A biohybrid that enhances extracellular electron transport, and methods of making and using the same

Abstract

This invention discloses a biological hybrid that enhances extracellular electron transport, its preparation method, and its application. The preparation method includes: adding iron salts and pyrrole to Clostridium yunnanense in its logarithmic growth phase under anaerobic conditions. Clostridium ljungdahlii The polymerization reaction was carried out in a medium containing iron salts and sulfur compounds. The intermediate product was centrifuged, washed, resuspended in YTF medium, and the pH was adjusted. Then, iron salts and sulfur-containing compounds were added to initiate a biomineralization reaction, yielding a Clostridium yongdarii-polypyrrole / ferrous sulfide biohybrid. This invention, through the synergistic modification of polypyrrole and ferrous sulfide, constructs highly efficient transmembrane electron transport channels on the surface and inside of Clostridium yongdarii, significantly improving the efficiency of extracellular electron transfer to intracellular regions in the microbial electrosynthesis system. This overcomes the bottleneck of slow electron transport rate in Clostridium yongdarii itself, thereby improving the efficiency of carbon dioxide fixation and chemical production in the microbial electrosynthesis system, and has promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial electrosynthesis technology, specifically relating to a biological hybrid that enhances extracellular electron transfer, its preparation method, and its application. Background Technology

[0002] Climate change, triggered by excessive emissions of greenhouse gases such as carbon dioxide, has become a major global ecological and environmental problem. Against this backdrop, capturing and converting carbon dioxide into high-value-added chemicals is considered one of the effective ways to achieve carbon neutrality. Microbial electrosynthesis (MES) technology offers a cutting-edge solution for this purpose. This technology uses electricity to drive electroautotrophic microorganisms, providing them with reducing power to efficiently reduce CO2 into multi-carbon chemicals. MES has the outstanding advantage of high energy conversion efficiency; its efficiency in converting electrical energy into chemical energy even exceeds that of green plants converting solar energy. Therefore, it is considered a typical representative of third-generation biorefining technology and shows broad application prospects in environmental remediation and energy conversion.

[0003] In MES systems, electron transfer between electroautotrophic microorganisms and the cathode mainly occurs through two pathways: direct electron transfer (DET) and indirect electron transfer (IET). To improve electron transfer efficiency, current research primarily focuses on modifying electrode materials. For example, increasing the cathode's specific surface area or enhancing its conductivity can accelerate direct electron transfer; or modifying the cathode using hydrogen evolution catalysts can reduce the hydrogen evolution overpotential, thereby increasing the rate of indirect electron transfer. The cell membrane, acting as a barrier to prevent extracellular substances from freely entering the cell, maintains the stability of the intracellular environment and ensures the orderly conduct of biochemical reactions. However, extracellular electrons must overcome this cell membrane barrier to be utilized by microorganisms, resulting in unsatisfactory electron transfer efficiency between electroautotrophic microorganisms and the electrode. This is the underlying reason for the low carbon fixation efficiency of the entire system.

[0004] In recent years, the emergence of biohybrid systems has provided new insights into overcoming the aforementioned bottlenecks. These systems significantly enhance the extracellular electron transport capabilities of microorganisms by combining electroactive microorganisms with artificially synthesized nanomaterials (such as organic conductive polymers and inorganic nanomaterials). Typical biohybrids are usually composed of nanomaterials adsorbed or deposited onto the surface or interior of microbial cells, possessing both the high catalytic selectivity and self-repair capabilities of biological components and the stability and efficient electron transport properties of synthetic materials. Currently, research on biohybrids largely focuses on photocatalytic materials, but their application is heavily reliant on light, with energy sources limited by time and space, and the separation and interfacial transfer efficiency of photogenerated charges still need improvement. Therefore, there is an urgent need to develop a biohybrid that does not rely on light and can significantly enhance the direct electron transport capabilities of microorganisms, and to apply it to microbial electrosynthesis systems to overcome existing technological bottlenecks and improve the bioconversion efficiency of CO2. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a bio-hybrid that enhances extracellular electron transport, its preparation method, and its application. By constructing a transmembrane electron transport channel synergistically modified with polypyrrole / ferrous sulfide, the extracellular electron capture and intracellular energy conversion efficiency of Clostridium yongdarii is significantly improved, thereby greatly enhancing the synthesis rate and yield of carbon dioxide reduction to chemicals in microbial electrosynthesis systems.

[0006] This invention is achieved through the following technical solution:

[0007] A method for preparing a biological hybrid that enhances extracellular electron transfer includes the following steps:

[0008] Step 1) Dissolve the iron salt and pyrrole in YTF medium and mix thoroughly;

[0009] Step 2) Under anaerobic conditions, the solution obtained in Step 1) is added to Clostridium yunnanense in the logarithmic growth phase (Clostridium yunnanense). Clostridium ljungdahlii In the bacterial culture, a polymerization reaction was carried out to obtain Clostridium yongdarii-polypyrrole biohybrid;

[0010] Step 3) After centrifuging and washing the Clostridium yongdarii-polypyrrole bio-hybrid obtained in Step 2), resuspend it in YTF medium and adjust the pH to neutral.

[0011] Step 4) Add iron salts and sulfur-containing compounds to the bacterial solution obtained in Step 3) to carry out a biomineralization reaction to obtain Clostridium yongdarii-polypyrrole / ferrous sulfide biohybrid.

[0012] Preferably, the iron salt in step 1) is ferric citrate or ferric chloride, with a concentration of 5-15 mM; and the amount of pyrrole added is 15-45 mM.

[0013] Preferably, the polymerization reaction temperature in step 2) is 37°C, the rotation speed is 100~180 r / min, and the reaction time is 24~36 h.

[0014] Preferably, the pH adjustment range in step 3) is 6.5 to 7.5.

[0015] Preferably, the iron salt in step 4) is ferric citrate or ferric chloride, with a concentration of 0.5-8 mM; the sulfur-containing compound is sodium thiosulfate or L-cysteine, with a concentration of 0.5-8 mM; and the biomineralization reaction time is 24-36 h.

[0016] Preferably, the YTF culture medium is composed of: 10 g / L tryptone, 16 g / L yeast extract, 10 g / L D-fructose, 0.2 g / L NaCl, 0.3 g / L cysteine ​​hydrochloride, 2 mL / L trace element solution, and 1 mL / L vitamin solution.

[0017] The composition of the trace element solution is as follows: 1.5 g / L nitric acid triacetic acid, 3 g / L MgSO4·7H2O, 0.5 g / L MnSO4·H2O, 1 g / L NaCl, 0.1 g / L FeSO4·7H2O, 0.1 g / L CoCl2·6H2O, 0.1 g / L CaCl2·2H2O, 0.1 g / L ZnSO4·7H2O, 0.01 g / L CuSO4·5H2O, 0.01 g / L AlK(SO4)2·12H2O, 0.01 g / L H3BO3, and 0.01 g / L Na2MoO4·2H2O.

[0018] The vitamin solution is composed of: biotin 2 mg / L, folic acid 2 mg / L, pyridoxine hydrochloride 10 mg / L, riboflavin 5 mg / L, thiamine 5 mg / L, niacin 5 mg / L, calcium pantothenate 5 mg / L, vitamin B12 0.1 mg / L, para-aminobenzoic acid 5 mg / L, and lipoic acid 5 mg / L.

[0019] A biological hybrid that enhances extracellular electron transport is prepared by the above-described method.

[0020] Preferably, the bio-hybrid is a Clostridium yongdarii-polypyrrole / ferrous sulfide bio-hybrid, wherein polypyrrole is attached to the surface of Clostridium yongdarii through a polymerization reaction, and ferrous sulfide nanoparticles are distributed on the surface, periplasmic space and intracellular space of Clostridium yongdarii through biomineralization, together constructing a transmembrane electron transport channel.

[0021] The aforementioned application of biohybrids in microbial electrosynthesis systems utilizes these biohybrids as biocatalysts to reduce carbon dioxide and produce chemicals.

[0022] Preferably, the microbial electrosynthesis system is a two-chamber reactor, with the anode being a platinum sheet or a ruthenium-iridium coated titanium mesh, the cathode being a carbon felt, the cathode chamber being inoculated with the bio-hybrid, the applied potential being -0.9 V to -1.2 V (vs. Ag / AgCl), the pH of the catholyte being 5.5 to 7.5, and carbon dioxide gas being introduced for electrocatalytic reduction.

[0023] The beneficial effects of this invention are as follows:

[0024] (1) This invention combines conductive polymer polypyrrole (PPy) with ferrous sulfide (FeS) nanoparticles in Clostridium yongdarii (… Clostridium ljungdahlii A continuous and efficient electron transport network was constructed on the surface and inside the microorganism. PPy significantly improved the overall conductivity of the microorganism by polymerizing to form a conductive layer on the cell surface; while FeS nanoparticles, due to their size diversity, not only distributed on the cell surface but also successfully entered the cell, establishing a rapid transmembrane direct electron transport channel. This synergistic modification of PPy and FeS effectively overcame the bottleneck of low electron transport efficiency in microorganisms, achieving efficient transmembrane transport of electrons from the extracellular to the intracellular space.

[0025] (2) In the biohybrid of the present invention, PPy is polymerized in situ on the surface of Clostridium yongdar to form a conductive layer, which significantly improves the interfacial contact performance between the bacteria and the electrode, enabling it to capture electrons from the cathode more effectively. Simultaneously, the introduction of FeS nanoparticles further reduces the interfacial impedance during electron transfer. Electrochemical characterization results show that, under optimized conditions (sodium thiosulfate addition of 2 mM), the Clostridium yongdar to FeS biohybrid exhibits the highest current response and the lowest charge transfer impedance, confirming the excellent interfacial electron transfer performance of this hybrid.

[0026] (3) This invention utilizes a constructed rapid electron transport channel, enabling the efficient conversion of extracellular electrons into intracellular energy flow (such as reducing power NADH). Experimental results show that the 7-day average current and NADH / NAD ratio of the Clostridium yongdarii-PPy / FeS biohybrid in the microbial electrosynthesis system are significantly improved. + The ratios were significantly higher than those of the Clostridium yongdarii-FeS hybrid, the Clostridium yongdarii-PPy hybrid, and Clostridium yongdarii alone. Correspondingly, the yields of acetic acid and 2,3-butanediol in this hybrid were significantly better than those in the control groups. This indicates that through synergistic modification with PPy and FeS, Clostridium yongdarii can more efficiently utilize cathode electrons to fix carbon dioxide or generate reducing power, thereby significantly improving the synthesis rate and yield of the target product.

[0027] (4) Unlike existing photocatalytic biohybrids that rely on light, the Clostridium yongdarii-PPy / FeS biohybrid constructed in this invention does not rely on an external light source and can be directly applied to microbial electrosynthesis systems to drive CO2 bioconversion using electrical energy. This strategy breaks through the inherent limitations of photocatalytic materials that are restricted by time and space, and provides new possibilities for the application of microbial electrosynthesis technology in continuous industrial production.

[0028] (5) By adjusting key process parameters such as the amount of pyrrole added during the PPy polymerization stage and the precursor concentration and catholyte pH during the FeS mineralization stage, the present invention can effectively control the synthesis rate of the target products (acetic acid and 2,3-butanediol). This provides a process basis for targeted optimization of product synthesis according to actual needs. Attached Figure Description

[0029] Figure 1 Electrochemical characterization of Clostridium yongdarii-FeS biohybrids with different sodium thiosulfate additions in Example 1: A is a linear scan; B is an impedance diagram.

[0030] Figure 2 The effect of different catholyte pH conditions on the production of chemicals by Clostridium yongdarii-FeS biohybrid in MES in Example 2: A is the average current; B is the acetic acid concentration;

[0031] Figure 3 The effect of different iron source precursors during the PPy formation stage on the production of chemicals by Clostridium yongdar in MES in Example 3: A is the concentration of acetic acid; B is the concentration of 2,3-butanediol.

[0032] Figure 4 The effect of different pyrrole addition amounts on Clostridium yongdarii during the PPy formation stage in Example 4: A is the acetic acid concentration during the synthesis stage; B is the butyric acid concentration during the synthesis stage; C is the acetic acid concentration in the MES; D is the 2,3-butanediol concentration in the MES.

[0033] Figure 5 Material characterization of Clostridium yongdarii-PPy / FeS in Example 5: A is SEM; B is TEM; C is EDS mapping; D is XPS image of Fe 2p; E is XPS image of S 2p; F is XPS image of N 1s;

[0034] Figure 6 For the MES performance of *Clostridium yongdarii*-PPy / FeS, *Clostridium yongdarii*-FeS, *Clostridium yongdarii*-PPy, and *Clostridium yongdarii* alone under the same conditions in Example 5: A is current; B is acetic acid concentration; C is 2,3-butanediol concentration; D is NADH / NAD ratio. + Ratio; E is ATP content; F is Faraday efficiency;

[0035] Figure 7 Electrochemical characterization of *Clostridium yongdarii*-PPy / FeS, *Clostridium yongdarii*-FeS, *Clostridium yongdarii*-PPy, and *Clostridium yongdarii* alone under the same conditions in Example 5: A is a CV scan; B is an impedance diagram; C is a biofilm SEM of *Clostridium yongdarii*-PPy / FeS; D is a biofilm SEM of *Clostridium yongdarii*-FeS; E is a biofilm SEM of *Clostridium yongdarii*-PPy; F is a biofilm SEM of *Clostridium yongdarii* alone. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0037] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] Example 1: Preparation of Clostridium yongdarii-FeS biohybrid and optimization of FeS precursor concentration

[0040] This embodiment investigates the effect of different sodium thiosulfate addition amounts on the electrochemical performance of Clostridium yongdarii-FeS biohybrid.

[0041] 1. Preparation of Clostridium yongdarii-FeS biohybrid

[0042] A method for in-situ synthesis of biological heterozygotes, the specific steps of which are as follows:

[0043] (1) Dissolve 2 mM ferric citrate and 0.5, 1, 2, 4, 6 and 8 mM sodium thiosulfate in 5 mL of YTF medium and stir until completely dissolved to obtain the precursor solution for FeS synthesis.

[0044] (2) During the logarithmic growth phase (OD) 600 Clostridium yongdarii (≈1.2) Clostridium ljungdahlii The above-mentioned precursor solution was added to the bacterial culture (purchased from the German Culture Collection Center, accession number ATCC 55383), and the pH of the solution was adjusted to 7.5.

[0045] (3) After 24 h of synthesis reaction, the solution turned black, indicating that FeS nanoparticles were synthesized in situ on the surface / periplasm / intracellular of Clostridium yongdar, thus obtaining Clostridium yongdar-FeS biohybrid.

[0046] The YTF culture medium is formulated as follows: tryptone 10 g / L, yeast extract 16 g / L, D-fructose 10 g / L, NaCl 0.2 g / L, cysteine ​​hydrochloride 0.3 g / L, trace element solution 2 mL / L, and vitamin solution 1 mL / L. To prepare, dissolve the above components in deionized water, sterilize at high temperature, and then cool before use.

[0047] The trace element solution is composed of: 1.5 g / L nitric acid, 3 g / L MgSO4·7H2O, 0.5 g / L MnSO4·H2O, 1 g / L NaCl, 0.1 g / L FeSO4·7H2O, 0.1 g / L CoCl2·6H2O, 0.1 g / L CaCl2·2H2O, 0.1 g / L ZnSO4·7H2O, 0.01 g / L CuSO4·5H2O, 0.01 g / L AlK(SO4)2·12H2O, 0.01 g / L H3BO3, and 0.01 g / L Na2MoO4·2H2O.

[0048] The vitamin solution is composed of: biotin 2 mg / L, folic acid 2 mg / L, pyridoxine hydrochloride 10 mg / L, riboflavin 5 mg / L, thiamine 5 mg / L, niacin 5 mg / L, calcium pantothenate 5 mg / L, vitamin B12 0.1 mg / L, para-aminobenzoic acid 5 mg / L, and lipoic acid 5 mg / L.

[0049] 2. Electrochemical performance testing

[0050] Electrochemical characterization was performed using a platinum sheet electrode (1 cm × 1 cm) as the anode, a carbon felt (1 cm × 1 cm) attached to the bio-hybrid obtained above as the cathode, and PBS (0.1 M, pH=7.0) as the electrolyte.

[0051] 3. Experimental Results and Analysis

[0052] like Figure 1 As shown in Figure A, within the sodium thiosulfate addition range of 0.5–2 mM, a higher addition results in a higher current response; conversely, within the range of 4–8 mM, a higher addition results in a lower current response. Figure 1 As shown in Figure B, the impedance change trend is consistent with the current response. When the sodium thiosulfate addition is 2 mM, the current response is highest and the impedance is lowest, indicating that FeS forms the optimal size under these conditions, exhibiting the best electron transport performance. Insufficient sodium thiosulfate addition cannot effectively utilize ferric citrate to form FeS, while excessive addition can easily inhibit bacterial activity.

[0053] Example 2: Effect of catholyte pH on the performance of Clostridium yongdarii-FeS biohybrid MES

[0054] This embodiment investigates the acid-producing performance of Clostridium yongdarii-FeS biohybrid in a microbial electrosynthesis system under different catholyte pH conditions.

[0055] 1. Preparation of Clostridium yongdarii-FeS biohybrid

[0056] A method for in-situ synthesis of biological heterozygotes, the specific steps of which are as follows:

[0057] (1) Dissolve 2 mM ferric citrate and 2 mM sodium thiosulfate in 5 mL of YTF medium and stir until completely dissolved to obtain the precursor solution for FeS synthesis.

[0058] (2) During the logarithmic growth phase (OD) 600 Add the above precursor solution to the Clostridium yongdarii bacterial culture (≈1.2), and adjust the pH of the solution to 7.5.

[0059] (3) After 24 h of synthesis reaction, the solution turned black, and the Clostridium yongdarii-FeS biohybrid was obtained.

[0060] (4) Centrifuge the above-mentioned bio-hybrid solution, wash it twice with PBS (0.1 M, pH=7.0), and connect it to the cathode chamber of the microbial electrosynthesis reactor.

[0061] 2. Application of Microbial Electrosynthesis (MES) System

[0062] A ruthenium-iridium coated titanium mesh was used as the anode, a carbon felt as the cathode, and a catholyte as the electrolyte. The pH of the catholyte was adjusted to 5.5, 6.0, 6.5, 7.0, and 7.5. The aforementioned bio-hybrid was inoculated into the cathode chamber, a potential of -1.0 V (vs. Ag / AgCl) was applied, a reference electrode was placed near the cathode, and 99.99% carbon dioxide gas was introduced for electrocatalytic reduction.

[0063] 3. Experimental Results and Analysis

[0064] like Figure 2 As shown in Figure A, within the pH range of 5.5 to 7.0, the higher the pH of the catholyte, the higher the average current, reaching a maximum of 38.24 ± 4.24 mA (pH 7.0). Figure 2 As shown in Figure B, acetic acid production increases with increasing pH, reaching a maximum of 4.3 ± 0.10 g / L at pH 7.0. When the pH rises to 7.5, although the current remains high, the acetic acid production decreases rapidly. This is because the excessively high pH combined with the high current leads to a continuous increase in the pH of the catholyte, which is unfavorable for microbial growth.

[0065] The experimental results of this embodiment show that when the catholyte pH is 7.0, the acetic acid yield of CO2 reduction by the Clostridium yongdarii-FeS hybrid in the MES is optimal.

[0066] Example 3: Effects of different iron sources on the MES performance of Clostridium yongdarii-PPy biohybrid

[0067] This embodiment investigates the effect of different iron sources during the polymerization stage of the conductive polymer polypyrrole (PPy) on the chemical properties produced by Clostridium yongdarii-PPy biohybrid.

[0068] 1. Preparation of Clostridium yongdarii-PPy biohybrid

[0069] A method for in-situ polymerization to form biological hybrids, comprising the following steps:

[0070] (1) Dissolve 10 mM FeCl3·6H2O or ferric citrate in 5 mL of YTF medium.

[0071] (2) Add the above precursor solution to the Clostridium yongdar bacteria solution in the logarithmic growth phase, and add 30 mM pyrrole solution. Place the mixture in a shaker at 37°C and 130 r / min for polymerization.

[0072] (3) After 24 h of polymerization, the solution color turned grayish-black, and the in-situ polymerized Clostridium yongdarii-PPy biohybrid was obtained.

[0073] (4) Centrifuge the above-mentioned bio-hybrid solution, wash it twice with PBS (0.1 M, pH=7.0), and connect it to the cathode chamber of the microbial electrosynthesis reactor.

[0074] 2. MES System Application

[0075] A ruthenium-iridium coated titanium mesh was used as the anode, a carbon felt as the cathode, and a catholyte as the electrolyte with a pH of 6.0. The aforementioned bio-hybrid was inoculated into the cathode chamber, and a potential of -1.0 V (vs. Ag / AgCl) was applied. The reference electrode was placed near the cathode, and carbon dioxide gas (99.99%) was introduced for electrocatalytic reduction.

[0076] 3. Experimental Results and Analysis

[0077] like Figure 3 As shown, PPy formed using FeCl3·6H2O as the iron source had an acetic acid yield of 2.79 ± 0.40 g / L. Figure 3 (A), the yield of 2,3-butanediol was 0.70 ± 0.06 g / L ( Figure 3 The PPy (Py) obtained using iron source ferric citrate (B) was significantly higher than that obtained using iron source ferric citrate (Ferric citrate).

[0078] The experimental results of this embodiment show that FeCl3·6H2O, as an iron source, is significantly superior to ferric citrate in terms of target product yield when forming Clostridium yongdarii-PPy biohybrids and applying them to MES for CO2 reduction.

[0079] Example 4: Effect of different pyrrole addition amounts on the MES performance of Clostridium yongdarii-PPy / FeS biohybrid.

[0080] This embodiment investigates the effect of different pyrrole addition amounts during the PPy polymerization stage on the chemical properties produced by the Clostridium yongdarii-PPy / FeS biohybrid.

[0081] 1. Preparation of Clostridium yongdarii-PPy / FeS biohybrid

[0082] A method for preparing a biological hybrid that enhances extracellular electron transfer, comprising the following specific steps:

[0083] (1) Dissolve 10 mM FeCl3·6H2O in 5 mL of YTF medium.

[0084] (2) During the logarithmic growth phase (OD) 600 The above precursor solution was added to the Clostridium yongdar bacterial culture (≈1.2), and 0, 15, 30, and 45 mM pyrrole solutions were added respectively. The mixture was placed in a shaker at 37℃ and 130 r / min for 24 h to carry out the polymerization reaction. The solution color turned gray-black, and the Clostridium yongdar-PPy biohybrid was obtained.

[0085] (3) Centrifuge the above Clostridium yongdarii-PPy biohybrid solution and wash it twice with PBS (0.1 M, pH=7.0).

[0086] (4) Dissolve 2 mM ferric citrate and 2 mM sodium thiosulfate in 5 mL of YTF medium and stir until completely dissolved to obtain the precursor solution for FeS synthesis.

[0087] (5) During the logarithmic growth phase (OD) 600 The above precursor solution was added to the Clostridium yongdarii-PPy hybrid bacterial culture (≈1.2), and the pH was adjusted to 7.5 with saturated sodium bicarbonate solution.

[0088] (6) After 24 h of synthesis reaction, the solution turned black, and the Clostridium yongdarii-PPy / FeS biohybrid was obtained.

[0089] (7) Centrifuge the above Clostridium yongdarii-PPy / FeS biohybrid solution, wash twice with PBS (0.1 M, pH=7.0), and connect it to the cathode chamber of the microbial electrosynthesis reactor.

[0090] 2. MES System Application

[0091] A ruthenium-iridium coated titanium mesh was used as the anode, a carbon felt as the cathode, and a catholyte as the electrolyte with a pH of 6.5. The above-mentioned Clostridium yongdarii-PPy / FeS biohybrid was inoculated into the cathode chamber, and a potential of -1.0 V (vs. Ag / AgCl) was applied. The reference electrode was placed near the cathode, and carbon dioxide gas (99.99%) was introduced for electrocatalytic reduction.

[0092] 3. Experimental Results and Analysis

[0093] like Figure 4 As shown in Figures A and B, the addition of 15–45 mM pyrrole to Clostridium yunnanense in its logarithmic growth phase did not affect its growth, and the concentrations of acetic acid and butyric acid in all four groups remained within the normal range. Figure 4 As shown in Figure C, the acetic acid yield of the Clostridium yongdarii-PPy / FeS biohybrid formed with 15 mM pyrrole was 3.93 ± 0.14 g / L, and the acetic acid yield of the hybrid formed with 30 mM pyrrole was 4.20 ± 0.08 g / L, both significantly higher than that of the hybrid formed with 45 mM pyrrole. Figure 4 As shown in Figure D, the hybrid 2,3-butanediol formed by 45 mM pyrrole yielded the highest amount (1.11 ± 0.12 g / L), followed by 30 mM (0.91 ± 0.10 g / L), both significantly higher than 15 mM.

[0094] The experimental results of this embodiment show that the yield of acetic acid is optimal when the amount of pyrrole added is 30 mM, and the yield of 2,3-butanediol is optimal when the amount of pyrrole added is 45 mM. The appropriate amount of pyrrole added can be selected according to the target product requirements.

[0095] Example 5: Comparison of MES performance between Clostridium yongdarii-PPy / FeS heterozygote and control group

[0096] This embodiment compares the performance of Clostridium yongdarii-PPy / FeS hybrid, Clostridium yongdarii-FeS hybrid, Clostridium yongdarii-PPy hybrid, and Clostridium yongdarii alone in the MES system.

[0097] 1. Preparation of Clostridium yongdarii-PPy / FeS hybrid

[0098] A method for preparing a biological hybrid that enhances extracellular electron transfer, comprising the following specific steps:

[0099] (1) Dissolve 10 mM FeCl3·6H2O in 5 mL of YTF medium.

[0100] (2) During the logarithmic growth phase (OD) 600The above precursor solution was added to the Clostridium yongdar bacterial culture (≈1.2), and 30 mM pyrrole solution was added. The mixture was placed in a shaker at 37℃ and 130 r / min for 24 h to carry out the polymerization reaction. The solution color turned gray-black, and the Clostridium yongdar-PPy hybrid was obtained.

[0101] (3) Centrifuge the above Clostridium yongdarii-PPy hybrid solution and wash it twice with PBS (0.1 M, pH=7.0).

[0102] (4) Dissolve 2 mM ferric citrate and 2 mM sodium thiosulfate in 5 mL of YTF medium and stir until completely dissolved to obtain the precursor solution for FeS synthesis.

[0103] (5) The above precursor solution was added to the Clostridium yongdarii-PPy hybrid bacterial solution and the Clostridium yongdarii bacterial solution alone in the logarithmic growth phase, the pH was adjusted to 7.5, and the reaction was carried out for 24 h to obtain Clostridium yongdarii-PPy / FeS hybrid and Clostridium yongdarii-FeS hybrid, respectively.

[0104] (6) Centrifuge the above hybrid solution and wash twice with PBS (0.1 M, pH=7.0).

[0105] 2. MES System Application

[0106] A ruthenium-iridium coated titanium mesh was used as the anode, a carbon felt as the cathode, and a catholyte as the electrolyte with a pH of 6.5. Clostridium yongdarii-PPy / FeS hybrids, Clostridium yongdarii-FeS hybrids, Clostridium yongdarii-PPy hybrids, and Clostridium yongdarii alone were inoculated into the cathode chamber. A potential of -1.0 V (vs. Ag / AgCl) was applied, and a reference electrode was placed near the cathode. Carbon dioxide gas (99.99%) was introduced for electrocatalytic reduction.

[0107] 3. Experimental Results and Analysis

[0108] like Figure 5 As shown, Clostridium yunnanense maintains a typical rod-shaped bacterial morphology. Figure 5 (A) The cell outline is intact, indicating that the biosynthesis and coating process is mild and does not significantly damage the physiological structure of the bacteria. The cell surface is composed of a large number of tightly packed PPy / FeS nanorods, which significantly improves the specific surface area and surface roughness of the material. The nanoparticles are uniformly distributed on the cell surface without obvious local aggregation. In the TEM image, due to the electron beam penetrating the entire cell, the nanorods overlap in the projection direction, resulting in a loose and interwoven network morphology of the functional layer on the cell surface. Figure 5 (B). Elements Fe, S, and N ( Figure 5The signal intensity (C) is uniformly distributed along the rod-shaped contour of the bacterial cell, exhibiting high intensity and a complete range, closely matching the bacterial morphology. This indicates that the biosynthesized PPy / FeS nanoparticles have been successfully loaded onto the bacterial surface, with the nitrogen element derived from the pyrrole ring nitrogen atom of PPy. The XPS of Fe 2p is shown in Figure 1. Figure 5 As shown in Figure D, its two main peaks are located at 710.9 eV and 724.2 eV, respectively, corresponding to Fe 2p. 3 / 2 and Fe2p 1 / 2 The XPS S2p is like... Figure 5 As shown in Figure E, its main peak is located at 161.5 eV, corresponding to S 2p. 3 / 2 The XPS of N 1s is as follows Figure 5 As shown in Figure F, its main peak is located at 399.3 eV, corresponding to pyrrole-type N. The above material characterization demonstrates that PPy / FeS has been successfully synthesized on Clostridium yongdarii.

[0109] like Figure 6 As shown, Clostridium yongdarii-PPy / FeS hybrid ( C.lj The 7-day average current of -PPy / FeS was 42.31 ± 2.39 mA. Figure 6 (Among the samples), the highest yield of acetic acid was 4.20 ± 0.08 g / L. Figure 6 The highest yield of 2,3-butanediol was 0.94 ± 0.11 g / L (in the middle B group). Figure 6 (C) Because the production of 2,3-butanediol requires a large supply of reducing power, the NADH / NAD ratio is... + The ratio was also as high as 0.65±0.06 ( Figure 6 (Middle D). Correspondingly, its ATP content is as high as 4.98±0.66 μmol ( Figure 6 (E). ATP is a prerequisite for anabolism; high ATP levels mean that cells have enough energy to fix CO2. In contrast, Clostridium yongdarii-FeS hybrids ( C.lj The highest acetic acid yield of NADH (FeS) reached 3.87 ± 0.15 g / L, but the yield of 2,3-butanediol was only 0.42 ± 0.01 g / L, and the NADH / NAD ratio was... + The ratio was 0.38±0.05, and the ATP content was 2.36±0.29 μmol, only higher than that of Clostridium yunnanense alone. C.lj Clostridium yongdarii-PPy heterozygote ( C.lj The highest 2,3-butanediol yield (-PPy) can reach 0.72 ± 0.05 g / L, NADH / NAD ratio +The ratio was also as high as 0.55±0.02, with an ATP content of 2.99±0.12 μmol, but the acetic acid production was only 2.58±0.01 g / L, only higher than that of Clostridium yunnanense alone. The Faraday efficiency of this reaction process was calculated (…). Figure 6 As shown in Figure F, the Clostridium yongdarii-PPy / FeS hybrid exhibits the highest Faradaic efficiency for 2,3-butanediol (23.71 ± 3.13%), indicating that the synergistic effect of PPy and FeS is more conducive to electron flow to the 2,3-butanediol synthesis pathway. Meanwhile, the Clostridium yongdarii-FeS hybrid exhibits the highest Faradaic efficiency for acetic acid (62.25 ± 4.60%), suggesting that FeS modification alone tends to promote acetic acid production.

[0110] like Figure 7 As shown in Figure A, Clostridium yongdarii-PPy / FeS hybrid ( C.lj The oxidation peak current density of -PPy / FeS was the highest, and the reduction peak current was also the most negative, indicating that it had the strongest electrochemical activity. The second strongest was the Clostridium yongdarii-PPy hybrid (…). C.lj -PPy) and Clostridium yongdarii-FeS hybrid ( C.lj -FeS), Clostridium yongdarii ( C.lj The current response of the redox current is the weakest and there are almost no redox peaks. The resistance is also consistent with the magnitude of the redox current; the larger the redox current, the smaller the resistance. Figure 7 Similarly, SEM images of the cathode biofilm after 7 days of MES reaction showed that the Clostridium yongdarii-PPy / FeS hybrid ( Figure 7 The C100 exhibits the densest and thickest biofilm, with the cathode surface covered by a large number of bacteria; in contrast, the Clostridium yongdarii-PPy hybrid (… Figure 7 The *Clostridium yongdarii*-FeS hybrid also exhibited good biofilm formation ability, with high cell loading and dense distribution. It is worth noting that although the *Clostridium yongdarii*-FeS hybrid (… Figure 7 The biofilm thickness formed by the modified Clostridium yongdarii was slightly lower than that of the former two, but its bacterial cell adhesion was still significantly better than that of the unmodified Clostridium yongdarii. Figure 7 (F). This series of comparisons shows that the introduction of PPy / FeS can significantly enhance the adhesion of bacteria to the cathode and their biofilm formation potential.

[0111] The experimental results of this embodiment show that the *Clostridium yongdarii*-PPy / FeS hybrid exhibits optimal electrochemical activity and product synthesis performance in MES. Material characterization confirmed that PPy / FeS forms a dense conductive network on the bacterial cell surface, effectively reducing interfacial charge transfer impedance and promoting biofilm formation. Intracellular NADH / NAD... +The increased ratio and ATP content further confirm the enhanced reducing power supply. Therefore, the synergistic modification of PPy and FeS significantly enhances the ability of Clostridium yongdarii to fix CO2 and synthesize acetic acid and 2,3-butanediol by optimizing the electron transfer efficiency at the microbial-electrode interface.

[0112] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a biological hybrid that enhances extracellular electron transport, characterized in that, Includes the following steps: Step 1) Dissolve the iron salt and pyrrole in YTF medium and mix thoroughly; Step 2) Under anaerobic conditions, the solution obtained in Step 1) is added to Clostridium yunnanense in the logarithmic growth phase (Clostridium yunnanense). Clostridium ljungdahlii In the bacterial culture, a polymerization reaction was carried out to obtain Clostridium yongdarii-polypyrrole biohybrid; Step 3) After centrifuging and washing the Clostridium yongdarii-polypyrrole biohybrid obtained in Step 2), resuspend it in YTF medium and adjust the pH to neutral. Step 4) Add iron salts and sulfur-containing compounds to the bacterial solution obtained in Step 3) to carry out a biomineralization reaction to obtain Clostridium yongdarii-polypyrrole / ferrous sulfide biohybrid.

2. The method for preparing a biological hybrid that enhances extracellular electron transport according to claim 1, characterized in that, Step 1) The iron salt is ferric citrate or ferric chloride, with a concentration of 5~15 mM; the amount of pyrrole added is 15~45 mM.

3. The method for preparing a biological hybrid that enhances extracellular electron transfer according to claim 1, characterized in that, Step 2) The polymerization reaction temperature is 37℃, the rotation speed is 100~180 r / min, and the reaction time is 24~36 h.

4. The method for preparing a biological hybrid that enhances extracellular electron transport according to claim 1, characterized in that, Step 3) The pH adjustment range is 6.5~7.

5.

5. The method for preparing a biological hybrid that enhances extracellular electron transport according to claim 1, characterized in that, Step 4) The iron salt is ferric citrate or ferric chloride, with a concentration of 0.5~8 mM; the sulfur-containing compound is sodium thiosulfate or L-cysteine, with a concentration of 0.5~8 mM; the biomineralization reaction time is 24~36 h.

6. The method for preparing a biological hybrid that enhances extracellular electron transfer according to claim 1, characterized in that, The YTF culture medium consists of: 10 g / L tryptone, 16 g / L yeast extract, 10 g / L D-fructose, 0.2 g / L NaCl, 0.3 g / L cysteine ​​hydrochloride, 2 mL / L trace element solution, and 1 mL / L vitamin solution. The composition of the trace element solution is as follows: 1.5 g / L nitric acid triacetic acid, 3 g / L MgSO4·7H2O, 0.5 g / L MnSO4·H2O, 1 g / L NaCl, 0.1 g / L FeSO4·7H2O, 0.1 g / L CoCl2·6H2O, 0.1 g / L CaCl2·2H2O, 0.1 g / L ZnSO4·7H2O, 0.01 g / L CuSO4·5H2O, 0.01 g / L AlK(SO4)2·12H2O, 0.01 g / L H3BO3, and 0.01 g / L Na2MoO4·2H2O. The vitamin solution is composed of: biotin 2 mg / L, folic acid 2 mg / L, pyridoxine hydrochloride 10 mg / L, riboflavin 5 mg / L, thiamine 5 mg / L, niacin 5 mg / L, calcium pantothenate 5 mg / L, vitamin B12 0.1 mg / L, para-aminobenzoic acid 5 mg / L, and lipoic acid 5 mg / L.

7. A biological hybrid that enhances extracellular electron transport, characterized in that, It is prepared by any one of the preparation methods described in claims 1-6.

8. A biological hybrid that enhances extracellular electron transport according to claim 7, characterized in that, The bio-hybrid is a Clostridium yongdarii-polypyrrole / ferrous sulfide bio-hybrid, wherein polypyrrole is attached to the surface of Clostridium yongdarii through a polymerization reaction, and ferrous sulfide nanoparticles are distributed on the surface, periplasmic space and intracellular space of Clostridium yongdarii through biomineralization, together constructing a transmembrane electron transport channel.

9. The application of the bio-hybrid as described in claim 7 or 8 in a microbial electrosynthesis system, characterized in that, The bio-hybrid is used as a biocatalyst to reduce carbon dioxide and produce chemicals.

10. The application according to claim 9, characterized in that, The microbial electrosynthesis system is a two-chamber reactor. The anode is a platinum sheet or a ruthenium-iridium coated titanium mesh, and the cathode is a carbon felt. The cathode chamber is inoculated with the bio-hybrid. The applied potential is -0.9 V to -1.2 V (vs. Ag / AgCl). The pH of the catholyte is 5.5 to 7.

5. Carbon dioxide gas is introduced for electrocatalytic reduction.

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