Gram-positive bacterium extracellular electron transport chain construction method and synthetic microbial agent

Abstract

The invention discloses a gram-positive bacterium extracellular electron transport chain construction method and a synthetic microbial agent. It is found that after a lipophilic material is added into an iron reduction system, the iron reduction speed and the iron reduction amount of gram-positive bacteria with weak electron transfer capacity are remarkably increased, and the iron reduction amount of the gram-positive bacteria can be consistent with the iron reduction magnitude of gram-negative strong iron reduction bacteria. The lipophilic degree and the iron reduction rate are subjected to correlation analysis and show significant positive correlation, which reveals that the lipophilic degree and the permeable membrane capacity of the material are key parameters for determining the iron reduction capacity of gram-positive bacteria. The lipophilic material not only can smoothly penetrate through the cell wall of gram-positive bacteria, but also can be enriched on a cell membrane in quantity, and gradually permeates into a cytoplasm matrix to generate electron exchange with NADH (Nicotinamide Adenine Dinucleotide Hormone) so as to construct a gram-positive bacteria extracellular electron transport chain. The invention provides a brand-new strategy for preparing the biological geological catalytic synthetic microbial inoculum for soil iron reduction.

Description

Technical Field

[0001] This invention belongs to the field of microbial electron transport technology, specifically relating to a method for constructing extracellular electron transport chains in Gram-positive bacteria and a synthetic bacterial agent. Background Art

[0002] Extracellular electron transfer (EET) is the process by which intracellular electrons produced by microorganisms are transferred across the membrane to extracellular solid electron acceptors such as iron oxide and manganese oxide. This process not only provides energy for the growth and reproduction of anaerobic microorganisms but is also a core driving force in the biogeochemical cycles of metals such as iron, manganese, and uranium. In anaerobic environments, this process drives a series of surface processes due to the continuous transfer of electrons from microorganisms to the extracellular environment. The most attention-grabbing process, dissimilar iron reduction, can couple with important environmental processes such as methane oxidation, ammonia oxidation, and interspecific methanogenesis, with extracellular electron transfer becoming the rate-limiting reaction in these processes. Extracellular electron transfer is a crucial link between the energy metabolism of anaerobic microorganisms and the cycling of carbon, nitrogen, and metals on the Earth's surface, and has become a cutting-edge field of interest for multiple disciplines, including soil science, environmental geochemistry, biogeochemistry, aquatic engineering, and even gut microbiology.

[0003] Since Lovley et al. reported on Geobacter ( Geobacter Shewanella ( Shewanella Since the discovery of extracellular respiration in Gram-negative bacteria, a theoretical system of extracellular electron transport, primarily driven by Gram-negative bacteria, has gradually developed and formed. This system includes the extracellular electron transport chain and functional proteins, electron shuttle mechanisms, and environmental effects. The key mechanism lies in the presence of redox-active cytochrome c on the periplasm and outer membrane of Gram-negative bacteria. This cytochrome c can accept electrons donated by inner membrane proteins and transfer them to the outside of the cell in a stepwise manner. The scientific discovery that intracellular electrons "pass through" the non-conductive cell membrane and cell wall to participate in geochemical reactions has led to the growing academic understanding that "Gram-negative bacteria such as *Geobacterium* and *Shewanella* dominate dissimilatory iron reduction." Microbially driven iron cycling plays a crucial role in the Earth's surface element cycle, and Gram-negative bacteria (such as *Geobacterium* and *Shewanella*) have become core participants in the iron reduction process due to their unique membrane structure and efficient electron transport capabilities. These strains achieve efficient extracellular electron transfer through a continuous electron transport protein network from the inner to the outer membrane (such as the MtrCAB complex). Shewanella MR-1 even exhibits bidirectional electron transfer capabilities, generating up to 3000 mW / m² after anodic film formation. 2 The current rapidly transports electrons to the extracellular iron minerals.

[0004] Gram-positive bacteria such as Bacillus and Clostridium have a 20-80 nm thick peptidoglycan cell wall barrier (accounting for 60% to 90%), lacking electron transport carriers such as outer membrane cytochrome c, making it difficult to achieve effective extracellular electron transport. Their extracellular electron transport capacity is typically low (their maximum biocurrent is only 92 mW / m). 2 Gram-negative bacteria, with an extracellular electron transport rate of less than 1% (as low as 1% of *Geobacterium*), are traditionally considered to have weak electroactivity and be unlikely to directly participate in iron reduction. This difference is mainly attributed to the fundamental differences in the cell structures of the two types of bacteria: the outer membrane of Gram-negative bacteria is rich in cytochrome c and channel proteins, forming a complete transmembrane electron transport chain, while the thick walls of Gram-positive bacteria, although providing mechanical protection, severely hinder electron transport. Therefore, although Gram-positive bacteria are diverse and abundant in nature—with *Bacillus* alone accounting for 5%–10% (flooded) or 40%–50% (dried) in paddy field soil, a difference of orders of magnitude compared to Gram-negative bacteria like *Geobacterium*—they have long been defined as "weak electron transport bacteria" or "non-iron reducing bacteria," and are not considered major drivers of iron reduction. Therefore, realizing extracellular electron transport and iron reduction in Gram-positive bacteria is of great significance for realizing and accelerating the iron cycle and even the broader element cycle.

[0005] The article "A New Electron Shuttling Pathway Mediated by Lipophilic Phenoxazine via the Interaction with Periplasmic and Inner Membrane Proteins of Shewanella oneidensis MR-1" discloses that knocking out the key cytochromes MtrC and OmcA on the outer membrane of Shewanella MR-1 significantly weakened protein-dependent electron transport, causing the biocurrent to plummet to 12% of the wild-type. However, when a lipophilic material (such as resazurite) was added, the current recovered to near the wild-type level. This indicates that lipophilic molecules can bypass the outer membrane protein transport channels of Gram-negative bacteria, directly harvesting electrons from the periplasm and transferring them to external receptors. However, there is currently no evidence to suggest whether Gram-positive bacteria can construct a ternary transport network of "cell-lipophilic material-mineral" by utilizing exogenous lipophilic shuttles. Especially given the thick cell walls of Gram-positive bacteria, whether lipophilic materials can overcome the physical limitations imposed by this thick-walled structure is crucial. Gram-positive bacteria have long been considered to have almost no extracellular electron transport capabilities. If they could penetrate cells and transfer electrons, it would enable highly efficient electron transport in Gram-positive bacteria, which would be significant for driving iron reduction in nature and even broader element cycles. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a method for constructing the extracellular electron transport chain of Gram-positive bacteria.

[0007] A second objective of this invention is to provide the application of lipophilic materials in enhancing the extracellular electron transport capacity of Gram-positive bacteria.

[0008] A third objective of this invention is to provide the application of lipophilic materials in enhancing the iron-reducing capacity of Gram-positive bacteria.

[0009] The fourth objective of this invention is to provide a synthetic bacterial agent system comprising lipophilic materials and Gram-positive bacteria.

[0010] The fifth objective of this invention is to provide a synthetic microbial agent prepared from the above-described synthetic microbial agent system.

[0011] The sixth object of the present invention is to provide the application of the above-mentioned microbial agent in accelerating iron reduction and increasing the amount of iron reduction, or in the preparation of products that accelerate iron reduction and increase the amount of iron reduction.

[0012] A seventh objective of this invention is to provide the application of the above-mentioned microbial agent in soil remediation or in the preparation of soil remediation products.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a method for constructing an extracellular electron transport chain in Gram-positive bacteria, by adding a lipophilic material to the bacterial culture of Gram-positive bacteria; the lipophilic material has the ability to pass through the cell wall of Gram-positive bacteria, permeate the cell membrane, and exchange electrons with intracellular electron donors.

[0014] For a long time, iron-reducing bacteria isolated from the natural environment have mainly been Gram-negative bacteria. Gram-positive bacteria, lacking a transmembrane protein-mediated electron transport chain, exhibit extremely slow extracellular iron reduction rates and are considered weak electron-transferring bacteria; their extracellular electron transport capabilities have long been neglected. This invention discovers that adding a lipophilic material to the iron reduction system significantly increases the rate and amount of iron reduction in Gram-positive bacteria with weak electron transport capabilities. Their iron reduction amounts are comparable to those of strong iron-reducing Gram-negative bacteria, indicating that the lipophilic material effectively mediates the iron reduction process in Gram-positive bacteria. Further investigation revealed that the material's ability to penetrate the cell membrane and transfer electrons across the membrane is closely related to its lipophilicity. The lipophilic material not only successfully penetrates the cell wall of Gram-positive bacteria but also accumulates extensively on the cell membrane and gradually penetrates into the cytoplasm, exchanging electrons with the intracellular electron donor NADH. Correlation analysis between lipophilicity and iron reduction rate revealed a significant positive correlation between the two. In other words, lipophilic materials can autonomously assemble with Gram-positive bacteria and embed themselves in their cell walls and intracellular membranes. As shuttle molecules, lipophilic materials work together with the intracellular electron transport chain of Gram-positive bacteria to construct a completely new extracellular electron transport chain. Without the participation of cytochrome c in the extracellular membrane as traditionally understood, Gram-positive bacteria are transformed from non-ferrogenic bacteria into strong ferrogenic bacteria, with the iron reduction rate increasing by two orders of magnitude, reaching the same level as strong ferrogenic bacteria such as Shewanella. This overturns the traditional understanding that Gram-positive bacteria have a slow rate of reducing solid iron oxide.

[0015] Based on this, the present invention also provides the application of lipophilic materials in improving the extracellular electron transfer ability of Gram-positive bacteria, wherein the lipophilic materials have the ability to pass through the cell wall of Gram-positive bacteria, permeate the cell membrane, and exchange electrons with intracellular electron donors.

[0016] This invention also provides the application of lipophilic materials in enhancing the iron-reducing capacity of Gram-positive bacteria. These lipophilic materials possess the ability to permeate the cell wall and cell membrane of Gram-positive bacteria and exchange electrons with intracellular electron donors. By adding lipophilic materials to the culture environment of Gram-positive bacteria, bacteria that originally had extremely low iron-reducing efficiency can acquire strong iron-reducing capabilities, thus becoming strong iron-reducing bacteria.

[0017] The present invention also provides a synthetic bacterial agent system, the system comprising a lipophilic material and Gram-positive bacteria; the lipophilic material has the ability to pass through the cell wall of Gram-positive bacteria, permeate the cell membrane, and exchange electrons with intracellular electron donors.

[0018] The present invention also provides a synthetic microbial agent prepared from the above-described synthetic microbial agent system.

[0019] Gram-positive bacteria are abundant and diverse in nature. Bacillus alone can reach 5%–10% (flooded) or 40%–50% (dried) in paddy field soil, a number order of magnitude lower than Gram-negative bacteria such as Geobacter. However, they have long been defined as "weak electron-transferring bacteria" and not considered major drivers of iron reduction. Therefore, providing a bacterial agent that leverages the efficient electron transfer capabilities of Gram-positive bacteria to drive the iron cycle and even broader elemental cycles is of great significance, especially in humus-rich or microbially interactive environments. This invention discovers that by adding lipophilic materials to a Gram-positive bacterial culture environment, weak electron-transferring Gram-positive bacteria can be transformed into bacteria with strong electron-transferring capabilities; and weak iron-reducing bacteria and non-iron-reducing bacteria can be transformed into strong iron-reducing bacteria. This invention has been extensively validated using a series of different genera of Gram-positive bacteria, including Clostridium, Bacillus, Staphylococcus, and Corynebacterium, to demonstrate that this effect is a common characteristic of a series of Gram-positive bacteria, providing a novel construction strategy for the synthesis of iron-reducing biocatalytic agents.

[0020] Furthermore, the method for preparing the synthetic bacterial agent involves adding a lipophilic material to the bacterial solution of Gram-positive bacteria.

[0021] The present invention also provides the application of the above-mentioned synthetic bacterial agent in accelerating iron reduction and increasing the amount of iron reduction, or in the preparation of products that accelerate iron reduction and increase the amount of iron reduction.

[0022] Furthermore, the accelerated iron reduction refers to accelerating the reduction of iron in soil or water.

[0023] The present invention also provides the application of the above-mentioned synthetic microbial agent in soil remediation or in the preparation of soil remediation products.

[0024] Furthermore, the Gram-positive bacteria include, but are not limited to, one or more of the genera Clostridium, Bacillus, Staphylococcus, or Corynebacterium.

[0025] Preferably, the Gram-positive bacteria are selected from... Bacillus subtilis , Clostridium sporogenes , Corynebacterium glutamicum , Staphylococcus xylosus or Bacillus megatherium One or more of them 。

[0026] Furthermore, the lipophilic material includes, but is not limited to, one or more of resazurin (RZ), phenazine-1-carboxylic acid (PCA), 2-hydroxy-1,4-naphthoquinone (2-HNQ), phenazine (PHZ), or 1-hydroxyphenazine (1-OHP).

[0027] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for constructing the extracellular electron transport chain of Gram-positive bacteria and a synthetic bacterial agent. It was found that adding a lipophilic material to an iron reduction system significantly increased the iron reduction rate and amount of Gram-positive bacteria with weak electron transport capabilities. The iron reduction amount reached a level comparable to that of Gram-negative, strongly iron-reducing bacteria, indicating that the lipophilic material effectively mediates the extracellular electron transport and iron reduction processes of Gram-positive bacteria. Further investigation revealed that the material's ability to penetrate the cell membrane and transfer electrons across the membrane is closely related to its lipophilicity. Correlation analysis between the degree of lipophilicity and the iron reduction rate showed a significant positive correlation, revealing that the degree of lipophilicity and membrane permeability of the material are key parameters determining the iron reduction capacity of Gram-positive bacteria. Lipophilic materials not only successfully penetrate the cell walls of Gram-positive bacteria but also accumulate in large quantities on the cell membrane and gradually permeate into the cytoplasm. There, they exchange electrons with the intracellular electron donor NADH, essentially undergoing autonomous assembly with the Gram-positive bacteria, embedding themselves in their cell walls and intracellular membranes. As shuttle molecules, these lipophilic materials, together with the intracellular electron transport chain of Gram-positive bacteria, construct a novel extracellular electron transport chain. Without the traditionally recognized participation of extracellular membrane cytochrome c, Gram-positive bacteria are transformed from non-ferrogenic bacteria into strong ferrogenic bacteria, with the iron reduction rate increasing by two orders of magnitude, reaching the same level as strong ferrogenic bacteria such as Shewanella. This overturns the traditional understanding that Gram-positive bacteria have a slow rate of reducing solid iron oxide. This invention provides a novel strategy for preparing biogeocatalytic synthetic agents for soil iron reduction, and has significant implications for driving the iron cycle and even the broader elemental cycle. Attached Figure Description

[0028] Figure 1 Lipophilic materials can effectively promote the iron reduction process in Gram-positive bacteria. Note: Shewanella oneidensis , Bacillus subtilis and Clostridium sporogenes The treatments were based on the iron reduction kinetics of the three microorganisms themselves, without the inclusion of materials; PHZ, 2-HNQ, PCA, RZ, and 1-OHP were lipophilic material treatments; AQDS, AQS, RF, and FMN were hydrophilic material treatments; CK was a blank control without materials; all iron reduction kinetic systems included OD. 600 =0.5 bacterial count, 50 μmol / L (hydrophilic / lipophilic) material, 5 mmol / L ferrohydrate (as Fe).

[0029] Figure 2 Lipophilic materials can effectively promote iron reduction in other Gram-positive bacteria.

[0030] Figure 3 Gram-positive bacteria Clostridium sporogenes Kinetics of reducing (lipophilic / hydrophilic) active materials.

[0031] Figure 4 The relationship between total iron reduction in Gram-positive bacteria and midpoint potential (Em) and lipophilicity index (logPe).

[0032] Figure 5 The cyclic voltammetry curves are for hydrophilic / lipophilic materials themselves.

[0033] Figure 6 To simulate the transcellular process of materials. Note: When peptidoglycan is loaded onto the surface of a glassy carbon electrode, the material is considered to be able to penetrate peptidoglycan if a redox peak characteristic of the material is detected; otherwise, it is considered that the material cannot penetrate peptidoglycan.

[0034] Figure 7 To simulate the transcellular membrane process of the material. Note: Lecithin was loaded onto the surface of a glassy carbon electrode. When a redox peak specific to the material was detected, it was considered that the material could penetrate lecithin; otherwise, it was considered that the material could not penetrate lecithin. In +Lecithin(t time), "t" refers to the cyclic voltammetric scan obtained at minute t after the material penetrates into the lecithin.

[0035] Figure 8 This is a schematic diagram illustrating the principle of direct interaction between lipophilic shuttles and NADH.

[0036] Figure 9 This demonstrates the enrichment of lipophilic materials into Gram-positive bacterial membrane structures. Note: The fluorescence colors of resazurin (RZ) and riboflavin (RF) in laser confocal microscopy are red and green, respectively. The left-hand images, from left to right, represent the in-situ fluorescence image and the high-resolution image, respectively; the right-hand images, from left to right, represent the fluorescence colors of resazurin (RZ) and riboflavin (RF), respectively. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

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

[0039] I. Experimental Materials This invention uses a total of 9 materials, namely flavins (FMN), riboflavin (RF), disodium anthraquinone 2,6-disulfonate (AQDS), sodium anthraquinone-2-sulfonate (AQS), resazurite (RZ), phenazine-1-carboxylic acid (PCA), 2-hydroxy-1,4-naphthoquinone (2-HNQ), phenazine (PHZ), and 1-hydroxyphenazine (1-OHP).

[0040] II. Experimental bacterial strains The experiments in this invention involve three types of microorganisms, namely... Bacillus subtilis ATCC 6051 (Bacillus subtilis 6051, Gram-positive bacterium) Clostridium sporogenes LHA6 (Clostridium LHA6, Gram-positive bacterium) and Shewanella oneidensis MR-1 (Shewanella MR-1, a Gram-negative bacterium, is the model strain of iron-reducing microorganisms).

[0041] Bacillus subtilis Cultivation of ATCC 6051: The culture medium was DMS liquid medium (pH = 7.0), containing: 8 g / L NB salt, 1 g / L KCl, 1 mmol / L MgSO4, 10 μmol / L MnCl2, 0.5 mmol / L CaCl2, and 1 μmol / L FeSO4. This strain is facultative (i.e., it can survive in both aerobic and anaerobic environments), therefore it can be grown aerobically in a shaker (200 rpm, 30℃). After 17 hours of cultivation (reaching the logarithmic growth phase), it is ready for use.

[0042] Clostridium sporogenes Cultivation of LHA6: The culture medium was TSB liquid medium, which consisted of 7.5 g / L tryptic soy broth (TSB) and 10 mM PIPES buffer (pH = 7.0). It is important to note that Clostridium requires a strictly anaerobic environment for growth; therefore, the TSB liquid medium was deoxygenated (by purging with high-purity nitrogen) and autoclaved (121°C, 20 min) before inoculation with 1% volume of Clostridium bacterial suspension. After static growth in a dark incubator at 30°C for 12 hours until it reached the logarithmic growth phase, it was ready for use.

[0043] Shewanella oneidensis Culture of MR-1: The isolation substrate for this strain was lake sediment, and the strain was purchased from the China Agricultural Microbial Culture Collection Center (MCCC). The culture medium was LB broth (pH = 7.0), which contained 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, and ultrapure water. Shewanella oneidensis MR-1 can be grown aerobically in a shaker (200 rpm, 30℃) and is ready for use after 17 hours of culture (reaching the logarithmic growth phase).

[0044] Example 1: Lipophilic materials can effectively promote the iron reduction process in Gram-positive bacteria. I. Experimental Methods 1. Construction of the iron reduction system The system consists of different types of 50 μmmol / L materials (FMN, RF, AQDS, AQS, RZ, PCA, 2-HNQ, PHZ, and 1-OHP). Bacillus subtilis ATCC 6051 or Clostridium sporogenes LHA6 or Shewanella oneidensis The reaction mixture consisted of MR-1, 4 mmol / L glucose (with 10 mmol / L sodium lactate added to the MR-1 system), 5 mmol / L Fe-ironite suspension, and 10 mM PIPES buffer (pH = 7.0). Bacterial strains in the logarithmic growth phase were selected as the reaction agent. The bacteria were washed three times with PIPES buffer to remove microbial metabolites and complex nutrients, then resuspended in PIPES buffer and quantitatively transferred to vials. The final bacterial count in the system was OD0.05. 600 =0.5. After deoxygenation with high-purity nitrogen, seal the vial and place it in a shaker at 30℃ and 200 rpm in the dark for incubation.

[0045] 2. Detection and analysis of iron reduction Extraction and determination of Fe(II): (1) Obtaining dissolved Fe(II): The sample in the experimental bottle was taken out evenly, and the mixed sample was filtered through a 0.22 μm aqueous filter membrane. After the sample was diluted in acetic acid-sodium acetate buffer (pH=5.0), the Fe(II) in the filtrate was determined by the o-phenanthroline colorimetric method and recorded as "Soluble Fe(II)". (2) Obtaining Fe(II) extracted by HCl: The sample in the experimental bottle was taken out evenly, and the mixed sample was mixed with 1 mol / L HCl at a volume ratio of 1:1. The sample was extracted by soaking in hydrochloric acid for 2 days until the sample was completely dissolved (the sample solution was clear). At this time, the sample was filtered, a certain amount of filtrate was taken out and diluted with acetic acid-sodium acetate buffer (pH=5.0). Then, the Fe(II) content in the sample was determined by the o-phenanthroline colorimetric method. Finally, the measured value was converted into the concentration of Fe(II) extracted by hydrochloric acid in the experimental bottle according to the dilution factor.

[0046] II. Experimental Results This invention selected three microorganisms to study their iron reduction capacity mediated by different (lipophilic or hydrophilic) materials (including four hydrophilic materials (AQDS, AQS, RF, and FMN) and five lipophilic materials (PHZ, 1-OHP, 2-HNQ, PCA, and RZ)). The iron reduction capacity was determined under the same bacterial count (OD). 600 When =0.5) and no materials are added, the result is as follows: Figure 1 As shown, Shewanella oneidensisThe amount of iron reduced (referring to ferrous iron extracted with 1 mol / L hydrochloric acid) can reach 106.26 mg / L; while for Bacillus subtilis and Clostridium sporogenes In contrast, their iron reduction capacity is significantly weaker than Shewanella oneidensis The iron reduction capacities were 4.21 and 21.26 mg / L, respectively. This result is consistent with the conventional view that Gram-positive bacteria are weakly electroactive.

[0047] After adding 9 different materials, the results were as follows: Figure 1 As shown, with the participation of 9 materials, S hewanella oneidensis The iron reduction reaction in the material reached equilibrium within 6 hours, more than four times faster than the equilibrium time of the control (CK). However, although the material accelerated the iron reduction reaction, the final amount of iron reduced in the material treatment was consistent with that in the control (CK) (at equilibrium). This indicates that the material can efficiently mediate the iron-reducing microorganisms (S... hewanella oneidensis It involves the extracellular electron transfer process, but has no effect on the iron reduction ability, a result consistent with existing research.

[0048] However, hydrophilic materials such as AQDS and FMN are... Bacillus subtilis and Clostridium sporogenes The iron reduction rate was almost unaffected. The difference was that the addition of lipophilic materials such as PHZ and 2-HNQ... Bacillus subtilis and Clostridium sporogenes The iron reduction capacity was significantly increased after the addition of 50 μmol / L PHZ, PCA, 2-HNQ, 1-OHP, and RZ. Bacillus subtilis The iron reduction capacity of the control group reached 146.83, 89.57, 72.31, 40.38 and 42.60 mg / L, respectively, which was 9.59 to 34.88 times higher than that of the control group. Clostridium sporogenes Similar phenomena were observed in iron reduction systems: the addition of 50 μmol / L PHZ, PCA, 2-HNQ, 1-OHP, and RZ resulted in... Clostridium sporogenes The iron reduction capacities of the lipophilic materials reached 100.22, 98.64, 72.07, 41.91, and 44.72 mg / L, respectively, which were 1.97–4.71 times higher than those of the control (CK). These results indicate that, compared to hydrophilic materials, lipophilic materials significantly improved iron reduction capacity. Bacillus subtilis and Clostridium sporogenes The iron reduction rate and the amount of iron reduced are related to material-mediated reduction. Shewanella oneidensis The reduction kinetics of iron are completely different.

[0049] Furthermore, the introduction of lipophilic materials significantly increased the iron reduction capacity of Gram-positive bacteria with weak electron transport capabilities, with iron reduction capacities (146.83 and 100.22 mg / L) comparable to those of electroactive microorganisms. Shewanella oneidensis The iron reduction capacity (106.26 mg / L) was on the same order of magnitude as that of the shuttle, revealing that the lipophilic material effectively mediated the iron reduction process of Gram-positive bacteria, converting non-iron-reducing bacteria into iron-reducing bacteria and weak iron-reducing bacteria into strong iron-reducing bacteria. The lipophilicity of the shuttle may be the key to promoting extracellular electron transfer in Gram-positive bacteria.

[0050] This phenomenon is observed in the genus Corynebacterium ( Corynebacterium glutamicum Staphylococcus spp. Staphylococcus xylosus ), Bacillus spp. Bacillus megatherium Both were observed in Bacillus megaterium, and the results were as follows: Figure 2 As shown, the iron reduction capacity of the three Gram-positive bacteria was significantly increased after the addition of the lipophilic material PHZ.

[0051] Example 2 Diffuse transmission spectroscopy scanning experiment Each material possesses a unique characteristic absorption peak, allowing for detection using a spectrophotometer. When a material gains electrons (i.e., transitions from an oxidized to a reduced state), its molecular structure changes, rendering the intensity of the characteristic peak undetectable. Based on this principle, diffuse transmission scanning spectroscopy can be used to monitor the kinetics of electron transfer from microbial cells to materials, providing a direct observation of the rate at which the material accepts intracellular electrons. If Gram-positive bacteria with thick-walled structures are selected, the results will reflect whether different materials can penetrate Gram-positive bacterial cells through their lipophilicity and accept electrons. The specific process is as follows: (1) Preparation of bacterial culture: Select Gram-positive bacteria in the logarithmic growth phase (C lostridium sporogenes LHA6), wash the bacterial culture three times with 10 mmol / L PIPES buffer, and finally resuspend it in PIPES buffer into a vial to achieve a bacterial concentration of OD0.05. 600 =0.5. High-purity nitrogen gas should be introduced during the preparation of the bacterial culture, and the container should be sealed and placed in an anaerobic glove box; (2) Material preparation: For PHZ, PCA, 1-OHP and 2-HNQ, a certain amount of anhydrous ethanol was used for dissolution, and the concentration of the mother liquor was 50 mmol / L; for RZ, FMN, RF, AQDS and AQS, ultrapure water was used for dissolution, and the concentration of the mother liquor was 3 mmol / L. The mother liquor was sealed after being purged with high-purity nitrogen and placed in an anaerobic glove box; (3) Testing: Step 1: Dark calibration. Use opaque cardboard to block the light source, ensuring the receiver cannot detect light intensity, and then perform dark calibration. Step 2: Blank calibration. Remove the cardboard and place the cuvette containing a certain amount of bacterial solution in the light path. After the light intensity signal stabilizes, perform blank calibration. Step 3: Quickly add a certain amount of material stock solution to the cuvette, making the liquid volume in the cuvette reach 3 mL, containing 50 μmol / L of material. Finally, set the instrument to perform automatic monitoring, and the output result is the kinetic curve of the material absorbance changing with time. Timing starts 30 seconds after the material is added.

[0052] II. Experimental Results To determine that lipophilicity is a key factor in material-mediated iron reduction by Gram-positive bacteria, experiments were conducted to measure the reception of typical Gram-positive bacteria by different types of active materials. Clostridium sporogenes Electron rate dynamics. The content of reduced-state active materials was determined using diffuse transmission scanning spectroscopy. Typical Gram-positive bacteria ( Clostridium sporogenes The kinetics of transferring electrons to an oxidized material and generating a reduced material are as follows: Figure 3 As shown, Clostridium sporogenes It can rapidly reduce lipophilic materials (PHZ, 1-OHP, PCA, 2-HNQ, and RZ) within 7–20 minutes, with the reduced content increasing rapidly from 0 μmol / L to 41.29, 34.42, 32.70, 28.07, and 48.88 μmol / L (the theoretical maximum yield is 50 μmol / L). In stark contrast, the reduction of hydrophilic materials is extremely slow. The formation of AQS reduced, RF reduced, FMN reduced, and AQDS reduced materials requires 220, 90, 230, and 160 minutes, respectively, to reach equilibrium, with the yield of AQDS reduced material being only 6.84 μmol / L.

[0053] The above results confirm that lipophilicity is a key factor in the material-mediated iron reduction of Gram-positive bacteria.

[0054] Example 3: Membrane Permeation Experiment of Materials I. Experimental Methods Parallel Artificial Membrane Permeability Assay (PAMPA) is a general-purpose high-throughput drug permeability screening tool. This model comprises a donor chamber and a recipient chamber, separated by an artificial membrane simulating a lipid bilayer of the cell membrane. The artificial membrane is constructed by coating a lecithin organic solution onto PVDF (polyvinyl alcohol), where the lecithin forms a stable film within the membrane pores, effectively mimicking the lipid bilayer. The test drug is added to the donor chamber, and after several hours at a specific temperature, the drug concentrations in the recipient and donor chambers are determined using LCMS / MS. The permeation rate and permeate volume are then calculated (J. Ponmozhi et al, 2021). This invention uses the Parallel Artificial Membrane Permeability Assay (PAMPA) model to evaluate the permeability of materials. Specifically, the following steps are included: (1) Preparation of lipid artificial membrane: First, a 1% (w / v) lecithin / dodecane solution was prepared and sonicated to ensure complete dissolution. Then, 5 mL of the prepared lecithin / dodecane solution was carefully transferred into each well of the donor plate, avoiding contact between the pipette tip and the membrane surface to ensure the integrity of the membrane.

[0055] (2) Preparation of working solution for test compound: The drug stock solution was diluted with 10 mM DMSO solution to prepare a 1 mM working solution. Subsequently, the working solution was further diluted with phosphate buffered saline (PBS, pH=7.0) to prepare a 10 µM donor solution.

[0056] (3) Incubation and Sample Processing: After the artificial membrane is applied, the donor solution must be added within 10 minutes. Add 150 µL of drug-containing donor solution to each well of the donor plate, and simultaneously add 300 µL of pH 7.0 phosphate buffer containing 1% DMSO (v / v) to each well of the receiver plate. Then, slowly and carefully place the donor plate into the receiver plate, ensuring that the bottom surface of the membrane in all wells is in full contact with the receiver buffer. After capping the plate, incubate it in a room at room temperature and humidity for 17 hours.

[0057] After incubation, samples were collected from the recipient and donor plates, respectively. 40 µL of the recipient sample, donor sample, propranolol, and methotrexate were mixed with 400 µL of acetonitrile containing the internal standard. The mixture was thoroughly mixed and centrifuged at 4000 rpm for 15 minutes. After centrifugation, 100 μL of the supernatant was transferred to a daughter plate containing 100 μL of H2O, mixed thoroughly, and analyzed using UPLC-MS / MS.

[0058] (4) Evaluation of the material's lipophilicity: If the material is detected on the receptor side, it indicates that the substance can permeate the artificial membrane and has lipophilic properties. Permeation rate (Pe) (×10) -6 cm·s -1 It can be calculated using the following formula: Pe = C × -ln(1 - [drug]) Receiver / drug] Equilibrium ) logPe=log{C×-ln(1-[drug] Receiver / [drug] Equilibrium )} Where, C = VD × VR / {(VD + VR) × Area × Time} = 0.15 × 0.3 / {(0.15 + 0.3) × 0.24 × 3600 × 17} = 6.81 × 10 -6 VD: Donor cavity volume (cm²) 3 ).

[0059] VR: Receptor cavity volume (cm) 3 ).

[0060] Area: The effective area of ​​the membrane, defined as membrane area × porosity. The membrane area of ​​the MultiScreen-IP plate is 0.24 cm². 2 The porosity is 100%, therefore Area = 0.24 cm. 2 .

[0061] Time: Incubation time (in seconds).

[0062] [drug] Receiver The drug concentration in the receptor cavity at the end of the experiment.

[0063] [drug] Equilibrium Drug concentration at theoretical equilibrium.

[0064] The purpose of standard reference materials (propranolol and methotrexate) is to determine whether the measurement results are scientifically sound. The evaluation criteria for permeability (or lipophilicity) are as follows: low permeability (or low lipophilicity): logPe < -6; medium permeability (or medium lipophilicity): -6 ≤ logPe ≤ -5; high permeability (or high lipophilicity): logPe > -5.

[0065] II. Experimental Results This invention designed a confirmatory experiment to determine whether different materials could permeate an artificial membrane (lecithin / dodecane). The lipophilicity of the materials (logPe, as shown in Table 1) was actually measured, and the lipophilicity was evaluated using standard substances (propranolol and methotrexate). The results are shown in Table 2. The permeability (logPe) of the materials was in the following order: 1-OHP>PHZ>PCA>2-HNQ>RZ>AQS ≈ RF ≈ FMN ≈ AQDS. This result is completely consistent with the order of electron reception rates from Gram-positive bacteria (1-OHP>PHZ>PCA>2-HNQ>RZ>AQS ≈ RF ≈ FMN ≈ AQDS), revealing the importance of the material's lipophilicity for extracellular electron transfer in Gram-positive bacteria.

[0066] Table 1. Midpoint potential (Em) and lipophilic coefficient (Pe, logPe) of the materials

[0067] Note: Em refers to the equilibrium position of an electrochemical reaction, that is, the potential at which a redox reaction is most likely to occur; Pe and logPe both represent indicators of a substance's lipophilicity, similar to Kow (the larger the value, the stronger the lipophilicity of the substance).

[0068] Table 2. Membrane permeability of materials (logPe)

[0069] Note: The lipophilicity of the material is reflected by the rate at which the material passes through the artificial membrane (lecithin / dodecane). Propranolol and methotrexate are used as control groups to verify the scientific validity of the test results.

[0070] Based on the above results, it can be concluded that: Clostridium sporogenes In the extracellular electron transport process, lipophilic materials exhibit a much stronger membrane permeability than hydrophilic materials, confirming that the lipophilicity of shuttles is a key factor in whether they can pass through the cell membrane to mediate electron transport.

[0071] By performing correlation analysis between lipophilicity and iron reduction rate, the results are as follows: Figure 4 As shown, a significant positive correlation was observed in Gram-positive bacteria. This confirms the importance of lipophilicity in the construction of electron transport chains.

[0072] Example 4: Investigation into the electron transfer through the cell membrane of lipophilic materials I. Experimental Methods Previous studies have shown that lipophilic materials can significantly enhance the iron reduction capacity of Gram-positive bacteria, specifically by significantly increasing the reduction rate and degree. Further experiments revealed that lipophilicity is a key factor in the efficient absorption of electrons from Gram-positive bacteria by the material. Because Gram-positive bacteria have thick cell walls (reaching tens of micrometers) and lack electron transport-related pigment proteins on their membranes, the electrons acquired by the material mainly originate from the cell interior, rather than the cell wall or the extracellular environment. This phenomenon suggests that the core mechanism of lipophilic materials mediating electron transport in Gram-positive bacteria may include the following two aspects: 1. Penetration of cell walls: Small molecule materials can easily penetrate the thick cell walls of Gram-positive bacteria.

[0073] 2. Penetration of the cell membrane: Due to its lipophilicity, the material can further penetrate the cell membrane and gain electrons in the quinone pool or during the reaction with NADH.

[0074] To verify the above hypotheses, this study employed electrochemical analysis methods to evaluate the material's transmembrane permeation capability. The specific experimental design is as follows: 1. Simulating cell structure: Peptidoglycan and lecithin were used to simulate the cell wall and cell membrane of Gram-positive bacteria, respectively, and were loaded onto the surface of glassy carbon electrode.

[0075] 2. Electrochemical detection: A voltage is applied in an electrochemical reaction cell to detect whether the material can penetrate the simulated cell structure and produce oxidation and reduction peaks. If a current signal is detected and the material's characteristic peaks appear, it indicates that it has successfully penetrated the simulated structure.

[0076] The specific experimental method is as follows: The experiment was conducted in an electrochemical reactor, equipped with a working electrode (glassy carbon electrode), a reference electrode (calomel electrode), and a counter electrode (platinum wire), using 10 mmol / L PIPES buffer. To ensure an oxygen-free reaction system, high-purity nitrogen was continuously purged into the reactor.

[0077] The working electrode is completely encapsulated by peptidoglycan (to simulate the thick cell wall of Gram-positive bacteria) or lecithin (to simulate the inner cell membrane of Gram-positive bacteria or the outer cell membrane of Gram-negative bacteria). During cyclic voltammetry (input voltage: -0.7 to +0.0 V or -0.5 to +0.2 V, scan rate: 0.01 V / s), the material produces oxidation / reduction peaks at specific voltages. When the material can penetrate the cell wall or cell membrane, i.e., when it comes into contact with the electrode, the cyclic voltammetry will display the corresponding oxidation / reduction peak signal of the shuttle; conversely, if the material cannot penetrate these structures, it cannot transfer electrons to the electrode, resulting in no oxidation / reduction peak signal.

[0078] The loading process for peptidoglycan or lecithin is as follows: 0.01 g of peptidoglycan or lecithin is added to 0.5 mL of DuPont membrane solution (containing 5% Nafion), and the mixture is then ultrasonically dispersed. Next, 10 μL of the mixture is pipetted and evenly added to the surface of the glassy carbon electrode, ensuring complete coverage. Finally, the peptidoglycan or lecithin membrane is allowed to solidify completely.

[0079] II. Experimental Results This study tested the properties of four hydrophilic materials (AQDS, AQS, FMN, and RF) and five lipophilic materials (1-OHP, 2-HNQ, PHZ, PCA, and RZ). Figure 5 ) and permeable peptidoglycan ( Figure 6 ), lecithin ( Figure 7 The cyclic voltammetric scan results are as follows: 1. Penetration of peptidoglycan (cell wall simulation): All materials (whether hydrophilic or lipophilic) can penetrate the peptidoglycan structure and generate corresponding electrical signals. This may be related to the small molecular weight of the materials.

[0080] 2. Penetration of lecithin (cell membrane simulation): All lipophilic materials were able to gradually penetrate lecithin within 0-30 minutes, showing a trend of gradually increasing characteristic peaks; while within the experimental time (30 minutes), the four hydrophilic materials were unable to penetrate lecithin and generate corresponding signals.

[0081] Although the results may be affected by the following factors: (1) Effect of lecithin layer thickness: The thickness of the lecithin layer loaded on the glassy carbon electrode in the experiment may be higher than that of the actual intracellular membrane of Gram-positive bacteria, but this does not affect the conclusion of the permeability test. Although the time for lipophilic materials to penetrate lecithin is relatively long (about 20 to 30 minutes), they can still gradually penetrate the structure due to their lipophilicity.

[0082] (2) Factors affecting the permeation rate: The permeation rate of a material is affected by a variety of factors, including the thickness of the cell structure, the diffusion rate of the shuttle in the solution, and the voltage intensity. Therefore, electrochemical analysis is mainly used to confirm whether a material has the ability to penetrate the cell wall or cell membrane, rather than to accurately measure the permeation rate.

[0083] This result confirms that lipophilic materials can not only permeate cell membranes but also facilitate transmembrane electron transfer. The ability of a material to penetrate the cell membrane and transfer electrons transmembranely is closely related to its lipophilicity, providing important experimental evidence for understanding the mechanism by which lipophilic materials enhance the iron-reducing capacity of Gram-positive bacteria.

[0084] Including Bacillus subtilis ATCC 6051 or Clostridium sporogenesIn the iron reduction system of LHA6, using rotenone to inhibit NADH dehydrogenase or quinacrine to inhibit succinate dehydrogenase, the study found that in the presence of phenazine (PHZ), the addition of inhibitors had almost no effect on electron transport when NADH dehydrogenase was inhibited, and the iron reduction rate remained unchanged; however, inhibition of succinate dehydrogenase had a slight effect on the iron reduction rate. This indicates that lipophilic materials can directly acquire electrons from NADH at a more upstream level, without the need for the participation of downstream electron transport chains. Figure 8 Therefore, it has been demonstrated that lipophilic materials can, by virtue of their lipophilicity, penetrate the cell wall and intracellular membrane, and acquire electrons during the NADH reaction, mediating electron transfer, thereby transforming non-iron-reducing bacteria into iron-reducing bacteria and weak iron-reducing bacteria into strong iron-reducing bacteria.

[0085] Example 5: Evidence that lipophilic materials can self-assemble within microbial cells I. Experimental Methods To more intuitively verify whether lipophilic materials can penetrate the interior of Gram-positive bacteria, their presence in a typical Gram-positive bacterium—Bacillus subtilis—was observed using laser confocal microscopy. Bacillus subtilis The distribution of ) in ).

[0086] like Figure 9 As shown, the lipophilic material RZ can penetrate extensively into the cell interior and emits a distinct red fluorescence, indicating that it is heavily enriched in lipid-soluble structures, especially the cell membrane. However, no obvious green fluorescence signal was observed in the hydrophilic material RF, suggesting that RF is mainly dispersed in the culture medium and does not effectively penetrate cell structures. This observation is consistent with the conclusions of the lecithin electrochemical osmosis experiment, further demonstrating that lipophilic materials can not only successfully penetrate the cell walls of Gram-positive bacteria but also accumulate in large quantities on the cell membrane and gradually penetrate into the cytoplasm, exchanging electrons with intracellular electron donors.

[0087] In summary, the electrochemical experimental results indicate that the material may penetrate the cell wall of Gram-positive bacteria due to its small molecule properties, while its ability to further penetrate the inner cell membrane depends on its lipophilicity. Direct observation using laser confocal microscopy confirmed this phenomenon, showing that the lipophilic material was abundantly accumulated on the cell membrane of Gram-positive bacteria. This accumulation and penetration characteristic may be a key mechanism for the efficient mediation of electron transport in Gram-positive bacteria by lipophilic materials, providing important experimental evidence for a deeper understanding of this electron transport process.

Claims

1. A method for constructing an extracellular electron transport chain in Gram-positive bacteria, characterized in that, A lipophilic material is added to the bacterial culture of Gram-positive bacteria; the lipophilic material has the ability to pass through the cell wall of Gram-positive bacteria, permeate the cell membrane, and exchange electrons with intracellular electron donors.

2. The application of lipophilic materials in improving the extracellular electron transport capacity of Gram-positive bacteria, characterized in that, The lipophilic material has the ability to pass through the cell wall of Gram-positive bacteria, permeate the cell membrane, and exchange electrons with intracellular electron donors.

3. The application of lipophilic materials in enhancing the iron-reducing ability of Gram-positive bacteria, characterized in that, The lipophilic material has the ability to pass through the cell wall of Gram-positive bacteria, permeate the cell membrane, and exchange electrons with intracellular electron donors.

4. A synthetic microbial agent system, characterized in that, The system comprises a lipophilic material and Gram-positive bacteria; the lipophilic material has the ability to pass through the cell wall of Gram-positive bacteria, permeate the cell membrane, and exchange electrons with intracellular electron donors.

5. A synthetic microbial agent prepared according to the synthetic microbial agent system according to claim 4.

6. The synthetic microbial agent according to claim 5, characterized in that, The method for preparing the synthetic bacterial agent is to add a lipophilic material to the bacterial solution of Gram-positive bacteria.

7. The use of the synthetic microbial agent according to claim 5 in accelerating iron reduction and increasing the amount of iron reduction, or in preparing products that accelerate iron reduction and increase the amount of iron reduction.

8. The use of the synthetic microbial agent according to claim 5 in soil remediation or in the preparation of soil remediation products.

9. The construction method according to claim 1, or the application according to any one of claims 2 or 3, or the synthetic microbial agent according to claim 5, characterized in that, The Gram-positive bacteria are selected from one or more of the genera Clostridium, Bacillus, Staphylococcus, or Corynebacterium.

10. The construction method according to claim 1, or the application according to any one of claims 2 or 3, or the synthetic microbial agent according to claim 5, characterized in that, The lipophilic material is selected from one or more of the following: razor violet, 2-hydroxy-1,4-naphthoquinone, phenazine-1-carboxylic acid, phenazine, or 1-hydroxyphenazine.

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