Method for improving continuous fermentation efficiency of clostridium aerovorans

Abstract

The invention discloses a method for improving the continuous fermentation efficiency of clostridium aerovorans. According to the method, a continuous fermentation system in which a first fermentation tank and a second fermentation tank are connected in series is adopted, clostridium aerovorans is used as a production strain, and synthesis gas is used as a raw material. The method is characterized in that after thalli in a first fermentation tank enter a stable phase, a continuous process is started, the dilution degree is optimized to be 0.15-0.7, the nutrient concentration of a supplemented fresh culture medium is adjusted to be 0.65-1 time of the standard concentration, and growth and metabolism of the thalli are cooperatively regulated. According to the invention, the thallus delay period of the second fermentation tank is effectively shortened, decayed thalli are replaced, the overall flora activity is enhanced, the continuous fermentation period is obviously prolonged to more than 200 hours, the yields of ethanol and butanol are greatly improved, meanwhile, foaming in the fermentation process is effectively inhibited, and the system is simple in structure, convenient to operate and suitable for industrial application.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Clostridium eatenii fermentation, and particularly relates to a method for improving the continuous fermentation efficiency of Clostridium eatenii. BACKGROUND

[0002] With the increasing depletion of fossil resources and the environmental pollution problems caused thereby, developing green and sustainable energy and chemical production technologies has become a global consensus. Using microbial fermentation technology to convert renewable resources into fuels and chemicals is one of the important ways to realize the transition from a fossil economy to a green economy. Among them, syngas (main components are CO, CO2 and H2) fermentation technology shows great application potential because it can use non-grain resources such as industrial waste gas and urban solid waste gasification gas. Compared with traditional catalytic conversion methods such as Fischer-Tropsch synthesis, syngas biological fermentation has the outstanding advantages of high product specificity, good raw material flexibility, low energy demand and strong tolerance to gas impurities.

[0003] Among syngas fermentation microorganisms, Clostridium is a representative anaerobic bacteria that can directly ferment high-value chemicals such as ethanol and butanol using syngas as carbon and energy sources. However, this technology still faces significant bottlenecks in the process of actual industrial application: first, Clostridium generally has low biomass and a long lag phase after inoculation during fermentation, resulting in slow fermentation start; second, the traditional batch fermentation mode has short cycle and low efficiency, and frequent auxiliary operations such as tank washing, sterilization and inoculation are required, resulting in low equipment utilization; finally, in the middle and late stages of fermentation, with the decline of bacterial activity and the accumulation of metabolic by-products (such as organic acids), the bacterial activity rapidly decreases, accompanied by severe foaming phenomenon, which leads to unstable fermentation process and even interruption.

[0004] To overcome the shortcomings of batch fermentation, continuous fermentation technology was introduced into this field. There are many technical solutions in the prior art for continuous fermentation to produce organic acids or solvents. For example, CN102925495A discloses a method for producing butanol by multi-stage continuous fermentation of sugar raw materials, which uses four or more fermentation tanks in series and controls the dilution rate to improve productivity. However, this method is aimed at sugar fermentation strains and liquid sugar raw materials, and its process conditions and challenges (such as substrate inhibition) are fundamentally different from the gas-feeding Clostridium fermentation based on gaseous substrates, and cannot be directly applied to syngas fermentation systems. CN118207062A provides a packed multi-stage simulated moving bed continuous fermentation system, which enriches bacteria by filling fillers in the tank to form a biofilm, and improves butanol yield by online adsorption of products. Although this scheme mentions gas-feeding Clostridium, its system composition is complex, relies on specific fillers and adsorption devices, has high investment and operating costs, and its starting mode of parallel connection followed by series connection is complicated and not suitable for application scenarios sensitive to operation simplicity and cost. CN102250971A discloses a continuous fermentation method for producing citric acid, which uses multiple fermentation tanks in series and controls the pH and ventilation rate of different tanks. However, this technology is aimed at aerobic Aspergillus niger and citric acid production, and its control parameters (such as pH = 1.3-4.5, forced ventilation) are completely different from the strict anaerobic and neutral pH fermentation environment of gas-feeding Clostridium, and the technical means cannot be borrowed from each other.

[0005] In summary, there is a lack of a continuous fermentation method in the prior art that is specifically designed for the characteristics of Clostridium autoethanogenum syngas fermentation, has a simple system structure, is easy to operate, and can effectively prolong the fermentation period and improve product yield. Therefore, there is an urgent need to develop a new technical solution to solve the key problems of Clostridium autoethanogenum in continuous fermentation, such as maintenance of cell activity, fermentation stability, and production efficiency. SUMMARY

[0006] To overcome the shortcomings of batch fermentation, continuous fermentation technology was introduced into this field. There are many technical solutions in the prior art for continuous fermentation to produce organic acids or solvents. For example, CN102925495A discloses a method for producing butanol by multi-stage continuous fermentation of sugar raw materials, which uses four or more fermentation tanks in series and controls the dilution rate to improve productivity. However, this method is aimed at sugar fermentation strains and liquid sugar raw materials, and its process conditions and challenges (such as substrate inhibition) are fundamentally different from the gas-feeding Clostridium fermentation based on gaseous substrates, and cannot be directly applied to syngas fermentation systems. CN118207062A provides a packed multi-stage simulated moving bed continuous fermentation system, which enriches bacteria by filling fillers in the tank to form a biofilm, and improves butanol yield by online adsorption of products. Although this scheme mentions gas-feeding Clostridium, its system composition is complex, relies on specific fillers and adsorption devices, has high investment and operating costs, and its starting mode of parallel connection followed by series connection is complicated and not suitable for application scenarios sensitive to operation simplicity and cost. CN102250971A discloses a continuous fermentation method for producing citric acid, which uses multiple fermentation tanks in series and controls the pH and ventilation rate of different tanks. However, this technology is aimed at aerobic Aspergillus niger and citric acid production, and its control parameters (such as pH = 1.3-4.5, forced ventilation) are completely different from the strict anaerobic and neutral pH fermentation environment of gas-feeding Clostridium, and the technical means cannot be borrowed from each other.

[0007] The present application is achieved by the following technical solutions:

[0008] A method for improving the efficiency of continuous fermentation of Clostridium autoethanogenum, comprising the following steps:

[0009] Step 1) connecting the first fermentor and the second fermentor in series to form a double-tank continuous fermentation system;

[0010] Step 2) inoculating Clostridium carboxidivorans in the first fermentor and feeding synthetic gas containing CO, CO2 and H2 as carbon source and energy source for fermentation;

[0011] Step 3) when the bacteria in the first fermentor enter the stable growth phase, starting the continuous fermentation process, adding fresh medium to the first fermentor at a dilution rate of 0.15-0.7, and at the same time, transporting the fermentation liquid in the first fermentor to the second fermentor at the same flow rate; by optimizing within the dilution rate range and / or adjusting the nutrient concentration of the fresh medium to 0.65-1 times the standard concentration, the growth of the bacteria and the synthesis of the product are controlled, thereby prolonging the continuous fermentation time and increasing the yield of ethanol and butanol;

[0012] wherein the dilution rate is defined as the ratio of the volume of fresh medium added to the volume of fermentation liquid in the fermentor.

[0013] Preferably, in step 2), the Clostridium carboxidivorans is Clostridium carboxidivorans GG6, with the preservation number CGMCC No. 34975.

[0014] Preferably, in step 2), the synthetic gas consists of 30% CO, 55% CO2 and 15% H2 by volume fraction; the gas flow rate of the synthetic gas is controlled at 50-150 mL / min.

[0015] Preferably, in step 2), the fermentation temperature is controlled at 35-38℃, and the stirring speed is controlled at 80-120 rpm.

[0016] Preferably, in step 3), the dilution rate is 0.5.

[0017] Preferably, in step 3), the nutrient concentration of the fresh medium is 0.7 times the standard concentration.

[0018] Preferably, in step 3), the fresh medium contains the following components: yeast powder 0.5 g / L, ammonium chloride 1 g / L, L-cysteine hydrochloride 0.2 g / L, sodium sulfide 0.2 g / L, potassium dihydrogen phosphate 0.5 g / L, trace element mother liquor 5 mL / L, vitamin solution mother liquor 5 mL / L, and phosphoric acid 1 mL / L.

[0019] Preferably, the trace element mother liquor comprises the following components: 10 g / L nitrilotriacetic acid, 0.5 g / L cobalt chloride hexahydrate, 2 g / L manganese sulfate, 0.1 g / L nickel chloride hexahydrate, 0.2 g / L iron sulfate, 0.1 g / L sodium selenite, 1 g / L zinc sulfate, 0.1 g / L copper chloride, 0.1 g / L sodium molybdate, and 0.1 g / L sodium tungstate.

[0020] Preferably, the vitamin solution mother liquor comprises the following components: 50 mg / L calcium pantothenate, 50 mg / L lipoic acid, 100 mg / L vitamin B6, 50 mg / L thiamine, 50 mg / L vitamin B2, 20 mg / L biotin, 20 mg / L folic acid, 50 mg / L p-aminobenzoic acid, 50 mg / L nicotinic acid, and 50 mg / L vitamin B12.

[0021] The beneficial effects of the present application are as follows:

[0022] (1) The present application effectively promotes the biomass accumulation and metabolic activity of Clostridium carboxidivorans by adopting a two-tank series continuous fermentation system and optimizing the synergistic control strategy of dilution (0.15-0.7) and medium nutrient concentration (0.65x-1x). Experimental data show that the method of the present application can extend the continuous fermentation cycle to more than 200 h and significantly improve the final yield of ethanol and butanol, greatly improving the production efficiency compared with traditional batch fermentation or continuous fermentation with fixed parameters.

[0023] (2) The present application creatively realizes the continuous renewal of decaying bacteria and the stability of the nutritional environment by supplementing fresh medium and replacing the fermentation broth, thereby overcoming the problem of reduced bacterial activity of Clostridium carboxidivorans in long-term fermentation. At the same time, by optimizing the nutrient concentration of the feed medium to 0.7x, the foaming phenomenon caused by excessive biomass is effectively inhibited while ensuring product accumulation, ensuring the long-term stable operation of the fermentation process and laying a foundation for industrialized continuous production.

[0024] (3) The present application only uses two fermentation tanks in series, and the system configuration is extremely simple, without the need for expensive additional equipment, greatly reducing the initial investment cost and the difficulty of later maintenance. At the same time, the simplified process also makes the operation control more convenient and reliable, easy to realize industrialization and application. The method of the present application is specially designed for Clostridium carboxidivorans fermentation system with syngas as substrate. Through specific dilution control and nutrition optimization, the core pain points of slow bacterial growth, short cycle, and easy foaming in this system are directly addressed, providing an efficient and special solution for the gas fermentation field.

[0025] (4) The present application determines the key process parameter range and optimal value (such as optimal dilution 0.5, optimal nutrient concentration 0.7x) through a large number of experiments, which is clear and controllable, and is convenient for automatic monitoring and adjustment in actual production through conventional sensors and control systems (such as pH electrode, peristaltic pump), ensuring the reproducibility and stability of the process. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The schematic diagram of the fermentation device used in the method for improving the continuous fermentation efficiency of Clostridium alimentans of the present application;

[0027] Figure 1 Medium: 1, anaerobic culture medium storage tank; 2, first fermentation tank; 3, second fermentation tank; 4, peristaltic pump;

[0028] Figure 2 The biomass graph of the first fermentation tank and the second fermentation tank after adopting the series fermentation process in Example 1;

[0029] Figure 3 The product result graph of the first fermentation tank and the second fermentation tank after adopting the series fermentation process in Example 1: A is the first fermentation tank; B is the second fermentation tank;

[0030] Figure 4 The microscope graph of the first fermentation tank and the second fermentation tank after adopting the series fermentation process in Example 1: A is the first fermentation tank; B is the second fermentation tank. DETAILED DESCRIPTION

[0031] The present application will be further described in detail below in combination with the drawings and specific examples.

[0032] Unless otherwise specified, the technical means used in the following examples are conventional means known to those skilled in the art, and the experimental methods not specified are conventional methods in the art.

[0033] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0034] The definition of dilution in the following examples: the ratio of the volume of fresh medium supplement to the volume of fermentation broth in the fermentation tank.

[0035] Example 1

[0036] A method for improving the continuous fermentation efficiency of Clostridium alimentans, such as Figure 1As shown, a dual-tank series fermentation method is adopted. The first fermenter 2 cultivates the bacteria and, after reaching stability, begins to discharge the mash into the second fermenter 3, thus shortening the growth time of the strains in the second fermenter 3. The first fermenter 2 is adjusted using fresh culture medium in the anaerobic culture medium storage tank 1, while the second fermenter 3 ferments simultaneously, thereby increasing the continuous fermentation time and product yield.

[0037] The fermentation apparatus used in this embodiment is as follows: Figure 1 As shown, the system includes an anaerobic culture medium storage tank 1, a first fermenter 2, and a second fermenter 3. The size and volume of the anaerobic culture medium storage tank 1 are configured according to the fermentation volume. The tank opening has three connections: an air inlet, an air outlet, and a culture medium outlet. Nitrogen gas is introduced through the air inlet to replace the gas inside the tank, ensuring an anaerobic environment. The culture medium outlet is connected to the first fermenter 2 via a rubber hose, and a peristaltic pump 4 is used to adjust the process during fermentation. The first fermenter 2 and the second fermenter 3 have identical structures, with their size and volume determined by the fermentation scale. They are equipped with a stirring paddle, temperature control, pH control, and an oxidation-reduction potential (ORP) device to control temperature, pH, and ORP during fermentation. The upper end of the fermenter has an air inlet, an air outlet, a culture medium replenishment inlet, and a fermentation discharge outlet. The culture medium replenishment inlet of the first fermenter 2 is connected to the culture medium outlet of the anaerobic culture medium storage tank 1, and its fermentation discharge outlet is connected to the culture medium replenishment inlet of the second fermenter 3. All interfaces are connected via a peristaltic pump 4. The fermentation broth from the first fermenter 2 is discharged into the second fermenter 3, and then fresh culture medium is added to continue fermentation. Because the strains from the first fermenter 2 are in good condition, the fermentation delay time in the second fermenter 3 is shortened, and the yield of the product is increased, thus extending the continuous fermentation time.

[0038] 1. Experimental Methods

[0039] (1) Culture of strains

[0040] One mL of frozen Clostridium carboxidivorans GG6 (deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, June 23, 2025, accession number: CGMCC No. 34975) was taken from -80℃ and transferred entirely to a 4 mL test tube. The culture was then anaerobic at 37℃ for 2–3 days. The grown strain was then inoculated at a 3% inoculation rate into a blue glass bottle containing 200 mL of culture medium and anaerobically cultured at 37℃ for 16–18 h.

[0041] (2) Constructing a fermentation system

[0042] In 1 L fermentation system, add yeast powder 0.5 g, ammonium chloride 1 g, L-cysteine hydrochloride 0.2 g, sodium sulfide 0.2 g, potassium dihydrogen phosphate 0.5 g, trace element mother liquor 5 mL, vitamin solution mother liquor 5 mL, phosphoric acid 1 mL, 121 ℃ sterilization for 20 min.

[0043] Trace element mother liquor (1 L): nitrilotriacetic acid 10 g, cobalt chloride hexahydrate 0.5 g, manganese sulfate 2 g, nickel chloride hexahydrate 0.1 g, iron sulfate 0.2 g, sodium selenite 0.1 g, zinc sulfate 1 g, copper chloride 0.1 g, sodium molybdate 0.1 g, sodium tungstate 0.1 g.

[0044] Vitamin mother liquor (1 L): calcium pantothenate 50 mg, thioctic acid 50 mg, vitamin B6 100 mg, thiamine 50 mg, vitamin B2 50 mg, biotin 20 mg, folic acid 20 mg, p-aminobenzoic acid 50 mg, nicotinic acid 50 mg, vitamin B12 50 mg.

[0045] After sterilization, replace the gas in the first fermentation tank 2 and the second fermentation tank 3 and the anaerobic medium storage tank 1 with nitrogen gas from the gas inlet, wait for the temperature to drop to about 37 ℃, install the stirring controller and pH electrode, close the nitrogen gas connected to the fermentation tank, and then introduce simulated synthesis gas (volume fraction: 30% CO, 55% CO2, 15% H2) into the first fermentation tank from the gas inlet at a flow rate of 100 mL / min, and control the fermentation temperature at 37 ℃ and the stirring speed at 100 rpm.

[0046] (3) After the fermentation system is constructed, 100 mL of the above-mentioned Clostridium alimentans cultured to the logarithmic growth phase is inoculated into the first fermentation tank 2, and samples are taken at regular intervals for determination of biomass and fermentation products.

[0047] (4) When the first fermentation tank 2 reaches the stationary phase, start the first fermentation tank 2 discharge and simultaneously add fresh medium to it at a dilution rate of 0.15, and then discharge the mash into the second fermentation tank 3 at the same flow rate through the peristaltic pump 4 to start fermentation.

[0048] The biomass is determined by ultraviolet spectrophotometry, and the absorbance at 600 nm is measured. The fermentation product is determined by liquid chromatography using an Amine HPX-87H column (7.8 mm x 300 mm) purchased from Bio-Rad Company. The chromatographic conditions are as follows: the mobile phase is 5 mM dilute sulfuric acid, the injection volume is 10 µL, the column temperature is 30 ℃, the sample room temperature is 25 ℃, the flow rate is 0.9 mL / min, the elution time is 30 min, and the elution mode is isocratic elution.

[0049] 2、Experimental results

[0050] As Figure 2 shown, the first fermenter was fermented for 24 h, and the biomass was only 0.223. The strain began to recover at 72 h, and the biomass reached 1.26. At the same time, continuous fermentation was started, and the fermentation liquid of the first fermenter was directly flowed into the second fermenter at a dilution of 0.15. After the cascade of the two fermenters, the strain in the second fermenter could proliferate rapidly, and the biomass could reach about 1.0 at 24 h, and the product was accumulated more, as shown in B of Figure 3 , the highest ethanol in the second fermenter could reach 6.96 g / L at 144 h, and the butanol could also reach 2.45 g / L. After the continuous fermentation was started, the fermentation period was prolonged to 200 h, and as shown in Figure 4 , the strain in the first fermenter and the second fermenter was observed under a microscope, and it was found that the strain morphology was relatively healthy, which was undoubtedly of reference significance for industrial production.

[0051] Example 2

[0052] A method for improving the continuous fermentation efficiency of Clostridium autoethanogenum, the medium preparation method and steps, and the device connection method and operation steps are consistent with those of Example 1, except that the first fermenter process is explored to explore the dilution limit of the first fermenter in continuous fermentation. The strain is fermented for a long time, and the nutrients in the medium will also be depleted under the condition of strain growth. If you want to maintain long-term continuous fermentation of the strain, you can supplement fresh medium while discharging part of the fermentation liquid to improve the activity of the strain, maintain the biomass and increase the product.

[0053] By adjusting different dilutions (0.15, 0.3, 0.4, 0.5, 0.6, 0.7), the growth of the strain and the product results in the continuous fermentation process were explored, and the experimental results are shown in Table 1.

[0054] Table 1 Influence of dilution on fermentation performance

[0055]

[0056] Table 1 Note: The first fermenter was continuously started when the strain grew up at 48 h.

[0057] As shown in Table 1, as the dilution increased, the fresh medium was injected into the fermenter, and the activity of the strain increased. The biomass in the first fermenter increased all the time. When the dilution was 0.5, the continuous production capacity of the fermenter reached the highest, the biomass of the strain in the first fermenter was 4.32 at 216 h, the ethanol was 6.43 g / L, and the butanol reached 3.02 g / L. The biomass in the second fermenter was 7.24 at 168 h, the ethanol was 9.12 g / L, and the butanol reached 5.31 g / L. When the dilution was further increased to 0.6, the biomass decreased, and the OD600 decreased to 4.02, and with further dilution, OD 600 continued to decrease, so we chose dilution 0.5 in consideration of all factors.

[0058] Example 3

[0059] A method for improving the efficiency of continuous fermentation of Clostridium autoethanogenum, the medium preparation method and steps, and the device connection method and operation steps are consistent with Example 1, the difference is that the first fermentation tank process is explored, and the fresh medium nutrient composition adjustment of the first fermentation tank under continuous fermentation is explored. Because in the fermentation process, with the increase of biomass, acetic acid and butyric acid will also increase, and high acid in the later stage will cause rancidity and affect the continuous fermentation of the strain, and the higher the biomass, the more the foaming phenomenon in the fermentation process, affecting the mass transfer of fermentation, leading to the collapse of the fermentation system, the biomass of the strain decreases, and the product result also shows a downward trend. The purpose of continuous fermentation is also to ensure that the strain can be stably fermented for a long time. The current medium composition is relatively rich, so gradually reducing the medium nutrition without affecting the product accumulation is adopted to control OD 600 , and reduce the foaming phenomenon.

[0060] Adjust the coefficient of the fresh medium added, and prepare the medium according to 1x, 0.9x, 0.8x, 0.7x, 0.65x of the standard concentration. The experimental results are shown in Table 2.

[0061] Table 2 Influence of fresh medium nutrient concentration on fermentation performance

[0062]

[0063] As can be seen from Table 2, after reducing the nutrition, the OD 600 of the first fermentation tank will be affected, when the nutrient concentration is adjusted to 0.7x, the biomass of the first fermentation tank is 3.3 after 216 h of fermentation, the foaming phenomenon is obviously reduced, and the content of ethanol and butanol also decreases slightly, the ethanol content is 5.38 g / L, and the butanol content is 2.26 g / L. The biomass of the second fermentation tank is 5.59 after 168 h of fermentation, the ethanol content is 8.801 g / L, and the butanol content is 4.46 g / L. Continue to reduce the nutrient concentration to 0.65x, the biomass and product decrease more, the OD 600 of the first fermentation tank is 2.1 after 264 h of fermentation, the ethanol is 2.28 g / L, and the butanol is 1.02 g / L. The biomass of the second fermentation tank is reduced to 4.308 after 216 h of fermentation, the ethanol content is 8.003 g / L, and the butanol content is 3.869 g / L. In addition to reducing the risk of foaming, continuous fermentation also needs to consider that the longer the fermentation time, the higher the final product yield should be kept at a relatively high level, so the nutrient concentration is selected to be 0.7x of the standard concentration in consideration of all factors.

[0064] The embodiments described above are only some of the embodiments of the present application, not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but merely represents selected embodiments of the application. The scope of protection of the present application is defined by the scope of the claims, and all other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present application without creative labor fall within the scope of protection of the present application.

Claims

1. A method for improving the continuous fermentation efficiency of Clostridium aeruginosa, characterized in that, Includes the following steps: Step 1) Connect the first fermenter and the second fermenter in series to form a two-tank continuous fermentation system; Step 2) Inoculate Clostridium aerobicans into the first fermenter and introduce syngas containing CO, CO2 and H2 as carbon and energy sources for fermentation; Step 3) Once the cells in the first fermenter have entered the stable growth phase, start the continuous fermentation process. Add fresh culture medium to the first fermenter at a dilution of 0.15 to 0.7, and simultaneously transfer the fermentation broth from the first fermenter to the second fermenter at the same flow rate. By optimizing within the dilution range and / or adjusting the nutrient concentration of the fresh culture medium to 0.65 to 1 times the standard concentration, control cell growth and product synthesis, thereby extending the continuous fermentation time and increasing the yield of ethanol and butanol. The dilution is defined as the ratio of the volume of fresh culture medium added to the volume of fermentation broth in the fermenter.

2. The method for improving the continuous fermentation efficiency of Clostridium aeruginosa according to claim 1, characterized in that, Step 2) The aerobic Clostridium carboxidivorans is Clostridium carboxidivorans GG6, with accession number CGMCC No. 34975.

3. The method for improving the continuous fermentation efficiency of Clostridium aeruginosa according to claim 1, characterized in that, Step 2) The synthesis gas, by volume fraction, consists of 30% CO, 55% CO2 and 15% H2; the gas flow rate of the synthesis gas is controlled at 50~150 mL / min.

4. The method for improving the continuous fermentation efficiency of Clostridium aeruginosa according to claim 1, characterized in that, Step 2) The fermentation temperature is controlled at 35~38℃, and the stirring speed is controlled at 80~120 rpm.

5. The method for improving the continuous fermentation efficiency of Clostridium aeruginosa according to claim 1, characterized in that, Step 3) The dilution is 0.

5.

6. The method for improving the continuous fermentation efficiency of Clostridium aeruginosa according to claim 1, characterized in that, Step 3) The nutrient concentration of the fresh culture medium is 0.7 times the standard concentration.

7. The method for improving the continuous fermentation efficiency of Clostridium aeruginosa according to claim 1, characterized in that, Step 3) The fresh culture medium contains the following components: yeast extract 0.5 g / L, ammonium chloride 1 g / L, L-cysteine ​​hydrochloride 0.2 g / L, sodium sulfide 0.2 g / L, potassium dihydrogen phosphate 0.5 g / L, trace element stock solution 5 mL / L, vitamin solution stock solution 5 mL / L, and phosphate 1 mL / L.

8. The method for improving the continuous fermentation efficiency of Clostridium aeruginosa according to claim 7, characterized in that, The trace element mother liquor contains the following components: 10 g / L nitrilotriacetic acid, 0.5 g / L cobalt chloride hexahydrate, 2 g / L manganese sulfate, 0.1 g / L nickel chloride hexahydrate, 0.2 g / L ferric sulfate, 0.1 g / L sodium selenite, 1 g / L zinc sulfate, 0.1 g / L copper chloride, 0.1 g / L sodium molybdate, and 0.1 g / L sodium tungstate.

9. A method for improving the continuous fermentation efficiency of Clostridium aeruginosa according to claim 7, characterized in that, The vitamin solution stock solution contains the following components: calcium pantothenate 50 mg / L, lipoic acid 50 mg / L, vitamin B6 100 mg / L, thiamine 50 mg / L, vitamin B2 50 mg / L, biotin 20 mg / L, folic acid 20 mg / L, para-aminobenzoic acid 50 mg / L, nicotinic acid 50 mg / L, and vitamin B12 50 mg / L.

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