Continuous fermentation method of clostridium aerovorans based on multi-parameter regulation and control

Abstract

The invention discloses a clostridium aerovorans continuous fermentation method based on multi-parameter regulation and control, and belongs to the technical field of microbial fermentation. The method comprises the following parameter control: fermentation culture medium concentration control: in a biomass stabilization stage, gradually reducing the fermentation culture medium concentration from an initial concentration so as to reduce an acid production ratio and improve an alcohol production ratio; the CO content in the tail gas is controlled, wherein the volume fraction of CO in the tail gas is controlled to be 5-10%, and therefore the growth rate of the clostridium aerovorans is increased; dilution degree control: gradually improving the dilution degree so as to improve the fermentation yield; wherein the dilution is equal to the product of the fermentation culture medium feeding speed and 24 / fermentation liquid volume; a ceramic membrane is added, and thalli are intercepted by adding the ceramic membrane, so that the biomass is improved. According to the method, the fermentation acid-alcohol ratio can be regulated and controlled, and the fermentation rate of the clostridium aerovorans is increased.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a continuous fermentation method for Clostridium aeruginosa based on multi-parameter regulation. Background Technology

[0002] Due to the depletion of fossil fuels and growing concerns about environmental issues, biofuels such as bioethanol and biobutanol are receiving increasing attention. As an alternative fuel for vehicles, butanol shows more promise than ethanol, with advantages including lower volatility, higher energy content, lower corrosiveness, and better compatibility with gasoline and diesel, thus requiring less modification to existing engine structures. However, the industrialization of biobutanol faces technological bottlenecks due to its higher cost, preventing it from achieving the same market competitiveness as bioethanol. Currently, domestic biobutanol production primarily uses corn and other grains as raw materials, but the use of these raw materials is restricted by national policies, and their cost is also high. In the ABE fermentation of Clostridium difficile, the mass ratio of acetone, butanol, and ethanol is 3:6:1. While butanol and ethanol can be used as fuel, acetone, due to its low boiling point, corrodes pipes and rubber, making it unsuitable for fuel use. Further distillation separation is required, increasing production costs. However, butanol is toxic, and its toxic effects on the producing strains lead to low product concentrations and low production efficiency during acetone-butanol (ABE) fermentation, significantly limiting the final butanol yield. Therefore, improving ABE production intensity through fermentation process control or enhancing the butanol tolerance of the producing strains through mutagenesis to mitigate butanol damage are key focuses of ABE fermentation research.

[0003] Acetogenic bacteria utilize the reducing acetyl-CoA synthesis pathway (also known as the Wood-Ljungdahl pathway, or WL pathway for short) to convert CO or CO2 into acetyl-CoA, which is then used to synthesize their own biomass or generate metabolites such as organic alcohols and organic acids. Acetyl-CoA is the central metabolite of Clostridium aerogenes, and under the action of phosphoacetyltransferase (Pta) and acetate kinase (Ack), it can be further converted into acetic acid, accompanied by the synthesis of one molecule of ATP, which further produces ethanol. Acetyl-CoA undergoes two steps of oxidation to produce acetic acid, butyryl-CoA to produce butyrate, and hexyl-CoA to produce hexanoate. All three processes involve substrate-level phosphorylation (PTA), producing one ATP molecule. However, because the WL pathway fixes one CO2 molecule, consuming one ATP molecule, the entire process does not produce a net ATP. The butanol production pathway is less efficient than that of ethanol. Improving the conversion efficiency and yield of butanol is a key determinant of the fermentation process, highly dependent on appropriate bacterial growth conditions and suitable substrate concentrations. Insufficient substrate will slow microbial growth; excessive substrate may lead to poor microbial growth and / or cell death.

[0004] Therefore, a method is needed to regulate the continuous fermentation of Clostridium aeruginosa to improve the continuous fermentation rate and yield. Summary of the Invention

[0005] Technical problem to be solved: In view of the above-mentioned technical problem, the present invention provides a continuous fermentation method of Clostridium aeruginosa based on multi-parameter control, which can control the ratio of fermentation acid to alcohol and improve the fermentation rate of Clostridium aeruginosa.

[0006] Technical solution: A continuous fermentation method for Clostridium aeruginosa based on multi-parameter control, including the following parameter control: Fermentation medium concentration control: During the biomass stabilization stage, the concentration of the fermentation medium is gradually reduced from the initial concentration to reduce the acid production ratio and increase the alcohol production ratio. CO content control in exhaust gas: Control the volume fraction of CO in exhaust gas to 5-10% to increase the growth rate of Clostridium aeruginosa. Dilution control: Gradually increase the dilution to improve fermentation yield; where, dilution = fermentation medium feeding rate × 24 / fermentation broth volume; Adding ceramic membranes: By adding ceramic membranes, bacterial cells are retained, thereby increasing biomass.

[0007] Preferably, the initial concentration of the fermentation medium is as follows: ammonium chloride 1 g / L, potassium chloride 0.1 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 0.8 g / L, potassium dihydrogen phosphate 0.1 g / L, yeast extract 1 g / L, calcium chloride dihydrate 0.04 g / L, sodium selenite 0.2 mg / L, nickel chloride hexahydrate 0.2 mg / L, sodium tungstate 0.17 mg / L, ferrous sulfate 1 mg / L, sodium molybdate 0.17 mg / L, biotin 0.02 mg / L, folic acid 0.02 mg / L, pyridoxine hydrochloride 0.1 mg / L, thiamine 0.05 mg / L, riboflavin 0.05 mg / L, niacin 0.05 mg / L, D-calcium pantothenate 0.05 mg / L, cobalamin 0.001 mg / L, p-aminobenzoic acid 0.05 mg / L, and lipoic acid 0.05 mg / L.

[0008] Preferably, the CO content in the exhaust gas is controlled by controlling the gas supply rate of the syngas source: when the volume fraction of CO in the exhaust gas is within 5-10%, the gas supply rate of the syngas source is controlled at 0.6 L / min; when the volume fraction of CO in the exhaust gas is less than 5%, the gas supply rate of the syngas source is increased; when the volume fraction of CO in the exhaust gas is greater than 10%, the gas supply rate of the syngas source is decreased; wherein, the components of the syngas source, by volume fraction, include 30% CO, 10% CO2, 35% H2 and 25% N2.

[0009] Preferably, the dilution is controlled as follows: starting from 0.5, increasing by 0.2 every 12 hours, gradually increasing to 1.8.

[0010] Preferably, the parameter control further includes controlling the pH to be 5.1~4.9.

[0011] Preferably, the volume fraction of CO in the exhaust gas is monitored using a Raman spectroscopy device.

[0012] Preferably, the content of metabolites from continuous fermentation is detected by HPLC, with the following specific parameters: hydrogen ion chromatography column, mobile phase: 5 mM H2SO4, column temperature: 30℃, RID detector temperature: 45℃, mobile phase flow rate: 0.9 mL / min, and injection volume: 10 µL.

[0013] Beneficial effects: Compared with the prior art, the present invention can effectively regulate the fermentation acid-alcohol ratio and improve the fermentation rate of Clostridium aeruginosa by controlling the concentration of fermentation medium, CO content in exhaust gas, dilution and adding ceramic membrane. Attached Figure Description

[0014] Figure 1 The graph shows the content of OD600 and metabolites in Example 1, with the vertical axis in g / L. Figure 2 This is a graph showing the CO content in the exhaust gas in Example 2; Figure 3 This is a graph showing the content of OD600 and metabolites in Example 2; Figure 4 This is a graph showing the content of OD600 and metabolites in Example 3; Figure 5 This is a graph showing the dilution and acid-alcohol molar ratio parameters in Example 3; Figure 6 The graph shows the content of OD600 and metabolites under the condition of adding a ceramic membrane in Example 4; Figure 7 The graph shows the contents of OD600 and metabolites in Example 4 without adding a ceramic membrane; Figure 8 This is the parameter control chart for Example 5; Figure 9 This is a graph showing the content of OD600 and metabolites in Example 5; In the above figures, the horizontal axis is in the unit of h. Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0016] The experimental materials used in the following examples are as follows: YTF medium: yeast extract 5 g / L, peptone 16 g / L, fructose 10 g / L, sodium chloride 0.2 g / L, L-cysteine ​​0.3 g / L, resazurin 500 μL / L, trace metals 2 mL / L, vitamins 1 mL / L, adjust pH to 6.0 with NaOH solution, sterilize at 121°C for 20 min.

[0017] Fermentation medium: ammonium chloride 1g / L, potassium chloride 0.1g / L, magnesium sulfate heptahydrate 0.2g / L, sodium chloride 0.8g / L, potassium dihydrogen phosphate 0.1g / L, yeast extract 1g / L, calcium chloride dihydrate 0.04g / L, sodium selenite 0.2mg / L, nickel chloride hexahydrate 0.2mg / L, sodium tungstate 0.17mg / L, ferrous sulfate 1mg / L, sodium molybdate 0.17mg / L, biotin 0.02mg / L, folic acid 0.02mg / L, pyridoxine hydrochloride 0.1mg / L, thiamine 0.05mg / L, riboflavin 0.05mg / L, niacin 0.05mg / L, D-calcium pantothenate 0.05mg / L, cobalamin 0.001mg / L, para-aminobenzoic acid 0.05mg / L, lipoic acid 0.05mg / L. Adjust the pH to 6.0 with NaOH solution and sterilize at 121℃ for 20 minutes.

[0018] The continuous fermentation process is as follows: (1) Seed culture: The carbon monoxide-eating Clostridium carboxidivorans GG6 (preservation number CGMCC 34975) preserved in glycerol at -80℃ was cultured in 3 mL of YTF medium for 24~48 h at a temperature of 37℃ to obtain the primary seed culture. The primary seed culture was then transferred to 250 mL of YTF medium and cultured anaerobically at 37℃ for 24 h to obtain the secondary seed culture.

[0019] (2) Fermentation culture in a bioreactor: A 5L bioreactor (Bailun) was used as the fermentation equipment. The fermentation medium was prepared in the reactor with a working volume of 3.5L and sterilized at 121℃ for 20min. After sterilization, a synthesis gas source (30% CO, 10% CO2, 35% H2 and 25% N2 by volume) was introduced into the reactor at a gas flow rate of 0.6L / min to remove oxygen from the fermentation equipment. The parameters of the fermentation equipment were set according to the initial stirring speed of 100rpm and the fermentation temperature of 37℃. In the first half hour before inoculation, 0.1g of sodium sulfide was added to the fermentation equipment. The secondary seed liquid obtained in step (1) was inoculated into the fermentation equipment at an inoculation rate of 10% v / v. Fermentation lasted for 24h. At this time, the pH of the fermentation liquid showed a downward trend, the inoculum began to recover, ammonia was automatically added to control the pH, and cysteine ​​was added to supply the sulfur source. After 72 hours of fermentation, start continuous fermentation mode with dilution D=0.5 (D is the dilution rate calculated based on the culture medium flow rate, dilution rate = fermentation medium feeding rate × 24 / fermentation liquid volume). At this time, the culture medium fed in is the basic component of the fermentation medium. When the biomass in the fermentation liquid is basically stable at around 1.2, adjust several parameters.

[0020] Determination of fermentation metabolites: Five substances, namely lactic acid, acetic acid, ethanol, butyric acid, and butanol, were simultaneously detected in the fermentation products by HPLC. A hydrogen ion chromatography column (Aminex HPX-87H Column, 300 x 7.8 mm) was used, with the following parameters: mobile phase: 5 mM H₂SO₄, column temperature: 30℃, RID detector temperature: 45℃, mobile phase flow rate: 0.9 mL / min, and injection volume: 10 µL.

[0021] Example 1: Control of Fermentation Medium Concentration During the growth phase (starting around day 0.5), substrate supply was increased by increasing agitation and / or gas flow rate, gradually increasing OD600 and metabolites. At 48 hours, continuous fermentation was initiated with a dilution of D=1.0, fed with fermentation medium. Gas rate, tank pressure, and agitation parameters were kept constant for 100 hours of fermentation. From 110 hours, the medium addition was reduced to 0.8x of the original fermentation medium concentration, and from 145 hours, the medium addition was reduced to 0.7x of the original fermentation medium concentration until 190 hours of fermentation.

[0022] The results are as follows Figure 1 Biomass gradually stabilized after 100 hours. Simultaneously, acetic acid levels were high (approximately 7 g / L). At this point, reducing the nutrient coefficient by 0.8x resulted in a decrease in acetic acid levels, while ethanol and butanol levels continued to increase. At 150 hours, reducing the culture medium composition by 0.7x followed a similar trend to 0.8x, with acetic acid decreasing to 5 g / L, ethanol to 7 g / L, and butanol above 2 g / L. This demonstrates that controlling nutrient supply can control acid production, resulting in ethanol production without accompanying acid production.

[0023] Example 2: Control of CO content in exhaust gas This embodiment is equipped with a Raman spectroscopy device for detecting exhaust gas components, which monitors the CO gas content in the exhaust gas in real time and detects the gas feedstock supply.

[0024] like Figure 2 and 3 As shown, in the early stages, the biomass was low, the gas supply was high, and the CO volume content in the exhaust gas was greater than 20%, resulting in slow and prolonged bacterial growth. As the biomass increased and gas utilization increased, the CO volume content in the exhaust gas gradually decreased by 5%, maintaining a high growth rate within the 5-10% CO volume fraction range. When the gas supply continued to increase, the CO volume fraction rose, and when it exceeded 15%, bacterial growth and the growth of metabolic products declined. Therefore, controlling the CO volume content in the exhaust gas between 5-10% can maintain a good carbon source supply.

[0025] Example 3 Dilution Control In this embodiment, when continuous fermentation is stable, the gas supply is adjusted to maintain the CO content in the exhaust gas at 10%. The dilution is gradually increased while maintaining a 1.0x addition of fermentation medium, and changes in biomass and metabolites are observed and compared. Figure 5 As shown, the dilution calculation method is: feeding rate (L / h) * 24 / fermentation broth volume (L). The dilution is maintained at 1 for 240-260 hours. In the second stage (260-270 hours), the dilution is increased to 1.3. In the third stage (290-308 hours), the dilution is increased to 1.5. According to... Figure 4The data results showed that, with increasing dilution and ensuring adequate gas supply, even at a higher dilution of 1.5, the OD600 remained at 5.61, acetic acid at 6.5 g / L, ethanol at 7.697 g / L, butyric acid at 0.624 g / L, and butanol at over 1 g / L. This indicates that, while maintaining constant biomass and product concentration, increasing the dilution can improve the yield.

[0026] Example 4: Adding a ceramic membrane This embodiment compares the impact of adding a ceramic membrane to retain microorganisms during continuous fermentation on the fermentation process.

[0027] The results are as follows Figure 6 and Figure 7 As shown, membrane retention rapidly increases bacterial biomass within 72 hours, while the absence of membrane retention does not increase the production of acetic acid, ethanol, butyric acid, and butanol.

[0028] Example 5: Multi-parameter control This embodiment attempts to control multiple aspects such as pH, gas supply, dilution, and nutrient supply to regulate fermentation production. It also utilizes real-time online pH and gas Raman spectroscopy equipment to monitor exhaust gas content and control the entire fermentation process. (See [link to relevant documentation]). Figure 8 Initially, the pH was around 5.2. As growth recovered, the pH began to decrease, and at 16 hours, it was controlled at 5.1. Simultaneously, the CO content in the exhaust gas began to decrease, and the gas velocity was gradually increased to maintain a CO content of 5-10%. With increasing biomass and metabolites, around 90 hours, when acetic acid > 3 g / L, the pH was lowered to 5.0, and continuous fermentation was initiated with a dilution of 0.5. The feed was the original culture medium, and the dilution was increased by 0.2 every 12 hours. After 110 hours of fermentation, when the biomass reached approximately 5 g / L, a ceramic membrane was added to assist fermentation, rapidly increasing both biomass and metabolite yield. At 190 hours, when acetic acid reached 5 g / L, the pH was lowered to 0.8x, and at 210 hours, the pH was automatically lowered to 4.9 until fermentation ended.

[0029] like Figure 9 As shown, under the condition of final dilution of 1.8, the biomass was around 18, acetic acid was controlled at around 5 g / L, ethanol at around 20 g / L, butyric acid at around 2 g / L and butanol yield was 7 g / L, with a butanol yield of 0.5 g / L / h.

Claims

1. A continuous fermentation method for Clostridium aeruginosa based on multi-parameter control, characterized in that, The following parameters are controlled: Fermentation medium concentration control: During the biomass stabilization stage, the concentration of the fermentation medium is gradually reduced from the initial concentration to reduce the acid production ratio and increase the alcohol production ratio. CO content control in exhaust gas: Control the volume fraction of CO in exhaust gas to 5-10% to increase the growth rate of Clostridium aeruginosa. Dilution control: Gradually increase the dilution to improve fermentation yield; where, dilution = fermentation medium feeding rate × 24 / fermentation broth volume; Adding ceramic membranes: By adding ceramic membranes, bacterial cells are retained, thereby increasing biomass.

2. The method for continuous fermentation of Clostridium aeruginosa based on multi-parameter control according to claim 1, characterized in that, The initial concentrations of the fermentation medium were as follows: ammonium chloride 1 g / L, potassium chloride 0.1 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 0.8 g / L, potassium dihydrogen phosphate 0.1 g / L, yeast extract 1 g / L, calcium chloride dihydrate 0.04 g / L, sodium selenite 0.2 mg / L, nickel chloride hexahydrate 0.2 mg / L, sodium tungstate 0.17 mg / L, ferrous sulfate 1 mg / L, sodium molybdate 0.17 mg / L, biotin 0.02 mg / L, folic acid 0.02 mg / L, pyridoxine hydrochloride 0.1 mg / L, thiamine 0.05 mg / L, riboflavin 0.05 mg / L, niacin 0.05 mg / L, D-calcium pantothenate 0.05 mg / L, cobalamin 0.001 mg / L, para-aminobenzoic acid 0.05 mg / L, and lipoic acid 0.05 mg / L.

3. The continuous fermentation method of Clostridium aeruginosa based on multi-parameter control according to claim 1, characterized in that, The CO content in the exhaust gas is controlled by adjusting the gas supply rate of the syngas source: when the volume fraction of CO in the exhaust gas is between 5% and 10%, the gas supply rate of the syngas source is controlled at 0.6 L / min; when the volume fraction of CO in the exhaust gas is less than 5%, the gas supply rate of the syngas source is increased; when the volume fraction of CO in the exhaust gas is greater than 10%, the gas supply rate of the syngas source is decreased; wherein, the components of the syngas source, by volume fraction, include 30% CO, 10% CO2, 35% H2, and 25% N2.

4. The continuous fermentation method of Clostridium aeruginosa based on multi-parameter control according to claim 1, characterized in that, The dilution was controlled as follows: starting from 0.5, increasing by 0.2 every 12 hours, gradually increasing to 1.

8.

5. The continuous fermentation method of Clostridium aeruginosa based on multi-parameter control according to claim 1, characterized in that, The parameter control also includes controlling the pH to be between 5.1 and 4.

9.

6. The continuous fermentation method of Clostridium aeruginosa based on multi-parameter control according to claim 1, characterized in that, The volume fraction of CO in the exhaust gas was monitored using a Raman spectroscopy device.

7. The continuous fermentation method of Clostridium aeruginosa based on multi-parameter control according to claim 1, characterized in that, The content of metabolites from continuous fermentation was detected by HPLC. The specific parameters were as follows: hydrogen ion chromatography column, mobile phase: 5 mM H2SO4, column temperature: 30℃, RID detector temperature: 45℃, mobile phase flow rate: 0.9 mL / min, injection volume: 10 µL.

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