EA036408B1 - Strain of heterotrophic bacterium stenotrophomonas acidaminiphila gbs-15-2 - associate for producing microbial protein mass - Google Patents

Abstract

The invention relates to the bioindustry, and more particularly to a heterotrophic bacterial strain Stenotrophomonas acidaminiphila GBS-15-2 for producing protein biomass by means of an associative culture comprising methane-oxidizing bacteria and bacterial heterotrophs. The microbial protein mass can be used in agriculture for feeding animals, and also as a feedstock for advanced processing. The technical result of the invention is the discovery of a new strain which is an associate of methane-oxidizing bacteria and is capable of utilizing methane homologue co-oxidation products present in natural gas, and which can also utilize proteins, amino acids and polysaccharides produced during lysis of the bacteria. The technical result is achieved by using the heterotrophic bacterial strain Stenotrophomonas acidaminiphila GBS-15-2, deposited in the All-Russian Collection of Microorganisms of the G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences under registration number VKM B-3264D, as a component of an associative culture of methane-oxidizing bacteria for producing microbial protein mass.

Description

The invention relates to the microbiological industry, in particular to a strain of heterotrophic bacteria Stenotrophomonas acidaminiphila GBS-15-2 for obtaining protein biomass by an associative culture, including methane-oxidizing bacteria and heterotrophic bacteria. Microbial protein mass can be used in agriculture for feeding animals, as well as raw materials for deep processing.

Today, the overall picture in Russia for the production of feed proteins is unfavorable. According to the doctrine signed by the President of Russia to provide 80-90% of its own food, the deficit of fodder products may be at least 2 million tons per year.

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

Among the producers of fodder biomass, various microorganisms belonging to different taxonomic groups are known that can grow on various substrates.

The use of bacteria as producers of fodder protein is more effective, since bacteria accumulate up to 79% of protein by weight, while yeast - no more than 60%.

As producers of protein and biomass, bacterial strains are used, known as protein producers based on methanol and belonging to the genus Acetobacter: Acetobacter methylicum VSB-924 CMPM B-2942 (RF patent No. 116363); Acetobacter methylicum VSB-867 TsMPM B-1947 (RF patent No. 925112); Acetobacter methylovorans VSB-914 TsMPM V-2479 (RF patent No. 1070916). All strains are characterized by a protein content of up to 76% by DIA.

The strains differ in sensitivity to typical phages and heat resistance: optimal growth in Acetobacter methylicum VSB-867 at 28-30 ° C, in Acetobacter methylicum VSB-924 - 30-36 ° C and Acetobacter methylovorans VSB-914 at 36-40 ° C.

However, upon non-sterile cultivation in a fermentor on bran and / or flour fermentolysates in an acidic medium at a pH below 6.0, experiments have shown that rapid infection with yeast and fungi and the displacement of the producer from the process by 30-40% or more of the total number of cells occurs. As a result, the protein content of the resulting product is greatly reduced.

One of the promising ways to obtain a complete protein feed product is the use of methane-oxidizing bacteria. Under suitable conditions, methanotrophic bacteria actively process methane from natural gas, multiply rapidly and build up a biomass rich in valuable protein, vitamins and other biologically active compounds.

The use of natural gas methane for the production of protein in unicellular organisms has a number of advantages over liquid hydrocarbons and other substrates, namely: large reserves of natural gas, its good transportability, the possibility of obtaining a feed product without additional purification from the substrate.

Considering that Russia has large gas reserves of subsoil, according to some sources, they account for up to 40% of the world, the introduction of microbiological production of single-celled protein at Russian enterprises promises not only an economic effect, but is also able to ensure the country's food security.

Obligate methane-oxidizing bacteria contain the enzyme methane monooxygenase, which is not substrate-specific and oxidizes not only methane, but also methane homologues (for example, ethane, propane, and butane) contained in natural gas.

Depending on the composition of natural gas, species characteristics of methane-oxidizing bacteria and cultivation conditions, among the products of incomplete oxidation of methane and its homologues, methanol, formaldehyde, formate, ethanol, acetaldehyde, acetate, propionaldehyde, propionic acid, butyraldehyde may be present in different ratios in the cultivation medium. ... These products, when accumulated in the cultivation medium at a certain concentration, have an inhibitory effect on the methane-oxidizing culture, on the oxidation of methane.

Stable continuous cultivation on natural gas is possible only when using an associative culture of methane-oxidizing microorganisms and heterotrophic satellite microorganisms using products of incomplete oxidation of methane homologues, possibly also products of autolysis of microbial cells.

It has now been established that even when using a monosubstrate, in the case of accumulation of incomplete oxidation products in the medium, it is advisable to use not a pure culture, but an associative one. When cultivating an associative culture, the growth activity of the main culture and its resistance to stressful effects of certain physicochemical parameters noticeably increase.

Known bacterial strain Stenotrophomonas maltophilia SNBS-3, which has the ability to quickly dispose of oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate) in a wide temperature range from +5 to + 37 ° C. The bacterial strain Stenotrophomonas maltophilia was deposited in the All-Russian Collection of Industrial Microorganisms under the registration number VKPM B-12247 and can be used both for soil cleaning and for cleaning

- 1 036408 water from pollution by oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate). The invention improves the efficiency and shortens the time for cleaning soil and water from oil and oil products in a wide temperature range from +5 to + 37 ° C (RF No. 2617950).

Known is the Stenotrophomonas acidaminiphila BJ1 strain used for biological control of apple diseases (CN No. 102851225).

Known strain Methylococcus capsulatus VSB-874 - producer of fodder biomass. The strain is stored in the collection of cultures of the Institute of VNIIGenetics under the collection number TsMPM B-1743 (ed. St. USSR No. 770200). The strain uses methane as a source of carbon and energy, both pure and natural gas. The disadvantage of this strain is its sensitivity to the products formed during the co-oxidation of methane homologues, which are invariably present in natural gas in greater or lesser amounts. The resulting products inhibit the growth of the producer.

The closest in technical essence and the achieved result is a strain of methane-oxidizing bacteria Methylococcus capsulatus GBS-15, registration number VKPM B-12549 in the All-Russian Collection of Industrial Microorganisms (RF No. 2613365).

The aim of the invention is to increase productivity and high productivity when cultivating methane-oxidizing bacteria on methane-containing gas in the presence of heterotrophic bacteria as an associate of a producer of microbial protein mass.

The technical result of the invention is the identification of a new strain, which is an associate of methane-oxidizing bacteria and is able to use the products of co-oxidation of methane homologues present in natural gas, and can also use proteins, amino acids and polysaccharides released during the lysis of bacteria.

The technical result was achieved by using the heterotrophic bacteria strain Stenotrophomonas acidaminiphila GBS-15-2, deposited in the All-Russian collection of microorganisms at the Institute of Biochemistry and Physiology of Microorganisms G.K. Skryabin RAS under registration number VKM B-3264D, as a component of the associative culture of methane-oxidizing bacteria for obtaining microbial protein mass.

The depositor's strain designation is GBS-15-2.

The Stenotrophomonas acidaminiphila GBS-15-2 strain is used as one of the components of an associative culture of methane-oxidizing bacteria for industrial production of fodder biomass based on natural gas for animal feeding, as well as as a raw material for deep processing.

Source of isolation of the strain: the strain was isolated from an association that includes the methane-oxidizing bacteria Methylococcus capsulatus GBS-15. For the selection of a rapidly growing mixed culture of bacteria, a mixture of active enrichment cultures isolated from groundwater in a number of gas and oil regions of the Russian Federation was used. During fermentation, the strain grows as an associate of the main producer of methane-oxidizing bacteria in a mineral medium and under physicochemical conditions optimal for the producer, consumes the products of co-oxidation of methane homologues present in natural gas, as well as metabolic products of the main producer.

The strain is not genetically modified. Information on the biological hazard (safety) of the strain: the species Stenotrophomonas acidaminiphila is not listed in the lists of 1-4 pathogenicity groups (Appendix 1 Classification of microorganisms - causative agents of infectious diseases ... to the Sanitary and Epidemiological Rules SP 1.3.2322-08 Safety of work with microorganisms, in ed.Additions and changes N 2, approved by the Resolution of the Chief State Sanitary Doctor of the Russian Federation of June 29, 2011 N 86).

Other information on the pathogenicity or virulence of the strain: The Stenotrophomonas acidaminiphila GBC-15-2 strain is avirulent, non-toxic, non-toxic and harmless.

Characteristic signs

I. Cultural and morphological: gram-negative. On nutrient agar (pH 7.0-7.2) forms opaque colonies of whitish-beige color with a smooth edge, 0 2-3 mm, soft consistency, easily removed from the agar. May form brown pigments with age. The shape of the cells can be straight or slightly curved rods, located singly or in pairs, from 0.5 to 1.5 μm long, non-spore-forming.

II. Physiological and biochemical: grows in the temperature range of 25-50 ° С, the optimum growth temperature is 32-35 ° С; pH range 5.0-8.0 optimal pH value 6.8-7.0. Obligate aerobic. It stably retains its characteristics with varying cultivation conditions and media - replacement of the carbon source, low or high values of the active reaction of the medium, increase in temperature, change in the flow rate (0.15-0.42 h ^-1 ). Possesses great potential. It uses lysis products of the producer strain (GBS-15) for growth: polypeptides, amino acids, organic acids, alcohols. Does not require specific growth factors.

- 2 036408

Recommended culture media and conditions:

Culture media options:

Option 1:

The composition of the mineral environment (g / l): (NH ₄ ) ₂ SO ₄ - 0.52; MgSO ₄ - 0.02; K ₂ SO ₄ - 0.06; NH ₄ H ₂ PO ₄ - 1.53. A solution of trace elements (prepared separately) (g / l): ZnSO ₄ - 0.43; MnSO ₄ 0.88; CuSO ₄ 0.78; H ₃ VO ₃ - 0.4; Na2MoO ₄ x2H2O 0.25; CoSO ₄ x7H ₂ O 0.25; FeSO ₄ x7H ₂ O - 4.97. Add 1 ml of the prepared solution of trace elements to 1000 ml of the mineral medium. Sterilize at 121 ° C for 15 minutes. The pH is adjusted to 6.0-6.2.

Butanol, propanol or ethanol (0.15-1.0%) can be used as a substrate (source of carbon and energy). Cultivation temperature 32-35 ° C; aerobic conditions.

Option 2: Nutrient broth or nutrient agar.

Nutrient agar, composition (in terms of 1 liter of prepared medium): enzymatic protein hydrolyzate, dry - 10.5 g; enzymatic peptone, dry - 10.5 g; clarified autolyzed yeast extract - 2.0 g; sodium chloride - 5.0 g; microbiological agar - 12.0 g. Sterilize at 121 ° C for 15 minutes.

Nutrient broth, composition (in terms of 1 liter of prepared medium): hydrolyzate fermentative protein, dry - 9.1 g; dry enzymatic peptone - 9.9 g; clarified autolyzed yeast extract 4.7 g; sodium chloride - 5.0 g; sodium carbonate - 0.3 g. Sterilize at 121 ° C for 15 minutes.

Recommended storage method and conditions: The strain is stored on nutrient agar (at a temperature of 4 ± 2 ° C), reseeding is carried out once every 3 months, it is possible to store the strain in a lyophilized state, as well as at the temperature of liquid nitrogen.

Thus, the inventive strain is characterized by stable productive growth on media of various compositions; the ability to maintain activity for a long time in a lyophilized state; lack of virulence, toxigenicity, toxicity and harmlessness.

The Stenotrophomonas acidaminiphila GBS-15-2 strain is used as a component of an associative culture of methane-oxidizing bacteria for the industrial production of feed biomass based on natural gas, as well as as a raw material for deep processing.

Heterotrophic bacteria Stenotrophomonas acidaminiphila GBS-15-2 use the resulting products (ethanol, propanol, butanol) as a carbon source, thereby removing the inhibitory effect on the main producer. In addition, the heterotroph can use proteins, amino acids, and polysaccharides released during the lysis of bacteria.

Methane-oxidizing bacteria develop due to natural gas methane, and another component of the associative culture - due to the products of co-oxidation of methane homologues in natural gas and due to the waste products of methanotroph.

In the process of co-cultivation, their content, regardless of the seeding value, is at a level determined by the amount of products used, formed as a result of co-oxidation of gaseous homologues of methane in natural gas, since in the mineral environment these products are the only available carbon source for the methane-free component of the associative culture. As a result of such self-regulation of the content of cultures in the process of cultivation, the initial ratio of culture cells in the inoculum is not significant, but it is recommended to introduce microorganisms that do not oxidize methane at least 0.1% and not more than 15% of the total number of cells. A deviation from these values in one direction or another can lead in the first case to a delay in the development of methane-oxidizing bacteria, and in the second to a delay in the growth of heterotrophic bacteria. In a mixture with selected bacteria that do not use methane, the biomass yield increases 3-5 times compared to a pure culture of methane oxidizing bacteria. Thus, the addition of selected methane-free heterotrophs growing due to the co-oxidation products of methane homologues to methane-oxidizing bacteria can increase the yield of biomass on methane. The addition of non-methane-oxidizing heterotrophic bacteria does not affect the quality of biomass, since even with the introduction of heterotrophs that do not use methane in a high concentration, their content in the growing culture is limited by the amount of available carbon source, which is the oxidation products of methane homologues, and usually does not exceed 1- 15% of the total number of cells.

Example 1.

The Stenotrophomonas acidaminiphila GBS-15-2 VKM-3264D strain is grown in a liquid mineral medium containing (g / l): (NH ₄ ) ₂ SO ₄ - 0.52; MgSO ₄ - 0.02; K ₂ SO ₄ - 0.06; NH ₄ H ₂ PO ₄ - 1.53.

The medium is sterilized at 121 ° C for 15 minutes.

A solution of trace elements (prepared and sterilized separately) (g / l): ZnSO ₄ - 0.43; MnSO ₄ 0.88; CuSO ₄ 0.78; H ₂ VO ₃ - 0.4; Na ₂ MoO ₄ x ₂ H ₂ O 0.25; CoSO ₄ x7H ₂ O 0.25; FeSO ₄ x7H ₂ O - 4.97. 1 ml of the prepared solution of trace elements is added to 1000 ml of the mineral medium. The pH is adjusted to 6.0-6.2.

Ethanol is used as a substrate (source of carbon and energy) - 1.0%.

The cultivation of the strain is carried out in periodic mode in flasks made of heat-resistant glass,

- 3 036408 with a volume of 1 liter with a filling factor of 0.3-0.4 in a thermostatically controlled rocker. The cultivation is carried out for 48 hours at 35 ° C and pH 6.2. At the end of cultivation, the concentration of dry matter g ASV / l. The resulting culture is used as an inoculum for the subsequent cultivation of bacteria in an automated fermentation complex with a volume of 10 l (working volume 7 l) or in an ejection-type fermenter with a volume of 40 l (working volume 28 l).

Example 2. Cultivation of the strain is carried out similarly to example 1, but propanol is used as a source of carbon and energy - 0.8%. Physicochemical conditions and cultivation time are the same, the concentration of dry substances is 2.5 g ASV / l.

Example 3. Cultivation of the strain is carried out analogously to example 1, but butanol is used as a source of carbon and energy - 0.15%. Physicochemical conditions and cultivation time are the same, the concentration of dry matter is 0.8 g ASV / l.

Example 4. An associative culture consisting of methane-oxidizing bacteria Methylococcus capsulatus GBS-15 and heterotrophic bacteria Stenotrophomonas acidaminiphila GBS-15-2 are grown in an automated fermentation complex with a volume of 10 l (working volume 7 l) on a mineral medium of the following composition (g / l): H ₃ PO4 (80%) - 17.2; K2SO4 5.0; MgSO ₄ x7H ₂ O 4.0; FeSO ₄ x7H ₂ O 0.21; CuSO ₄ 0.78; MpBO ₄ x4H ₂ O - 0.38; ZnSO ₄ x7H ₂ O 0.06; H ₃ VO ₃ - 0.25; Na ₂ MoO ₄ x ₂ H ₂ O 0.009; CoSO ₄ x7H ₂ O 0.0095.

Ammonia water is supplied as a source of nitrogen and titrating agent. The process is carried out at a temperature of 41-43 ° C, pH 5.5-5.7 and a continuous supply of a gas mixture containing methane and oxygen. When the biomass concentration reaches 10 g ASV / l, they switch to a continuous cultivation process with a dilution factor of the medium D = 0.25 h ^-1 . The dominance of the methane-oxidizing bacteria Methylococcus capsulatus GBS-15 was 92% at a biomass concentration of 16 g ASB / L.

Example 5. An associative culture consisting of methane-oxidizing bacteria Methylococcus capsulatus GBS-15 and heterotrophic bacteria Stenotrophomonas acidaminiphila GBS-15-2 are grown in an ejection-type fermenter with a volume of 40 l (working volume 28 l) on a mineral medium of the following composition (g / l): H3PO4 (80%) - 17.2; K2SO4 5.0; MgSO4x7H2O - 4.0; FeSO4x7H2O 0.21; CuSO4 0.78; MnSO4x4H ₂ O 0.38; ZnSO ₄ x7H ₂ O 0.06; H ₃ VO ₃ - 0.25; Na ₂ MoO ₄ x ₂ H ₂ O 0.009; CoSO ₄ x7H ₂ O - 0.0095. Ammonia water is supplied as a source of nitrogen and titrating agent. The process is carried out at a temperature of 41-45 ° C, pH 5.5-5.7 and a continuous supply of a gas mixture containing methane and oxygen. When the biomass concentration reaches 10 g ASV / l, the pressure is gradually increased to 0.8 MPa. With increasing pressure, the supply of gaseous food sources for microorganisms is gradually increased. The process indices were as follows: D = 0.28 h ^-1 , biomass concentration 43 g ASB / l, the dominance of methane-oxidizing bacteria Methylococcus capsulatus GBS-15 93%.

Claims (1)

CLAIM The strain of heterotrophic bacteria Stenotrophomonas acidaminiphila, deposited in the All-Russian collection of microorganisms at the Institute of Biochemistry and Physiology of Microorganisms G.K. Scriabin, Russian Academy of Sciences under the registration number VKM B-3264D, as a component of the associative culture of methane oxidizing bacteria to obtain a microbial protein mass.