Cupriavidus eutrophus vkpm b-10646 bacteria strain - producer of polyhydroxy alkanoates and production method thereof

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

 

The invention relates to biotechnology and related to strain-producer of polymers hydroxyethanoic acid (PGA) and the method of their derivation. These thermoplastic polymers are characterized by high biological compatibility and destruction in biological environments, so promising for various applications in medicine (surgical items, implants, matrices functioning cells), pharmacology (to create long-term medicinal systems with controlled release of drugs, hygiene and sanitation), in agriculture (biorstwami coating and packaging of fertilizers and pesticides with the aim of targeted delivery).

Among producers GAA known various prokaryotic microorganisms belonging to different taxonomic groups, able to grow on different substrates (sugars, organic acids, waste from agriculture and industry) and to synthesize these polymers [Madison L.L., Huisman G.V. Metabolic engineering of poly (3-hydroxyalkanoates): From DNA to plastic // Environ. And Molecular Biology Rev. - 1999. - Vol.63. - P.1-25]. However, for industrial applications selected several highly productive microorganisms. This odoratissima bacteria Alcaligenes latus, Alcaligenes eutrophus (renamed from Ralstonia and Wautersia eutropha, currently referred to the genus Cupriavidus), nitrogen-fixing Azotobacter vinelandii, methylotrophy Methylomonas, Methylobacterium organophlum [Madison L.L., Huisman G.V. Metabolic engineering of poly(3-hydroxyalkanoates): From DNA to plastic // Environ. And Molecular Biology Rev. - 1999. - Vol.63. - P.1-25], synthesizing, mainly polymer 3-hydroxybutiric acid (3-hydroxybutyrate (3-FCB), which is the most studied and widely used representative of the GAA. 3-FCB is vysokokritichnyh (degree of crystallinity is more than 70%) thermoplastic, high biocompatibility which is based on the fact that hydroxybutiric acid is a natural metabolite of cells and tissues of higher organisms and humans [R.N. Reusch, Sparrow A.W., J. Gardiner

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

Despite the fact that poly-3-FCB has a high biological compatibility, degradable in biological environments and can be used for various medical devices, its disadvantage is that it does not crystallize in an orderly fashion, so this type GAA difficult to recycle products that have low impact strength, rigidity and "grow old" in time [Lakshmi S., Laurencin C. Biodegradable polymers as biomaterials // Prog. Polym. Sci. - 2007. V.32. - P.762-798].

Especially valuable in GAA is the possibility of synthesis of polymers of different composition formed by monomers with different length of the C-chain from C4to C12and above. Monomeric composition GAA determines their basic properties. However, the synthesis of heteropolymeric GAA (e polymers formed by monomers with different length of the C-chain) is a complex technological problem, the solution of which is possible in several ways. The first is the search for new strains - producers GAA and creation of special conditions for their cultivation. The second way involves the construction of genetically modified organisms, which combine genes controlling the synthesis of PGA from different natural strains.

Known strains-producers of multicomponent and diverse GAA formed not only monomers 3-hydroxybutyrate, but also other monomers: 2-hydroxybutyrate, 2-hydroxyvalerate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyoctanoic, 3-hydroxydecanoate and 4-hydroxybutyrate, 4-hydroxyvalerate and their copolymers. These GAA, in contrast to the homogeneous 3-FCB, are characterized by a higher rate of biodegradation, greater elasticity and ability to be processed into a variety of products with high physical-mechanical characteristics [U.S. patent # :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 strains-producers heteropolymeric GAA, are that they are genetically modified organisms require for ROS is expensive and specialized environments as well as instability in the cultivation process and the possible reduction or loss of ability to synthesize PGA desired composition.

Known natural strain Alcaligenes eutrophus B-5786 (deposited in Russian national collection of industrial microorganisms, PMBC), capable of high outputs to synthesize 3-polyhydroxybutyric on mixtures of carbon dioxide, oxygen, and hydrogen [RF patent №2053292, IPC C12N 1/20, publ. 27.01.1996,, (prototype)].

A known method of producing heteropolymer 3-hydroxybutyric and 3-oxovalerate acids, which lies in the cultivation of the strain Alcaligenes eutrophus VKPM B-5786 under conditions of aeration and mixing liquid salt medium containing a growth substrate hydrogen and carbon dioxide or salt of acetic acid with the addition of valerianic or propionic acid, at the limit of nitrogen [RF patent №2051968, IPC C12P 7/62, publ. 10.01.1996,, (prototype)].

Disadvantages of strain In-5786 and the method of obtaining on the basis of the GAA are in high sensitivity to salts alkanovykh acids with long-chain more than C5the inability to synthesize copolymers of 3-hydroxybutyrate, 4-hydroxybutyrate, and the inability to utilize glucose and other sugars as a growth substrate.

The technical result of the invention is the identification of a new productive strain, is capable of being the go steadily to synthesize high (70-90%) outputs tech heteropolymer GAA, containing as microflute (over 10-20 mol.%), in addition to 3-hydroxybutyrate, other monomers (3-hydroxyvalerate, 3-hydroxyhexanoate, 4-hydroxybutyrate)with a degree of crystallinity of not higher than 50%, growing on various substrates, including mixtures of carbon dioxide with oxygen and hydrogen of different origin, sugars (fructose, glucose) and a range of organic acids (acetate, butyrate, and valerate, hexanoate, butyrolactone and others) and with a broader limits of physiological actions in relation to the listed substrates, which are the precursors of monomers (4-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate) and reinforcing education heteropolymeric GAA.

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

The technical result is also achieved by the fact that in the method of obtaining polyhydroxyalkanoate according to claim 1, which lies in the cultivation of the producer strain in conditions of aeration and mixing liquid salt medium containing a growth substrate with an additional source of carbon, while the limit of nitrogen, what is new is that as a producer strain using the strain of bacteria Cupriavidus eutrophus VKPM B-10646, as the growth substrate using glucose or fructose, or 3-oil to the slot, or a gas mixture of hydrogen, oxygen and carbon dioxide, or synthesis gas is mixed with oxygen, and as an additional carbon source using a solution of 3-valerate potassium, or solutions of 3-valerate potassium and 3-hexanoate potassium, or solutions of 3-potassium valerate, 3-hexanoate and potassium acrylate, or solutions of 3-hexanoate and potassium acrylate, or solutions of 3-oil acid and 4-butanolate, or solutions of 3-oil acid, 4-butanolate and 3-valerate potassium, or solutions of 3-oil acid, 4-butanolate and 3-valerate potassium, or solutions of 3-oil acid, 4-butanolate and 3-hexanoate potassium, or solutions of 3-oil acid, 4-butanolate, 3-valerate potassium and 3-hexanoate potassium.

The inventive strain is one of the variants isolated from the culture glucosidase strain Ralstonia eutropha B-8562 (strain obtained in the result of a long selection of strain B-5786 on media with the sole carbon source is glucose, a strain deposited in PMBC) during long-term multi-stage selection on the effectiveness of multicomponent synthesis GAA formed by monomers with different length chains, the growth medium containing as a carbon source, glucose or a mixture of organic acids. For selection of strain is able to synthesize heteropolymer GAA, the composition of the growth medium was additionally introduced travie salt alkanovykh acids (pentanol, hexane, octane and others), stimulating the synthesis of 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyethanoic and others) or 4-butyrolactone, stimulating the formation of 4-hydroxybutyrate. Strain unlike the prototype resistant to higher concentrations of toxic substrates (CO, salt alkanovykh acid, 4-butyrolactone).

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

The resulting strain is characterized by the following features:

Cultural and morphological characteristics of strain: gram, the rod cells (young - short, stationary phase - different lengths, sizes of 0.3-0.5×1.2-2.0 MK), mobile (young, monotachi, with age - peritricha). Lobopodian. The optimum growth 30-31°C, pH of 6.7 to 7.2. On agar medium with peptone (heterotrophic growth conditions) constitute a morphologically homogeneous rounded colonies, light cream, opaque with a slightly wavy edge with a diameter of 2-4 mm On mineral agar medium (autotrophic growth conditions) colonies are small (1.5-2.5 mm), light-gray, semi-transparent. In the liquid nutrient medium is homogeneous suspension (mineral environment of Schlegel, containing as a carbon source mixture of monocarboxylic acids or sugars, organic acids, linakis is the notes, alcohols) during heterotrophic growth, if autotrophy - a mixture of carbon dioxide, hydrogen and oxygen. Obligate aerobe. Optional chemolithoautotroph. Oxidizability. Hydrolytic enzymes is not. Gelatine does not dilute. Starch is not hydrolyses. It has a wide organotrophic potential as a carbon source to use: sugar (glucose, fructose), amino acids (alanine, serine, leucine, histidine, tryptophan, glutamic, aspartic, lysine), organic acids (oxalic, citric, succinic, fumaric, acetic, 3-and 4-butyric acid, pentanoyl, hexane, octane, nonanoyl), alcohols (ethanol, glycerol), 4-butyrolactone, CO2and CO. As the nitrogen source uses nitrates, ammonium salts, urea, amino acids. Enzyme activity cycle GAA is (U/min×mg protein): β-ketothiolase 3.57-4.26, acetoacetyl-CoA-reductase 0.98-1.23, PGA-synthase 0.08-0.10 E., (D)-hydroxysteroiddehydrogenase 0.18-0.22. Content HZ nucleotides in DNA is 66%. Cloned and characterized a DNA fragment 1381 i.e. comprising the nucleotide sequence of 16S rRNA gene of strain In-10646.

Growth characteristics: strain grows on mineral medium with sugars or organic acids, as well as in the atmosphere of hydrogen, carbon dioxide and oxygen, specific growth factors and organic add is not required. The boundaries of the physiological effect of pH in the range of 4.4-8.6, strain retains the ability to grow in the temperature range 20-41°C. Stably retains its characteristics under varying culture conditions and environments (replacement source of carbon and energy, low or high values of the active reaction medium, the temperature increases to 35°C, using as the growth of toxic substrate - 4-butyrolactone and salts of monocarboxylic acids. Full nutrient medium specific growth rate to 0.45 1/h, the productivity - up to 2 g/l hour, the total nitrogen content of 12%, protein - up to 65%, with a deficit of nitrogen in the environment accumulates polymers hydroxy alkanovykh acids (polydioxanone, PGA), up to a maximum of 97% of different chemical composition (containing monomers with long-chain from C4to C9).

Cultivation of bacteria is carried out in a sterile mode using a liter flasks filled environment of Schlegel 40-50% by volume on a thermostatted shaker or using automated enzymatic complex BioFlo 110 ("New Brunswic, USA) with a volume of 15 l, which allows to implement aseptic mode when stabilization of the main culture parameters (pH, temperature, concentration of oxygen and nitrogen in the culture) in mineral salt medium of the following composition (g/l): Na2HPO4·H2O - 9.1, H 2PO4- 1.5; MgSO4H2O - 0.2, Fe3C6H5O7·7H2O - 0.25, NH4Cl - 1.0. Standard solution of trace elements on Hoagland at the rate of 3 ml per 1 l culture medium, which contains: H3BO3- 0.228, CoCe2×6H2O - 0.030, CuSO4×5H2O - 0.008, MnCe2×4H2O - 0.008, ZnSO4×7H2O - 0.176, NaMoO4×2H2O - 0.050, NiCe2- 0.008 (g/l). The fill factor of the fermenter is from 0.3 to 0.5, the number of revolutions of the stirrer 1000-1500 (rpm).

When heterotrophic mode - initial concentration of sugars - 10-15 g/l with water culture substrate using a peristaltic dosing pump, aeration culture - sterile air. In autotrophic mode as a source of carbon and energy using a mixture of gases (CO2H2O2) with a mixing ratio of 1:2:7 by volume. The gas mixture using a membrane type compressor is continuously pumped through the culture with the consumption of 10-14 l/min. control of the composition of the gas mixture is carried out in a continuous mode using a serial analyzers, as well as on the chromatograph LHM-80 MD ("GC", Russia) (detector katharometer, carrier gas - argon).

The optimal growth rate of pH 6.7-7.2, the optimum temperature for reproduction 29.5-31.5°C. the Ratio of gases in the mixture during autotrophic growth (% vol.): dioxide is gerada 5-10, oxygen 10-25 (depending on the density of the culture), hydrogen from 30 to 70 (no inhibition). Salt environment Schlegel (composition specified above). When heterotrophic growth - all parameters and environment Schlegel similar to autotrophic growth, the carbon source (sugar and/or organic acids) - 10-15 g/l

Thus, for the proposed strain characteristic:

- sustainable productive growth on media of different composition,

the long - term ability to maintain activity in dried form,

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

- ability to synthesis of PGA with high yields,

- resistance to impact With the substrate - stimulants synthesis heteropolymeric GAA,

- ability to synthesis heteropolymeric GAA formed of various monomers with macrostructure last,

- ability to synthesize PGA having a degree of crystallinity less than 50%.

For realization of this mode of synthesis of PGA declare strain, strain Cupriavidus eutrophus VKPM B-10646 grown in periodic mode on the environment Schlegel in autotrophic mode (mixed gas of hydrogen:oxygen:carbon dioxide) or heterotrophic on sugars or organic acids: in the first phase with an excess of carbon substrate on a complete nutrient medium; the second stage - in a nitrogen-free environment. For education GAA with different composition of the monomers inof the environment using a peristaltic pump dispenser in culture contribute additional carbon substrate in the form of salts of acids (oily or valerianic, hexane, octane, nonanol), or 4-butanolate, etc. depending on the dose supplied additional carbon substrate and fermentation time, the ratio of monomers with different long-chain polymer varies widely, with synthesized heteropolymer GAA, having a crystallinity of 50% or less.

The concentration of PGA in cell biomass and composition of monomers was determined after preliminary methanolysis samples on / mass spectrometer Agilent 5975Inert, firms Agilent (USA). The selection of the polymer from a biomass conducted dichloromethane, the extract obtained after concentration on a rotary evaporator the rotavapor R-210 (Switzerland), precipitated with isopropanol. To obtain highly purified samples of the reconstitution procedure and deposition was performed several times. The polymer was dried in a box-the kelp and analyzed physico-chemical characteristics.

Using gel permeation chromatography Waters Breeze System" company "Waters" (USA) determined the molecular mass and molecular mass distribution of polymers relative to polystyrene standards "Sigma-Aldrich". X-ray analysis and the determination of the degree of crystallinity of the samples was performed on rentgenospektralny D8 ADVANCE "Bruker (Germany) (graphite monochromator on the reflected beam). To define which of the temperature characteristics of the GAA conducted a comprehensive thermal analysis of the samples using simultaneous thermoanalyzer STA 449 Jupiter firm NETZCSH (Germany), combining simultaneous measurement of mass changes (Tg) and heat flow (differential scanning calorimetry).

Getting polyhydroxyalkanoates based on strain Cupriavidus eutrophus B-10646 illustrated by the following examples:

Example 1.

The Museum culture of the producer strain Cupriavidus eutrophus VKPM B-10646 suspended in a liquid salt medium containing (g/l): glucose 10 g/l, Na2HPO4·H2O - 9.1, KH2PO4- 1.5; MgSO4·H2O - 0.2, Fe3C6H5O7·7H2O - 0.25, NH4Cl - 1.0. Standard solution of trace elements on Hoagland at the rate of 3 ml per 1 l culture medium, which contains: H3BO3- 0.228, Sosa2×6H2O - 0.030, CuSO4×5H2O - 0.008, MnCe2×4H2O - 0.008, ZnSO4×7H2O - 0.176, NaMoO4×2H2O - 0.050, NiCe2- 0.008 (g/l). The cultivation of the producer strain is carried out in periodic mode in glass flasks with a volume of 1 liter with a fill factor of 0.3-0.5 in a thermostatted shaker. The cultivation is carried out for 16 h at 30°C and pH 7.0. The resulting culture used as inoculum for the next growing bacteria in an automated fermentation complex BioFlo 110 ("New Brunswic, USA) with a volume of 15 l, which allows to implement aseptic mode when stabilization of the main culture parameters (pH, temperature, oncentrate oxygen and nitrogen in the culture) in mineral salt medium of the following composition (g/l): the fill Factor of the fermenter is from 0.3 to 0.5, adjustable number of revolutions of the stirrer 1000-1500 (rpm). Cultivation of bacteria is carried out at 30°C and pH 7.0, at the current concentration of glucose in the culture of 5-10 g/l and NH4Cl - 0.1-0.2 g/l, the solutions of which are served in the culture of NASA-dispenser separate threads. After 16 h after stopping the feed of nitrogen in the culture, the cultivation continued for 24 h at the feed in the culture of glucose solution (the current concentration in the culture - not more than 5 g/l). The total time of cultivation of the producer strain is 40 h when the 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 GAA presented in table (No. 1).

Example 2.

Cultivation of the strain is carried out analogously to Example 1, but after 16 h after stopping the feed of nitrogen in the culture, the cultivation continued for 24 h at the feed in the culture of separate streams of glucose solution (the current concentration in the culture - not more than 5 g/l) and a solution of 3-valerate potassium as an additional carbon source (the current concentration of 3 g/l). The total time of cultivation of the producer strain is 40 h when the total polymer yield 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 about the clients (mol.%). Physico-chemical characteristics of this type GAA presented in table (No. 2).

Example 3.

Cultivation of the strain is carried out analogously to Example 1, after 16 h the supply of nitrogen in the culture cease, and the cultivation continued for 16 h at the feed in the culture of separate streams of glucose solution (the current concentration in the culture - 5 g/l) and a solution of 3-valerate potassium as an additional carbon source (the current concentration of 2 g/l). The total time of cultivation of the producer strain is 32 hours when the total polymer yield 87.9% (by weight of dry matter 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 GAA presented in table (No. 3).

Example 4.

Cultivation of the strain is carried out analogously to Example 1, after 16 h after stopping the feed of nitrogen in the culture, the cultivation continued for 24 h at the feed in the culture of separate streams of glucose solution (the current concentration in the culture - not more than 5 g/l)solution of 3-valerate potassium and a solution of 3-hexanoate potassium as an additional carbon source (current concentrations in culture, respectively, 3-valerate and 3-hexanoate, 3 g/l and 1 g/l). The total time of cultivation of the producer strain is 40 h when the total polymer yield 74.6% (by weight of dry matter of the cell). SOS the AB polymer: 3-hydroxybutyrate - 60.5; 3-hydroxyvalerate - 33.0; 3-hydroxyhexanoate - 6.5 molar percent (mol.%). Physico-chemical characteristics of this type GAA is presented in table No. 4).

Example 5.

Cultivation of the strain is carried out analogously to Example 1, after 16 h after stopping the feed of nitrogen in the culture, cultivation continue 34 h at the feed in the culture of separate streams of glucose solution (the current concentration in the culture - not more than 5 g/l)solution of 3-valerate potassium solution of 3-hexanoate potassium as an additional carbon source (current concentrations in culture, respectively, 3-valerate and 3-hexanoate, 3 g/l and 1 g/l)and solution of acrylate (the current concentration in the culture - 1.5 g/l). The acrylate used as an inhibitor of the cycle of beta-oxidation of fatty acids to avoid the destruction of the C-chain is added to the culture 3-hexanoate and, thereby, increasing its inclusion in the GAA. The total time of cultivation of the producer strain is 50 h when the total polymer yield 64.6% (by weight of dry matter 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 GAA is presented in table No. 5).

Example 6.

Cultivation of the strain is carried out analogously to Example 1, after 16 h after stopping the feed of nitrogen in the culture, cultivar is of continued 34 h at the feed in the culture of separate streams of glucose solution (the current concentration in the culture not more than 5 g/l)solution of 3-valerate potassium (the current concentration of 2.5 g/l)solution of 3-hexanoate potassium (the current concentration in the culture 2.0 g/l), solution of acrylate (the current concentration in the culture - 1.5 g/l). The total time of cultivation of the producer strain is 50 h when the total polymer yield 75.0% (by weight of dry matter 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 GAA presented in table (No. 6).

Example 7.

Cultivation of the strain is carried out analogously to Example 1, after 16 h after stopping the feed of nitrogen in the culture, the cultivation continued for 30 h at the feed in the culture of separate streams of glucose solution (the current concentration in the culture - not more than 5 g/l)solution of 3-hexanoate potassium as an additional carbon source (the current concentration in the culture 2.5 g/l) and a solution of acrylate (the current concentration in the culture - 1.5 g/l). The total time of cultivation of the producer strain is 46 h under the total polymer yield 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 GAA presented in table (No. 7).

Example 8.

Cultivation of the strain of spending is similar to Example 1, but as the growth substrate using fructose at current concentrations in the culture of 5-10 g/l and NH4Cl - 0.1-0.2 g/l After 12 h the supply of nitrogen in the culture cease, and the cultivation continued for 22 h at the feed in the culture of separate streams of solution of 3-butyric acid (the current concentration in the culture - not more than 5 g/l) and a solution of 4-butanolate as an additional source of carbon and precursor formation of monomers 4-hydroxybutyrate (the current concentration of 2 g/l). The total time of cultivation of the producer strain is 44 hours when the total polymer yield 87.2% (by weight of dry matter 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 GAA presented in table (No. 8).

Example 9.

Cultivation of the strain is carried out analogously to Example 1, as the growth substrate using fructose at current concentrations in the culture of 5-10 g/l and NH4Cl - 0.1-0.2 g/l After 16 h the supply of nitrogen in the culture cease, and the cultivation continued for 22 h at the feed in the culture of separate streams of solution of 3-butyric acid (the current concentration in the culture - not more than 5 g/l)solution of 4-butanolate and a solution of 3-valerate potassium as an additional carbon source (current concentration is AI which culture are 2.5 g/l). The total time of cultivation of the producer strain is 38 hours when the total polymer yield 80.0% (by weight of 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 GAA presented in table (No. 9).

Example 10.

Cultivation of the strain is carried out analogously to Example 1, as the growth substrate using fructose at current concentrations in the culture of 5-10 g/l and NH4Cl - 0.1-0.2 g/l After 16 h the supply of nitrogen in the culture stop and cultivation continued for 36 h at the feed in the culture of separate streams of solution of 3-butyric acid (the current concentration in the culture - not more than 5 g/l)solution of 4-butyrolactone (the current concentration in the culture at 2.5 g/l) and a solution of 3-valerate potassium (the current concentration in the culture - 3 g/l). The total time of cultivation of the producer strain is 52 h when the total polymer yield 76.9% (by weight of dry matter 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 GAA presented in table (No. 10).

Example 11.

Cultivation of the strain is carried out analogously to Example 1, but as the growth substrate using 3-butyric acid at a current the concentration of butyric acid in the culture of 5 g/l and NH 4Cl - 0.1-0.2 g/l After 16 h the supply of nitrogen in the culture stop and cultivation continued for 26 h at the feed in the culture of separate streams of solution of 3-butyric acid (the current concentration in the culture - not more than 5 g/l) and a solution of 4-butanolate as an additional carbon source (the current concentration of 2 g/l). The total time of cultivation of the producer strain is 42 h when the total polymer yield 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 GAA presented in table (No. 11).

Example 12.

Cultivation of the strain is carried out analogously to Example 1, as the growth substrate using 3-butyric acid at the current concentration of butyric acid in the culture of 5 g/l and NH4Cl - 0.1-0.2 g/l After 20 h the supply of nitrogen in the culture stop and cultivation continued for 26 h at the feed in the culture of separate streams of solution of 3-butyric acid (the current concentration in the culture - not more than 5 g/l) and a solution of 4-butyrolactone as an additional carbon source (the current concentration of 2 g/l) and a solution of 3-hexanoate potassium (current concentration 1.5 g/l). The total time of cultivation of the producer strain is 46 h under the total polymer yield 70% (in the su 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 GAA presented in table (No. 12).

Example 13.

Cultivation of the strain is carried out analogously to Example 1, as the growth substrate using 3-butyric acid at the current concentration of butyric acid in the culture of 5 g/l and NH4Cl - 0.1-0.2 g/l After 20 h the supply of nitrogen in the culture stop and cultivation continued for 30 h at the feed in the culture of separate streams of solution of 3-butyric acid (the current concentration in the culture - not more than 5 g/l) and a solution of 4-butyrolactone as an additional carbon source (the current concentration of 2 g/l) and a solution of 3-valerate potassium (current concentration 1.5 g/l) and a solution of 3-hexanoate potassium (current concentration 1.5 g/l). The total time of cultivation of the producer strain is 50 h when the total polymer yield 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 GAA presented in table (No. 13).

Example 14.

Cultivation of the strain is carried out analogously to Example 1, but as the growth substrate using a mixture of gases sledujushih the composition (in vol.%): hydrogen 70, oxygen 20 and the carbon dioxide 10, from which gas tank volume of 50 l through the system microbiological filters pumped continuously through culture compressor at a rate of 8 l/min at 24 h after stopping the feed of nitrogen in the culture, the cultivation continued for another 26 hours when applying to the culture solution of 3-valerate potassium as an additional carbon source (the current concentration of 2 g/l). The total time of cultivation of the producer strain is 50 h when the total polymer yield 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 GAA presented in table (No. 14).

Example 15.

Cultivation of the strain is carried out analogously to Example 1, but as the growth substrate using synthesis gas produced by gasification of brown coal [according to the patent of Russian Federation №2207375, IPC C12P 7/42, publ. 27.06.2003,] or the conversion of natural gas [A.S. USSR №1233483, publ. 1985 - No. 32] and oxygen gas mixture contains (in vol.%): the 60 hydrogen, oxygen 20 and the carbon dioxide 10, carbon monoxide 10, from which gas tank volume of 50 l through the system microbiological filters pumped continuously through culture compressor at a rate of 8 l/min After 30 h after stopping the feed of nitrogen in the culture, Kul is the illicit cultivation continued for another 30 h when submitting to the culture solution of 3-valerate potassium as an additional carbon source (the current concentration of 2 g/l) and 3-hexanoate potassium (current concentration 1.5 g/l). The total time of cultivation of the producer strain is 60 h when the total polymer yield 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 GAA presented in table (No. 15).

The use of strain Cupriavidus eutrophus VKPM B-10646 as a producer GAA, varying the concentration and type of carbon substrate, and duration of cultivation, allows to obtain a variety of PGA formed of different monomers and significantly different physico-chemical properties (table 1).

td align="center"> 168
Table 1
Composition and physico-chemical properties of PGA synthesized Cupriavidus eutrophus VKPM B-10646
No.The composition of the GAA is the ratio of the monomers (mol.%)Properties GAA
3-GB3-HS3-YY4 GBMinYesPD(n)Cx, %Tpl.,°Cdegr.,°C
199.40.40.2-13403292.6478*182286
26.493.10.5-11114463.4941159260
373.026.11.09-15016222.2750176272
460.533.06.5-12004512.6740167276
537.826.0 36.2-9869122.8632166277
633.930.136.0-8128922.6436169281
743.70.156.2-12408543.1230167280
870.11.40.612.08442562.3138170278
955.436.6CL.8.011138662.3243278
1034.846.60.618.09654012.5925166274
1170.30.80.428.55684121.9112167272
1249.30.828.421.513492152.9845171283
1336.326.820.416.512160902.6746177278
1446.1 53.00.9-12302182.4644164267
1542.836.020.9-11092162.8950176280
Note:
* in the example of the homopolymer poly 3-hydroxybutyrate having a high degree of crystallinity 3-GB-3 hydroxybutyrate; 3-GW-3 hydroxyvalerate; 3-GG-3 hydroxyhexanoate; 4-GB-4 hydroxybutyrate;
Min- molecular mass, Da - Dalton; PD (n) - polydispersity; Cx, % - crystallinity; Tpl.°C - melting point; Tdegr.°C - temperature thermal degradation.

1. The strain of bacteria Cupriavidus eutrophus VKPM B-10646 - producer of polyhydroxyalkanoates.

2. The method of receiving polyhydroxyalkanoates, which lies in the cultivation of the producer strain in conditions of aeration and mixing liquid salt environment, with the containing a series of the growth substrate with an additional source of carbon, when the limit of nitrogen, characterized in that as a producer strain using the strain of bacteria Cupriavidus eutrophus VKPM B-10646, as the growth substrate using glucose or fructose, or 3-butyric acid, or a gas mixture of hydrogen, oxygen and carbon dioxide, or synthesis gas is mixed with oxygen, and as an additional carbon source using a solution of 3-valerate potassium, or solutions of 3-valerate potassium and 3-hexanoate potassium, or solutions of 3-potassium valerate, 3-hexanoate and potassium acrylate, or solutions of 3-hexanoate and potassium acrylate, or solutions of 3-butyric acid and 4-butanolate, or solutions of 3-butyric acid, 4-butanolate and 3-valerate potassium, or solutions of 3-butyric acid, 4-butanolate and 3-hexanoate potassium, or solutions of 3-butyric acid, 4-butanolate, 3-valerate potassium and 3-hexanoate potassium.



 

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6 tbl, 2 ex

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2 tbl, 3 ex

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1 tbl, 3 ex

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5 cl, 8 dwg, 10 ex

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