RU2439143C1 - Cupriavidus eutrophus VKPM B-10646 BACTERIA STRAIN - PRODUCER OF POLYHYDROXY ALKANOATES AND PRODUCTION METHOD THEREOF - Google Patents

Abstract

FIELD: chemistry. ^ SUBSTANCE: group of inventions relates to biotechnology and a Cupriavidus eutrophus VKPM B-10646 bacteria strain which produces polyhydroxy alkanoates (PHA) and a method of producing said strain. The strain is isolated from Ralstonia eutropha VKPM B-8562 during long multi-step selection based on effectiveness of synthesis of multicomponent PHA. PHA is obtained by culturing the strain in conditions of aeration and mixing on a liquid salt medium with limited nitrogen. The medium contains a growing substrate with an additional carbon source. The growing substrate used is glucose or fructose or 3-butyric acid or a gaseous mixture - hydrogen, oxygen and carbon dioxide, or synthetic gas mixed with oxygen. The additional carbon source used is a solution of potassium 3-valarate or solution of potassium 3-valerate and potassium 3-hexanoate, or solution of potassium 3-valerate, potassium 3-hexanoate and acrylate, or solution of potassium 3-valerate, or solution of potassium 3-hexanoate and acrylate, or solution of 3-butyric acid and 4-butyrolactone, or solution of 3-butyric acid, 4- butyrolactone and potassium 3-valerate, or solution of 3-butyric acid, 4- butyrolactone and potassium 3-hexanoate, or solution of 3-butyric acid, 4- butyrolactone, potassium 3-valerate and potassium 3-hexanoate. ^ EFFECT: invention enables to obtain polyhydroxy alkanoate producer with high output. ^ 2 cl, 1 tbl, 15 ex

Description

The invention relates to biotechnology and relates to a producer strain of polymers of hydroxyalkanoic acids (PHA) and a method for their preparation. These polymers are thermoplastic, characterized by high biocompatibility and destructibility in biological environments, therefore they are promising for various fields of application: in medicine (surgical elements, implants, matrices of functioning cells), in pharmacology (for creating long-term drug systems with a controlled release of drugs, hygiene items and sanitation), in agriculture (biodegradable coatings and packaging of fertilizers and pesticides for targeted delivery).

Various prokaryotic microorganisms belonging to different taxonomic groups are known among PHA producers, capable of growing on various substrates (sugars, organic acids, agricultural and industrial wastes) and synthesizing these polymers [Madison L.L., Huisman G.V. Metabolic engineering of poly (3-hydroxyalkanoates): From DNA to plastic // Microbiol. And Molecular Biology Rev. - 1999. - Vol. 63. - P.1-25]. However, several highly productive microorganisms have been isolated for industrial use. These are hydrogen-oxidizing bacteria Alcaligenes latus, Alcaligenes eutrophus (renamed from Ralstonia and Wautersia eutropha, currently assigned to the genus Cupriavidus), nitrogen fixers Azotobacter vinelandii, methylotrophs Methylomonas, Methylobacterium organophilum.V. L. L. Madison Metabolic engineering of poly (3-hydroxyalkanoates): From DNA to plastic // Microbiol. And Molecular Biology Rev. - 1999. - Vol. 63. - P.1-25], synthesizing mainly the polymer of 3-hydroxybutyric acid (3-hydroxybutyrate (3-PHB)), which is the most studied and common representative of PHA. 3-PHB is a highly crystalline (crystallinity degree of over 70%) thermoplastic, whose high biocompatibility is based on the fact that hydroxybutyric acid is a natural metabolite of cells and tissues of higher organisms and humans [Reusch R.N., Sparrow A.W., Gardiner J.

Transport of poly-beta-hydroxybutyrate in human plasma // Biochim. Biophys Acta. - 1992. - V.1123. - P.33-40].

Despite the fact that poly-3-PHB is highly biocompatible, degrades in biological media and can be used to produce various medical devices, its disadvantage is that it does not crystallize in an orderly manner; therefore, this type of PHA is rather difficult to process into products that characterized by low impact strength, stiffness and “age” in time [Lakshmi S., Laurencin C. Biodegradable polymers as biomaterials // Prog. Polym. Sci. - 2007. V.32. - P.762-798].

Especially valuable in PHA is the ability to synthesize polymers of various compositions formed by monomers with different lengths of the C chain, from C ₄ to C ₁₂ and higher. The monomer composition of PHA determines their basic properties. However, the synthesis of heteropolymer PHAs (i.e., polymers formed by monomers with different lengths of the C chain) is a complex technological problem, which can be solved in several ways. The first is to search for new strains - producers of PHA and create specialized conditions for their cultivation. The second way involves the construction of genetically modified organisms, which combine genes that control the synthesis of PHA from various natural strains.

The known producer strains of multicomponent and diverse PHA compositions formed not only by 3-hydroxybutyrate monomers, but also by other monomers: 2-hydroxybutyrate, 2-hydroxyvalerate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 4-hydroxybutyroate, 4-hydroxyvalerate and their copolymers. These PHA, in contrast to homogeneous 3-PHB, are characterized by a higher rate of biodegradation, greater elasticity and the ability to be processed into a variety of products with high physical and mechanical characteristics [US patents: 5,245,023 (September 14, 1993); 6,323,010 (November 27, 2001); 6,316,262 (November 13,2001); 6,593,116 (Jule 15, 2003); 6,689,589 (February 10, 2004); 6,838,493 (January 4, 2005); 7,229,804 (June 12, 2007)].

The disadvantages of these producer strains of heteropolymer PHA are that they are genetically modified organisms, require specialized expensive media for growth, and are also characterized by instability during cultivation and the possibility of reducing or losing the ability to synthesize PHA of the required composition.

Known natural strain Alcaligenes eutrophus B-5786 (deposited in the All-Russian collection of industrial microorganisms, VKPM), capable of synthesizing high-yield 3-polyhydroxybutyrate in mixtures of carbon dioxide, oxygen and hydrogen [RF patent No. 2053292, IPC C12N 1/20, publ. 01/27/1996, (prototype)].

A known method of producing a heteropolymer of 3-hydroxybutyric and 3-hydroxyvaleric acids, which consists in cultivating a strain of Alcaligenes eutrophus VKPM B-5786 under aeration and mixing in a liquid salt medium containing a growth substrate - hydrogen and carbon dioxide or acetic acid salt with the addition of valerianic or propionic acid acid, with a limit of nitrogen [RF patent No. 2051968, IPC C12P 7/62, publ. 01/10/1996, (prototype)].

The disadvantages of strain B-5786 and the method for producing PHA based on it are their high sensitivity to alkanoic acid salts with a C chain length of more than C ₅ , the inability to synthesize copolymers of 3-hydroxybutyrate with 4-hydroxybutyrate, and the inability to use glucose and other sugars as growth substrate.

The technical result of the invention is the identification of a new productive strain that is capable of stably synthesizing with high (70-90%) yields technologically advanced heteropolymer PHAs containing, in addition to 3-hydroxybutyrate, other monomers (3-hydroxyvalerate) as macroinclusions (over 10-20 mol.%) , 3-hydroxyhexanoate, 4-hydroxybutyrate), having a crystallinity of not higher than 50%, growing on various substrates, including mixtures of carbon dioxide with oxygen and hydrogen of various origins, sugar (fructose, glucose) and an organic spectrum acids (acetate, butyrate, as well as valerate, hexanoate, buturolactone, etc.) and having wider physiological effects in relation to the listed substrates, which are the precursors of monomers (4-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate) and enhance the formation of heteropolymer PGA.

The technical result is achieved by the fact that the identified strain Cupriavidus eutrophus VKPM B-10646, which is a producer of polyhydroxyalkanoates.

The technical result is also achieved by the fact that in the method for producing polyhydroxyalkanoates according to claim 1, which consists in cultivating a producer strain under conditions of aeration and mixing in a liquid salt medium containing a growth substrate with an additional carbon source, with a nitrogen limit, new is that the bacterial strain Cupriavidus eutrophus VKPM B-10646 is used as a producer strain, glucose or fructose, or 3-butyric acid, or a gas mixture of hydrogen, oxygen and carbon dioxide are used as a growth substrate or synthesis gas mixed with oxygen, and a solution of potassium 3-valerate, or solutions of potassium 3-valerate and potassium 3-hexanoate, or solutions of potassium 3-valerate, potassium 3-hexanoate and acrylate, or solutions are used as an additional carbon source Potassium 3-hexanoate and acrylate, or solutions of 3-butyric acid and 4-butorolactone, or solutions of 3-butyric acid, 4-butorolactone and 3-potassium valerate, or solutions of 3-butyric acid, 4-butorolactone and 3-potassium valerate, or solutions of 3-butyric acid, 4-butorolactone and potassium 3-hexanoate, or 3- butyric acid, 4-butorolactone, potassium 3-valerate and potassium 3-hexanoate.

The inventive strain is one of the variants isolated from the culture of glucose-assimilating strain Ralstonia eutropha B-8562 (the strain was obtained as a result of long-term selection of strain B-5786 on media with a single carbon source - glucose, the strain was deposited in VKPM) during long-term multi-stage selection for synthesis efficiency multicomponent PHAs formed by monomers with different C-chain lengths on a growth medium containing glucose or a mixture of organic acids as a carbon source. To select a strain capable of efficiently synthesizing heteropolymeric PHAs, sodium salts of alkanoic acids (pentanoic, hexanoic, octanoic, etc.) were additionally introduced into the growth medium, stimulating the synthesis of 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyheptanoate, etc.) or 4 -buturolactone, stimulating the formation of 4-hydroxybutyrate. The strain, in contrast to the prototype, is resistant to higher concentrations of toxic substrates (CO, alkanoic acid salts, 4-buturolactone).

The inventive strain Cupriavidus eutrophus deposited in the All-Russian collection of industrial microorganisms (VKPM), collection number VKPM B-10646.

The resulting strain is characterized by the following features:

Cultural and morphological features of the strain: gram-negative, rod cells (young - short, in the stationary phase - different lengths, sizes 0.3-0.5 × 1.2-2.0 microns), motile (young - monotrich, with age - mash). Inactive. The optimum growth is 30-31 ° C, pH 6.7-7.2. On an agar medium with peptone (heterotrophic growth conditions) form morphologically homogeneous, rounded colonies, light cream, opaque with a slightly wavy edge with a diameter of 2-4 mm. On a mineral agarized medium (autotrophic growth conditions), the colonies are small (1.5-2.5 mm), light gray, translucent. In a liquid nutrient medium, it is a homogeneous suspension (Schlegel mineral medium containing a mixture of monocarboxylic acids (or sugars, organic acids, amino acids, alcohols) as a carbon source during heterotrophic growth, and during autotrophy, a mixture of carbon dioxide, hydrogen and oxygen. Obligatory aerob. Optional chemolithoautotroph. Oxide-positive. Does not possess hydrolytic enzymes. Does not dilute gelatin. Does not hydrolyze starch. Has wide organotrophic potential and is capable as a source carbon use: sugars (glucose, fructose), amino acids (alanine, serine, leucine, histidine, tryptophan, glutamic, aspartic, lysine), organic acids (oxalic, citric, succinic, fumaric, acetic, 3 and 4-butyric acid, pentapene, hexane, octane, nonanoic), alcohols (ethanol, glycerin), 4-buturolactone, CO ₂ and CO. Nitrates, ammonium salts, urea, amino acids are used as a nitrogen source. The activity of PHA cycle enzymes is (U / min × mg protein): β-ketothiolase 3.57-4.26, acetoacetyl-CoA reductase 0.98-1.23, PHA synthase 0.08-0. 10 E., (D) -hydroxybutyrate dehydrogenase 0.18-0.22. The content of HC nucleotide pairs in DNA is 66%. A 1381 bp DNA fragment was cloned and characterized, including the nucleotide sequence of the 16S rRNA gene of strain B-10646.

Growth characteristics: the strain grows on a mineral medium with sugars or organic acids, as well as in an atmosphere of hydrogen, carbon dioxide and oxygen, specific growth factors and organic additives are not required. The boundaries of the physiological effect of pH in the range 4.4–8.6, the strain retains the ability to grow in the temperature range 20–41 ° C. It stably maintains its characteristics under varying cultivation conditions and media (replacing the carbon source and energy, low or high values of the active reaction of the medium, increasing the temperature to 35 ° C, using 4-buturolactone and monocarboxylic acid salts as a toxic growth substrate. specific growth rate - up to 0.45 1 / h, productivity - up to 2 g / l hour, total nitrogen content - up to 12%, protein - up to 65%, with a nitrogen deficiency in the medium it accumulates polymers of hydroxy derivatives of alkanoic acids (half oksialkanoaty, PHA), 97% to a maximum of different chemical composition (monomers containing a C-chain length from C ₄ to C _9).

Bacterial cultivation is carried out in a sterile mode using liter flasks filled with Schlegel medium 40-50% by volume on a thermostatically controlled rocking chair or using an automated fermentation complex BioFlo 110 (New Brunswic, USA) with a volume of 15 l, which allows for the implementation of aseptic mode stabilization of the main parameters of the culture (pH, temperature, oxygen and nitrogen concentration in the culture) on the mineral salt medium of the following composition (g / l): Na ₂ HPO ₄ · H ₂ O - 9.1, KH ₂ PO ₄ - 1.5; MgSO ₄ H ₂ O - 0.2, Fe ₃ C ₆ H ₅ O ₇ · 7H ₂ O - 0.25, NH ₄ Cl - 1.0. A Hoagland standard trace element solution at the rate of 3 ml per 1 liter of nutrient medium, which contains: H ₃ BO ₃ - 0.228, CoCe ₂ × 6H ₂ O - 0.030, CuSO ₄ × 5H ₂ O - 0.008, MnCe ₂ × 4H ₂ O - 0.008, ZnSO ₄ × 7H ₂ O - 0.176, NaMoO ₄ × 2H ₂ O - 0.050, NiCe ₂ - 0.008 (g / l). The fill factor of the fermenter is from 0.3 to 0.5, the number of revolutions of the mixer 1000-1500 (rpm).

In the heterotrophic regime, the initial concentration of sugars is 10-15 g / l with feed culture with a substrate using a peristaltic dosing pump, aeration of the culture with sterile air. In the autotrophic mode, a mixture of gases (CO ₂ , H ₂ , O ₂ ) with a component ratio of 1: 2: 7 by volume is used as a source of carbon and energy. The gas mixture using a membrane-type compressor is continuously pumped through the culture at a flow rate of 10-14 l / min. The composition of the gas mixture is monitored continuously using serial gas analyzers, as well as on a LKhM-80 MD chromatograph (Chromatograph, Russia) (detector - katharometer, carrier gas - argon).

Optimal for the growth rate are pH values of 6.7–7.2; optimum medium temperature for propagation is 29.5–31.5 ° C. The ratio of gases in the mixture during autotrophic growth (% vol.): Carbon dioxide 5-10, oxygen 10-25 (depending on the density of the culture), hydrogen from 30 to 70 (no inhibition). Schlegel salt medium (composition indicated above). With heterotrophic growth, all parameters and Schlegel’s medium are similar to autotrophic growth, the carbon source (sugars and / or organic acids) is 10-15 g / l.

Thus, the claimed strain is characterized by:

- sustainable productive growth on media of various compositions,

- the ability to maintain activity for a long time in lyophilized form,

- high activity of enzymes that control the synthesis of PHA,

- the ability to synthesize PHA with high yields,

- resistance to the effects of C-substrates - stimulators of the synthesis of heteropolymer PHA,

- the ability to synthesize heteropolymer PHAs formed by various monomers with macroinclusions of the latter,

- the ability to synthesize PHA having a degree of crystallinity below 50%.

To implement the PHA synthesis regime by the claimed strain, the Cupriavidus eutrophus VKPM B-10646 strain is grown in batch mode on Schlegel medium in an autotrophic mode (on a gas mixture of hydrogen: oxygen: carbon dioxide) or heterotrophically on sugars or organic acids: at the first stage with an excess of carbon substrate in full culture medium; at the second stage - in a nitrogen-free environment. For the formation of PHA with a different composition of monomers, an additional carbon substrate in the form of acid salts (oil or valerianic, hexanoic, octanoic, nonanoic), or 4-butorolactone, etc. is added to the medium using a peristaltic pump of a metering device. Depending on the dose applied additional carbon substrate and fermentation time, the ratio of monomers with different lengths of the C-chain in the polymer varies widely, while heteropolymer PHAs with a crystallinity of 50% and less.

The concentration of PHA in the cell biomass and the composition of monomers in it were determined after preliminary methanolysis of the samples on an Agilent 5975Inert chromatomass spectrometer (Agilent, USA). The polymer was isolated from biomass by dichloromethane, the obtained extract after its concentration on a Rotavapor R-210 rotary evaporator (Switzerland) was precipitated with isopropanol. To obtain highly purified samples, the re-dissolution and deposition procedure was performed several times. The polymer was dried in a laminar box and the physicochemical characteristics were analyzed.

Using a Waters Breeze System gel permeation chromatography system (Waters, USA), the molecular weight and molecular weight distribution of polymers were determined relative to Sigma-Aldrich polystyrene standards. X-ray diffraction analysis and determination of the degree of crystallinity of the samples were performed on a D8 ADVANCE X-ray spectrometer (Bruker, Germany) (graphite monochromator on a reflected beam). To determine the temperature characteristics of PHA, a complex thermal analysis of the samples was carried out using a synchronous thermal analyzer STA 449 Jupiter from NETZCSH (Germany), combining the simultaneous measurement of changes in mass (thermogravimetry) and heat fluxes (differential scanning calorimetry).

The preparation of polyhydroxyalkanoates based on the Cupriavidus eutrophus B-10646 strain is illustrated by the following examples:

Example 1

The museum culture of the producer strain Cupriavidus eutrophus VKPM B-10646 is suspended in a liquid salt medium containing (g / l): glucose - 10 g / l, Na ₂ HPO ₄ · H ₂ O - 9.1, KH ₂ PO ₄ - 1.5; MgSO ₄ · H ₂ O - 0.2, Fe ₃ C ₆ H ₅ O ₇ · 7H ₂ O - 0.25, NH ₄ Cl - 1.0. A Hoagland standard trace element solution at the rate of 3 ml per 1 liter of nutrient medium, which contains: H ₃ BO ₃ - 0.228, CoCe ₂ × 6H ₂ O - 0.030, CuSO ₄ × 5H ₂ O - 0.008, MnCe ₂ × 4H ₂ O - 0.008, ZnSO ₄ × 7H ₂ O - 0.176, NaMoO ₄ × 2H ₂ O - 0.050, NiCe ₂ - 0.008 (g / l). The cultivation of the producer strain is carried out periodically in glass flasks with a volume of 1 l at a fill factor of 0.3-0.5 in a thermostatically controlled rocking chair. Cultivation is carried out for 16 hours at 30 ° C and pH 7.0. The resulting culture is used as an inoculum for subsequent bacterial growth in an automated fermentation complex BioFlo 110 (New Brunswic, USA) with a volume of 15 l, which allows the implementation of an aseptic regime while stabilizing the main parameters of the culture (pH, temperature, oxygen and nitrogen concentration in the culture) on mineral salt medium of the following composition (g / l): The fermenter filling factor is from 0.3 to 0.5, the adjustable speed of the stirrer is 1000-1500 (rpm). Bacterial cultivation is carried out at 30 ° С and pH 7.0 at a current glucose concentration in the culture of 5-10 g / l and NH ₄ Cl - 0.1-0.2 g / l, the solutions of which are supplied to the culture by the metering pump in separate streams. 16 hours after turning off the nitrogen supply to the culture, cultivation is continued for 24 hours when a glucose solution is fed into the culture (current concentration in the culture is not higher than 5 g / l). The total cultivation time of the producer strain is 40 hours with a total polymer yield of 96% (by weight of dry matter of the cell). The composition of the polymer: 3-hydroxybutyrate - 99.4; 3-hydroxyvalerate - 0.4; 3-hydroxyhexanoate - 0.2 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 1).

Example 2

The cultivation of the strain is carried out similarly to Example 1, but after 16 hours after turning off the nitrogen supply to the culture, cultivation continues for 24 hours when the glucose solution is fed into the culture in separate streams (current concentration in the culture is not higher than 5 g / l) and potassium 3-valerate solution as an additional carbon source (current concentration of 3 g / l). The total cultivation time of the producer strain is 40 hours with a total polymer yield of 87% (by weight of dry matter of the cell). The composition of the polymer: 3-hydroxybutyrate - 6.4; 3-hydroxyvalerate - 93.1; 3-hydroxyhexanoate - 0.5 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 2).

Example 3

The cultivation of the strain is carried out similarly to Example 1, after 16 hours, the nitrogen supply to the culture is stopped, and cultivation is continued for 16 hours when the glucose solution (current concentration in the culture is 5 g / l) and a solution of potassium 3-valerate are added to the culture as an additional source carbon (current concentration of 2 g / l). The total cultivation time of the producer strain is 32 hours with a total polymer yield of 87.9% (based on the dry weight of the cell). The composition of the polymer: 3-hydroxybutyrate - 73.0 mol.%; 3-hydroxyvalerate - 26.1 mol%; 3-hydroxyhexanoate - 1.09 mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 3).

Example 4

The cultivation of the strain is carried out similarly to Example 1, 16 hours after turning off the nitrogen supply to the culture, cultivation is continued for 24 hours when the glucose solution is fed into the culture in separate streams (current concentration in the culture is not higher than 5 g / l), potassium 3-valerate solution and solution Potassium 3-hexanoate as an additional carbon source (current concentrations in the culture, respectively, of 3-valerate and 3-hexanoate, 3 g / l and 1 g / l). The total cultivation time of the producer strain is 40 hours with a total polymer yield of 74.6% (based on the dry weight of the cell). The composition of the polymer: 3-hydroxybutyrate - 60.5; 3-hydroxyvalerate - 33.0; 3-hydroxyhexanoate - 6.5 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 4).

Example 5

The cultivation of the strain is carried out similarly to Example 1, 16 hours after turning off the nitrogen supply to the culture, cultivation is continued for 34 hours when the glucose solution is fed into the culture in separate streams (current concentration in the culture is not higher than 5 g / l), potassium 3-valerate solution, solution Potassium 3-hexanoate as an additional carbon source (current concentrations in the culture, respectively, of 3-valerate and 3-hexanoate, 3 g / l and 1 g / l), as well as an acrylate solution (current concentration in the culture is 1.5 g / l ) Acrylate is used as an inhibitor of the beta oxidation of fatty acids in order to avoid the destruction of the C-chain of 3-hexanoate added to the culture and, thereby, increase its inclusion in PHA. The total cultivation time of the producer strain is 50 hours with a total polymer yield of 64.6% (based on the dry weight of the cell). The composition of the polymer: 3-hydroxybutyrate - 37.8; 3-hydroxyvalerate - 26.0; 3-hydroxyhexanoate - 36.2 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 5).

Example 6

The cultivation of the strain is carried out similarly to Example 1, 16 hours after turning off the nitrogen supply to the culture, cultivation continues for 34 hours when the glucose solution is fed into the culture in separate streams (current concentration in the culture is not higher than 5 g / l), potassium 3-valerate solution (current concentration of 2.5 g / l), potassium 3-hexanoate solution (current concentration in the culture is 2.0 g / l), acrylate solution (current concentration in the culture is 1.5 g / l). The total cultivation time of the producer strain is 50 hours with a total polymer yield of 75.0% (based on the dry weight of the cell). The composition of the polymer: 3-hydroxybutyrate - 33.9; 3-hydroxyvalerate - 30.1; 3-hydroxyhexanoate - 36.0 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 6).

Example 7

The cultivation of the strain is carried out similarly to Example 1, 16 hours after turning off the nitrogen supply to the culture, cultivation is continued for 30 hours when the glucose solution is fed into the culture in separate streams (current concentration in the culture is not higher than 5 g / l), potassium 3-hexanoate solution is used as an additional carbon source (current concentration in the culture is 2.5 g / l) and an acrylate solution (current concentration in the culture is 1.5 g / l). The total cultivation time of the producer strain is 46 hours with a total polymer yield of 79.0% (by weight of dry matter of the cell). The composition of the polymer: 3-hydroxybutyrate - 43.7; 3-hydroxyvalerate - traces; 3-hydroxyhexanoate - 56.3 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 7).

Example 8

The cultivation of the strain is carried out similarly to Example 1, but fructose is used as a growth substrate at a current concentration in the culture of 5-10 g / l and NH ₄ Cl - 0.1-0.2 g / l. After 12 hours, the flow of nitrogen into the culture is stopped, and cultivation is continued for 22 hours when the solution of 3-butyric acid (current concentration in the culture is not higher than 5 g / l) and a solution of 4-butorolactone as an additional carbon source and precursor are fed into the culture in separate streams formation of 4-hydroxybutyrate monomers (current concentration of 2 g / l). The total cultivation time of the producer strain is 44 hours with a total polymer yield of 87.2% (based on the dry weight of the cell). The composition of the polymer: 3-hydroxybutyrate - 70.1; 4-hydroxybutyrate - 12.0; 3-hydroxyvalerate - 1.4; 3-hydroxyhexanoate - 0.6 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 8).

Example 9

The cultivation of the strain is carried out similarly to Example 1, fructose is used as a growth substrate at a current concentration in the culture of 5-10 g / l and NH ₄ Cl - 0.1-0.2 g / l. After 16 hours, the flow of nitrogen into the culture is stopped, and cultivation is continued for 22 hours when the solution of 3-butyric acid (current concentration in the culture is not higher than 5 g / l), a solution of 4-butorolactone and a solution of potassium 3-valerate as an additional source of carbon (current concentrations of which in the culture are 2.5 g / l). The total cultivation time of the producer strain is 38 hours with a total polymer yield of 80.0% (by weight of the dry matter of the cell). The composition of the polymer: 3-hydroxybutyrate - 55.4; 4-hydroxybutyrate - 8.0; 3-hydroxyvalerate - 36.6; 3-hydroxyhexanoate - traces (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 9).

Example 10

The cultivation of the strain is carried out similarly to Example 1, fructose is used as a growth substrate at a current concentration in the culture of 5-10 g / l and NH ₄ Cl - 0.1-0.2 g / l. After 16 hours, the flow of nitrogen into the culture is stopped and cultivation is continued for 36 hours when a solution of 3-butyric acid (current concentration in the culture is not higher than 5 g / l) and a solution of 4-buturolactone (current concentration in the culture are 2.5 g) are fed into the culture by separate streams / l) and a solution of potassium 3-valerate (current concentration in the culture is 3 g / l). The total cultivation time of the producer strain is 52 hours with a total polymer yield of 76.9% (based on the dry weight of the cell). The composition of the polymer: 3-hydroxybutyrate - 34.8; 4-hydroxybutyrate - 18.0; 3-hydroxyvalerate - 46.6; 3-hydroxyhexanoate - 0.6 (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 10).

Example 11

The cultivation of the strain is carried out analogously to Example 1, but 3-butyric acid is used as a growth substrate with a current concentration of butyric acid in the culture of 5 g / l and NH ₄ Cl - 0.1-0.2 g / l. After 16 hours, the nitrogen supply to the culture is stopped and cultivation is continued for 26 hours when the solution of 3-butyric acid (current concentration in the culture is not higher than 5 g / l) and a solution of 4-butorolactone as an additional carbon source (current concentration) are fed into the culture in separate streams 2 g / l). The total cultivation time of the producer strain is 42 hours with a total polymer yield of 73% (by weight of dry matter of the cell). The composition of the polymer: 3-hydroxybutyrate - 70.3; 4-hydroxybutyrate - 28.5; 3-hydroxyvalerate - 0.8; 3-hydroxyhexanoate - 0.4 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 11).

Example 12

The cultivation of the strain is carried out similarly to Example 1, 3-butyric acid is used as a growth substrate with a current concentration of butyric acid in the culture of 5 g / l and NH ₄ Cl - 0.1-0.2 g / l. After 20 hours, the nitrogen supply to the culture is stopped and cultivation is continued for 26 hours when the solution of 3-butyric acid (current concentration in the culture is not higher than 5 g / l) and a solution of 4-buturolactone as an additional carbon source (current concentration) are fed into the culture in separate streams 2 g / l) and a solution of potassium 3-hexanoate (current concentration of 1.5 g / l). The total cultivation time of the producer strain is 46 hours with a total polymer yield of 70% (by weight of dry matter of the cell). The composition of the polymer: 3-hydroxybutyrate - 49.3; 4-hydroxybutyrate - 21.5; 3-hydroxyvalerate -0.8; 3-hydroxyhexanoate - 28.4 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 12).

Example 13

The cultivation of the strain is carried out similarly to Example 1, 3-butyric acid is used as a growth substrate with a current concentration of butyric acid in the culture of 5 g / l and NH ₄ Cl - 0.1-0.2 g / l. After 20 hours, the flow of nitrogen into the culture is stopped and cultivation is continued for 30 hours when a separate solution of 3-butyric acid (current concentration in the culture is not higher than 5 g / l) and a solution of 4-buturolactone as an additional carbon source (current concentration) are fed into the culture in separate streams 2 g / l) and a solution of potassium 3-valerate (current concentration of 1.5 g / l) and a solution of potassium 3-hexanoate (current concentration of 1.5 g / l). The total cultivation time of the producer strain is 50 hours with a total polymer yield of 75% (by weight of dry matter of the cell). The composition of the polymer: 3-hydroxybutyrate - 36.3; 4-hydroxybutyrate 16.5; 3-hydroxyvalerate -26.8; 3-hydroxyhexanoate - 20.4 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 13).

Example 14

The cultivation of the strain is carried out similarly to Example 1, but as a growth substrate using a mixture of gases of the following composition (in vol.%): Hydrogen 70, oxygen 20 and carbon dioxide 10, which from a gas tank with a volume of 50 l through a system of microbiological filters is pumped continuously through a culture with a compressor with speed of 8 l / min. 24 hours after the nitrogen supply to the culture was turned off, cultivation continued for another 26 hours when a solution of potassium 3-valerate was added to the culture as an additional carbon source (current concentration of 2 g / l). The total cultivation time of the producer strain is 50 hours with a total polymer yield of 82% (by weight of dry matter of the cell). The composition of the polymer: 3-hydroxybutyrate - 46.1; 3-hydroxyvalerate - 53.0; 3-hydroxyhexanoate - 0.9 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 14).

Example 15

The cultivation of the strain is carried out similarly to Example 1, but the synthesis gas obtained by gasification of brown coals is used as a growth substrate [according to RF patent No. 2207375, IPC C12P 7/42, publ. June 27, 2003] or conversion of natural gas [as USSR No. 1233483, publ. 1985 - No. 32] and the oxygen used gas mixture contains (in vol.%): Hydrogen 60, oxygen 20 and carbon dioxide 10, carbon monoxide 10, which is pumped continuously from the gas tank with a volume of 50 l through a system of microbiological filters through a compressor at a speed 8 l / min 30 hours after turning off the nitrogen supply to the culture, cultivation is continued for another 30 hours when a solution of potassium 3-valerate is added to the culture as an additional carbon source (current concentration of 2 g / l) and potassium 3-hexanoate (current concentration of 1.5 g / l) . The total cultivation time of the producer strain is 60 hours with a total polymer yield of 80% (by weight of dry matter of the cell). The composition of the polymer: 3-hydroxybutyrate - 42.8; 3-hydroxyvalerate - 36.0; 3-hydroxyhexanoate - 20.9 molar percent (mol.%). Physico-chemical characteristics of this type of PHA are presented in the table (No. 15).

The use of the Cupriavidus eutrophus VKPM B-10646 strain as a producer of PHA, varying the concentration and type of carbon substrate, as well as the duration of cultivation, allows one to obtain a spectrum of various PHA formed by various monomers and significantly differing in physicochemical properties (table 1).

Table 1 Composition and physicochemical properties of PHA synthesized by Cupriavidus eutrophus VKPM B-10646 No. The composition of the PHA - the ratio of monomers (mol.%) PHA Properties 3 GB 3-GW 3-gg 4 GB M _in , yes PD (n) C _x ,% T _pl. ° C T ° _deg. ° C one 99.4 0.4 0.2 - 1340329 2.64 78 * 182 286 2 6.4 93.1 0.5 - 1111446 3.49 41 159 260 3 73.0 26.1 1.09 - 1501622 2.27 fifty 176 272 four 60.5 33.0 6.5 - 1200451 2.67 40 167 276 5 37.8 26.0 36.2 - 986912 2.86 32 166 277 6 33.9 30.1 36.0 - 812892 2.64 36 169 281 7 43.7 0.1 56.2 - 1240854 3.12 thirty 167 280 8 70.1 1.4 0.6 12.0 844256 2.31 38 170 278 9 55.4 36.6 next 8.0 1113866 2.32 43 168 278 10 34.8 46.6 0.6 18.0 965401 2.59 25 166 274 eleven 70.3 0.8 0.4 28.5 568412 1.91 12 167 272 12 49.3 0.8 28.4 21.5 1349215 2.98 45 171 283 13 36.3 26.8 20.4 16.5 1216090 2.67 46 177 278 fourteen 46.1 53.0 0.9 - 1230218 2.46 44 164 267 fifteen 42.8 36.0 20.9 - 1109216 2.89 fifty 176 280 Note: * - as an example, homopolymer poly 3-hydroxybutyrate having a high degree of crystallinity 3-GB-3 hydroxybutyrate; 3-HB-3 hydroxyvalerate; 3-GG-3 hydroxyhexanoate; 4-GB-4 hydroxybutyrate; M _in - molecular weight, Yes - daltons; PD (n) - polydispersity; C _x ,% is the degree of crystallinity; T _pl. ° C - melting point; T _deg. ° C - temperature of thermal degradation.

Claims (2)

1. The bacterial strain Cupriavidus eutrophus VKPM B-10646 - producer of polyhydroxyalkanoates. 2. The method of producing polyhydroxyalkanoates, which consists in cultivating the producer strain under aeration and mixing on a liquid salt medium containing a growth substrate with an additional carbon source, with a nitrogen limit, characterized in that the bacterial strain Cupriavidus eutrophus VKPM B is used as the producer strain -10646, glucose or fructose, or 3-butyric acid, or a gas mixture of hydrogen, oxygen and carbon dioxide, or synthesis gas mixed with oxygen, is used as a growth substrate, and as a supplement For a carbon source, use a solution of potassium 3-valerate, or solutions of potassium 3-valerate and potassium 3-hexanoate, or solutions of potassium 3-valerate, 3-potassium hexanoate and acrylate, or solutions of potassium 3-hexanoate and acrylate, or 3-oil solutions acids and 4-butorolactone, or solutions of 3-butyric acid, 4-butorolactone and 3-valerate of potassium, or solutions of 3-butyric acid, 4-butorolactone and 3-hexanoate of potassium, or solutions of 3-butyric acid, 4-butorolactone, 3 potassium valerate and potassium 3-hexanoate.