A method for controlling carbon dioxide feeding based on sugar consumption

Abstract

The present invention discloses a method for controlling carbon dioxide feeding based on sugar consumption. The method uses an online carbon dioxide electrode to analyze the concentration of carbon dioxide in water, uses the consumption rate of glucose, and feedback controls the flow of carbon dioxide into a fermentation tank to adjust the concentration of carbon dioxide in water, thereby increasing the yield and conversion rate of succinic acid and reducing production costs.

Description

Technical Field

[0001] The invention relates to a carbon dioxide feeding control method based on sugar consumption, belonging to the field of biotechnology. Background Art

[0002] Succinic acid, also known as succinic acid, is a dicarboxylic acid. Succinic acid is a colorless crystal with a sour taste. It is soluble in water, ethanol and ether, but insoluble in chloroform and dichloromethane. Its relative density is 1.572 (25 / 4°C), its melting point is 188°C, and its boiling point is 236.15°C. As an important chemical raw material, succinic acid is widely used in food, medicine, chemical industry and materials. In recent years, with the intensification of greenhouse effect and environmental pollution, polybutylene succinate (PBS) materials prepared by polymerization of succinic acid and butanediol have attracted widespread attention. In order to meet the growing demand for succinic acid raw materials for polymerization-grade succinic acid, it is urgent to develop a production process for polymerization-grade succinic acid. There are two methods for producing succinic acid, one is chemical synthesis and the other is microbial synthesis. Since the biological fermentation of succinic acid is carried out at room temperature and pressure, weak acid and weak alkali conditions, it does not involve high temperature and high pressure, flammable and explosive, and highly toxic conditions. Therefore, various equipment does not have to consider explosion-proof issues. In addition, the material requirements for various equipment are also relatively low, which reduces investment costs. Second, the raw material for biological fermentation of succinic acid is simple and easily available glucose, and its production cost is less restricted by raw materials. Therefore, the use of biological fermentation to produce succinic acid has attracted much attention.

[0003] In the current succinic acid fermentation process, carbonate is used as the carbon dioxide donor. Carbonate is used to supplement carbon dioxide. The carbon dioxide concentration in the fermentation broth is easy to control, and solid carbonate is easy to store. However, when carbonate is added to provide carbon dioxide, a large amount of metal ions will be added. A large amount of metal ions will not only increase the osmotic pressure in the fermentation broth, but also need to be removed during the downstream separation process, which will further increase the cost. If gaseous carbon dioxide is introduced, too little carbon dioxide will affect the yield and conversion rate of succinic acid. Too much carbon dioxide will form carbonic acid in the fermentation broth, and the presence of carbonate will inhibit the activity of the bacteria. How to more accurately control the amount of carbon dioxide introduced has become a difficult problem that needs to be solved in the industry. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for controlling carbon dioxide feeding based on sugar consumption. The method of the present invention can accurately control the amount of carbon dioxide introduced, so that the yield of succinic acid and the activity of the bacteria are within the optimal range, thereby increasing the total yield of succinic acid.

[0005] In order to solve the above technical problems, the present invention provides a carbon dioxide feeding control method based on sugar consumption. After the succinic acid producing strain is activated, the succinic acid producing strain is inoculated into a fermentation medium for fermentation, and then a pH regulator is used to adjust the pH, and the air volume, rotation speed or tank pressure is adjusted to maintain the dissolved oxygen at 15%. After the OD reaches the requirement, anaerobic acid production is started, and the air supply is stopped at this time, and glucose is added. At this time, carbon dioxide is introduced to control the amount of carbon dioxide introduced.

[0006] Taking 10-15min fermentation as a control cycle, the concentration of carbon dioxide (in g / L) in each cycle is calculated according to the formula:

[0007]

[0008] Among them, t0 is the start time of the calculation period, and t1 is the end time of the calculation period.

[0009] a is a coefficient, and its specific value is related to the mass conversion rate of succinic acid to glucose and the carbon dioxide consumed by the strain to produce unit weight of succinic acid. The value of a is equal to the product of the mass conversion rate of succinic acid to glucose and the mass of carbon dioxide consumed by the strain to produce unit mass of succinic acid during the control period. The mass conversion rate of succinic acid to glucose is equal to the mass of succinic acid produced during the control period divided by the mass of glucose added; the mass of carbon dioxide consumed by the strain to produce unit mass of succinic acid = (m2-m3-m4) / m1, m1 is the mass of succinic acid produced during the control period, m2 is the mass of carbon dioxide introduced into the fermentation tank, m3 is the mass of carbon dioxide in the tail gas, and m4 is the mass of succinic acid dissolved in water. The value range of a is generally 0.2-0.35, preferably 0.3-0.33.

[0010] -b ln(t)+c is the glucose consumption rate during fermentation, where the values ​​of b and c can be obtained by curve fitting based on the glucose consumption rate during fermentation. With fermentation time as the horizontal axis and glucose consumption rate (g / L.min) as the vertical axis, the glucose consumption rate at each time point after fermentation is stable is measured to obtain the values ​​of b and c.

[0011] Preferably, b=0.1-5.8, c=3-30.

[0012] The concentration calculated by the above formula is the current most suitable carbon dioxide concentration of the fermentation medium. Then the carbon dioxide concentration in the current fermentation liquid is measured. If the carbon dioxide concentration in the current fermentation liquid is less than the calculated concentration, the flow rate of carbon dioxide is increased; otherwise, the flow rate of carbon dioxide is reduced; if the calculated value exceeds the saturated solubility of carbon dioxide in the current state, the pressure of the fermentation tank is increased. The maximum pressure of the fermentation tank is 0.15MPa.

[0013] The specific derivation process of the above formula is as follows, where the formula -b ln(t)+c is the glucose consumption rate curve during fermentation. After integration, the total amount of glucose consumed in the time period from t0 to t1 is obtained in g / L, and after multiplying by the volume of the fermentation tank, the total amount of glucose consumed in the control period is obtained in g; a is a specific coefficient, and its specific value is related to the mass conversion rate of succinic acid to glucose in the control period and the carbon dioxide consumed by the strain to produce unit weight of succinic acid. The obtained value divided by the volume of the fermentation liquid is the optimal carbon dioxide concentration of the fermentation medium. The formula is as follows:

[0014]

[0015] The above formula can be simplified.

[0016] V is the volume of the fermentation liquid, in L. As the temperature rises, the solubility of carbon dioxide decreases rapidly. Generally, the fermentation temperature is between 30-45°C. If the carbon dioxide solubility is low at this time, the pressure of the fermentation tank needs to be increased to increase the saturated solubility of carbon dioxide. For this patent, if the calculated carbon dioxide concentration in the fermentation liquid exceeds the saturated solubility at the current temperature, the pressure of the fermentation tank needs to be increased to increase its solubility. The maximum pressure of the fermentation tank does not exceed 0.15MPa.

[0017] Preferably, the carbon dioxide is introduced into the fermentation medium in the form of gaseous carbon dioxide. The concentration of carbon dioxide in the gas is between 50-100% (volume fraction), preferably the concentration of carbon dioxide is between 70-100% (volume fraction), more preferably the concentration of carbon dioxide is between 80-100% (volume fraction), and most preferably the concentration of carbon dioxide is between 90-100% (volume fraction).

[0018] A carbon dioxide feeding control method based on sugar consumption, wherein the succinic acid production strain is Escherichia coli, succinic acid producing actinomycetes, Corynebacterium glutamicum, lipolytic yeast or cerevisiae.

[0019] A method for controlling carbon dioxide feeding based on sugar consumption, wherein the succinic acid producing strain is a genetically engineered strain that is modified to produce succinic acid using Escherichia coli, Actinomycetemcomitans succinogenes, Corynebacterium glutamicum, Saccharomyces lipolytica or Saccharomyces cerevisiae as a chassis strain.

[0020] A method for controlling carbon dioxide feeding based on sugar consumption, wherein the pH value of the fermentation liquid during fermentation and acid production is 6.0-7.2.

[0021] The present invention provides application of the method in producing succinic acid by fermentation.

[0022] The present invention provides a carbon dioxide feeding control method based on sugar consumption, which is used for the carbon dioxide feeding control method for fermentation production of succinic acid, and controls the flow amount of carbon dioxide by detecting the glucose consumption rate during the fermentation process, thereby greatly improving the yield and conversion rate of succinic acid during the fermentation process. By using this method, the genetically engineered strain of Escherichia coli is inoculated into a 50L fermentation tank for fermentation, and the yield of succinic acid can reach 110g / L, and the conversion rate can reach 1.05. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the glucose consumption rate of the fermentation system of Example 1.

[0024] Figure 2 This is a graph showing the glucose consumption rate of the fermentation system of Example 2. DETAILED DESCRIPTION

[0025] The following embodiments describe the present invention in detail, but do not limit the present invention.

[0026] Example 1

[0027] Bacterial strain: The bacterial strain used was E. coli AFP111, which was obtained by using E. coli W1485 as the chassis cell and knocking out the Ldha, pflB and ptsG genes.

[0028] LB solid medium (g / L): yeast extract 5, tryptone 10, NaCl 10, agar powder 15. pH 7.0, sterilized at 121°C for 20 min.

[0029] LB medium (g / L): yeast extract 5, tryptone 10, NaCl 10. pH 7.0, sterilized at 121°C for 20 min.

[0030] Trace elements (g / L): FeCl3·6H2O 2.4, CoCl2·6H2O 0.3, CuCl2·2H2O 0.15, ZnCl20.3, Na2MO4·2H2O 0.3, H3BO3 0.075, MnCl2·4H2O 0.495, sterilization at 121℃ for 20min.

[0031] Fermentation medium (g / L): glucose 40 (g / L), Na2HPO4·12H2O 15.11, KH2PO4 3, NH4Cl1, NaCl 0.5, sterilized at 121℃ for 20 min, 1 / 1000 (volume fraction, relative to the initial volume of fermentation) of MgSO4 (1 mol / L) and 1 / 1000 (volume fraction, relative to the initial volume of fermentation) of trace elements were added before the start of fermentation.

[0032] Activation of bacterial strains: Take the bacterial strains stored at -80℃ and streak them on LB solid culture medium, then place them in an incubator at 37℃ for activation and culture for 10-15 hours, then pick a single colony and inoculate it into the seed culture medium.

[0033] Seed liquid culture: The seed culture medium is LB medium. The seed culture medium after inoculation is cultured in a shaker at 220r / min and 37°C for 9.5-12h, so that the OD of the seed culture medium reaches above 2. At this time, the seed liquid can be inoculated into the fermentation medium.

[0034] 50L fermentation tank culture: The liquid volume of the 50L fermentation tank is 25L, and the pH of the sterilized fermentation medium is about 7. Before fermentation, the DO (dissolved oxygen) value in the fermentation tank is calibrated to 100%. At this time, the seed liquid is inoculated into the fermentation medium with an inoculation amount of 5%. Initially, the conditions are set to a speed of 100r / min, a ventilation volume of 0.5VVM, a temperature of 37°C, and a pH value of 7.0. During the fermentation process, ammonia water is used to maintain the pH at 7.0. Due to the continuous growth of the bacteria, the dissolved oxygen will continue to decrease. During this period, the ventilation volume and speed are associated with DO so that DO is maintained above 20%, the maximum ventilation volume is 3VVM, and the maximum speed is 500r / min. After the OD (Optical Density) reaches 30, the fermentation is switched to anaerobic fermentation, the pH regulator ammonia water is replaced with 2mol / L sodium hydroxide solution, 600g / L glucose is added to the fermentation tank, so that the concentration of glucose in the fermentation tank is between 20-30g / L, and the speed is reduced to 100r / min. The valve for air inlet is closed, and 90% of the volume fraction of carbon dioxide (the rest is nitrogen) is introduced at a flow rate of 2L / min. The pressure of the fermentation tank is 0.01MPa. After 10 minutes, the flow rate of carbon dioxide, the tank pressure of the fermentation tank and the consumption rate of glucose are related. At this time, the carbon dioxide inlet flow rate is controlled by a computer, and each 10 minutes is a control cycle. According to the formula Calculate the concentration of carbon dioxide in each cycle and measure the concentration of carbon dioxide in real time. When the measured concentration of carbon dioxide in the current fermentation liquid is less than the calculated concentration, increase the flow rate of carbon dioxide; otherwise, reduce the flow rate of carbon dioxide; PID will gradually adjust the flow rate of carbon dioxide so that the concentration of carbon dioxide in the fermentation tank is within the optimal range.

[0035] The value of a is equal to the product of the mass conversion rate of succinic acid to glucose during the control period and the mass of carbon dioxide consumed by the strain to produce unit mass of succinic acid. The mass conversion rate of succinic acid to glucose is equal to the mass of succinic acid produced during the control period divided by the mass of glucose added; the mass of carbon dioxide consumed by the strain to produce unit weight of succinic acid = (m2-m3-m4) / m1, m1 is the mass of succinic acid produced during the control period, m2 is the mass of carbon dioxide introduced into the fermentation tank, m3 is the mass of carbon dioxide in the tail gas, and m4 is the mass of succinic acid dissolved in water.

[0036] In this embodiment, the value of a in a control cycle is calculated. With 10 minutes as a control cycle, the total carbon dioxide content introduced is 200 g (m2), the carbon dioxide dissolved in water is 25 g (m4), the carbon dioxide in the tail gas is 167.5 g (m3), the mass of succinic acid produced in the control cycle is 25 g (m1), so the mass of carbon dioxide consumed by the strain to produce unit mass of succinic acid is 0.3, and the conversion rate of succinic acid produced in the control cycle to glucose is 1.1, so the value of a is 0.33

[0037] In this embodiment, according to Figure 1 The consumption rate of glucose during the fermentation shown was subjected to curve fitting to obtain the values ​​of b and c, b = 4.048, c = 19.135.

[0038] Glucose measurement: After centrifuging the fermentation broth, take the supernatant, dilute it to the range of 0-1g / L, and measure it using the biosensor SBA-40D.

[0039] OD measurement: In the aerobic growth stage, take the fermentation liquid and dilute it to a suitable multiple and use a UV spectrophotometer to measure it, the wavelength is 600nm. In the anaerobic stage, take the fermentation liquid and dilute it to a suitable multiple with dilute hydrochloric acid and use a UV spectrophotometer to measure it, the wavelength is 600nm.

[0040] Measurement of succinic acid: Take the fermentation liquid in the anaerobic culture stage, centrifuge and take the supernatant, dilute the supernatant to an appropriate multiple, and use high performance liquid chromatography for measurement. The measurement conditions of liquid chromatography are: the chromatographic column is Aminex HPX-87H organic acid analysis column, the mobile phase is 5mM sulfuric acid, the mobile phase flow rate is 1mL / min, the column temperature is 40℃, and the running time is 1h. The corresponding succinic acid concentration is obtained by comparing the peak area with the concentration and peak area of ​​the standard sample.

[0041] After the fermentation, the concentration of succinic acid was tested to be 110 g / L and the conversion rate was 1.05.

[0042] The conversion rate is the mass of succinic acid produced divided by the mass of glucose added.

[0043] Example 2

[0044] Bacterial strain: The bacterial strain used in this example is Actinobacillus succinogenes, and the preservation center is the China Industrial Microbiological Culture Collection Center, and the bacterial strain number is CICC 11014.

[0045] Seed culture medium (g / L): glucose 10 (diluted), yeast extract 5, NaHCO3 10, NaH2PO4·2H2O 9.6, K2HPO4·3H2O 15.5; pH 6.8, sterilization at 121℃ for 15 min.

[0046] Fermentation medium (g / L): glucose 100, yeast extract 10, KH2PO4 3, MgCl2·6H2O 0.3, CaCl2 0.3, NaCl 1, Na2HPO4 0.31, NaH2PO4 1.6, sterilized at 121℃ for 15min. Glucose was added separately after being sterilized at 121℃ for 15min.

[0047] Seed culture: Take the frozen bacteria at -80 degrees Celsius and inoculate them into the seed culture medium. The culture medium conditions are 120r / min, 37°C. After 10-12 hours, the culture medium is used as the seed solution.

[0048] Fermentation in a 5L fermenter: The liquid volume of a 5L fermenter is 2.5L, and the pH of the sterilized fermentation medium is about 7. The seed liquid is inoculated into the fermentation medium with an inoculation amount of 5%. The fermentation conditions are set to a speed of 200r / min, a temperature of 37°C, the valve for air inlet is closed, and a volume fraction of 90% carbon dioxide (the rest is nitrogen) is introduced at a flow rate of 2L / min. The pressure of the fermenter is 0.01MPa. After 180 minutes, the flow rate of carbon dioxide, the tank pressure of the fermenter and the consumption rate of glucose are associated. At this time, the carbon dioxide inlet flow rate is controlled by a computer, and each 10 minutes is a control cycle. According to the formula Calculate the concentration of carbon dioxide in each cycle, where the value of a may be different in each control cycle, and the specific calculation method is the same as in Example 1. Measure the carbon dioxide concentration in real time. When the measured carbon dioxide concentration in the current fermentation liquid is less than the calculated concentration, increase the flow rate of carbon dioxide; otherwise, reduce the flow rate of carbon dioxide; PID will gradually adjust the carbon dioxide flow rate so that the carbon dioxide concentration in the fermentation tank is within the optimal range. During the fermentation process, due to the accumulation of succinic acid, the pH value in the fermentation liquid continues to decrease. At this time, basic magnesium carbonate needs to be added to maintain the pH value above 6.6.

[0049] In this embodiment, according to Figure 2The consumption rate of glucose during the fermentation shown was subjected to curve fitting to obtain the values ​​of b and c, b = 0.924, c = 4.9313.

[0050] Taking 10 minutes as a control cycle, the total carbon dioxide content introduced is 40g (m2), the carbon dioxide dissolved in water is 2.5g (m4), the carbon dioxide in the exhaust gas is 36.9g (m3), and the mass of succinic acid produced within the control cycle is 1.25g (m1). Therefore, the mass of carbon dioxide consumed by the strain to produce unit mass of succinic acid is 0.403. The conversion rate of succinic acid produced within the control cycle to glucose is 0.62, so the value of a is 0.25.

[0051] Glucose measurement: After centrifuging the fermentation broth, take the supernatant, dilute it to the range of 0-1g / L, and measure it using the biosensor SBA-40D.

[0052] Measurement of succinic acid: Take the fermentation liquid in the anaerobic culture stage, centrifuge and take the supernatant, dilute the supernatant to an appropriate multiple, and use high performance liquid chromatography for measurement. The measurement conditions of liquid chromatography are: the chromatographic column is Aminex HPX-87H organic acid analysis column, the mobile phase is 5mM sulfuric acid, the mobile phase flow rate is 1mL / min, the column temperature is 40℃, and the running time is 1h. The corresponding succinic acid concentration is obtained by comparing the peak area with the concentration and peak area of ​​the standard sample.

[0053] After the fermentation, the concentration of succinic acid was tested to be 54 g / L and the conversion rate was 0.56.

[0054] Comparative Example 1

[0055] The same bacterial strains, fermentation conditions and experimental methods as those in Example 1 were used.

[0056] After the 50L fermenter entered the anaerobic stage, 90% carbon dioxide was introduced, and the flow rate of carbon dioxide was 2L / min during the entire anaerobic fermentation process until the fermentation was completed. After the fermentation was completed, the concentration of succinic acid was tested to be 83g / L and the conversion rate was 0.9.

[0057] Comparative Example 2

[0058] This embodiment is basically the same as embodiment 2, except that the amount of carbon dioxide introduced during the fermentation process is 1 L / min. Other fermentation conditions are the same.

[0059] After the fermentation, the concentration of succinic acid was tested to be 30 g / L and the conversion rate was 0.42.

[0060] According to the comparison of the experimental results of Example 1 and Example 2, it is found that after the sugar consumption is associated with the carbon dioxide feeding rate, the yield of succinic acid is increased by 32.5% and the conversion rate is increased by 15%. This shows that the present invention provides a carbon dioxide feeding control method based on sugar consumption, which can effectively improve the yield and conversion rate of succinic acid.

Claims

1. A method for controlling carbon dioxide feeding based on sugar consumption, characterized in that: After the succinic acid producing strain is activated, the succinic acid producing strain is inoculated into the fermentation medium for fermentation, and then the pH is adjusted using a pH regulator, and the air volume, rotation speed or tank pressure is adjusted to maintain the dissolved oxygen at 15%. After the OD reaches the requirement, it is switched to anaerobic acid production, at which time the air is stopped and glucose is added. At this time, carbon dioxide is introduced to control the amount of carbon dioxide introduced. Taking 10-15min fermentation as a control cycle, the concentration of carbon dioxide in each cycle is calculated according to the formula: Where t0 is the start time of the calculation period, and t1 is the end time of the calculation period; a is a coefficient, and the value of a is equal to the product of the mass conversion rate of succinic acid to glucose and the mass of carbon dioxide consumed by the strain to produce unit mass of succinic acid during the control period; -bln(t)+c is the glucose consumption rate during fermentation, where the values ​​of b and c can be obtained by curve fitting according to the glucose consumption rate during fermentation; with fermentation time as the horizontal axis and glucose consumption rate g / L·min as the vertical axis, the glucose consumption rate at each time point after fermentation is stable is measured to obtain the values ​​of b and c; The concentration of carbon dioxide is in g / L; The concentration calculated by the above formula is the most suitable carbon dioxide concentration of the fermentation medium at present. Then the carbon dioxide concentration in the current fermentation liquid is measured. If the carbon dioxide concentration in the current fermentation liquid is less than the calculated concentration, the flow rate of carbon dioxide is increased; otherwise, the flow rate of carbon dioxide is reduced; if the calculated value exceeds the saturated solubility of carbon dioxide in the current state, the pressure of the fermentation tank is increased. The maximum pressure of the fermentation tank is 0.15MPa. The succinic acid producing strain is Escherichia coli, Actinobacillus succinogenes, Corynebacterium glutamicum, Saccharomyces lipolytica or Saccharomyces cerevisiae.

2. The control method according to claim 1, characterized in that: The value of a is equal to the product of the mass conversion rate of succinic acid to glucose during the control period and the mass of carbon dioxide consumed by the strain to produce unit mass of succinic acid; the mass conversion rate of succinic acid to glucose is equal to the mass of succinic acid produced during the control period divided by the mass of glucose added; the mass of carbon dioxide consumed by the strain to produce unit weight of succinic acid = (m2-m3-m4) / m1, m1 is the mass of succinic acid produced during the control period, m2 is the mass of carbon dioxide introduced into the fermentation tank, m3 is the mass of carbon dioxide in the exhaust gas, and m4 is the mass of succinic acid dissolved in water.

3. The control method according to claim 1, characterized in that: a＝0.2-0.35。 4. The control method according to claim 3, characterized in that: a＝0.3-0.33。 5. The control method according to claim 1, characterized in that: b=0.1-5.8,c=3-30.

6. The control method according to claim 1, characterized in that: If the calculated carbon dioxide concentration in the fermentation liquid exceeds the saturated solubility at the current temperature, the pressure of the fermentation tank needs to be increased to improve its solubility. The maximum pressure of the fermentation tank shall not exceed 0.15MPa.

7. The control method according to claim 1, characterized in that: The carbon dioxide is introduced into the fermentation medium in the form of gaseous carbon dioxide. The volume concentration of carbon dioxide in the gas is 50%-100%.

8. The control method according to claim 7, characterized in that: The volume concentration of carbon dioxide in the gas is 70%-100%.

9. The control method according to claim 8, characterized in that: The volume concentration of carbon dioxide in the gas is 80%-100%.

10. The control method according to claim 1, characterized in that: The succinic acid producing strain is a genetic engineering strain that is modified to produce succinic acid using Escherichia coli, succinic acid producing actinomycetes, Corynebacterium glutamicum, lipolytic yeast or cerevisiae yeast as a base strain.

11. The control method according to claim 1, characterized in that: During the fermentation process to produce acid, the pH value of the fermentation liquid is 6.0-7.2.

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