RU2720121C1 - Method of producing microbial protein based on hydrocarbon material - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to the microbiological industry. Method of producing microbial protein based on methane-oxidising bacteria Methylococcus capsulatus GBS-15 provides for preparation of nutrient medium consisting of potassium, magnesium, iron (II), copper, manganese, zinc, cobalt and sodium molybdate of specified concentration with addition of phosphoric acid, fermentation of bacterial cultures with continuous supply of culture liquid, ammonia solution and gas mixture at temperature of 40–45 °C in continuous flow of fermenter capacity 0.2–0.3 per hour. Then one performs separation to produce the ready product. After the spent culture liquid is returned after separation through the storage container to the fermentation stage in amount of 10 to 95 % of the total amount of used water, enriched with the missing mineral salts to the specified concentrations in the culture liquid with subsequent continuation of the bacterial culture fermentation.

EFFECT: invention increases output of the finished product.

1 cl, 1 tbl, 2 ex

Description

The invention relates to the microbiological industry, namely: to obtain a microbial protein mass from a methane-oxidizing bacteria strain Methylococcus capsulatus GBS-15, which can be used in agriculture for animal feeding.

To date, the overall picture in Russia on the production of feed proteins is not favorable. The shortage of feed products is at least 2 million tons per year.

One of the promising ways to obtain a complete protein feed product is methanotrophic bacteria that produce protein. Under suitable conditions, methanotrophic bacteria actively process natural gas, multiply rapidly and build up their biomass, which is rich in valuable protein, vitamins and other biologically active substances.

The use of methane to produce unicellular protein has several advantages compared to liquid hydrocarbons: large reserves of natural gas, its good transportability, the ability to obtain a finished product without additional purification from the substrate.

Given that Russia has large gas reserves of the bowels, according to some sources, they make up 40% of the world. The introduction of microbiological production of unicellular protein in Russian enterprises promises not only economic benefits, but also can ensure food security of the country.

There are various strains of bacteria producing feed protein related to different types of methane-oxidizing bacteria (methanotrophs), for example, strains of Methylococcus capsulatus VKM B-2116, Methylocystis parvus VKM B-2129, Methylosinus sporium VKM B-2123, Methylosinus tr Methylomonas rubra VSB-901, Methylococcus sp. FM-9, Methylococcus capsulatus VSB-874, Methylococcus minimus, Methylomonas methanica.

They are the products of microbial synthesis resulting from the cultivation of methane-oxidizing bacteria on natural gas as the sole source of carbon and energy. These strains are characterized by different growth rates and biomass yields.

The disadvantages of these strains include a low growth rate and an unstable protein content, which in most cases is lower than 70%, as well as the need for increased amounts of a biostimulator, in particular an autolysate, which is manifested when the strain is grown under aseptic laboratory conditions.

A known strain producing biomass Methylococcus capsulatus VSB-874. It is stored in the Museum of Cultures of the Institute "VNIIgenetika" under the collection number TsMPM V-1743 (auth. Certificate. USSR No. 770200). The strain uses methane as a carbon source, both pure and as part of natural gas. The disadvantage of this strain is the impossibility of its cultivation when returning the spent culture fluid.

Known bacterial strain Cupriavidus eutrophus VKPM B-10646 - producer of polyhydroxyalkanoates and method for their preparation (RF №2439143). The method consists in cultivating the producer strain under aeration and mixing on a liquid salt medium containing a growth substrate with an additional carbon source, with a nitrogen limit, using the Cupriavidus eutrophus VKPM B-10646 bacterial strain as the producer strain, using the growth substrate glucose or fructose, or 3-butyric acid, or a gas mixture of hydrogen, oxygen and carbon dioxide, or synthesis gas mixed with oxygen, and a 3-valerate solution is used as an additional carbon source and potassium, or solutions of potassium 3-valerate and potassium 3-hexanoate, or solutions of potassium 3-valerate, potassium 3-hexanoate and acrylate, or solutions of potassium 3-hexanoate and acrylate, or solutions of 3-butyric acid and 4-butorolactone, or solutions of 3-butyric acid, 4-butorolactone and 3-valerate potassium, or solutions of 3-butyric acid, 4-butorolactone and 3-hexanoate potassium, or solutions of 3-butyric acid, 4-butorolactone, 3-valerate potassium and 3-hexanoate potassium.

The closest in technical essence and the achieved result is a method of growing a culture strain Methylococcus capsulatus GBS-15 (RF No. 2613365) in a continuous mode in an ejection-type fermenter with a volume of 40 l (working volume - 25 l) with a continuous supply of a nutrient medium containing the following components ( per 1 liter of medium):

Phosphoric acid H ₃ PO ₄ (70%) - 0.35 ml

Potassium chloride KCl - 0.125 g

Magnesium sulfate MgSO ₄ × 7H ₂ O - 0.105 g

Iron sulfate FeSO ₄ × 7H ₂ O - 10.75 mg

Copper sulfate CuSO ₄ × 5H ₂ O - 10 mg

Manganese sulfate MnSO ₄ × 5H ₂ O - 9.5 mg

Boric acid H ₃ BO ₃ - 6.25 mg

Zinc sulfate ZnSO ₄ × 7H ₂ O - 1.5 mg

Cobalt sulfate CoSO ₄ × 7H ₂ O - 0.25 mg

Molybdenum sodium Na ₂ MoO ₄ × 2H ₂ O - 0.25 mg

The growing process is carried out at a temperature of 42 ° C and a pH of the growing medium of 5.6-5.8. The pH value was maintained with a 10% solution of ammonia water. The process temperature is controlled by supplying cooling water to the apparatus heat exchanger. The consumption of natural gas and air per 1 liter of culture medium was 15 and 45 l / h, respectively. The culture is grown at atmospheric pressure, a temperature of 42 ° C and at a temperature difference of 40 ° to 45 ° C, a pH of 5.6 and a flow rate coefficient of 0.25 h ^-1 , the biomass concentration in the fermenter was 10-11 g / l . When using this mode of cultivation for a long time, using natural gas of various compositions (with methane contents from 85% vol. To 99.9% vol.) And water from various sources, culture phagolysis was not observed.

The objective of the invention is the creation of a method for growing a methane-oxidizing strain of culture Methylococcus capsulatus GBS-15, which has a higher technological potential, in particular:

- returning to the process from the spent culture fluid of various compositions instead of prepared water, which will increase the yield of the finished product, increase the productivity of the fermentation process, reduce the consumption of salts for fermentation, reduce the consumption of prepared water supplied to the process.

The technical result obtained as a result of using the proposed technical solution is: increasing the productivity of the process of obtaining microbial protein based on hydrocarbon feeds from 10 to 30%, solving the problem of purification and complete utilization of spent culture fluid. At the same time, the quality of the obtained product meets all the requirements for a microbial protein based on hydrocarbon raw materials produced using purified water.

This goal is achieved through the use of a method of producing a microbial protein based on hydrocarbon feeds from a methane-oxidizing bacteria strain Methylococcus capsulatus GBS-15, including the preparation of a nutrient medium consisting of potassium, magnesium, iron (II), copper, manganese, zinc, cobalt and sodium molybdate predetermined concentration with the addition of phosphoric acid, fermentation of bacterial cultures with a constant supply of waste liquid, ammonia solution, and gas mixture at a temperature of 40-45 ° C in a continuous flow of 0.2-0.3 volume of the enzyme Fera per hour, separation, production of finished biomass and its drying. In this case, the spent culture fluid after separation is returned through the storage tank to the fermentation stage in a volume of 10 to 95% of the total amount of water used, enriched with the missing mineral salts to the specified concentrations in the culture fluid, and then the fermentation of the bacterial culture is continued until the finished product is obtained.

Obtaining a microbial protein based on natural gas is an enzymatic process on a mineral nutrient medium. The process of preparing the nutrient medium consists of several stages (see the process diagram in Figure 1):

- the preparation of individual solutions of mineral salts of a given concentration on the prepared water (potassium, magnesium, iron (II) sulfates, copper, manganese, zinc, cobalt, boric acid and sodium molybdate);

- preparation of a concentrated solution of salts by dosing in a separate container the calculated amount of ready-made individual solutions of mineral salts, phosphoric acid and prepared water;

- the preparation of water is carried out by cleaning it from mechanical impurities, certain undesirable components and microorganisms.

Inoculation of the fermenter is carried out with a strain of culture Methylococcus capsulatus GBS-15 by the association of microorganisms that allow the disposal of organic metabolic products of the main culture.

The process is conducted in a duct (0.2-0.3 fermenter volumes per hour) with a continuous supply of a solution of mineral salts and phosphoric acid, a solution of ammonia, solutions of calcium salts, sodium and a gas mixture at a temperature of 40-45 ° C.

The suspension formed during the fermentation process is separated, separating the biomass and the spent culture fluid, consisting of unused elements of mineral nutrition, bacteria vital products, such as peptides, amino acids, soluble carbohydrates, etc.

A process has been developed for returning spent culture fluid from 10 to 95% to a fermentation process instead of water. At the same time, both the supply of the spent culture fluid and the supply of a concentrated solution of salts to balance the composition of the mineral nutrition are controlled. In addition, the living microflora remaining after separation, consisting of both the main culture and the satellites processing the metabolites of the main culture, can increase the productivity of the process by 10-30%. The quality of the resulting product meets all the requirements for a microbial protein based on hydrocarbon materials produced using purified water.

Table 1 shows the finished product of the microbial protein obtained by returning a different amount of spent culture fluid.

The invention is illustrated by the following examples.

Example 1. The return of 10% of the spent culture fluid

A culture of the Methylococcus capsulatus GBS-15 strain was grown continuously in an ejection-type fermenter with a volume of 40 l (working volume 25 l) with a continuous supply of concentrated salt solution, prepared water and spent culture fluid in an amount of 0.625 l / h.

The growing process was carried out at a temperature of 41-43 ° C and a pH of the growing medium of 5.6-5.8. The pH value was maintained with a 10% solution of ammonia water. The process temperature was controlled by supplying cooling water to the apparatus heat exchanger.

The consumption of natural gas and air per 1 liter of nutrient medium in the fermenter was 15 and 45 l / h, respectively.

The culture was grown at atmospheric pressure, with a flow rate coefficient of 0.25 h ^-1 , the biomass concentration in the fermenter was 10-11 g / L.

When using this mode of cultivation for a long time, using natural gas of various compositions (with methane content from 85% vol. To 99.9% vol.), The process was stable and the quality of the resulting biomass corresponded to the composition of the biomass obtained by cultivation without return waste culture fluid.

Example 2. The return of 95% of the spent culture fluid

The process was carried out analogously to example 1, the amount of returned spent culture fluid was 95%.

A culture of the Methylococcus capsulatus GBS-15 strain was grown in continuous mode in an ejection-type fermenter with a volume of 40 l (working volume 25 l) with a continuous supply of a concentrated salt solution, spent culture fluid in an amount of 5.9 l / h.

The process was stable, the quality of the obtained biomass corresponded to the composition of the biomass obtained by cultivation without returning the spent culture fluid.

The results are shown in table 1.

The use of the association of bacterial cultures in conjunction with the strain Methylococcus capsulatus GBS-15 allows the fermentation process to return the spent culture fluid from 10 to 95% of the total amount of prepared water used. This makes it possible to solve the problem of cleaning and complete disposal of waste culture fluid. Correction of the mineral components supplied to the fermenter as part of the spent culture fluid can increase the productivity of the process of obtaining a microbial protein based on natural gas (methane mixed with its low molecular weight homologs) from 10 to 30% at atmospheric pressure. The quality of the resulting product meets all the requirements for a microbial protein based on natural gas.

Claims (1)

A method of producing a microbial protein based on methane-oxidizing bacteria Methylococcus capsulatus GBS-15, comprising preparing a nutrient medium consisting of potassium, magnesium, iron (II), copper, manganese, zinc, cobalt and sodium molybdate of a given concentration with the addition of phosphoric acid, fermentation of bacterial cultures with a constant supply of culture fluid, a solution of ammonia and a gas mixture at a temperature of 40-45 ° C in a continuous flow of 0.2-0.3 volume of the fermenter per hour, separation, obtaining the finished biomass and its drying, characterized in that After separation, the recovered culture liquid is returned through the storage tank to the fermentation stage in a volume of 10 to 95% of the total amount of water used, enriched with the missing mineral salts to the specified concentrations in the culture liquid, and then the bacterial culture is fermented to obtain the finished product.

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