Method for enhancing microbial extracellular electron transfer

Abstract

The invention relates to a method for enhancing microbial extracellular electron transfer. The invention aims to solve the problem that the existing extracellular electron transfer efficiency is low, so that the organic matter degradation effect of microorganisms is poor. The method comprises the following steps: adding single-cell green algae into a specific culture medium, and culturing in a photobioreactor to obtain algae liquid; adding the mixed bacteria into the prepared artificial simulation wastewater for domestication to obtain a bacteria solution; mixing the algae liquid and the bacteria liquid with the prepared artificial simulation wastewater; respectively carrying out effluent collection on the photobioreactor in the step 3 at the initial stage and the final stage of the experiment; the method comprises the following steps: taking 40mL of a collected sample, and extracting loose type-extracellular polymeric substances and compact type-extracellular polymeric substances in an algae-bacterium system by adopting a heat extraction method; the electrochemical property of the extracellular polymeric substance is detected by adopting an electrochemical workstation; taking 30mL of the collected sample, and rapidly putting the collected sample into liquid nitrogen; and carrying out metagene detection by adopting an Illumina NovaSeq PE150 platform. The invention belongs to the technical field of biological environmental protection and bioelectrochemistry.

Description

Technical Field

[0001] The invention relates to a method for strengthening extracellular electron transfer of microorganisms, and belongs to the technical fields of biological environmental protection and bioelectrochemistry. Background Art

[0002] Extracellular electron transfer is a ubiquitous process in water, soil, and sediment, forming the foundation of biogeochemical cycles. Extracellular electron transfer involves microorganisms utilizing electron donors for their own metabolism, or transferring electrons generated through intracellular metabolism to extracellular acceptors. Electron transfer from the cell membrane to extracellular electron acceptors can occur via various pathways, including cytochrome c, nanowires, and electron shuttles. The fastest and most efficient way for microorganisms to transfer electrons to extracellular electron acceptors is through direct contact with cytochrome c on the cell membrane. Nanowires are conductive protein microfilaments synthesized by microorganisms and grown around their cells, similar to cilia. They form a "bridge" between microorganisms and extracellular electron acceptors, mediating long-distance electron transfer. Studies have confirmed that cytochrome OmcZ and the pili-related gene piliA are key components of long-distance electron transfer (Energy Environmental Science, 2009, 2, 506-516).

[0003] Electron shuttles are a type of redox mediator, small molecules that can undergo reversible conversion between oxidized and reduced states. Electron shuttles can be divided into (1) endogenous electron shuttles and (2) exogenous electron shuttles according to their source. Endogenous electron shuttles are mainly secreted by microorganisms. The endogenous electron shuttles discovered so far include flavin and certain quinone compounds. Flavin can bind to the microbial extracellular protein OmcA to enhance the electron transfer between microorganisms and difficult-to-degrade substances. Humic acid is an exogenous electron shuttle with a certain degree of water solubility (Science of The Total Environment, 2023, 899, 165682). It has aromatic rings, quinone groups, carboxyl groups, carbonyl groups, hydroxyl groups, etc. The quinone groups and phenazine groups are important receptors involved in the electron transfer process, and the hydroxyl groups give it a certain degree of hydrophilicity. The surface groups of water-soluble humic acid play an important role in the electron shuttle mediated by it.

[0004] Notably, electron shuttles are an important pathway for enhancing extracellular electron transport. Previous studies have demonstrated that increasing the synthesis and secretion of electron shuttles in microorganisms significantly promotes electron transport and can effectively enhance extracellular electron transport capacity (Science of The Total Environment, 2024, 949, 175222). Furthermore, the effective diffusion of electron shuttles outside the cell is a key issue, making the promotion of transmembrane efflux of electron transport carriers in microorganisms a major scientific challenge to be addressed.

[0005] However, the slower extracellular electron transfer efficiency significantly affects the performance of microorganisms in degrading organic matter. Improving the efficiency of extracellular electron transfer is of great significance to promoting the large-scale application of microbial electrochemical technology.

[0006] Patent publication number CN107342428A, filed on June 8, 2017, discloses a method for enhancing extracellular electron transfer in a microbial electrochemical system. The method involves using graphene or nitrogen-doped graphene loaded onto a carbon electrode as an anode. The graphene or nitrogen-doped graphene is directly loaded onto the carbon electrode via electrophoretic deposition. The invention also provides a modified carbon electrode for use as an anode in a microbial electrochemical system, a method for preparing the carbon electrode, and a microbial electrochemical system including the carbon electrode. However, the document does not disclose how to address the low efficiency of existing extracellular electron transfer, which results in poor microbial degradation of organic matter. Summary of the Invention

[0007] The present invention aims to solve the problem that the existing extracellular electron transfer efficiency is low, resulting in poor effect of microbial degradation of organic matter, and further proposes a method for strengthening microbial extracellular electron transfer.

[0008] The technical solution adopted by the present invention to solve the above problems is: the steps of the present invention include:

[0009] Step 1: adding unicellular green algae to a specific culture medium, and then placing it in a photobioreactor for cultivation until the logarithmic phase to obtain an algae solution;

[0010] Step 2: adding the mixed bacteria into the prepared artificial simulated wastewater for acclimation to obtain a bacterial solution;

[0011] Step 3: mixing the algae solution obtained in step 1 and the bacterial solution obtained in step 2 with the prepared artificial simulated wastewater, and placing the mixture into a photobioreactor;

[0012] Step 4: collecting effluent from the photobioreactor in step 3 at the beginning and end of the experiment;

[0013] Step 5: Take 40 mL of the sample collected in step 4 and extract the loose extracellular polymeric substances and tight extracellular polymeric substances in the algae-bacteria system using a thermal extraction method;

[0014] Step 6: The electrochemical properties of the extracellular polymers in step 5 are detected using an electrochemical workstation;

[0015] Step 7: Take 30 mL of the sample collected in step 4, quickly place it in liquid nitrogen, and then perform metagenomic testing;

[0016] Step 8: Use the Illumina NovaSeq PE150 platform to detect the metagene in step 7.

[0017] Furthermore, in step 1, the culture temperature in the photobioreactor is room temperature, the culture speed is 250 rpm to 300 rpm, and the culture light intensity is 200 μmol / m 2 / s~250μmol / m 2 / s, CO2 gas is introduced, and the CO2 ventilation rate is 2.5%; the unicellular green algae is Chlorella pyrenoidosa.

[0018] Furthermore, the mixed bacterial community in step 2 is: Vampirovibrio, Caulobacter, Micractinium, Pseudacidovorax, Pannonibacter, Phenylobacterium, Dyadobacter, Paenibacillus, and Sphingopyxis.

[0019] Furthermore, in step 3, the temperature of the algae solution, bacterial solution, and simulated wastewater in the photobioreactor is room temperature, the rotation speed is 250-300 rpm, and the light intensity is 200 μmol / m 2 / s~250μmol / m 2 / s, a mixture of CO2 and air with a content of 2.5%.

[0020] Furthermore, the composition of the artificial simulated wastewater in step 3 is: 0.6g of C6H 12 O6·H2O, 0.255g of NaNO3, 1.623g of NH4Cl, and 0.0845g of K2HPO4;

[0021] The initial concentration of algal cells obtained in step 1 is 0.5 g / L, and the initial inoculation concentration of the mixed bacteria obtained in step 2 is 0.1 g / L.

[0022] Furthermore, the thermal extraction step in step 5 is:

[0023] Step 501: Take 40 mL of sample and put it into a centrifuge tube, and centrifuge it at 7000 rpm / min for 10 minutes;

[0024] Step 502: The supernatant after separation includes most of the extracellular polymers separated from the microorganisms. The supernatant is filtered through a 0.22 μm filter membrane to remove the residual bacteria in the solution to obtain LB-EPS;

[0025] Step 503: Add distilled water to the centrifuge tube to restore the original volume, vortex evenly, place in a 60°C water bath and heat for 30 minutes, take out, vortex evenly, and high-speed centrifuge at 7000 rpm / min for 10 minutes, and then filter with a 0.22 μm filter membrane to obtain TB-EPS;

[0026] Step 504: The protein and polysaccharide contents in the EPS are determined using the Coomassie brilliant blue method and the anthrone-sulfuric acid method, respectively;

[0027] Step 505: Use the modified Folin-Lowry method to detect the humic acid content.

[0028] Furthermore, the electrochemical property detection step in step 5 is: using an electrochemical workstation to detect the redox activity of EPS; a platinum sheet is used as a working electrode, Ag / AgCl is used as a reference electrode, and a platinum wire is used as a counter electrode; cyclic voltammetry measurement is performed in a potential range of -700 to +700 mV at a scan rate of 100 mV / s; and electrochemical impedance spectroscopy is performed under conditions of an initial voltage of 5 mV and a frequency range of 105 Hz to 0.1 Hz.

[0029] Furthermore, the steps of the metagene detection in step 7 are:

[0030] Step 701: DNA samples that pass the test are randomly fragmented into fragments of approximately 350 bp in length using a Covaris ultrasonic disruptor. The entire library is prepared through steps such as end repair, A-tailing, addition of sequencing adapters, purification, and PCR amplification.

[0031] Step 702: Use MetGeneMark to predict ORFs on the scaftigs of the sample, and filter out information with a length of less than 100 nt in the prediction results;

[0032] Step 703: Using CD-HIT software to remove redundancy from the ORF prediction results to obtain a non-redundant initial gene catalogue. Here, the nucleic acid sequences encoded by non-redundant continuous genes are referred to as genes.

[0033] Step 704: Use Bowtie2 to compare the clean data of each sample to the initial gene catalog, and calculate the number of reads of the gene in each sample;

[0034] Step 705: Filter out genes with a read count of <= 2 in each sample to obtain the final gene catalogue for subsequent analysis;

[0035] Step 706: Based on the number of reads and gene lengths in the comparison, calculate the abundance information of each gene in each sample.

[0036] The beneficial effects of the present invention are:

[0037] 1. The green algae and mixed bacteria used in the present invention are widely distributed in nature and are easy to culture;

[0038] 2. The present invention is simple and easy to operate, and the preparation cost is relatively low;

[0039] 3. The present invention has the potential for large-scale promotion and application;

[0040] 4. The present invention improves extracellular electron transfer and also purifies wastewater;

[0041] 5. The present invention can significantly increase the expression of genes related to cytochrome C, nanowires, electron shuttles, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a schematic diagram of water treatment performance after improving extracellular electron transfer;

[0043] Figure 2 is a schematic diagram of the electrochemical properties of LB-EPS and TB-EPS;

[0044] Figure 3 is a schematic diagram of the CV characteristics of LB-EPS at the beginning and end of the experiment;

[0045] Figure 4 Schematic diagram of annotation of metagenomic data according to the eggNOG database at the cutoff of E value <10-5;

[0046] Figure 4 Among them, (A) some important functional descriptions involving electron transfer activity, polysaccharide biosynthesis and other functions;

[0047] (B) Description of genes related to cytochrome c;

[0048] (C) Description of genes related to flavin; the Y-axis represents annotations, and the X-axis represents the absolute abundance of single genes;

[0049] Figure 5 (A) is a schematic diagram of the expression levels of genes encoding cytochrome C, type IV pili, and electron transport proteins;

[0050] Figure 5 (B) is a heat map of genes related to nicotinamide adenine dinucleotide (NADH), cell membrane, energy, flavin extracellular electron transport, and tricarboxylic acid cycle (TCA). DETAILED DESCRIPTION

[0051] Specific implementation method 1: Figures 1 to 5 As shown, a method for enhancing extracellular electron transfer of microorganisms comprises the following steps:

[0052] Step 1: adding unicellular green algae to a specific culture medium, and then culturing in a photobioreactor until the logarithmic phase to obtain an algae solution; the initial algae cell concentration is 0.5 g / L;

[0053] The culture temperature in the photobioreactor was room temperature, the culture speed was 250 rpm to 300 rpm, and the culture light intensity was 200 μmol / m 2 / s~250μmol / m 2 / s, CO2 gas is introduced, and the CO2 ventilation rate is 2.5%; the unicellular green algae is Chlorella pyrenoidosa;

[0054] Step 2: adding the mixed bacteria into the prepared artificial simulated wastewater for acclimation to obtain a bacterial solution; the initial inoculation concentration of the mixed bacteria is 0.1 g / L;

[0055] Among them, the mixed flora includes: Vampirovibrio, Caulobacter, Micractinium, Pseudacidovorax, Pannonibacter, Phenylobacterium, Dyadobacter, Paenibacillus, and Sphingopyxis;

[0056] Step 3: mixing the algae solution obtained in step 1 and the bacterial solution obtained in step 2 with the prepared artificial simulated wastewater, and placing the mixture into a photobioreactor;

[0057] The temperature of algae solution, bacterial solution and simulated wastewater in the photobioreactor was room temperature, the rotation speed was 250-300 rpm, and the light intensity was 200 μmol / m 2 / s~250μmol / m 2 / s, a mixture of CO2 and air with a content of 2.5%;

[0058] The composition of artificial simulated wastewater is: 0.6g of C6H 12 O6·H2O, 0.255g of NaNO3, 1.623g of NH4Cl, and 0.0845g of K2HPO4;

[0059] Step 4: collecting effluent from the photobioreactor in step 3 at the beginning and end of the experiment;

[0060] Step 5: Take 40 mL of the sample collected in step 4 and extract the loose extracellular polymeric substances and tight extracellular polymeric substances in the algae-bacteria system using a thermal extraction method;

[0061] The steps of thermal extraction are:

[0062] Step 501: Take 40 mL of sample and put it into a centrifuge tube, and centrifuge it at 7000 rpm / min for 10 minutes;

[0063] Step 502: The supernatant after separation includes most of the extracellular polymers separated from the microorganisms. The supernatant is filtered through a 0.22 μm filter membrane to remove the residual bacteria in the solution to obtain LB-EPS;

[0064] Step 503: Add distilled water to the centrifuge tube to restore the original volume, vortex evenly, place in a 60°C water bath and heat for 30 minutes, take out, vortex evenly, and high-speed centrifuge at 7000 rpm / min for 10 minutes, and then filter with a 0.22 μm filter membrane to obtain TB-EPS;

[0065] Step 504: The protein and polysaccharide contents in the EPS are determined using the Coomassie brilliant blue method and the anthrone-sulfuric acid method, respectively;

[0066] Step 505: using the modified Folin-Lowry method to detect the humic acid content;

[0067] The electrochemical properties were tested using an electrochemical workstation to detect the redox activity of EPS. A platinum sheet was used as the working electrode, Ag / AgCl was used as the reference electrode, and a platinum wire was used as the counter electrode. Cyclic voltammetry was performed over a potential range of -700 to +700 mV at a scan rate of 100 mV / s. Electrochemical impedance spectroscopy was performed at an initial voltage of 5 mV and a frequency range of 105 Hz to 0.1 Hz.

[0068] Step 6: The electrochemical properties of the extracellular polymers in step 5 are detected using an electrochemical workstation;

[0069] Step 7: Take 30 mL of the sample collected in step 4, quickly place it in liquid nitrogen, and then perform metagenomic testing;

[0070] The steps of metagenomic testing are:

[0071] Step 701: DNA samples that pass the test are randomly fragmented into fragments of approximately 350 bp in length using a Covaris ultrasonic disruptor. The entire library is prepared through steps such as end repair, A-tailing, addition of sequencing adapters, purification, and PCR amplification.

[0072] Step 702: Use MetGeneMark to predict ORFs on the scaftigs of the sample, and filter out information with a length of less than 100 nt in the prediction results;

[0073] Step 703: Using CD-HIT software to remove redundancy from the ORF prediction results to obtain a non-redundant initial gene catalogue. Here, the nucleic acid sequences encoded by non-redundant continuous genes are referred to as genes.

[0074] Step 704: Use Bowtie2 to compare the clean data of each sample to the initial gene catalog, and calculate the number of reads of the gene in each sample;

[0075] Step 705: Filter out genes with a read count of <= 2 in each sample to obtain the final gene catalogue for subsequent analysis;

[0076] Step 706: Based on the number of reads and gene lengths compared, calculate the abundance information of each gene in each sample.

[0077] Step 8: Use the Illumina NovaSeq PE150 platform to detect the metagene in step 7.

[0078] Wherein, the composition of the culture medium in step 1 is shown in Table 1:

[0079] Table 1 Composition of microalgae culture medium

[0080]

[0081] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the present profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical content disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent replacement and improvement of the above embodiments made according to the technical essence of the present invention, within the spirit and principles of the present invention, without departing from the content of the technical solution of the present invention, shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for enhancing extracellular electron transfer in microorganisms, characterized in that: The specific steps include: Step 1: adding unicellular green algae to a specific culture medium, and then placing it in a photobioreactor for cultivation until the logarithmic phase to obtain an algae solution; Step 2: adding the mixed bacteria into the prepared artificial simulated wastewater for acclimation to obtain a bacterial solution; Step 3: mixing the algae solution obtained in step 1 and the bacterial solution obtained in step 2 with the prepared artificial simulated wastewater, and placing the mixture into a photobioreactor; Step 4: collecting effluent from the photobioreactor in step 3 at the beginning and end of the experiment; Step 5: Take 40 mL of the sample collected in step 4 and extract the loose extracellular polymeric substances and tight extracellular polymeric substances in the algae-bacteria system using a thermal extraction method; Step 6: The electrochemical properties of the extracellular polymers in step 5 are detected using an electrochemical workstation; Step 7: Take 30 mL of the sample collected in step 4, quickly place it in liquid nitrogen, and then perform metagenomic testing; Step 8: Use the Illumina NovaSeq PE150 platform to detect the metagene in step 7.

2. The method for enhancing extracellular electron transfer of microorganisms according to claim 1, wherein: In step 1, the culture temperature in the photobioreactor is room temperature, the culture speed is 250 rpm to 300 rpm, and the culture light intensity is 200 μmol / m 2 / s~250μmol / m 2 / s, CO2 gas is introduced, and the CO2 ventilation rate is 2.5%; the unicellular green algae is Chlorella pyrenoidosa.

3. The method for enhancing extracellular electron transfer of microorganisms according to claim 1, wherein: The mixed bacterial community in step 2 is: Vibrio batiflora, Bacillus cereus, Microcystis microphylla, Pseudomonas aeruginosa, Bacillus panlongianus, Pseudomonas phenylene oxide, Entamoeba bacillus, Paenibacillus, and Sphingobacterium spp.

4. The method for enhancing extracellular electron transfer of microorganisms according to claim 1, wherein: In step 3, the temperature of the algae solution, bacterial solution, and simulated wastewater in the photobioreactor is room temperature, the rotation speed is 250-300 rpm, and the light intensity is 200 μmol / m 2 / s~250μmol / m 2 / s, a mixture of CO2 and air with a content of 2.5%.

5. The method for enhancing extracellular electron transfer of microorganisms according to claim 1, characterized in that: The composition of artificial simulated wastewater in step 3 is: 0.6g of C6H 12 O6·H2O, 0.255g of NaNO3, 1.623g of NH4Cl, and 0.0845g of K2HPO4; The initial concentration of algal cells obtained in step 1 is 0.5 g / L, and the initial inoculation concentration of the mixed bacteria obtained in step 2 is 0.1 g / L.

6. The method for enhancing extracellular electron transfer of microorganisms according to claim 1, characterized in that: The thermal extraction steps in step 5 are: Step 501: Take 40 mL of sample and put it into a centrifuge tube, and centrifuge it at 7000 rpm / min for 10 minutes; Step 502: The supernatant after separation includes most of the extracellular polymers separated from the microorganisms. The supernatant is filtered through a 0.22 μm filter membrane to remove the residual bacteria in the solution to obtain LB-EPS; Step 503: Add distilled water to the centrifuge tube to restore the original volume, vortex evenly, place in a 60°C water bath and heat for 30 minutes, take out, vortex evenly, and high-speed centrifuge at 7000 rpm / min for 10 minutes, and then filter with a 0.22 μm filter membrane to obtain TB-EPS; Step 504: The protein and polysaccharide contents in the EPS are determined using the Coomassie brilliant blue method and the anthrone-sulfuric acid method, respectively; Step 505: Use the modified Folin-Lowry method to detect the humic acid content.

7. The method for enhancing extracellular electron transfer of microorganisms according to claim 1, characterized in that: The electrochemical property detection step in step 5 is as follows: the redox activity of EPS is detected using an electrochemical workstation; a platinum sheet is used as a working electrode, Ag / AgCl is used as a reference electrode, and a platinum wire is used as a counter electrode; cyclic voltammetry is performed in a potential range of -700 to +700 mV at a scan rate of 100 mV / s; and electrochemical impedance spectroscopy is performed under conditions of an initial voltage of 5 mV and a frequency range of 105 Hz to 0.1 Hz.

8. The method for enhancing extracellular electron transfer of microorganisms according to claim 1, characterized in that: The steps for metagene detection in step 6 are: Step 701: DNA samples that pass the test are randomly fragmented into fragments of approximately 350 bp in length using a Covaris ultrasonic disruptor. The entire library is prepared through steps such as end repair, A-tailing, addition of sequencing adapters, purification, and PCR amplification. Step 702: Use MetGeneMark to predict ORFs on the scaftigs of the sample, and filter out information with a length of less than 100 nt in the prediction results; Step 703: Using CD-HIT software to remove redundancy from the ORF prediction results to obtain a non-redundant initial gene catalogue. Here, the nucleic acid sequences encoded by non-redundant continuous genes are referred to as genes. Step 704: Use Bowtie2 to compare the clean data of each sample to the initial gene catalog, and calculate the number of reads of the gene in each sample; Step 705: Filter out genes with a read count of <= 2 in each sample to obtain the final gene catalogue for subsequent analysis; Step 706: Based on the number of reads and gene lengths in the comparison, calculate the abundance information of each gene in each sample.

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