A method for producing hexanoic acid by enhancing the biocarbon chain lengthening reaction with a recyclable biochar-based material

Abstract

The application discloses a method for producing hexanoic acid by recycling biochar-based material to strengthen the carbon chain lengthening reaction, wherein magnetic Fe3O4 nanoparticles are loaded on the surface of biochar by a chemical deposition method to endow the biochar with recyclable characteristics, significantly reduce the addition frequency and cost of the biochar, and further improve the yield of hexanoic acid compared with non-recyclable biochar. The application has the advantages of simple synthesis method, low raw material price, wide adaptability, no need to use pure bacterial strains, and strong anti-interference ability of the strengthened system, and can meet the demand of high-value utilization of putrescible organic solid waste. Moreover, the recycled biochar still has excellent strengthening performance, solves the problems of high addition amount, difficulty in recycling and relatively poor promotion effect in the traditional carbon material strengthening system, significantly improves the economy, and provides a new technical approach for the high-value utilization of putrescible organic solid waste.

Description

Technical fields:

[0001] This invention relates to a method for enhancing the production of hexanoic acid through the reaction of biochar chain lengthening using recyclable biochar-based materials. Background technology:

[0002] Medium-chain fatty acids are a class of straight-chain monocarboxylic acids with 6-12 carbon atoms. They possess high energy density and strong hydrophobicity, and can be directly used as antibacterial agents and food additives. They are also important platform compounds that can be further converted into high-value-added products such as pharmaceuticals, fragrances, lubricants, rubber, dyes, and biofuels. Hexanoic acid, a representative medium-chain fatty acid, is traditionally produced using animal and vegetable oils or petroleum resources, but the high cost of raw materials and equipment limits its large-scale application. In recent years, the technical route for synthesizing hexanoic acid through anaerobic fermentation of biodegradable organic solid waste (such as crop straw, livestock and poultry manure, and sewage sludge) has attracted widespread attention.

[0003] However, the efficiency of bio-carbon chain elongation in the synthesis of hexanoic acid is mainly limited by the metabolic activity of functional microorganisms. Studies have shown that the activity of functional microorganisms is easily inhibited by the toxicity of fermentation substrates and products; excessively high substrate or product concentrations can significantly reduce microbial activity, thus affecting hexanoic acid yield. Furthermore, the low electron transfer efficiency between organic matter in the substrate and functional microorganisms, as well as the disorderly competition from side reactions, also severely restrict the production efficiency of hexanoic acid. Therefore, developing effective technical means to enhance the bio-carbon chain elongation reaction to improve hexanoic acid yield has become a key issue that urgently needs to be addressed in the industry.

[0004] Biochar, as a novel functional material, exhibits significant advantages in promoting the activity of functional microorganisms, enhancing electron transfer efficiency, and inhibiting competitive reactions. In the prior art, CN117701644A discloses a method for producing medium-chain fatty acids from crop straw, achieving in-situ production of ethanol and acetic acid through two-step fermentation, providing electron donors and acceptors for carbon chain elongation without the need for exogenous addition; the preparation of biochar from the solid residue of straw fermentation effectively increases hexanoic acid yield, but this biochar is unrecoverable and requires continuous addition. CN116640808A discloses a method for converting methane fermentation into medium-chain fatty acid fermentation, adding biochar loaded with anthraquinone-2-carboxylic acid at a final concentration of 5–10 g / L, but the extremely high price of anthraquinone-2-carboxylic acid (150,000,000-200,000,000 RMB / ton for industrial grade) and the unrecoverable nature of the loaded biochar greatly reduce the economic viability of this invention. CN118497292A discloses a method for enhancing the single-phase synthesis of hexanoic acid from agricultural waste through anaerobic fermentation. The method involves mixing agricultural waste, alginate microsphere hydrogel, and modified biochar, followed by anaerobic fermentation to obtain hexanoic acid. The modified biochar comprises biochar and sulfite loaded onto the biochar. Sulfite-modified biochar improves the synthesis efficiency of hexanoic acid, but this invention also suffers from the drawback of excessively high biochar addition (generally exceeding 10 g / L) and the inability to recycle it. In addition, CN118272458A discloses a method for producing medium-chain fatty acids by anaerobic fermentation of microorganisms with biochar-supported zero-valent iron immobilized. This method uses sludge as a substrate and mainly promotes the hydrolysis and acidification stage of sludge through the strong reducing properties of zero-valent iron, producing more small molecule acids. Then, by adding electron donor ethanol, the above-mentioned small molecule acids and ethanol are converted into hexanoic acid under the action of functional microorganisms, thereby increasing the yield of hexanoic acid. However, zero-valent iron is a consumable, and its high cost (6,000-20,000 yuan / ton for industrial grade) and non-recyclable characteristics seriously restrict the economic feasibility of the technology.

[0005] Addressing the technical bottlenecks of unrecyclable biochar and the cumbersome and costly nature of traditional biochar modification methods, developing a method for producing hexanoic acid based on the enhanced biochar chain lengthening reaction using recyclable biochar has significant practical implications and application value. Summary of the Invention:

[0006] The purpose of this invention is to provide a method for enhancing the production of hexanoic acid through the reaction of biochar chain lengthening using recyclable biochar-based materials, thereby solving the problem of the inability to recycle and utilize biochar in the prior art.

[0007] This invention is achieved through the following technical solutions:

[0008] A method for enhancing the production of hexanoic acid through a biochar chain-lengthening reaction using recyclable biochar-based materials, the method comprising the following steps:

[0009] 1) Modify biochar particles by loading Fe3O4 nanoparticles onto their surface to prepare magnetic modified biochar that is easy to recycle.

[0010] 2) Add the modified biochar obtained in step 1) to the domesticated inoculum, add a culture medium solution containing ethanol and acetic acid, and construct a fermentation system for producing hexanoic acid with an extended biochar chain. The reaction temperature is 30℃~40℃ and the pH is 5~7. After the reaction is completed, the modified biochar is recovered and recycled.

[0011] Preferably, the biochar particles have a particle size of 75 μm to 300 μm, more preferably 75 μm to 150 μm. The preparation method of the biochar particles is as follows: the biomass raw material is crushed, dried, and pyrolyzed at 400℃ to 900℃ (preferably 400℃ to 800℃) under an inert atmosphere, followed by grinding and sieving. The biomass raw material includes one or more of anaerobic fermentation residue, agricultural and forestry waste, and livestock and poultry manure.

[0012] Preferably, step 1) specifically involves: placing biochar particles in an aqueous solution and stirring to mix them evenly to obtain a biochar solution; then adding ferrous salts and ferric salts, and adding ammonia solution to maintain the pH of the solution between 9 and 10, stirring the reaction, and after the reaction is completed, separating the solid and liquid, washing the precipitate and drying it at 60°C to 110°C in an inert atmosphere to obtain recyclable biochar.

[0013] Preferably, in step 1), the reaction is carried out in an inert atmosphere, the reaction temperature is maintained at 70°C, the molar ratio of ferrous ions to trivalent ions is 2:1 to 3:1, and the reaction time is 60 to 180 min.

[0014] Preferably, the solution solid-liquid separation method in step 1) includes centrifugation, static precipitation, and magnetic attraction.

[0015] Preferably, in step 2), the mass ratio of modified biochar to acclimatized inoculum is 1:16 to 2:1, the molar ratio of ethanol to acetic acid is 1:1 to 5:1, and the total carbon atom concentration is 20 mM to 1000 mM.

[0016] Preferably, step 2) involves recovering the modified biochar by using a strongly magnetic object to absorb it, washing it with an aqueous solution, and then drying it in air at 60°C to 110°C to obtain the recovered biochar.

[0017] The specific method for obtaining the domesticated inoculum in step 2) is as follows:

[0018] a. Pretreatment of anaerobic microbial inoculum to inhibit the activity of methanogens;

[0019] b. The anaerobic microbial inoculum after the pretreatment in step a is mixed with the culture medium solution and inoculated into the reactor. Ethanol and acetic acid are added as substrates, and the mixture is acclimatized and cultured at 30℃~40℃ and pH 5~6. The sources of anaerobic microbial inoculum include municipal anaerobic sludge, sewer sludge, effluent from anaerobic fermenters, and one or more mixtures of livestock and poultry manure.

[0020] Step a involves pretreating the anaerobic microbial inoculum using one or more of the following methods: (1) autoclaving at 100℃ to 120℃ for 10 min to 60 min; (2) adding 5 g / L to 20 g / L of sodium 2-bromoethylsulfonate (2-BES); and (3) adjusting the pH of the inoculum solution to 5 to 6.

[0021] Preferably, 1 L of culture medium solution contains: 1450 mg K2HPO4, 1910 mg NH4Cl, 1020 mg MgCl2, 734 mg CaCl2·2H2O, 110 mg Na2SO4, 6700 mg KCl, 9.6 mg FeCl2, 0.7 mg ZnCl2, 1.3 mg MnSO4·H2O, 0.06 mg H3BO3, 1.9 mg CoCl2·6H2O, 0.02 mg CuCl2·2H2O, 0.24 mg NiCl2·6H2O, 0.31 mg Na2MoO4·2H2O, 0.03 mg Na2SeO3·5H2O, 1 mg glutamic acid, 0.25 mg ascorbic acid, 0.25 mg riboflavin, 0.2 mg citrate monohydrate, 0.1 mg folic acid, 0.1 mg para-aminobenzoic acid, and 0.25 mg cysteine.

[0022] Preferably, in step b, the volatile solids content of the inoculum after mixing the pretreated anaerobic microbial inoculum with the culture medium solution is 0.5% to 5%, more preferably 0.5% to 3%.

[0023] Preferably, in step b, the molar ratio of ethanol to acetic acid is 1:1 to 5:1, and the total carbon atom concentration is 20 mM to 550 mM.

[0024] Preferably, the criteria for judging the successful acclimatization and cultivation of the inoculum in step b are 5-30 days, hexanoic acid carbon atom conversion rate ≥10%, and no methane production.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] 1. The biochar raw materials used in this invention are widely available, and the biochar synthesis method is simple and does not require high temperature and high pressure conditions. The chemical raw materials used are inexpensive, such as industrial-grade ferrous sulfate (heptahydrate) at 800-1,200 yuan / ton and ferric sulfate (nonahydrate) at 2,000-2,800 yuan / ton. No precious metals or rare elements are required, and the modified biochar material can be recycled, solving the problem that traditional carbon material strengthening systems with high addition levels (>10g / L) cannot be recycled.

[0027] 2. The anaerobic microbial strains for producing hexanoic acid in this invention are widely available and can be obtained directly through cultivation and domestication without the need for expensive pure cultures. The inoculum after cultivation and domestication has strong resistance to interference, and the reaction is carried out at room temperature and pressure.

[0028] 3. The method proposed in this invention achieves recyclability of biochar materials while further improving hexanoic acid production efficiency compared to traditional biochar. More importantly, the promoting effect of the recycled biochar material remains unaffected, thus balancing recyclability, stability, and superior performance. Furthermore, the ethanol and acetic acid used can be obtained through other mature anaerobic fermentation processes, fulfilling the need for high-value utilization of biodegradable organic solid waste.

[0029] In summary, this invention loads magnetic Fe3O4 nanoparticles onto the surface of biochar via chemical deposition, endowing the biochar with recyclable properties. This significantly reduces the frequency and cost of biochar addition, while further improving the hexanoic acid yield compared to traditional biochar. This invention features a simple synthesis method, inexpensive raw materials, wide applicability to various systems, no need for pure microbial strains, and strong anti-interference capabilities in the enhanced system, meeting the needs for high-value utilization of biodegradable organic solid waste. Furthermore, the recovered biochar retains excellent enhancement properties, solving the problems of high addition amounts, difficult recovery, and relatively poor promoting effects in traditional char material enhancement systems, significantly improving economic efficiency and providing a new technical approach for the high-value utilization of biodegradable organic solid waste. Attached image description:

[0030] Figure 1 This is a process flow diagram of the present invention. Detailed implementation method:

[0031] The following is a further description of the invention, but not a limitation thereof.

[0032] Example 1:

[0033] (a) Biochar preparation: Waste wood was crushed to a size of 2-3 cm and dried at 105°C for 6 hours; then pyrolyzed at 700°C in an oxygen-free atmosphere for 1 hour, followed by grinding and sieving to obtain biochar particles with a particle size of 75 μm-150 μm.

[0034] (b) Biochar modification: Weigh 3.29 g of biochar particles, disperse them in 400 mL of deionized water, and stir until homogeneous; add 9.8 mmol FeSO4·7H2O and 4.9 mmol FeCl3·6H2O sequentially, and stir for 10 minutes; slowly add 11 mL of 25% ammonia solution, at which point the pH is 9.4; react for 70 minutes under a nitrogen atmosphere and at 70 °C; after the reaction is complete, let the precipitate stand for 30 minutes, discard the supernatant, and wash the precipitate three times with deionized water; dry at 105 °C for 12 hours under vacuum to obtain recyclable biochar.

[0035] (c) Inoculum pretreatment: Anaerobic microbial inoculum was taken from the anaerobic reactor used to treat cheese wastewater. The pH of the inoculum was adjusted to 5.5 to inhibit the activity of methanogens.

[0036] (d) Inoculum Acclimation and Cultivation: Pretreated inoculum and culture medium solution (1L culture medium solution contains: 1450mg K2HPO4, 1910mg NH4Cl, 1020mg MgCl2, 734mg CaCl2·2H2O, 110mg Na2SO4, 6700mg KCl, 9.6mg FeCl2, 0.7mg ZnCl2, 1.3mg MnSO4·H2O, 0.06mg H3BO3, 1.9mg CoCl2·6H2O, 0.02mg CuCl2·2H2O, 0.24mg NiCl2·6H2O, 0.31mg Na2MoO4·2H2O, 0.03mg... Na2SeO3·5H2O, 1 mg glutamic acid, 0.25 mg ascorbic acid, 0.25 mg riboflavin, 0.2 mg citric acid monohydrate, 0.1 mg folic acid, 0.1 mg para-aminobenzoic acid, and 0.25 mg cysteine ​​were mixed, and the volatile solids mass fraction of the mixed inoculum was 2.7%. Ethanol and acetic acid with a total carbon atom concentration of 100 mM and a molar ratio of 2:1 were added. The mixture was acclimated for 15 days at 37℃, pH 5.5, and a rotation speed of 150 rpm. After acclimation, the carbon atom conversion rate of hexanoic acid reached 20%, and no methane was produced, indicating that the acclimation was successful.

[0037] (e) Construction of the fermentation system: Recyclable biochar and domesticated inoculum were added to the reactor at a mass ratio of 2:1; ethanol and acetic acid with a total carbon number concentration of 522 mM and a molar ratio of 2:1 were added; the reaction was carried out at 37 °C, pH 6.0, and 150 rpm; the concentration of hexanoic acid was determined by gas chromatography. After 13 days of reaction, the hexanoic acid yield reached a maximum of 56.0 mM, and the carbon atom conversion rate of hexanoic acid was 64.4%. The sample preparation and detection methods were based on the literature [Wu, B.; Lin, R.; Gu, J.; Yuan, H.; Murphy, JDB Biochar confers significant microbial resistance to ammoniatoxicity in n-caproic acid production. Water Res. 2024, 266, 122367.].

[0038] (f) Recovery of biochar material: A magnet is placed at the bottom of the reactor, and the biochar material at the bottom of the reactor is collected after the solution inside the reactor is poured out. The above steps are repeated twice for the poured-out solution to obtain recyclable biochar with a recovery rate of over 95%.

[0039] (g) Enhanced production of hexanoic acid by lengthening the biochar chain after recycling: The experimental conditions in this step are the same as those in step (c), except that the biochar is replaced with the recyclable biochar recycled in step (f); after 25 days of reaction, the hexanoic acid yield reaches the maximum value of 51.2 mM, and the carbon atom conversion rate of hexanoic acid is 58.9%.

[0040] Table 1 Comparison of key indicators for hexanoic acid production enhanced by recyclable biochar and increased biochar chain length. *

[0041]

[0042]

[0043] * The data in the examples are all average values ​​obtained from three repeated measurements.

[0044] Comparative Example 1:

[0045] Referring to Example 1, the difference is the absence of step (b), and the biochar recoverable in step (e) is replaced with the biochar obtained in step (a). In this comparative example, after 10 days of reaction, the hexanoic acid yield reached a maximum of 34.3 mM, with a carbon atom conversion rate of 39.4%. The biochar after the reaction could not be recovered using a magnet.

[0046] Comparative Example 2:

[0047] Referring to Example 1, the difference is that FeCl3·6H2O was not added in step (b). After 10 days of reaction, the hexanoic acid yield reached a maximum of 31.3 mM, with a carbon atom conversion rate of 36.0%. The resulting biochar could not be recovered using a magnet.

[0048] Comparative Example 3:

[0049] Referring to Example 1, the difference is that no biochar material is added in step (e). In this comparative example, after 38 days of reaction, the hexanoic acid yield reached a maximum of 19.4 mM, with a carbon atom conversion rate of 22.3%.

[0050] By comparing steps (e) and (g) of Example 1 with Comparative Examples 1 and 3, it can be seen that the addition of unmodified biochar resulted in a significantly higher hexanoic acid yield compared to the group without added biochar. By loading magnetic Fe3O4 nanoparticles onto the surface of biochar through chemical deposition, the biochar was modified, not only acquiring recyclable properties but also exhibiting a higher hexanoic acid yield than the unmodified biochar group. Furthermore, the recyclable biochar can be easily recovered and reused using magnets, demonstrating a stable promoting effect. This is because the biochar loaded with Fe3O4 nanoparticles has higher electrical conductivity and specific surface area. By enhancing intracellular and extracellular electron transport in microorganisms, it improves the electron transport activity of the respiratory chain in functional microorganisms and the expression of related genes, thereby directionally promoting the hexanoic acid production reaction of ethanol and acetic acid and reducing other side reactions. This indicates that the present invention, while endowing biochar with recyclability, further improves the hexanoic acid yield-promoting effect of biochar.

[0051] A comparison of Example 1 and Comparative Examples 1-2 shows that the biochar obtained by simply adding ferrous salt in step (b) of biochar modification not only failed to achieve recovery characteristics but also reduced the yield of hexanoic acid. This indicates that the present invention requires the simultaneous addition of both ferrous and ferric salts to achieve the unexpected technical effect. Furthermore, a comparison of steps (e) and (g) of Example 1 and Comparative Examples 2 and 3 shows that the recyclable biochar achieved a significant promoting effect, which further demonstrates the superiority of the present invention.

[0052] Example 2:

[0053] Referring to Example 1, the difference lies in that the biochar feedstock in step (a) is taken from the sludge of an anaerobic willow pulp methanogenization reactor; the pyrolysis temperature is 400°C; and in step (e), ethanol and acetic acid are added in a mixture with a total carbon atom concentration of 544 mM and a molar ratio of 2:1. In this example, after 7 days of reaction, the hexanoic acid yield reached its maximum of 54.7 mM, and the carbon atom conversion rate of hexanoic acid was 60.3%.

[0054] Example 3:

[0055] Referring to Example 1, the difference lies in that the biochar feedstock in step (a) is taken from the sludge of an anaerobic willow pulp methanogenization reactor; the pyrolysis temperature is 800℃; and in step (e), ethanol and acetic acid with a total carbon atom concentration of 594 mM and a molar ratio of 2:1 are added. In this example, after 7 days of reaction, the hexanoic acid yield reached its maximum value of 58.0 mM, and the carbon atom conversion rate of hexanoic acid was 58.6%.

[0056] Example 4:

[0057] Referring to Example 1, the difference lies in that, in step (c), the pH of the inoculum was adjusted to 5.5 and 10.5 g / L of sodium 2-bromoethylsulfonate was added during the anaerobic microbial inoculum pretreatment; in step (e), the mass ratio of recyclable biochar to successfully acclimatized inoculum was 1:16; and the total carbon number concentration of ethanol and acetic acid was 532 mM, with a molar ratio of 2:1. In this example, after 10 days of reaction, the hexanoic acid yield reached its maximum of 27.8 mM, and the carbon atom conversion rate of hexanoic acid was 31.4%.

[0058] Example 5:

[0059] Referring to Example 1, the difference lies in that, in step (c), the pH of the inoculum was adjusted to 5.5 and 10.5 g / L of sodium 2-bromoethylsulfonate was added during the pretreatment of the anaerobic microbial inoculum; and in step (e), the total carbon number concentration of ethanol and acetic acid was 450 mM, with a molar ratio of 1:1. In this example, after 7 days of reaction, the hexanoic acid yield reached its maximum of 33.4 mM, and the carbon atom conversion rate of hexanoic acid was 44.5%.

[0060] Example 6:

[0061] Referring to Example 1, the difference lies in that, in step (c), the pH of the inoculum was adjusted to 5.5 and 10.5 g / L of sodium 2-bromoethylsulfonate was added during the pretreatment of the anaerobic microbial inoculum; and in step (e), the total carbon number concentration of ethanol and acetic acid was 344 mM, with a molar ratio of 5:1. In this example, after 7 days of reaction, the hexanoic acid yield reached its maximum of 38.9 mM, and the carbon atom conversion rate of hexanoic acid was 67.8%.

[0062] By comparing Examples 2-6 with Comparative Examples 1-3, it can be seen that the method proposed in this invention can achieve significant promoting effects under different addition amounts, different biochar sources, different ethanol to acetic acid molar ratios, and different ethanol to acetic acid addition amounts. This further demonstrates that this invention has the characteristics of wide applicability and high economy.

Claims

1. A method for enhancing the production of hexanoic acid through a biochar chain-lengthening reaction using recyclable biochar-based materials, characterized in that, The method includes the following steps: 1) Modify biochar particles by loading Fe3O4 nanoparticles onto their surface to prepare magnetic modified biochar that is easy to recycle. 2) Add the modified biochar obtained in step 1) to the domesticated inoculum, add a culture medium solution containing ethanol and acetic acid, and construct a fermentation system for producing hexanoic acid with an extended biochar chain. The reaction temperature is 30℃~40℃ and the pH is 5~7. After the reaction is completed, the modified biochar is recovered and recycled.

2. The method according to claim 1, characterized in that, The biochar particles have a particle size of 75μm to 300μm. The preparation method of biochar particles is as follows: the biomass raw material is crushed, dried, pyrolyzed at 400℃ to 900℃ in an inert atmosphere, and then ground and sieved. The biomass raw material includes one or more of anaerobic fermentation biogas residue, agricultural and forestry waste, and livestock and poultry manure.

3. The method according to claim 2, characterized in that, The biochar particles have a particle size of 75μm to 150μm. The preparation method of biochar particles is as follows: the biomass raw material is crushed, dried, pyrolyzed at 400℃ to 800℃ in an inert atmosphere, and then ground and sieved.

4. The method according to claim 1, characterized in that, Step 1) The specific steps are as follows: place the biochar particles in an aqueous solution and stir to mix them evenly to obtain a biochar solution; then add ferrous salt and ferric salt, add ammonia solution to maintain the pH of the solution between 9 and 10, stir the reaction, and after the reaction is completed, separate the solid and liquid, wash the precipitate and dry it in an inert atmosphere at 60℃ to 110℃ to obtain recyclable biochar.

5. The method according to claim 4, characterized in that, Step 1) The reaction is carried out in an inert atmosphere and the reaction temperature is maintained at 70℃; the molar ratio of divalent iron ions to trivalent iron ions is 2:1 to 3:1, and the reaction time is 60 to 180 min; the solid-liquid separation methods include centrifugation, static precipitation, and magnetic adsorption.

6. The method according to claim 1, characterized in that, In step 2), the mass ratio of modified biochar to acclimatized inoculum is 1:16 to 2:1, the molar ratio of ethanol to acetic acid is 1:1 to 5:1, and the total carbon number concentration is 20 mM to 1000 mM.

7. The method according to claim 1, characterized in that, Step 2) The method for recovering modified biochar is to use a strongly magnetic object to absorb it, wash it with an aqueous solution, and then dry it in an air atmosphere at 60℃~110℃ to obtain the recovered biochar.

8. The method according to claim 1, characterized in that, The specific method for obtaining the domesticated inoculum in step 2) is as follows: a. Pretreatment of anaerobic microbial inoculum; b. The anaerobic microbial inoculum after the pretreatment in step a is mixed with the culture medium solution and inoculated into the reactor. Ethanol and acetic acid are added as substrates, and the mixture is acclimatized and cultured at 30℃~40℃ and pH 5~6. The sources of anaerobic microbial inoculum include municipal anaerobic sludge, sewer sludge, effluent from anaerobic fermenters, and one or more mixtures of livestock and poultry manure.

9. The method according to claim 8, characterized in that, Step a involves pretreatment of the anaerobic microbial inoculum using one or more of the following methods: (1) autoclaving at 100℃ to 120℃ for 10 min to 60 min; (2) adding 5 g / L to 20 g / L of sodium 2-bromoethylsulfonate; and (3) adjusting the pH of the inoculum solution to 5 to 6.

10. The method according to claim 8, characterized in that, In step b, 1 L of culture medium solution contains: 1450 mg K₂HPO₄, 1910 mg NH₄Cl, 1020 mg MgCl₂, 734 mg CaCl₂·2H₂O, 110 mg Na₂SO₄, 6700 mg KCl, 9.6 mg FeCl₂, 0.7 mg ZnCl₂, 1.3 mg MnSO₄·H₂O, 0.06 mg H₃BO₃, 1.9 mg CoCl₂·6H₂O, 0.02 mg CuCl₂·2H₂O, 0.24 mg NiCl₂·6H₂O, 0.31 mg Na₂MoO₄·2H₂O, 0.03 mg Na2SeO3·5H2O, 1 mg glutamic acid, 0.25 mg ascorbic acid, 0.25 mg riboflavin, 0.2 mg citric acid monohydrate, 0.1 mg folic acid, 0.1 mg p-aminobenzoic acid, and 0.25 mg cysteine; the volatile solids content of the inoculum after pretreatment and mixing with the culture medium solution was 0.5%–5%; the molar ratio of ethanol to acetic acid was 1:1–5:1, and the total carbon number concentration was 20 mM–550 mM; the criteria for successful inoculum acclimatization and cultivation in step b were 5–30 days, hexanoic acid carbon number conversion rate ≥10%, and no methane production.

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