JP6919910B2 - Symbiotic Bacteria for Obtaining Microbial Proteins Heterotrophomonas acidaminifila GBS-15-2 Strain - Google Patents

Description

The present invention relates to the microbial industry and, in particular, to strains of the heterotrophic bacterium Klebsiella pneumonia 1-17 for obtaining protein biomass by co-culture containing methaneoxidizing bacteria and heterotrophic bacteria. Microbial proteins can be used as livestock feed in agriculture and as a raw material for advanced processing.

Today's feed protein production in Russia is not in good shape overall. According to the doctrine signed by the Russian President to bring food self-sufficiency to 80-90%, the shortage of feed products could amount to at least 2 million tonnes per year.

The main ingredient in protein products is soybean meal. However, the natural conditions of Japan are not suitable for cultivating a sufficient amount of soybeans. Therefore, those engaged in this field must look for other ways to produce feed protein.

Among the producers of feed biomass, various taxa of microorganisms that can grow on various substrates are known.

Using yeast as a producer of feed protein is more efficient than using yeast, as yeast stores less than 60% of its protein by weight, while bacteria store 79% of its mass of protein. good.

As a producer of proteins and biomass, a bacterial strain that produces proteins based on methanol and the genus Acetobacter VSB-924 CMPM V-2942 (Russian Patent No. 116363, C12 No. 15/00, 1984) (Patent Document 1), Acetobacter methylicum VSB-867 CMPM V-1947 (Russian Patent No. 925112, C12 No. 15/00, 1982) (Patent Document 2), Acetobacter methylovorans VSB-914 CMPM V-24 National Patent No. 1070916, C12 No. 15/00, 1983) (Patent Document 3) is used. All these strains are characterized by a protein content of up to 76% in absolute desiccant.

The sensitivity and heat resistance of these strains to typical phages vary widely. The optimum growth temperature for Acetobacter methylicum VSB-867 is 28 ° C. to 30 ° C., 30 to 36 ° C. for Acetobacter methylicum VSB-924, and 36 to 40 ° C. for Acetobacter methylovorans VSB-914.

However, in non-sterile cultures with acidic cultures below pH 6.0 in fermenters with wheat bran and / or wheat flour, as experiments show, yeast and fungal contamination occurs quickly and the total cell count is 30. Producer substitution reactions occurred in ~ 40% or more. As a result, the protein content level of the resulting product is significantly reduced.

One of the promising ways to obtain a complete feed protein product is to utilize methane-oxidizing bacteria. Methane-utilizing bacteria actively convert natural gas methane under conditions suitable for them and proliferate rapidly to produce biomass rich in useful proteins and vitamins and other bioactive compounds.

The use of natural gas methane to obtain single-cell proteins has several advantages over liquid hydrocarbons and other substrates, especially the high reserves and portability of natural gas. It has the advantages of height and the ability to obtain feed protein without further purification from the substrate.

Considering that Russia has a large amount of underground gas reserves, which is as high as 40% of the world's reserves according to multiple data, it is not possible to introduce the production of single-celled proteins using microorganisms. Not only can it bring economic benefits to Russian companies, but it also contributes to ensuring food stability in the country.

Absolute methane-oxidizing bacteria contain the enzyme methane monooxygenase, which is not highly substrate-specific, and can oxidize not only methane but also methane homologues (eg, ethane, propane and butane) contained in natural gas.

Methanol, formaldehyde, formic acid, ethanol, acetaldehyde, acetate, propionaldehyde, among the products of methane and its homologues that are incompletely oxidized, depending on the composition of the natural gas, the characteristics of the species of methane-oxidizing bacteria and the culture conditions. , Propionic acid, oil aldehyde may be present in the culture group in various proportions. Accumulation of these products in culture media at specific concentrations exerts an inhibitory effect on methane oxidation cultures and methane oxidation.

Stable continuous culture in natural gas is possible only by co-culture of methane-oxidizing microorganisms and their heterotrophic microorganisms, which utilize the product of incomplete oxidation of methane homologues and also. Products produced by self-degradation of microbial cells may also be used.

It is now established that co-cultivation rather than pure culture is more appropriate when incompletely oxidized products accumulate in the culture medium, even when using a single substrate. During co-culture, the growth of the main culture is noticeably active, and the resistance of the main culture to the stress action of some physicochemical indicators is also noticeably increased.

Stenotrophomonas maltophilia SNBS-3 bacterial strains are known that quickly dispose of petroleum and petroleum products (diesel fuels, motor oils, hydraulic hydraulic oils, condensates) over a wide temperature range of + 5 to + 37 ° C. A strain of the bacterium Stenotrophomonas maltophilia has been deposited in the All-Russian Industrial Microbial Collection under registration number "VKPM V-12247" to purify soil contaminated with petroleum and petroleum products (diesel fuel, motor oil, hydraulic oil, condensate). And water purification. According to the present invention, soil and water contaminated with petroleum and petroleum products in a wide temperature range of + 5 to + 37 ° C. (Russian Patent No. 2617950) (Patent Document 4). It is possible to improve the purification efficiency of the product and shorten the period.

Strains of Stenotrophomonas acidaminifila BJ1 (Chinese Patent No. 102851225) (Patent Document 5) used for ecologically controlling diseases of apple trees are known.

A strain of Methylococcus capsulatus VCB-874, which is a producer of feed biomass, is known. The strain is stored in the culture collection of the "Institute for All-Russian Genetics and Industrial Microbial Breeding" as the collection number "CMPM V1743 (Авт. свид. Magazine Soviet Union No. 770200) (Non-Patent Document 1)". There is. This strain utilizes methane as a carbon source and an energy source, whether it is pure methane or methane as a component in natural gas. The disadvantage of this strain is that it is highly sensitive to the products produced by the co-oxidation of methane homologues, which may be large or small, but are always present in natural gas. It is a thing. The product produced inhibits the growth of the grower.

The closest technical essence and results to be achieved are strains of the methane-oxidizing bacterium Methylococcus capsaturus GBS-15 for obtaining microbial proteins under All-Russian Industrial Microbial Collection Registration No. VKPM V-12549 (Russian Patent No. 2613365). ) (Patent Document 6).

Russian Patent No. 116363 Russian Patent No. 925112 Russian Patent No. 1070916 Russian Patent No. 2617950 Chinese Patent No. 102851225 Russian Patent No. 2613365

Авт. свид. Magazine Soviet Union No. 770200

An object of the present invention is to increase the production efficiency and obtain high productivity for culturing methane-oxidizing bacteria in natural gas in the presence of heterotrophic bacteria as cohabitants of microbial protein producers.

The technical result of the present invention is that methane-oxidizing bacteria co-existing bacteria that can utilize co-oxidized products of methane homologues in natural gas, as well as proteins, amino acids, produced during the bacterial lysis process. To identify new strains that can utilize polysaccharides.

The technical results of the present invention are described in Russian Academy of Sciences G.M. K. The heterotrophomas acidaminifila GBS-15-2, which was deposited under the registration number VKM B-3264D in the All-Russian Microbial Collection attached to the Skryabin Memorial Institute for Biochemical Physiology, was used as a co-culture of methane-oxidizing bacteria for the purpose of obtaining microbial proteins. Achieved when used.

The code of the strain given by the depositor is GBS-15-2.

The Stenotrophomonas acidaminifila GBS-15-2 strain is one of the co-cultured bacteria of methane-oxidizing bacteria for industrially producing feed biomass for livestock feed based on natural gas, and as a raw material for advanced processing. Available.

Strain Extraction Source: Strains are extracted from co-cultures containing the methane-oxidizing bacterium Methylococcus capsaturus GBS-15. To obtain fast-growing mixed-culture bacteria, a mixture of active stock-cultured bacteria extracted from natural gas and groundwater in oil-producing areas within the Russian Federation was used. Bacterial strains grow as co-cultures of the main producers of methane-oxidizing bacteria during fermentation in mineral culture media, and in the optimal physicochemical environment for the producers, co-oxidation by methane homologues present in natural gas. It requires products, as well as products from the material metabolism of the main producer.

Bacterial strains are those that have not been genetically modified.

Information on the biological risk (safety) of bacterial strains: Stenotrophomonas acidaminifila species are pathogenic classification 1st to 4th groups (Health and Epidemiology Regulations SP 1.3.2322-08 "Pathogenicity (risk) classification No. 1" Safety of Work Using Pathogens of Group III-IV Microorganisms and Parasite Diseases "Attachment No. 1" Classification of pathogenic microorganisms, protozoa, parasites, and biological toxins that cause infectious diseases to humans , No. 86 Addition and Revision No. 86 of the National Health Doctor Certification of the Russian Federation dated June 29, 2011) is not on the list.

Other information regarding pathogenicity or toxicity: Stenotrophomonas acidaminifila GBS-15-2 strain is non-pathogenic, non-toxin-producing, non-toxic and harmless.

Characteristic:
I. Culture and morphological features : Gram-negative. On nutrient agar (pH 7.0-7.2), opaque, white-beige, 2-3 mm in diameter, with flat edges, soft solid, forming colonies that can be easily peeled off from the agar. .. As it matures, it may produce a brown pigment. The shape of the cells is straight or slightly curved rod-like, present alone or in pairs, having a length of 0.5-1.5 μm and not forming spores.

II. Physiological and biochemical characteristics : It grows in the temperature range of 25 to 50 ° C, and the optimum growth temperature is 32 to 35 ° C. It grows in the pH range 5.0-8.0 and the optimum pH is 6.8-7.0. Absolutely aerobic bacteria. Stable retention of its own characteristics even under fluctuations in culture conditions and culture media, that is, changes in carbon source, fluctuations in the activity reaction value of culture media, temperature rise, and flow velocity fluctuations (0.15 to 0.42 h- ^1). do. It has a wide range of potential in terms of ability. Polypeptides, amino acids, organic acids and alcohols produced by lysis of the producer strain (GBS-15) are utilized for growth. Does not require specific growth factors.

Recommended culture media and conditions for culturing:
Proposal of nutrient medium:
Proposal 1:
Mineral culture group components (g / l): (NH ₄ ) ₂ SO ₄ 0.52, sulfonyl ₄ 0.02, K ₂ SO ₄ 0.06, NH ₄ H ₂ PO ₄ 1.53. Microbial solution (prepared separately) (g / l): ZnSO ₄ 0.43, MnSO ₄ 0.88, CuSO ₄ 0.78, H ₃ BO ₃ 0.4, Na ₂ MoO ₄ × 2 H ₂ O 0. 25, CoSO ₄ × 7H ₂ O 0.25, FeSO ₄ × 7H ₂ O 4.97. Add 1 ml of the prepared microbial solution to 1000 ml of mineral culture medium.

Sterilize at 121 ° C for 15 minutes. Set the pH to 6.0-6.2.

Butanol, propanol or ethanol (0.15-1.0%) can be used as the substrate (carbon source and energy source). Culture temperature 32 to 35 ° C, aerobic culture.

Proposal 2:
Nutrient solution or nutritional agar.
Nutritional agar components (1 liter of prepared medium): 10.5 g of dried protein enzyme hydrolyzate, 10.5 g of dried peptone for fermentation medium, 2.0 g of filtered autolyzed yeast extract, 5.0 g of sodium chloride, microbial culture agar 12.0 g. Sterilize at 121 ° C for 15 minutes.

Nutrient solution components (1 liter of prepared medium): 9.1 g of dried protein enzyme hydrolyzate, 9.9 g of dried peptone for fermentation medium, 4.7 g of filtered autolyzed yeast extract, 5.0 g of sodium chloride, 0 of sodium carbonate .3g. Sterilize at 121 ° C for 15 minutes.

Recommended storage method and storage conditions: Strains are stored on nutrient agar (temperature 4 ± 2 ° C), transplanted once every three months, and the strains can be stored in a lyophilized state or at the temperature of liquid nitrogen.

As described above, the strain of the present invention grows stably and productively in the culture medium of various components, can maintain its activity for a long period of time in a lyophilized state, and is non-pathogenic, toxin-producing, and non-toxic. It has the characteristic of being harmless.

The Stenotrophomonas acidaminifila GBS-15-2 strain can be used as a co-cultivator in co-culture of methane-oxidizing bacteria with natural gas for the purpose of industrial production of feed biomass, and can also be used as a raw material for advanced processing. ..

The heterotrophomonas acidaminifila GBS-15-2 utilizes the products produced (methanol, ethanol, propanol) as a carbon source, thereby removing the inhibitory effect on the main producer. In addition, heterotrophs can utilize proteins, amino acids, and polysaccharides produced in the bacterial lysis process.

Methane-oxidizing bacteria grow on natural gas methane, and the other element of co-culture grows on the co-oxidation products of methane homologues in natural gas and on the bacterial activity of methane-utilizing bacteria.

In the process of co-culture, the content level of co-culture bacteria is determined by the number of products produced and available as a result of the co-oxidation of the gaseous homologues of methane in the gas, regardless of the initial planting amount. This is because in mineral culture groups, these products are the only carbon source for co-culture elements that do not utilize methane. Since the cultured bacteria perform such self-regulation, the proportion of cultured cells in the initial planting of the culture is not significant, but the number of non-methane-oxidizing microorganisms added is 0.1% or more of the total number of cells. It is recommended to keep it within 15%. Outside this range, it first leads to stagnation of growth of methane-oxidizing bacteria, and secondly, stagnation of growth of heterotrophic bacteria. In the mixed culture in which bacteria that do not assimilate methane were selected and added, the amount of biomass produced increased 3 to 5 times as compared with the pure culture in which only methane-oxidizing bacteria were added. Thus, the biomass production in methane can be increased by selecting heterotrophs that grow on the co-oxidation products of the methane homologue and do not assimilate methane and add them to the methane-oxidizing bacteria. Even with high concentrations of heterotrophs that do not assimilate methane, their content is limited by the number of carbon sources, or co-oxidized products of the methane homologue, obtained in the growth of the cultured bacterium, and is usually limited to the total cells. Adding heterotrophic bacteria that do not oxidize methane does not affect the quality of the biomass, as it does not exceed 1-1.5% of the number.

Example 1
Liquid mineral culture group components (g / l) for culturing the Stenotrophomonas acidaminifila GBS-15-2 strain of VKM-3264D: (NH ₄ ) ₂ SO ₄ 0.52, sulfonyl ₄ 0.02, K ₂ SO ₄₀ . 06, NH ₄ H ₂ PO ₄ 1.53.

Sterilize the culture medium at 121 ° C. for 15 minutes.

Microbial solution (prepared separately from the culture group and sterilized) (g / l): ZnSO ₄ 0.43, MnSO ₄ 0.88, CuSO ₄ 0.78, H ₃ BO ₃ 0.4, Na ₂ MoO ₄ × 2H ₂ O 0.25, CoSO ₄ × 7H ₂ O 0.25, FeSO ₄ × 7H ₂ O 4.97. Add 1 ml of the prepared microbial solution to 1000 ml of mineral culture medium. Set the pH to 6.0-6.2.

Ethanol 1.0% is used as the substrate (carbon source and energy source).

The strains are cultivated in a heat-resistant glass flask with a volume of 1 liter at a filling rate of 0.3 to 0.4 in a constant temperature shaking incubator by a method for a fixed period. Culturing is carried out at 35 ° C. and pH 6.2 for 48 hours. The dry matter concentration at the end of culturing is 7 g of absolute dry matter per liter. The obtained cultured bacteria are used as inoculum for subsequent bacterial culture in an automatic fermentation system having a capacity of 10 liters (working capacity of 7 liters) or a discharge fermenter having a capacity of 40 liters (working capacity of 28 liters).

Example 2 The strain is cultivated in the same manner as in Example 1, but 0.8% propanol is used as a carbon source and an energy source. The physicochemical conditions and culture time are the same, and the dry matter concentration is 2.5 g of absolute dry matter per liter.

Example 3 The strain is cultivated in the same manner as in Example 1, but 0.15% butanol is used as a carbon source and an energy source. The physicochemical conditions and culture time are the same, and the dry matter concentration is 0.8 g of absolute dry matter per liter.

Example 4 A co-culture composed of the methane-oxidizing bacterium Methylococcus capsaturus GBS-15 and the heterotrophic bacterium Stenototropomonas acidaminifila GBS-15-2 in an automatic fermentation system having a capacity of 10 liters (actual capacity of 7 liters) has the following configuration. Perform on a mineral culture medium. (Unit: g / l) H ₃ PO ₄ (80%) 17.2, K ₂ SO ₄ 5.0, 0073 ₄ × 7H ₂ O 4.0, FeSO ₄ × 7H ₂ O 0.21, CuSO ₄ 0. 78, MnSO ₄ × 4H ₂ O 0.38, ZnSO ₄ × 7H ₂ O 0.06, H ₃ BO ₃ 0.25, Na ₂ MoO ₄ × 2 H ₂ O 0.009, CoSO ₄ × 7H ₂ O 0. 0095

Ammonia water is supplied as a nitrogen source and a titrator. The culturing process is carried out at 41 to 43 ° C. and pH 5.5 to 5.7 while constantly supplying a mixed gas containing methane and oxygen. When the biomass concentration reaches 10 g of the absolute dry matter per liter, the ^{process proceeds to} a continuous culture process in which the dilution ratio of the culture medium is D = 0.25 h-1. When the biomass concentration was 16 g of absolute dry matter per liter, the predominance of Methylococcus capsaturus GBS-15 was 92%.

Example 5 A co-culture composed of the methane-oxidizing bacterium Methylococcus capsulatus GBS-15 and the heterotrophonas acidaminifila GBS-15-2 is arranged in a discharge-type fermenter having a capacity of 40 liters (actual capacity of 28 liters) as follows. Performed in the mineral culture medium of. (Unit: g / l) H ₃ PO ₄ (80%) 17.2, K ₂ SO ₄ 5.0, 0073 ₄ × 7H ₂ O 4.0, FeSO ₄ × 7H ₂ O 0.21, CuSO ₄ 0. 78, MnSO ₄ × 4H ₂ O 0.38, ZnSO ₄ × 7H ₂ O 0.06, H ₃ BO ₃ 0.25, Na ₂ MoO ₄ × 2 H ₂ O 0.009, CoSO ₄ × 7H ₂ O 0. 0095

Ammonia water is supplied as a nitrogen source and a titrator. The culturing process is carried out at 41 to 45 ° C. and pH 5.5 to 5.7 while constantly supplying a mixed gas containing methane and oxygen. When the biomass concentration reaches 10 g of absolute dry matter per liter, the atmospheric pressure is gradually increased to 0.8 MPa. As the air pressure rises, the supply of gas, which is a nutrient source for microorganisms, is gradually increased. At that time, the index of the culture process was D = 0.28 h- ¹ , the biomass concentration was 43 g of absolute dry matter per liter, and the dominant rate of the methane-oxidizing bacterium Methylococcus capsaturus GBS-15 was 93%.

Claims (1)

Registration number VKM B-3264D, Russian Academy of Sciences G.M. K. A strain of the heterotrophomas acidaminifila, a heterotrophic bacterium deposited in the All-Russian Microbial Collection attached to the Skryabin Memorial Institute for Biochemical Physiology, which is a bacterial strain as a co-culture component with methane-oxidizing bacteria for obtaining microbial proteins.