Method for removing antibiotic resistance genes in sludge by photocatalytic coupling biological electrochemical system

Abstract

The application discloses a method for removing antibiotic resistance genes in sludge by a photocatalysis coupling bio-electrochemical system, which comprises the following steps: S1, preparing a K-g-C3N4 photocatalyst; S2, preparing a photocatalysis anode of the photocatalysis coupling bio-electrochemical system; and S3, removing antibiotic resistance genes in a sludge mixture to be treated by the photocatalysis coupling bio-electrochemical system. The method for removing antibiotic resistance genes in sludge by the photocatalysis coupling bio-electrochemical system disclosed in the application effectively reduces the abundance of antibiotic resistance genes after the treatment of the ICPBES system, wherein the removal rates of tetracycline, sulfonamide, macrolide and beta-lactam resistance genes are 84.78%, 71.40%, 82.57% and 84.28% respectively; the ecological pollution risk of the antibiotic resistance genes is reduced; the ICPBES system reduces the abundance of mobile genetic elements, the removal rate reaches 69.65%, the further spread of the antibiotic resistance genes in the environment is inhibited, and the pollution and spread risk of the antibiotic resistance genes is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of removing antibiotic resistance genes in sludge, and particularly relates to a method for removing antibiotic resistance genes in sludge by coupling photocatalysis and a biological electrochemical system. BACKGROUND

[0002] Antibiotics in the environment can promote the generation and spread of antibiotic resistance genes (ARGs). Sewage treatment plants are the largest source of ARGs spread, and ARGs enter the natural world with effluent and residual sludge. Current ARGs removal processes include biological treatment, disinfection processes, advanced oxidation, and composite processes. The commonly used biological treatment technology is activated sludge process. The mechanism of removing ARGs by activated sludge process is that under high load of activated sludge, activated sludge can adsorb a large amount of ARGs in sewage. After the activated sludge process, the abundance of ARGs is reduced by 2-3 orders of magnitude.

[0003] Extracellular polymeric substances (EPS) in sludge of sewage treatment plants are the main components of activated sludge flocs. EPS directly covers the cell membrane, and the amount and composition of EPS can affect the surface properties of activated sludge flocs, and further affect the adsorption and flocculation properties of activated sludge. Sludge is mainly composed of cell bodies and EPS. EPS is mainly composed of proteins and polysaccharides. Polysaccharide molecules are rich in hydroxyl, carboxyl and other hydrophilic functional groups, which can form a stable and elastic network structure through adsorption and bridging, and cross-link and fix free cells, which is conducive to sludge flocculation. Proteins and lipids can maintain the stability of the floc structure and improve the biological flocculation of sludge. These substances form a compact and high-density network structure through electrostatic force, hydrogen bond combination, ionic attraction and biochemical action, which can act as a protective layer for microorganisms to resist external heavy metals and toxic compounds. Antibiotic resistance genes exist in a large amount in EPS. EPS absorbs a large amount of ARGs through bridging effect, hydrophobic effect and entanglement, and the proportion of ARGs in total ARGs can be as high as 74.2%. In addition, some ARGs in sludge are also distributed in the genetic material of bacteria. Since a large amount of ARGs exist in EPS in sludge of sewage treatment plants, ARGs are wrapped in EPS to form a high-molecular polymer structure, and the removal of this part of ARGs is the core and difficulty of sludge ARGs removal and degradation.

[0004] The existing sludge ARGs reduction technology has poor effect and cannot effectively reduce ARGs in sludge, so there is a need to provide a method for efficiently reducing sludge ARGs to solve the problem. SUMMARY

[0005] The present application provides a method for removing antibiotic resistance genes in sludge by coupling photocatalysis and a biological electrochemical system to solve the above problems.

[0006] To achieve the above object, the technical scheme of the present application is:

[0007] A method for removing antibiotic resistance genes in sludge by a photocatalytic coupling bio-electrochemical system, comprising the following steps:

[0008] S1: preparing a K-g-C3N4 photocatalyst:

[0009] Take the ground g-C3N4 catalyst, add KCl for mixing and grinding, the mass ratio of the g-C3N4 catalyst to KCl is 10:1, after mixing, high-temperature calcination and grinding are performed to obtain a K-g-C3N4 photocatalyst;

[0010] S2: preparing a photocatalytic anode of the photocatalytic coupling bio-electrochemical system:

[0011] Take the K-g-C3N4 photocatalyst, add 5% Nafion solution and 1 mL of isopropanol, mix uniformly by ultrasonic, and then coat on a foam nickel carrier, and after drying, a K-g-C3N4 photocatalyst-loaded foam nickel electrode is obtained; the mass ratio of the K-g-C3N4 photocatalyst to the Nafion solution and the volume ratio of isopropanol are: 400 mg: 2 mL: 1 mL;

[0012] The K-g-C3N4 photocatalyst-loaded foam nickel is used as an anode, and a pretreated carbon brush is used as a cathode, which are installed on a biofilm culture reactor, a cathode solution is introduced into a cathode chamber of the reactor, a mixed solution containing anode sludge is introduced into the cathode chamber of the reactor, and the anode and the cathode are electrically connected to form a loop, so that electric energy is generated by the chemical potential difference of the system itself, a biofilm is formed in the pores of the K-g-C3N4 photocatalyst-loaded foam nickel, after the maximum voltage is measured and stabilized, the biofilm is domesticated, and a photocatalytic anode of the photocatalytic coupling bio-electrochemical system is prepared;

[0013] S3: removing antibiotic resistance genes in the sludge mixture to be treated by the photocatalytic coupling bio-electrochemical system:

[0014] The photocatalytic anode prepared in S2 is connected to a single-chamber photocatalytic electrochemical reactor, and a modified air cathode is connected to the single-chamber photocatalytic electrochemical reactor as a cathode, the photocatalytic anode and the modified air cathode are connected to an external circuit to form a loop, forming a photocatalytic coupling bio-electrochemical system, a diluted sludge mixture to be treated is introduced into the single-chamber photocatalytic electrochemical reactor, and a light source is used to irradiate above the photocatalytic anode, so that the photocatalytic coupling bio-electrochemical system removes antibiotic resistance genes in the sludge mixture to be treated.

[0015] Further, the preparation method of the g-C3N4 catalyst is as follows: 15 g of urea is added into a 50 mL alumina crucible with a cover, and then the mixture is placed into a muffle furnace and calcined at 550 DEG C for 3 h at a temperature rising rate of 5 DEG C / min. After the product is naturally cooled, it is washed with water, dried, and ground into powder, to obtain the g-C3N4 catalyst.

[0016] Further, in S2, the mixed solution containing the anode sludge is a mixed solution of sludge from a sewage treatment plant and a sodium acetate solution, and the content of the sludge is 2.0 g / L.

[0017] Further, in S3, the diluted sludge mixture to be treated is a mixture formed by diluting the sludge to be treated with a 0.1 mol / L glucose solution, wherein the pH value of the sludge to be treated is 7.58±0.01, the moisture content is 98.64±0.06%, the total suspended solids (TSS) is 8.07±0.03 g / L, the volatile suspended solids (VSS) is 3.65±0.02 g / L, the soluble chemical oxygen demand (SCOD) is 284±27 mg / L, and the total organic carbon (TOC) is 105±17 mg / L.

[0018] Further, in S1, the high-temperature calcination condition is that the g-C3N4 catalyst is uniformly mixed with KCl, and then placed in a muffle furnace and calcined at 500 DEG C for 3 h at a temperature rising rate of 2.5 DEG C / min.

[0019] Further, in S2, the pretreated carbon brush is a carbon brush pretreated with a acetone solution.

[0020] Further, the cathode solution is a mixed solution of 0.2 mol / L potassium dihydrogen phosphate and 0.1 mol / L potassium ferricyanide.

[0021] Further, in S2, the maximum voltage plateau is determined after the biofilm is formed for 1 month, and the voltage reaches 0.6-0.7 V.

[0022] Further, in S3, the preparation method of the modified air cathode is as follows: carbon felt is used as the material of the air cathode, 10 g of 60% PTFE is weighed, added with water to 120 g, and coated on one side of the carbon felt as a carbon-based layer, and then dried at room temperature for two hours, and then placed into a 370 DEG C oven and heated for 30 min; four layers of 60% PTFE emulsion are coated on the carbon-based layer as an air diffusion layer, and each layer is dried in a 370 DEG C oven for 10-15 min; 318 mg of activated carbon and 13.25 mg of conductive carbon black are weighed and added with 1.48 ml of isopropanol and 1 mL of Nafion reagent, and then mixed uniformly and coated on the other side of the carbon felt as a catalytic layer, and the other side is the side that contacts the electrolyte, and then dried in a 60 DEG C oven for 6 h.

[0023] Furthermore, in S2, the cathode solution and the mixed solution containing anode sludge are replaced every three days.

[0024] The beneficial effects of the present invention are:

[0025] (1) The present invention discloses a method for removing antibiotic resistance genes from sludge using a photocatalytic coupled bioelectrochemical (ICPBES) system. Kg-C3N4 catalyst and biofilm are simultaneously loaded onto a porous nickel foam anode to construct an ICPBES photocatalytic anode. K doping improves the photoelectric performance of g-C3N4. In this method, the photocatalytic anode generates electrons and transfers them to the anode through two pathways, thereby increasing the amount of electron transfer. Oxidizing substances such as O2 generated during the anode and cathode reactions - ·, H2O2 and ·OH can destroy the structure of potential host bacteria, causing the release of intracellular antibiotic resistance genes (iARGs) in sludge and the degradation of iARGs, the enrichment of ARGs in extracellular polymers (EPS), the massive degradation of mobile genetic elements (MGEs) and the death of host bacteria, thereby blocking the horizontal transfer of iARGs between bacteria;

[0026] (2) The reactive oxygen species (ROS) generated by photocatalysis in the present invention can directly oxidize intracellular DNA (iDNA) and extracellular DNA (e-DNA), thereby degrading iARGs and ARGs in EPS;

[0027] (3) The ICPBES used in the present invention reshapes the EPS structure by acting on potential host bacteria or changing metabolic pathways, leading to the degradation of intracellular ARGs (iARGs) and the migration and transformation of ARGs in EPS; oxidative free radicals can regulate metabolism-related genes (secE, rstA, virB2, virB6, virB8), reduce the gene expression levels of the two-component regulatory system, bacterial secretion system and type ⅵ secretion system, thereby reducing ARGs, or can affect the core microbial strains related to metabolism ( Promineofilum Piscinibacter, Accumulibacter ), affecting the EPS structure and thus changing the distribution of ARGs in EPS, causing AGRs to migrate to the outer layer of cells, thereby promoting the degradation of ARGs;

[0028] (4) After treatment with the ICPBES system, the abundance of antibiotic resistance genes was effectively reduced. The removal rates of tetracycline, sulfonamide, macrolide, and β-lactam resistance genes reached 84.78%, 71.40%, 82.57%, and 84.28%, respectively, which can effectively reduce the ecological pollution risk of antibiotic resistance genes. The ICPBES system reduced the abundance of mobile genetic elements, with a removal rate of 69.65%, inhibiting the further spread of antibiotic resistance genes in the environment and reducing the risk of antibiotic resistance gene pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 A diagram of a small test device used in an embodiment of the present invention;

[0031] Figure 2 Schematic diagram of a photocatalytic anode in an embodiment of the present invention;

[0032] Figure 3 Characterization diagram of the catalyst of the present invention, wherein, 3 (a) is the XPS spectrum of the prepared Kg-C3N4 and g-C3N4; 3 (b) is the XRD spectrum of Kg-C3N4 and g-C3N4; 3 (c) is the UV-visible diffuse reflectance spectrum of Kg-C3N4 and g-C3N4; 3 (d) is the DMPO-·O2EPR spectrum of Kg-C3N4; 3 (e) is the DMPO-·OH EPR spectrum of Kg-C3N4; 3 (f) is the PL photoluminescence spectrum of Kg-C3N4 and g-C3N4; 3 (g) is the SEM image of the nickel foam electrode without attached microorganisms and catalysts; 3 (h) is the SEM image of the PES anode; 3 (i) is the SEM image of the BES anode; 3 (j) is the SEM image of the ICPBES anode;

[0033] Figure 4 Figure 4 (a) shows the concentration change of polysaccharides and proteins in EPS after treatment with different systems (4 (a) shows the concentration change of polysaccharides, and 4 (b) shows the concentration change of proteins);

[0034] Figure 5 The relative abundance results of various ARGs in sludge after treatment by different systems (5 (a) is the total relative abundance accumulation diagram of various ARGs in sludge after treatment by different systems, and 5 (b) is the relative abundance heat map);

[0035] Figure 6 Figure 6 is a result of absolute abundance of various ARGs in sludge after different system treatment (6(a) is a pile diagram of total absolute abundance of various ARGs in sludge after different system treatment, and 6(b) is a heat map of absolute abundance);

[0036] Figure 7 Figure 7 is a heat map of absolute abundance of various MGEs in sludge after different system treatment of the application;

[0037] Figure 8 Figure 8 is a relative abundance diagram of microbial community in different treatment groups of the application (8(a) is at the level of door, 8(b) is at the level of horizontal, and 8(c) is at the level of genus)

[0038] Figure 9 Figure 9 is a result of ARGs propagation related key metabolic pathway expression (9(a) is relative abundance (%) of KO number, 9(b) is metabolic function based on KEGG, and 9(c) is expression of typical genes in ARGs propagation related metabolic pathway);

[0039] Figure 10 Figure 10 is a network diagram of ARGs, microbial community, metabolic pathway and EPS structure (in the diagram, the network interaction of microorganisms (green circle), metabolic pathway (blue circle), EPS structure (orange circle) and ARGs (pink circle) in SEPS (S), LB-EPS (L) and TB-EPS (T) of each group);

[0040] Figure 11 Figure 11 is a result of Mantel test of ARGs (ARGs, MGEs, EPS structure, bacterial diversity, bacterial community and metabolic pathway).

[0041] In the figure: 1, photocatalytic anode; 2, modified air cathode; 3, external resistance; 4, xenon lamp light source 5, reaction chamber; 6, K-g-C3N4 catalyst; 7, electrochemically active bacteria. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the embodiments of the application clearer, the technical scheme in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0043] The application relates to the principle:

[0044] Doping K improves the photoelectric performance of g-C3N4, K-g-C3N4 shows lower band gap energy after doping, resulting in higher visible light absorption efficiency Figure 3 a), increasing the interlayer distance, which promotes the charge transfer in the photocatalytic process Figure 3 b), and reduces the emission peak in the PL spectrum, indicating a decrease in the electron-hole recombination rate Figure 3 c). In the photocatalytic coupling BES system (ICPBES), the K-g-C3N4 catalyst and the biofilm are simultaneously loaded onto the porous nickel foam anode to construct a photocatalytic anode, and the photocatalytic anode is enlarged as shown in Figure 2 , Figure 2 6 is the K-g-C3N4 catalyst, and 7 is the electrochemically active bacteria. The photocatalytic anode produces electrons by two pathways, and then transfers to the cathode through an external circuit. Under visible light irradiation, the K-g-C3N4 photocatalyst loaded on the surface of the nickel foam anode is excited to produce holes and photoelectrons (equation (1)). These holes react with water to produce ·OH (equation (2)), and the generated electrons are transferred to the cathode, the electron-hole recombination rate is reduced, and the photocatalytic efficiency is improved. In addition, electrochemically active bacteria, such as Rhodopseudomonas palustris, Pseudomonas aeruginosa, Escherichia coli, Enterococcus, Geobacillus, Arcobacter, Clostridium and Geosiphon, exist in the biofilm in the pores of the nickel foam, and oxidize organic substrates by enzymes to release intracellular electrons. These generated electrons are further transferred to the anode through direct pathways (outer membrane cytochrome C or flagella) and indirect pathways (redox shuttle substances). Then the electrons are transferred to the cathode through an external circuit, and react with oxygen to generate O2 - ·, further obtaining electrons to form H2O2 (equation (4)).

[0045] Anode: (1)

[0046] (2)

[0047] Cathode: (3)

[0048] (4)

[0049] (5)

[0050] Oxidizing substances such as O2 - ·, H2O2 and ·OH produced in the anode and cathode reaction process can be used to Bacteria , Accumulibacter , Nannocystis , Burkholderiaceae , Promineofilum Rhodoferax) disrupts the structure of potential host bacteria, such as sludge, leading to the release of intracellular antibiotic resistance genes (iARGs), which degrade the iARGs and enrich them within the EPS. The released iARGs further migrate and transform within the EPS layers. iARGs are significantly correlated with intracellular mobile genetic elements (iMGEs) and share the same potential hosts. Extensive degradation of MGEs and the death of host bacteria block the horizontal transfer of iARGs between bacteria. Furthermore, direct oxidation of intracellular DNA (iDNA) and extracellular DNA (e-DNA) by photocatalytically generated reactive oxygen species (ROS) is a primary factor in the degradation of iARGs and ARGs in EPS. These oxidative free radicals can also directly act on ARGs and MGEs in EPS, causing severe oxidative base damage and 2-deoxyribose modification, leading to gene damage. In addition, oxidative free radicals can regulate metabolism-related genes (secE, rstA, virB2, virB6, virB8), reduce the expression levels of genes in the two-component regulatory system, bacterial secretion system, and type VI secretion system, thereby reducing ARGs, or can affect metabolism-related core microbial strains ( Promineofilum 、 Piscinibacter, Accumulibacter ), affecting the EPS structure and thus changing the distribution of ARGs in the EPS. For example, network analysis found that Promineofilum It is positively correlated with the bacterial secretion system and the two-component system, and is also related to the polysaccharides in the inner EPS (TB-EPS), ereA 、 tetO 、 tetS 、 strB 、 intI3 Related; OLB14 、 Piscinibacter Also relevant for two-component systems, and OLB14 There was a significant negative correlation between the protein in soluble extracellular polymeric substances (S-EPS), loosely bound extracellular polymeric substances (LB-EPS), and polysaccharides in S-EPS. Piscinibacter It was positively correlated with protein in S-EPS and LB-EPS, and polysaccharide in S-EPS; Accumulibacter There was a significant negative correlation between the two-component system and the polysaccharides, tetO 、 strB , LB-EPS tetQ 、 tetS 、 intl3 and S-EPS intl3 Positive correlation. AccumulibacterAs a bacterium, it may play a mediating role in photocatalytic coupling biological electrochemical systems, and environmental factors affect the expression of EPS-related ARGs. In summary, ICPBES remodels the EPS structure by acting on potential host bacteria or changing metabolic pathways, leading to the degradation of intracellular ARGs (iARGs) and the migration and transformation of ARGs in EPS. The biofilm culture reactor used in the step of forming a biofilm inside the pores of the K-g-C3N4 photocatalyst-loaded nickel foam in this application, i.e., the BES reactor, is composed of two identical 216 mL organic glass cubes (6 cm x 6 cm x 6 cm), which are used as the cathode chamber and anode chamber of the BES reactor, respectively, and a proton exchange membrane is used as a separator. The reaction conditions are as follows: the anode is a nickel foam electrode loaded with 400 mg of K-g-C3N4, the cathode is a carbon brush pretreated with a ketone solution, the electrodes are connected by titanium wires with a diameter of 0.3 mm, an external resistance of 100 Ω is loaded, and the voltage is 14 V. When removing antibiotic resistance genes in the sludge mixture to be treated by a photocatalytic coupling biological electrochemical system, a small-scale test device as shown in Figure 1 is used for treatment, i.e., a single-chamber reactor, which includes a photocatalytic anode 1, a modified air cathode 2, an external resistance 3, a xenon lamp light source 4, and a reaction chamber 5. When treating sludge, the sludge is diluted with a 0.1 mol / L glucose solution and then injected into the reaction chamber. After the voltage stabilizes, the photocatalytic experiment is performed. The light group uses a xenon lamp to irradiate the upper part of the photocatalytic anode, with a light intensity of 4500 Lux. After nine hours, the sludge sample is taken from the reaction chamber of the BES. The reaction is carried out at room temperature.

[0051] The sludge treated in this application is sludge that has adsorbed ARGs in wastewater. The activated sludge method is to continuously introduce air into wastewater, and after a certain period of time, a sludge-like flocculant is formed due to the reproduction of aerobic microorganisms. The treatment object of the technology of this application is sludge from a wastewater treatment plant, so the sludge of this application is activated sludge flocculant that has adsorbed ARGs in wastewater.

[0052] Example 1:

[0053] A method for removing antibiotic resistance genes in sludge by a photocatalytic coupling biological electrochemical system, denoted as ICPBES, includes the following steps:

[0054] S1: Preparation of K-g-C3N4 photocatalyst:

[0055] S11: Preparation of g-C3N4 catalyst: weigh 15 g of urea and add it to a 50 mL alumina crucible with a lid. Place it in a muffle furnace at 550 ℃ for 3 h, with a heating rate of 5 ℃ / min. After the product is naturally cooled, wash it with water, dry it, and grind it into a powder to obtain the g-C3N4 catalyst;

[0056] S12: Preparation of K-g-C3N4 photocatalyst: 2 g of g-C3N4 catalyst ground in step S11 was weighed, 0.2 g of KCl was added, and mixed and ground for 30 min. After being ground uniformly, it was placed in a muffle furnace and calcined at 500°C for 3 h, with a heating rate of 2.5°C / min. The K-g-C3N4 photocatalyst was obtained by grinding;

[0057] S2: Preparation of a photocatalytic anode for a photocatalytic bioelectrochemical system:

[0058] S21: Preparation of the anode: The prepared K-g-C3N4 photocatalyst was coated on a nickel foam anode: The nickel foam was cut to the desired size, and cleaned with acetone, alcohol, and ultrapure water for ten minutes each, and then dried in an oven to obtain a K-g-C3N4 photocatalyst-loaded nickel foam electrode, which was ready for use;

[0059] S21: Preparation of a K-g-C3N4 photocatalyst-loaded nickel foam electrode:

[0060] 400 mg of K-g-C3N4 photocatalyst was weighed into a beaker, 2 ml of 5% Nafion solution and 1 mL of isopropyl alcohol were added, and ultrasonic treatment was performed for ten minutes. After mixing uniformly, it was coated on the cut nickel foam and dried in an oven to obtain a K-g-C3N4 photocatalyst-loaded nickel foam electrode;

[0061] S22: Preparation of a modified air cathode:

[0062] Carbon felt was used as the material for the air cathode, 10 g of 60% PTFE was weighed and added to 120 g of water and coated on one side of the carbon felt as a carbon-based layer. After drying at room temperature for two hours, it was placed in a 370°C oven and heated for 30 min. Four layers of 60% PTFE emulsion were then brushed on the carbon-based layer as an air diffusion layer, and each layer was dried in a 370°C oven for 10-15 min. Then 318 mg of activated carbon and 13.25 mg of conductive carbon black were weighed and added to 1.48 ml of isopropyl alcohol and 1 ml of Nafion reagent, and mixed uniformly before being coated on the other side of the carbon felt (the side that contacts the electrolyte) as a catalytic layer. It was dried in a 60°C oven for 6 h.

[0063] S23: Domestication of the photocatalytic anode biofilm:

[0064] A double-chamber reactor configuration is used to construct a special reactor for anodic biofilm culture. The anode is a prepared nickel foam electrode, and the cathode is a carbon brush that is pre-treated with acetone solution (the carbon brush size is 3 cm x 5 cm x 15 cm; the treatment method of the carbon brush is: the carbon brush is placed in a beaker containing acetone solution (13.5 mol / L), the beaker is covered with tin paper, and ultrasonic treatment is performed for 10 min). The electrode is connected with a titanium wire with a diameter of 0.3 mm, and an external load of 100 Ω resistor is used. The anode chamber is inoculated with a mixture of sludge from a sewage treatment plant and sodium acetate solution (2.0 g / L), and the cathode solution is a mixture of 0.2 mol / L potassium dihydrogen phosphate and 0.1 mol / L potassium ferricyanide as an electron acceptor. The output voltage value is recorded daily with a digital multimeter. The anode sludge and the cathode solution are replaced every three days. After about one month, the maximum voltage reaches 0.6-0.7 V, at which time it is considered that the biofilm is domesticated. A biofilm is formed inside the pores of the K-g-C3N4 photocatalyst-loaded nickel foam anode, and a photocatalytic anode of a photocatalytic coupled bioelectrochemical system is prepared.

[0065] S3: Removal of antibiotic resistance genes in the sludge mixture to be treated by the photocatalytic coupled bioelectrochemical system:

[0066] The domesticated reactor is changed to a single-chamber photocatalytic coupled bioelectrochemical system, the cathode (carbon brush pre-treated with acetone solution) in S23 is replaced with a modified air cathode, and the external circuit is connected. The sludge to be treated (the parameters of the sludge to be treated are: pH value is 7.58±0.01, water content is 98.64±0.06%, total suspended solids (TSS) is 8.07±0.03 g / L, volatile suspended solids (VSS) is 3.65±0.02 g / L, soluble chemical oxygen demand (SCOD) is 284±27 mg / L, and total organic carbon (TOC) is 105±17 mg / L) is diluted with 0.1 mol / L glucose solution and injected into the reaction chamber. After the voltage is stable again, the photocatalytic experiment is performed. The reactor is placed on a magnetic stirrer, and a xenon lamp is used to irradiate the anode chamber. After nine hours, the sludge sample is taken from the reaction chamber.

[0067] Example 2:

[0068] A method for removing antibiotic resistance genes in sludge by a bioelectrochemical system, which is also named as BES in this embodiment, comprises the following steps:

[0069] A double-chamber bioelectrochemical system (double-chamber reactor) is used, the anode is the uncoated nickel foam anode prepared in Example 1, the cathode is the modified air cathode as in Example 1, and after the anode biofilm is domesticated according to Example 1, the sludge is diluted with 0.1 mol / L glucose solution and injected into the anode reaction chamber. After the voltage is stable again, the sludge sample is taken.

[0070] Example 3

[0071] A method for removing antibiotic resistance genes in sludge by a catalyst coupled bioelectrochemical system, the embodiment is also named as ICPBES-Dark, comprising the following steps:

[0072] A double-chamber catalyst coupled bioelectrochemical system (double-chamber reactor) is adopted, the anode is the photocatalytic anode prepared in Example 1, after the anode biofilm is domesticated according to Example 1, the domesticated reactor is changed into a single-chamber catalyst coupled bioelectrochemical system (single-chamber reactor), the original cathode is replaced by the modified air cathode, the sludge is diluted by 0.1 mol / L glucose solution and then injected into the reaction chamber, the external circuit is connected, after the voltage is stable again, the experiment is carried out. The reactor is placed on a magnetic stirrer and wrapped with tin paper to create a dark environment, and the sludge sample is taken out from the reaction chamber after nine hours.

[0073] Example 4

[0074] A method for removing antibiotic resistance genes in sludge by an open circuit photocatalytic coupled bioelectrochemical system, the embodiment is also named as ICPBES-OC, comprising the following steps:

[0075] A double-chamber catalyst coupled bioelectrochemical system (double-chamber reactor) is adopted, the anode is the photocatalytic anode prepared in Example 1, after the anode biofilm is domesticated according to Example 1, the domesticated reactor is changed into a single-chamber catalyst coupled bioelectrochemical system (single-chamber reactor), the original cathode is replaced by the modified air cathode, the sludge is diluted by 0.1 mol / L glucose solution and then injected into the reaction chamber, the external circuit is disconnected, and the experiment is carried out. The reactor is placed on a magnetic stirrer, the upper side of the photocatalytic anode is irradiated by a xenon lamp, and the sludge sample is taken out from the reaction chamber after nine hours.

[0076] Example 5

[0077] A method for repairing antibiotic resistance genes in sludge by a photocatalytic coupled bioelectrochemical system without biofilm connection, the embodiment is also named as PES, comprising the following steps:

[0078] A single-chamber catalyst coupled bioelectrochemical system is adopted, the anode is the photocatalytic anode prepared in Example 1, and the cathode is the modified air cathode prepared in Example 1, the sludge is diluted by 0.1 mol / L glucose solution and then injected into the reaction chamber, the external circuit is connected, after the voltage is stable again, the experiment is carried out. The reactor is placed on a magnetic stirrer and wrapped with tin paper to create a dark environment, and the sludge sample is taken out from the reaction chamber after nine hours.

[0079] Performance test:

[0080] (1) The K-g-C3N4 photocatalytic material prepared in the examples was subjected to UV / visible diffuse reflection (UV-Vis DRS) test, XRD (X-ray diffraction) test and EPR spectrum characterization, and pure g-C3N4 was used as a comparison, and the results are shown in Figure 3

[0081] As Figure 3 a is the UV / visible diffuse reflection spectrum of g-C3N4 and K-g-C3N4, and by converting UV-vis DRS into Tacu graph, it is found that the UV-vis absorption of K-g-C3N4 has red shift compared with pure g-C3N4, and the band gap of K-g-C3N4 is 2.78 eV, which is lower than that of pure g-C3N4 (2.79 eV), therefore, K-g-C3N4 has wider visible light absorption range, and K-g-C3N4 has higher visible light absorption efficiency, the utilization rate of light is improved, and K-g-C3N4 has higher photocatalytic activity than g-C3N4; and the luminescence intensity of K-g-C3N4 is also lower, so the recombination rate of electron-hole is slowed down, the carrier lifetime is prolonged, and the interlayer spacing of K-g-C3N4 is increased, which is beneficial to the transfer of electric charge, so the photocatalytic efficiency is improved, under xenon lamp irradiation, K-g-C3N4 is excited to generate electrons and holes, the holes react with water to generate ·OH, and the electrons are transferred to the cathode through the external circuit of the BES system, and further generate O2 - and H2O2 in the cathode, and these oxidative free radicals directly act on extracellular ARGs or on host cells.

[0082] In addition, the XRD spectrum of g-C3N4 and K-g-C3N4 was also characterized (as Figure 3 b), and in pure g-C3N4, an obvious diffraction peak was found at 27°, and with the addition of K, the diffraction peak in K-g-C3N4 slightly moved (from 27.900 to 27.618), indicating that the interlayer distance of g-C3N4 was increased, which was beneficial to improve the charge transfer in the photocatalytic reaction. As Figure 3 c is the photoluminescence PL spectrum of K-g-C3N4 and g-C3N4 excited at room temperature 400 nm. The luminescence intensity of pure g-C3N4 is higher than that of K-g-C3N4, and there is no difference in the position of the luminescence peak. Because lower peak intensity indicates lower photo-bio-recombination rate, K-g-C3N4 can effectively hinder the recombination rate of photo-generated electron-hole pairs relative to pure g-C3N4; as Figure 3 d is the XPS spectrum of g-C3N4 and K-g-C3N4, and from the figure it can be seen that K-gC3N4 appears K peak, indicating that K is successfully doped.

[0083] In order to further verify the degradation mechanism of k-g-C3N4 in the photocatalytic degradation system of ARGs, EPR spectrum characterization was used for exploration, and it was verified that O2 - ​and the presence of ·OH, EPR spectra were characterized in aqueous solution and methanol solution respectively with DMPO as a trapping agent to detect whether DMPO-·OH and DMPO-·O2 - signal peaks. As shown in Figure 3 e, 3f, no signal peaks were detected under dark conditions, and obvious DMPO-·OH and DMPO-·O2 were detected after 10 min of visible light irradiation - characteristic signal peaks, indicating that the catalysis of light excited K-g-C3N4 to produce a large number of oxidative free radicals.

[0084] (2) Explore the effect of the method of examples 1-4 on polysaccharide and protein in sludge extracellular polymer substance EPS:

[0085] The sludge sample treated by the method of examples 1-4 was layered and extracted by centrifugation S-EPS, LB-EPS (TB-EPS (tightly bound cell polymer), as follows: take 25 mL of sludge, centrifuge for 15 min (4℃, 4000 r·min -1 ), the supernatant was filtered by 0.45 μm filter membrane, and the filtrate was S-EPS; the remaining sludge was supplemented to 25 mL with 0.9% NaCl by mass fraction, and centrifuged for 20 min (4℃ 8000 r min -1 ), the supernatant was filtered by 0.45 μm filter membrane, and the filtrate was S-EPS; the remaining sludge was supplemented to 25 mL with 0.9% NaCl by mass fraction, and centrifuged for 20 min (4℃ 8000 r min -1 ), the supernatant was filtered by 0.45 μm filter membrane, and the filtrate was S-EPS; the remaining sludge was supplemented to 25 mL with 0.9% NaCl by mass fraction, and centrifuged for 20 min (4℃ 8000 r min

[0086] The effect of the photo-catalytic coupling bio-electrochemical system on the characteristics of the biofilm EPS was investigated by detecting the content and composition of the EPS. The content of the EPS was equal to the total of the protein and polysaccharide. The content of the polysaccharide was determined by the phenol-concentrated sulfuric acid method, and the content of the protein was determined by the coomassie brilliant blue method.

[0087] As shown in Figure 4 , the concentrations of the soluble polysaccharide and protein derived from the EPS in the original sludge were 47.19 mg / L and 5.61 mg / L respectively. The content of the polysaccharide in the TB-EPS was 19.78 mg / L, and the content of the protein was 1.83 mg / L. The content of the polysaccharide and protein in the LB-EPS was 16.41 mg / L and 1.73 mg / L respectively, and the content in the S-EPS was even lower. However, their concentrations were significantly improved in each treatment group (especially in the ICPBES group), indicating that each treatment changed the structure of the sludge.

[0088] In the BES group of Example 2, the total content of polysaccharides and proteins in each layer was slightly increased compared with the original sludge, and the polysaccharides in S-EPS and LB-EPS did not change significantly, and increased by 36.20% in TB-EPS, and the proteins in S-EPS increased by 50.50%, and in LB-EPS increased by 71.43%, and in TB-EPS was not detected.

[0089] In the PES group of Example 5, the polysaccharide and protein contents were also increased compared with the original sludge, and in S-EPS, the polysaccharide and protein contents increased by 209.55% and 25.25% respectively, and in LB-EPS, they increased by 11.90% and 45.16% respectively, and in TB-EPS, there was no significant change, indicating that the free radicals generated by photocatalysis can significantly change the structure of EPS, mainly affecting S-EPS and LB-EPS, and the degree of damage to the inner EPS is smaller.

[0090] In the ICPBES-dark group of Example 3, the polysaccharide content in S-EPS decreased slightly, and the protein content increased by 10.10%, and in LB-EPS, the polysaccharide and protein contents increased, and increased by 37.42% and 20.81% respectively compared with the original sludge, and in TB-EPS, the polysaccharide content increased by 17.29% and the protein content did not change significantly. It shows that the catalyst coupled with the bio-electrochemical system can effectively destroy the EPS structure, and even can degrade the polysaccharides in the outer S-EPS.

[0091] In the ICPBES group in Example 1, the polysaccharide and protein concentrations reached 150.06 mg / L and 15.71 mg / L respectively. In S-EPS, the polysaccharide and protein increased significantly by 804.18% and 303.03% respectively, and in LB-EPS, they increased by 45.16% and 172.62% respectively, and in TB-EPS, they only increased by 35.39% and 50.56% respectively, which may be due to the different changes of substances in the inner and outer EPS, and the distance between TB-EPS and the cell is closer, and SEPS and LB-EPS are farther away from the cell, and the change of substances is greatly affected by the reaction mechanism.

[0092] These results show that the photocatalytic coupling of bio-electrochemical treatment is effective in destroying the EPS of each layer of sludge, and the effect on the outer EPS is the most significant, and the effect of each part is ICPBES > PES > ICPBES-dark > BES. Overall, the effective destruction of EPS in sludge by photocatalytic coupling of bio-electrochemical system exposes the bacterial cells previously protected by EPS, making them more susceptible to increased microbial permeability.

[0093] (3) Study on the removal effect of antibiotic resistance genes ARGs and mobile genetic elements MGEs using the methods of Examples 1-5:

[0094] Changes in the abundance of total ARGs and MGEs

[0095] To investigate the role of each component in the photocatalytically coupled bioelectrochemical system, the relative abundance of ARGs in samples treated using the methods provided in Examples 1-5 was compared. DNA was extracted from sludge samples treated using the Power Soil DNA Isolation Kit (MoBio, USA). High-throughput quantitative polymerase chain reaction (HT-qPCR) was performed using the SmartChip Real-Time PCR System (Clontech, China). A total of 48 genes were detected, including 40 major ARGs, 7 MGEs, 2 insertion sequences, 2 transposase genes, and 3 integrase genes. All quantifications were performed in triplicate.

[0096] like Figure 5 As shown in (a), the total relative abundance of ARGs in RS was 0.28. After treatment with the different systems, the relative abundance of ARGs in sludge decreased compared to RS. The relative abundances of ARGs after treatment with the BES and ICPBES systems were 0.09 and 0.07, respectively. ICPBES achieved removal rates of 84.78%, 71.40%, 82.57%, and 84.28% for tetracycline-resistance genes, 71.40%, 82.57%, and 84.28% for β-lactam-resistance genes, respectively. The removal rate of MGEs reached 69.65%, indicating that the BES system combined with photocatalysis promoted the reduction of ARGs. The relative abundances of sludge ARGs in ICPBES-Dark and ICPBES-OC were 0.18 and 0.17, respectively. Comparison of these values ​​with the relative abundances of sludge ARGs after ICPBES treatment indicates that photocatalysis and extracellular electron transfer in ICPBES contributed 0.11 and 0.10, respectively. Comparing the relative abundance of ARGs in sludge treated by PES and ICPBES systems, the contribution of ICPB was calculated to be 0.15. These results indicate that photocatalysis, bioelectrochemical processes, and ICPB in the ICPBES system all contribute to the attenuation of ARGs.

[0097] The abundance of ARGs in BES-treated sludge reflects the effect of bioelectrochemical processes on the elimination of ARGs. The relative abundance of macrolide, sulfonamide, and tetracycline resistance genes decreased more significantly, decreasing by 6.41×10 -2 , 5.63×10 -2 and 1.45×10 -2 ( Figure 5 a).ereA 、 ereB 、 ermB and ermF macrolides decreased by 1.66 x 10 -2 , 5.00 x 10 -3 , 2.85 x 10 -2 and 7.63 x 10 -3 , respectively. sul1 and sul2 sulfonamides decreased by 1.27 x 10 -2 and 4.19 x 10 -2 , respectively. Among the tetracycline resistance genes, all decreased except tetC which slightly increased, with tetM decreasing most significantly by 1.29 x 10 -2 ( Figure 5 a). In addition, the relative abundances of blaOXY-1 , blaOXY-1 and cphA β-lactams decreased by 2.59 x 10 -3 , 1.69 x 10 -3 and 1.54 x 10 -3 , respectively. aacA / aphD , aadA2-1 , aadA5 and ampC aminoglycosides also slightly decreased. The relative abundances of all MGEs subtypes decreased, with IS26 and tnpA-5 decreasing by 2.26 x 10 -2 and 2.00 x 10 -3 ( Figure 5 b), respectively.

[0098] Further investigation of the differences in the relative abundances of ARGs in the BES and ICPBES systems after treatment and the effect of photocatalytic elimination of ARGs showed that the relative abundances of aminoglycosides, β-lactams and macrolides ARGs decreased by 4.46 x 10 -3 , 2.85 x 10 -4 and 8.41 x 10 -5 , respectively. In addition, the total relative abundance of MGEs decreased by 7.31 x 10 3 . Among them, ermF and mefA macrolides decreased by 1.59 x 10 -2 and 1.53 x 10 -3 , respectively. strB and sul1 sulfonamides decreased by 1.01 x 10 -3 and 4.24 x 10 -3 , respectively.​​tetQ and tetS (Tetracyclines) decreased by 1.03 x 10 -5 and 1.49 x 10 -5 respectively. Among β-lactams, except for blaTEM , cphA and ampC slightly enriched, other subtypes decreased, blaCMY , blaCTX-M and blaMOX / blaCMY disappeared in ICPBES system. aac(6')-Ib and aadA2-1 (Aminoglycosides) significantly decreased by 1.83 x 10 -3 . Among MGEs, only IS26 and intl2 increased by 2.03 x 10 -3 and 2.27 x 10 -5 respectively, other subtypes decreased, int1 and tnpA-7 decreased by 1.36 x 10 -3 and 1.20 x 10 -3 respectively. This indicated that the synergistic effect of photocatalysis and bioelectrochemical reaction had a significant elimination effect on ARGs and MGEs, especially targeting macrolide, sulfonamide and tetracycline resistance genes Figure 5 b).

[0099] Changes in EPS-related and intracellular ARGs abundance

[0100] As Figure 6 a, in ICPBES, the abundance of i-ARGs and EPS-ARGs were significantly reduced (1 order of magnitude) compared with raw sludge (RS). The total absolute abundance of EPS-ARGs ranged from 3.48 x 10 7 to 9.91 x 10 7 copies / mL, and the total absolute abundance of i-ARGs was 7.25 x 10 7LB-ARGs was the lowest (decreased by 95.05% compared with the original sludge), while TB-ARGs was the highest (decreased by 74.12% compared with the original sludge), and i-ARGs only decreased by 41.03%, indicating that the photocatalysis coupled with bioelectrochemical system mainly destroyed LB-ARGs, making ARGs migrate and transform in the cell and EPS. The reason may be that the photocatalysis coupled with bioelectrochemical system first destroyed the outer S-ARGs (ARGs in S-EPS), and degraded them, resulting in a decrease in the absolute abundance of S-ARGs; then it destroyed LB-ARGs and TB-ARGs from the outside to the inside, directly degrading them or degrading them after migrating to the outer layer; finally, it destroyed i-ARGs, releasing them from the cell to the EPS for further migration and transformation. However, due to the limited capacity of the photocatalysis coupled with bioelectrochemical system, the destruction of i-ARGs in the inner layer was less, and the destroyed i-ARGs were first released into TB-EPS. At the same time, the photocatalysis coupled with bioelectrochemical system had limited effect on the inner TB-ARGs, so TB-ARGs showed a higher abundance. In addition, the abundance of S-ARGs was higher than that of LB-ARGs, which may be due to the rate of LB-ARGs and TB-ARGs in the inner layer transferring to S-ARGs being greater than their degradation rate. For example, the total absolute abundance of aminoglycoside ARGs decreased in each layer, but S-ARGs decreased the least, and aac (6')-II was not detected in LB-EPS and TB-EPS, and the absolute abundance in S-EPS was 8.38×10 4 copies / mL; in addition, the total absolute abundance of tetracycline ARGs in S-EPS, LB-EPS, TB-EPS and the cell decreased by 19.16%, 99.55%, 95.60% and 38.71%, respectively, among which tetM 、 tetT In S-EPS, it was significantly higher than that in other layers, tetM The absolute abundance in S-EPS, LB-EPS, TB-EPS and the cell was 7.55×10 6 copies / mL, 1.69×10 5 copies / mL, 2.62×10 5 copies / mL, 2.05×10 6 copies / mL, respectively. tetT 1.14×10 6 copies / mL, 5.54×10 3 copies / mL, 5.90×10 4 copies / mL, 0 copies / mL, respectively. In addition, under the action of the photocatalysis coupled with bioelectrochemical system, the absolute abundance of ARGs in each layer of EPSaacA / aphD 、 blaCTX-M-1,3,15 、 ermB 、 ermX The degradation rates of all ARGs were more than 90%, and the degradation rates in cells were 70.49%-90.38%. In summary, the photocatalysis coupled with bioelectrochemical systems can destroy ARGs in each layer, but the migration and transformation of LB-ARGs and TB-ARGs are the most significant. And the removal effect of aacA / aphD 、 blaCTX-M-1,3,15 、 ermB 、 ermX is better than that of other genes ( Figure 6 b).

[0101] In the BES group, the absolute abundance of LB-ARGs and TB-ARGs decreased by 19.23% and 52.00% compared with RS, while i-ARGs and S-ARGs increased, indicating that the bioelectrochemical system alone can act on ARGs in EPS, but it is not enough to destroy i-ARGs, and the oxidation ability of S-ARGs formed by the migration and transformation of inner layers in EPS is also limited, so the absolute abundance of S-ARGs increases compared with the original mud, for example, the absolute abundance of tetracycline ARGs increases in S-EPS, and decreases by 88.27%, 7.01% and 9.39% in LB-EPS, TB-EPS and cells, respectively. Among them tetM 、 tetQ and tetX slightly increase in S-EPS, and decrease in other layers, tetS slightly increase in S-EPS and cells, and decrease in other layers. strB 、 sul1 、 sul2 sulfonamides slightly increase in S-EPS and cells, while decrease in other layers ( Figure 6 b). In summary, the bioelectrochemical system has no obvious effect on i-ARGs, but can cause the migration and transformation of ARGs in EPS, and shows a good removal effect on LB-ARGs and TB-ARGs.

[0102] In the PES group, compared with RS, the absolute abundance of LB-ARGs and TB-ARGs decreased by 83.54% and 30.20%, respectively, while i-ARGs and S-ARGs increased, indicating that photocatalysis can only reduce ARGs in EPS, and has no destructive effect on i-ARGs. For example, the total absolute abundance of β-lactam antibiotics decreased by 26.37%, 84.71% and 58.19% in S-EPS, LB-EPS and TB-EPS, respectively, and increased by 15.25% in cells, among which blaPSE and blaTEM slightly increase in cells, and decrease in each layer in EPS, cphASlightly increased in S-EPS and decreased in other layers. strB decreased in S-EPS and cells, sul2 Slightly increased in cells and decreased in other layers ( Figure 6 b). In summary, photocatalysis is not sufficient to destroy i-ARGs, but it can cause the migration of ARGs within the EPS, shifting LB-ARGs and TB-ARGs to S-ARGs, which increases the absolute abundance of S-ARGs. Furthermore, the photocatalytic effect on EPS-associated ARGs is stronger than that of the bioelectrochemical system.

[0103] In the ICPBES-Dark group, compared with RS, the absolute abundance of TB-ARGs and i-ARGs decreased by 87.58% and 9.04%, respectively, while the absolute abundance of LB-ARGs and S-ARGs increased. This may be because the catalyst-coupled bioelectrochemical system can destroy i-ARGs and release them into EPS, and migrate the original TB-ARGs and TB-ARGs converted from i-ARGs to the outer layer. However, since the rate of migration of some ARGs from the inner layer is greater than the degradation rate, the absolute abundance of LB-ARGs and S-ARGs increased. For example, in aminoglycosides aac(6')-Ib 、 aac(6')-II 、 aacA / aphD The absolute abundance of β-lactamase decreased in TB-EPS and cells but increased in S-EPS and LB-EPS. blaPSE 、 cphA It also decreased in TB-EPS and cells but increased in S-EPS and LB-EPS, among which blaPSE Almost undetectable in cells ( Figure 6 b). In summary, the catalyst-coupled bioelectrochemical system can destroy i-ARGs and TB-ARGs, but is insufficient to completely degrade LB-ARGs and S-ARGs that migrated to the outer layer.

[0104] Therefore, the degree of effect on total ARGs is ICPBES>ICPBES-Dark>PES>BES, and only the photocatalytic coupled bioelectrochemical system can significantly destroy i-ARGs and transfer them out of cells.

[0105] Changes in EPS-related and intracellular MGEs abundance

[0106] like Figure 7 As shown, compared with RS, the abundance of TB-MGEs in the BES group decreased by 9.93%, while that in other layers increased. tnpA-5 and tnpA-7 A decrease of 82.82% and 48.39% respectively. ISCR1The above results indicate that BES is not sufficient to destroy i-MGEs, and the destroyed TB-MGEs may migrate to LB-EPS and S-EPS, thereby increasing their abundance in the outer layer of EPS.

[0107] In the PES group, compared with the original mud, the removal effect of LB-MGEs was the most obvious, with the absolute abundance reduced by 37.14%, TB-MGEs slightly increased, and i-MGEs and S-MGEs increased significantly, indicating that the photocatalytic reaction was not enough to destroy i-MGEs, and mainly destroyed LB-MGEs. IS26 , and its absolute abundance decreased by 60.74% and 8.96% in LB-EPS and TB-EPS, respectively. intl3 In LB-EPS, it decreased by 46.62% and in TB-EPS, it disappeared. ISCR1 It disappeared in all layers, indicating that the photocatalytic reaction had no significant effect on i-MGEs and S-MGEs, but could cause the migration and transformation of LB-MGEs and some TB-MGEs.

[0108] In the ICPBES-dark group, the absolute abundances of TB-MGEs and i-MGEs decreased by 85.58% and 11.51%, respectively, compared with the original mud, but the absolute abundances of LB-MGEs and S-MGEs increased. int1 、 tnpA-7 and ISCR1 The effect was more obvious in TB-MGEs and i-MGEs. int1 The absolute abundance decreased by 84.91% and 16.27%, respectively. tnpA-7 They decreased by 95.19% and 22.56% respectively. ISCR1 The reductions in LB-MGEs, TB-MGEs, and i-MGEs were 19.38%, 83.67%, and 7.91%, respectively, indicating that the catalyst-coupled bioelectrochemical system can destroy i-ARGs and TB-ARGs, but is insufficient to completely degrade LB-ARGs and S-ARGs that migrated to the outer layer.

[0109] Therefore, the photocatalytic coupled bioelectrochemical system also has a significant effect on the migration and transformation of MGEs in each layer, and the action mechanism of its various parts is similar to that of ARGs, indicating that the photocatalytic coupled bioelectrochemical system inhibits the reproduction of ARGs by reducing extracellular MGEs.

[0110] (4) Effects of the methods of Examples 1-4 on microbial communities

[0111] Bacteria are important carriers of ARGs, and bacterial communities are key drivers of the fate of ARGs. This study observed the changes in bacterial communities in reactors under different pretreatment conditions.

[0112] The V3-V4 region of the 16S rRNA gene was amplified by PCR using universal primers 341F and 806R. The PCR products were sequenced on the Illumina Hiseq2500 platform (Novogene, China). Quality control procedures (including trimming barcodes and primers, filtering low-quality reads and chimeras) were then performed using Quantitative Insights Into Microbial Ecology (QIIME, version 1.9.1). Operational taxonomic units (OTUs) were defined using Uparse (version 7.0.1001) based on 97% identity. The classification and quantification of OTUs were performed using the Mothur software against the Silva SSU rRNA database. The Shannon index was calculated to quantify the microbial a-diversity of each sample. According to the data in Table 1, the lowest level of the Coverage index of the five groups of samples reached 0.99, indicating that the data of the microbial samples in this determination were reliable.

[0113] Table 1 The a-diversity index of microorganisms in sludge after different system treatments

[0114]

[0115] According to Table 1, in each treatment group, the Chao1 index and the Ace index were less than those of the RS group, and the Simpson index was greater than that of the RS group while the Shannon index was less than that of the RS group, indicating that the abundance and diversity of the microbial community decreased after treatment and decreased most significantly in the ICPBES, and the decrease in richness and diversity may inhibit or affect the proliferation of potential hosts.

[0116] Comparing the ICPBES-dark, PES, and BES groups, it was found that the richness of microorganisms was the highest in the ICPBES; the Shannon index and the Simpson index were also greater than those of the other treatment groups, indicating that the diversity of microorganisms was also the highest in the ICPBES. This is because the photocatalysis coupled with the bioelectrochemical system first degrades the strong pollutants into intermediate products with lower toxicity, which is conducive to the growth of the microbial community. It is confirmed that the ICPBES can indeed enhance the degradation efficiency and power generation performance of the traditional BES while having little effect on microorganisms, and to some extent, it can increase the abundance of the microbial community. In the RS group, the Chao1 index was lower than that of each treatment group, and the Simpson index was higher than that of each treatment group, indicating that after treatment by the photocatalysis coupled with the bioelectrochemical system, the richness of the microbial community increased and the diversity decreased, which may be due to the screening of potential ARGs hosts by the photocatalysis coupled with the bioelectrochemical system.

[0117] As Figure 8a is the percentage of microbial communities at the phylum level in each group. The dominant phyla mainly include the following: Proteobacteria (16.08%-23.86%), Chloroflexota (16.08%-24.34%), Bacteroidota (10.79%-15.91%), Acidobacteriota (2.94%-4.79%), Actinobacteriota (1.52%-7.06%). Their abundances were significantly different in different treatment groups. Chloroflexi is a member of some ARGs ( tetM 、 tetQ 、 tetX, sul1 ), with relative abundances decreasing by 31.75%, 33.96%, 14.12%, and 7.96% in ICPBES, BES, PES, and ICPBES-dark, respectively, compared with RS. Firmicutes, also a key host of ARGs, is considered the source of class I integrons. Its relative abundance decreased by 30.30%, 40.98%, and 14.65% in ICPBES, BES, and PES, respectively. Actinobacteriota, Acidobacteriota, Nitrospirota, Planctomycetota The relative abundances of β-catenin and β-catenin in ICPBES were significantly reduced by 65.28%, 21.19%, 61.30%, and 11.94%. Proteobacteria, Bacteroidota In summary, the photocatalytic coupled bioelectrochemical system significantly changed the microbial community structure at the phylum level, among which Chloroflexi, Firmicutes, Actinobacteriota, Acidobacteriota, Nitrospirota, Planctomycetota The gate has an inhibitory effect on Proteobacteria, Bacteroidota The door is for promotion.

[0118] like Figure 8 As shown in b, Anaerolineae, Polyangia, Verrucomicrobiae, Alphaproteobacteria, Planctomycetia As the dominant bacteria at the class level, their relative abundances in ICPBES decreased by 32.73%, 6.67%, 4.61%, 41.77% and 30.29% respectively compared with RS, while they decreased by 4.61%-76.40% in other groups. Gammaproteobacteria and Bacteroidia Gang increased by 9.24% and 48.76%, 78.59% and 48.16%, 73.40% and 19.47%, 42.82% and 23.12% in the ICPBES-dark, ICPBES, BES, and PES groups, respectively.

[0119] Microbial community analysis at the genus level also showed similar trends. OLB14 、 PHOS-HE28 、 UBA12294 、 Nannocystis 、 PromineofilumThe relative abundance of the genus in the ICPBES group decreased by 42.51%, 3.37%, 19.69%, 62.09%, and 60.00%, respectively; OLB14 and Promineofilum The relative abundance of the genus in the ICPBES-Dark, BES, and PES groups decreased by 2.23% and 56.70%, 17.53% and 51.02%, and 83.83% and 49.49%, respectively. In the ICPBES group EnvOPS12 , OLB15 , JJ008 The relative abundance of the genus decreased by 31.62%, 37.06%, and 22.61%, respectively, while M3007 , Accumulibacter The relative abundance of the genus increased by 41.51% and 124.13%. M3007 and Accumulibacter The relative abundance of the genus also increased in the ICPBES-Dark and PES groups. The above results indicate that ICPBES had the most significant impact on the microbial community structure at the genus level, inhibiting OLB14 , PHOS-HE28 , UBA12294 , Nannocystis , Promineofilum and promoting the relative abundance of the genus, which was consistent with the changes in the microbial community at the phylum level (c). M3007 , Accumulibacter Figure 8 c).

[0120] (5) Effects on metabolism

[0121] Previous studies have shown that the spread of ARGs is highly dependent on related biological pathways. To understand the different effects of different treatments on functional modules, the functions of the gene KO and KEGG databases were predicted by PICRUSt 2. The expression levels of genes related to the type IV secretion system (T4SS) and the two-component system (TCs) and bacterial secretion system were assessed by KEGG orthologs, and the metabolic function results based on KEGG are shown in Figure 9 b. As shown in Figure 9 a, 21 metabolic pathways were significantly different in each treatment group compared to RS. Compared to RS, the abundance of membrane transport, signal transduction, and infectious diseases: bacterial in each group decreased, except for the BES group. In addition, carbohydrate metabolism, other amino acid metabolism, and translation decreased in each treatment group. This indicates that the photocatalysis coupled bioelectrochemical system may exert stress on the microorganisms, causing them to shift in these reactors. The following further investigates the changes in genes related to membrane transport, signal transduction, and infectious diseases: bacterial.

[0122] Two-component regulatory system

[0123] ​The two-component system is an important mechanism for bacteria to perceive and respond to environmental signals. It can transmit cell signals through the cytoplasm to the bacterial nucleoid to regulate gene expression and mediate drug resistance. The relative abundance of key genes in the two-component regulatory system decreased in all groups, and was most obvious in the ICPBES group. Figure 9 As shown in (c), the relative abundance of rstA decreased by 22.22%, 55.56%, 11.11%, and 33.33% in the ICPBES-dark, ICPBES, BES, and PES groups, respectively. The relative abundances of chpB, chpC, and chpE, which are involved in pili and flagella synthesis, decreased by 25%, 20%, and 13.56%, 25%, 10%, and 44.07%, 37.50%, 50%, and 18.64%, 12.50%, and -20%, and 23.73%, respectively. The relative abundances of degU and desR, which are involved in cell communication, decreased by 20%, 10.71%, 40%, and 14.29%, 33.33%, and 28.57%, and 26.67%, and 35.71%, respectively. In summary, the photocatalytic coupled bioelectrochemical system inhibits transduction of cell signals and gene expression by reducing the abundance of genes in the two-component regulatory system, thereby hindering the proliferation of ARGs.

[0124] Bacterial secretion system

[0125] Bacterial secretion systems can not only transfer proteins across cell membranes, but also transfer DNA, playing an important role in HGT. Figure 9 As shown in Figure c, their relative abundance decreased in all treatment groups, with the most significant decrease in the ICPBES group. Compared with RS, secD and secE decreased by 67.02% and 93.26%, respectively, while tatA and tatC decreased by 11.56% and 9.62%, respectively. Furthermore, the downregulation of bacterial secretion systems is consistent with the lower abundance of MGEs.

[0126] Type IV secretion system

[0127] The type IV secretion system is considered to be an important mechanism for the transfer of genes and substances between microorganisms. It enables the transfer of genetic material between microorganisms through the construction of channels and participates in the horizontal transfer of ARGs. The relative abundance of genes encoding T4SS (VirB1-6, Vir8-11 and VirD4) in the original mud was 8.26×10 -4 , in each treatment group was 5.64×10 -4 -8.35×10 -4 ( Figure 9c) VirD4, which plays a key role in substrate binding; VirB1, which facilitates DNA release; VirB2, VirB3, and VirB5, which are responsible for genetic material exchange; VirB4 and VirB11, which are involved in energy synthesis; VirB6, VirB8, and VirB10, which facilitate ARGs horizontal transfer; the total relative abundance decreased by 13.46%, 6.38%, 33.06%, and 1.46% in the ICPBES-dark, ICPBES, BES, and PES groups, respectively. Thus, ICPBES inhibited the expression of each module gene of T4SS, thereby weakening the spread and proliferation of ARGs in the system.

[0128] Obviously, the photocatalytic coupling of the bioelectrochemical system helps to reduce the genetic expression levels of the two-component regulatory system, bacterial secretion system, and type IV secretion system, thereby inhibiting the spread of ARGs.

[0129] (6) Microorganisms, metabolic pathways, EPS structure, MGEs, and ARGs analysis

[0130] Figure 10 The network analysis shown describes the process by which microorganisms affect the EPS structure through metabolic pathways, as shown in Figure 10 In ICPBES, EPS proteins and polysaccharides significantly affect the movement of ARGs and MGEs, and ARGs and MGEs are significantly correlated with proteins in S-EPS, LB-EPS, and TB-EPS and polysaccharides in S-EPS, especially proteins in S-EPS (p < 0.05, r > 0.95). This indicates that ICPBES can change the composition of EPS in sludge, thereby changing the distribution of ARGs and MGEs.

[0131] The microbial community also showed a similar trend, being significantly correlated with polysaccharides in S-EPS and LB-EPS and TB-EPS and proteins in S-EPS and TB-EPS, for example, OLB14 and significantly negatively correlated with proteins in S-EPS and LB-EPS and polysaccharides in S-EPS and TB-EPS (P < 0.05). Rubrivivax , UBA1229 4 was also related to proteins and polysaccharides in EPS. At the same time, EPS-ARGs were also related to OLB14 , Accumulibacter , Sulfuritalea , Azonexus , Rubrivivax

[0132] In addition, Promineofilum significantly negatively correlated with the bacterial secretion system; Sulfuritalea , Rhodoferax related to T4SS, which was related to intracellular ereA ,​tetS, intl3 、 ISCR1 , LB- ereA , LB- intl3 , S- ISCR1 related, indicating that ICPBES affected the permeability of ARGs or MGEs through the cell membrane by affecting T4SS, thereby inhibiting the HGT of ARGs; TCs were significantly related to OLB14 、 Accumulibacter 、 Azonexus significantly related (P < 0.05), and Accumulibacter polysaccharides in TB-EPS, tetO 、 strB LB-EPS tetQ 、 tetS 、 intl3 and S- intl3 related, indicating that Accumulibacte r is the key node connecting sludge EPS structure, metabolic pathways and EPS-ARGs, plays a mediating role in ICPBES, and makes environmental factors affect the expression of EPS-ARGs.

[0133] Therefore, the photocatalysis coupled bioelectrochemical system can change the EPS structure by changing the core microbial strains involved in the two-component system, T4SS and bacterial secretion system, thereby changing the distribution of ARGs in the photocatalysis coupled bioelectrochemical system.

[0134] (7) Determine the potential environmental impact of ARGs

[0135] As mentioned above, the changes in EPS structure, microbial state, bacterial diversity, dominant microbial community, metabolic pathway, MGEs and other parameters will affect the changes of ARGs in ICPBES. Therefore, partial mantel test was used to show the relationship between ARGs and diversity parameters (P < 0.05). Figure 11 As shown in Figure 11 , ARGs were significantly related to proteins and polysaccharides, two-component systems, and Chao1, Simpson, ACE index, dominant microbial community and two-component system.

[0136] In addition, there is an inherent correlation between each factor. Proteins and polysaccharides are positively correlated with MGEs, Simpson index and T4SS. Bacterial secretion system is significantly related to Chao1, Shannon, ACE index and two-component system (r > 0.6). Dominant microbial community is related to Chao1, Shannon and ACE index. Therefore, these influencing factors work together to achieve the removal of ARGs.

[0137] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for removing antibiotic resistance genes from sludge using a photocatalytic coupled bioelectrochemical system, characterized in that: The following steps are involved: S1: Preparation of Kg-C3N4 photocatalyst: The ground g-C3N4 catalyst was taken and KCl was added for mixing and grinding. The mass ratio of the g-C3N4 catalyst to KCl was 10:

1. After the two were mixed, they were calcined at high temperature and ground to obtain Kg-C3N4 photocatalyst; S2: Preparation of photocatalytic anode for photocatalytic coupled bioelectrochemical system: Take Kg-C3N4 photocatalyst, add 5% Nafion solution and 1 mL of isopropanol, mix thoroughly by ultrasonication, and coat the mixture on a nickel foam support. After drying, a nickel foam electrode loaded with Kg-C3N4 photocatalyst is obtained. The volume ratio of Kg-C3N4 photocatalyst mass to Nafion solution and isopropanol is: 400 mg:2 mL:1 mL. A nickel foam loaded with Kg-C3N4 photocatalyst is used as an anode, and a pretreated carbon brush is used as a cathode. The two are installed on a biofilm culture reactor. A cathode solution is introduced into the cathode chamber of the reactor, and a mixed solution containing anode sludge is introduced into the cathode chamber of the reactor. The anode and cathode are electrically connected to form a loop. Electric energy is generated by the chemical potential difference of the system itself, and a biofilm is formed inside the pores of the nickel foam loaded with Kg-C3N4 photocatalyst. After the maximum voltage measured stabilizes, the acclimation of the biofilm is completed, and a photocatalytic anode of a photocatalytic coupled bioelectrochemical system is prepared. S3: Removal of antibiotic resistance genes from treated sludge mixed liquor by photocatalysis coupled with bioelectrochemical system: The photocatalytic anode prepared in S2 is connected to a single-chamber photocatalytic electrochemical reactor, and the modified air cathode is connected to the single-chamber photocatalytic electrochemical reactor as a cathode. The photocatalytic anode and the modified air cathode are connected to an external circuit to form a loop to form a photocatalytic coupled bioelectrochemical system. The diluted sludge mixture to be treated is introduced into the single-chamber photocatalytic electrochemical reactor, and a light source is used to irradiate the top of the photocatalytic anode. The antibiotic resistance genes in the sludge mixture to be treated are removed by the photocatalytic coupled bioelectrochemical system.

2. The method for removing antibiotic resistance genes in sludge using a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: The g-C3N4 catalyst was prepared by adding 15 g of urea to a 50 mL alumina crucible with a lid, calcining the crucible at 550°C for 3 h in a muffle furnace at a heating rate of 5°C / min. After the product was naturally cooled, it was washed with water, dried, and fully ground into powder to obtain the g-C3N4 catalyst.

3. The method for removing antibiotic resistance genes in sludge using a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: In S2, the mixed solution containing anode sludge is a mixture of sludge from a sewage treatment plant and sodium acetate solution, and the content of the sludge is 2.0 g / L.

4. The method for removing antibiotic resistance genes in sludge using a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: In S3, the diluted sludge mixed liquor to be treated is: a mixed liquor formed by diluting the sludge to be treated with 0.1 mol / L glucose solution, wherein the pH value of the sludge to be treated is 7.58±0.01, the moisture content is 98.64±0.06%, the total suspended solids TSS is 8.07±0.03 g / L, the volatile suspended solids VSS is 3.65±0.02 g / L, the soluble chemical oxygen demand SCOD is 284±27 mg / L, and the total organic carbon TOC is 105±17 mg / L.

5. The method for removing antibiotic resistance genes in sludge using a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: In S1, the high-temperature calcination conditions are as follows: after mixing the g-C3N4 catalyst with KCl, the mixture is placed in a muffle furnace and calcined at 500 °C for 3 h with a heating rate of 2.5 °C / min.

6. The method for removing antibiotic resistance genes in sludge using a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: In S2, the pretreated carbon brush is a carbon brush pretreated with an acetone solution.

7. The method for removing antibiotic resistance genes in sludge using a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: In S2, the cathode solution is a mixed solution of 0.2 mol / L potassium dihydrogen phosphate and 0.1 mol / L potassium ferrocyanide.

8. The method for removing antibiotic resistance genes in sludge using a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: In S2, the maximum voltage plateau measured was 1 month after the biofilm was formed, and the voltage reached 0.6-0.7 V.

9. The method for removing antibiotic resistance genes in sludge using a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: In S3, the preparation method of the modified air cathode is: using carbon felt as the material of the air cathode, weighing 10g of 60% PTFE, adding water to 120g, and applying it on one side of the carbon felt as a carbon base layer, drying it at room temperature for two hours, and then placing it in a 370°C oven and heating it for 30 minutes, and then applying four layers of 60% PTFE emulsion on the carbon base layer as an air diffusion layer, and drying each layer in a 370°C oven for 10-15 minutes; then weighing 318mg of activated carbon and 13.25mg of conductive carbon black and adding 1.48ml of isopropyl alcohol and 1ml of Nafion reagent, mixing them evenly, and then applying them on the other side of the carbon felt as a catalytic layer, the other side being the side in contact with the electrolyte, and drying it in an oven at 60°C for 6h.

10. The method for removing antibiotic resistance genes in sludge using a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: In S2, the cathode solution and the mixed solution containing anode sludge are replaced every three days.

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