Method for producing polyhydroxyalkanoic acid aggregate

Abstract

To provide a method for efficiently producing a high-purity aggregate of PHA from a culture solution containing polyhydroxyalkanoic acid (PHA) accumulated in bacterial cells.　An aggregate of PHA is obtained by a very simple method in which hydrogen peroxide and a surfactant are added to a culture solution containing bacterial cells containing PHA and stirred under prescribed conditions.

Description

Method for producing polyhydroxyalkanoic acid aggregates

[0001] The present invention relates to a method for producing polyhydroxyalkanoic acid aggregates from a culture medium by culturing a microorganism that produces polyhydroxyalkanoic acid within the cells, and to the aggregates obtained thereby.

[0002] Polyhydroxyalkanoic acids (hereinafter referred to as "PHAs") are produced and accumulated within the cells of many microbial species. PHAs are biodegradable thermoplastic polyesters, and with growing concern about environmental issues, they are attracting attention as non-petroleum-derived plastics, and their practical application is anticipated. Representative examples of PHAs include polyhydroxyalkanoic acids (PHAs) containing polyhydroxybutyric acid (PHB, poly(3-hydroxybutyric acid)).

[0003] However, for practical use, it is necessary to develop an efficient manufacturing method on an industrial scale. PHA produced by microorganisms usually forms granules and accumulates within the cells of the microorganisms, so a process is required to separate and purify the PHA from the cells of the microorganisms. In other words, in order to use it as a plastic, it is necessary to remove as many components other than PHA as possible and increase the purity of the PHA.

[0004] Various methods for separating and purifying PHA have been proposed, including a method that combines disruption of PHA-containing microbial cells with surfactant treatment (Patent Document 1), a method that involves heat treatment under alkaline conditions followed by disruption treatment (Patent Document 2), and a method that involves solubilizing biological components other than PHA by treatment with sodium hypochlorite, enzymes, or the like, and then separating the PHA (Patent Document 3).

[0005] The means for separating PHA from the suspension obtained by the above-mentioned treatments include separation procedures such as centrifugation or filtration, or drying procedures such as spray drying. However, if the PHA particles produced by the bacterial cells are separated without agglomeration, there is a problem that the amount of fine powder increases, making it difficult to handle as a product.

[0006] To address the handling issue, it is conceivable to flocculate PHA by adding salt or the like. However, it is extremely difficult to flocculate and separate high-purity PHA from an aqueous suspension containing a large amount of microbial components such as proteins. Even if aluminum sulfate, which is widely used in activated sludge treatment, is used, it flocculates almost all components in the aqueous suspension, making it impossible to selectively flocculate PHA. Furthermore, even if it is possible to selectively flocculate PHA using a polymer flocculant or the like, it is difficult to separate these additives from PHA, which affects the quality of the polymer material.

[0007] Other methods include heating a PHA suspension (Patent Document 4) and repeating heating and cooling (Patent Document 5), but these involve heating to near the melting point of the PHA, which poses the problem of a decrease in the molecular weight of the PHA.

[0008] After dissolving PHA in an organic solvent, adding an organic solvent or water with low solubility allows for precipitation to obtain a highly pure PHA. Examples of extracting solvents that can be used include lower ketones (Patent Document 6) and tetrahydrofuran (Patent Document 7). However, the dissolution-precipitation method has problems such as the need for a large amount of organic solvent and the fact that the molecular weight of the PHA decreases due to heating during dissolution.

[0009] In addition, a method has been reported in which PHA aggregates are obtained by carrying out a purification process to remove bacterial cell-derived impurities from the bacterial cell culture solution to a certain concentration or below, and then adjusting the PHA aqueous suspension to an acidic range (Patent Document 8). This method has the advantage of reducing the amount of organic solvent used and not requiring the addition of salts or polymer flocculants or high-temperature treatment, but it requires a purification process and must be made more efficient as an industrial process. Further improvements in handling are also considered necessary.

[0010] Japanese Patent Publication No. 08-502415 International Publication No. 2004 / 065608 Japanese Patent Application Laid-Open No. 2005-348640 Japanese Patent Application Laid-Open No. 2000-502399 Japanese Patent Application Laid-Open No. 2002-517582 Japanese Patent Application Laid-Open No. 10-504460 Japanese Patent Application Laid-Open No. 07-79788 International Publication No. 2010-067543

[0011] Although various methods have been reported as described above, no method for efficiently obtaining high-purity PHA particle aggregates in an industrial process is known, partly because the parameters governing PHA aggregation were unknown.

[0012] The problem to be solved by the present invention is to provide a method for efficiently producing a highly pure PHA aggregate from a culture solution containing PHA accumulated within bacterial cells, and to provide a highly pure PHA aggregate by such an efficient method.

[0013] The present inventors have conducted extensive research to solve the above problems and have found a solution. Specifically, when hydrogen peroxide and a surfactant are added to a culture solution containing PHA-containing bacteria, and the pH is controlled to 8 or less, the required stirring power calculated by the Kamei-Hiraoka formula is 2 W / m ３ It was found that PHA aggregates could be obtained by the very simple method of stirring the above.

[0014] More specifically, the present invention provides the following: (1) A method for producing an aggregate of polyhydroxyalkanoic acid, comprising the steps of culturing a microorganism capable of accumulating polyhydroxyalkanoic acid within its cells, adding hydrogen peroxide and a surfactant to the obtained culture solution, and stirring the culture solution under conditions controlled at pH 8 or less, until the required stirring power calculated by the Kamei-Hiraoka formula is 2 W / m ３(2) The method according to (1), further comprising a recovery step of recovering aggregates of polyhydroxyalkanoic acid after the stirring step. (3) The method according to (1) or (2), further comprising a sterilization step of sterilizing the microorganisms by heat treatment after the culturing step. (4) The method according to any one of (1) to (3), wherein the pH in the stirring step is controlled between 4.0 and 7.0. (5) The method according to any one of (1) to (4), wherein the surfactant is sodium dodecyl sulfate. (6) The method according to any one of (1) to (5), wherein the amount of hydrogen peroxide added is 2 to 10% by weight. (7) The method according to any one of (1) to (5), wherein the required stirring power calculated by the Kamei-Hiraoka formula is 5 W / m for 5 hours or more from the time of adding hydrogen peroxide and surfactant. ３ (8) The method according to any one of (1) to (6), wherein the required stirring power calculated by the Kamei-Hiraoka formula is 10 W / m or more for 5 hours or more from the time of adding hydrogen peroxide and a surfactant. ３(9) The method according to any one of (1) to (8), wherein the liquid temperature in the stirring step is 40°C to 100°C. (10) The method according to any one of (1) to (8), wherein the liquid temperature in the stirring step is 50°C to 80°C. (11) The method according to any one of (1) to (10), wherein the median diameter of the obtained polyhydroxyalkanoic acid aggregates is 1000 μm or more. (12) The method according to any one of (1) to (11), wherein the total nitrogen concentration of the obtained polyhydroxyalkanoic acid aggregates is 500 ppm or less. (13) The method according to any one of (1) to (12), wherein the polyhydroxyalkanoic acid contains 3-hydroxybutyric acid as a monomer unit. (14) The method according to any one of (1) to (13), wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxybutyric acid and 4-hydroxybutyric acid as monomer units. (15) The microorganism is Cupriavidus, Alcaligenes, Ralstonia, Delftia, Comamonas, Hydrogenophaga, Burkholderia, Escherichia, Azotobacter, Methylobacterium, Paracoccos, Pseudomonas, Acinetobacter, Aeromonas, Allochromat. (14) The method according to any one of (1) to (14), wherein the microorganism is a microorganism belonging to a genus selected from the group consisting of the genera Rhodobacter, RhodoCOCCuS, Rhodospirillum, Rickettsia, Sinorhizobium, Sphingomonas, Synechocystis, Thiococcus, Thiocystis, Vibrio, and Wautersia. (15) The method according to (14), wherein the microorganism is Cupriavidus necator. (16) A polyhydroxyalkanoic acid aggregate having a median diameter of 1000 μm or more and a bulk density in a dry state of 0.06 g / mL to 0.20 g / mL.(18) The polyhydroxyalkanoic acid aggregate according to (17), having a bulk density in a dry state of 0.08 g / mL to 0.10 g / mL. (19) The polyhydroxyalkanoic acid aggregate according to (17) or (18), having a total nitrogen concentration of 500 ppm or less.

[0015] According to the present invention, PHA aggregates can be produced from a bacterial culture solution containing PHA by a simple method. The method of the present invention is particularly useful in industrial processes because it significantly reduces the complexity of the process. In addition, since it produces aggregates with large particle sizes that are easy to handle, it is also a great advantage in subsequent processing steps. Furthermore, the PHA aggregates of the present invention have a low amount of impurities and can be used for various purposes, including medical applications.

[0016] 1 shows a scanning electron microscope photograph of a PHA aggregate obtained by the method of the present invention. In Experiment 1, the particle size and coefficient of variation of the PHA aggregate obtained by only one stirring step are measured in a dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.). In Experiment 2, the particle size and coefficient of variation of the PHA aggregate obtained by the dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.) are measured in a dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.). In Experiment 3, the particle size and coefficient of variation of the PHA aggregate obtained by the dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.) are measured in a dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.). In Experiment 4, the particle size and coefficient of variation of the PHA aggregate obtained by the dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.) are measured in a dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.). This shows a chart of the particle size and coefficient of variation of the PHA aggregate obtained in Experiment 5, measured in a dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.). This shows a chart of the particle size and coefficient of variation of the PHA aggregate obtained in Experiment 6, measured in a dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.). This shows a chart of the particle size and coefficient of variation of the PHA aggregate obtained in Experiment 7, measured in a dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.). This shows a chart of the particle size and coefficient of variation of the PHA aggregate obtained in Experiment 8, measured in a dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.). This shows a chart of the particle size and coefficient of variation of the PHA aggregate obtained in Experiment 10, measured in a dry state using a particle size analyzer (LA-960N2, manufactured by Horiba, Ltd.). The particle size and coefficient of variation of the PHA aggregates obtained in Experiment 1 through two stirring steps were measured in a dry state using a particle size measuring device (LA-960N2, manufactured by Horiba, Ltd.) and a chart is shown below.

[0017] The method according to the present invention will be specifically described below.

[0018] The method of the present invention comprises the steps of culturing a microorganism capable of accumulating polyhydroxyalkanoic acid within its cells, adding hydrogen peroxide and a surfactant to the culture solution obtained, and cultivating the culture solution under conditions where the pH is controlled at 8 or less and the required stirring power calculated by the Kamei-Hiraoka formula is 2 W / m ３The process includes a step of stirring the mixture as described above. <PHA> In the present invention, PHA is a general term for polymers having 3-hydroxyalkanoic acid or 4-hydroxyalkanoic acid as a monomer unit. The constituent hydroxyalkanoic acid is not particularly limited, but examples include 3-hydroxypropionate, 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate, and 4-hydroxybutyrate. PHA includes not only homopolymers but also copolymers composed of two or more hydroxyalkanoic acids selected from the group consisting of 3-hydroxypropionate, 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate, and 4-hydroxybutyrate.

[0019] As the copolymer, a two-component copolymer of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) is preferred in terms of physical properties. The composition ratio of each monomer unit constituting the two-component copolymer of 3HB and 4HB is not particularly limited, but when the total of all monomer units is taken as 100 mol%, the 4HB unit is 1 to 99 mol%, preferably 1 to 50 mol%, and more preferably 5 to 25 mol%. <Culturing Step> The method of the present invention uses a microorganism that accumulates PHA. Any microorganism that accumulates PHA can be used without limitation. Microorganisms isolated from nature or deposited in a depository institution for strains (e.g., IFO, ATCC, etc.), or mutants or transformants that can be prepared from them, can be used. Examples of suitable microorganisms include those belonging to the genera Cupriavidus, Alcaligenes, Ralstonia, Pseudomonas, Bacillus, Azotobacter, Nocardia, and Aeromonas. Microorganisms belonging to the genera Aeromonas, Alcaligenes, Ralstonia, and Cupriavidus are particularly preferred. Strains such as A. lipolytica, A. latus, A. caviae, A. hydrophila, and C. necator are particularly preferred, with C. necator being the most preferred.

[0020] Furthermore, when a microorganism does not inherently have the ability to produce PHA or when the production amount is low, the gene encoding the desired PHA synthase and / or its mutant can be introduced into the microorganism, and the resulting transformant can be used.

[0021] In the culturing step, the PHA-accumulating microorganism is cultured in a medium containing a nutrient source. The medium used for culturing can be any medium that allows the microorganism to grow. For example, a medium containing a carbon source such as glucose, fructose, methanol, acetic acid, or butyric acid, a nitrogen source such as ammonium sulfate, ammonium nitrate, or peptone, a phosphate source such as potassium phosphate or sodium phosphate, various minerals, and trace nutrient sources can be used.

[0022] In addition, an antifoaming agent may be added to suppress foaming.

[0023] The culturing conditions may be any conditions that allow the microorganisms used to grow and accumulate PHA. From this viewpoint, the culturing time, aeration conditions, etc. can be appropriately set. The PHA content in the microbial cells after the culturing is not particularly limited, but is preferably 20% or more, more preferably 30% or more, even more preferably 40% or more, and even more preferably 50% or more.

[0024] After the culture is completed, the mixture may be sterilized by heating for several hours at a temperature that will kill the microbial cells. <Agitation Step> The agitation step is carried out by adding hydrogen peroxide and a surfactant.

[0025] The surfactant used is not particularly limited, but an anionic surfactant is preferred, and sodium dodecyl sulfate is particularly preferred. The concentration of the surfactant used is preferably at least twice the net dry cell weight (the cell weight obtained by subtracting the dry weight of PHA from the dry cell weight including PHA) by weight, more preferably at least three times, and particularly preferably at least four times. The upper limit is not particularly limited, but from a cost perspective, it is preferably 10 times or less by weight. As such, the surfactant concentration is determined by measuring the dry cell weight including PHA and the dry weight of PHA. However, the surfactant concentration can also be simply determined from an estimated net dry cell weight. For example, if the net dry cell weight is estimated to be 10 g / L, the surfactant amount can be in the range of 2% to 10% by weight.

[0026] The amount of hydrogen peroxide added is about 2 to 10% by weight, preferably 4 to 8% by weight, based on the amount of the solution. It can also be added in several portions rather than all at once.

[0027] The pH of the liquid in the stirring step is preferably controlled to 8 or less, more preferably within the range of pH 4.0 to 7.0. The liquid temperature in the stirring step is preferably 40 to 100°C, and more preferably 50 to 80°C.

[0028] The type of stirring blade is not limited, but examples include flat paddle blades, inclined paddle blades, Rushton turbine (disk turbine) blades, propeller blades, three-bladed swept (Faudler) blades, anchor blades, helical ribbon blades, screw blades, etc. Regarding the stirring speed, the required stirring power calculated from the Kamei-Hiraoka formula is set to 2 W / m ３ More than 5 W / m is preferable. ３ More preferably, 10 W / m or more ３ More preferably, 20 W / m ３ The above is particularly preferred.

[0029] An antifoaming agent may also be added to suppress foaming.

[0030] The stirring step can be performed two or more times, for example, to further remove impurities derived from the bacterial cells. The second and subsequent stirring steps can be performed under the same conditions as the first, but different conditions may also be used. For example, surfactants may not be used in the second and subsequent stirring steps, and the stirring time may be shorter than the first. By performing the stirring step two or more times, PHA aggregates of even higher purity can be obtained, while maintaining the size (median particle size) and shape of the aggregates. <PHA Aggregate Recovery Step> PHA aggregates can be appropriately recovered from the liquid after the stirring step by a method such as filtration. Since aggregates with large particle sizes are obtained, they are easy to handle and to recover. After recovery, the PHA can be washed with water or the like and dried to obtain dried aggregates, if necessary. <PHA Aggregates> The method of the present invention makes it possible to produce the above-mentioned high-purity PHA aggregates from a bacterial cell culture solution containing PHA by a simple method. The amount of organic nitrogen contained in the PHA aggregate can be measured, for example, using a trace total nitrogen analyzer (TN-2100H, manufactured by Nitto Seiko Analytech Co., Ltd.). The amount of organic nitrogen contained in the PHA aggregate is 500 ppm or less, preferably 400 ppm or less, more preferably 300 ppm or less, even more preferably 200 ppm or less, and particularly preferably 100 ppm or less.

[0031] In the present invention, the PHA aggregates can be prepared by a simple method, and the decrease in molecular weight of the PHA during the separation and purification process is suppressed. The molecular weight of the PHA aggregates is preferably 500,000 or more, more preferably 600,000 or more, and even more preferably 700,000 or more.

[0032] The particle size of the PHA aggregates can be measured in a dry state using, for example, a particle size measuring device (LA-960N2, manufactured by Horiba, Ltd.). The median diameter of the PHA aggregates is preferably 1000 μm or more, and the 10% particle size is preferably 500 μm or more. Although there are no particular upper limits, the present invention can provide PHA aggregates with a median diameter of approximately 10 mm or less.

[0033] The large particle size of the aggregates obtained provides a great advantage in terms of ease of handling in the subsequent processing steps.

[0034] Furthermore, the PHA aggregates of the present invention contain a small amount of impurities and can be used in a variety of applications, including medical applications.

[0035] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0036] (Method of Measuring Viscosity) Viscosity was measured by the following method.

[0037] The liquid was sampled in a 1 L container for measurement. A rotor (Rotor No. 19L / ADP) was attached to a viscometer (TVB-10M manufactured by Toki Sangyo Co., Ltd.), and the rotor was immersed in the measurement liquid and rotated at 100 rpm to measure the viscosity. The measurement was carried out at a liquid temperature of 65°C.

[0038] (Method of Calculating Required Power for Mixing) The required power for mixing (P) is calculated by the following formula.

[0039] The power number (Np) was calculated using the Kamei-Hiraoka formula below.

[0040] The equipment conditions are as follows: d: blade diameter = 0.168 m b: blade width = 0.025 m D: tank diameter = 0.33 m H: liquid depth = 0.093 m n ｐ : Number of blades = 2 θ: Blade installation angle = 90°

[0041] (Method for Measuring Bulk Density) The bulk density was measured by the following method.

[0042] The dried PHA aggregates were passed through a sieve with a mesh size of 4 mm to remove aggregates of 4 mm or larger. Then, 100 mL of the PHA aggregates were filled into a 100 mL measuring cylinder, and the weight of the filled PHA aggregates was measured using a precision balance. The measured weight (g) was divided by 100 (mL) to calculate the bulk density (g / mL).

[0043] <Experiment 1> [Preparation of culture medium] Preparation of a culture medium containing PHA (P(3HB-co-4HB)) consisting of 3-hydroxybutyric acid and 4-hydroxybutyric acid using Cupriavidus necator H16 strain (ATCC 17696) KH２ P.O. ４ 2.72g / L, Na ２ HPO ４ 4.26g / L, NaHCO ３ 0.3g / L, (NH ４ ) ２ SO ４ 2 g / L, MgSO ４ ・7H ２ Fructose and ε-caprolactone were added to six 1-L Erlenmeyer flasks containing 420 mL of a sterilized medium consisting of 0.2 g / L of ethanol, 0.2 g / L of yeast extract, and 3.5 mL / L of mineral solution, so that the concentrations were 7.25 g / L and 5 g / L, respectively, and 2 mL of the frozen bacterial cell suspension was inoculated thereto. The mixture was cultured at 30°C and 252 rpm for 96 hours to prepare a main culture medium (preculture medium).

[0044] The mineral solution is: FeC ６ H ５ O ７ ・xH ２ O6g / L, ZnSO ４ ・7H ２ O2g / L, CuSO ４ ・5H ２ O0.1g / L, MnCl ２ ・4H ２ O1g / L, KI0.1g / L, (NH ４ ) ６ Mo ７ O ２４ ・4H ２ O0.1g / L, CoCl ２ ・6H ２ O 0.1 g / L, H ３ BO ３ 0.2g / L, NaCl5g / L, CaCl ２ ・2H ２ The medium was prepared by dissolving 04 g / L in water. ４ ) ２ SO ４The amount of carbon source was changed to 7.3 g / L, and Pluronic L101 (antifoaming agent: manufactured by ADEKA) was added to a concentration of 0.08 wt %. 250 L of this medium was prepared in a 500 L jar fermenter and sterilized. 2.52 L of the culture medium was inoculated, and 42 mass % fructose solution and ε-caprolactone were aseptically fed through a sterile filter (PTFE 0.2 μm pores). The feed rate and feeding ratio of the carbon source could be set arbitrarily, but to avoid the microorganisms not consuming all the carbon source and leaving excess carbon source in the culture tank, which could cause bacterial growth to stop, the culture was started at a low flow rate of approximately 0.2 to 2.0 g / h L for the fructose solution and 0.03 to 0.4 g / h L for the ε-caprolactone, and the feed rates were then increased stepwise or continuously in accordance with the growth of the bacterial cells. The aeration rate was 50 to 100 L / min, the stirring speed was 130 to 153 rpm, the culture temperature was 36°C, the lower limit of culture pH was 6.0, and 4 M aqueous sodium hydroxide solution was used as an alkali for pH adjustment. The culture was completed approximately 170 hours after the start of culture.

[0045] The resulting culture solution was heated and stirred at 60° C. for 80 minutes to sterilize it.

[0046] During or after the cultivation, the cells and the culture supernatant were collected by centrifugation, and the cells were frozen at -20°C and then freeze-dried.

[0047] The freeze-dried cells were used for PHA composition analysis and PHA molecular weight analysis.

[0048] The PHA composition analysis and PHA content in the cells were carried out by analyzing the methyl esters derived from the monomer units constituting the PHA by gas chromatography after methyl esterification.

[0049] A portion of the sterilized bacterial culture was sampled and centrifuged to remove the supernatant, recovering the bacterial cells and PHA. The samples were frozen at -20°C and then lyophilized. The total nitrogen concentration in the PHA aqueous suspension was measured using an organic elemental analyzer (FlashSmart, Thermo Fisher Scientific). The organic nitrogen concentration in the PHA aqueous suspension was 63,500 ppm per polymer weight. [Production of PHA Aggregates] The sterilized bacterial culture was transferred to a 30-L glass-lined reactor, immersed in a water bath set at 75.0°C, and the culture temperature was increased. When the internal temperature reached 70°C, sodium dodecyl sulfate (hereinafter referred to as "SDS") was added in an amount four times the net bacterial cell weight (the bacterial cell weight calculated by subtracting the weight of polyhydroxyalkanoic acid from the bacterial cell weight including polyhydroxyalkanoic acid). Next, 35% aqueous hydrogen peroxide was added to a concentration of 6% by weight. The reaction started when the addition of hydrogen peroxide water was completed, and was continued for 21 hours. From the start of heating to the end of the reaction, the temperature was maintained at around 70°C, and the required stirring power calculated by the Kamei-Hiraoka formula was 120 W / m ３ The mixture was stirred at 80°C, and the pH was controlled at 6.0 by adding a 15% by weight aqueous solution of sodium hydroxide.

[0050] Five hours after the start of the reaction, the reaction solution was sampled, (1+4) sulfuric acid was added, and the hydrogen peroxide concentration was measured by oxidation-reduction titration using 0.02 mol / L potassium permanganate solution. The amount of hydrogen peroxide consumed was added to the reaction solution so that the hydrogen peroxide concentration in the reaction solution became 6 wt%.

[0051] 21 hours after the start of the reaction, the aeration rate was 15 cc / cm ２ The reaction solution was subjected to solid-liquid separation using a filter cloth of 1 / sec, and the PHA aggregates were recovered. The recovered PHA was washed with pure water and dried, and the recovery rate was calculated.

[0052] In Experiment 1, a second stirring step was performed.

[0053] The recovered PHA and 5 L of pure water were transferred to a 10 L medium bottle, and the medium bottle was immersed in a hot water bath set to 75.0 ° C. to begin heating the PHA aqueous suspension. When the internal temperature reached 70 ° C., 35% hydrogen peroxide solution was added to make the concentration 6 wt %. The reaction started when the addition of hydrogen peroxide solution was completed, and the reaction was allowed to proceed for 7 hours. From the start of heating until the end of the reaction, the temperature was maintained at around 70 ° C., and the pH was controlled at 7.5 by adding a 5 wt % sodium hydroxide aqueous solution.

[0054] 7 hours after the start of the reaction, the aeration rate was 15 cc / cm ２ The reaction solution was subjected to solid-liquid separation using a filter cloth of 1 / sec, and the PHA aggregates were recovered. The recovered PHA was washed with pure water until the hydrogen peroxide concentration in the filtrate was 2% or less, and then washed with ethanol and dried, and the recovery rate was calculated.

[0055] The dried PHA aggregates were passed through a sieve with a mesh size of 4 mm to remove aggregates of 4 mm or more. The particle size and coefficient of variation of the PHA aggregates were then measured in a dry state using a particle size measuring device (LA-960N2, manufactured by Horiba, Ltd.).

[0056] The total nitrogen concentration of the PHA aggregates was measured using a trace total nitrogen analyzer (TN-2100H, manufactured by Nitto Seiko Analytech).

[0057] <Experiment 2> The required stirring power calculated from the Kamei-Hiraoka formula in the stirring process was 18 W / m ３ The same procedure as in Experiment 1 was carried out except that the stirring step was performed once and the amount of SDS added in the stirring step was three times the net bacterial mass by weight, and the required stirring power calculated by the Kamei-Hiraoka formula was 831 W / m ３ The same experiment as in Experiment 1 was carried out except that the pH control value in the stirring step was set to 4.0, and the required stirring power calculated from the Kamei-Hiraoka formula was set to 831 W / m ３ The same experiment as in Experiment 1 was carried out except that the pH control value in the stirring step was set to 5.0, and the required stirring power calculated from the Kamei-Hiraoka formula was set to 831 W / m ３Experiment 6: The same experiment as experiment 1 was carried out except that the pH control value in the stirring process was set to 7.0 and the stirring process was carried out once. Experiment 7: The required stirring power calculated from the Kamei-Hiraoka formula in the stirring process was set to 2.7 W / m ３ The same experiment as in Experiment 1 was carried out except that the stirring step was performed once. <Experiment 8> The required stirring power calculated from the Kamei-Hiraoka formula in the stirring step was 0.7 W / m ３ Experiment 9 was performed in the same manner as Experiment 1, except that the amount of SDS added in the stirring step was 1 times the net bacterial weight by weight, and the stirring step was performed once. Experiment 10 was performed in the same manner as Experiment 1, except that the pH control value in the stirring step was set to 7.5, and the stirring step was performed once.

[0058] Detailed conditions and measurement results for each experimental system are shown in Tables 1 and 2.

[0059] High-purity PHA aggregates were obtained in all experiments except for Experiment 9. In particular, high-purity PHA aggregates with a median diameter of 1500 μm or more and a total nitrogen concentration of 100 ppm were obtained in Experiments 1 to 3 and 5 to 6. Thus, it was confirmed that high-purity PHA aggregates can be obtained by a very simple method.

[0060] In Experiment 1, the stirring process was carried out twice, and the median diameter and total nitrogen concentration of the resulting PHA aggregates are shown in Table 3. It was confirmed that carrying out the stirring process twice did not significantly change the median diameter of the PHA aggregates, but could significantly reduce the total nitrogen concentration.

[0061]

[0062]

[0063]

Claims

1. A method for producing an aggregate of polyhydroxyalkanoic acid, comprising: a culturing step of culturing a microorganism that accumulates polyhydroxyalkanoic acid within its cells; and a step of adding hydrogen peroxide and a surfactant to the resulting culture solution, and controlling the pH to 8 or less, so that the required stirring power calculated from the Kamei-Hiraoka formula is 2 W / m. ３ a stirring step of stirring the above, in which the surfactant is added in an amount of at least twice the weight of the microbial cell mass obtained by subtracting the dry weight of the polyhydroxyalkanoic acid from the dry cell mass of the microbial cell mass containing the polyhydroxyalkanoic acid.

2. The method of claim 1, further comprising the step of recovering the polyhydroxyalkanoic acid aggregates after the stirring step.

3. The method according to claim 1 or 2, further comprising a sterilization step of sterilizing the microorganism by heat treatment after the culturing step.

4. The method according to claim 1 or 2, wherein the pH in the stirring step is controlled between 4.0 and 7.

0.

5. The method of claim 1 or 2, wherein the surfactant is sodium dodecyl sulfate.

6. The method according to claim 1 or 2, wherein the amount of hydrogen peroxide added is 2 to 10% by weight.

7. For more than 5 hours after adding hydrogen peroxide and surfactant, the required stirring power calculated by Kamei-Hiraoka's formula is 5 W / m ３ The method according to claim 1 or 2.

8. For more than 5 hours after adding hydrogen peroxide and surfactant, the required stirring power calculated by Kamei-Hiraoka's formula is 10 W / m ３ The method according to claim 1 or 2.

9. The method according to claim 1 or 2, wherein the liquid temperature in the stirring step is 40°C to 100°C.

10. The method according to claim 1 or 2, wherein the liquid temperature in the stirring step is 50°C to 80°C.

11. The method according to claim 1 or 2, wherein the median diameter of the resulting polyhydroxyalkanoic acid aggregates is 1000 μm or more.

12. The method according to claim 1 or 2, wherein the total nitrogen concentration of the resulting polyhydroxyalkanoic acid aggregate is 500 ppm or less.

13. The method of claim 1 or 2, wherein the polyhydroxyalkanoic acid contains 3-hydroxybutyric acid as a monomer unit.

14. The method according to claim 1 or 2, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxybutyric acid and 4-hydroxybutyric acid as monomer units.

15. The microorganism is of the genus Cupriavidus, the genus Alcaligenes, the genus Ralstonia, the genus Delftia, the genus Comamonas, the genus Hydrogenophaga, the genus Burkholderia, the genus Escherichia, the genus Azotobacter, the genus Methylobacterium, the genus Paracoccos, the genus Pseudomonas, the genus Acinetobacter, the genus Aeromonas, and the genus Allochromat.

3. The method according to claim 1 or 2, wherein the microorganism is a microorganism of a genus selected from the group consisting of the genera Streptococcus, Azorhizobium, Bacillus, Caulobacter, Chromobacterium, Ectothiorhodospira, Klebsiella, Nocardia, Rhodobacter, Rhodococcus, Rhodospirillum, Rickettsia, Sinorhizobium, Sphingomonas, Synechocystis, Thiococcus, Thiocystis, Vibrio, and Wautersia.

16. The method of claim 15, wherein the microorganism is Cupriavidus necator.

17. A polyhydroxyalkanoic acid aggregate having a median diameter of 1000 μm or more and a bulk density in a dry state of 0.06 g / mL to 0.20 g / mL.

18. The polyhydroxyalkanoic acid aggregate of claim 17, having a bulk density in the dry state of 0.08 g / mL to 0.10 g / mL.

19. The polyhydroxyalkanoic acid aggregate according to claim 17 or 18, having a total nitrogen concentration of 500 ppm or less.

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