RU2613365C1 - Strain of methane-oxidizing bacteria methylococcus capsulatus gbs-15 for obtaining of microbial protein mass - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the biotechnologies and may be used for obtaining of a microbial protein mass. The strain of the methane-oxidizing bacteria Methylococcus capsulatus GBS-15 having the high growth rate under the conditions of continuous culturing, the resistance to methane homologs in natural gas, and the capability for heterotrophic fixation of carbon dioxide and for the growth at excessive pressure (up to 16 atm) is deposited into the All-Russian Collection of the Industrial Microorganisms under the number All Russian Collection of Industrial Microorganisms B-12549.

EFFECT: strain of the methane-oxidizing bacteria methylococcus capsulatus GBS-15 providing the accumulation of biomass up to 32 g/l.

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Description

The invention relates to the microbiological industry, and in particular to a strain of methane-oxidizing bacteria Methylococcus capsulatus GBS-15, a producer of microbial protein mass, and can be used in agriculture for animal feeding.

To date, the overall picture in Russia on the production of feed proteins is not favorable. According to the doctrine signed by the president of Russia, in order to provide 80-90% of own food, the shortage of feed products can be at least 2 million tons per year.

The main source of protein product is soybean meal. However, the natural conditions of our country are not suitable for growing soybeans in sufficient quantities. Professionals have to look for other ways to produce feed protein.

It is known that the traditional raw materials for the production of fodder yeast were hydrolysates of wood and other plant wastes, vinasse of hydrolysis and distilleries, liquors of pulp and paper mills, wastes of caprolactam production, milk whey, sugar beet molasses, molasses and grain distillery distilleries, liquid hydrocarbons and methane.

So, technologies have been developed for the production of feed protein based on the waste of alcohol (post-alcohol stillage) and flour (bran, flour) industries and a mixture of stillage with waste of grain raw materials (RF patent No. 2159287; RF patent No. 2140449; USSR copyright certificate No. 1595900; USSR copyright certificate No. 1571061; USSR copyright certificate No. 1532580; USSR copyright certificate No. 1507787; RF patent No. 2054881, etc.). As producer cultures in the above inventions, yeast-like imperfect fungi of the genus Candida are used. A disadvantage of known strains of microorganisms is the lack of ability to actively grow in a long non-sterile cultivation process. In addition, the use of yeast of the genus Candida as a producer culture requires the complication of production technology by the obligatory introduction of the heat treatment stage of the resulting feed biomass to ensure the absence of living cells in the finished product.

Known strain of yeast Candida tropicalis VSB-928K - producer of feed protein (RF Patent No. 2042713.) The disadvantage of this known strain is the use of yeast of the genus Candida to produce feed protein, which is a conditionally pathogenic microorganism, which requires an additional thermolysis process and the availability of special equipment, a complex system for the treatment of wastewater and air emissions with significant volumes of used air.

Sanitary and epidemiological rules SanPiN 2.3.2.1078-01 yeast of the genus Candida are included in the list of substances that have a harmful effect on human health. The cell growth rate of the known strain is low, which lengthens the production cycle and reduces the specific yield of the target product at the stage of biosynthesis. In addition, in the preparation of feed protein using this known strain of yeast, a large consumption of heat and energy for drying the biomass and a significant consumption (1.5 g / l) of ammonium sulfate are required.

Known yeast strain Saccharomyces cerevisiae VKPM U-760 - a source of protein and biologically active substances (Aut. St. USSR N 1575570). The disadvantage of the strain S. cerevisiae VKPM U-760 is the ability to grow at a low concentration of the substrate in the medium, which is non-technological and reduces the economic efficiency of the cultivation process. Another disadvantage of the strain is its low productivity and high expenditure coefficient (about 2), which reduces the performance of the strain. The disadvantages of the strain, based on its systematic position, include the ability to ferment various sugars (glucose, galactose, sucrose, maltose and raffinose), which can lead to bloating when feeding animals.

The use of bacteria as a producer of protein feed is more effective, since bacteria form up to 75% of the protein by weight, while yeast - not more than 60%.

Various bacterial strains that are protein producers are known.

The use of strain Lactobacillus acidophilus 1660/08 for the preparation of protein feed does not require air consumption and energy consumption for its supply, since this strain of lactic acid bacteria is an anaerobic.

The strain Lactobacillus acidophilus VGNKI - 04/03/10 - DEPT - producer of feed protein (RF patent No. 224000). Used production waste - flour, starch, fruit squeezes, crushed grain.

The known strain of Lactobacillus plantarum 578/26 is a producer of protein feed, deposited in the GNU VNIIIPBT of the Russian Academy of Agricultural Sciences (RF patent 2390554) and strain Lactobacillus plantarum 578/25 is a producer of feed protein (RF patent No. 224004001).

A common drawback of these strains is the insufficiently high concentration of biomass accumulated in the presence of 2.5% lactic acid, and consequently, the insufficient amount of protein that could be formed by the culture.

Known producer protein strains: Propionibacterium freudenreichii subsp. shermanii VGNKI-04.04.12. - DEPT - (RF patent No. 227149), strain Propionibacterium acnes VGNKI-03.04.11. - DEPT - producer of feed protein (RF patent No. 2250258).

The known strain of acidophilic methylotrophic bacteria Acetobacter methylicum VSB-867 (collection of the Central Museum of Industrial Microorganisms of the Institute "VNIIgenetika" N CMPM B-1947) is a producer of protein-vitamin biomass (ed. St. USSR No. 925112).

Known strain producing protein Flammulina velutipes NSC F-112, which is grown in both deep and surface culture. The resulting biomass can be used as a protein-complete feed additive (ed. Certificate of the USSR No. 727687). However, the disadvantage of the strain is the use for its cultivation of mineral media with a high glucose content, as well as waste from the food industry, which themselves are feed additives.

The strain Pleurotus ostreatus Kummer IMV F-1330, capable of growing both in deep and surface cultures, contains a significant amount of protein (ed. St. USSR No. 427993). The disadvantage is the need to use complex multicomponent nutrient media and the low productivity of the strain.

A common disadvantage of the bacteria listed above is an unstable biomass yield, and not enough high strain productivity.

One of the promising ways to obtain a complete protein feed product is methanotrophic bacteria that produce protein. Under suitable conditions, methanotrophic bacteria actively process natural gas, multiply rapidly and build up their biomass, which is rich in valuable protein, vitamins and other biologically active substances.

The use of methane to produce unicellular protein has several advantages over liquid hydrocarbons: large reserves of natural gas, its good transportability, and the possibility of obtaining the finished product without additional purification from the substrate.

Given that in Russia there are large gas reserves of the subsoil, according to some reports, they make up 40% of the world. The introduction of microbiological production of unicellular protein at Russian enterprises promises not only economic benefits, but also can ensure food security of the country.

Various strains of fodder protein producing bacteria are known that are related to different types of methane-oxidizing bacteria (methanotrophs), for example, strains of the genus Pseudomonas methanica, Methylococcus capsulatus BKM B-2116, Methylocystis parvus BKM B-2129, Methylosinus sporium Bchum23 Bichum BKh23 Bichum bichi BKM B-2117, Methylobacter acidophilus, Methylomonas rubra VSB-90, Methylococcus sp. FM-9, Methylococcus capsulatus VSB-874, Methylococcus minimus, Methylomonas methanica, Methylomonas agile.

They are the products of microbial synthesis obtained by culturing methane-oxidizing bacteria on natural gas. These strains are characterized by different growth rates and biomass yields.

The disadvantages of these strains include a low growth rate and an unstable protein content, which in most cases is lower than 70%, as well as the need for increased amounts of a biostimulator, in particular an autolysate, which is manifested when the strain is grown under aseptic laboratory conditions.

The closest in technical essence and the achieved result is a strain of Methylococcus capsulatus VSB-874 - a biomass producer. It is stored in the Museum of Cultures of the Institute "VNIIgenetika" under the collection number TsMPM V-1743 (auth. Certificate. USSR No. 770200). Methane, both pure and natural gas, and methanol are used as a carbon source. The disadvantages of the prototype are not high productivity (8-10 g / l h), low crude protein content in biomass (77%), unstable biomass yield (30-60 g / l).

The objective of the invention is the creation of a new methane-oxidizing strain of microorganisms with a higher technological potential, in particular:

- increased resistance to homologues of natural gas methane, which allows the use of natural gas of various compositions and to expand raw materials for the industrial process;

- capable of heterotrophic fixation of carbon dioxide, which provides a lower coefficient of economic consumption of the substrate due to autotrophic absorption of carbon from carbon dioxide at its high concentrations (above 30% vol.) with highly productive fermentation processes;

- phage resistance;

- resistance to short-term changes in temperature and pressure.

This goal is achieved using a new strain of methane-oxidizing bacteria Methylococcus capsulatus GBS-15, registration number VKPM B-12549 in the All-Russian Collection of Industrial Microorganisms to obtain microbial protein mass.

The strain Methylococcus capsulatus GBS-15 was selected from an accumulative culture of obligant methane-oxidizing microorganisms of the strains Methylococcus capsulatus ATCC 33009, Methylococcus capsulatus BKM 2116 and samples of the associative culture of methane-oxidizing microorganisms from various laboratories, which were kept in laboratory settings ( specific studies (for example, studies of individual enzymes), followed by stepwise auto-selection in a continuous cultivation process with an increasing flow rate to 0.35 -0.36 h ^-1 on a mineral medium without the addition of growth factors and other organic compounds in a methane: air atmosphere in a ratio of 1: 2.5 to 1: 3 at a temperature of 42-45 ° C and a pH of 5.6-5 ,9.

As a result of the selection, a strain was obtained which, when cultivated under industrial conditions on natural gas, can be used both as part of the association and individually.

Cultural and morphological features of the strain

The culture is gram-negative, aerobic. The cells of the strain are in the form of cocci or diplococcus, with a diameter of 0.9-1.4 μm, motionless, form a microcapsule. The cell wall has smooth contours and fits snugly on the cytoplasmic membrane. The cytoplasm is filled with tightly packed stacks of intraplasmic membranes (VIC type).

By cell structure and morphology, it belongs to group I of the X-type of methane-oxidizing microorganisms (according to the classification of Whittenbury. R). It grows weakly on an agarized mineral medium, forms colonies 1-2 mm in diameter in diameter, creamy, translucent colonies are rounded with a smooth edge and smooth surface, convex. The strain is phage resistant. The culture is able to grow in a wide temperature range of 30-55 ° C and pH 4.0-8.0, is able to form micro and macro capsules. The optimum temperature for growth is 40-45 ° C and a pH value of 5.5-5.9. Ubiquinone is synthesized in cells (mainly Q ₇ and Q ₈ ). No plasmids were detected. The strain refers to obligant methane-oxidizing bacteria, capable of autotrophy, nitrogen fixation. Does not grow on organic substrates. It has an increased sensitivity to organic compounds of both endo- and exo-origin, which indicates an increase in the strain of obligatoriness of the strain with respect to methane. The strain does not need vitamins and other growth stimulants. Resistant to admixtures of methane homologs and sulfur compounds contained in natural gas.

Genetic manipulations with the strain were not performed.

The strain is able to form resting forms such as immature Azotobacter cysts, however, it does not develop and can remain viable for 2-6 months in the absence of methane.

Selected strain is characterized by the following industrial valuable features:

- the strain is thermotolerant (can withstand temperatures up to 55 ° C, the optimum temperature for growth is 40-45 ° C);

- the strain is acid tolerant (for bacterial cultures) - pH 5.3-5.9.

- the activity of the strain is determined by the high growth rate under conditions of continuous cultivation, resistance to methane homologs in natural gas, and the ability to grow at high flow rates;

- the strain is barotolerant, capable of growth at elevated pressures (up to 16 atm), which allows to increase the productivity of the process up to 15 kg / m ³ * hour.

The systematic position of the strain was confirmed using not only morphophysiological, but also genetic methods using analysis of 16S RNA.

The ability of cells of methane-oxidizing bacteria to assimilate carbon dioxide as a carbon source along with methane was discovered. This process is called heterotrophic fixation of carbon dioxide in the working gas mixture in the range of 30-55%. The level of carbon fixation of CO ₂ reaches 10-12% of the mass of carbon in bacterial cells, which leads to a decrease in the specific consumption coefficient for methane and a decrease in the heat release by bacterial cells that use the excess energy generated during the oxidation of a methane molecule to activate the carbon dioxide molecule and its inclusion in the metabolism of bacterial cells.

The strain is non-pathogenic and competitive with other types of methane-oxidizing bacteria.

The product formed by the strain is microbial protein mass.

The scope of the strain is the production of protein-vitamin complexes for balancing animal feed.

The method for determining the activity of the strain is when cultured on a simple mineral medium in the presence of methane and air in a ratio of 1: 3.

The method, conditions and composition of the media for long-term storage of the strain:

The strain is stored for 1.5-3.0 months. at a temperature of + 2 ° - + 10 ° C without natural gas on a mineral agar medium.

Storage in suspension culture with different cell densities and high cell densities under a layer of liquid paraffin is allowed for 4-8 months.

The method, conditions and composition of the media for the propagation of the strain on a mineral medium in the presence of a gas-air mixture of methane: air in a ratio of 1: 2.5-1: 3, pH 5.3-6.2, when the flasks are thermostated on a rocking chair at 160-280 rpm at a temperature of 40-45 ° C.

The optimal conditions and composition of the medium for the cultivation of Methylococcus capsulatus GBS-15 is pH 5.6-5.8, temperature 41-44 ° C.

The composition of the mineral medium per 1 liter:

Phosphoric acid H ₃ PO ₄ (70%) - 0.35 ml

Potassium chloride KCl - 0.125 g

Magnesium sulfate MgSO ₄ × 7H ₂ O - 0.105 g

Iron sulfate FeSO ₄ × 7Н ₂ О - 10.75 mg

Copper sulfate CuSO ₄ × 5H ₂ O - 10 mg

Manganese sulfate MnSO ₄ × 5H ₂ O - 9.5 mg

Boric acid H ₃ BO ₃ - 6.25 mg

Zinc sulfate ZnSO ₄ × 7Н ₂ О -1.5 mg

Cobalt sulfate CoSO ₄ × 7Н ₂ О - 0.25 mg

Molybdenum sodium Na ₂ MoO ₄ × 2H ₂ O - 0.25 mg

Genetic features of the strain

The strain is not genetically modified and does not contain the genes of other organisms; transferred resistance genes; genetic changes associated with the use of genetic engineering techniques.

1) Mutations, deletions, inversions - not identified

2) Phage resistance - phage resistant strain

3) Plasmids - not detected

4) Profages - not identified

Strain Safety Information

The strain is not zoopathogenic, phytopathogenic and not pathogenic to humans. Not dangerous for any other reason.

EXAMPLE 1. The culture of Methylococcus capsulatus strain GBS-15 was grown continuously in an ejection-type fermenter with a volume of 40 l (working volume 25 l) with a continuous supply of a nutrient medium containing the following components (per 1 l of medium):

Phosphoric acid H ₃ PO ₄ (70%) - 0.35 ml

Potassium chloride KCl - 0.125 g

Magnesium sulfate MgSO ₄ × 7H ₂ O - 0.105 g

Iron sulfate FeSO ₄ × 7Н ₂ О - 10.75 mg

Copper sulfate CuSO ₄ × 5H ₂ O - 10 mg

Manganese sulfate MnSO ₄ × 5H ₂ O - 9.5 mg

Boric acid H ₃ BO ₃ - 6.25 mg

Zinc sulfate ZnSO ₄ × 7Н ₂ О - 1.5 mg

Cobalt sulfate CoSO ₄ × 7Н ₂ О - 0.25 mg

Molybdenum sodium Na ₂ MoO ₄ × 2H ₂ O - 0.25 mg

The growing process was carried out at a temperature of 42 ° C and a pH of the growing medium of 5.6-5.8. The pH value was maintained with a 10% solution of ammonia water. The process temperature was controlled by supplying cooling water to the apparatus heat exchanger.

The consumption of natural gas and air per 1 liter of culture medium was 15 and 45 l / h, respectively.

The culture was grown at atmospheric pressure, a temperature of 42 ° C and a temperature difference of 40 ° to 45 ° C, a pH of 5.6 and a flow rate coefficient of 0.25 h ^-1 , the biomass concentration in the fermenter was 10-11 g / l .

When using this mode of cultivation for a long time, using natural gas of various compositions (with methane contents from 85% vol. To 99.9% vol.) And water from various sources, culture phagolysis was not observed.

EXAMPLE 2. Continuous cultivation of a culture of Methylococcus capsulatus strain GBS-15 was carried out in an ejection-type fermenter with a volume of 40 l (working volume - 25 l) using a nutrient medium and culture conditions, as in example 1.

Cultivation was carried out using natural gas of various compositions with different contents of methane homologs.

1. When using natural gas with a methane content of 96% vol., Ethane - 2% vol., Propane - 3% vol. and butane - 0.1% vol. and a pressure in the fermenter of 2 atm, a biomass concentration of 12 g / l was obtained at a flow rate of 0.25 h ^-1 . The crude protein content in biomass is 79%.

2. When using natural gas with a methane content of 92% vol., Ethane 3% vol., Propane 1.5% vol., Butane 0.4% vol. and a pressure in the fermenter of 2 atm, a biomass concentration of 11.9 g / l was obtained at a flow rate of -0.25 h ^-1 . The crude protein content in biomass is 77.9%.

3. When using natural gas with a methane content of 89% vol., Ethane -4% vol., Propane - 2.0% vol., Butane -0.8% vol. and a pressure in the fermenter of 2 atm, a biomass concentration of 11.5 g / l was obtained at a flow rate of 0.25 h ^-1 . The crude protein content in biomass is 78%.

The data obtained indicate the resistance of the strain Methylococcus capsulatus strain GBS-15 to lower methane homologs in the studied concentrations.

EXAMPLE 3. A culture of Methylococcus capsulatus strain GBS-15 was grown continuously in an ejection-type fermenter of 40 l volume (working volume 25 l) with a continuous supply of nutrient medium (as in the previous example).

The growing process was carried out at a temperature of 42 ° C and a pH of 5.6–5.8; the pH was maintained with a 10% solution of ammonia water. Natural gas (with a methane content of 90% vol.) And technical oxygen with a content of 95% vol. Were fed into the fermenter. oxygen.

Cultivation was carried out in two experiments, both at atmospheric pressure and at a pressure of 6 atm.

1. Growing at atmospheric pressure

When growing microorganisms Methylococcus capsulatus strain GBS-15 at atmospheric pressure, the biomass concentration was 8 g / l at a flow rate of 0.25 h ^-1 . The gas consumption was respectively: natural gas - 240 l / h, oxygen (95%) - 280 l / h. 80 l / h of carbon dioxide are discharged from the off-gas process.

2. Growing under high pressure (6 atm)

When growing microorganisms Methylococcus capsulatus strain GBS-15 at a pressure of 6 atm, the biomass concentration was 32 g / l at a flow rate of 0.25 h ^-1 . The gas consumption was respectively: natural gas (90%) - 330 l / h, oxygen (95%) - 450 l / h. 120 l / h of carbon dioxide is discharged from the process with the exhaust gas, which indicates the stability of the culture to high concentrations of carbon dioxide. A lower specific amount of carbon dioxide released in the exhaust gas stream during cultivation of the strain at a pressure of 6 atm, compared with atmospheric pressure, indicates the ability of the culture to heterotrophic fixation of carbon dioxide.

Claims (1)

The strain of methane oxidizing bacteria Methylococcus capsulatus VKPM B-12549 to obtain microbial protein mass.