JP6966096B2 - Symbiotic Bacteria for Obtaining Microbial Proteins Heterotrophic Bacteria Cupriavidus guilardii GBS-15-1 Strain - Google Patents

Description

The present invention relates to the microbial industry and, in particular, to a strain of the heterotrophic bacterium Cupriavidus gilardii GBS-15-1 for obtaining protein biomass by co-culture containing a methane-oxidizing bacterium and a heterotrophic bacterium. 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 source of 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 of these strains are characterized by a protein content of up to 76% in absolute dry matter.

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 natural gas reserves and portability. 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 reserve of underground gas, which is as high as 40% of the world's reserves according to multiple data, it is not possible to introduce the production of unicellular proteins using microorganisms. Not only can it bring economic benefits to Russian companies, but it also contributes to ensuring the stability of domestic food.

Absolute methane-oxidizing bacteria contain the enzyme methane monooxygenase, which has low substrate specificity, 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 incompletely oxidized methane and homologues of methane, 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.

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.

Cupriavidus europhos VKPM V-10646 (Russian Patent No. 2439143) (Patent Document 4), which is a producer of polyhydroxyalkanoate, is known. Glucose or fructose, or 3-butyric acid, or a mixed gas of hydrogen, oxygen, and carbon dioxide is used as the growth substrate.

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 5).

Russian Patent No. 116363 Russian Patent No. 925112 Russian Patent No. 1070916 Russian Patent No. 2439143 Russian Patent No. 2613365

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 dependent bacterium as a symbiotic bacterium of a microbial protein producer.

The technical result of the present invention is a co-existing bacterium of methane-oxidizing bacteria, which can utilize the products produced by the co-oxidation of methane homologues in natural gas, as well as proteins, amino acids, which are produced during the lysis process of the bacteria. 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. Cupriavidus gilardii GBS-15-1, a heterotrophic bacterium deposited under the registration number VKM B-3265D in the All-Russian Microbial Collection attached to the Skryabin Memorial Institute for Biochemical Physiology, has been 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-15-1.

Cupriavidus gilardii GBS-15-1 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.

The Cupriavidus gilardii GBS-15-1 strain of the present invention is extracted from a co-culture 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. As a result, a strain that can be used as a culture bacterium when culturing methane-oxidizing bacteria in natural gas in an industrial environment and as a component of various co-cultures was obtained.

Bacterial strains grow as co-cultures of the main producers of methane-oxidizing bacteria during fermentation in mineral culture media, and in the physicochemical environment optimal 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 recombined.

The Cupriavidus gilardii species is a work that uses pathogens of pathogenicity classifications 1 to 4 (Health and Epidemiology Regulations SP 1.3.2322-08 "Pathogenicity (risk) classification III-IV groups". Safety ”Attachment No. 1“ Classification of pathogenic microorganisms, protozoa, parasites, and biological toxins that cause infectious diseases to humans by pathogenicity ”, certified by the Chief of the National Health Doctor of the Russian Federation on June 29, 2011 No. 86 Addition and revision No. 2) is not on the list.

The Cupriavidus guilardii GBS-15-1 strain is non-pathogenic, non-toxin-producing, non-toxic and harmless.

The resulting heterotrophic bacterium Cupriavidus guilardii GBS-15-1 strain is characterized as follows.

Culture and morphological features : Gram-negative. On vegetative agar (pH 7.0-7.2), it is opaque, light cream, 2-4 mm in diameter, has flat edges, is a soft solid, and forms colonies that can be easily peeled off from the agar. The cell shape is rod-shaped with a length of 0.3-0.5 × 0.9-1.2 μm and does not form spores.

Physiological and biochemical characteristics : The strain grows in the temperature range of 30 to 47 ° C., and the optimum growth temperature is 32 to 35 ° C. The limit pH of physiological action is in the range of 5.0 to 7.2. The optimum pH range is 6.8 to 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 rises, and flow velocity fluctuations (0.15 to 0.42 h- ^1). do. It has a wide range of potential in terms of ability. Sugars, amino acids, organic acids and alcohols are used as carbon sources. Nitrate, ammonium salt and amino acids are used as nitrogen sources. Does not require specific growth factors.

Recommended culture media and conditions for culturing:
Proposal 1: Mineral culture group Mineral culture group (culture group component, 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 .. Trace element solution (separately prepared) (г / л): 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 trace element solution to 1000 ml of mineral culture medium.

Sterilize at 121 ° C for 15 minutes. Set the pH to 6.0-6.2. Methanol (less than 1.5% by volume), ethanol (less than 2% by volume), and propanol (less than 1% by volume) can be used as substrates (carbon source and energy source). Culture temperature 32-35 ° C, aerobic culture.

Proposal 1: Nutritional agar or nutrient solution Nutritional agar component (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, chloride 5.0 g of sodium, 12.0 g of microbial culture agar. 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.

The strain is stored on nutrient agar (temperature 4 ± 2 ° C) and transplanted once every three months, and the strain can be stored in lyophilized state or in liquid nitrogen.

Characteristics of the strain of the present invention-Stable and productive growth in culture media of various components-Can maintain activity for a long period of time in a freeze-dried state-Non-pathogenic, non-toxic, non-toxic and harmless

The strain is used in the field of producing protein-vitamin concentrated feeds by methane-oxidizing bacteria that grow in natural gas.

The dependent bacterium Cupriavidus gilardii GBS-15-1 is co-cultured with the methane-oxidizing bacterium Methylococcus capsaturus GBS-15 in natural gas methane and mineral culture groups, where the methane-oxidizing bacterium is the only natural gas source of carbon and energy. Uses methane. At the same time, methane-oxidizing bacteria co-oxidize _{methane homologues (C 2} , C ₃ , C ₄ ) that produce products that inhibit the growth of methane-utilizing bacteria. Cupriavidus guilardii GBS-15-1 uses the product 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 lysis process of the main producer.

Culturing is carried out mainly by a continuous method, in which the culture medium is aerated with a mixed gas of natural gas and oxygen-containing gas in an optimum environment of pH 5.6 to 5.8 and a temperature of 42 to 45 ° C. The culture medium is inoculated with a heterotrophic bacterium Cupriavidus gilardii GBS-15-1, which is 0.1 to 0.15% of the total cell count. When the constant concentration of biomass is up to 32 g / l, the content ratio of the methane-oxidizing bacterium Methylococcus capsulatus GBS-15 in the co-cultured bacteria is 85 to 99.9%. Methane-oxidizing bacteria grow on natural gas methane, and the other element of co-culture grows on co-oxidation products of methane homologues in natural gas and products of bacterial activity of methane-utilizing bacteria.

Methane-oxidizing bacteria and heterotrophic bacteria that do not utilize methane are separately collected in advance by a usual method premised on the culture of inoculum, and are simultaneously planted in a vegetative culture medium. The number of heterotrophic bacteria that are planted at this time and do not oxidize methane shall be in the range of 0.1 to 15% of the total number of cells. 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 to 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-oxidized 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 methane homologues, of co-oxidation products obtained in the growth of the cultured bacterium, and is usually limited by the total number of 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.

The present invention can be carried out in the following examples.

Example 1 Liquid mineral culture group component (g / l) for culturing Cupriavidus gilardii GBS-15-1 strain: (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.

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. 0.5% methanol 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 6.8 for 48 hours. The concentration of the dried product at the end of culturing is 2-2.5 g of the absolute dried product 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 type fermenter having a capacity of 40 liters (working capacity of 28 liters).

Example 2 The culture of the Cupriavidus gilardii GBS-15-1 strain is carried out in the same manner as in Example 1, but 0.8% of ethanol is used as a carbon source and an energy source, and the following components are contained as a nutrient culture medium (culture medium). (1 liter equivalent): 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, 0.3 g of sodium carbonate. The physicochemical conditions and culture time are the same, and the dry matter concentration is 4 g of absolute dry matter per liter.

Example 3 Cupriavidus guilardii GBS-15-1 strain is cultured in a culture medium supplemented with butanol in an amount such that the concentration is 0.5% with respect to the whole. 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 6.8 for 48 hours. The concentration of the dried product at the end of culturing is 2.5 to 3.0 g of the absolute dried product per liter. The resulting suspension is centrifuged at 8000 grams for 7 minutes. The precipitate is then centrifuged again in a small amount of saline and subsequent bacterial culture in an automatic fermentation system with a capacity of 10 liters (working capacity 7 liters) or a discharge fermenter with a capacity of 40 liters (working capacity 28 liters). Used as an inoculum for.

Example 4 A co-culture composed of the methane-oxidizing bacterium Methylococcus capsaturus GBS-15 and the heterotrophonas acidaminifila GBS-15-2 in an automatic fermentation system with 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 x 4H ₂ O 0.38, ZnSO ₄ x 7H ₂ O 0.06, H ₃ BO ₃ 0.25, Na ₂ MoO ₄ x 2H ₂ O 0.009, CoSO ₄ x 7H ₂ O 0. 0095

Ammonia water is supplied as a nitrogen source and a titrator. The culturing process is carried out at 40 to 45 ° C. and pH 5.0 to 6.0 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 15 g of absolute dry matter per liter, the dominance of Methylococcus capsaturus GBS-15 was 90%.

Claims (1)

Registration number VKM B-3265D, Russian Academy of Sciences G.M. K. A strain of the dependent bacterium Cupriavidus gilardii deposited in the All-Russian Microbial Collection attached to the Scriabin Memorial Institute for Biochemical Physiology, and a bacterial strain as a co-culture component with methane-oxidizing bacteria for obtaining microbial proteins.