RU2742472C1 - Nutrient medium for the cultivation of yeast biomass and a method for its production - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: group of inventions proposes a nutrient medium for the cultivation of yeast, containing methanol, dimethyl ether, aqueous ammonia, mineral salts and yeast extract, and a method for obtaining the proposed nutrient medium. The method includes dissolving in water a certain concentration of raw methanol and ammonia hydroxide obtained from the process gas by purifying associated petroleum gas, as well as mineral salts and yeast extract.EFFECT: inventions provide expansion of the range of nutrient media for the cultivation of methylotrophic yeast in production of protein fodder product.2 cl, 1 ex

Description

The invention relates to biotechnology and is intended for the use of associated petroleum gas as a source of production of a nutrient medium based on methanol for the production of a protein feed product - yeast biomass.

The constantly growing population of the world makes it increasingly important to provide the population with adequate nutrition, in particular, in terms of protein content. Feed used in agriculture must also have an optimal composition of nutrients, especially protein. The lack of protein only for the purposes of fodder production for agriculture in the Russian Federation is estimated at 2 million tons. One of the protein sources can be the biomass of microorganisms (for example, yeast) obtained using biotechnological processes carried out in an industrial environment.

A possible source of non-food raw materials for the production of protein in the form of biomass of microorganisms is natural gas containing methane.

The source of natural gas containing methane is natural gas produced from gas fields and transported to consumers either through gas transmission pipelines or in liquefied form. Another source of natural gas containing methane is associated gas from oil fields. While the first source of natural gas is widely used as a fuel and raw material for the chemical industry, the second source of natural gas obtained in oil production sites is usually not used, due to the lack of the necessary infrastructure for its transportation to consumers. Therefore, associated gas is burnt in oil production areas, being a source of environmental pollution by combustion products.

The claimed method is aimed at the use of associated gas from oil fields to obtain the main component of the nutrient medium used for the cultivation of microorganisms - yeast of the genus Hansenula polymorphs. At the same time, the technical task of creating the most simple for operation, using the minimum infrastructure of the technological process of obtaining from the associated methanol.

It is known that the production of fodder protein is carried out using controlled cultivation of microorganisms in a nutrient medium containing sources of carbon, nitrogen, phosphorus, potassium, magnesium, iron, calcium, growth substances (amino acids and vitamins). When protein is obtained on a nutrient medium from natural gas, methane (CH4) is used as a carbon source, ammonia (NH3), ammonium salts or nitrates are used as a nitrogen source, phosphoric acid or phosphates are used as a source of phosphorus, as a source of potassium potassium salts (for example, phosphates, sulfates, chlorides), magnesium sulfates are used as a source of magnesium, iron sulfates are used as a source of iron, and calcium chloride is used as a source of calcium.

In particular, a nutrient medium for the cultivation of microorganisms containing methane gas is known. This nutrient medium is used to obtain a protein product - haprin ("gas protein"), which includes B vitamins, amino acids and trace elements (see article Deaf S. Methane from coal seams and mines - a source of feed protein. "Compound feed", No. 5, 2015, pp. 84-87). To ensure the main technological stages of the process, auxiliary stages are provided: reception, preparation of solutions and supply of ammonia water, orthophosphoric acid, salts and other chemicals in the required concentrations to the fermenters; preparation and supply of air oxygen. The main continuous technological process of growing haprin takes place in fermenters. Mixing and aeration in them is carried out with the help of circulation pumps with ejectors on the pressure side of the pumps for air injection and organization of gas phase recirculation. Natural gas and, if necessary, additional air are introduced through the bubblers. The composition of the gas phase (oxygen, methane, carbon dioxide) at the outlet of the fermenter is monitored by gas analyzers.

Other methods are also known (RU 2064016, 20.07.1996; RU 2699986, 11.09.2019) for producing protein on a nutrient medium obtained from natural gas containing methane, in which methane is fed in a mixture with air into an aqueous solution of mineral salts containing nitrogen sources , phosphorus, potassium, magnesium, iron, calcium, and some other trace elements necessary for the growth of microorganisms. The supply of the specified gas mixture to an aqueous solution of salts serves to enrich this solution with a carbon source, which is methane, and atmospheric oxygen. Thus, a liquid nutrient medium is obtained containing all the necessary components for the growth of microorganisms.

These methods have a number of disadvantages.

First, methane gas has very low solubility in aqueous media. Because of this, the gas mixture of methane with air is repeatedly circulated through the liquid nutrient medium in order to ensure a more complete utilization of methane by microorganisms. Because of this, the design of fermenters (biorectors), in which microorganisms are cultivated, is greatly complicated. Moreover, the control of the process of cultivation of microorganisms becomes much more complicated, because during the growth of the latter, gaseous waste products (metabolism) of microorganisms are formed, which gradually accumulate in a circulating mixture of methane with air and, at a certain concentration, can inhibit the growth of a microbial culture. For this reason, the circulating methane-air mixture must be periodically renewed. For the timely renewal of the gas-air mixture, it is necessary to constantly monitor its composition. This leads to the complication of both the equipment and the control system for the cultivation of microorganisms. Moreover, periodic renewal of the gas-air mixture leads to the release of a significant part of the methane undissolved in the nutrient medium into the atmosphere. The consequence of this is the low efficiency of using natural gas as the basis of a nutrient medium as a source of carbon for the growth of microorganisms, while the environment is polluted with unused methane.

Secondly, a significant disadvantage of the known methods for producing a nutrient medium from natural gas is that methane gas forms an explosive mixture with air. To avoid an explosion, fermenters (bioreactors) have to be manufactured in an explosion-proof design and equipped with gas analyzers for the methane content in the air and with special automation equipment to avoid the formation of an explosive concentration of methane in the air. Moreover, the composition of the gas mixture must be continuously monitored and controlled not only inside the equipment, but also in the production area where microbial protein is produced from natural gas. This leads to a strong increase in the complexity and cost of equipment, as well as a process control system.

A known method of cultivating methanotrophic bacteria Pseudomonas methylotropha and Psendomonas rosea on a nutrient medium containing methanol (GB 13708992 A, US 3989594 A). The disadvantage of this method is the use of bacteria as microorganisms-producers of protein, which grow on a nutrient medium with a pH of 6.8 and a temperature close to 37 ° C. The indicated pH and temperature values are favorable for the growth of foreign microorganisms. For this reason, the production of protein by the mentioned method is carried out under sterile conditions and in a nutrient medium prepared in demineralized water. These conditions greatly complicate and increase the cost of the process of protein production from methanol, require the presence of highly qualified production personnel who can carry out industrial production of protein in sterile conditions. This disadvantage of methanol-acid bacteria makes it especially difficult to use the known method for the production of protein from associated gas in remote oil production areas with undeveloped infrastructure and in the absence of highly qualified biotechnologists.

The closest analogue of the claimed invention is a nutrient medium containing methanol and used for the cultivation of methanotrophic bacteria Methylocystis or Methylosinus, and the method for its preparation (SU 770174, 07.09.1981). The culture medium contains sources of nitrogen, phosphorus, mineral salts and methanol as a source of carbon and energy, while carbonic acid is additionally introduced into the culture medium in the form of sodium bicarbonate at a concentration of 0.1-0.25% or in the form of carbon dioxide to a concentration of gas phase 3-10%.

This nutrient medium and the method for its preparation are intended for use in the mining industry to reduce the methane content of coal seams, i.e. has a limited field of application, therefore, the present invention is not intended for industrial production of a protein product.

The technical problem solved by the claimed group of inventions is the creation of a method for producing a nutrient medium obtained from associated gas of oil fields (hereinafter referred to as associated gas for short) and a nutrient medium obtained by this method. The inventive method can also be applied to obtain a nutrient medium from pipeline natural gas.

To solve this technical problem, the claimed method is characterized by the following operations.

A preliminary analysis of the associated gas for a specific field is carried out in order to determine the chemical composition of the gas, using known methods. Taking into account the results of the analysis of the associated gas composition, the associated gas is purified from water, inorganic sulfur compounds (including hydrogen sulfide and mercaptan), iron pentacarbonyl and heavy hydrocarbons, with the receipt of process gas. The purification of associated gas from the above contaminants is carried out by known methods in a complex gas treatment unit (GPP) (Russian Gas Encyclopedia. M., 2004. S. 462-464. - ISBN 5-85270-327-3).

The process gas obtained after cleaning the associated gas in the gas processing plant is used to obtain liquid components of the nutrient medium, which are sources of carbon and nitrogen, and are readily soluble in water, unlike the methane gas contained in the associated gas.

The carbon source component of the culture medium is obtained in the form of raw methanol.

The crude methanol used in the claimed method and which is the basis for the nutrient medium is a colorless or slightly yellowish transparent liquid containing methanol, dimethyl ether and water. The raw methanol used in the inventive method may contain isobutyl alcohol and olefins as impurities.

The component of the nutrient medium, which is a source of nitrogen, is obtained in the form of ammonia, followed by dissolution of ammonia in water. Raw methanol and ammonia are produced from associated gas together. In this case, one of the known methods is used, for example, the method described in RF patent No. 2174953. At the first stage of the technological process, crude methanol is obtained in the form of a mixture of methanol (CH3OH), dimethyl ether (CH3OCH3) and water. In this case, raw methanol, as a component of the nutrient medium, which is a source of carbon, is obtained with a methanol content from 84.0% to 96.0%, dimethyl ether from 0.1% to 3.6%, water from 3.0% to 15.0%.

At the second stage of the technological process, ammonia is obtained as a component of the nutrient medium, which is a source of nitrogen. For the production of a protein product (yeast biomass), ammonia is dissolved in water to obtain an aqueous solution of ammonia - ammonium hydroxide (NH4OH) with a content of 5% to 22%.

A nutrient medium for the cultivation of microorganisms is prepared by dissolving in water at a certain concentration a carbon-containing component (raw methanol), a nitrogen-containing component (ammonia hydroxide), mineral salts that are sources of potassium, phosphorus, magnesium, iron and calcium, as well as a component that is a source growth properties of the nutrient medium, for example, yeast extract.

The culture medium is prepared with the addition of the following mineral salts. KH2PO4 and / or KCl are used as a source of potassium, KH2PO4 and / or Na2HPO4 as a source of phosphorus, MgSO4 * 7H2O as a source of magnesium, FeSO4 * 7H2O as an iron source, and CaCl2 and / or CaHCO3 as a source of calcium , as a source of growth properties of the nutrient medium - yeast extract.

The culture medium is prepared with the following composition containing 1 liter of medium:

- methanol from 3.00 g to 30.00 g,

- dimethyl ether from 0.01 g to 0.90 g,

- an aqueous solution of ammonia (NH4OH) from 0.50 g to 5.00 g,

-K2HPO4 1.50 g to 3.50 g,

- MgSO4 * 7H2O from 0.5 to 2.50 g,

- FeSO4 * 7H2O from 0.01 g to 0.02 g,

- CaCl2 from 0.05 to 0.20 g,

- yeast extract from 5.00 g to 30.00 g

For cultivation on the above nutrient medium, the methylotrophic yeast Hansenula polymorpha is mainly used.

For cultivation on the claimed nutrient medium, any yeast strain belonging to the genus Hansenula polymorpha can be used. The fact that yeast belonging to the genus Hansenula polymorpha is successfully cultivated both on pure methanol and on a nutrient medium containing methanol has long been widely known in the art. This is indicated by the following publications:

Подгорский B.C. Физиология и метаболизм метанолусваивающих дрожжей. Киев: Наукова думка, 1982, с. 48-51;

Podgorskiy BC Physiology and metabolism of methanol-assimilating yeast. Kiev: Naukova Dumka, 1982, p. 48-51;

Троцеико Ю.А., Торгонская М.Л. Метилотрофные дрожжи. Москва: «ТР-Принт», 2011, с. 9-10.

Troceiko Yu.A., Torgonskaya M.L. Methylotrophic yeast. Moscow: "TR-Print", 2011, p. 9-10.

A feature of this kind of yeast is that it actively grows on a nutrient medium with methanol. As a result, there is no need to purify (rectify) the raw methanol to use it as a carbon source in the preparation of a nutrient medium for the growth of microorganisms. Therefore, it is possible to exclude the rectification of the raw methanol, as is done in the prior art methods, and in which pure methanol is used as a carbon source for the cultivation of other methanol-oxidizing microorganisms that produce protein.

The technical result obtained in the implementation of the claimed method is the possibility of using associated gas as a source of raw materials for the production of a nutrient medium on which the cultivation of methylotrophic yeast is carried out as a source of fodder protein. At the same time, the main components of the nutrient medium used for the cultivation of methylotrophic yeast in order to obtain protein are simultaneously produced from the associated gas, namely, the associated gas is also a source of carbon and a source of nitrogen. The presence of dimethyl ether in the composition of the nutrient medium, as well as the introduction into its composition of an aqueous solution of ammonia and yeast extract as a source of growth properties, increases the growth rate of yeast and the volume of the final product - yeast biomass. For the culture medium, the technical result is also a simplification of technology and a reduction in the time for its production due to the use of raw methanol (excluding the rectification operation) and drinking water (no special water treatment is required). At the same time, obtaining a nutrient medium is entirely based on the processing of associated petroleum gas (or natural gas).

An essential feature of the claimed method and the resulting nutrient medium is:

- simplification of the technology for obtaining a nutrient medium by eliminating the stage of purification of raw methanol from impurities (rectification stage),

- preparation of the culture medium not with drinking water instead of distilled or deionized water.

The carbon source and nitrogen source obtained from the associated gas are completely water-soluble carbon sources (methanol and dimethyl ether) and a nitrogen source (ammonium hydroxide). They are included as necessary components in the nutrient medium, and their presence allows you to reduce the amount of additives in the form of minerals. Associated petroleum gas and conventional natural gas differ in composition, although they are based on methane. Associated gas contains less methane than natural gas, but more impurities, which is an advantage for the claimed method. Nevertheless, it is necessary to purify associated gas from some of the impurities, namely, from inorganic sulfur compounds (including hydrogen sulfide and mercaptan), as well as from iron pentacarbonyl, if any.

The advantages of the claimed method also include the simultaneous production of ammonia with the synthesis of methanol, an aqueous solution of which is used in the nutrient medium as a nitrogen-containing component, which, in addition to simplifying the method, makes it possible to reduce the cost of purchasing, transporting and storing salts, which are a source of nitrogen. Thus, the main components necessary for obtaining a nutrient medium and cultivating microorganisms are produced within a single technological process at the oil production site.

In addition, the proposed use of associated gas makes it possible to reduce the volume of its combustion, i.e. reduce the harmful effects of this incineration process on the environment.

The Hansenula polymorpha yeast cultivated on the proposed nutrient medium utilizes raw methanol obtained from associated gas with a high yield of yeast biomass containing microbial protein.

With regard to the nutrient medium, it should also be noted that the liquid components of the nutrient medium used do not form explosive mixtures with air, for this reason, to implement the claimed method, equipment in a special explosion-proof design with a complex system of continuous monitoring and control of the composition of the methane-air gas mixture is not required, on the contrary , for the implementation of the proposed method, simple equipment is used, traditionally used in the production of products using microorganisms (fermenters for conducting the process in non-sterile conditions and other technological equipment

An example of the implementation of this method:

For the preparation of the nutrient medium, crude methanol was used, obtained from the associated gas of an oil field, having the following composition:

From the associated gas of the above composition, crude methanol was obtained, containing:

Ammonia was obtained from the same associated gas, which was dissolved in water to obtain ammonium hydroxide (NH4OH) to a concentration of 22 wt%.

The nutrient medium was prepared using drinking-quality tap water: using raw methanol, ammonium hydroxide and mineral salts of the following composition per 1 liter:

The process of obtaining a microbial fodder protein product according to the claimed method was carried out in a standard fermenter with a heat exchange jacket and a stirrer made of stainless steel, and having a working volume of 30 liters (the volume occupied by the nutrient medium).

The inoculum of microorganisms was introduced into the specified nutrient medium in the form of a seed culture of the Hansenula polymorpha yeast culture strain obtained on the above nutrient medium in shaking flasks on a thermostatted shaker at a temperature of 42 +/- 1 ° C. The volume of inoculated material was 10% of the working volume of the fermenter. The cultivation process was carried out in a continuous mode with a dilution rate of 0.12 +/- 0.02 h-1. The cultivation process was carried out at a temperature of 42 +/- 1 ° С, a pH value of 4.0 +/- 0.1, a dissolved oxygen concentration of 25 +/- 5%, a stirrer speed of 500-800 rpm, a volumetric air flow rate of aeration of the culture 40-50 +/- 2 l / min, the pressure in the fermenter vessel is 1.2-2.7 +/- 0.2 kg / cm2. The duration of the continuous cultivation process was 458 hours. The average specific productivity of the process in terms of yeast biomass in terms of absolutely dry weight was 14.8 g / l / h.

The obtained yeast biomass was concentrated using centrifugal separation to a biomass concentration of 15 +/- 2% by absolute dry weight. The concentrated biomass was granulated in a granulator with the addition of potato starch with a starch to wet biomass ratio in the range of 20-30% (weight).

The granulated yeast biomass was dried in a vacuum drying oven to a residual moisture content of 8-10%. The content of microbial protein in the dry granular product ranged from 28 to 36%.

Claims (10)

1. Nutrient medium for yeast cultivation, containing methanol and mineral salts, which are sources of potassium, phosphorus, magnesium, iron and calcium, characterized in that it additionally contains an aqueous solution of ammonia, dimethyl ether and yeast extract, while methanol, dimethyl ether and ammonia for an aqueous solution is obtained from associated petroleum gas, with the following content of components in 1 liter of medium: - methanol 23.10 g; - dimethyl ether 0.07 g; - an aqueous solution of ammonia (NH4OH) 3.50 g; - K2HPO4 2.00 g; - MgSO4 * 7H2O 0.50 g; - FeSO4 * 7H2O 0.01 g; - CaCl2 0.10 g; - yeast extract 10.00 g. 2. A method of obtaining a nutrient medium according to claim 1 for cultivating yeast, characterized in that the nutrient medium is obtained from associated petroleum gas, while the associated petroleum gas is purified from inorganic sulfur compounds and gasoline to obtain a process gas, from which methanol is then obtained; crude in the form of a mixture of methanol, dimethyl ether and water with a methanol content from 84.0 to 96.0%, dimethyl ether from 0.1% to 3.6%, water from 3.0% to 15%, and a nitrogen-containing component in the form an aqueous solution of ammonia - ammonium hydroxide with an ammonia content of 5% to 30%, then prepare a nutrient medium for the cultivation of yeast biomass, dissolving in water at a certain concentration the carbon-containing component raw methanol, nitrogen-containing component ammonia hydroxide, mineral salts, which are sources of potassium, phosphorus , magnesium, iron and calcium, as well as yeast extract, which is the source of the growth properties of the nutrient medium.