MXPA06009257A - Process for producing polyhydroxyalkanoate - Google Patents

Abstract

A process for easily producing a biodegradable polyhydroxyalkanoate according to a solvent extraction technique without inviting of a substantial decrease of molecular weight. There is provided a process for producing polyhydroxyalkanoate, characterized in that polyhydroxyalkanoate is extracted from a polyhydroxyalkanoate-containing biomass of 5 wt.%or less water content with the use of an extraction solvent, crystallized and recovered.

Description

METHOD TO PRODUCE PO IHIDROXIA CANOATO Field of the Invention The present invention relates to a method for producing a polyhydroxyalkanoate.

Background of the Invention A polyhydroxyalkanoate (hereinafter briefly referred to as "PHA") is a biodegradable, thermoplastic polyester that is synthesized and accumulated as an energy storage substance in cells of a variety of microorganisms. A PHA, which is produced by microorganisms using natural organic acids or oils as carbon sources, is completely biodegraded by a microorganism in soil or water to be assimilated into the carbon cycle of the natural world. Therefore, it can be said that a PHA is a non-aggressive plastic material for the environment which hardly causes adverse effects for the ecological system. In these years, a synthetic plastic became a serious social problem in view of environmental pollution, residual waste and oil resources, and thus a PHA has attracted attention as a green plastic not aggressive to the environment and its practical applications are expected. As a PHA producing biomass, there is a REF .: 174661 microorganism that innately produces a PHA, or a transformant that can be obtained by recombining a PHA synthase gene in a microorganism or a plant. In both cases, since a PHA is accumulated in the biomass, the PHA will be produced by recovering the biomass containing PHA, and separating and purifying the PHA from the biomass. Regarding the separation and purification of PHA from a biomass, a method is known as the most convenient which comprises extracting a PHA using a solvent soluble in PHA, crystallizing the result using a poor solvent, and recovering the PHA as a crystal. For example, a method is described which comprises drying a biomass in which a PHA is accumulated, the PHA is extracted using a halogen-containing organic solvent such as chloroform and methylene chloride, and then the extract is mixed with a poor solvent such as methanol and hexane to precipitate and recover the PHA (see Japanese publication Kokai Sho-59-205992 and U.S. Patent No. 4,324,907). However, since these halogen-containing organic solvents are related to environmental regulations, the restrictions on use are strict, and thus can not be used substantially in commercial scale production. Accordingly, a study has been made for an extraction using a halogen-free organic solvent without using an organic solvent containing halogens. However, since a PHA has a fairly low solubility in halogen-free solvents (patent of United States No. 6,043,063), a large amount of solvent is required in a commercial scale production. Thus, a study has been conducted aimed at reducing the amount of solvent t as much as possible by extracting at high temperature to increase solubility (U.S. Patent No. 6,043,063, U.S. Patent No. 5,894,062 and U.S. Pat. No. 6,087,471). However, notwithstanding the solvent species, a high temperature heat extraction tends to significantly reduce the molecular weight of the PHA with the extraction time (U.S. Patent No. 4,101,533). Although many of the prior art documents do not relate to the reduction of molecular weight at the time of extraction, it is assumed that a problem has not been caused since the extraction is carried out in a fairly short time (patent No. 6,043,063 and U.S. Patent No. 5,894,062), or a purified polymer is used for extraction (U.S. Patent No. 6,043,063). However, the present inventors experienced that when, for example, an extraction was carried out from a cell as biomass, the molecular weight of a PHA was significantly reduced after extraction for several or many hours, and the PHA is disqualified as a product. When mass production is carried out on a commercial scale, it is sufficiently presumed that a polymer is exposed to high temperature for several hours from the start of extraction through the removal of residues other than PHA for crystallization. During this time, it is sufficiently possible that the molecular weight of the polymer is reduced to a degree where processing becomes impossible. Within the cells, there are many impurities such as a polymer degrading enzyme, cytoplasmic membrane, cell wall components, lipids, nucleic acids and proteins. In the extraction by heating, it is considered that a molecular weight of a polymer is reduced by a synergistic interaction between these impurities and a solvent. In U.S. Patent No. 5,821,299, a PHA is extracted using a solvent and then the lipid-soluble impurities are washed and removed with a solvent. By this operation, the reduction in molecular weight can possibly be suppressed to a certain extent. However, this method is inadequate since operations are complicated on a commercial scale, a separate solvent batch is required and equipment costs are raised. Since a PHA is low in solubility, it is desirable to carry out an extraction at the highest possible concentration. However, the higher the concentration, the greater the sequence of operations including a removal of residues after extraction, and this tends to be complicated and requires a long time, and thus the significant reduction in molecular weight is involved. However, a technology to suppress molecular weight reduction in a sequence of operations using current equipment has not yet been found. Therefore, the current state is that the solvent extraction method that is considered to be substantially convenient has not been put into practical use.

Brief Description of the Invention Accordingly, the object of the present invention is to provide a commercially preferable method for producing a polyhydroxyalkanoate having high processability while suppressing the reduction in molecular weight of the polyhydroxyalkanoate when the polyhydroxyalkanoate is extracted from a biomass that contain polyhydroxyalkanoate using a solvent. The present inventors have investigated enthusiastically about the objective mentioned above. As a result, they have surprisingly found that, for the first time, the significant reduction in molecular weight of a polyhydroxyalkanoate at the time of a solvent extraction can be suppressed by controlling the water content in a biomass up to 5% by weight or less. Therefore, they completed the present invention. By this method, it becomes possible to extract a high molecular weight polyhydroxyalkanoate directly from a biomass without removing impurities by a solvent wash before extracting the polyhydroxyalkanoate. That is, the present invention relates to a method for producing a polyhydroxyalkanoate from a polyhydroxyalkanoate-containing biomass, which comprises extracting a polyhydroxyalkanoate from a polyhydroxyalkanoate-containing biomass whose water content has been controlled at 5% by weight or less using an extraction solvent, crystallizing and recovering the result.

Detailed Description of the Invention The present invention is described in detail below. According to the present invention, the water content in a biomass containing polyhydroxyalkanoate (PHA) is 5% by weight or less. It is preferably 3% by weight or less and still more preferably 2% by weight or less, with a view to obtaining a more preferable suppressive effect of the reduction in molecular weight of the PHA. The lower limit that is preferred is 0% by weight, and a lower limit of 1% by weight is more preferred. The water content in a biomass is preferably measured by, for example, an infrared water weight manufactured by companies such as Kett Electric Laboratory or Sanko Electric Laboratory Co., Ltd., since the measurement can be carried out in a manner fast and simple However, the team is not restricted to these. In the present invention the infrared water weight manufactured by Kett Electric Laboratory is used, and the measurement is carried out by the following method. That is, 1 to 2 g of a sample are prepared on the weight, and dried at 130 ° C for about 15 minutes until the weighting value is balanced. The same sample is weighed 3 times and the average of the three values is determined as the water content. To control the water content in the biomass used for the present invention up to 5% by weight or less, a method comprising drying by heating, or a method comprising reducing the water content in a biomass by concentration and / or azeotropic dehydration under the coexistence of a solvent. However, these methods are not restricted in any way. As a heating drying equipment that can be used for the purpose of the present invention, for example, a spray dryer, vacuum incubation dryer, drum dryer, high temperature heating oven, water heater, etc. can be used suitably. ceramic, silicone rubber heater, high frequency continuous heating equipment, far infrared radiation heater, high frequency continuous heating equipment, far infrared radiation heater, microwave heating equipment, etc. However, the equipment is not restricted to these, and those with which the desired water content can be achieved can be used. In a safe way, it is also possible to use this equipment in combination to achieve the desired water content. In case of drying by heating, a biomass containing PHA is preferably exposed to a heat environment of 40 ° C or higher, and most preferably 50 ° C or higher. The drying by heating above is preferably carried out within the temperature and time scales in which the reduction of the molecular weight by heating does not occur. The preferable upper limit of the temperature is 100 ° C, and most preferably an upper limit of 90 ° C. Further, drying by previous heating is preferably carried out under reduced pressure. When the water content in a biomass is reduced by the concentration and / or azeotropic dehydration under the coexistence of a solvent, such solvent can be used, for example, toluene, butanol, ethyl acetate, etc. In addition, azeotropic concentration and / or dehydration under the coexistence of a solvent can be carried out either at 40 ° C or more, or less than 40 ° C. In the present invention, in the process for controlling the water content in a biomass up to 5% by weight or less, when the biomass is a cell, the cultured broth can be used as such. Alternatively, a wet cell recovered by a method such as centrifugation or membrane separation can also be used. In the present invention, by controlling the water content in a biomass up to 5% by weight or less, the reduction in molecular weight of a PHA in extraction with solvent at high temperature can be suppressed. Therefore, an extraction can be completed in one step without removing impurities other than the polyhydroxyalkanoate prior to extraction. In the production method of the present invention, a polyhydroxyalkanoate is extracted by adding an extraction solvent to a biomass containing PHA having a water content of 5% by weight or less. The weight ratio of a PHA in solvent extraction is not particularly restricted, but is preferably 1 to 20% by weight, relative to the total amount of the PHA and extraction solvent. More preferred is a lower limit of 2% by weight, and still more an upper limit of 15% by weight. An even more preferable lower limit is 3% by weight, and an even more preferable upper limit is 10% by weight in order to reduce as much as possible the amount of a solvent that will be used, and to carry out the extraction with a high efficiency. The temperature for extracting a PHA is preferably higher than 50 ° C, most preferably higher than 55 ° C and still more preferably higher than 60 ° C. However, to avoid as much as possible the decomposition of a PHA, it is preferable that the extraction temperature does not substantially exceed 100 ° C for 3 hours. Moreover, when using an extraction solvent with a low boiling point, it is preferable to carry out the extraction under a pressurized condition at a temperature below the boiling point. The duration for extracting a PHA is not particularly restricted, but preferably 10 to 150 minutes and most preferably 30 to 120 minutes with a view to obtaining a preferable extraction efficiency and preventing the decomposition of a PHA. In the production method of the present invention, it is preferable to separate a PHA and insoluble biomass after extraction. The separation of a PHA and insoluble biomass can be carried out by methods well known to the person skilled in the art. In this case, it is preferable to use a closed pressurized filter, reduced pressure filter, centrifugal separator, decanter type separator and the like. In accordance with the preferred embodiment of the present invention, after a PHA is extracted using an extraction solvent, the PHA solution is preferably kept warm. The temperature for the heating mentioned above is preferably 50 ° C or higher, most preferably 55 ° C or higher, and even more preferably 60 ° C or higher. If the temperature drops below 50 ° C, a PHA starts to gel without fluidity, solidifies later and becomes a state that can not be recovered. However, to avoid decomposition of a PHA, it is preferable that the temperature for heating mentioned above does not substantially exceed 100 ° C. Further, the above heating is preferably continued until the subsequent crystallization is completed. Further, in accordance with the preferred embodiment of the present invention, the crystallization of a PHA from a PHA solution is preferably carried out by gradually adding a poor solvent for crystallization to the above-heated PHA solution, and cooling the solution at less than 50 ° C, or preferably up to near room temperature while stirring vigorously to precipitate the dissolved PHA almost completely. The amount of the lean solvent that will be added for the crystallization is preferably such that the weight ratio of the solvent poor for crystallization in relation to the total amount of the solvent poor for crystallization and the extraction solvent is 10 to 70% by weight . A more preferable lower limit is 20% by weight, and a more preferable upper limit is 60% by weight. By means of this method, it is possible to obtain a PHA that is fluid, capable of being removed by brushing and that also has a low liquid content, which conventionally has been very difficult to obtain. In the present invention, to be put into practical use, the PHA preferably has the weight average molecular weight determined by a gel chromatography method, in which polystyrene is established as a molecular weight parameter of 10,000 or more. It is natural that an adequate molecular weight varies according to various applications. However, taking into consideration the molecular weight reduction by heat in the pellet or in the subsequent processing step, the weight average molecular weight of the PHA which is recovered after crystallization and dried is preferably 400,000 or more, and particularly preferably 500,000 or more. The extraction solvent used in the present invention is a solvent that dissolves 3% by weight or more of a PHA at this boiling point, but preferably one having a solubility of 4% by weight or more, most preferably 5% by weight. weight or more, and particularly preferably 6% by weight or more. The extraction solvent is preferably at least one species selected from the group consisting of monohydric alcohols having 1 to 10 carbon atoms, aromatic hydrocarbons having 6 to 10 carbon atoms, ketones having 4 to 7 carbon atoms and esters fatty acid alkyl having 5 to 8 carbon atoms. As the monohydric alcohols having 1 to 10 carbon atoms, there may be mentioned methanol, ethanol, propanol, butanol, pentanol, hexanol, cyclohexanol, 1-methylcyclohexanol, 2-ethylhexanol, benzyl alcohol, heptanol, octanol, nonanol, decanol, isomers thereof (e.g., n-butanol, isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol, n-pentanol, 2-pentanol, 3-pentanol, 1-hexanol, 2-hexanol, 1-heptanol, 2-heptanol, 3-heptanol, 1-octanol, 2-octanol, 1-nonanol, 2-nonanol, 1-decanol, 2-decanol, etc.), and the like. As the monohydric alcohols mentioned above having 1 to 10 carbon atoms, at least one species selected from the group consisting of butanol, pentanol, hexanol, cyclohexanol, 1-methylcyclohexanol, 2-ethylhexanol, benzyl alcohol, heptanol, octanol, nonanol, decanol and isomers thereof. As the aromatic hydrocarbons having 6 to 10 carbon atoms, benzene, toluene, xylene, ethylbenzene, dimethoxybenzene, trimethylbenzene, eumeno, butylbenzene, cymene and isomers thereof (eg, 1,3-dimethoxybenzene, 1,2) are preferred. , 3-trimethylbenzene, 1,2,4-trimethylbenzene, o-xylene, m-xylene, p-xylene, o-cymene, m-cimeno, p-cymene, etc.). As the ketones having 4 to 7 carbon atoms, methyl ethyl ketone, methyl butyl ketone, pentanone, hexanone, cyclohexanone, heptanone and isomers thereof are preferred (eg, methyl isobutyl ketone, methyl n-amyl ketone, 2-hexanone, 3-hexanone, 5-methy1-2-hexanone, etc.). Moreover, as the fatty acid alkyl esters having 5 to 8 carbon atoms, propyl acetate, butyl acetate, pentyl acetate, hexyl acetate and isomers thereof (eg, isobutyl acetate, butyrate) are preferred. of ethyl, isoamyl acetate, propyl propionate, butyl propionate, pentyl propionate, butyl butyrate, isobutyl isobutyrate, ethyl butyrate, ethyl valerate, methyl valerate, etc.). The extraction solvent mentioned above can be used as a species, or two or more. Among these extraction solvents, n-butanol, isobutanol, n-pentanol, 2-pentanol, 3-pentanol, toluene, benzene, methyl ethyl ketone, butyl acetate, butyl propionate and ethyl acetate as the extraction solvents are particularly preferred. the present invention, with a view to having high solubility of a PHA. Among these extraction solvents, aromatic hydrocarbons and ketones, ie, toluene, benzene and methyl ethyl ketone, are preferred, with a view to preventing the reduction in the molecular weight of a PHA in solution, but toluene is more preferred due to its comparative cost low. The poor solvent for crystallization used in the present invention is a solvent that does not dissolve 0.5% or more of a PHA at 15 to 25 ° C, and is preferably a solvent that does not dissolve 0.3 wt% or more of a PHA. As the solvent poor for the crystallization mentioned above, aliphatic hydrocarbons having 6 to 12 carbon atoms with a boiling point of 60 ° C or more are preferred. For example, there may be mentioned hexane, heptane, methylcyclohexane, octane, nonane, decane, dodecane, undecane, isomers thereof (e.g., n-heptane, isoheptane, etc.), and the like. Among these poor crystallization solvents, heptane and methylcyclohexane are preferred as the poor solvent for the crystallization of the present invention. More heptane is preferred, and n-heptane such as heptane is particularly preferred. Recovery of PHA after crystallization is carried out by methods well known to the person skilled in the art such as a liquid filtration or centrifugation of a PHA solution after crystallization. The recovered PHA is preferably washed with a suitable poor solvent. As this poor solvent to be used in the above washing, for example, solvents such as water, methanol, ethanol, acetone and hexane, or a mixture thereof, can be used with the extraction solvents mentioned above. The drying of a recovered PHA is carried out by methods well known to the person skilled in the art such as, for example, air-jet drying and vacuum drying. However, the method is not restricted to these. The PHA of the present invention is not particularly restricted in terms of its hydroxyalkanoate components, but specifically can be mentioned 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxypropionate, 4-hydroxyvalerate, 5-hydroxyvalerate, 3-hydroxyhexanoate (3HH), 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3- hydroxynonanoate, 3-hydroxydecanoate, etc. "The PHA of the present invention may be a homopolymer of one or more of these hydroxyalkanoates or a copolymer obtainable by copolymerizing at least two or more species thereof." As specific examples of the PHA, there may be mentioned PHB (a homopolymer of 3HB). ), PHBV (a binary copolymer composed of 3HB and 3HV), PHBH (a binary copolymer composed of 3HB and 3HH, see Japanese Patent Publication No. 2,777,757), PHBHV (a ternary copolymer composed of 3HB, 3HV and 3HH, see publication of Japanese Patent No. 2,777,757), etc. The PHA of the present invention is preferably a copolymer obtainable by copolymerizing at least two species of the hydroxyalkanoates mentioned above, particularly among them, a copolymer comprising 3HH is preferred, and less a species of other hydroxyalkanoates as monomeric components, since it has a degradation capacity as a biodegradable polymer and softness, and In this case, the compositional ratio of monomer units constituting the PHBH is not particularly restricted, but those containing 40 mol% or less of 3HH units are preferred, and those containing 30 mol% or more are preferred. less than 3HH units, and those containing 20 mol% or less of 3HH units are particularly preferred in view of the crystallinity preferable in crystallization. In the case of PHBHV, the compositional ratio of monomeric units constituting PHBHV is not particularly restricted, but for example, those containing 1 to 95 mole% of 3HB units, 1 to 96 mole% of 3HV units and 1 to 30% molar of 3HH units. The biomass to be used in the present invention is not particularly restricted as long as it is a living organism capable of accumulating a PHA in cells. However, a microorganism is preferred. For example, microorganisms that prefer the genus Alcaligenes such as Alcaligenes lipolytica and Alcaligenes latus, the genus Ralstonia such as Ralstonia eutropha, the genus Pseudomonas such as Pseudomonas oleovorance and Pseudomonas resinovorans, the genus Bacillus, the genus Azotobacter, the genus Nocardia as Nocardia salmonicolur, the genus Aeromonas such as Aeromonas caviae, the genus Rhodospirillum such as Phodospirillum rubrum, the genus Zoogloea such as Zoogloea ramigera, the genus Methylobacterium, the genus Paracoccus, the genus Clostridium, the genus Halobacterium, the genus Candida, the genus Yarrowia , the genus Saccharomyces and the like, can accumulate a PHA in cells by controlling the culture conditions. The PHA of the present invention is preferably produced by at least one cell species selected from the group of biomasses consisting of the genus Aeromonas, the genus Alcaligenes, the genus Azotobacter, the genus Bacillus, the genus Clostridium, the genus Halobacterium, the genus Nocaria genus, the genus Rhodospirillum, the genus Pseudomonas, the genus Ralstonia, the genus Zoogloea, the genus Candida, the genus Yarrowia and the genus Saccharomyces. Alternatively, the biomass used in the present invention can also be a transformant obtainable by introducing a gene group involved with a PHA synthesis of these microorganisms. In that case, the host is not particularly restricted, and microorganisms such as Bscherichia coli and yeast may be mentioned (see WO 01/88144), and additional plants may be mentioned in addition to the microorganisms mentioned above. Among these, Aeromonas caviae belonging to the genus Aeromonas and the transformed cell obtainable by introducing a gene from the PHA synthase group derived from the Aeromonas cavieae are preferred., since they have excellent capacitive synthesizer of PHBH as a polymer. In particular, Ralstonia eutropha obtainable by introducing a gene from the PHA synthase group of Aeromonas caviae is more preferred. An example of these microorganisms is deposited internationally based on the Budapest Treaty at the National Institute of Industrial Science and Advanced Technology and International Depositary of Patent Organizations, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan with the name of Alcaligenes eutrophus AC32 (registration date: August 7, 1997, registration number: FERM BP-6038). A method for cultivating the PHA-producing microorganisms mentioned above during the present is not particularly restricted, but for example, the method well known to the person skilled in the art described in the Japanese publication Kokai 2001-340078 can be used. To recover a PHA, it is naturally preferable that the content of the PHA in the cultured microbial cell be higher. In the application for commercial scale production, the PHA content is preferably 50% by weight or more in a biomass containing PHA whose water content has been controlled at 5% by weight or less. The content of PHA is most preferably 60% by weight or more, and particularly preferably 70% by weight or more. The biomass residues containing PHA after having been treated according to the present invention are preferably used as animal feed, food for microorganisms or vegetable fertilizer. Accordingly, the extraction solvent to be used in the present invention is preferably in an amount such that it is permissible as a food or fertilizer. However, it is preferable to substantially remove the solvent from the biomass substances. The PHA obtained by the production method according to the present invention can be configured in various forms, such as fibers, strands, ropes, textiles, fabrics, non-woven fabrics, papers, films, sheets, tubes, boards, sticks, containers, bags, parts and foamed bodies. Moreover, it can also be processed in a biaxial stretched film. The products formed can be used appropriately for fields such as agriculture, fishing, forestry, gardening, medicine, sanitary products, clothing, non-clothing, packaging and others. By the production method of the present invention, it becomes possible to produce and provide a biodegradable polyhydroxyalkanoate having preferable processing capacity on a commercial scale.

Examples Hereinafter, the present description will be described in more detail by means of examples. In each example, poly (3-hydroxybutyrate-co-3-hydroxyethanoate) (hereinafter briefly referred to as "PHBH") was produced as a PHA. Surely, the present invention is not limited to these examples in its technical scope, and is not restricted to the production of PHBH. In the examples, the water content is measured using the infrared water weight FD230 manufactured by Kett Electric Laboratory. That is, 1 to 2 g of a sample are prepared in that weight, and dried at 130 ° C until the weighting value is balanced (approximately for 15 minutes). The same sample is weighed 3 times and the average of the three values is determined as the water content. The weight average molecular weight of a PHA was determined using a gel chromatography system of Shimadzu (Rl detection) equipped with Shodex K806L column (300 x 8 mm, 2 columns connected) (product of Showa Denko K.K.) with chloroform as a mobile phase. As the molecular weight parameter sample, a commercially available standard polystyrene was used. In addition, the purity of PHBH was determined by gas chromatography after methyl esterification of PHBH.

Comparative Example 1 PHBH was produced in the cultivar Ralstonia eutropha which was deposited internationally on August 7, 1997 as Alcaligenes eutrophus AC32 (deposit number FERM BP-6038) obtained by introducing a gene of the PHA synthase group derived from Aeromonas caviae in accordance with the method described in Example 1 of the Japanese Kokai publication 2001-340078. After concluding the culture, the cell broth was sterilized at 60 ° C for 20 minutes. At the conclusion of the culture, the PHBH had a weight average molecular weight of 1,000,000 and a 3-hydroxyhexanoate (hereinafter briefly referred to as "3HH") of 6.2 molar%. The culture broth was subjected to spray drying using a "Pulvis GB22" spray dryer manufactured by Yamato Scientific Co., Ltd., under the conditions of the 150 ° C heat stream inlet temperature, and the output of the 60 ° C heat stream The molecular weight of the PHBH was not reduced at the time of spray drying under this condition. The water content of the dried cells obtained was 8.2% by weight. Ten grams of these dried cells were added to 92 g of toluene heated to 90 ° C (PHBH 6% by weight), and an extraction was carried out for one hour. After the extraction was complete, the solution was transferred to a pressurized cover type filter kept warm at 90 ° C, and an extract of PHBH was recovered by filtration. The recovered extract was kept warm at 90 ° C and 30 g of n-heptane were added gradually while stirring the solution vigorously. After the addition was complete, the solution was gradually cooled to room temperature with vigorous stirring, and then white PHBH was precipitated. The PHBH could be easily recovered by filtration, and the recovered PHBH was washed with methanol and dried under vacuum at 45 ° C after washing. The recovery amount was 5.4 g (recovery rate 90%), and the purity was 98%. The weight average molecular weight was 390,000, that is, as much as 61% reduction in one hour.

Example 1 The dried cells obtained in comparative example 1 were dried under vacuum at 50 ° C for 5 hours. The water content of the dried cells obtained was 4.9% by weight. Ten grams of the dried cells were subjected to an extraction with toluene in the same manner as in Comparative Example 1 to recover PHBH. The recovery amount was 5.5 g (recovery rate 92%), and the purity was 99%. The weight average molecular weight was 750,000, that is, 25% reduction in one hour, but it was a sufficient molecular weight for processing.

Example 2 The dried cells obtained in comparative example 1 were dried under vacuum at 50 ° C for 10 hours. The water content of the dried cells obtained was 2.6% by weight. 10 g of the dried cells were subjected to an extraction with toluene in the same manner as in Comparative Example 1 to recover PHBH. The recovery amount was 5.5 g (recovery rate 92%), and the purity was 99%. The weight average molecular weight was 800,000, that is, only 20% reduction in one hour.

Example 3 The dried cells obtained in comparative example 1 were dried under vacuum at 50 ° C for 20 hours.

The water content of the dried cells obtained was 1.8% by weight. Ten grams of the dried cells were subjected to an extraction with toluene in the same manner as in Comparative Example 1 to recover PHBH. The recovery amount was 5.5 g (recovery rate 92%), and the purity was 99%. The weight average molecular weight was 850,000, that is, only 15% reduction in one hour. From Examples 1 to 3, it was shown that the reduction in molecular weight of PHA can be substantially avoided by reducing the water content in dry cells.

Industrial Applicability By the production method of the present invention, it is possible to produce and provide a biodegradable polyhydroxyalkanoate having preferable processing capacity on a commercial scale. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (23)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for producing a polyhydroxyalkanoate, characterized in that it comprises extracting a polyhydroxyalkanoate from a biomass containing polyhydroxyalkanoate and having a water content of 5% by weight or less using an extraction solvent, crystallizing and recovering the result.

2. The method for producing a polyhydroxyalkanoate according to claim 1, characterized in that the water content in the biomass is controlled at 5% by weight or less by drying the biomass by heating.

3. The method for producing a polyhydroxyalkanoate according to claim 2, characterized in that drying by heating the biomass is carried out at 40 ° C or more.

The method for producing a polyhydroxyalkanoate according to claim 1, characterized in that the biomass containing polyhydroxyalkanoate and having a water content of 5% by weight or less is prepared by azeotropic concentration and / or dehydration under the coexistence of a solvent.

5. The method for producing a polyhydroxyalkanoate according to any of claims 1 to 4, characterized in that the extraction of a polyhydroxyalkanoate is carried out in one step without removing impurities other than the polyhydroxyalkanoate in the biomass.

The method for producing a polyhydroxyalkanoate according to any of claims 1 to 5, characterized in that a polyhydroxyalkanoate crystal is precipitated by dissolving a polyhydroxyalkanoate in an extraction solvent, keeping the solution hot at 50 ° C or more, adding to the same a poor solvent for crystallization and further cooling the solution to less than 50 ° C.

The method for producing a polyhydroxyalkanoate according to any of claims 1 to 6, characterized in that the extraction solvent is at least one species selected from the group consisting of monohydric alcohols having 1 to 10 carbon atoms, aromatic hydrocarbons having 6 to 10 carbon atoms, ketones having 4 to 7 carbon atoms and alkyl esters of fatty acid having 5 to 8 carbon atoms.

8. The method for producing a polyhydroxyalkanoate according to claim 7, characterized in that the monohydric alcohols having 1 to 10 carbon atoms are at least one species selected from the group consisting of butanol, pentanol, hexanol, cyclohexanol, 1- methylcyclohexanol , 2-ethylhexanol, benzyl alcohol, heptanol, octanol, nonanol, decanol and isomers thereof.

The method for producing a polyhydroxyalkanoate according to claim 7, characterized in that the aromatic hydrocarbons having 6 to 10 carbon atoms are at least one species selected from the group consisting of benzene, toluene, xylene, ethylbenzene, dimethoxybenzene, trimethylbenzene , eumeno, butylbenzene, cymene and isomers thereof.

The method for producing a polyhydroxyalkanoate according to claim 7, characterized in that the ketones having 4 to 7 carbon atoms are at least one species selected from the group consisting of methyl ethyl ketone, methyl butyl ketone, pentanone, hexanone, cyclohexanone, heptanone and isomers thereof.

The method for producing a polyhydroxyalkanoate according to claim 7, characterized in that the alkyl esters of fatty acid having 5 to 8 carbon atoms are at least one species selected from the group consisting of propyl acetate, butyl acetate, pentyl acetate, hexyl acetate and isomers thereof.

The method for producing a polyhydroxyalkanoate according to any of claims 6 to 11, characterized in that the solvent poor for crystallization is an aliphatic hydrocarbon having 6 to 12 carbon atoms with a boiling point of 60 ° C or more high.

13. The method for producing a polyhydroxyalkanoate according to claim 12, characterized in that the poor solvent for crystallization is at least one species selected from the group consisting of hexane, heptane, methylcyclohexane, octane, nonane, decane, dodecane, undecane and isomers thereof.

The method for producing a polyhydroxyalkanoate according to any of claims 1 to 13, characterized in that the weight ratio of the polyhydroxyalkanoate in relation to the total amount of the polyhydroxyalkanoate and the extraction solvent in the extraction of the polyhydroxyalkanoate is within the scale from 1 to 20% by weight.

15. The method for producing a polyhydroxyalkanoate according to any of claims 6 to 14, characterized in that the poor solvent for crystallization is added to an amount such that the weight ratio of the solvent poor for crystallization in relation to the total amount of the poor solvent for crystallization and extraction solvent becomes from 10 to 70% by weight.

16. The method for producing a polyhydroxyalkanoate according to any of claims 6 to 15, characterized in that the extraction solvent is toluene and the poor solvent for the crystallization is heptane.

The method for producing a polyhydroxyalkanoate according to any of claims 1 to 16, characterized in that the polyhydroxyalkanoate is a copolymer obtainable by copolymerizing at least two species of monomers selected from the group consisting of 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxypropionate, 4-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxynonanoate and 3-hydroxydecanoate.

18. The method for producing a polyhydroxyalkanoate according to any of claims 1 to 17, characterized in that the polyhydroxyalkanoate is a copolymer composed of 3-hydroxyhexanoate and at least one species of hydroxyalkanoate that is not 3-hydroxyhexanoate.

19. The method for producing a polyhydroxyalkanoate according to any of claims 1 to 18, characterized in that the polyhydroxyalkanoate is a copolymer composed of 3-hydroxyhexanoate and 3-hydroxybutyrate.

20. The method for producing a polyhydroxyalkanoate according to any of claims 1 to 19, characterized in that the biomass is a microorganism.

The method for producing a polyhydroxyalkanoate according to any of claims 1 to 20, characterized in that the polyhydroxyalkanoate is produced by at least one cell species selected from the group consisting of species belonging to the genus Aeromonas, Alcaligenes, Azotobacter, Bacillus , Clostridium, Halobac terium, Nocaria, Rhodospirillum, Pseudomonas, Ralstonia, Zoogloea, Candida, Yarrowia and Saccharomyces.

22. The method for producing a polyhydroxyalkanoate according to any of claims 1 to 21, characterized in that the polyhydroxyalkanoate-containing biomass is a transformant obtainable by introducing a synthetic gene group of polyhydroxyalkanoate derived from Aeromonas caviae.

23. The method for producing a polyhydroxyalkanoate according to claim 22, characterized in that the polyhydroxyalkanoate-containing biomass is Ralstonia eutropha which can be obtained by introducing a synthetic gene group of polyhydroxyalkanoate derived from Aeromonas caviae.