Construction method and application of microorganism-driven activation-circulation system

Abstract

The invention discloses a construction method and application of a microorganism-driven activation-circulation system, the construction method of the microorganism-driven activation-circulation system comprises the following steps: step 1, screening and adaptive domestication of metal reducing flora; step 2, carrying out carrier pretreatment so as to inoculate the metal reducing flora in the step 1; 3, constructing an MPCS system.According to the method, a catalyst does not need to be prepared manually, the MPCS system is formed by adding microbiota and through microorganism metabolism coupling, biodegradation and PS-AOP are tightly coupled through MPCS, the biodegradability of pollutants is effectively improved, degradation and removal of macromolecular organic matter are achieved, and the method has the advantages of being environmentally friendly and free of pollution. And the risk of drug resistance diffusion in the biochemical treatment process is effectively reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of pollutant degradation, and in particular to a construction method and application of a microorganism-driven activation-circulation system. Background Art

[0002] With the rapid development of my country's social economy and the rapid development of industrialization, the production of various types of refractory organic pollutants is increasing. Existing advanced technologies such as advanced oxidation processes are used to treat pollutants, among which persulfate advanced oxidation process (PS-AOP) is widely used to remove organic pollutants. However, the disadvantage of this method is that its catalytic conditions (such as light, heat, ultrasound, transition metal ions such as Fe²⁺, Mn²⁺ and metal oxides such as Fe3O4, FeS, MnO, Mn2O3 and Co3O4) require external energy input, and may produce a large amount of heavy metal high-valent ions, causing secondary pollution. In addition, the preparation of catalysts usually relies on physical and chemical methods such as chemical precipitation or hydrothermal methods, with complex operation procedures and high material costs. At the same time, this method may also result in the inability to completely degrade pollutants, generate toxic by-products, and the metal oxide catalyst is lost after use, which will reduce the subsequent catalytic efficiency.

[0003] In addition to advanced oxidation processes, microbial treatment is also popular. This method usually does not produce harmful byproducts and is environmentally friendly. Microorganisms are widely available and easy to cultivate, and the operating costs are relatively low. Many microorganisms can effectively degrade a variety of organic pollutants, and they can survive and reproduce under a variety of environmental conditions, showing good adaptability. In addition, microorganisms can continue to survive after treatment and have the ability to regenerate, removing pollutants through natural processes, which helps achieve sustainable development goals.

[0004] However, the microbial method also has some disadvantages. Microbial degradation usually takes a long time, and it is difficult to degrade cyclic compounds with large molecular weight, which makes it difficult to meet the needs of rapid treatment. In addition, the requirements for environmental conditions are relatively strict, and the degradation ability of certain organic pollutants to microorganisms is limited, which limits its scope of application. High concentrations or toxic pollutants may inhibit the growth of microorganisms, thereby reducing the treatment effect.

[0005] Therefore, a method for constructing a microbial-driven activation-circulation system and its application are provided to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing and applying a microbial-driven activation-cycling system, wherein the microorganisms can mediate the reduction of high-valent metal ions and convert them into catalysts for PS (such as converting Fe2O3 into Fe3O4) and deposit them on the biofilm, naturally and tightly coupling the two methods of advanced oxidation (AOP) and microbial treatment for the removal of pollutants. Therefore, a microbial advanced oxidation cyclic system (MPCS) for deep degradation of antibiotics is constructed by utilizing the metabolism of microorganisms (metal-reducing bacteria). MPCS tightly couples biodegradation with AOP, and the activation of oxidizing substances such as PS and H2O2 effectively improves the biodegradability of pollutants, which not only alleviates the survival pressure of microorganisms, but also ensures the activity of microorganisms by the biological metal shell (such as magnetite).

[0007] The object of the present invention is achieved in that: A method for constructing a microbial-driven activation-circulation system comprises the following steps: Step 1: screening and adaptive domestication of metal-reducing bacteria to improve the tolerance of metal-reducing bacteria to pollutants, thereby increasing the survival rate and pollutant degradation capacity in the presence of pollutants; Step 2: pre-treating the carrier for inoculation with the metal-reducing bacteria in step 1; Step 3, constructing the MPCS system, taking out the carrier treated in step 2, and selecting different microbial groups for inoculation according to different pollutants. The specific operations are as follows: Step 3.1 Place the carrier treated in step 2 into a vial containing culture medium, and inoculate different microbial groups according to different pollutants; Step 3.2 Seal the vial with a pressure cap, draw a vacuum with a vacuum pump for 10-30 minutes, and then use high-purity nitrogen with a nitrogen content greater than 98% to remove residual air, thereby simulating the anaerobic environment in sewage; Step 3.3 The microorganisms in the vial use hydrated iron oxide to generate Fe3O4, which has PS catalysis, thereby generating free radicals to crack complex pollutants such as OTC, and then form small molecule compounds used by microorganisms, which are finally decomposed into non-toxic CO2 and H2O. Free radicals can effectively destroy extracellular resistance genes to prevent horizontal gene transfer, effectively reducing the risk of resistance spread during biochemical treatment.

[0008] The catalyst precursor in step 3.2 includes oxides or hydroxides of transition metal elements such as iron and copper, which are converted into catalysts with peroxide activation ability through the dissimilatory reduction of metal-reducing bacteria. The PS added in step 3.2 generates sulfate radicals, and the added amount varies accordingly with the pollutant concentration.

[0009] The specific operation of step 1 is as follows: using anaerobic activated sludge and laboratory preserved dissimilatory metal-reducing bacteria / electrochemically active bacteria as the source of functional bacteria, adding a set concentration of hydrated iron oxide and OTC to the sodium acetate culture medium for subculture; gradually increasing the pollutant concentration during the culture process to adaptively domesticate the microorganisms.

[0010] During the culture process of step 1, the pollutant concentration is gradually increased, and the pollutant concentration is 5 mg / L, 10 mg / L, and 20-100 mg / L from low to high.

[0011] The carrier in step 2 is any one of biochar, porous ceramic particles, polyurethane sponge and calcium alginate microspheres.

[0012] The carrier is a polyurethane sponge, and the specific operation of step 2 is as follows: the polyurethane sponge is sequentially ultrasonically washed with deionized water for 30 minutes, ultrasonically washed with anhydrous ethanol for 30 minutes, ultrasonically washed with anhydrous acetone for 30 minutes, soaked in a 0.5M HCl solution for 6 hours, and soaked in a 0.5M NaOH solution for 6 hours; the polyurethane sponge is used to remove inorganic and organic residues on the polyurethane sponge, and the treated polyurethane sponge is sterilized with high-pressure steam. After the sterilization is completed, the polyurethane sponge is taken out and placed in a suspension containing hydrated iron oxide to fully adsorb the hydrated iron oxide. After drying, the polyurethane sponge is added to a syringe bottle containing a sodium acetate culture medium and microorganisms are added to load the microorganisms on the sponge and fully contact with the catalyst precursor material, so as to drive the subsequent synthesis of the catalyst and peroxide activation.

[0013] An application of a microbial-driven activation-circulation system, which is used to treat organic pollutants and control the risk of resistance gene transmission.

[0014] The beneficial effects of the present invention are:

[0015] 1. Compared with the prior art, the present invention does not require artificial preparation of catalysts. By adding microbial communities and coupling the metabolism of microorganisms themselves, the MPCS system is formed. MPCS tightly couples biodegradation with PS-AOP, effectively improving the biodegradability of pollutants and achieving the degradation and removal of macromolecular organic matter.

[0016] 2. Compared with the prior art, the bio-generated catalyst in the present invention can be regenerated under the metabolism of microorganisms after the catalytic oxidation substance is activated, and no supplementation is required.

[0017] 3. Compared with the prior art, the catalyst self-generated by microorganisms of the present invention has the following advantages over artificially synthesized catalysts: high biocompatibility. The existing artificial exogenous addition of catalysts will significantly destroy the physiological structure of the loaded part of the microorganisms, causing the death of the microorganisms. The biogenic catalyst generated by the present invention can effectively reduce the toxic effect of the catalyst on the microorganisms.

[0018] 4. Compared with the prior art, the PS-AOP of the present invention is more closely integrated with the microbial degradation process, as shown by SEM images (see Figure 2 AC) It can be seen that there are biogenic magnetite nanoparticles on the surface of microorganisms, forming a "protective film" that can not only protect microorganisms from free radicals, but also effectively resist the toxic effects of environmental pollutants on microorganisms.

[0019] 5. In the present invention, since metal-reducing bacteria can spontaneously produce trace amounts of hydrogen peroxide to drive a Fenton-like reaction, even without adding exogenous hydrogen peroxide and persulfate, a good catalytic degradation effect can be achieved.

[0020] 6. The present invention effectively shields microorganisms from direct contact with organic pollutants (antibiotics) and intermediate metabolites, thereby reducing the selection pressure. The expression of drug-resistant genes in the degradation process is significantly downregulated compared with simple microbial degradation, and free radicals can effectively destroy extracellular drug-resistant genes to prevent horizontal gene transfer, effectively reducing the risk of drug resistance spread during biochemical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a real-life picture of the MPCS system of the present invention; Figure 2 Figure 1 is a characterization diagram of the MPCS system of the present invention. Figure A is a diagram of the microorganism-biogenic nanomaterial loading on the carrier surface; Figure B is a partial enlarged diagram of the carrier, in which the microorganism self-generated catalyst is not only loaded on the carrier, but also loaded on the microorganism surface (iron shell); Figure C is a diagram of the biofilm loaded on the carrier; Figure D is an X-ray diffraction spectrum; Figure E is a full spectrum of X-ray photoelectron energy spectrum; Figure F is an X-ray photoelectron energy spectrum Fe2P spectrum; Figure 3 It is the effect diagram of COD removal rate and OTC removal rate of the present invention; Figure 4 It is a bar graph of the ferric iron and ferrous iron redox cycle; Figure 5 This is a graph showing the decrease in the expression level of drug-resistant genes; Figure 6 Figure 1 is the mechanism of MPCS for removing pollutants and protecting microorganisms; Figure A is a schematic diagram of the formation of biogenic magnetite protective film; Figure B is a diagram of the specific operating mechanism of MPCS. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0023] A method for constructing a microbial-driven activation-circulation system comprises the following steps: Step 1: Screening and adaptive domestication of metal-reducing bacteria. The screened and domesticated metal-reducing bacteria can achieve resource recycling through biotechnology, and can improve their tolerance to pollutants, thereby increasing their survival rate and pollutant degradation ability in the presence of pollutants, so as to enhance their actual application effect in pollution control. This process can help develop more efficient microbial strains for different remediation scenarios; The specific operation is as follows: anaerobic activated sludge and laboratory preserved dissimilatory metal-reducing bacteria / electrochemically active bacteria are used as the source of functional bacteria, and a set concentration of hydrated iron oxide (1g / L) is added to the sodium acetate medium for subculture; during the culture process, the concentration of OTC pollutants is gradually increased (5, 10, 20-100mg / L) to adapt the microorganisms; taking 1L sodium acetate medium as an example, NaCH3COO·3H2O10.93g, NaCl2.32g, NH4Cl1.12g, MgSO4·7H200.1g, HEPES2.38g, Traceelement0.4ml, and Fe in the culture medium are detected by inductively coupled plasma mass spectrometry (ICP-MS) and o-phenanthroline method. 2+ Concentration changes, COD detector and liquid chromatography (LC) detect organic matter and OTC removal, LC-MS determines intermediate degradation products. During the cultivation process of step 1, the pollutant concentration is gradually increased, and the pollutant concentration from low to high is 5mg / L, 10mg / L, and 20-100mg / L.

[0024] Step 2: pre-treat the carrier for inoculation with the metal-reducing bacteria in step 1. The carrier is any one of biochar, porous ceramic particles, polyurethane sponge and calcium alginate microspheres.

[0025] The carrier is a polyurethane sponge with a size of 1cm×1cm×1cm; the specific operation of step 2 is as follows: the polyurethane sponge is sequentially ultrasonically washed with deionized water for 30 minutes, ultrasonically washed with anhydrous ethanol for 30 minutes, ultrasonically washed with anhydrous acetone for 30 minutes, soaked in a 0.5M HCl solution for 6 hours, and soaked in a 0.5M NaOH solution for 6 hours; in order to remove inorganic and organic residues on the polyurethane sponge, the treated polyurethane sponge is sterilized with high-pressure steam (121°C, 30 minutes), and after sterilization, it is taken out and placed in a suspension containing hydrated iron oxide to fully absorb the hydrated iron oxide, and then dried for use.

[0026] Step 3, constructing an MPCS system, taking out the carrier treated in step 2, and selecting different microbial groups for inoculation according to different pollutants. The specific operations are as follows: Step 3.1 placing the carrier treated in step 2 into a vial containing a culture medium, and inoculating different microbial groups according to different pollutants; Step 3.2 sealing the vial with a pressure cap, drawing a vacuum with a vacuum pump for 10-30 minutes, and then using high-purity nitrogen (nitrogen content greater than 98%) to remove residual air, thereby simulating the anaerobic environment in sewage; the catalyst precursor in step 3.2 includes oxides or hydroxides of transition metal elements such as iron and copper, which are converted into catalysts with peroxide activation ability through the dissimilatory reduction of metal-reducing bacteria.

[0027] There is no specific requirement for the type of PS added in step 3.2, as long as it can generate sulfate radicals, and the amount of PS added increases accordingly with the concentration of pollutants. The type of PS added in this patent is sodium persulfate, the amount added is between 0-3mM, and the concentration of pollutant OTC is 20mg / L; in addition, this method still has a certain removal effect on pollutants without adding oxidants such as PS.

[0028] Step 3.3 The microorganisms in the vial use hydrated iron oxide to generate Fe3O4, which has PS catalysis, thereby generating free radicals to crack OTC, and then forming small molecular compounds used by microorganisms, which are finally decomposed into non-toxic CO2 and H2O. Free radicals can effectively destroy extracellular resistance genes to prevent horizontal gene transfer, effectively reducing the risk of resistance spread during biochemical treatment. For wastewater contaminated by OTC. The basic formula of the culture medium is consistent with the adaptive culture of the bacterial flora. The difference is that the amount of hydrated iron oxide added is 2 g / L, the concentration of OTC is fixed at 20 mg / L, and OTC is used as the only external carbon source for the degradation of OTC-contaminated wastewater. Since OTC has a photolysis effect, the vial should be wrapped in tin foil when adding OTC to avoid the influence of light. The specific operation is to put the treated polyurethane sponge into the vial containing the above culture medium, inoculate different microbial communities according to different pollutants, seal the cap, and use a vacuum pump to draw a vacuum for 10-30 minutes, and then use high-purity nitrogen to remove the residual air to simulate the anaerobic environment in the sewage. The microorganisms in the bottle can use hydrated iron oxide to generate Fe3O4. Fe3O4 has PS catalysis, which can generate free radicals to crack OTC, and then form small molecular compounds that can be used by microorganisms, which are finally decomposed into non-toxic CO2 and H2O.

[0029] Targeting wastewater contaminated by polychlorinated phenols. Polychlorinated phenols are relatively stable in the environment and can exist for a long time, and the cleavage intermediates may be more toxic. It should be noted that when polychlorinated phenols are the only external carbon source, there is no need to add them in a dark place like oxytetracycline. The specific system construction operation is the same as steps 3.1 to 3.3. The polyurethane sponge carrier, due to its loose and porous characteristics, not only provides a relatively safe environment for microorganisms to avoid being damaged by external high-concentration pollutants and free radicals, but also provides attachment sites for microorganisms and self-generated catalysts of microorganisms, realizing external catalytic cleavage of OTC. Internal microorganisms use cleavage products to maintain life activities, forming a virtuous circle, and realizing the close coupling of PS-AOP and biodegradation.

[0030] 3.3 Regarding wastewater contaminated with polycyclic aromatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants widely distributed in water bodies and soils. They have a stable chemical structure, are highly hydrophobic, difficult to degrade, and have tri-toxic properties. Therefore, they are also used as model pollutants for treatment. It should be distinguished that, for PAHs, in the present invention, PAHs can be used as the only carbon source for microbial metabolism, or a carbon source such as sodium acetate can be added for co-metabolism to accelerate the degradation of PAHs. The specific system construction operation is the same as steps 3.1 to 3.3. The large porosity of the polyurethane sponge is conducive to the generation of the MPCS system. More biomass and bio-generated catalysts can be loaded per unit area, thereby improving the degradation efficiency of PAHs.

[0031] Example 1 1. Targeting OTC polluted wastewater 2. Microbial adaptive domestication 3. Add 50-100 g of anaerobic activated sludge and existing known electrochemically active microorganisms such as dissimilatory metal-reducing bacteria / electrochemically active bacteria preserved in the Resource and Environmental Microbiology Technology Laboratory of Southwest Jiaotong University as functional bacteria sources (Pseudomonas, Geobacter, Shewanella, etc.) to 1 L of culture medium for subculture, and add filtered and sterilized organic pollutants thereto; as the culture proceeds, the concentration of pollutants is gradually increased, and the pollutant concentrations are 5 mg / L, 10 mg / L, and 20-100 mg / L from low to high, respectively, to adaptively acclimate the microorganisms, and the culture is completed when the microorganisms can adapt to the added pollutant concentration (i.e., can grow normally under the added pollutant concentration).

[0032] 4. Culture medium. Take 1L sodium acetate culture medium as an example, NaCH3COO·3H2O 10.93g, NaCl 2.32g, NH4Cl1.12g, MgSO4·7H20 0.1g, HEPES 2.38g, Trace element 0.4mL, and add a set concentration of hydrated iron oxide (1g / L) for subculture; during the culture process, gradually increase the concentration of OTC pollutants (5, 10, 20-100mg / L) to adapt the microorganisms; 5. Pretreatment of sponge carrier (same as step 2). The carrier is a polyurethane sponge with a size of 1cm×1cm×1cm; the specific operation is as follows: the polyurethane sponge is ultrasonically washed with deionized water for 30 minutes, anhydrous ethanol for 30 minutes, anhydrous acetone for 30 minutes, soaked in 0.5M HCl solution for 6 hours, and soaked in 0.5M NaOH solution for 6 hours; it is used to remove inorganic and organic residues on the polyurethane sponge, and the treated polyurethane sponge is sterilized by high-pressure steam (121°C, 30 minutes). After sterilization, it is taken out and placed in a suspension containing hydrated iron oxide to fully absorb the hydrated iron oxide, and then dried for use.

[0033] 6. Construct the MPCS system, take out the treated carrier, and select different microbial groups for inoculation according to different pollutants. The specific operations are as follows: Step 3.1 Place the carrier treated in Step 2 into a vial containing culture medium, and inoculate different microbial groups according to different pollutants; Step 3.2 Seal the vial with a pressure cap, use a vacuum pump to draw a vacuum for 30 minutes, and then use high-purity nitrogen (nitrogen content greater than 98%) to remove residual air, so as to simulate the anaerobic environment in sewage and construct the MPCS system ( Figure 1 ).

[0034] Example 2 For polychlorinated phenol wastewater: Polychlorinated phenols are relatively stable in the environment and can exist for a long time, and their cleavage intermediates may be more toxic.

[0035] This embodiment is substantially the same as embodiment 1, except that the organic pollutant is polychlorinated phenol.

[0036] Example 3 For wastewater containing polycyclic aromatic hydrocarbons (pyrene): PAHs are a class of persistent organic pollutants widely distributed in water and soil. It is stable, highly hydrophobic, difficult to degrade and tertiary toxic, so it is also used as a model pollutant for treatment.

[0037] This embodiment is basically the same as Embodiment 1, except that the organic pollutant is pyrene.

[0038] Example 4 For wastewater with complex organic pollutants: This embodiment is basically the same as Embodiment 1, except that the organic pollutant is a mixture of oxytetracycline, polychlorophenols and pyrene, and the molar ratio thereof is 1:1:1.

[0039] Experimental example The MPCS morphology obtained in Example 1 was photographed. Figure 1 As shown in the figure, the original sponge cube is white and has a smooth porous fiber structure. When loaded with hydrated iron oxide, the sponge cube is reddish brown, and under the action of metal-reducing bacteria, the sponge finally turns black. The MPCS is further enlarged and the morphology is photographed. Figure 2 As shown in AC.

[0040] like Figure 2 As shown in A, SEM shows that the sponge cubes that produce biogenic magnetite are black and rough. After the sponge is loaded with hydrated iron oxide, its structure becomes rough and is conducive to bacterial loading. Figure 2 BC showed that the nanoparticles were evenly distributed on the biofilm and carrier surface, and the nanoparticles attached to the bacteria to form a shell, showing a tendency to film formation, which may be due to the negatively charged extracellular secretions of microorganisms (such as polysaccharides, proteins, and peptides), which promoted the heterogeneous nucleation of the inorganic phase, while hindering the homogeneous nucleation and promoting the growth of crystals.

[0041] Extract the biogenic magnetite particles in the MPCS in Example 1, and make X-ray diffraction patterns and X-ray photoelectron spectroscopy patterns, such as Figure 2 As shown in DF.

[0042] like Figure 2 As shown in D, the XRD spectrum of biogenic magnetite shows several obvious diffraction peaks at 30°, 35° and 63° of 2θ, corresponding to the (220), (311) and (440) planes of magnetite (PDF#85-1436). Figure 2 As shown in EF, the Fe2p spectrum of biogenic magnetite is shown by XPS analysis. 3 / 2 (711.17 eV) and Fe2p 1 / 2 (710.10 eV) close to standard Fe2p 3 / 2 (710.80 eV) and Fe2p 1 / 2 (709.60 eV), Fe 2+ The signal is clear. The above results mutually confirm the successful synthesis of biogenic magnetite (Fe3O4).

[0043] For the wastewater contaminated by OTC in Implementation 1, the basic formula of the culture medium is consistent with the adaptive domestication culture of the bacterial flora. The difference is that the amount of hydrated iron oxide added is 2 g / L, and OTC is added to the MPCS successfully constructed in Example 1. The OTC concentration is fixed at 20 mg / L, and OTC is used as the only external carbon source for the degradation of OTC-contaminated wastewater. Since OTC has a photolysis effect, the vial should be wrapped in tin foil when adding OTC to avoid the influence of light. After adding 0.6mMPDS, the culture was carried out at 30°C and 100rpm for 24 hours, and water samples were taken dynamically. The reduction of pollutants was measured by liquid chromatography and COD detector. The experimental results are shown in Figure 3 shown.

[0044] like Figure 3 As shown in A, the removal effects of CK, biological removal and physical removal on OTC were not obvious (less than 10% after 8 h), indicating that natural degradation and adsorption did not play a significant role. The OTC removal rates of sterile MPCS and MPCS within 8 h were 55.45% and 64.80%, respectively. MPCS performed better than sterile MPCS. In addition to biogenic magnetite, biodegradation was also reflected in MPCS. After the introduction of PDS, MPCS-PDS removed more than 80.0% of OTC within 30 minutes and 98.78% within 2 hours, proving that biogenic magnetite can activate PDS to produce free radicals to destroy OTC. The removal rate of OTC by sterilized MPCS-PDS was similar to that of MPCS-PDS (P>0.05), indicating that the degradation of OTC was mainly driven by PDS activation rather than biodegradation. As shown in Figure 3 As shown in Figure B, the COD removal rates of CK, physical removal, and biological removal at 72 h were 2.92%, 4.16%, and 6.72%, respectively, indicating that the biological and physical removal of OTC were weak. The COD removal rate of sterilized MPCS was 6.20%, which may be related to the adsorption and catalytic effect of biogenic magnetite, while the COD removal rate of MPCS was 7.82%, slightly higher than that of sterile MPCS, which may also be related to biological removal. With the participation of PDS, the COD removal rate of MPCS-PDS was 108.96% higher than that of sterilized MPCS-PDS, confirming the synergistic effect of microbial metabolism and PDS activation, using the intermediates produced by PDS activation as an energy source, which greatly promoted the elimination of intermediates.

[0045] like Figure 4 In order to verify the regenerability of the catalyst of Example 1, the iron morphology was determined. 2+ and Fe 3+ The content ratios were 49.64% and 50.35% respectively. After the introduction of PDS, Fe 3+After 2 hours, it rose to 70.80%, while Fe 2+ It dropped to 29.20%, indicating that Fe 2+ PDS activated Fe 3+ After 72 h of repair, Fe 2+ It rebounded to 53.94%, indicating that the microorganisms survived and functioned well under the dual stress of OTC and PDS. The good iron cycle performance of MPCS ensured the feasibility and stability of long-term operation.

[0046] qPCR assay was performed before and after MPCS reaction and other control groups to detect the changes in ARGs. The results are as follows Figure 5 shown.

[0047] like Figure 5 As shown, qPCR analysis showed that among the 16 ARGs, Shewanella oneidensis MR-4, (Accession No.: CICC 25104, NCBI No.: NC_008321.1) expresses a certain level of tetA (P) , but expressed at very low levels tg , tetL , w and tetM , while other ARGs were not detected, indicating that there was a certain background resistance. S.oneidensis Similar situation to MR-4, but ARGs ( tetL , tetM ) was slightly higher, probably because iron mineral stimulation improved cellular energy metabolism. S.oneidensis MR-4 not only expressed 4 additional ARGs ( Q , tetS , tetX and tetH ), and the expression levels of other ARGs were also significantly upregulated (131.34% ~24973.16%), indicating that the risk of ARG transmission is greater during pure biodegradation. S.oneidensis Compared with MR-4+OTC, the abundance of ARG expression in MPCS+OTC was significantly reduced (62.94% ~ 99.91%), and the diversity also showed similar results ( tetS and tetH disappeared), which may be due to the isolation and protection of the biogenic magnet protective film ( Figure 2B). In addition, the expression of ARG in MPCS+OTC+PDS was further reduced (19.39% to 97.47%) compared with MPCS+OTC, indicating that the introduction of PDS prevented the diffusion of ARG. The synergistic effect of AOPs and biodegradation can effectively degrade antibiotics and remove toxic intermediates, thereby reducing potential ARG induction.

[0048] Therefore, based on the above results, it is speculated that the operating mechanism of MPCS is as follows Figure 5 Shown Figure 6 The operating mechanism of MPCS is vividly illustrated: (1) Free radicals generated by AOPs can degrade extracellular ARGs and pollutant molecules, reducing HGT; (2) Free radical stress can prevent excessive accumulation of ARBs and reduce VGT; (3) The biogenic magnetic protective membrane isolates bacterial cells from free radicals, OTCs, and degradation intermediates to prevent potential selection pressure; (4) The synergistic effect of AOPs and biodegradation effectively destroys antibiotics and removes toxic intermediates, thereby reducing the induction of ARBs and ARGs.

[0049] The present invention also lies in the recycling of catalyst by microorganisms: 1. Microorganisms metabolize and transform precursor substances to produce catalysts. The catalysts activate oxidizing substances such as PDS and are then consumed, regenerating precursor substances, which are then metabolized by microorganisms to regenerate catalysts. The cycle repeats and no re-addition is required. 2. During the metabolism of metal-reducing bacteria, trace amounts of hydrogen peroxide are spontaneously produced, driving a Fenton-like reaction and generating free radicals to destroy organic pollutants. Therefore, even without the addition of exogenous oxidizing substances, a good degradation effect can be achieved. This process is significantly different from the existing advanced oxidation process that requires repeated addition of catalysts and oxidizing substances.

[0050] In summary, the preparation method provided in the present application targets the shortcoming that organic pollutants inhibit the activity of microorganisms. According to the types of target organic pollutants in wastewater, the anaerobic activated sludge is domesticated (adding wastewater containing target pollutants) and bio-enhanced (adding metal-reducing bacteria) to obtain a microbial community that can degrade the target pollutants. Different microbial communities are constructed for different pollutants to improve their capabilities in practical applications. On this basis, the obtained microbial community is loaded on a porous carrier to form a biofilm, thereby further improving the resistance of the microbial community to different pollutants. Furthermore, the preparation method provided in the present application utilizes a microorganism-advanced oxidation coupling system, through the spontaneous metabolic activities of microorganisms, using metal minerals, and self-assembly. It does not require human intervention, does not consume chemical substances, and is green and environmentally friendly. MPCS can catalyze oxidants such as persulfate and hydrogen peroxide to form active species for efficient degradation of complex organic pollutants. When no exogenous substances are added, MPCS itself also has a certain degradation ability for pollutants by producing trace hydrogen peroxide and undergoing Fenton-like reactions. The degradation intermediates are once again utilized by microorganisms and deeply mineralized and degraded into CO2 and H2O. Free radicals can effectively destroy extracellular resistance genes to prevent horizontal gene transfer, effectively reducing the risk of resistance spread during biochemical treatment.

[0051] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a microbial-driven activation-circulation system, characterized in that: The following steps are involved: Step 1: screening and adaptive domestication of metal-reducing bacteria to improve the tolerance of metal-reducing bacteria to pollutants, thereby increasing the survival rate and pollutant degradation capacity in the presence of pollutants; Step 2: pre-treating the carrier for inoculation with the metal-reducing bacteria in step 1; Step 3, constructing the MPCS system, taking out the carrier treated in step 2, and selecting different microbial groups for inoculation according to different pollutants. The specific operations are as follows: Step 3.1 Place the carrier treated in step 2 into a vial containing culture medium, and inoculate different microbial groups according to different pollutants; Step 3.2 Seal the vial with a pressure cap, draw a vacuum with a vacuum pump for 10-30 minutes, and then use high-purity nitrogen with a nitrogen content greater than 98% to remove residual air, thereby simulating the low oxygen environment in sewage; Step 3.3 The microorganisms in the vial use catalyst precursors to generate substances with the ability to activate PS oxidants, thereby generating free radicals to crack complex pollutants, and then forming small molecule compounds used by microorganisms, which are finally decomposed into non-toxic CO2 and H2O. Free radicals can effectively destroy extracellular resistance genes and prevent horizontal gene transfer, effectively reducing the risk of resistance spread during biochemical treatment.

2. The method for constructing a microbial-driven activation-circulation system according to claim 1, characterized in that: In step 3.2, the catalyst precursor material includes oxides or hydroxides of transition metal elements such as iron and copper, which are converted into catalysts with peroxide activation ability through the dissimilatory reduction of metal-reducing bacteria.

3. The method for constructing a microbial-driven activation-circulation system according to claim 1, characterized in that: The PS added in step 3.2 generates sulfate radicals, and the added amount varies accordingly with the pollutant concentration.

4. The method for constructing a microbial-driven activation-circulation system according to claim 1, characterized in that: The specific operation of step 1 is as follows: using anaerobic activated sludge and laboratory preserved dissimilatory metal-reducing bacteria / electrochemically active bacteria as the source of functional bacteria, adding a set concentration of hydrated iron oxide and OTC to the sodium acetate culture medium for subculture; gradually increasing the pollutant concentration during the culture process to adaptively domesticate the microorganisms.

5. The method for constructing a microbial-driven activation-circulation system according to claim 4, characterized in that: During the culture process of step 1, the pollutant concentration is gradually increased, and the pollutant concentration is 5 mg / L, 10 mg / L, and 20-100 mg / L from low to high.

6. The method for constructing a microbial-driven activation-circulation system according to claim 1, characterized in that: The carrier in step 2 is any one of biochar, porous ceramic particles, polyurethane sponge and calcium alginate microspheres.

7. The method for constructing a microbial-driven activation-circulation system according to claim 6, characterized in that: The carrier is a polyurethane sponge, and the specific operation of step 2 is as follows: the polyurethane sponge is sequentially ultrasonically washed with deionized water for 30 minutes, ultrasonically washed with anhydrous ethanol for 30 minutes, ultrasonically washed with anhydrous acetone for 30 minutes, soaked in a 0.5M HCl solution for 6 hours, and soaked in a 0.5M NaOH solution for 6 hours; the polyurethane sponge is used to remove inorganic and organic residues on the polyurethane sponge, and the treated polyurethane sponge is sterilized with high-pressure steam. After the sterilization is completed, the polyurethane sponge is taken out and placed in a suspension containing hydrated iron oxide to fully adsorb the hydrated iron oxide. After drying, the polyurethane sponge is added to a syringe bottle containing a sodium acetate culture medium and microorganisms are added to load the microorganisms on the sponge and fully contact with the catalyst precursor material, so as to drive the subsequent synthesis of the catalyst and peroxide activation.

8. Application of a microorganism-driven activation-circulation system, wherein the microorganism-driven activation-circulation system is any one of claims 1 to 7, and the microorganism-driven activation-circulation system is applied to the treatment of organic pollutants and the control of the risk of resistance gene transmission.

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