WO2024158272A1

WO2024158272A1 - Method for producing a protein feed concentrate from microbial protein

Info

Publication number: WO2024158272A1
Application number: PCT/KZ2023/000002
Authority: WO
Inventors: Никита Константинович МИРОНОВ; Есенбай Исраилович ИСЛАМОВ; Кайрат Куандыкович САДВАКАСОВ; Ерлан Алимжанович ДЖУМАБАЕВ
Original assignee: Нао "Казахский Национальный Аграрный Исследовательский Университет"
Priority date: 2023-01-23
Filing date: 2023-01-23
Publication date: 2024-08-02

Abstract

The invention relates to the field of agrotechnology and can be used for producing a protein feed concentrate, while also protecting the environment. The technical result is that of improving the composition and nutritional value of a feed concentrate, as well as reducing harmful emissions into the environment by applying automated processes for degassing a gas phase in a fermenter and using closed cyclic systems for circulating said gas phase during cultivation processes. This result is achieved in that in the present method for producing a protein feed concentrate consisting of microbial protein, which includes cultivating bacteria in a bioreactor supplied with a gas medium, cultivation of the methanotrophic bacteria Methylococcus capsulatus VKM В-2990 is carried out with the cyclic use of a gas mixture, wherein the gas mixture is continuously fed from the fermenter into a degassing chamber.

Description

METHOD FOR OBTAINING PROTEIN FEED CONCENTRATE FROM MICROBIAL PROTEIN

The invention relates to the field of agricultural technology and can be used to obtain protein feed concentrate and protect the environment.

There is a known method for fermentation of biomass with gas supply using a vertical flow zone, which includes repeated transfer of gas of a multiphase mixture into a liquid culture medium of a multiphase mixture in a loop section, separation of a multiphase mixture of gas and liquid culture medium into a gaseous phase and a liquid phase in a gas-liquid separator, removal of the liquid phase and delivery of the removed liquid phase to the inlet of the loop section of the loop reactor. The system contains a loop reactor with a gas-liquid separator having inlet and outlet openings for separating a multiphase mixture of gas and liquid culture medium, as well as a loop section /WO 2018/132379 A1, publ. 07/19/2018/.

The disadvantage of this analogue is the presence of losses in the form of methane emissions into the environment, insufficient opportunities for the use of closed cyclic systems during cultivation processes.

A method is known for producing a first reaction product through a first fermentation process conducted in a first loop reactor, the method comprising the steps of: (i) adding an inoculum containing one or more methanogenic microorganisms to a first loop reactor providing a first inoculated fermentation medium; (j) adding hydrogen gas (Hg) to the first inoculated fermentation medium; (iii) adding a first carbon source to the first inoculated fermentation medium; (iv) ensuring the possibility of fermentation of the first fermentation environment to obtain the first reaction product; and (y) isolating the first reaction product obtained in step (iv) /WO 2022/008478 A2, publ. 01/13/2022/.

The disadvantage of this analogue is the presence of losses in the form of methane emissions into the environment, insufficient opportunities for the use of closed cyclic systems in cultivation processes.

The closest in essence to the present invention is a method for obtaining microbial biomass of methane-oxidizing microorganisms, which consists in supplying water in an amount of 70 to 80% of the nominal volume into a clean, sterilized reactor, after which a solution of orthophosphoric acid is supplied until the required phosphorus concentration is achieved, then the concentration nitrogen is brought to the required value with ammonia water, after which the liquid flow stimulator is started to circulate the liquid phase, after which, by controlling the temperature of the coolant, a stable temperature of the liquid phase is achieved in the range of 42-44 ° C, then, according to the readings of temperature sensors, the temperature of the liquid phase is controlled , thermostatting of the liquid phase is carried out continuously in automatic mode in a heat exchanger, after which the reactor is purged with an inert gas to reduce the oxygen concentration to no more than 5% of the volume, while the oxygen concentration is controlled in the gas mixture leaving the reactor, at the same time, using a liquid flow stimulator, the liquid phase is taken from degasser and feeds it through a heat exchanger into the overflow chamber of the aerator, while using the ejection effect, the gas phase is supplied to the aerator from the upper part of the working area, then the liquid phase is directed through the aerator into the liquid layer in the working area, thereby saturating it with gases, simultaneously with This monitors the concentration of dissolved oxygen in the liquid phase with dissolved oxygen sensors immersed in the working medium. oxygen, at the same time, aqueous solutions of mineral nutrition are supplied inside the working chamber until the required concentrations are achieved, while by introducing titrating solutions, the required pH = 5.7 is achieved, then, if there is an excess of nitrogen, titration is carried out with a solution of sodium hydroxide, while ensuring composition of the gas mixture of the gas phase, adding methane and air in the required proportions at the required pressure, provided that the required environmental parameters are achieved, seeding biomass is supplied inside the housing to create the required concentration, after which the level of the liquid phase is brought to 100% of the working volume, while providing oxygen microbial culture synthesized in the reactor, which is determined by the residual working concentration of dissolved oxygen, which is regulated by increasing the partial pressure in the cultivation system, increasing the operating pressure in the reactor or increasing the air flow, while the supply of the nutrient medium is carried out continuously according to the need of microorganisms in mineral nutrition sources from the calculation of residual concentrations, which are determined by the amount of nitrogen and phosphorus, their concentration in solutions is adjusted if necessary, the operating pressure is changed by gas flow regulators on the inlet pipelines into the reactor, and the composition of the gas mixture is controlled under conditions of pressure changes, the gas mixture is monitored and regulated continuously, simultaneously adjusting the flow rate of water and nutrient solutions, while a certain amount of bacterial suspension is continuously taken from the reactor with a continuous supply of all components of the nutrient medium, natural gas and air; when taking the microbial suspension from the reactor, it is allowed to settle for degassing of dissolved gases /RU 2766708 C1, publ. 03/15/2022/. The disadvantage of this analogue is the presence of losses in the form of methane emissions into the environment, insufficient opportunities for the use of closed cyclic systems during cultivation processes.

The objective of the invention is to develop a method for producing protein feed concentrate from microbial protein using a degassing chamber and cyclic use of a gas mixture with improved technical characteristics.

The technical result is an improvement in the composition and value of the feed concentrate, as well as a reduction in harmful emissions into the environment, thanks to automated gas phase degassing processes in the fermenter and the use of closed cyclic gas phase circulation systems during cultivation processes.

This is achieved by the fact that the method of producing a protein feed concentrate consisting of microbial protein, including the cultivation of microorganisms in a bioreactor with the supply of a gas mixture, according to the invention, the cultivation of methanotrophic microorganisms Methylococcus capsulatus VKM No. B 2990 is carried out with the cyclic use of a gas mixture, in which a gas mixture is continuously supplied mixture from the fermenter to the degassing chamber, in which: i) the stock solution of methanotrophic microorganisms Methylococcus capsulatus VKM No. B 2990 is first cultivated in the fermenter with the supply of a gas mixture of oxygen and methane at a ratio of 1: 1 for 165-170 hours, then further cultivation of methanotrophic microorganisms is carried out microorganisms Methylococcus capsulatus VKM No. B 2990 on a nutrient medium in a fermenter for 46-50 hours at a temperature of 40-45°C, pressure 1.8-2.2 atm., pH 5.5-6.0 in a gas mixture of oxygen and methane at a ratio of 1:5 to 1:6, ii) a gas mixture from metabolic products is supplied to the degassing chamber connected to the above-mentioned fermenter of the above-mentioned methanotrophic microorganisms at a circulation rate of the gas mixture of 3500-3700 l/hour, then the concentration of the gas mixture in the degassing chamber is analyzed, and if the concentration of the gas mixture decreases, the gas mixture is supplied from the fermenter through the automated valves of the degassing chamber.

As a nutrient medium, a nutrient medium of the following composition is used: KCl-0.125 g/l, MgSO4*7HiO - 0.125 g/l, FeSO4*7H2O - 10.75 mg/l, CUSC>4*5H2O - 10 mg/l, Mn8O4* 5H2O - 9.5 mg/l, H3BO3 - 6.25 mg/l, ZnSC>4*7H2O - 1.5 mg/l, Na2MoO4*2H2O - 0.25 mg/l, CoC12*6H2O - 0.25 mg /l, NzPO ₄ (70%) - 0.35 ml/l.

The proposed method involves the cyclic use of a gas mixture for the growth of methanotrophic microorganisms, in which the gas phase, when cultivating methanotrophic organisms in a bioreactor, is constantly removed from it into a degassing chamber due to a pressure difference of 0.1 kPa, where the concentration of methane and oxygen is analyzed, from the gas degassing chamber phase is pumped back into the bioreactor, while methane and oxygen are automatically dosed, due to this the content of methane and oxygen consumed during the growth of bacteria increases to the concentrations necessary for their optimal growth, the discharge of unspent methane is carried out only once at the end of the cultivation process, thus reducing thus the consumption of methane for protein production and environmental pollution.

Figure 1 shows a diagram showing the dynamics of the growth of raw biomass of Methylococcus Capsulatus in the culture liquid.

The invention works as follows.

A laboratory model experiment was carried out to obtain a protein feed concentrate in a fermenter by cultivating methanotrophic microorganisms, in which, unlike other approaches, the cyclic use of a gas mixture was used, consisting of 15% oxygen and 85% methane in order to save methane and eliminate its emissions into the atmosphere on the basis of the laboratory of the Kazakh National Agrarian Research University.

In this experiment, the microorganism Methylococcus Capsulatus VKM No. B 2990 was cultivated.

At the first stage, the mother culture of Methylococcus Capsulatus VKM No. B 2990 was grown, for which the culture obtained from the bank was sown on liquid medium “P” (KCl-0.125 g/l; MgSO ₄ *7H ₂ O - 0.125 g/l ; FeSO ₄ *7H ₂ O - 10.75 mg/l; CuSO ₄ *5H ₂ O - 10 mg/l; MnSO ₄ *5H ₂ O - 9.5 mg/l; ZnSO ₄ *7H ₂ O - 1.5 mg/l; Na ₂ _MoO ₄ *2H ₂ O - 0.25 mg/l; CoC1 ₂ *6H ₂ O - 0.25 mg/l; - 0.35 ml/l) in a volume of 5% of the working volume of liquid in the fermenter. Cultivation of the mother culture was carried out in a thermoshaker type fermenter for about 7 days (168 hours). The gas mixture in the flask was brought to a ratio of 50% oxygen and 50% methane (1:1). After 3 days (72 hours), the gas mixture was purged again. Since the working volume of the fermenter used is 25 liters, it required 1.25 liters of mother liquor. In the process of preparing the mother culture, a fermenter with a total volume of 45 liters was prepared. Sterilization of the fermenter was carried out in 2 stages: washing with sterilizing solutions (6% hydrogen peroxide solution) for 45 minutes, stirrer speed 150 rpm, and steam sterilization of the culture liquid and the bioreactor itself with a PGE 50 steam generator at a temperature of 121°C for 50 minutes.

After all the preparatory measures, the culture was seeded into the fermenter. Further cultivation in the fermenter was carried out: at a pressure in the fermenter of 2 atm., a stirrer speed of 3500 rpm, a temperature of 42°C and a pH of 5.8 for 48 hours on a nutrient gas mixture with a total volume of 40 l, consisting of 15% oxygen and 85% methane (ratio 1:5.6). Composition of the gas phase in the fermenter supported by methane and oxygen sensors. A gas mixture from the metabolic products of the above-mentioned methanotrophic microorganisms is supplied to the degassing chamber connected to the above-mentioned fermenter at a circulation rate of the gas mixture of 3600 l/hour. Upon reaching a certain level of gas phase concentration in the degassing chamber, the gas phase is pumped back into the fermenter at a speed of 3600 l/hour. Due to this, the content of methane and oxygen consumed during the growth of Methylococcus Capsulatus bacteria is compensated to concentrations of 15% oxygen and 8% methane required for their optimal growth. Methane consumption is 1 normal liter (0.715 g) per 0.59 g of raw biomass, oxygen consumption is 1.2 normal liter (1.7 g).

The gas phase of the 15 liter fermenter is discharged only once at the end of the cultivation process after 48 hours.

As a result, the average growth rate of Methylococcus Capsulatus was 0.12 g/l per hour. That is, the average methane consumption per liter of culture liquid is 0.2 l/hour, the peak is 0.6 l/hour, there are virtually no methane emissions.

Table 1

Under completely similar cultivation conditions without using a closed cycle, the average value of methane utilization 5 microorganisms is 0.2 l/hour, peak 0.6 l/hour, and methane emissions

2855 l/hour.

Claims

CLAIM

1. A method for producing protein feed concentrate from microbial protein, including the cultivation of microorganisms in a bioreactor with the supply of a gas mixture, characterized in that the cultivation of methanotrophic microorganisms Methylococcus capsulatus VKM No. B 2990 is carried out with the cyclic use of a gas mixture, in which the gas mixture is continuously supplied from the fermenter to a degassing chamber in which: i) the mother solution of methanotrophic microorganisms Methylococcus capsulatus VKM No. B 2990 is first cultivated in the fermenter with the supply of a gas mixture of oxygen and methane at a ratio of 1: 1 for 165-170 hours, then further cultivation of methanotrophic microorganisms Methylococcus capsulatus VKM is carried out No. B 2990 on a nutrient medium in a fermenter for 46-50 hours at a temperature of 40-45°C, pressure 1.8-2.2 atm., pH 5.5-6.0 in a gas mixture of oxygen and methane at a ratio of 1:5 to 1:6, ii) a gas mixture from the metabolic products of the above-mentioned methanotrophic microorganisms is supplied to the degassing chamber connected to the above-mentioned fermenter at a circulation rate of the gas mixture of 3500-3700 l/hour, then the concentration of the gas mixture in the degassing chamber is analyzed, and in the event of a decrease in the concentration of the gas mixture, a gas mixture is supplied from the fermenter through automated valves of the degassing chamber.

2. The method according to claim 1, characterized in that the following composition is used as a nutrient medium: KCl-0.125 g/l, MgSO ₄ *7H ₂ O - 0.125 g/l, FeSO ₄ *7H ₂ O - 10, 75 mg/l, CuSO ₄ *5H ₂ O - 10 mg/l, MnSO ₄ *5H ₂ O - 9.5 mg/l, H3BOz - 6.25 mg/l, ZnSO ₄ *7H ₂ O - 1.5 mg/l, Na ₂ MoO ₄ *2H ₂ O - 0.25 mg/l, CoC1 ₂ *6H ₂ O - 0.25 mg/l, H ₃ PO ₄ (70%) - 0.35 ml/l.

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