RU2706074C9 - Methylococcus capsulatus concept-8 bacteria strain – producer of protein biomass - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to microbiological industry and can be used for production of protein biomass. Disclosed is a strain of bacteria Methylococcus capsulatus, deposited in the All-Russian Collection of Microorganisms IBPM them. G.K. Skryabin RAS under registration number VKM V-3289D.

EFFECT: strain produces a protein biomass with protein content higher than 79 %, has methane-oxidyzing properties, resistance to external effects in a wide range of cultivation parameters, high growth rate under optimal conditions.

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Description

The invention relates to the microbiological industry and can be used for the biosynthesis of high-protein premix for feed of agricultural, domestic and captive animals. The claimed bacterial strain Methylococcus capsulatus CONCEPT-8, with methane-oxidizing properties, resistance to external influences over a wide range of cultivation parameters, high growth rate under optimal conditions, was deposited in the All-Russian collection of microorganisms under the number VKM B-3289D

Methane-oxidizing bacteria using methane and its derivatives as a source of carbon and energy are used to reduce the concentration of methane in coal mine gases, in the biosynthesis of protein-rich biomass, which has found application as a premix to animal feed.

Methane-oxidizing bacteria using methane and its derivatives as a source of carbon and energy are widespread in nature, being a biofilter that reduces the likelihood of methane entering the planet's troposphere and ozone layer disturbance.

Of particular interest is the use of Methylococcus capsulatus bacteria as a biosynthetic system that actively processes methane contained in natural gas, including mine gas, and quickly builds up its biomass, which is characterized by a high content of valuable protein, as well as biologically active substances, including vitamins. The biomass of Methylococcus capsulatus is characterized by a high level of crude protein (at least 70%), as well as a ratio of amino acids similar to fishmeal, which allows it to be used as a protein premix to feed.

Tests of feed with the inclusion of biomass of methane-oxidizing bacteria conducted on farm animals, birds and fish have confirmed the high biological value of this biomass as a source of protein, vitamins and other nutrients. The safety of using biomass of methane-oxidizing bacteria has long been proven (1-5). Veterinary and sanitary-hygienic studies have established the benignness and harmlessness of livestock products, poultry farming and fish farming, obtained using feed with biomass of methane-oxidizing bacteria. (6-8).

Despite the fact that, according to a number of indicators, bacterial biomass differs from traditional protein premixes, in particular, by a high content of nucleic acids, the absence of polyunsaturated fatty acids and the presence of fatty acids (9), the use of biomass of methane-oxidizing bacteria does not adversely affect the growth and development of animals. in particular fry and commercial fish. (10, 11).

Various types of microorganisms produce high-grade protein and vitamins. Many of them have found application in feed production due to the developed and tested technologies (12).

In a number of technologies, highly productive Candida mushrooms are used as protein producers. (13-16). (RF patent No. 2042713, No. 2090610, Aut. St. USSR No. 3221059, 1817471). Others successfully use the yeast Saccharomyces cerevisiae (Aut. St. USSR No. 1575570; RF Patent No. 2650689), which differ, however, by low productivity and capable of developing only at low substrate concentrations in the medium, which reduces the economic efficiency of their use (17-18) .

Known strains of lactic acid and propionic acid bacteria used as protein producers (RF patents No. 224000; 2390554; No. 22400400; No. 2250258 and several others). (19-22). A common drawback of these strains is the insufficiently high concentration of biomass, the biosynthesis of which is suppressed by the resulting lactic or propionic acids.

Known cap mushrooms Flammulina velutipes NSC F-112 (A.S. No. 727687) and Pleurotus ostreatus Kummer (A.S. No. 427993) are producers of protein whose biomass can be used as a complete protein premix (23-24). However, the use for their cultivation of complex environments with a high glucose content and food industry wastes directly used as additives in feed of farm animals makes their use practically meaningless.

The known strain of acidophilic methylotrophic bacteria Acetobacter methylicum VSB-867 is a producer of protein-vitamin biomass (AS No. 925112), characterized, however, by insufficiently high productivity and unstable yield of the final product (25)

Known strains of methanotrophic bacteria cultivated on natural gas and actively propagating, increasing their biomass, producing a protein enriched with biologically active compounds and suitable for use as a premix to feed (26-32). (RF patent No. 2613365, 206416; Autost. Of the USSR No. 770200, 908085, 501681, 1072815, 615871).

The most active producer of protein biomass in natural gas is the species Methylococcus capsulatus. The disadvantages of this type of methanotroph include the instability of its growth on natural gas during prolonged cultivation under flow conditions, which reduces the productivity of the process and the yield of the finished product.

Closest to the claimed invention is a methane-oxidizing bacteria strain Methylococcus capsulatus GBS-15, described in RF patent No. 2613365 (33). The obtained strain Methylococcus capsulatus GBS-15 has a number of properties that provide it with more stable growth during cultivation. The authors of the patent show the phage resistance of the strain when growing it in an atmosphere of natural gas of different composition (with methane content from 85% vol. To 99.9% vol.) And in different water, which are potential sources of external phages to the system, and can also provoke the activity of prophages of the culture itself. The authors also showed the resistance of the strain Methylococcus capsulatus GBS-15 to methane homologs entering the culture medium together with methane in the composition of natural gas, in particular ethane (2-4%), propane (1.5-3%) and butane (0 , 1-0.8%). The patent also notes the growth of the strain Methylococcus capsulatus GBS-15 under high pressure up to 6 atm. In this case, a biomass concentration of 32 g / l and a flow rate of 0.25 hour ^{-1 are} achieved.

However, it should be noted that in example No. 1, the authors do not indicate the duration of the cultivation process, which complicates the assessment of the phage resistance of the strain. In addition, depending on the natural gas field (especially gas condensate fields), it contains higher amounts of methane homologues, and pentane is also found. In associated gases, higher hydrocarbons can be found in small quantities. Mine gas contains, among other things, CO ₂ , the concentration of which is 3-6%. It should be noted that during the cultivation of methane-oxidizing microorganisms, the products of incomplete oxidation of methane (methanol, formaldehyde, formate), organic acids, carbohydrates, etc., which can also have an inhibitory effect on bacterial growth, can accumulate in the medium.

The problem to which the invention is directed, is to obtain a new strain of methane-oxidizing bacteria with a wider potential for stability, maintaining stable growth and high productivity during long-term cultivation on natural gas.

The technical result, the achievement of which the present invention is directed, is to increase the productivity of the methane-oxidizing microorganism strain Methylococcus capsulatus, to achieve a stable yield of the final product, and also to provide resistance to variations in environmental conditions, including:

- mechanical resistance, which allows to intensify flows in the culture medium, reduce diffusion restrictions and increase the concentration of methane to activate aeration;

- bar resistance, which allows, if necessary, to regulate the solubility of gases in the culture fluid and increase the yield of biomass by varying the pressure;

- resistance to increased concentrations of methane homologs contained in natural gas, including mine gas, which allows to reduce its concentration in mine gas, as well as to use protein raw materials for biosynthesis of methane-oxidizing microorganisms Methylococcus capsulatus from various sources and to reduce the variability of the yield of the final product;

- increased resistance to the inhibitory effect of metabolic products in the spent culture fluid returned to the cultivation process, which allows to reduce the loss of mineral nutrition components and, accordingly, increase the efficiency of the process.

To achieve the indicated technical result, the strain Methylococcus capsulatus CONCEPT-8 was obtained from the collection strain Methylococcus capsulatus VKM B - 2116. The strain was preliminarily adapted to potential inhibitors under conditions of rocking flasks under sequential repeated passivation of the strain under given conditions. Further strain selection was carried out by sequential multi-stage selection under conditions of flow cultivation of the strain in natural gas under unprotected fermentation in the presence of selected potential inhibitors with a gradual increase in flow rate from 0.05 h-1 to 0.35 h-1. Technological parameters were used as selection criteria: growth rate and protein content in biomass.

As a result of the selection, the methane-oxidizing bacteria strain Methylococcus capsulatus (CONCEPT-8) VKM B-3289D was isolated, which can be used in technologies for producing high protein biomass when cultured on methane,

contained both in natural gas and in mine gas.

The strain Methylococcus capsulatus VKM B-3289D is stored in a liquid mineral medium at a temperature of 4 ° C, reseeding 1 time in 3 months on a mineral medium in a methane atmosphere.

Cultural and morphological characteristics of the strain.

The cells of the strain are in the form of cocci and diplococcus, with a diameter of 0.8-1.0 μm, motionless. On an agarized medium it forms small transparent colonies (d - 1 mm on the 2nd day of cultivation), convex, shiny, smooth with a smooth edge. The strain remains viable in a wide range of temperatures (20 -0 ° C) and pH (4.5-8.0). The optimum temperature is 40-42 ° C and a pH of 6.6-6.7. The strain is an obligate methanotroph, uses methane as the sole source of carbon and energy. Does not need growth factors. Resistant to impurities of the homologues of methane and CO ₂ contained in natural gas. The strain is able to form resting forms such as immature Azotobacter cysts.

The strain is non-pathogenic.

Genetic manipulations with the strain were not performed. The strain Methylococcus capsulatus VKM B-3289D obtained as a result of breeding is characterized by:

- mechanically resistant, as a result of which the number of integers capable of normal cell activity practically does not decrease in the field of acoustic microflows with high gradients of velocities, which makes it possible to intensify mixing in the culture medium, reduce diffusion restrictions, accelerate the dissolution process and increase the concentration of dissolved in the culture gas environment;

- bar resistance, which allows to increase the concentration of gases dissolved in the culture medium;

- resistance to increased concentrations of methane homologs contained in natural gas, including mine gas, which allows to reduce its concentration in mine gas, as well as to use protein biosynthesis with methane-oxidizing microorganisms Methylococcus capsulatus VKM B-3289D from various sources and reduce output variability final product;

- increased resistance to the inhibitory effect of metabolic products in the spent culture fluid returned to the cultivation process, which allows to reduce the loss of mineral nutrition components and, accordingly, increase the efficiency of the process.

The final product obtained by culturing the strain Methylococcus capsulatus VKM B-3289D is a microbial biomass with

high in protein.

The scope of the strain is industrial biosynthesis of biomass with a high protein content.

The invention is illustrated by examples, which are not, however, restrictive.

Example 1. The strain Methylococcus capsulatus (CONCEPT-8) VKM B-3289D was grown on a fermenter with a working volume of 1.7 l on a mineral medium of the following composition g / l: KNO ₃ - l, 0; MgSO ₄ ⋅ 7H ₂ O-0.2; CaCl ₂ - 0.02;

Na ₂ HPO ₄ ⋅ 12H ₂ O -1.5; KH ₂ PO ₄ 0.7;

FeSO ₄ ⋅ 7H ₂ O-2.0 mg; ZnSO ₄ ⋅ 7H ₂ O - 0.1 mg;

MnCl ₂ ⋅ 4H ₂ O - 0.03 mg; CuCl ₂ ⋅ 5H ₂ O - 0.1 mg; CoCl ₂ ⋅ 6H ₂ O - 0.2 mg; NiCl ₂ ⋅ 6H ₂ O - 0.02 mg; Na ₂ MoO ₄ - 0.03 mg; H ₃ BO ₃ - 0.03 mg; EDTA-Na ₂ - 5.0.

pH 6.7

The process was conducted at a pressure of 0.2 MPa, the gas phase contained a methane: air mixture in a ratio of 1: 3.

Methane -99.9%

Cultivation temperature 42 ° C

The duration of the process is 336 hours.

The flow rate D = 0.28 hour ^-1

The biomass concentration was 11.8-12.3 g / l, the crude protein content in biomass was 79.4%

Example 2

Cultivation was carried out as described in example 1.

The gas mixture contained methane: ethane in a% ratio of 94: 6. Flow rate D = 0.28 hour -1

The biomass concentration was 11.7-12.0 g / l, the crude protein content of 78.1%

Example 3

Cultivation was carried out as described in example 1.

The gas mixture contained methane: ethane: pentane in a% ratio of 92: 6: 2. The flow rate D = 0.28 hour -1

The biomass concentration was 11, 5-11, 7. g / l, the crude protein content of 78%

Example 4

Cultivation was carried out as described in example 1. The process was conducted at a pressure of 0.2 MPa, the gas phase contained a methane: air mixture in a ratio of 1: 3.

Methane - 99.9%

In the culture medium was added waste culture fluid (OC) obtained after separation of methanotroph cells.

OKJ contains the whole complex of metabolic products of methanotroph, which are formed in the process of growing methanotroph in natural gas

including methanol, formaldehyde, formate, polysaccharides, organic acids, etc. (33-38).

The total quantitative characteristic of all organic substances formed in the process of cultivation are indicators of COD (mgO ₂ / l) and BOD (mgO ₂ / l).

OKJ was added to the culture medium with the following indicators:

COD = 1650 mgO ₂ / L

BOD = 1100 mgO ₂ / L

OKL in the culture medium was added in an amount of 1-10 vol.%.

Example 5

The cultivation of the strain Methylococcus capsulatus (CONCEPT-8) VKM B-3289D was carried out on a fermenter with a working volume of 1.7 l on a mineral medium as in example 1.

pH 6.7

The process was conducted at a pressure of 0.1 MPa, the gas phase at the inlet to the fermenter contained a mixture: methane -6%, oxygen - 19%, carbon dioxide - 1.5%, the rest was nitrogen.

Methane -99.9%

Cultivation temperature 42 ° C

The duration of the process is 336 hours.

The flow rate D = 0.25 hour -1

The biomass concentration was 0.7-0.8 g / l, the crude protein content in biomass was 78.0%.

The gas phase at the outlet of the fermenter contained a mixture: methane -3.7%, oxygen - 17%, carbon dioxide - 4.3%, the rest was nitrogen.

The presented examples characterize the strain Methylococcus capsulatus VKM B-3289D as a producer of a high protein content in microbial biomass, capable of ensuring its high performance in a wide range of methane concentration (from 6 to 99.9%) during cultivation and increased resistance to the inhibitory effect of metabolic products .

Claims (1)

Methylococcus capsulatus VKM B-3289D methane-oxidizing bacteria strain for protein biomass biosynthesis.