as in Example 4, except that in steps (a) and (b), washing water 6 was used as the washing water.

[0082] [Comparative Example 2] A polyhydroxybutyric acid-based resin powder was obtained in the same manner as in Example 4, except that in steps (a) and (b), washing water 7 was used as the washing water.

[0083] [Comparative Example 3] A polyhydroxybutyric acid-based resin powder was obtained in the same manner as in Example 4, except that washing water 4 was used as the washing water in steps (a) and (b).

[0084] The thermal stability and YI value of the polyhydroxybutyric acid-based resin powders obtained in the Examples and Comparative Examples were measured and evaluated as described above, and the results are shown in the following Table 2. In the following Table 2, the 3HH content before heating means the content of 3HH in the polyhydroxybutyric acid-based resin before heating.

[0085] [Table 2]

[0086] In the examples, by using washing water having a calcium ion concentration of 4.5 mg / L or less in steps (a) and (b), the polyhydroxybutyric acid-based resin powder has high thermal stability and good color tone. In addition, if the calcium ion concentration in the washing water is low, the polyhydroxybutyric acid-based resin powder tends to have excellent thermal stability and color tone. In Comparative Example 2, water with high transparency was used, but the YI value exceeded the standard value, which showed that the calcium ion concentration has a stronger effect on the YI value of the polyhydroxybutyric acid-based resin powder. In particular, in Examples 1 to 5, in which treated water obtained by treating wastewater discharged in the polyhydroxybutyric acid-based resin production process was used as the washing water, the polyhydroxybutyric acid-based resin powder had high thermal stability and good color tone, making it possible to reduce the amount of water used, and it was possible to produce a polyhydroxybutyric acid-based resin with good color tone and thermal stability while reducing the environmental load.

[0087] The present invention includes, but is not limited to, one or more of the following embodiments. [1] A step (a) of disrupting or solubilizing microbial cells containing a polyhydroxybutyrate-based resin; and A step (b) of separating the polyhydroxybutyric acid-based resin in the composition obtained in the step (a), A method for producing a polyhydroxybutyric acid-based resin, comprising the steps of: (a) and (b) using water having a calcium ion concentration of 4.5 mg / L or less. [2] The method for producing a polyhydroxybutyric acid-based resin according to [1], wherein the water used in steps (a) and (b) has a sodium ion concentration of 450 mg / L or less. [3] The method for producing a polyhydroxybutyric acid-based resin described in [1] or [2], wherein the water used in step (a) and step (b) is obtained by subjecting wastewater discharged from the production process of a polyhydroxybutyric acid-based resin to anaerobic and aerobic treatment using microorganisms, followed by a pretreatment filtration process using a membrane separation activated sludge method and a filtration process using a calcium ion removal membrane. [4] The method for producing a polyhydroxybutyric acid-based resin described in any one of [1] to [3], wherein the calcium ion removal membrane is one or more selected from the group consisting of an NF membrane and an RO membrane. [5] The method for producing a polyhydroxybutyric acid-based resin according to [4], wherein the NF membrane or RO membrane has an MgSO4 rejection rate of 60% or more and 100% or less when subjected to a pressure of 3000 kPa at 20°C. [6] A method for producing a polyhydroxybutyric acid-based resin according to any one of [1] to [5], wherein step (a) includes at least one treatment selected from the group consisting of a chemical treatment and a physical crushing treatment. [7] The method for producing a polyhydroxybutyric acid-based resin described in [6], wherein the chemical treatment is a chemical treatment with at least one selected from the group consisting of alkaline compounds, proteolytic enzymes, and cell wall-degrading enzymes. [8] A method for producing a polyhydroxybutyric acid-based resin according to any one of [1] to [7], wherein the polyhydroxybutyric acid-based resin is poly(3-hydroxybutyric acid-co-3-hydroxyhexanoic acid). [9] A method for producing a polyhydroxybutyric acid-based resin described in any one of [1] to [8], wherein the polyhydroxybutyric acid-based resin has a weight average molecular weight retention of 70% or more when heat-treated at 160°C for 20 minutes.

[10] A method for producing a polyhydroxybutyric acid-based resin according to any one of [1] to [9], wherein a sheet having a thickness of 5 mm obtained by press-molding the polyhydroxybutyric acid-based resin at 160°C has a yellowness index (YI value) of 20 or less.

Claims

1. (a) 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), In the steps (a) and (b), water having a calcium ion concentration of 4.5 mg / L or less is used; The method for producing a polyhydroxybutyric acid-based resin is characterized in that the water used in steps (a) and (b) is obtained by subjecting wastewater discharged from a production process of a polyhydroxybutyric acid-based resin to anaerobic and aerobic treatment using microorganisms, followed by a pretreatment filtration process using a membrane separation activated sludge method and a filtration process using a calcium ion removal membrane.

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

3. 3. The method for producing a polyhydroxybutyric acid-based resin according to claim 1, wherein the calcium ion-removing membrane is at least one selected from the group consisting of an NF membrane and an RO membrane.

4. The NF membrane or RO membrane has a MgSO 4 4. The method for producing a polyhydroxybutyric acid-based resin according to claim 3, wherein the rejection rate is 60% or more and 100% or less.

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

6. 6. The method for producing a polyhydroxybutyric acid resin according to claim 5, wherein the chemical treatment is a chemical treatment with at least one selected from the group consisting of alkaline compounds, proteolytic enzymes and cell wall decomposing enzymes.

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

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

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

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