Method for obtaining protein from methanotrophic bacteria and their associates

Abstract

FIELD: microbiology.SUBSTANCE: method for obtaining protein from methanotrophic bacteria and their associates is proposed. The method consists in the fact that, during the operating mode of biomass cultivation, after reaching a concentration of 1.0–1.5 g / l, methane and compressed air are fed into the main fermentation apparatus at a pressure at the point of entry of methane and compressed air of 4.00–4.20 atm. g and providing a gradient along the length of the apparatus of 0.22–0.26 atm. / m and a residual pressure at the outlet of the gas phase not exceeding 0.1 atm. g. The process is continued in the periodic cultivation mode until the biomass concentration reaches 6–7 g / l, while at the initial stages of cultivation the flow rate is set at 0.05–0.10 h-1 and the composition of the gas phase methane: compressed air is increased from 1:1 to 1:7 as the biomass concentration increases, and the full flow rate is set at 0.35-0.42 h-1 upon reaching a cell concentration of at least 10–11 g / l. The culture liquid separated from the gas phase is subjected to vacuuming at a residual pressure of at least 0.1 atm. to prevent boiling of the water contained in the culture liquid.EFFECT: increasing the biomass growth rate to 0.42 h-1, increasing productivity to 5.4 g / l·h and ensuring process safety.2 cl, 2 ex

Description

[0001] The invention relates to the field of microbiology, specifically to a method for producing biomass of methane-oxidizing microorganisms. The invention can find application in the industrial production of a bioproduct in the form of aerobic microbial biomass using a gaseous substrate, such as natural methane-containing gas, as the organic raw material, and cultures of Methylococcus capsulatus, Methylococcus, Methylosinus, Methymoncy, and others, as well as symbiotic cultures, as the methane-oxidizing microorganisms.

[0002] A key aspect of culturing process intensification, in addition to maintaining optimal parameters, is overcoming limiting factors, including gas solubility, high concentrations of byproducts of protein biosynthesis, and neutralization products of nutrient medium components, as well as avoiding stressful conditions for the culture / producer association at risk points in the technological process. Patent literature contains information on various technological processes where removing the limit on a particular technological parameter plays a key role.

[0003] Methanotrophic protein production methods are known (patent RU2064016C1). The bacterial culture Methylococcus capsulatus strain VSB-874 was grown continuously in a two-story fermenter with an ejector mixing and gas distribution system and a growth medium volume of 350 cubic meters of monolithic liquid. The process temperature of 42-43°C was maintained by circulating recycled water through the fermenter heat exchanger. The pH of the growth medium, 5.6-5.8, was stabilized by feeding it an aqueous ammonia solution.

[0004] The carbon feed source was natural gas with a methane content of 95%, the oxygen source was process oxygen with an oxygen content of 95% (removing the oxygen limit).

[0005] The values ​​of the natural gas, oxygen, and mineral nutrient solution flows were set proportionally to the flow rate of ammonia water with the proportionality coefficient adjusted based on the readings of gas analyzers for oxygen and methane in the outgoing gas phase flow and measurements of the phosphorus concentration in the growing medium.

[0006] The cultivation process was carried out under pressure, with continuous withdrawal of the biomass-containing suspension from the fermenter to centrifugal separators. The concentrated biomass was heat-treated and dried in spray dryers, fueled by the off-gas from the cultivation process. The spent cultivation medium after the separators was divided into two streams, one of which was returned to the cultivation process, and the other was sent to the biological water treatment system.

[0007] In the control process, the flow rate (70 m3 / h), the flow rate of returned spent growth medium (21 m3 / h), and the pressure in the fermenter (0.3 MPa) were maintained at a constant level for 5 days. Power supplies were supplied automatically.

[0008] During the control period, the biomass concentration in the suspension stream withdrawn from the fermenter varied from 11 to 16 g / L (average 15 g / L), which resulted in a change in the biomass concentration in the spent growth medium stream after separation from 0.15 to 0.4 g / L (average 0.25 g / L). The average daily amount of biomass entering the drying process in the control process was 25.0 tons, and losses amounted to 0.25 tons (10%). A disadvantage is the need for pure oxygen.

[0009] Also known is a method for obtaining biomass of methane-oxidizing bacteria, which involves cultivating them in a fermenter under aeration conditions on a nutrient medium containing methane as a carbon source, sources of nitrogen, phosphorus, mineral salts and microelements, separating the biomass from the culture liquid, partially returning the spent culture liquid to the growing stage, dehydrating and drying the biomass. A distinctive feature of the known method is that the biomass is cultivated in a mode of alternating aeration to activate molecular oxygen using the reduced coenzyme NADH (nicotinamide adenine dinucleotide) with the biomass being maintained without oxygen supply and with oxygen supply in a ratio of 1:4 minutes to 1:6 minutes, while methane is continuously supplied to the most turbulent aerated zone of the fermenter (see Russian patent No. 2 699 293, published 09 / 04 / 2019).

[0010] A disadvantage of this method is the complication of the process for producing methane-oxidizing bacteria biomass, due to the use of reduced coenzyme and the need for frequent, real-time oxygen supply to the biomass. Considering that even at a biomass concentration of 7–8 g / L, oxygen in the medium is depleted within 20–30 seconds, methane oxidation without oxygen becomes impossible. Therefore, the process will periodically proceed under anaerobic conditions, with increased foaming and the release of metabolic products into the culture fluid. This will prevent high biomass production productivity. Furthermore, the increased concentration of metabolic products in the spent culture fluid (SCF) will require a reduction in the amount of SCF returned.

[0011] A method is known for obtaining biomass of methane-oxidizing microorganisms, which includes their continuous cultivation in a water-mineral nutrient medium with the supply of methane-containing gas and gas containing free oxygen, using the spent medium after concentrating the biomass and drying it (see Russian Federation Patent No. 2064016, published July 20, 1996).

[0012] The peculiarity of the known method is that the process of growing microorganisms is carried out at a pressure in the nutrient medium higher than atmospheric, with partial use of the spent medium after concentration of the biomass, while the rate of supply of nutrient components for microorganisms in the process and the selection of a suspension containing biomass from the process of continuous cultivation of microorganisms is set proportionally to the supplied flow to stabilize the pH of the nutrient medium.The ratio between the flow of spent medium returned to the cultivation process after concentration and the flow of suspension withdrawn from the process is set to ensure the maximum flow rate to stabilize the pH of the nutrient medium. The pressure in the nutrient medium is set to a minimum level that ensures an optimal concentration of dissolved oxygen in the nutrient medium, but not lower than the pressure required to transport and utilize the energy of the gas flows exiting the process. A device implementing the known method includes means for preparing the nutrient medium, a bioreactor for cultivating microorganisms using methane- and oxygen-containing gases, and means for concentrating and drying the biomass.

[0013] One of the key features of this known technical solution is the partial reuse of the spent medium during microorganism cultivation after biomass concentration. Patent RU (11) 2 699 986(13) C1 provides for the movement of the culture biomass through a recirculation loop from the portion of the fermenter with minimal oxygen content, which serves as the deaeration zone, to the portion of the fermenter with maximum dissolved oxygen content, which serves as the aeration and liquid phase feed zone. The resulting product (the biomass of methane-oxidizing bacteria Methylococcus capsulatus VSB 874) contained 70% protein and 6.7% ash.

[0014] The closest in technical essence and achieved result is patent RU 2607782 from 10.01.2017. Bioreactor for growing meta-nutritive microorganisms. The description provides a method for continuous fermentation of the meta-nutritive bacterial culture Methylococcus capsulatus, indicating temperature conditions and other conditions for implementing the method. In a bioreactor with a volume of 150 m 3 The process of continuous fermentation of the meta-nutritive bacterial culture Methylococcus capsulatus is carried out, maintaining a temperature of 36-52°C, preferably 40-46°C, and a pH of the medium of 4.5-7.5, preferably 5.5-6.0.

[0015] The process is carried out using natural gas containing 95% methane, supplied to the appropriate sectors of the bioreactor under a pressure of 0.3 MPa, and technical oxygen (96%), also supplied under pressure.

[0016] The mineral medium used is sulfate salts of magnesium, copper, iron, potassium, zinc, magnesium, and cobalt in the prescribed ratio, as well as boric and phosphoric acids and molybdenum salt. A 25% ammonia water solution is used to stabilize the pH of the medium. The internal reaction volume of the bioreactor is 110 m3 of liquid. 3 and the reaction volume of each sector of the bioreactor is 2.5 m 3 The rotation speed of the stirrers in each sector is 1200 rpm. The average flow rate of the medium through the bioreactor is D - 30 m 3 / hour (flow rate 0.27 h -1 ), while the average concentration of bacterial cells in the collected biosuspension is 17 g / l, calculated as dry biomass. The specific productivity of the process in the bioreactor is 4.4 kg biomass / m3. 3hour. The specific energy consumption for fermentation in the bioreactor averages 1.1 kWh / kg of biomass. The bioreactor's biomass capacity is approximately 12 tons of biomass per day, with a crude protein content of 74%.

[0017] The disadvantages of the prototype are:

[0018] 1. Insufficient specific productivity of the process in the bioreactor.

[0019] 2. The device is constantly under high pressure, preventing the removal of carbon dioxide.

[0020] 3. Increased fire hazard of the process due to the use of 96% oxygen.

[0021] 4. It is impossible to use oil and fatty acid based defoamers due to the presence of 96% oxygen.

[0022] The technical problem that the invention is aimed at solving is the development of a method for obtaining protein from methanotrophic bacteria and their associates under conditions of maintaining optimal parameters during protein biosynthesis, as well as avoiding stressful conditions for the producer culture / association at risk points of the technological process.

[0023] The technical result is an increase in the biomass growth rate to 0.42 h -1 , increasing the process productivity to 5.4 g*l / h, ensuring the safety of the method.

[0024] The technical problem is solved and the technical result is achieved by the fact that in the method for obtaining protein from methanotrophic bacteria and their associates, which includes continuous cultivation in a water-mineral nutrient medium, using ammonia water as a source of nitrogen and for stabilizing the pH of the medium, and with the supply of methane-containing gas and air containing free oxygen, under increased pressure, the method is carried out as follows:

[0025] 1. To combine methane and oxygen saturation, compressed air is used instead of technical oxygen (96%), and a pressure gradient along the length of the primary fermentation apparatus is applied to ensure reliable removal of carbon dioxide and nitrogen. The pressure at the methane and compressed air inlet point is 4.00-4.20 atm.g, the pressure gradient along the apparatus length is 0.22-0.26 atm / m; the pressure at the gas phase outlet from the gas separator should not exceed 0.1 atm.g.

[0026] 2. The gas phase composition during the methane:compressed air cultivation operating mode varies from 1:1 to 1:7 as the biomass concentration increases (the amount of compressed air supplied is adjusted based on the need to maintain the dissolved oxygen concentration in the medium at a level of 0.5 to 5.0%).

[0027] 3. The culture liquid separated from the gas phase is subjected to vacuum at a residual pressure of at least 0.1 atm. to prevent boiling of the water.

[0028] 4. The primary fermentation apparatus is seeded with a pure microbial culture at a concentration of 1.0-1.5 g / L based on absolutely dry biomass and a growth rate of at least 0.28-0.32 (higher growth rates are difficult to achieve in a typical apparatus with a stirrer). A low seed concentration is required because high culture densities result in high oxygen and methane consumption. Pumping the seed through a pipeline does not involve gas supply, intensive mixing, or monitoring of nitrogen content and pH, which can lead to reduced growth rates and partial cell death.

[0029] 5. The primary fermentation apparatus is brought into operation after seeding the pure culture in the following order:

[0030] a) After inoculation, the primary fermentation apparatus operates in batch mode. The gas phase is introduced into the apparatus, which is 90% filled with the liquid phase, under a pressure of 2 atm. excess. Ammonium nitrogen is introduced as sulfate (chloride is acceptable). The pH of the medium is adjusted at this stage by feeding a sodium hydroxide solution. The methane:compressed air ratio is 1:1. As the dissolved oxygen concentration decreases to 0.5-5.0% of saturation, stirring is stopped, and a gas "cap" forms in the upper part of the apparatus, which is vented onto a diffusion candle.

[0031] b) Then the main fermentation apparatus is closed and mixing and the supply of methane and compressed air are resumed until a pressure of 2 atmospheres is reached. The cycle is repeated until the biomass concentration reaches 1.0-1.5 g / l, after which the methane and compressed air flow is turned on, the pressure of the supplied gases is increased to 4.0-4.2 atmospheres, the pressure gradient along the length of the apparatus is 0.22-0.26 atm / m; the pressure at the outlet of the gas phase from the gas separator should not exceed 0.1 atm. excess. In addition, the nitrogen source is replaced from ammonium salts to ammonia water. To save methane, the exhaust mixture of methane and carbon dioxide can be collected in a gas holder and, after purification from carbon dioxide, fed back to the main fermentation apparatus (bioreactor). The process continues in the periodic cultivation mode until the biomass concentration reaches 6-7 g / l, after which the liquid phase flow is turned on and the process switches to continuous mode.

[0032] c) At the initial stages, the flow rate is set at 0.05-0.10 h-1 for, which ensures the removal of metabolites and the accumulation of biomass. At full flow rate (determined during the technological process and is 0.35-0.42 h-1) -1 ) the apparatus is deactivated when the cell concentration reaches at least 10-11 g / L. To minimize the transition of the main fermentation apparatus to continuous operation, a gradient is set to increase the current flow rate to standard values.

[0033] Example 1

[0034] The seed material (monoculture of Methylococcus capsulatus) was grown in a fermenter-inoculator (apparatus with a stirrer) in a volume of 10 liters. The growth rate was 0.27 h -1. Removable-refill mode. After the setup reached the required operating conditions for all parameters: nutrient medium temperature – 41°C, pH of the medium – was adjusted to 6.8. After inoculating the apparatus (5% of the seed volume), 1 atmosphere of methane and 1 atmosphere of compressed air were introduced. Sensor readings were then monitored. Methane and compressed air were then periodically supplied and released to the diffusion candle in a 1:1 ratio, and cultivation proceeded in a closed-loop gas-phase mode.

[0035] The volume of the main fermentation apparatus is 220 liters; the filling factor is 90%; the gas phase during cultivation is methane: compressed air from 1:1 to 1:7 as the biomass concentration increases (while the amount of compressed air supplied is adjusted based on the need to maintain the concentration of dissolved oxygen in the environment at a level of 0.5 to 5.0%).

[0036] At the initial stages, the flow rate is set at 0.05-0.10 h -1, which ensures the removal of metabolites and the accumulation of biomass. At full flow rate (determined during the technological process and is 0.35-0.42 h -1 ) the apparatus is removed when the cell concentration reaches at least 10-11 g / l. The methane content in the gases leaving the gas separator is 0-0.2% by volume. The pressure at the methane and air inlet point is 4.0-4.2 atm. excess, the pressure gradient along the length of the apparatus is 0.22-0.26 atm. / m; the pressure at the outlet of the gas phase should not exceed 0.1 atm. excess.

[0037] The pressure at the exhaust fitting was 0.01-0.02 atm. excess.

[0038] After gas separation, the culture liquid was evacuated. The vacuum was created using a water-jet pump at a residual pressure of at least 0.1 atm by discharging the gas phase into the sewer through a water seal.

[0039] Growth speed reached 0.40h -1The cell suspension density achieved was 13.1 g / l (productivity 5.24 g*l / h). The protein mass fraction according to Barshtein was 64.10±1.85% by weight, and the crude protein mass fraction according to Kjeldahl was 72.0±2.1% by weight.

[0040] Example 2

[0041] The seed material (mixed culture / association / Methylococcus capsulatus satellite culture Cupriavidus gilardii) was grown in a fermenter-inoculator (apparatus with a stirrer) in a volume of 10 liters. The growth rate is 0.28 h -1 . Removable-refill mode. After the setup reached operating conditions according to all parameters: nutrient medium temperature – 41°C, medium pH – 6.8. After inoculating the apparatus (5% of the seed volume), 1 atmosphere of methane and 1 atmosphere of air were introduced. Sensor readings were then monitored. Methane and compressed air were then periodically supplied and released to the diffusion candle in a 1:1 ratio, and cultivation proceeded in a closed-loop gas-phase mode.

[0042] The primary fermentation apparatus has a capacity of 220 liters; the filling factor is 90%; the gas phase during cultivation is methane:compressed air in a ratio of 1:1 to 1:7 as the biomass concentration increases. At the initial stages, the flow rate is set at 0.05-0.10 h -1 , which ensures the removal of metabolites and the accumulation of biomass. At full flow rate (determined during the technological process and is 0.35-0.42 h -1 ) the apparatus is removed when the cell concentration reaches at least 10-11 g / l. The methane content in the gases leaving the gas separator is 0-0.2% by volume. The pressure at the methane and air inlet point is 4.0-4.2 atm. excess; the pressure gradient along the length of the apparatus is 0.22-0.26 atm. / m.

[0043] Growth speed reached 0.42h -1 The cell suspension density achieved was 12.9 g / l (productivity 5.4 g*l / h). The protein mass fraction according to Barshtein was 63.5±3.5% by weight, and the crude protein mass fraction according to Kjeldahl was 70.5±2.5% by weight.

[0044] As can be seen from the description of the method and the examples given, the technical problem is solved and the technical result is achieved by eliminating the inhibition of microorganisms during the process of pumping the seed material from the pure culture apparatus to the main fermentation apparatus, eliminating the inhibition of the culture by oxygen at the initial stages of the main fermentation, minimizing inhibition by carbon dioxide and limiting methane and oxygen at stages characterized by a high concentration of microorganisms in the culture liquid.

Claims

1. A method for obtaining protein from methanotrophic bacteria and their associates, including continuous cultivation of biomass in a water-mineral nutrient medium in a primary fermentation apparatus using ammonia water as a source of nitrogen and for stabilizing the pH of the medium, feeding methane-containing gas and oxygen-containing air into the apparatus at elevated pressure, characterized in that in the operating mode of cultivation, after reaching a biomass concentration of 1.0-1.5 g / l, methane and compressed air are fed into the primary fermentation apparatus at a pressure at the point of introduction of methane and compressed air of 4.00-4.20 atm excess, while ensuring a pressure gradient along the length of the apparatus of 0.22-0.26 atm / m and a residual pressure at the outlet of the gas phase not exceeding 0.1 atm excess, the process is continued in the periodic cultivation mode until the accumulation of a biomass concentration of 6-7 g / l, while at the initial At the cultivation stages, the flow rate is set at 0.05-0.10 h -1and the composition of the gas phase methane: compressed air as the concentration of biomass increases is increased from 1:1 to 1:7, and the full flow rate is set at 0.35-0.42 h -1 upon reaching a cell concentration of at least 10-11 g / l.

2. The method according to paragraph 1, characterized in that the culture liquid separated from the gas phase is subjected to vacuuming at a residual pressure of at least 0.1 atm to prevent the boiling of the water contained in the culture liquid.