JP2009195179A - Method for extracting polyhydroxyalkanoic acid - Google Patents

Abstract

The present invention provides a polyhydroxyalkanoic acid extraction method that is simple and inexpensive to operate and is suitable for mass production.
A polyhydroxyalkanoic acid extraction method according to the present invention is a polyhydroxyalkanoic acid extraction method for extracting polyhydroxyalkanoic acid produced by a microorganism from the microorganism, wherein the polyhydroxyalkane is extracted using a cooling medium. Freezing step S1 for freezing the microorganism that has produced the acid, thawing step S2 for thawing the frozen microorganism, and crushing the thawed microorganism with a cell crushing device, and removing insoluble matter containing the polyhydroxyalkanoic acid Crushing step S3 to be obtained, and extraction step S4 for washing the obtained insoluble matter and extracting the polyhydroxyalkanoic acid.
[Selection] Figure 1

Description

  The present invention relates to a polyhydroxyalkanoic acid extraction method for extracting a polyhydroxyalkanoic acid produced by a microorganism from the microorganism.

  General plastics are widely used because they are manufactured from fossil raw materials such as petroleum and are chemically stable. However, since it is a chemically stable substance, when it is discarded, it is difficult to be biodegraded by microorganisms in the soil or water and remains in the environment for many years. Moreover, since it is manufactured from petroleum etc., if it incinerates, a toxic gas will be discharged | emitted or a lot of carbon dioxide will be discharged | emitted, and it is unpreferable from a viewpoint of environmental conservation.

  In view of such a situation, in recent years, biodegradable plastics that are relatively easily biodegraded by microorganisms and the like have attracted attention. Biodegradable plastics have the same functions as general plastics when they are used, and after use, they are biodegraded into low molecular weight compounds by microorganisms that live in the natural soil and water. Biodegraded to carbon dioxide.

  One such biodegradable plastic is an aliphatic polyester such as polyhydroxyalkanoate. Among the polyhydroxyalkanoic acids, polyhydroxybutyrate is highly biodegradable by microorganisms. For example, the genus Ralstonia, the genus Sinorhizobium, the genus Athiorhodium, the genus Azotobacter, and the Bacillus ( Since it can be produced relatively easily and in large quantities by various microorganisms such as the genus Bacillus, Nocardia, Pseudomonas, Rhizobium, and Spirrillum, it will be used in various fields in the future. It is thought to be used greatly in.

  Here, as a general method for extracting polyhydroxyalkanoic acid produced by a microorganism from the microorganism, there is a Soxhlet extraction method using an organic solvent such as chloroform. In addition, simpler polyhydroxyalkanoic acid extraction methods are described in Patent Documents 1 to 3, for example.

  In Patent Document 1, a microorganism suspension containing polyhydroxybutyric acid is treated with a high-pressure homogenizer to crush the microorganism to cause polyhydroxybutyric acid granules to leak out of the microorganism, and then from this high-pressure homogenizer treatment solution. It describes that microbial constituents other than polyhydroxybutyric acid are separated to obtain a polyhydroxybutyric acid fraction, and the resulting polyhydroxybutyric acid fraction is treated with an oxygen bleach.

  In Patent Document 2, polyhydroxyalkanoic acid (PHA) is produced by a microorganism to obtain a PHA-containing microorganism, and a basic component is added to the PHA-containing microorganism to separate the first PHA fraction. And a step of adding hypochlorite to the first PHA fraction and separating the second PHA fraction.

  Patent Document 3 discloses a method for separating and purifying a polyhydroxy organic acid ester corresponding to the polyhydroxyalkanoic acid of the present invention from a polyhydroxy organic acid ester-accumulating microorganism, which comprises adding a lytic enzyme to a microorganism suspension. After the step of lysing the cell wall, the step of separating and collecting the polyhydroxy organic acid ester granules having a particle size of 0.1 μm or more covered with the granule film present in the cytoplasm, and then proteolysis A method for separating and purifying a polyhydroxy organic acid ester comprising a step of removing a granular film by enzymatic treatment is described.

JP 7-177894 A JP 2005-348640 A JP-A-5-336882

However, the Soxhlet extraction method, for example, requires at least 1 kg of chloroform to extract 1 kg of polyhydroxyalkanoic acid, which is not only expensive, but also uses a large amount of organic solvent such as chloroform. There is a problem that it is not suitable for mass production from the viewpoint of maintenance.
The technique described in Patent Document 1 has a problem that it is not preferable from the viewpoint of environmental protection and mass production because it uses chemicals such as hydrogen peroxide and sodium persulfate and an enzyme bleach.

In the technique described in Patent Document 2, for example, basic components such as sodium hydroxide and potassium hydroxide, and hypochlorites such as sodium hypochlorite and potassium hypochlorite are used. There is a problem that it is not preferable for mass production from the viewpoint of maintenance.
The technique described in Patent Document 3 has a problem that it is not only suitable for mass production but also expensive because a lytic enzyme such as lysozyme which is not expensive and is not mass-produced must be used.
In addition, in the Soxhlet extraction method and Patent Documents 1 and 2, as described above, in order to use an organic solvent and various chemicals, industrial waste production must be performed when mass production is performed. There is a problem that it becomes complicated.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a polyhydroxyalkanoic acid extraction method that is simple and low in cost and suitable for mass production.

(1) The polyhydroxyalkanoic acid extraction method according to the present invention that has solved the above-mentioned problems is a polyhydroxyalkanoic acid extraction method for extracting from a microorganism the polyhydroxyalkanoic acid produced by the microorganism, using a cooling medium. Freezing step of freezing the microorganism that produced the polyhydroxyalkanoic acid, thawing step of thawing the frozen microorganism, crushing the thawed microorganism with a cell crushing device, and containing the polyhydroxyalkanoic acid A crushing step for obtaining a product and an extraction step for extracting the polyhydroxyalkanoic acid by washing the obtained insoluble matter.

  As described above, the microorganisms that have produced polyhydroxyalkanoic acid can be frozen and thawed before being crushed by the cell crushing apparatus, so that the microorganisms can be lysed (killed). Therefore, it becomes possible to crush microorganisms and polyhydroxyalkanoic acid contained therein more finely than before by lysis and subsequent crushing with a cell crushing apparatus. And the high purity polyhydroxyalkanoic acid can be obtained by wash | cleaning the insoluble matter containing the polyhydroxyalkanoic acid obtained by crushing.

(2) In this invention, it is preferable to include the liquid-medium removal process which removes the liquid medium which performed the centrifugation and culture | cultivated the said microorganisms before the said freezing process.
Thus, by removing unnecessary liquid medium in advance before the freezing step, freezing of the microorganism in the freezing step and thawing of the microorganism in the thawing step can be performed in a short time. Moreover, it becomes possible to efficiently perform crushing with a cell crushing apparatus. Furthermore, since the unnecessary liquid medium is removed before the microorganisms and polyhydroxyalkanoic acid are finely crushed by the cell crushing device, the liquid medium is mixed with finely crushed polyhydroxyalkanoic acid when the liquid medium is later removed. Can be prevented and the yield of polyhydroxyalkanoic acid can be further prevented from decreasing.

(3) In the present invention, the thawing in the thawing step is preferably performed at room temperature. In this way, the operation is simple because the frozen microorganisms are simply left at room temperature and thawed.

(4) In the present invention, after the thawing step, it is preferable to perform a washing step of suspending the thawed microorganism in water and centrifuging it. In this way, impurities derived from microorganisms other than polyhydroxyalkanoic acid generated during lysis can be removed.

(5) In this invention, it is preferable that the washing | cleaning in the said extraction process is performed by centrifuging the said insoluble matter once or more. In this way, impurities derived from microorganisms other than polyhydroxyalkanoic acid produced by crushing with a cell crushing device can be removed.

(6) In the present invention, the cooling medium is preferably liquid nitrogen. As described above, when liquid nitrogen is used as a cooling medium, rapid freezing is possible, so that cell walls and cell membranes are easily destroyed by rapidly growing ice crystals. Therefore, lysis of microorganisms can be performed reliably. Further, liquid nitrogen is suitable not only for its low cost, but also from the viewpoint of environmental protection because it is vaporized into nitrogen gas.

(7) In the present invention, the cell disruption apparatus is preferably a high-pressure homogenizer. Thus, when a high-pressure homogenizer is used, microorganisms can be crushed reliably and efficiently, so that polyhydroxyalkanoic acid can be extracted with high purity.

(8) In the present invention, the microorganism is preferably Ralstonia eutropha. Thus, when Ralstonia eutropha is used, it can be easily lysed by freezing and thawing, and polyhydroxyalkanoic acid can be reliably extracted with high purity.

(9) In the present invention, the polyhydroxyalkanoic acid is preferably polyhydroxybutyric acid. In this way, polyhydroxybutyric acid, which is a biodegradable plastic, can be reliably extracted.

According to the method for extracting polyhydroxyalkanoic acid according to the present invention, the microorganism that produced polyhydroxyalkanoic acid is frozen using a cooling medium, thawed, crushed, and then washed to extract the polyhydroxyalkanoic acid. Therefore, the operation is simple and low cost, and it is suitable for mass production.
In particular, since no organic solvent or chemicals are used, it is very suitable from the viewpoint of environmental conservation, and since no enzyme is used, the cost is very low.

  A feature of the present invention is that the cultured microorganism can be frozen at a room temperature after being frozen using a cooling medium such as liquid nitrogen, so that the microorganism can be lysed. An alkanoic acid (polyhydroxyalkanoate; hereinafter simply referred to as “PHA”) can be easily extracted.

  In the present invention, PHA is extracted using a microorganism that has produced PHA. However, the best operation for carrying out the invention is necessary for handling of microorganisms and necessary operations to be performed when extracting PHA from microorganisms. J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York, unless otherwise stated in the form and examples (2001) and standard protocols such as FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. The methods described in the collection, or modified or modified methods thereof can be used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them can be used. Further, those skilled in the art can easily reproduce the present invention from the description of the present specification and the description of the standard protocol collection described above.

Hereinafter, the PHA extraction method according to the present invention will be described in detail with reference to FIG. FIG. 1 is a flowchart showing the flow of the PHA extraction method according to the present invention.
The PHA extraction method according to the present invention is a method for extracting PHA produced by a microorganism from the microorganism, as shown in FIG. 1, a freezing step S1, a thawing step S2, a crushing step S3, and an extraction step. S4 is included.

  Here, the PHA extracted by the PHA extraction method of the present invention includes, for example, polyhydroxybutyric acid, polyhydroxyvaleric acid, polylactic acid, polycaprolactone, polyethylene succinate, polybutylene succinate, polybutylene adipate, polymalic acid and the like. Is included. Of these, polyhydroxybutyrate (hereinafter simply referred to as “PHB”) is preferred in the present invention. Among PHAs, PHB, which is industrially in high demand, can be reliably extracted and obtained. The PHA only needs to be produced by the microbial strain used. For example, the PHA may be produced by a microbial strain transformed so as to be able to produce PHA using a technique such as genetic recombination.

As microorganisms that can be used in the PHA extraction method of the present invention, for example, microorganisms belonging to the genus Ralstonia can be used, and among them, PHB that is industrially in high demand can be obtained. (Ralstonia eutropha) is preferably used. When such microorganisms are used, industrially demanding PHB can be obtained.
The microorganisms that can produce PHA that can be used in the present invention are not limited to those described above. For example, the genus Sinorhizobium, the genus Athiorhodium, the genus Azotobacter, (Bacillus) genus, Nocardia genus, Pseudomonas genus, Rhizobium genus, Spirilum genus and the like can also be used. Furthermore, it is also possible to use a microorganism transformed by a technique such as genetic recombination.
In addition, the production of PHA by these microorganisms can be appropriately produced by culturing under culture conditions and culture methods suitable for various microorganisms, or by performing necessary operations during the culture. Therefore, the method for producing PHA is not particularly limited.

Returning to FIG. 1, the description will be continued.
The freezing step S1 shown in FIG. 1 is a step of freezing a microorganism that has produced PHA using a cooling medium. In addition, since the microorganism which produced PHA is used in freezing process S1, it cannot be overemphasized that there exists culture | cultivation process S11 for making a microorganism produce PHA before the said freezing process S1. In the culturing step S11, culturing can be performed under the culturing conditions and culturing method suitable for the microorganism used as described above.

  When freezing microorganisms in the freezing step S1, the microorganisms may be put in a container and frozen so that the outer surface of the container is in contact with the cooling medium. The cooling medium used for freezing the microorganism is preferably, for example, liquid nitrogen from the viewpoint of freezing speed and cost, but is not limited thereto. For example, it may be left in a freezer at about −20 to −80 ° C. and frozen. In this case, air cooled to about −20 to −80 ° C. becomes a cooling medium.

  The freezing in the freezing step S1 is not limited to a specific temperature or freezing time as long as the microorganism can be frozen. If the amount of microorganisms increases, the temperature of the cooling medium can be lowered or the freezing time can be increased in order to ensure that the microorganisms are frozen. Moreover, when liquid nitrogen is used, it can be reliably frozen by lengthening the immersion time in liquid nitrogen. Freezing conditions can be arbitrarily set by conducting a test or the like in advance.

In addition, it is preferable to perform liquid medium removal process S12 which removes the liquid medium which culture | cultivated microorganisms after culture | cultivation process S11 and before freezing process S1. This is because freezing of microorganisms in the freezing step S1 and thawing of microorganisms in the thawing step S2 described later can be performed quickly.
In the liquid medium removal step S12, for example, centrifugation is performed at 4 ° C. to about 15,000 rpm / minute (rpm) for 3 minutes to precipitate the microorganisms that produced PHA, and the resulting supernatant, ie, liquid medium Can be done by decanting. The above-described centrifugation is not limited to the above-described conditions, as long as the microorganism that has produced PHA and the liquid medium can be separated and only the liquid medium can be discarded. Further, in the liquid medium removing step S12, it is sufficient if the liquid medium can be removed, and the liquid medium removing step S12 can be performed by other means such as filtration.

The next thawing step S2 is a step of thawing the microorganism frozen in the freezing step S1.
The thawing of microorganisms in the thawing step S2 can be performed, for example, by leaving at room temperature. Further, for example, in order to shorten the thawing time, the thawing can be performed by heating to about 30 to 40 ° C. Thus, when the frozen microorganism is thawed, the microorganism is killed and lysed. Therefore, PHA encapsulated in the microorganism can be efficiently released outside the microorganism by the crushing step S3 described later. Note that if the thawing temperature in the thawing step S2 is higher than the above-described temperature, the PHA may be reduced in molecular weight. Therefore, it is preferable to prevent the temperature from becoming too high.
Further, in order to ensure that the microorganisms are lysed in the thawing step S2, after thawing the microorganisms, it is preferable to leave them at room temperature for about 1 hour. It should be noted that the standing time is not limited as long as the microorganism can be lysed, and can be appropriately changed depending on the amount of the frozen microorganism.

In addition, it is preferable to perform washing | cleaning process S21 which suspends the microorganisms thawed | decompressed with water after this thawing | decompression process S2, and centrifuges.
In the washing step S21, for example, about 1 to 5 times the amount of bacterial cells, specifically about 2 times the amount of distilled water is added and suspended, and centrifuged at 4 ° C to about room temperature at 15000 rpm for 3 minutes. It is good to carry out once or more. In addition, the above-mentioned distilled water should just add the quantity which can suspend microorganisms, and can be suitably changed with the kind and microbial cell quantity of the microorganisms to suspend. Although the purity of the obtained PHA may increase as the number of times of performing the washing step S21 by centrifugation increases, the yield may decrease. Therefore, the washing step S21 by centrifugation is preferably performed about 1 to 5 times, specifically about 3 times.

The next crushing step S3 is a step of crushing the microorganisms thawed in the thawing step S2 with a cell crushing device to obtain an insoluble matter containing PHA. The insoluble matter refers to a substance that does not dissolve in the suspension when the microorganisms are crushed in the crushing step S3. At this stage, the microorganisms are only crushed and are not washed. include.
The cell crushing apparatus used in the crushing step S3 may be any device as long as it can destroy the cell wall or cell membrane of the microorganism and release the PHA contained in the microorganism to the outside of the microorganism. A high-pressure homogenizer that shears and crushes microorganisms by passing through a small fixing hole at high speed can be suitably used. When such a high-pressure homogenizer is used, the microorganisms can be quickly crushed, processed in large quantities, and inexpensively. For example, when a high-pressure homogenizer is used, the thawed microorganism is suspended or concentrated in water to obtain a suspension with an appropriate concentration according to the attached instruction. For example, the crushing pressure is about 276 MPa (40 kpsi). Can be carried out by repeating crushing for 5 minutes twice.
The cell crushing apparatus that can be used in the present invention is not limited to this, and an ultrasonic cell crushing apparatus that crushes microorganisms by ultrasonic waves, a bead-type cell crushing apparatus that crushes microorganisms by beads, and the like are also suitable. Can be used.

The extraction step S4 to be performed next is a step of extracting PHA by washing the insoluble matter containing PHA obtained in the crushing step S3.
The washing in the extraction step S4 is preferably performed by centrifuging the obtained insoluble matter at 15,000 rpm for 3 minutes at 4 ° C. to room temperature at least once as described above. When the purity of the PHA washed in this way is low, washing may be repeated a plurality of times if necessary by adding distilled water again to the PHA obtained by the above-mentioned centrifugation and suspending it. In addition, although the purity of obtained PHA may become high, so that the frequency | count of washing | cleaning by centrifugation increases, there exists a possibility that a yield may become low. Therefore, it is better to perform washing by centrifugation in the extraction step S4 about 1 to 3 times.

Next, examples in which the effects of the present invention have been confirmed will be described.
(1) Culture of Ralstonia eutropha Ralstonia eutropha was used as a microorganism producing PHB. Ralstonia Eutropha was purchased from Technosurga.
As a liquid medium for cultivating Ralstonia eutropha, NB medium (meat extract 1%, bactopeptone 1%, NaCl 0.5%, pH 7.0) was used for pre-culture with 5 mL and pre-culture with 100 mL. Further, the main culture in the fermenter used an MSM medium having the composition shown in Table 2 to which TES having the composition shown in Table 1 was added. The MSM medium was adjusted to pH 7 using an aqueous hydrochloric acid solution or an aqueous sodium hydroxide solution. Such NB medium and MSM medium were autoclaved at 120 ° C. for 20 minutes and used for cultivation of Ralstonia / eutropha.

  Ralstonia eutropha was precultured at 5 mL in an NB medium prepared by dissolving 8 g of Nutrient Browth reagent per liter, and precultured at 100 mL. The pre-culture and the pre-culture were both performed at 34 ° C. for 24 hours with 120 rpm swirling.

  Three 100 mL of the preculture solution obtained was put into a fermenter (CULLAN CLN-10000 manufactured by MBS), and main culture was performed with the fermenter. In the main culture using a fermenter, 1.7 L of the aforementioned MSM medium was used, and the total culture volume was adjusted to 2.0 L with 300 mL of the preculture solution. The conditions of the main culture in the fermenter were 32 ° C. and 48 hours. The stirring speed was adjusted in the range of 200 to 350 rpm so that the dissolved oxygen was sufficiently distributed in the MSM medium.

The main culture in fermenter is based on fed-batch culture. The main nutrient carbon source is fed with 40% Na-Gluconate aqueous solution so that the concentration in the MSM medium is maintained at about 5%, and the nitrogen source is MSM. A 20% NH ₄ Cl aqueous solution was fed to maintain the concentration in the medium at about 0.05%. A 4N hydrochloric acid aqueous solution and a 4N NaOH aqueous solution were appropriately added so as to maintain a pH of about 7 at all times to adjust the pH.

(2) Freezing, thawing, crushing and extracting PHB of Ralstonia / eutropha After completion of the main culture in the fermenter, 500 mL of the main culture solution is taken and centrifuged at 15,000 rpm for 3 minutes at 4 ° C. to produce PHB. We obtained wet cells of Ralstonia eutropha.
Thereafter, the centrifuge tube containing the wet cells was immersed in liquid nitrogen for 1 minute to freeze the wet cells.
After immersion in liquid nitrogen, the centrifuge tube containing the frozen wet cells was left at room temperature for 60 minutes to thaw the wet cells. With the thawing, the wet cells were lysed and became highly viscous wet cells.

Then, the wet cells sufficiently left at room temperature were suspended in twice the amount of distilled water, and the wet cells were washed by repeating the centrifugation at 15000 rpm for 3 minutes at 4 ° C. three times.
Thereafter, distilled water in an amount sufficient to suspend wet cells was added, and crushing pressure was 276 MPa (40 kpsi) for 5 minutes using a constant system shearing crusher twice.
The insoluble matter containing PHB obtained by crushing was suspended in distilled water, and the centrifugal separation was repeated 3 times at 15000 rpm at 4 ° C. for 3 minutes to extract PHB (Sample 1).

  In addition, in order to measure and compare the purity of PHB in each step, a wet cell body (sample 2) subjected to centrifugation after the completion of the main culture, and a wet cell body (sample 3) that was thawed and lysed after freezing An appropriate amount was taken out. In addition, after centrifugation after the completion of the main culture, without disrupting the freezing and thawing of the wet cells, it is immediately subjected to a crushing treatment under the same conditions as described above using a constant system shearing crusher, and PHB is contained. An insoluble material (sample 4) was obtained.

(3) Measurement of PHB purity The PHB of the obtained samples 1 to 4 was subjected to gas chromatography to determine the purity of PHB. According to 37 (1978), the measurement was performed as follows. First, 1 mL of methyl ester reaction solution prepared in advance (97 mL of methanol produced by Nacalai Tesque, 3 mL of concentrated sulfuric acid, 0.29 g of benzoic acid (both manufactured by Nacalai Tesque)), and 1 mL of chloroform (manufactured by Nacalai Tesque) Then, accurately weighed 5 mg of PHB of Samples 1 to 4 were put in a container, sealed, and reacted at 100 ° C. for 4 hours. After cooling with water, 1 mL of distilled water was added and stirred vigorously. Then, after leaving still and isolate | separating a water layer and an organic-solvent layer, the organic-solvent layer was analyzed by the gas chromatography. The column used in the gas chromatography was Aminex HPX-87H Ion Exclusion Column 300 mm x 7.8 mm manufactured by Bio-Rad. Gas chromatography conditions were an injection temperature of 250 ° C., a column temperature of 160 ° C., FID (hydrogen, air), a carrier gas using helium, and a sample amount of about 3 μL.

The measured PHB purity is 92% for sample 1, 62% for sample 2, 68% for sample 3, and 78% for sample 4. The purity of PHB for sample 1 is 14 to It was as high as 30%.
Sample 1 was a very simple PHB, despite the fact that the operation of extracting PHB was very simple, in which wet cells were frozen, thawed, crushed, washed and extracted. Could get. It was also found that the organic solvent, chemicals and the like are not used, which is very suitable from the viewpoint of environmental conservation, and the enzyme and the like are not used, so that the cost is very low and it is suitable for mass production.

  The polyhydroxyalkanoic acid extraction method of the present invention has been specifically described with reference to the best mode for carrying out the invention and examples, but the gist of the present invention is not limited to these descriptions. Rather, its scope of rights should be construed broadly based on the claims. In addition, those skilled in the art can easily change and modify the method based on the description of the best mode for carrying out the invention and the examples of the present specification to obtain a method equivalent to the polyhydroxyalkanoic acid extraction method of the present invention. Such are also included in the polyhydroxyalkanoic acid extraction method of the present invention.

It is a flowchart which shows the flow of the polyhydroxyalkanoic acid extraction method which concerns on this invention.

Explanation of symbols

S1 Freezing step S11 Incubation step S12 Liquid medium removal step S2 Thawing step S21 Washing step S3 Crushing step S4 Extraction step

Claims (9)

A polyhydroxyalkanoic acid extraction method for extracting a polyhydroxyalkanoic acid produced by a microorganism from the microorganism,
A freezing step of freezing the microorganism that produced the polyhydroxyalkanoic acid using a cooling medium;
A thawing step of thawing the frozen microorganism;
A crushing step of crushing the thawed microorganism with a cell crushing device to obtain an insoluble matter containing the polyhydroxyalkanoic acid,
An extraction step of washing the obtained insoluble matter and extracting the polyhydroxyalkanoic acid;
A method for extracting polyhydroxyalkanoic acid, comprising:   The polyhydroxyalkanoic acid extraction method according to claim 1, further comprising a liquid medium removing step of removing the liquid medium in which the microorganisms are cultured by centrifuging before the freezing step.   The polyhydroxyalkanoic acid extraction method according to claim 1 or 2, wherein the thawing in the thawing step is performed at room temperature.   The polyhydroxyalkanoic acid extraction method according to any one of claims 1 to 3, wherein after the thawing step, a washing step is performed in which the thawed microorganism is suspended in water and centrifuged.   The polyhydroxyalkanoic acid extraction method according to any one of claims 1 to 4, wherein the washing in the extraction step is performed by centrifuging the insoluble matter one or more times.   The method for extracting polyhydroxyalkanoic acid according to any one of claims 1 to 5, wherein the cooling medium is liquid nitrogen.   The polyhydroxyalkanoic acid extraction method according to any one of claims 1 to 6, wherein the cell disruption device is a high-pressure homogenizer.   The method for extracting polyhydroxyalkanoic acid according to any one of claims 1 to 7, wherein the microorganism is Ralstonia eutropha.   The polyhydroxyalkanoic acid extraction method according to any one of claims 1 to 8, wherein the polyhydroxyalkanoic acid is polyhydroxybutyric acid.

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