RU2728345C1 - Methylococcus capsulatus vkpm b-13479 strain - producer of microbial protein mass, resistant to aggressive medium - Google Patents

Abstract

FIELD: microbiology.SUBSTANCE: invention relates to microbiological industry and can be used for production of microbial protein mass. Strain of methane-oxidising bacteria Methylococcus capsulatus LBTI 028 is able to produce a microbial protein mass. Strain is deposited in the Russian National Collection of Industrial Microorganisms under registration number VKPM B-13479.EFFECT: invention increases output of microbial protein mass.1 cl, 4 ex

Description

The invention relates to the microbiological industry, concerns a new culture of microorganisms, which can be used to obtain a microbial protein mass.

One of the methods of obtaining a microbial protein mass is the cultivation of methane-oxidizing microorganisms on natural gas. Under suitable conditions, methanotrophic bacteria actively process natural gas, multiply rapidly and build up their biomass rich in valuable protein, vitamins and other biologically active substances.

Research in the field of microbiological protein synthesis on methane began almost simultaneously with the development of processes for the cultivation of microorganisms on liquid hydrocarbons. However, due to low biomass yields and difficulties in constructive design of the technological process, the development of methods for microbiological production of protein using natural gas was at the stage of laboratory research for a long time.

Currently known yeast strain Candida tropicalis VSB-942 - producer of biomass enriched in protein (RF Patent No. 2031115). The strain was deposited in the All-Russian Collection of Industrial Microorganisms under the number VKPM Y-1987.

The disadvantage of this invention is the ability of the producer to grow at low concentrations of n-paraffins (0.8%), low productivity of the fermentation process, the need to clean the biomass from residues of unused paraffins, and the content of crude protein in biomass does not exceed 60-65%.

The intensity of the fermentation process can be increased by applying oxidative stress. Oxidative stress is understood in particular as the formation of reactive oxygen species and free radicals, which are highly reactive. Such forms of substances can react with environmental components, oxidizing them, which leads to damage to cells of microorganisms or their death.

As a factor of oxidative stress, exposure to hydrogen peroxide is used. This effect simulates the real conditions of the culture of microorganisms in unfavorable conditions of the cultivation environment, and also acts as a selection factor aimed at the selection and adaptation of a more active population of microorganisms.

This approach makes it possible to mobilize the adaptive resources of microorganisms at the level of stimulating their metabolism. As a result of stress, microorganisms acquire a more powerful oxidative system, which can manifest itself in the ability to consume a difficult-to-digest substrate or in an increase in the productivity of cell mass and valuable metabolic products (RF Patent No. 2553213, RF Patent No. 2268924, RF Patent No. 2394098).

The closest analogue (prototype) of the claimed invention is a strain of methane-oxidizing bacteria Methylococcus capsulatus GBS-15 for obtaining a microbial protein mass (RF Patent No. 2613365). The strain of methane-oxidizing bacteria Methylococcus capsulatus GBS-15 was deposited in the All-Russian Collection of Industrial Microorganisms under the number VKPM B-12549. The strain is resistant to homologues of methane in natural gas, the ability to heterotrophic fixation of carbon dioxide and to grow at elevated pressure (up to 16 atm), but the disadvantage of the prototype is the insufficiently high growth rate of the culture (0.25 h ^-1 ) in a continuous cultivation mode, and also the use of a nutrient medium with a high content of mineral components for cultivation of the strain. The components of mineral nutrition are not completely consumed by the culture of methanotrophs; their residual concentrations in the culture liquid are quite high. This, in turn, leads to the need for additional purification of biomass (as a product) from mineral salts, and the resulting effluents have a large load on the treatment facilities.

The objective of the present invention is to create a new strain of methane-oxidizing microorganism with a higher technological potential, namely, with higher growth characteristics.

The technical result of the claimed invention is to increase the efficiency of the newly obtained strain for obtaining a microbial protein mass by increasing the growth rate, reducing the consumption of mineral components for shoving and increasing resistance to aggressive environments.

The technical result is achieved by creating a new strain of Methylococcus capsulatus VKPM B-13479 to obtain a microbial protein mass resistant to aggressive environments, registration number VKPM B-13479 in the All-Russian Collection of Industrial Microorganisms. This strain is resistant to aggressive environments, has a high growth rate and is capable of producing a large amount of biomass when cultivated on a nutrient medium with low concentrations of mineral nutrients.

The strain of methane-oxidizing bacteria Methylococcus capsulatus VKPM B-13479 has the following advantages:

- high growth rate (0.27 h ^-1 ) under conditions of continuous cultivation in a methane-air atmosphere at atmospheric pressure;

- growth on nutrient media with a lower content of mineral nutritional components (compared to the prototype) without reducing the growth characteristics and productivity of the process;

- resistance to aggressive environments without reducing the growth rate, which is confirmed by laboratory tests.

The cultivation of methane-oxidizing microorganisms often occurs under suboptimal conditions, and often even in an aggressive environment. The aggressiveness of the environment can manifest itself, in particular, in the formation of reactive oxygen species and free radicals, which are the result of oxidative processes occurring as a result of the vital activity of microorganisms. The aggressiveness of the cultivation environment can be caused by a number of factors of both natural and technogenic origin. A generally accepted model of an aggressive environment can be the introduction of hydrogen peroside into the culture liquid in a certain concentration range. Within the framework of laboratory tests, this technique simulates the real conditions of the culture of microorganisms in aggressive environmental conditions, and also acts as a selection factor aimed at the selection and adaptation of more stable and active populations of microorganisms.

The strain Methylococcus capsulatus VKPM B-13479 was isolated from the soil of the Bovanenkovo oil and gas condensate field, the Yamal Peninsula, by the method of enrichment cultures by repeated reseeding in a methane-air environment. Further, the strain was selected in a continuous process of cultivation at a flow rate of 0.30-0.35 h ^-1 on a mineral medium in an atmosphere of methane: air in a ratio from 1: 1 to 1: 3 at a temperature of 41-45 ° C and a pH of 5 , 5-5.8.

Cultural and morphological characteristics of the strain.

The strain is aerobic. The gram-type strain is gram-negative. The cells of the strain have a spherical shape in the form of a coccus or diplococcus, 0.7-1.0 microns in diameter, immobile, form a microcapsule. In terms of cell structure and morphology, it belongs to the I group of the X-type of methane-oxidizing microorganisms (according to the classification of Whittenbury R.).

Growth on agar mineral medium: colonies are round 1.0-1.5 mm in diameter, the shape of the colonies is convex, drop-shaped with an even smooth edge, the structure is homogeneous, dense, the color of the colonies is beige, water-soluble pigment is not emitted. The optimum temperature for growth is 41-45 ° C and a pH value of 5.5-5.8.

The strain belongs to obligate methane-oxidizing bacteria. It assimilates methane as the only source of carbon, including in the composition of natural gas. As a source of nitrogen, ammonium nitrogen assimilates nitrate nitrogen. Does not grow on organic substrates. Resistant to hydrogen peroxide.

The strain was not genetically manipulated.

In a resting state, the strain forms cysts of the Azotobacter type, can remain viable for 5-6 months in the absence of methane in the form of a suspension in a cooled state.

The selected strain is characterized by the following industrial characteristics:

- the strain can withstand temperatures up to 50 ° C, the optimum temperature for growth is 41-45 ° C);

- the activity of the strain is determined by the high growth rate under conditions of continuous cultivation, resistance to methane homologues in natural gas, and resistance to hydrogen peroxide.

The systematic position of the strain was confirmed by a genetic method using 16S RNA analysis.

The strain is not pathogenic and is competitive with other types of methane-oxidizing bacteria.

The product formed by the strain is a microbial protein mass.

The area of application of the strain is the production of protein feed biomass for balancing feed for agricultural animals and aquaculture.

The method for determining the activity of a strain is sieving into mineral (mineral-agarized) media in a methane-air atmosphere with a methane (natural gas): air ratio from 1: 1 to 1: 3.

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

The strain is stored for 4-5 months. at a temperature of + 4 ° ± + 6 ° С without natural gas on a mineral-agar medium.

Storage in suspension culture for 5-6 months is allowed.

Method, conditions and composition of media for the propagation of the strain - sieving on a liquid mineral medium in a methane-air atmosphere. Cultivation is carried out in flasks with bumpers with a volume of 750-1000 ml filled with a mineral medium by no more than 15%, in a methane-air atmosphere 1: 2, with constant shaking at an intensity of 200 rpm, at a temperature of 41-45 ° C, within 5-7 days.

Optimal conditions and composition of the culture medium

Methylococcus capsulatus VKPM B-13479 - pH 5.5-5.8, temperature 41-45 ° C.

The composition of the mineral medium per liter: (NH4) ₂ SO ₄ - 0.56 g; MgSO ₄ × 7H ₂ O - 0.02 g; KCl - 0.025 g; (NH4) ₂ HPO ₄ 0.04 g; (NH ₄ ) H ₂ PO ₄ 0.04 g; ZnSO ₄ × 7H ₂ O 0.3 mg; MnSO ₄ × 5H ₂ O - 0.54 mg; CuSO ₄ × 5H ₂ O - 0.54 mg; H ₃ BO ₃ - 0.6 mg; FeSO ₄ × 7H ₂ O - 2.0 mg; Na ₂ MoO ₄ × 2H ₂ O - 0.045 mg; CoSO ₄ × 7H ₂ O - 0.048 mg; Trilon B - 5.0 mg.

Genetic features of the strain:

a) mutations, deletions, inversions - not identified;

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

c) plasmids - not identified;

d) prophages - not identified.

Information on 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.

Below are examples confirming the effectiveness of the proposed strain.

Example 1.

The cultivation of the Methylococcus capsulatus strain VKPM B-13479 was carried out in a continuous mode in a fermenter with mechanical stirring with a volume of 50 liters (working volume - 30 liters) with a continuous supply of a nutrient medium containing the following mineral components (per 1 liter of water): (NH4) ₂ SO ₄ - 0.56 g; MgSO ₄ × 7H ₂ O - 0.02 g; KCl - 0.025 g; (NH ₄ ) HPO ₄ 0.04 g; (NH ₄ ) H ₂ PO ₄ 0.04 g; ZnSO ₄ × 7H ₂ O 0.3 mg; MnSO ₄ × 5H ₂ O - 0.54 mg; CuSO ₄ × 5H ₂ O - 0.54 mg; H ₃ BO ₃ - 0.6 mg; FeSO ₄ × 7H ₂ O - 2.0 mg; Na ₂ MoO ₄ × 2H ₂ O - 0.045 mg; CoSO ₄ × 7H ₂ O - 0.048 mg; Trilon B - 5.0 mg; The pH of the medium is 5.6-5.8.

The process was carried out under non-sterile conditions:

- at a temperature of 41-42 ° С;

- at a pH of 5.6-5.8;

- at atmospheric pressure;

- when the consumption of natural gas and air per 1 liter of fermentation medium is 15 and 45 l / h, respectively;

- at a flow rate of the nutrient medium of 0.27 h ^-1 .

With prolonged performance of such a cultivation regime with a continuous flow of a nutrient medium of 0.27 h ^{-1 of the} specified composition, the concentration of biomass in the fermentation medium remained constant and amounted to 10.2-11.3 g / l. The content of crude protein in biomass was 76.0-78.0%.

The data obtained indicate that the cultivation of the Methylococcus capsulatus VKPM B-13479 strain on a nutrient medium of the specified mineral composition, with a lower concentration (in comparison with the prototype, approximately 5 times) with a continuous flow allows to obtain a stable biomass yield comparable to the prototype.

Example 2.

Cultivation of the Methylococcus capsulatus VKPM B-13479 strain was carried out as described in example 1, but hydrogen peroxide was added to the cultivation medium. Hydrogen peroxide was supplied to the fermentation apparatus in the amount necessary to ensure the concentration of H ₂ O ₂ in the culture medium of 0.005 g / L (in terms of 100% H ₂ O ₂ ).

Under these conditions of cultivation, the concentration of biomass in the fermentation medium was 10.1-11.5 g / l at a flow rate of 0.27 h ^-1 . The crude protein content in the biomass was 76.5-77.8%.

Example 3.

Continuous cultivation of the strain Methylococcus capsulatus VKPM B-13479, obtained according to example 2. Hydrogen peroxide was introduced into the fermentation apparatus in the amount necessary to ensure the concentration of H ₂ O ₂ in the cultivation medium of 0.01 g / L (in terms of 100% H ₂ O ₂ ).

Under these conditions of cultivation, the concentration of biomass in the fermentation medium was 10.0-11.2 g / l at a flow rate of 0.27 h ^-1 . The content of crude protein in biomass was 76.8-77.9%.

Example 4.

Continuous cultivation of the strain Methylococcus capsulatus VKPM B-13479, obtained according to example 3. Hydrogen peroxide was introduced into the fermentation apparatus in the amount necessary to ensure the concentration of H ₂ O ₂ in the cultivation medium 0.015 g / l (in terms of 100% H ₂ O ₂ ) ...

After the introduction of hydrogen peroxide into the fermentation apparatus, the concentration of biomass decreased to 6.0-6.5 g / l at a flow rate of 0.27 h ^-1 . With continued cultivation with an alternate decrease and increase in the flow rate of the nutrient medium, the concentration of biomass in the fermenter reached 10.0-11.0 g / l and remained constant at a flow rate of 0.27 h ^-1 . The content of crude protein in biomass was 77.0-78.0%.

The results obtained during laboratory studies according to examples 2-4 indicate that the Methylococcus capsulatus strain VKPM B-13479 is resistant to hydrogen peroxide in concentrations up to 0.01 g (in terms of 100% H ₂ O ₂ ) in a liter fermentation medium without reducing the growth rate under conditions of continuous cultivation with a medium flow of 0.27 h ^-1 .

The introduction of hydrogen peroxide into the fermentation medium simulates the real conditions of culture in unfavorable environmental conditions. Thus, the effect of an aggressive environment does not impair the properties of the proposed strain and allows maintaining a stable biomass yield. The resistance of this strain of methane-oxidizing bacteria to hydrogen peroxide may turn out to be a valuable and decisive property when choosing a producer microorganism and its cultivation conditions.

Claims (1)

Strain of methane-oxidizing bacteria Methylococcus capsulatus VKPM B-13479 for obtaining microbial protein mass, resistant to aggressive environment.