Method for constructing three-bacterium synergistic microbial complex and converting CO2 to electrically synthesize acetic acid

Abstract

The invention belongs to the technical field of bioelectrochemistry, and particularly relates to a method for electrically synthesizing acetic acid by converting CO2 based on construction of a three-bacterium synergistic microbial complex. Microorganisms with different functional characteristics are used for constructing a microbial complex for electrochemical synthesis of acetic acid, so that the problems of low acetic acid conversion rate and unstable product existing in a microbial electrosynthesis system are solved. And furthermore, interaction and electron transfer modes among microorganisms are regulated and controlled through a specific adding sequence, the spatial distribution of each flora of the system is optimized, and synergistic interaction is realized. The high-conversion-rate synthesis of acetic acid by CO2 is realized. According to the system constructed by the invention, the net conversion rate of reducing CO2 into acetic acid is also up to 87.04%, and the highest concentration of acetic acid can reach 1448mg / L. And the stability of the three-bacterium combined system is obviously improved, the cycle operation period is prolonged to 720 hours or above, and the performance is not attenuated. An innovative technical scheme is provided for producing acetic acid by efficiently and stably converting CO2, efficient and stable CO2 resource utilization can be achieved without electrode modification, and the method has high application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioelectrochemistry, and particularly relates to a method for electrochemically synthesizing acetic acid by converting carbon dioxide based on a microbial consortium. Background Art

[0002] Carbon capture and utilization (CCU) technology is one of the core paths to achieve the carbon neutrality goal. Microbial electrosynthesis (MES) technology has attracted much attention due to its unique bio-electrochemical synergistic effect. It catalyzes the cathodic reduction reaction through electroactive microorganisms to convert CO2 into high-value-added chemicals such as acetic acid and butyric acid, with dual values of carbon emission reduction and resource utilization. Among them, acetic acid, as the basic product of microbial electrosynthesis, is not only an important representative of short-chain fatty acids but also a key precursor for synthesizing long-chain compounds such as ethanol and butanol. Its synthesis efficiency directly affects the economy and large-scale potential of MES technology.

[0003] In recent years, MES technology has improved the acetic acid yield through cathode material modification (such as modifying the electrode with redox substances, CN116641072A), reactor configuration optimization (such as single-tank membrane-free design, CN112899156A), or addition of conductive media (CN108277239A). However, these methods rely on complex processes and are costly, and are prone to performance degradation due to electrode passivation or microbial community imbalance during long-term operation. In addition, although the traditional mixed microbial community strategy (such as CN111961691A) can improve stability through domestication, there are still two major bottlenecks: First, the composition of the mixed microbial community is complex, the CO2 conversion efficiency is low, and the acetic acid synthesis efficiency is not high; Second, the disordered distribution of electroactive bacteria and non-electroactive bacteria in the biofilm and suspension leads to limited electron transfer efficiency; Third, the relationship between the microbial communities within the mixed microbial community is complex, and the metabolic competition of the microbial communities causes secondary degradation of acetic acid, resulting in insufficient product accumulation. Despite the above-mentioned various methods for enhancing electron transfer through electrode modification to improve acetic acid synthesis, the CO2 conversion efficiency still needs to be improved. Recently, it has been reported that the acid production effect is enhanced through the multi-layer biofilm electrode technology (CN116288426A), but it only focuses on the biofilm on the electrode surface and does not fully utilize the metabolic potential of the cathodic suspension area.

[0004] Therefore, it is necessary to develop a new method to solve the problems existing in the prior art. Summary of the Invention

[0005] Aiming at the technical bottlenecks of the existing microbial electrosynthesis system, such as relying on electrode modification, complex composition of mixed microbial communities, disordered spatial distribution, and unstable acetic acid synthesis; the present invention proposes a method and device for electrochemically synthesizing acetic acid from carbon dioxide based on a microbial consortium constructed according to the functional characteristics of strains. Further, through specific sequential inoculation of strains, the colonization order and niche distribution of functional strains are regulated to achieve synergistic enhancement of electron transfer and metabolism.

[0006] The first aspect of the present invention provides a method for electrochemically synthesizing acetic acid from carbon dioxide based on a microbial consortium, wherein the microbial consortium includes a bidirectional electron transfer assisting bacterium, an electroactive acetic acid-producing bacterium, and a non-electroactive acetic acid-producing bacterium;

[0007] The method includes the following steps:

[0008] S1. Provide an H-type two-chamber electrolytic cell composed of a cathode chamber, an anode chamber, and a proton membrane isolation, and simultaneously configure a working electrode, a counter electrode, and a reference electrode to form a microbial electrosynthesis reactor system;

[0009] S2. Add a liquid medium to the cathode chamber, add a phosphate buffer solution to the anode chamber, and purge with nitrogen to provide an anaerobic environment;

[0010] S3. Connect the anode electrode and the cathode electrode to the positive and negative poles of a DC power supply respectively, inoculate the microbial consortium into the medium in the cathode chamber, and add a carbon source. Acetic acid is synthesized under the action of an applied voltage.

[0011] Optimizing the microbial population composition and improving the electro-synthesis conversion efficiency are the keys to breaking through the bottlenecks of the efficiency and stability of the existing CO2 electro-synthesis of acetic acid. Based on the functional characteristics of the strains, the present invention constructs a microbial consortium, and can realize efficient and stable CO2 resource utilization without electrode modification.

[0012] Furthermore, the bidirectional electron transfer assisting bacterium is used to preferentially colonize the electrode to construct a conductive biofilm, and is selected from Shewanella oneidensis MR-1 and Geobacter sulfurreducens PCA.

[0013] The electroactive acetic acid-producing bacterium is used to form an electron relay station at the biofilm-suspension interface, and is selected from Clostridium aceticum, Sporomusa ovata, and Moorella thermoacetica.

[0014] The non-electroactive acetic acid-producing bacterium is selected from Acetobacterium woodii and Eubacterium limosum.

[0015] In some embodiments of the present invention, a method for electrochemically synthesizing acetic acid using a microbial consortium composed of electroactive acetic acid-synthesizing microorganism Acetobacterium woodii (DSM 1030), non-electroactive acetic acid-producing bacterium Clostridium aceticum (DSM 1496), and bidirectional electron transfer-assisted bacterium Shewanella oneidensis MR-1 (MCCC ATCC 700550) is selected.

[0016] The core of this solution lies in utilizing the natural spatial differentiation characteristics of the microbial consortium to synchronously optimize the electron utilization efficiency of the biofilm and the metabolic complementarity in the suspension area. The specific technical solution is as follows:

[0017] Construction and functional division of the microbial consortium: The non-electroactive acetic acid-producing bacterium Acetobacterium woodii dominates the synthesis of acetic acid through the Wood-Ljungdahl pathway (WLP). Since it cannot directly utilize electrode electrons, it preferentially colonizes in the cathode suspension area and can interact with electroactive bacteria through metabolic intermediates (such as formic acid). The electroactive acetic acid-producing bacterium Clostridium aceticum has both the WLP pathway and the ability of direct electron transfer. It colonizes in the transition area between the electrode biofilm and the suspension, consumes by-products, and strengthens the electron supply. The electroactive bacterium Shewanella oneidensis MR-1 can perform bidirectional electron transfer and direct electron transfer through cytochrome C. It preferentially colonizes on the electrode surface to form a conductive biofilm, providing a stable electron flow for acetic acid-producing bacteria.

[0018] Furthermore, the inoculated microorganisms are microorganisms grown to the logarithmic phase.

[0019] Furthermore, the concentration of the microbial consortium in the cathode suspension is 10 6 to 10 9 CFU / mL.

[0020] The inoculation ratio of the bidirectional electron transfer-assisted bacterium, electroactive acetic acid-producing bacterium, and non-electroactive acetic acid-producing bacterium is 1-10% (v / v), and further preferably 2-5% (v / v) to construct a microbial consortium.

[0021] The present invention provides a better method for electrochemically synthesizing acetic acid, which is optimized on the basis of the foregoing solution. In a preferred embodiment of the present invention, the ecological niche distribution of the microbial community is regulated by a staged inoculation sequence; this solution makes full use of the biofilm and suspension spaces to further improve the electron utilization efficiency and CO2 conversion efficiency of the electrosynthesis system.

[0022] Specifically, in step S3, when inoculating the microbial consortium, the bidirectional electron transfer-assisted bacterium, the electroactive acetate-producing bacterium, and the non-electroactive acetate-producing bacterium are inoculated sequentially. The inoculation time interval is adjusted according to specific culture conditions and the growth of the bacterial strains. The next strain can be inoculated after the previously inoculated strain has fully grown. In this preferred scheme, by inoculating in a specific strain order, the colonization order and niche distribution of the functional strains are regulated to achieve synergistic enhancement of electron transfer and metabolism.

[0023] The strategy of this scheme is that electroactive bacteria colonize preferentially. First, S. oneidensis is inoculated. This bacterium has strong electrode attachment ability, and the direct electron transfer efficiency mediated by its cytochrome C is higher than the indirect electron transfer efficiency. After culturing for a period of time, the electroactive acetate-producing bacterium C. aceticum is inoculated to form an "electron relay station" in the biofilm-suspension transition zone. After another period of time, the non-electroactive acetate-producing bacterium A. woodii is inoculated. Its growth rate is faster than that of C. aceticum. Inoculating later can avoid occupying the suspension space prematurely and rely on the indirect electron transfer provided by S. oneidensis and C. aceticum for acetic acid synthesis. The mechanism of action of this scheme: S. oneidensis preferentially constructs the electrode biofilm conductive network, C. aceticum strengthens the electron transfer in the biofilm-suspension area, and A. woodii mainly uses the metabolic intermediates in the suspension area to enhance acetic acid accumulation.

[0024] Furthermore, the inoculation ratio of the three bacterial strains is 5% (v / v);

[0025] In some embodiments of the present invention, the interval time for sequential inoculation is 24 h.

[0026] In some embodiments, the liquid medium of Acetobacterium woodii is DSMZ-Medium135;

[0027] In some embodiments, the liquid medium of Clostridium aceticum is DSMZ-Medium135;

[0028] In some embodiments, the medium of Shewanella oneidensis MR-1 is tryptic soy broth or Luria-Bertani medium.

[0029] Furthermore, an electrolyte containing a redox mediator and a buffer is provided in the anode chamber. The redox mediator is a transition metal cyanide complex, and the buffer is used to maintain the pH of the electrolyte. In some embodiments, the anolyte is 50 mM PBS containing 20 g / L of potassium ferrocyanide.

[0030] Further, in step S1, the working electrode and the counter electrode are selected from copper sheets, carbon cloths or carbon felts; the reference electrode is selected from Ag / AgCl electrodes, calomel electrodes or hydrogen electrodes.

[0031] Further, in step S2, the liquid medium is a modified DSMZ-Medium135 medium.

[0032] Further, each 1 L of the modified DSMZ-Medium135 basal medium contains: 0.25 g of NH4Cl, 0.31 g of KH2PO4, 0.33 g of K2HPO4, 0.2 g of MgSO4·7H2O, 1 mL of trace elements, 1 mL of vitamins, 0.10 - 0.25 g of yeast powder, 3.5 g of NaHCO3, 0.25 g of L-cysteine, 0.25 g of Na2S·9H2O, 0.5 mL of 1% (w / v) resazurin.

[0033] Further, in step S3, after inoculation, the pH of the catholyte is maintained at 7.0 ± 0.3;

[0034] Further, in step S3, the temperature is controlled at 36 ± 2 °C.

[0035] Further, the target products mainly include acetic acid, and a small amount of propionic acid and butyric acid.

[0036] Even further, for the sequentially inoculated microbial consortium, the pH is regulated daily to be maintained at 6.8 - 7.2 to adapt to the growth and metabolic requirements of the three bacteria.

[0037] In some embodiments of the present invention, the carbon source is HCO3 - or gaseous CO2 is directly introduced.

[0038] Further, in step S3, the applied voltage is -0.8 V to -1.2 V;

[0039] Preferably, the applied voltage is -0.88 V. It better balances the electron supply and the H2 inhibition effect.

[0040] Further, the working electrode is disposed in the cathode chamber, and the counter electrode is disposed in the anode chamber.

[0041] In some embodiments of the present invention, an H-type reactor composed of two chambers separated by a proton exchange membrane is used. The effective volumes of the anode chamber and the cathode chamber are both 300 mL. The anode and cathode working electrodes both use unmodified copper sheets, carbon cloths or carbon felts. The working electrode is fixed with titanium wire; the reference electrode is placed in the catholyte.

[0042] In some embodiments of the present invention, the concentration of NaHCO3 in the cathode-improved culture medium is 3.5 g / L, serving as the source of CO2.

[0043] In some embodiments of the present invention, the cathode chamber and the anode chamber are separated by a proton membrane (Nafion 117).

[0044] In some embodiments of the present invention, the steps for the activation and cultivation of microorganisms are as follows: (1) Acetobacterium woodii (DSM 1030), Clostridium aceticum (DSM 1496), and Shewanella oneidensis MR-1 (MCCC ATCC 700550) stored at -80 °C are inoculated into a liquid culture medium for cultivation to obtain a seed solution. (2) The seed solution obtained in step (1) is inoculated into a fresh liquid culture medium for scale-up cultivation until they grow to the logarithmic phase respectively. The bacterial solution is centrifuged at 8000 rpm / min for 5 min, and the bacterial cells are collected and washed 3 times with phosphate buffer solution (1×PBS). PBS is added to adjust and prepare a bacterial suspension with an OD 600nm of 0.3, and the bacterial concentration is 10 6 to 10 9 CFU / mL.

[0045] The second aspect of the present invention lies in providing an electrochemical synthesis device for implementing the method. In some embodiments, an aeration device is provided in the cathode chamber of the electrochemical synthesis device.

[0046] Beneficial effects: Compared with the prior art, the technical solution of the present application has at least the following advantages:

[0047] (1) The technical solution of the present invention can efficiently obtain electrons from the cathode biofilm and fully utilize the cathode suspension space to continuously synthesize acetic acid; without relying on electrode engineering transformation, by regulating the colonization sequence and spatial niche distribution of microorganisms, the MES technology of synergistically enhancing electron transfer and metabolism is realized;

[0048] (2) Based on the solution of the present invention, high-efficiency acid production can be achieved: the acetic acid production in the three-bacteria co-culture system reaches 1.45 g / L, which is 136.06% - 747.06% higher than that in the single-bacteria system, and the acetic acid is 42.6% higher than that in the group with simultaneous inoculation of the three bacteria.

[0049] (3) The present invention further improves the capacitance of the biofilm and enhances the utilization rate of the suspension space by microorganisms through sequential inoculation. The overall microbial metabolic activity is improved, and the carbon conversion efficiency of synthesizing acetic acid from CO2 is also increased to 87.0%, avoiding redundant energy loss;

[0050] (4) High stability: After continuous operation for 240 h, the acetic acid degradation rate is <5%, and the system can be repeatedly operated for more than 720 h without significant performance degradation;

[0051] (5) Cost advantage: Simplify the system construction process, without the need for electrode modification or addition of exogenous conductive media; The process flow is simplified, and the equipment investment cost is reduced; It is easier to scale up and promote the application, with significant economic benefits. Description of the Drawings

[0052] The drawings are used to provide a further understanding of the present application, and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the present application, and do not constitute a limitation to the present application; In the drawings:

[0053] Figure 1 is a schematic diagram of the microbial electrosynthesis reactor device of the present invention;

[0054] Figure 2 is the change in acetic acid concentration of different microbial consortia constructed in Example 1 and Comparative Example 1 of the present invention;

[0055] Figure 3 is the acid production situation (a - e) and carbon conversion rate (f) of the system in Examples 1 - 2 and Comparative Examples 1 - 2 of the present invention, where the figures (a - e) are stacked graphs;

[0056] Figure 4 is the CV curve of the cathode biofilm under different inoculation conditions in Examples 1 - 2 of the present invention;

[0057] Figure 5 is the three - dimensional fluorescence spectrum of the cathode bio - suspension under different inoculation conditions in Examples 1 - 2 of the present invention. Detailed Embodiments

[0058] In order to make the above - mentioned objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention is made in conjunction with the embodiments of the specification. Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0059] The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; The reagents and materials described, unless otherwise specified, can all be obtained from commercial sources.

[0060] For the schematic diagram of the microbial electrosynthesis system used in the embodiments of the present invention, see Figure 1, a typical H-type dual-chamber microbial electrosynthesis reactor was used. An H-type reactor composed of two chambers separated by a proton exchange membrane was adopted, and the effective volume of both the anode chamber and the cathode chamber was 300 mL. The unmodified carbon cloth was used for both the anode and cathode working electrodes. The working electrodes were fixed with titanium wires; the reference electrode was a saturated calomel electrode placed in the cathode solution.

[0061] In the embodiment of the present application, the anolyte was a 50 mM phosphate buffer solution containing 20 g / L of potassium ferrocyanide.

[0062] In the embodiment of the present application, the composition of the cathode culture solution was: K2HPO4 0.31 g / L, KH2PO4 0.23 g / L, NH4Cl 0.25 g / L, MgSO4·7H2O 0.20 g / L, NaHCO3 3.5 g / L, yeast powder 0.10 - 0.25 g / L, L-cysteine 0.25 g / L, Na2S·9H2O 0.25 g / L, 0.5 mL of 1% (w / v) resazurin. Trace element solution 1.00 mL / L, vitamin solution 1.00 mL / L. The trace element solution and the vitamin solution were prepared according to the formula of DSMZ141 medium preserved by the German Collection of Microorganisms.

[0063] Among them, each 1 L of the trace element solution contained: 1.50 g of nitrilotriacetic acid, 0.10 g of CaCl2·2H2O, 3.00 g of MgSO4·7H2O, 0.01 g of CuSO4·5H2O, 0.50 g of MnSO4·H2O, 0.01 g of H3BO3, 1.00 g of NaCl, 0.03 g of NiCl2·6H2O, 0.10 g of FeSO4·7H2O, 0.40 mg of Na2WO4·2H2O, 0.18 g of CoSO4·7H2O, 0.18 g of ZnSO4·7H2O, 0.02 g of KAl(SO4)2·12H2O, 0.01 g of Na2MoO4·2H2O, 0.30 mg of Na2SeO3·5H2O.

[0064] Among them, each 1 L of the vitamin solution contained: 2 mg of biotin, 10.00 mg of pyridoxine hydrochloride, 2 mg of folic acid, 5.00 mg of p-aminobenzoic acid, 5.00 mg of thiamine hydrochloride·2H2O, 5.00 mg of D-calcium pantothenate, 5.00 mg of riboflavin, 0.10 mg of vitamin B 12 , 5.00 mg of nicotinic acid, 5.00 mg of lipoic acid.

[0065] Test methods used in the examples:

[0066] Method for determining acetic acid: The obtained sample was filtered through a 0.22 μm aqueous filter membrane, thoroughly mixed with phosphoric acid at a concentration of 3 mM in a ratio of 1:1 (v / v), and then added to a sample vial. The acetic acid concentration in the sample was determined using a Shimadzu GC-2030 gas chromatograph. The chromatographic column was of the Shimadzu SK-WAX type, and the operating parameters of the gas chromatograph were: split mode, injection volume 1 μL, split ratio 20.0; temperature programming 60 °C - 0.5 min, 130 °C - 20 °C / min, 200 °C - 15 °C / min - 5 min, FID detector temperature 250 °C, and chromatographic column flow rate 1 mL / min. The peak emergence times of acetic acid, propionic acid, and butyric acid were 7.234 min, 8.052 min, and 8.755 min, respectively. The measurement accuracy calibration of the standard curve correlation coefficient of the standard sample was 0.999 or above.

[0067] Calculation of acetic acid conversion rate: Moles of carbon in the product / Moles of carbon added * 100%.

[0068] Biological preservation information:

[0069] All the listed strains are publicly available preserved strains, Acetobacterium woodii (DSM 1030): Clostridium aceticum (DSM 1496): Shewanella oneidensis MR-1 (MCCC ATCC 700550):

[0070] Preservation institutions: MCCC: China Center for Marine Microbial Culture Collection; DSM: German Collection of Microorganisms and Cell Cultures. Activation culture of the strains:

[0071] (1) Acetobacterium woodii (DSM 1030), Clostridium aceticum (DSM1496), and Shewanella oneidensis MR-1 (MCCC ATCC 700550) stored at -80 °C were inoculated into a liquid medium for cultivation to obtain seed solutions. The liquid media for A. woodii and C. aceticum were DSMZ-Medium135; the medium for S. oneidensis MR-1 was tryptic soy broth (TSB) liquid medium or Luria-Bertani (LB) medium.

[0072] (2) The seed solutions obtained in step (1) were inoculated into fresh liquid media for scale-up cultivation until they grew to the logarithmic phase. The bacterial solutions were centrifuged at 8000 rpm / min for 5 min, the cells were collected, washed 3 times with phosphate buffer solution (1×PBS), and PBS was added to prepare OD 600nmThe bacterial suspension with an OD of 0.3 - 0.5 and a bacterial concentration of 10 6 to 10 9 CFU / mL.

[0073] In the examples of this application, inoculation of microorganisms in the cathode medium: In a sterilized (121°C, 20 min) assembled H-type two-chamber reactor, N2 gas was introduced for 30 min. Subsequently, it was placed in a laminar flow hood and sterilized with ultraviolet light for 30 min. After sterilization, 2 - 5% (v / v) of S. oneidensis MR-1, C. aceticum, and A. woodii that had grown to the logarithmic phase were inoculated into the cathode chamber. The reactor was operated at a voltage of 0.88 V and a temperature of 36 ± 2°C. The pH of the cathode solution was adjusted with 2 M hydrochloric acid every day to control the pH of the cathode solution at 7.0 ± 0.2.

[0074] For sequential inoculation, first, 2 - 5% (v / v) of S. oneidensis MR-1 that had grown to the logarithmic phase was inoculated. After a 24-hour interval, an equal proportion of the homoacetogen C. aceticum was inoculated, and the culture was continued at the same potential for 24 h; after another 24-hour interval, an equal proportion of the homoacetogen A. woodii was inoculated. The reactor was operated at a voltage of 0.88 V and a temperature of 36 ± 2°C. The pH of the cathode solution was adjusted with 2 M hydrochloric acid every day to control the pH of the cathode solution at 7.0 ± 0.2.

[0075] Example 1: Simultaneous inoculation of three bacteria

[0076] In the MES system of the method of this patent, 50 mM PBS containing 20 g / L of potassium ferrocyanide was configured as the anode solution, and the cathode culture medium with 3.5 g / L NaHCO3 as the sole carbon source. S. oneidensis, C. aceticum, and A. woodii were respectively cultured to the logarithmic phase, the bacterial cells were washed with PBS buffer and resuspended with PBS buffer. The OD of the resuspended solution 600 was adjusted to 0.3, and a bacterial suspension of a 1:1:1 (v / v) combination of S. oneidensis, C. aceticum, and A. woodii was prepared. A total of 15% of the bacterial suspension (the inoculation amount of each bacterium was 5%) was inoculated into the cathode culture medium. Then, N2 was bubbled into the cathode solution and the anode solution respectively to make both chambers in a fully anaerobic condition. An external voltage of -0.88 V and an initial pH = 7.0 were set. It was run in a constant temperature incubator at 36 ± 2°C for 7 days. The pH of the cathode solution was adjusted with 2 M hydrochloric acid every day to control the pH of the cathode solution at 7.0 ± 0.3. 2 mL of the cathode solution was collected from the sampling port of each treatment cathode chamber every 2 days for product concentration determination.

[0077] Comparative Example 1: Simultaneous inoculation of two bacteria

[0078] In the MES system, 50 mM PBS containing 20 g / L of potassium ferrocyanide was configured as the anodic solution, and the cathodic culture medium with 3.5 g / L NaHCO3 as the sole carbon source. S. oneidensis, C. aceticum, and A. woodii were respectively cultured to the logarithmic phase, the cells were washed with PBS buffer, and various cell precipitates were resuspended with PBS buffer. The OD 600 of the resuspension was adjusted to 0.3, and cell suspensions with a ratio of 1:1 in pairwise combinations were prepared. The following 3 combinations of cell suspensions (inoculation ratio: 10% v / v) were respectively inoculated into the cathodic chamber: Combination 1: S. oneidensis + C. aceticum; Combination 2: S. oneidensis + A. woodii; Combination 3: C. aceticum + A. woodii. Then, N2 was respectively bubbled into the catholyte and anolyte to make both chambers in a fully anaerobic condition. An external voltage of -0.88 V and an initial pH = 7.0 were set. It was run in a constant temperature incubator at 36 ± 2 °C for 7 days. The pH was adjusted with 2 M hydrochloric acid every day to control the catholyte pH at 7.0 ± 0.3. 2 mL of catholyte was collected from the sampling port of each treated cathodic chamber every 2 days for product concentration determination.

[0079] The experimental results of Example 1 and Comparative Example 1 are as Figure 2 shown. Within 240 h of the operation of the two-bacteria consortium and the three-bacteria consortium, no acetic acid degradation phenomenon occurred (degradation rate < 5%), and the ability of the consortium with three bacteria inoculated simultaneously to electrosynthesize acetic acid was better than that of the two-bacteria combined system, indicating that the acid production performance of the three-bacteria consortium was more efficient and stable.

[0080] Comparative Example 2 Single-bacteria inoculation

[0081] In the MES system, 50 mM PBS containing 20 g / L of potassium ferrocyanide was configured as the anodic solution, and the cathodic culture medium with 3.5 g / L NaHCO3 as the sole carbon source. Cell suspensions of S. oneidensis, C. aceticum, and A. woodii grown to the logarithmic phase were respectively inoculated into different cathodic culture media (inoculation ratio: 5% v / v, OD 600 = 0.3). Then, N2 was respectively bubbled into the catholyte and anolyte to make both chambers in a fully anaerobic condition. An external voltage of -0.88 V and an initial pH = 7.0 were set. It was run in a constant temperature incubator at 36 ± 2 °C for 7 days. The pH was adjusted with 2 M hydrochloric acid every day to control the catholyte pH at 7.0 ± 0.2. 2 mL of catholyte was collected from the sampling port of each treated cathodic chamber every 2 days for product concentration determination.

[0082] Example 2 Three bacteria were inoculated sequentially

[0083] The sequential inoculation method is as follows: inoculate S. oneidensis grown to the logarithmic phase into the cathode culture solution (inoculation ratio is 5% v / v, OD 600 = 0.3). Then, bubble N2 into the cathode solution and the anode solution respectively to make both chambers in a fully anaerobic condition. Set an external voltage of -0.88V and an initial pH = 7.0. After 24h, inoculate C. aceticum (inoculation ratio is 5% v / v, OD 600 = 0.3), and then inoculate A. woodii (inoculation ratio is 5% v / v, OD 600 = 0.3) at an interval of 24h. After completing the sequential inoculation in the first 3 days, run it in a constant temperature incubator at 36 ± 2°C for 7 days. Adjust the pH with 2M hydrochloric acid every day to control the pH of the cathode solution at 7.0 ± 0.3. Collect 2 mL of the cathode solution from the sampling port of each treatment cathode chamber every 2 days for product concentration determination.

[0084] The experimental results of Example 1 and Comparative Example 1 are as Figure 2 shown. The highest acetic acid concentration in the three - strain co - culture system with simultaneous inoculation can reach 1016 mg / L, which is superior to the product synthesis ability of the two - strain consortium.

[0085] The experimental results of Example 1, Example 2, and Comparative Example 2 are as Figure 3 shown. As Figure 3 (a - e) shows, the highest acetic acid concentration in the three - strain co - culture system with sequential inoculation can reach 1448 mg / L, which is 41.73% higher than the system with three - strain simultaneous inoculation (1016 mg / L); and the highest propionic acid production detected is 159 mg / L, and the highest butyric acid production is 111 mg / L. Compared with the highest acetic acid production of single - strain, it has also increased by 136.06% - 747.06%.

[0086] As Figure 3 f shows, in the system of Example 2, the carbon conversion rate in the sequential inoculation system of the present invention can reach more than 87%; significantly higher than other systems.

[0087] Capacitance test of the systems of Example 1 and Example 2:

[0088] After the experiments of Example 1 - 2 are completed, perform cyclic voltammetry (CV) tests on the sequential inoculation experimental group and the simultaneous inoculation experimental group to measure capacitance on a multi - channel Chenhua electrochemical workstation (CHI660E, Shanghai).

[0089] Test method: A three-electrode system was adopted, including a carbon cloth working electrode, a platinum wire counter electrode, and a reference electrode. The reference electrode was based on a saturated calomel electrode. The electrolyte was 50 mM PBS buffer (pH = 7.0), which was purged with N2 and saturated for 1 hour to remove oxygen. The scanning potential range of the CV curve was -1.2 V to 1 V (vs SCE), and the scanning rate was 10 mV / s. After obtaining the CV curve, the area specific capacitance value (Ca, mF / cm 2 ) of the system could be calculated according to the formula. The test temperature was 30 °C.

[0090]

[0091] In the formula, S (cm 2 ) is the projected area of the biocathode, v (V / s) is the scanning rate, V (V1) is the starting potential, and V (V2) is the ending potential.

[0092] As Figure 4 shown, for the cathode biofilm electrode constructed by the strategy of the inoculation sequence described in the present invention, its area specific capacitance reached 1.54 times that of Example 1, and the peak current of the redox peak in the cyclic voltammetry curve increased significantly, indicating that the electron transfer efficiency at the electrode-microbe interface was substantially improved. This result confirmed the strengthening effect of the sequential inoculation strategy on the biofilm electrochemistry activity.

[0093] In addition, after obtaining the cathode suspension after the reaction, 10 mL of samples were taken from each system and centrifuged at 8000 rpm for 10 min. The supernatant was filtered through a 0.22 μm aqueous filter membrane, and the three-dimensional excitation-emission matrix (3D-EEM) spectrum of a fluorescence spectrometer (Horiba JY Aqualog) was used. The emission wavelength (E m ) range was 250 - 500 nm, with an increment of 4.48 nm, the excitation wavelength (E x ) range was 200 - 500 nm, with an interval of 5 nm, and the scanning rate was 12000 nm / min.

[0094] As Figure 5 shown, for the cathode suspension constructed by the sequential inoculation strategy in Example 2 of the present invention, the fluorescence intensities in the protein-like region (E x / E m = 280 / 350 nm) and the fulvic acid-like region (E x / E m = 320 / 420 nm) were increased compared with the synchronous inoculation system in Example 1; the significant increase in the intensity of the above characteristic peaks confirmed that the sequential inoculation strategy effectively enhanced the metabolic activity of the suspended microorganisms and reduced the substrate competition among bacteria, enabling the microorganisms to make more full use of the suspended space resources.

[0095] Example 3 Cyclic Performance Test

[0096] In the microbial electrosynthesis system of the present invention, 50 mM PBS containing 20 g / L of potassium ferrocyanide was configured as the anodic solution, and the cathodic culture medium with 3.5 g / L NaHCO3 as the sole carbon source was used. S. oneidensis (5% v / v) grown to the logarithmic phase was inoculated into the cathodic culture medium. Then, N2 was bubbled into the catholyte for 30 min to make both chambers in a fully anaerobic condition. The applied voltage was set to 0.88 V and the initial pH = 7.0. After 24 h, C. aceticum was inoculated (in equal proportion), and then A. woodii was inoculated (in equal proportion) at an interval of 24 h. After the sequential inoculation was completed in the first 3 days, it was run in a constant temperature incubator at 36 ± 2 °C for 7 days. The pH was adjusted with 2 M hydrochloric acid every day to control the pH of the catholyte at 7.0 ± 0.3. After 10 days of operation, the new medium was replaced and added to the microbial electrosynthesis system again according to the inoculation sequence in Example 2, and it was continued to run for 7 days. A total of 3 cycles were carried out. 2 mL of catholyte was sampled from the sampling port of each treatment cathodic chamber every 2 days for product concentration determination. During the continuous operation process, the acetic acid degradation rate was <5%, and the system could be repeatedly run for ≥3 cycles (total duration 720 h) without significant performance decay.

[0097] Table 1 Maximum acetic acid production and acetic acid conversion rate of the experimental group in Example 3 of the present invention

[0098]

[0099] The present invention discloses a system and method for electrochemically synthesizing acetic acid based on the synergistic conversion of carbon dioxide by three bacteria. Microbial consortia with different functional characteristics are constructed for the electrochemical synthesis of acetic acid, which solves the problems of low acetic acid conversion rate and unstable products existing in the microbial electrosynthesis system. The natural spatial differentiation characteristics of functional strains are fully utilized to synchronously optimize the electron utilization efficiency of biofilms and suspension areas; and further, the competition among bacteria is inhibited by sequential inoculation to maintain the long-term stability of the acetic acid synthesis pathway. By regulating the interspecies interaction and electron transfer mode of microorganisms through a specific dosing sequence, the spatial distribution of each bacterial community in the system is optimized to achieve synergistic enhancement. The high-conversion synthesis of acetic acid from CO2 is realized. The microbial consortia constructed by the present invention have excellent CO2 electro-synthesis activity. The net conversion rate of reducing CO2 to acetic acid in the three-bacteria co-culture system by sequential inoculation is as high as 87.04%, and the highest concentration of acetic acid can reach 1448 mg / L, which is increased by 136.06% - 747.06% compared with the single-bacteria system. Moreover, the system stability of the three-bacteria combination is significantly improved, and the cycle operation period is extended to more than 720 h without performance decay. The present invention provides an innovative technical solution for the efficient and stable conversion of CO2 to produce acetic acid, and can realize the efficient and stable resource utilization of CO2 without electrode modification, having high application value.

[0100] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the present invention.

Claims

1. A method for electrochemically synthesizing acetic acid by converting carbon dioxide based on a microbial consortium, characterized in that, The microbial consortium includes bidirectional electron transfer-assisted bacteria, electroactive acetate-producing bacteria, and non-electroactive acetate-producing bacteria; The method includes the following steps: S1. Provide an H-type two-chamber electrolytic cell composed of a cathode chamber, an anode chamber, and a proton membrane isolation, and at the same time configure a working electrode, a counter electrode, and a reference electrode to form a microbial electrosynthesis reactor system; S2. Add a liquid medium to the cathode chamber, add an electrolytic solution containing a redox mediator and a buffer to the anode chamber, and purge nitrogen to provide an anaerobic environment; S3. Connect the anode electrode and the cathode electrode to the positive and negative poles of a DC power supply respectively, inoculate the microbial consortium into the medium in the cathode chamber, and add a carbon source. Acetic acid is synthesized under the action of an applied voltage.

2. The method for electrochemically synthesizing acetic acid according to claim 1, wherein In step S3, when inoculating the microbial consortium, the bidirectional electron transfer-assisted bacteria, the electroactive acetate-producing bacteria, and the non-electroactive acetate-producing bacteria are inoculated sequentially.

3. The method for electrochemically synthesizing acetic acid according to claim 1 or 2, characterized in that, The concentration of the described microbial consortium in the cathode suspension is 10 6 to 10 9 CFU / mL.

4. The method for electrochemically synthesizing acetic acid according to claim 1 or 2, characterized in that, In step S1, the working electrode and the counter electrode are selected from copper sheets, carbon cloths, or carbon felts; The reference electrode is selected from an Ag / AgCl electrode, a calomel electrode, or a hydrogen electrode.

5. The method for electrochemically synthesizing acetic acid according to claim 1 or 2, characterized in that, In step S2, the liquid medium is a modified DSMZ-Medium135 medium.

6. The method for electrochemically synthesizing acetic acid according to claim 1 or 2, characterized in that, In step S3, the pH of the cathode solution is adjusted to 7.0 ± 0.2 after inoculation; and / or, the carbon source is HCO3 - or directly introduce gaseous CO2 by aeration.

7. The method for electrochemically synthesizing acetic acid according to claim 1 or 2, characterized in that, The applied voltage is 0.8V to 1.2V.

8. The method for electrochemically synthesizing acetic acid according to claim 1 or 2, wherein The bidirectional electron transfer-assisted bacteria are used to preferentially colonize the electrode to construct a conductive biofilm, and are selected from Shewanella and Geobacter sulfurreducens; The electroactive acetate-producing bacteria are used to form an electron relay station at the biofilm-suspension interface, and are selected from Clostridium aceticum, Sporomusa ovata, and Moorella thermoacetica; The non-electroactive acetate-producing bacteria are selected from Acetobacterium woodii and Eubacterium limosum.

9. The method for electrochemically synthesizing acetic acid according to claim 1 or 2, characterized in that, The working electrode is arranged in the cathode chamber, and the counter electrode is arranged in the anode chamber.

10. An electrochemical synthesis device based on the method according to any one of claims 1-9.

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