RU2796376C1 - Methylobacterium extorquens strain вкпм в-14231 as producer of microbial protein mass - Google Patents

Abstract

FIELD: microbiological industry.

SUBSTANCE: invention can be used to obtain protein biomass used in feeds for aquaculture, agricultural and domestic animals. The invention relates to the field of biotechnology and is Methylobacterium extorquens BF 19-02M strain of methanol-oxidizing bacteria deposited in the Bioresource Center All-Russian Collection of Industrial Microorganisms (BRTS VKPM) of the National Research Center “Kurchatov Institute” — GosNIIgenetika under registration number ВКПМ В-14231, as a producer for obtaining microbial protein mass. The activity of the strain is up to 5.2 kg×m³/hour. The concentration of biomass at the outlet of the fermenter is 15–19 g/l with a crude protein content in the biomass of 62–65%.

EFFECT: invention allows to expand the arsenal of means for the production of microbial protein mass, creating a new highly productive strain of Methylobacterium extorquens methanol-oxidizing bacteria used to obtain microbial protein mass with the ability to utilize hazardous organic waste (methanol) during cultivation without harming the environment, with strain resistance to changing conditions cultivation.

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Description

The invention relates to the microbiological industry and can be used to obtain protein biomass. This product is used as a source of protein in protein-vitamin complexes designed to balance feed for aquaculture, farm and domestic animals.

Various types of microorganisms are used as producers of complete protein and vitamins.

Producers of fodder biomass include microorganisms of various taxonomic groups that can grow on various substrates: fungi, yeast, bacteria, algae.

It is known to use methane-oxidizing bacteria as producers of protein-enriched biomass, which use methane as a carbon source in the biosynthesis. Methanotrophic bacteria, under suitable conditions, actively process methane, multiply rapidly and increase biomass, rich in valuable protein, vitamins, amino acids and other biologically active substances.

Known strain Methylococcus capsulatus VKPM B-13479 - producer of microbial protein mass, resistant to aggressive environments [RF patent RU2728345, IPC: C12N 1/20; C12R 21/00; publ. July 29, 2020]. The strain belongs to obligate methane-oxidizing bacteria. As the only source of carbon, it assimilates methane, including in the composition of natural gas. As a source of nitrogen, it absorbs ammonium and nitrate nitrogen. Does not grow on organic substrates. It is resistant to hydrogen peroxide. The strain is stored for 4-5 months at a temperature of +4 - +6°C without natural gas on a mineral agar medium. Storage in suspension culture is allowed for 5-6 months.

Known strain Methylococcus capsulatus MC19, which has the ability to produce protein mass, deposited in the Institute of Biochemistry and Physiology of Microorganisms. G.K. Skryabin of the Russian Academy of Sciences (IBFM RAS) under the registration number VKM B-3493D. [RF patent RU 2760288, IPC: C12N 1/20; C12R 21/00; A23K 10/10, publ. November 23, 2021]. The strain makes it possible to obtain biomass, which, in addition to protein, contains antioxidants and B vitamins that increase the biological value of the protein mass.

A known strain of methane-oxidizing bacteria Methylococcus capsulatus BF19-07 is a producer for obtaining microbial protein mass [RF patent RU 2745093, IPC: C12N 1/20; C12R 21/00; publ. March 19, 2021]. This strain was deposited at the Bioresource Center of the All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the National Research Center "Kurchatov Institute - GosNIIGenetika" under the registration number VKPM V-13764, as a microbial protein producer for the production of protein-vitamin complexes in the composition of feed for farm animals. The activity (productivity) of the strain is up to 12 kg×m ³ /hour.

It is known to use genetically modified microorganisms Methylobacterium extorquens for the production of proteins, feed compositions and food additives [US application US2020/0224236, IPC: C12P 21/00, C12N 1/20; publ. 07/16/2020; US application US2015/0044327, IPC: A23K 1/16, A23K 1/18, C12N 15/74; publ. February 12, 2015].

As the closest analogue, an unmodified strain of methanol-oxidizing bacteria Acidomonas methanolica BF 21-05M, a producer for obtaining microbial protein mass according to RF patent RU2773502 [IPC: C12N 1/20; C12R 21/00; publ. 06/07/2022]: The indicated strain was deposited at the Bioresource Center of the All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the National Research Center "Kurchatov Institute" - GosNIIgenetika under the registration number VKPM V-14051, and can be used as a producer for obtaining microbial protein mass and balancing feed for farm animals. The activity (productivity) of the strain is up to 9.3 kg×m ³ /hour. The concentration of biomass during cultivation for a long time was 13-15 g/l with a crude protein content in the biomass of 60-68%. However, when cultivating this strain, it is necessary to use a yeast autolysate as a growth factor. The addition of yeast autolysate can provoke the development of extraneous microflora. In addition, the cultivation of the strain without loss of productivity is possible in a narrow range of pH values (optimal growth at pH 4.0-4.5).

The technical problem, the solution of which is provided by the implementation of the invention, is to create a new highly productive strain of methanol-oxidizing bacteria for the production of fodder biomass.

The technical result consists in expanding the arsenal of means for the production of microbial protein mass, creating a new highly productive strain of methanol-oxidizing bacteria Methylobacterium extorquens used to obtain microbial protein mass with the ability to utilize hazardous organic waste (methanol) during cultivation without harming the environment. The technical result also consists in increasing the resistance of the strain to changing cultivation conditions without reducing productivity when the temperature regime changes from 28°C to 32°C and pH values from 6.0 to 8.0.

To achieve this technical result, it is proposed to use a strain of methanol-oxidizing bacteria Methylobacterium extorquens BF 19-02M, deposited in the Bioresource Center All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the National Research Center "Kurchatov Institute" - GosNIIgenetika under the registration number VKPM B-14231, as a producer for obtaining microbial protein mass. The strain designation assigned by the depositor is BF 19-02M.

The Methylobacterium extorquens BF 19-02M strain was isolated from soddy-podzolic soil (Moscow region, Russia). A sample of soddy-podzolic soil was used to obtain an enrichment culture of methanol-oxidizing bacteria. After incubation in a liquid mineral medium with methanol as the sole carbon source, the culture was subcultured using the serial dilution method. After several passages, the culture was sown on an agar mineral medium to obtain isolated colonies of a pure culture of methylotrophic bacteria. The thus obtained strain Methylobacterium extorquens BF 19-02M was selected in the process of long-term (more than six months) passaging on liquid and agar media with methanol.

The strain of the bacterium Methylobacterium extorquens VKPM B-14231 was identified by cultural-morphological, physiological-biochemical properties and genetic characteristics and has the following characteristics.

The systematic position was confirmed by the results of the analysis of the 16S rRNA gene, as well as by the decoding of the genome. The work was carried out by SciBear OU (Tartu mnt 67/1-13b, Kesklinna linnaosa, Tallinn, 10115 Harju maakond, Reg. No. 16221849). The resulting sequences were deposited with NCBI under numbers MT346348 and JAHKKE000000000, respectively.

Genetic features of the strain:

a) mutations, deletions, inversions - not detected;

b) resistance (sensitivity) to antibiotics, phages, etc. - not identified;

c) plasmids - not detected;

d) prophages - not detected.

Information about the safety of using the strain:

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

b) the strain is not zoopathogenic, phytopathogenic and does not pose a danger for any other reason.

The culture is gram negative. Cells have the form of pleomorphic sticks, ranging in size from 0.5 to 1.5 × 1.0-10.0 μm, do not form spores, are immobile.

Colonies on the 5th day of cultivation on a mineral agar medium with methanol (1% vol.) at 30°C are shiny, smooth, convex, spherical, pink or red with a diameter of 1-3 mm. Contains terminally located poly-β-hydroxybutyrate granules.

Growth at a temperature of 15-37°C (optimum 28-32°C) and pH 4.0-10.0 (optimum 6.0-8.0).

Does not reduce nitrates to nitrites.

Indole and hydrogen sulfide does not form.

Hydrolysis of gelatin and starch is not observed.

Does not produce ammonia.

Strict aerobe, chemoorganotroph and facultative methylotroph, assimilating C1 compounds along the serine pathway.

It uses succinic acid, ethanol, acetic acid, betaine, methanol, methylamine as sources of carbon and energy.

Does not utilize L-arabinose, D-xylose, D-glucose, D-mannose, D-fructose, galactose, maltose, sucrose, lactose, trehalose, D-sorbitol, D-mannitol, inositol, soluble starch, citrate, or methane.

Tests for oxidase, catalase and urease are positive.

Does not need vitamins and other growth stimulants.

It uses ammonium salts as a source of nitrogen, worse than nitrates.

Ubiquinone is synthesized in cells (mainly Q ₁₀ ).

The activity (productivity) of the strain is up to 5.2 kg×m ³ /hour. A method for determining the activity of a strain indicating the method: on a liquid mineral medium with the addition of methanol in an amount of 20 ml/l.

Method, conditions and composition of media for long-term storage of the strain: in a lyophilized state; cryopreservation at -80°C 30% glycerol solution is used as a cryoprotectant.

Method, conditions and composition of media for propagation of the strain:

Option 1

The composition of the mineral medium for growing culture in flasks (g/l): (NH ₄ ) ₂ SO ₄ - 0.5; MgSO ₄ × 7H ₂ O - 0.2; NaCl - 0.1; CaCl ₂ - 0.05; K ₂ HPO ₄ - 1.2; KH ₂ PO ₄ - 0.62. Trace elements (mg/l): MnSO ₄ × 5H ₂ O - 0.2; FeSO ₄ ×7H ₂ O - 1; H ₃ BO ₃ - 0.2; Na ₂ MoO ₄ × 2H ₂ O - 0.2; KI - 0.2. Methanol 10 ml/l, pH 6.8-7.0, cultivation temperature 30°C.

Option 2

Optimal conditions and composition of the fermentation medium (g/l methanol):

H ₃ PO ₄ (70%) - 40.6 ml;

KCl - 48.0 g;

MgSO ₄ × 7H ₂ O - 12.7 g;

FeSO ₄ × 7H ₂ O - 3.4 g;

CuSO ₄ × 5H ₂ O - 0.28 g;

MnSO ₄ × 5H ₂ O - 2.5 g;

ZnSO ₄ × 7H ₂ O - 2.3 g;

CoCl ₂ ×2H ₂ O - 0.00012 g;

Na ₂ MoO ₄ × 2H ₂ O - 0.002 g.

Genetic features of the strain:

a) there are no mutations, deletions, inversions;

b) phage-resistant;

c) no plasmids were found;

d) no prophages were found.

The proposed strain is characterized by:

- sustainable productive growth on media of different composition, in a wide range of pH and temperature;

- the ability to maintain activity for a long time in a lyophilized state;

- lack of virulence, toxigenicity, toxicity and harmlessness.

The cultivation is carried out mainly in a continuous way.

The invention is illustrated by the following examples.

Example 1

Cultivation of the strain Methylobacterium extorquens BF 19-02M was carried out in periodic mode in heat-resistant glass flasks with a volume of 0.7 l at a filling factor of 0.3-0.4 in a thermostatically controlled shaker-incubator at 30°C. Cultivation was carried out for 24 hours. At the end of cultivation, the concentration of solids was 1.2 g/l. The resulting culture was used as seed material for the subsequent cultivation of bacteria in an automated fermentation complex with a volume of 30 l (working volume 20 l).

Example 2

The culture of Methylobacterium extorquens BF 19-02M was grown continuously in a fermenter with a magnetic stirrer with a volume of 30 l (working volume - 20 l) with a constant supply of a mineral medium containing the following components (g/l of methanol):

H ₃ PO ₄ (70%) - 40.6 ml;

KCl - 48.0 g;

MgSO ₄ × 7H ₂ O - 12.7 g;

FeSO ₄ × 7H ₂ O - 3.4 g;

CuSO ₄ × 5H ₂ O - 0.28 g;

MnSO ₄ × 5H ₂ O - 2.5 g;

ZnSO ₄ × 7H ₂ O - 2.3 g;

CoCl ₂ ×2H ₂ O - 0.00012 g;

Na ₂ MoO ₄ × 2H ₂ O - 0.002 g.

The cultivation process was carried out under non-sterile conditions, with varying cultivation conditions: changing the temperature regime from 28°C to 32°C and pH 6.0-8.0. The studies were carried out under the following conditions: t=28°С pH=7.0; t=30°С pH 6.0; t=32 °С pH 7.0; t=30°С pH 8.0. The methanol feed rate was 100 ml/h. With a flow rate coefficient of 0.25 h ^-1 . The pH value was adjusted with a 5% ammonia water solution or a 70% phosphoric acid solution. The process temperature was controlled by supplying cooling water to the apparatus heat exchanger. The air flow rate per 1 liter of the culture medium was 60 l/hour.

As a result of applying such a cultivation mode for a long time, using water from different sources, the culture productivity remained stable, the biomass concentration in the fermenter was 15-19 g/l with a crude protein content in the biomass of 62-65%.

Example 3

The culture of Methylobacterium extorquens BF 19-02M was grown continuously in a fermenter with a magnetic stirrer with a volume of 30 l (working volume - 20 l) with a continuous supply of mineral salt solutions (as in the previous example). The growing process was carried out at a temperature of 30°C and pH 7.0. The pH value was maintained with a 10% ammonia water solution. The fermenter was supplied with air in the amount of 360 l/h. The flow rate coefficient was 0.25 h ^-1 . The culture fluid at the outlet of the fermenter was separated from the cells in a non-sterile mode and returned to the fermentation process instead of water. The share of accompanying microflora with this method of cultivation was unchanged and did not exceed 20% of the total number of cells. The concentration of biomass at the outlet of the fermenter was 15-19 g/L. The content of crude protein in biomass is 62-65%. The data obtained indicate that the cultivation of the strain Methylobacterium extorquens BF 19-02M in a mineral medium of the specified composition with continuous flow and in a wide range of cultivation conditions (temperature, pH,) allows you to obtain a stable biomass yield comparable to the prototype.

Claims (1)

The strain of methylotrophic bacteria Methylobacterium extorquens VKPM B-14231 is a producer of microbial protein mass.