RU2199587C2 - Method of isolation of polyhydroxyalkanoate from biomass and polyhydroxyalkanoate prepared by this method - Google Patents

Abstract

FIELD: biotechnology, microbiology. SUBSTANCE: method involves extraction of polyhydroxyalkanoate from biomass with at least one solvent of polyhydroxyalkanoate, for example, acetone, acetonitrile, benzene, butyl acetate, butyl propionate, β-butyrolactone, γ-butyrolactone, diethylcarbonate, diethylformamide, dimethylcarbonate, dimethylsuccinate, dimethylsulfoxide, dimethylformamide, ethyl acetate, ethylene glycol diacetate, methyl acetate, methylethylketone, 1,4-dioxane, tetrahydro-furan, toluene, xylene and their mixture. Biomass presents vegetable material containing oil or biomass of bacteria. Precipitation of poly-hydroxyalkanoate from extract is carried out using precipitating agent. Invention presents also polyhydroxyalkanoate prepared, its melting point is about 80 C and above. Method provides simple and ecologically pure technology for preparing highly pure bioplastics from biological sources. EFFECT: improved method of isolation. 22 cl, 3 dwg, 3 ex

Description

 The invention relates to methods for extracting specific components from other components of biomass. More specifically, the present invention relates to the extraction of a polyhydroxyalkanoate from a biological system such as a plant or bacteria, by performing solvent extraction.

 Commercial polymers are usually obtained from petrochemical sources by well-known synthetic methods. However, recent advances in technology are creating prospects for new sources of commercial polymers. Particularly promising is the production of plastic resins using living organisms (“bioplastics”) including genetically manipulated bacteria and cultivated plants that are designed to produce polymers such as polyhydroxyalkanoate (PHA); a number of bacteria that produce PHA in vivo are also a promising source of PHA (see, for example, Poirier, Y., DEDennis, K. Klomparens and C. Somerville, "Polyhydroxybutyrate, a biodegradable thermoplastic, produced in transgenic plants". Science, T. 256, pp. 520-523 (1992); Published International Patent Application 95/05472 of February 23, 1995; Published International Patent Application 93/02187 of February 4, 1993; Novel Biodegradable Microbial Polymers, EA Dawes, eds., NATO ASI Series, Series E: Applied Sciences - Vol. 186, Kluwer Academic Publishers (1990)). In large-scale production, for example, in agricultural production, the collection and cleaning of such bioplastics from biomass residues is a crucial stage that determines the practical applicability of this technology.

 Isolation of polymer lipids such as PHA from a large-scale biological source, for example from crops, is a non-trivial task. Traditional methods of isolation, widely used in the extraction of low molecular weight lipids, do not have sufficient practical cost-effectiveness for use in the process of isolation of resins. For example, simple mechanical pressing is not suitable for this purpose, because, unlike the isolation of vegetable oils from oilseeds, hard plastics cannot be squeezed out of crops by mechanical pressing.

 In principle, it is possible to isolate PHA by sedimentation methods. However, simple gravitational (by force in IG) deposition in a liquid suspending medium is practically inapplicable. The deposition rate is extremely low. In addition, such slow deposition is easily disrupted by the Brownian motion of small PHA particles induced by thermal fluctuation of the suspending liquid molecules surrounding the particles. In addition, the long periods of time required to deposit very fine PHA particles pose a problem associated with bacterial contamination and subsequent biodegradation of the particle suspension.

 Known solvent extraction methods are also not suitable for large-scale isolation of PHA from crops. The traditionally used solvent for the extraction of PHA from bacteria is chloroform. Other halogenated hydrocarbons such as dichloromethane, dichloroethane and chloropropane are also described for the same purpose (see, for example, U.S. Patent 4,562,245, Stademan, issued December 31, 1985; US Patent 4,324,907, Senior, Wright and Alderson, issued April 13, 1982 g .; US patent 4,310,684, Vanlautem and Gilain, issued January 12, 1982; US patent 4,705,604, Vanlautem and Gilain, issued November 10, 1987; European patent application 036 699, Holmes and Wright, published September 3, 1981. ; German patent application 239,609, Schmidt, Schmiechen Rehm and Trennert, published January 10, 1986). However, such solvents are potentially harmful to health and the environment. Therefore, the use of a large number of such solvents, especially near the collection site, is undesirable.

 Based on the foregoing, it is obvious that there is a need for a simple and economical method for isolating bioplastics from a large-scale biological source. Preferably, such a method is easily adaptable as an integral part of the agricultural production of related products, for example, oil and flour in the case of oilseeds.

 An object of the present invention is to provide a method for isolating bioplastics from biomass.

 This and other objectives of the present invention will become apparent to a person skilled in the art of the present description and the attached claims.

 A method for isolating a polyhydroxyalkanoate from biomass is proposed, which comprises extracting a polyhydroxyalkanoate with at least one PHA solvent selected from the group consisting of acetone, acetonitrile, benzene, butyl acetate, butyl propionate, β-butyrolactone, γ-butyrolactone, diethyl methyl carbonate, diethyl methyl carbonate, diethyl methyl carbonate, dimethyl succinate, dimethyl sulfoxide, dimethylformamide, ethyl acetate, ethylene glycol diacetate, methyl acetate, methyl ethyl ketone, tetrahydrofuran, toluene, xylene and mixtures thereof.

 This method satisfies the need for a relatively simple, environmentally friendly and economical process for the isolation of bioplastics from a large-scale biological source.

Brief Description of the Drawings
FIG. 1 depicts a diagram of a technical solution of the invention, including a single-stage extraction of oil and PHA followed by evaporation of the solvent.

 FIG. 2 depicts a diagram of a technical solution of the invention, including a single-stage extraction of oil and PHA followed by precipitation of PHA using a PHA precipitant.

 FIG. 3 depicts a diagram of a technical solution of the invention, including a two-stage sequential extraction of oil and PHA.

 The following is a list of definitions used in the text of the terms.

The term "alkenyl" means a carbon chain, preferably from about C ₂ to about C ₂₄ carbon atoms, more preferably from about C ₂ to about C ₁₉ ; moreover, such a chain may be straight, branched or cyclic, preferably straight or branched, more preferably straight; may be substituted (mono- or poly-) or unsubstituted; and monounsaturated (i.e., the chain has one double or triple bond), or polyunsaturated (i.e., the chain contains two or more double bonds, two or more triple bonds or one or more double and one or more triple bonds) preferably monounsaturated.

The term “alkyl” means a carbon chain, preferably from about C ₁ to about C ₂₄ , more preferably from about C ₁ to about C ₁₉ carbon atoms; which may be straight, branched or cyclic, preferably straight or branched, more preferably straight; the chain may be substituted (mono- or poly-) or unsubstituted; and saturated.

 The term “comprising” means that other steps and other ingredients may be added that do not affect the end result. This term covers the terms “consisting of” and “essentially consisting of”.

 The term "extraction of polyhydroxyalkanoate from biomass" in addition to the fact that it refers to the extraction of a particular PHA produced by the biomass that forms a single PHA, also refers to the extraction of one or more types of PHA when the biomass forms more than one type of PHA.

в котором R, предпочтительно, представляет собой Н, алкил, или алкенил; a m имеет значение от примерно 1 до примерно 4. Термины полигидроксиалканоат и РНА включают полимеры, содержащие одно или более различных повторяющихся звеньев.The term "polyhydroxyalkanoate" and "PHA" means a polymer containing the following repeating unit:

in which R is preferably H, alkyl, or alkenyl; am has a value of from about 1 to about 4. The terms polyhydroxyalkanoate and PHA include polymers containing one or more different repeating units.

в которой R¹ представляет собой Н или C₁-C₂ алкил; а n равно 1 или 2; второе статически повторяющееся мономерное звено имеет структуру

в которой R² представляет собой С₃-С₁₉ алкил или С₃-C₁₉ алкенил; и в которых, по крайней мере, 50% статистически повторяющихся мономерных звеньев имеют структуру первого статистически повторяющегося мономерного звена. Более предпочтительно, примеры высококристаллических РНА, экстрагируемых настоящим способом, включают вещества, раскрытые в заявке на патент США с серийным 08/465,046, Noda от 6 июня 1995 г.; заявке на патент США с серийным 08/422,008, Noda от 13 апреля 1995 г.; заявке на патент США с серийным 08/422,009, Noda от 5 июня 1995 г. ; заявке на патент США с серийным 08/467,373, Noda от 6 июня 1995 г. ; заявке на патент США с серийным 08/188,271, Noda от 28 января 1994 г.; заявке на патент США с серийным 08/469,969, Noda от 6 июня 1995 г. ; заявке на патент США с серийным 08/472,353, Noda от 7 июня 1995 г. ; заявке на патент США с серийным 08/469,269, Noda от 6 июня 1995 г.; и патенте США 5,292,860, Shiotani и Kobayashi, выданном 8 марта 1994 г.PHAs extracted by the method of the present invention preferably have a melting point (“T _m ”) of about 80 ^{° C.} or higher. Preferably, such PHA contain at least two statically repeating monomer units in which the first statistically repeating monomer unit has the structure

in which R ¹ represents H or C ₁ -C ₂ alkyl; and n is 1 or 2; the second statically repeating monomer unit has the structure

in which R ² represents C ₃ -C ₁₉ alkyl or C ₃ -C ₁₉ alkenyl; and in which at least 50% of the statistically repeating monomer units have the structure of the first statistically repeating monomer unit. More preferably, examples of highly crystalline PHAs extracted by the present method include those disclosed in US Patent Application Serial No. 08 / 465,046, Noda June 6, 1995; U.S. Patent Application Serial 08 / 422,008, Noda April 13, 1995; U.S. Patent Application Serial 08 / 422,009, Noda June 5, 1995; U.S. Patent Application Serial 08 / 467,373, Noda June 6, 1995; US Patent Application Serial 08 / 188,271, Noda January 28, 1994; U.S. Patent Application Serial 08 / 469,969, Noda June 6, 1995; U.S. Patent Application Serial 08 / 472,353, Noda June 7, 1995; U.S. Patent Application Serial 08 / 469,269, Noda June 6, 1995; and U.S. Patent 5,292,860 to Shiotani and Kobayashi, issued March 8, 1994.

 By the term “solvent” is meant a substance capable of dissolving another substance (soluble substance) to form a uniformly dispersed mixture (solution) of molecular or ionic particles.

 The term "non-solvent" means a substance that is unable to significantly dissolve another substance.

 The term “precipitant” means a substance that is capable of causing precipitation of another substance and / or weakening the solvating ability of the solvent. Although the precipitant is also considered a non-solvent, however, the non-solvent is not always a precipitant. So, for example, methanol and hexane are precipitators of PHA and non-solvents of PHA; whereas oil is a PHA non-solvent, but is not always an effective PHA precipitant (although at very high concentrations, the oil precipitates PHA from solution).

 All percentages are given in mol.% Based on the entire composition, unless otherwise indicated.

 All relations are given in mass terms, unless otherwise indicated.

 Further, the present invention in its aspects regarding the product and method is described in more detail.

Biomass
Sources from which PHA is extracted by the method of the present invention include such unicellular organisms as bacteria or fungi, as well as higher organisms such as plants (referred to as “biomass”). Although such biomass can be represented by wild-type organisms, they are preferably genetically manipulated species specifically designed to produce a specific target PHA of interest to the producer. Such genetically manipulated organisms are obtained by introducing the genetic information necessary to obtain one or more types of PHA. Typically, such genetic information is extracted from bacteria that produce PHA in vivo.

 Plants used in the present invention include any genetically engineered plant for producing PHA. Preferred plants include crops such as cereals, oilseeds and tubers; more preferably, avocado, barley, various beets, fava beans, buckwheat, carrots, coconut, copra, corn (maize), cotton seed, bottle gourd, various types of lentils, Lima beans, millet, mung bean, oats, oil palm, various peas, peanuts, potatoes, common squash, rapeseed (e.g. canola), rice, sorghum, soybeans, sugar beets, sugarcane, sunflower, sweet potato, tobacco, wheat and yams. Such genetically variable fruiting plants used in the method of the present invention include, but are not limited to, apple, apricot, banana, cantaloupe, various varieties of cherries, grapes, kumquat, lemon, lime real, orange, papaya, various varieties of peaches, pear , pineapple, tangerines, tomato and watermelon. Preferably, such plants are genetically engineered to produce PHA according to the methods described by Poirier, Y., DEDennis, K. Klomparens and C. Somerville "Polyhydroxybutyrate, a biodegradable thermoplastic, produced in transgenic plants", Science, vol. 256, pp. 520-523 (1992); in published international patent application 95/05472 of February 23, 1995; published international patent application 93/02187 of February 4, 1993. Particularly preferred plants are soybeans, potatoes, corn and coconut plants that have been genetically engineered to produce PHA; more preferably soybean cultured.

 The bacteria used in the present invention include any genetically engineered bacteria to produce PHA, as well as bacteria that produce PHA in vivo. Examples of such bacteria are those described in Novel Biodegradable Microbial Polymers, E.A. Dawes, eds., NATO ASI Series, Series E: Applied Sciences - Vol. 186, Kluwer Academic Publishers (1990); U.S. Patent 5,292,860 to Shiotani and Kobayashi, issued March 8, 1994; U.S. Patent 5,250,430, Peoples and Sinskey, issued October 5, 1993; U.S. Patent 5,245,023, Peoples and Sinskey, issued September 14, 1993; U.S. Patent 5,229,279, Peoples and Sinskey, issued July 20, 1993.

Solvent Extraction
The present invention relates to a method for isolating a polyhydroxyalkanoate from biomass, the method comprising extracting a polyhydroxyalkanoate with at least one PHA solvent selected from the group consisting of acetone, acetonitrile, benzene, butyl acetate, butyl propionate, β-butyrolactone, γ-butyrolactone, diethyl carbonate, diethyl formamide, dimethyl carbonate, dimethyl succinate, dimethyl sulfoxide, dimethyl formamide, ethyl acetate, ethylene glycol diacetate, methyl acetate, methyl ethyl ketone, 1,4-dioxane, tetrahydrofuran, t Luola, xylene and mixtures thereof. A preferred PHA solvent is acetone, acetonitrile, γ-butyrolactone, 1,4-dioxane, methyl acetate, toluene, methyl ethyl ketone, ethyl acetate, and mixtures thereof. A more preferred PHA solvent is acetone or ethyl acetate; even more preferred is acetone.

 PHA precipitators can also be used at some stages of the process to effect the precipitation of PHA. The use of such precipitants in the method of the invention is discussed in more detail below. Preferred PHA precipitants include, but are not limited to, butane, diethyl ether, diglyceride, ethanol, fat, heptane, hexane, methanol, monoglyceride, octane, oil, pental, triglyceride, water, or mixtures thereof.

Preferably, the PHA solvent is used in the method of the invention at an elevated temperature, since it has been found that the rate of dissolution of PHA in the PHA solvent at elevated temperatures is significantly higher. The extraction of PHA can be carried out in the temperature range from about 20 ^o C to the melting temperature of PHA; more preferably in the range of from about 20 ^° C to about 80 ^° C; more preferably in the range of from about 45 ^{° C.} to the boiling point of the PHA solvent; and even more preferably in the range of from about 50 ^{° C.} to about 60 ^° C.

 Preferably, the solid mass containing PHA is subjected to stirring during extraction with the PHA solvent, since this also increases the rate of dissolution of the PHA.

 Removal of the PHA solvent from the solution containing PHA results in the final precipitation of PHA in the form of a crystalline solid. However, in the process of evaporation of the solvent, the concentrated PHA solution often forms a very highly viscous liquid or sometimes even a gel, which is difficult to process further. If the solution contains a relatively non-volatile non-solvent PHA in addition to the solvent PHA and PHA, PHA will precipitate after removal of the PHA solvent and form a suspension in the PHA non-solvent.

a. One-stage extraction of oil and PHA followed by evaporation of the solvent
In accordance with one embodiment of the invention, the method includes: a) treating biomass containing PHA and oil (for example, plant material) with a PHA solvent; c) removal of the existing insoluble plant material, resulting in a solution of polyhydroxyalkanoate and oil dissolved in the PHA solvent; c) removing the PHA solvent from the solution, which leads to the precipitation of polyhydroxyalkanoate in oil; and e) oil removal, after which polyhydroxyalkanoate remains in the system.

An example of such a technical solution is schematically presented in figure 1. This technical solution provides the simplest method for extracting both oil and PHA from biomass containing such components. Biomass (e.g. cultured soybeans containing oil and PHA) is exposed to a PHA solvent. After separation of the solid biomass, the PHA solvent is directly evaporated from the resulting solution containing the extracted oil and PHA. Since PHAs with a T _m value of about 80 ^{° C.} or higher are usually insoluble in oil, removal of solvent results in precipitation of PHA in the oil. Such oil and PHA are further separated by conventional methods, for example by filtration.

in. Single-stage oil and PHA extraction followed by non-solvent precipitation
In accordance with another technical solution of the invention, the method includes: a) treating biomass containing PHA and oil (for example, plant material) with a PHA solvent; c) removal of existing insoluble plant material, leaving a solution of polyhydroxyalkanoate and oil dissolved in a PHA solvent; c) treating the solution with PHA precipitant, resulting in the precipitation of polyhydroxyalkanoate; d) removal of the PHA precipitant and the oil dissolved in it, resulting in polyhydroxyalkanoate remaining. Optionally, the PHA solvent and the PHA precipitant may subsequently be evaporated from the oil.

 Such a technical solution is shown in diagrammatic form in FIG. 2. In accordance with such a technical solution, biomass (for example, soybean culture) containing oil and PHA is once exposed to a PHA solvent, such as acetone, which is a good solvent for both oil and PHA. The addition of a PHA precipitant (e.g., hexane) to an extraction solution containing oil and PHA results in precipitation of the PHA. The settled mixture containing PHA solvent, PHA precipitant and oil is removed to obtain PHA. The PHA solvent and PHA precipitant are then evaporated from the supernatant to give an oil. Then the PHA solvent and PHA precipitant can be separated from each other and recycled.

 Preferred PHA precipitants are mixed with the PHA solvent. Preferred PHA precipitants include diethyl ether, diglyceride, ethanol, fat, heptane, hexane, methanol, monoglyceride, oil, triglyceride, water, or mixtures thereof. More preferred precipitants are those substances which are miscible with oil, for example diethyl ether, diglyceride, ethanol, fat, heptane, hexane, methanol, monoglyceride, oil, triglyceride or mixtures thereof; most preferred are hexane and methanol.

from. Two-stage oil and PHA extraction sequence
In accordance with another technical solution of the invention, the method includes: a) treating biomass containing PHA and oil (for example, plant material) with a solvent that dissolves the oil and which does not dissolve polyhydroxyalkanoate; c) removal of the solvent and the oil contained in it, as a result of which the existing insoluble plant material remains in the system; c) treating the biomass treated with a solvent that dissolves the oil (i.e., non-fat biomass) with a PHA solvent; e) removal of insoluble plant material, leaving a solution of PHA solvent and PHA dissolved in it; and e) evaporating PHA from the solvent solution, leaving PHA remaining.

 An example of such a technical solution is schematically depicted in figure 3. Biomass (for example, soybean culture) is first exposed to a solvent that dissolves the oil (for example, hexane), which does not dissolve PHA. Then, in the first stage, only the oily part is extracted. Then the solvent, which dissolves the oil, is evaporated from the oil solution and it can be used again for additional oil extraction. Then, dry, fat-free soybean culture is affected by the PHA solvent. In the second extraction step, the PHA is removed from non-fat soy. Then, the PHA solvent is evaporated from the PHA extraction solution, resulting in solid PHA.

 Preferred oil dissolving solvents include butane, diethyl ether, diglyceride, ethanol, fat, heptane, hexane, methanol, monoglyceride, octane, oil (other than the oil to be extracted), pentane, triglyceride, or mixtures thereof. Preferably, the solvent of the oil has a higher volatility than the extracted oil, as a result of which its subsequent evaporation from the extracted oil is facilitated. More preferably, the solvent dissolving the oil is hexane.

The above-described embodiments of the present invention have many unexpected advantages, including the possibility of extracting crystallizable PHA with a high melting point (about 80 ^{° C.} or higher) without using a halogen-containing solvent, and, at the same time, the possibility of efficient co-extraction of oil. The PHA solvents used in the present invention, such as acetone and ethyl acetate, are inexpensive, safe and benign, and also readily available even from reduced sources. Such PHA solvents are also considered to be significantly less hazardous to the environment, especially for the ozone layer of the earth, than halogen-containing compounds that are commonly used to extract PHA from bacteria.

 Finding suitable solvents for crystalline polymers having relatively high melting points is not a trivial task. Unlike most low molecular weight compounds and non-crystallizable amorphous polymers, the solubility of crystalline polymers cannot be predicted on the basis of such commonly used simple criteria as the similarity of the chemical structure or the selection of refractive indices, dielectric constants or solubility parameters. A good example of the impossibility of predicting the solubility of crystalline polymers is the well-known insolubility of linear polyethylene in hexane, when both compounds are composed of identical hydrocarbon repeating units. Similarly, crystalline aliphatic polyesters such as isotactic poly (3-hydroxybutyrate) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) do not significantly dissolve in ethyl acetate or acetone, despite the fact that the chemical structures of such compounds suggest some molecular affinity. Thus, the accidental discovery of the fact that crystalline PHA containing a small number of medium-sized branches can easily dissolve in such solvents is unexpected.

 Additional unexpected advantages are realized when the extraction of PHA is carried out together with the extraction of oil. Initially, the oil functions as a miscible co-solvent promoting the extraction of PHA. However, after removal of the volatile PHA solvent, the oil becomes an effective suspending medium for the precipitation of PHA due to its limited PHA-solvating ability. Compared to a concentrated solution, a suspension of a polymer solid in a liquid non-solvent usually has a significantly lower apparent viscosity and thus has an improved processability. The elimination of the serious gelation problem that often occurs during the evaporation of the solvent from the concentrated polymer solution is an additional unexpected and important advantage of the method according to the present invention.

 The following examples further describe and demonstrate preferred technical solutions that are within the scope of the present invention. These examples are provided solely for purposes of illustration and should not be construed as limitations of the present invention, since various variants of the invention are possible without violating its essence and scope.

EXAMPLE 1
Extraction followed by evaporation of the solvent
A soybean sample with a culture weight of 60 g, containing vegetable oil and poly (3-hydroxybutyrate-co-3-hydroxyhexanoate), comprising 7.5% of the repeating units of 3-hydroxyhexanoate, was placed in a closed container loaded with 600 ml of acetone containing a very small amount ( about 0.1 g) of vegetable oil, and the contents were stirred for 3 hours at 55 ^° C. Then the acetone solution containing the extracted oil and PHA was poured from soybeans using a wire-mesh filter. The extraction solution containing vegetable oil, poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) and acetone were placed in a steam-heated reactor for evaporating volatile acetone, which was collected using a water-cooled refrigerator. After removal of acetone, solid flakes of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) appeared in the remaining oil, which was drained using a fine mesh filter to obtain 7 g of pure vegetable oil. The polymer flakes were washed with cold acetone pre-collected using a refrigerator to remove residual entrained oil and then dried to obtain 6 g of crystalline solid poly (3-hydroxybutyrate-co-3-hydroxyhexanoate). The acetone used for washing, which now contained a small amount of oil, was then used to further extract the oil and poly (3-hydroxybutyrate-co-3-hydroxyhexanoate).

EXAMPLE 2
Extraction followed by precipitation with a non-solvent
A 60 g sample of soybean containing vegetable oil and poly (3-hydroxybutyrate-co-3-hydroxyoctadecanoate) containing 5% of repeating units of 3-hydroxyoctadecanoate was placed in a closed container loaded with 600 ml of acetone, and the contents were stirred for 3 hours at 55 ^o C. Then the acetone solution containing the extracted oil and PHA was poured from soybeans using a wire-mesh filter. To the resulting solution was added 800 ml of hexane to precipitate poly (3-hydroxybutyrate-co-3-hydroxyoctadecanoate). Then the polymer flakes were isolated from the settled solution using a fine mesh filter and dried to obtain 5 g of poly (3-hydroxybutyrate-co-3-hydroxyoctadecanoate). The settled solution containing vegetable oil, acetone and hexane was placed in a steam heated reactor to evaporate the volatile mixture of hexane and acetone, which was collected using a water-cooled refrigerator, for additional separation into pure hexane and acetone. After removal of hexane and acetone, 7 g of vegetable oil was collected.

EXAMPLE 3
Two-stage extraction
A 60 g soybean sample containing vegetable oil and poly (3-hydroxybutyrate-co-3-hydroxyoctanoate) containing 6% of repeating units of 3-hydroxyoctanoate was placed in 600 ml of hexane and the resulting mixture was stirred for 2 hours. A hexane solution containing extracted oil was then poured from soy using a wire mesh filter.

Then, the resulting solution was placed in a steam-heated reactor to evaporate hexane, which was collected using a water-cooled refrigerator for further use. After hexane was removed, 6 g of vegetable oil was collected. Then defatted soybeans were placed in a closed container loaded with 600 ml of acetone, and the contents were stirred for 3 hours at 55 ^o C. After that, the acetone solution containing PHA was poured from soybeans using a wire-mesh filter. The resulting solution was placed in a steam-heated reactor to evaporate acetone, which was collected using a water-cooled refrigerator for further use. After acetone was removed, 6 g of solid crystalline poly (3-hydroxybutyrate-co-3-hydroxyoctanoate) was collected.

 All publications mentioned above, granted patents and patent applications, in their entirety, are referred to in the present description.

 It should be borne in mind that the examples and technical solutions described in the text are provided for illustrative purposes only and that, on their basis, a person skilled in the art can be offered various modifications or changes that are encompassed by the nature and scope of the present application and the attached claims.

Claims (22)

1. A method of isolating a polyhydroxyalkanoate from a biomass containing a polyhydroxyalkanoate, characterized in that the polyhydroxyalkanoate is extracted with at least one solvent of a polyhydroxyalkanoate selected from the group consisting of acetone, acetonitrile, benzene, butyl acetate, butyl propionate, β-butyrolactone , diethyl carbonate, diethyl formamide, dimethyl carbonate, dimethyl succinate, dimethyl sulfoxide, dimethyl formamide, ethyl acetate, ethylene glycol diacetate, methyl acetate, methyl ethyl ketone, 1,4-dioxane , Tetrahydrofuran, toluene, xylene, and mixtures thereof, wherein the polyhydroxyalkanoate has a melting point of about 80 ^o C or above.  2. The method according to claim 1, characterized in that the polyhydroxyalkanoate solvent is selected from acetone, methyl acetate, methyl ethyl ketone, ethyl acetate and mixtures thereof.  3. The method according to claim 2, characterized in that the solvent of the polyhydroxyalkanoate is acetone.  4. The method according to claim 2, characterized in that the solvent of the polyhydroxyalkanoate is ethyl acetate.  5. The method according to claim 1, characterized in that the biomass is a plant material.  6. The method according to claim 5, characterized in that the plant material contains oil.  7. The method according to claim 1, characterized in that the biomass is a bacterium. 8. The method according to claim 5, characterized in that the polyhydroxyalkanoate contains at least two statistically repeating monomer units, the first statistically repeating monomer unit having the structure

where R ¹ represents H, or C ₁ -C ₂ alkyl;
n = 1 or 2,
the second, statistically repeating monomer unit has the structure

where R ² represents C ₃ -C ₁₉ alkyl, or C ₃ -C ₁₉ alkenyl,
and in which at least 50% of the statistically repeating monomer units have the structure of the first, statistically repeating monomer unit.  9. The method according to p. 6, characterized in that after the extraction of the PHA the insoluble plant material is removed and the PHA solvent is removed from the remaining PHA solution and the oil in the PHA solvent, and the precipitated PHA is isolated by removing the oil. 10. The method according to claim 1, characterized in that the polyhydroxyalkanoate includes the following repeating unit having the structure

where R is preferably hydrogen, alkyl or alkenyl;
m is from about 1 to about 4.  11. The method according to claim 10, characterized in that the solvent of the polyhydroxyalkanoate is ethyl acetate. 12. The method according to claim 9, characterized in that the polyhydroxyalkanoate contains at least two statistically repeating monomer units, the first statistically repeating monomer unit having the structure

where R ¹ represents H, or C ₁ -C ₂ alkyl;
n = 1 or 2,
the second, statistically repeating monomer unit has the structure

where R ² represents C ₃ -C ₁₉ alkyl, or C ₃ -C ₁₉ alkenyl,
and in which at least 50% of the statistically repeating monomer units have the structure of the first, statistically repeating monomer unit.  13. The method according to p. 9, characterized in that the precipitation of PHA from the extract is carried out by treating it with precipitant PHA and PHA is isolated by removing the precipitant, solvent, oil. 14. The method according to item 13, wherein the polyhydroxyalkanoate contains at least two statistically repeating monomer units, the first statistically repeating monomer unit has the structure

where R ¹ represents H, or C ₁ -C ₂ alkyl;
n = 1 or 2,
the second, statistically repeating monomer unit has the structure

where R ² represents C ₃ -C ₁₉ alkyl, or C ₃ -C ₁₉ alkenyl,
and in which at least 50% of the statistically repeating monomer units have the structure of the first, statistically repeating monomer unit.  15. The method according to item 13, wherein the precipitant polyhydroxyalkanoate is selected from the group consisting of diethyl ether, ethanol, fat, heptane, hexane, methanol, oil, triglycerin, water and mixtures thereof.  16. The method according to p. 13, characterized in that it further includes evaporating the solvent and precipitant of polyhydroxyalkanoate from the oil.  17. The method according to claim 6, characterized in that before the extraction, the plant material is treated with an oil solvent that does not dissolve the polyhydroxyalkanoate, and the solvent in which the oil is dissolved and the oil dissolved in it and from the solution of the PHA solvent and the PHA dissolved in it are removed obtained by extraction, remove the solvent by evaporation, releasing PHA.  18. The method according to 17, characterized in that the solvent of the polyhydroxyalkanoate is acetone.  19. The method according to 17, characterized in that the solvent of the polyhydroxyalkanoate is ethyl acetate.  20. The method according to 17, characterized in that the solvent of the oil is butane, diethyl ether, diglycerides, ethanol, fat, heptane, hexane, methanol, monoglyceride, octane, oils of a different grade than oil from plant material, pentane, triglyceride and their mixtures. 21. The method according to 17, characterized in that the polyhydroxyalkanoate contains at least two statistically repeating monomer units, the first statistically repeating monomer unit having the structure

where R ¹ represents H, or C ₁ -C ₂ alkyl;
n = 1 or 2,
the second, statistically repeating monomer unit has the structure

where R ² represents C ₃ -C ₁₉ alkyl, or C ₃ -C ₁₉ alkenyl,
and in which at least 50% of the statistically repeating monomer units have the structure of the first, statistically repeating monomer unit.  22. The polyhydroxyalkanoate obtained by extraction by the method according to claim 1, or 8, or 12, or 16.

Images