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

Abstract

The invention discloses a method for removing antibiotic resistance genes in sludge by a photocatalysis coupled bioelectrochemical system. The method comprises the following steps: S1, preparing a K-g-C3N4 photocatalyst; s2, preparing a photocatalytic anode of the photocatalytic coupling bioelectrochemical system; S3, removing antibiotic resistance genes in the sludge mixed liquid to be treated through the photocatalytic coupling bioelectrochemical system; according to the method for removing the antibiotic resistance genes in the sludge through the photocatalysis coupled bioelectrochemical system, the abundance of the antibiotic resistance genes is effectively reduced after treatment of the ICPBES system, and the removal rates of tetracyclines, sulfonamides, macrolides and beta-lactam resistance genes reach 84.78%, 71.40%, 82.57% and 84.28% respectively; the ecological pollution risk of antibiotic resistance genes is reduced; the ICPBES system reduces the abundance of the mobile genetic element, the removal rate reaches 69.65%, further propagation of the antibiotic resistance gene in the environment is inhibited, and the pollution propagation risk of the antibiotic resistance gene is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of removing antibiotic resistance genes in sludge, and in particular to a method for removing antibiotic resistance genes in sludge by using a photocatalytic coupled bioelectrochemical system. Background Art

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

[0003] Extracellular polymers (EPS) in the sludge of sewage treatment plants are the main components of activated sludge flocs. EPS directly covers the outside of the cell membrane. The amount and composition of EPS will affect the surface characteristics of activated sludge flocs, and then affect the adsorption and flocculation performance of activated sludge. Sludge is mainly composed of two parts: cell body and EPS. EPS is mainly composed of protein and polysaccharide. Polysaccharide molecules are rich in hydrophilic functional groups such as hydroxyl and carboxyl groups. They can form a stable and elastic network structure through adsorption and bridging. Cross-linking and fixation of free cells is beneficial to sludge flocculation. Proteins and lipids can maintain the stability of floc structure and improve the biological flocculation of sludge accordingly. These substances form a compact and dense network structure through electrostatic force, hydrogen bonding, ion attraction, biochemistry and other effects, which can serve as a protective layer for microorganisms to resist the invasion of adverse factors such as external heavy metals and toxic compounds. Antibiotic resistance genes exist in large quantities in EPS. EPS absorbs a large number of ARGs through bridging effect, hydrophobic effect and entanglement, accounting for up to 74.2% of the total ARGs. In addition, some resistance genes of sludge are also distributed in the genetic material of bacteria. Since resistance genes in sewage treatment plant sludge exist in large quantities in EPS, EPS wraps ARGs to form a high molecular polymer structure that is difficult to degrade. The removal of these 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. Therefore, it is necessary to provide a method that can efficiently reduce sludge ARGs to solve this problem. Summary of the invention

[0005] The present invention provides a method for removing antibiotic resistance genes in sludge by using a photocatalytic coupled bioelectrochemical system to solve the above problems.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A method for removing antibiotic resistance genes in sludge by using a photocatalytic coupled bioelectrochemical system comprises the following steps:

[0008] S1: Preparation of Kg-C3N4 photocatalyst:

[0009] The ground g-C3N4 catalyst was taken, KCl was added for mixed grinding, the mass ratio of the g-C3N4 catalyst to KCl was 10:1, the two were mixed, high-temperature calcination and grinding were performed to obtain Kg-C3N4 photocatalyst;

[0010] S2: Preparation of photocatalytic anode for photocatalytic coupled bioelectrochemical system:

[0011] Take Kg-C3N4 photocatalyst, add 5% Nafion solution and 1mL isopropanol, mix by ultrasonication and coat on the nickel foam carrier, and dry to obtain the nickel foam electrode loaded with Kg-C3N4 photocatalyst; the mass ratio of Kg-C3N4 photocatalyst to Nafion solution and isopropanol is: 400mg:2mL:1mL;

[0012] A Kg-C3N4 photocatalyst loaded nickel foam 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 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 the cathode are electrically connected to form a loop, and electrical energy is generated through the chemical potential difference of the system itself. A biofilm is formed inside the pores of the Kg-C3N4 photocatalyst loaded nickel foam. After the maximum voltage measured is stable, the domestication of the biofilm is completed, and a photocatalytic anode of a photocatalytic coupled bioelectrochemical system is prepared.

[0013] S3: Removal of antibiotic resistance genes from the treated sludge mixed liquor by photocatalytic coupled bioelectrochemical system:

[0014] 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 to remove the antibiotic resistance genes in the sludge mixture to be treated through the photocatalytic coupled bioelectrochemical system.

[0015] Furthermore, the preparation method of the g-C3N4 catalyst is as follows: take 15g of urea, add it into a 50mL alumina crucible with a lid, place it in a muffle furnace and roast it at 550°C for 3h, with a heating rate of 5°C / min, and after the product is naturally cooled, it is washed with water, dried, and fully ground into powder to obtain the g-C3N4 catalyst.

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

[0017] Furthermore, 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 mpl / L glucose solution, wherein the pH value of the sludge to be treated is 7.58±0.01, the water 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] Furthermore, in S1, the high temperature calcination conditions are as follows: after the g-C3N4 catalyst is mixed with KCl, it is placed in a muffle furnace and calcined at 500°C for 3h with a heating rate of 2.5°C / min.

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

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

[0021] Furthermore, in S2, the maximum voltage steady state measured is 1 month after the biofilm is formed, and the voltage reaches 0.6-0.7V.

[0022] Furthermore, 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 heating it in a 370°C oven for 30min, 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 10-15min; then weighing 318mg of activated carbon and 13.25mg of conductive carbon black and adding 1.48ml of isopropanol and 1mL of Nafion reagent, mixing evenly, and applying it on the other side of the carbon felt (the side in contact with the electrolyte) as a catalytic layer, and drying it in an oven at 60°C for 6h.

[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 by 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, the bacterial secretion system, and the type ⅵ secretion system, thereby reducing ARGs, or can affect the core microbial strains related to metabolism (Promineofilum, Piscinibacter, Accumulibacter), affect the EPS structure and thus change the distribution of ARGs in EPS, causing AGRs to migrate to the outer layer of the cell, 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 briefly introduces 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 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 The concentration changes of polysaccharides and proteins in EPS after treatment with different systems (4(a) is the concentration change diagram of polysaccharides (a), and 4(b) is the concentration change diagram 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 The absolute abundance results of various ARGs in sludge after treatment by different systems (6(a) is the total absolute abundance accumulation diagram of various ARGs in sludge after treatment by different systems, and 6(b) is the absolute abundance heat map);

[0036] Figure 7 It is the absolute abundance heat map of various MGEs in the sludge after treatment by different systems of the present invention;

[0037] Figure 8 Figure 8 is the relative abundance diagram of microbial communities in different treatment groups of the present invention (8(a) is the phylum level; 8(b) is the level; 8(c) is the genus level)

[0038] Fig. 9 The expression results of key metabolic pathways related to ARGs propagation of the present invention (9(a) is the relative abundance of KO number (%); 9(b) is the metabolic function based on KEGG; 9(c) is the typical gene expression in ARGs propagation-related metabolic pathways);

[0039] Fig.10 The network diagram of ARGs and microbial communities, metabolic pathways, and EPS structures (the network interactions of microorganisms (green circles), metabolic pathways (blue circles), EPS structures (orange circles), and ARGs (pink circles) in the SEPS (S), LB-EPS (L), and TB-EPS (T) of each group);

[0040] Fig.11 Mantel test results for ARGs (ARGs, MGEs, EPS structure, bacterial diversity, bacterial community and metabolic pathways).

[0041] In the figure: 1. Photocatalytic anode; 2. Modified air cathode; 3. External resistor; 4. Xenon lamp light source 5. Reaction chamber; 6. Kg-C3N4 catalyst; 7. Electrochemically active bacteria. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] The principle involved in the present invention:

[0044] K doping improves the photoelectric properties of g-C3N4. After doping, Kg C3N4 exhibits a lower band gap energy, leading to higher visible light absorption efficiency ( Figure 3 a), increasing the interlayer distance, which facilitates charge transfer during photocatalysis ( Figure 3 b) and reduced the emission peak in the PL spectrum, indicating that the electron-hole recombination rate is reduced ( Figure 3 c). In the photocatalytic coupled BES system (ICPBES), Kg-C3N4 catalyst and biofilm are simultaneously loaded onto the nickel foam porous anode to construct a photocatalytic anode. The enlarged image of the photocatalytic anode is shown in Figure 2 As shown, Figure 2 6 is the Kg-C3N4 catalyst and 7 is the electrochemically active bacteria. The photocatalytic anode generates electrons through two pathways and transfers them to the anode, which are then transferred to the cathode through an external circuit. Under visible light irradiation, the Kg-C3N4 photocatalyst loaded on the surface of the nickel foam anode is excited to generate holes and photoelectrons (Equation (1)). These holes react with water to generate ·OH (Equation (2)), and the generated electrons are transferred to the cathode, reducing the electron-hole recombination rate and improving the photocatalytic efficiency. In addition, the electrochemically active bacteria present in the biofilm in the pores of the nickel foam, such as Rhizoctonia solani, Pseudomonas aeruginosa, Escherichia coli, Enterococcus, Geobacter, Arcobacter, Clostridium and Geotrichum, oxidize organic substrates through 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). The electrons are then transferred to the cathode through an external circuit, reacting with oxygen to generate O2-, and further gaining electrons to form H2O2 (Equation (4)).

[0045] Anode: g-C3N4+h 0 →e - +h + (1)

[0046] h + +H2O→H + + OH (2)

[0047] cathode:

[0048]

[0049] H202+e - → OH+OH - (5)

[0050] Oxidizing substances such as O2 are produced during the anode and cathode reactions. -·, H2O2 and ·OH can destroy the structure of potential host bacteria (Bacteria, Accumulibacter, Nannocystis, Burkholderiaceae, Promineofilum, Rhodoferax), resulting in the release of intracellular antibiotic resistance genes (iARGs) in sludge, which leads to the degradation of iARGs and the enrichment of ARGs in EPS. The released iARGs further migrate and transform in each layer of EPS. iARGs are significantly correlated with intracellular mobile genetic elements (iMGEs) and have the same potential hosts. The massive degradation of MGEs and the death of host bacteria will block the horizontal transfer of iARGs between bacteria. At the same time, the direct oxidation of intracellular DNA (iDNA) and extracellular DNA (e-DNA) by reactive oxygen species (ROS) generated by photocatalysis is also the main reason for the degradation of iARGs and ARGs in EPS. These oxidative free radicals can also directly act on ARGs in EPS and MGEs in EPS to cause 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 gene expression levels of 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), affect the EPS structure and thus change the distribution of ARGs in EPS. For example, network analysis found that Promineofilum was positively correlated with the bacterial secretion system and the two-component system, and was related to polysaccharides, ereA, tetO, tetS, strB, and intI3 in the inner EPS (TB-EPS); OLB14 and Piscinibacter were also related to the two-component system, and OLB14 was significantly negatively correlated with soluble extracellular polymers (S-EPS), proteins in loosely bound extracellular polymers (LB-EPS), and polysaccharides in S-EPS, while Piscinibacter was positively correlated with S-EPS, proteins in LB-EPS, and polysaccharides in S-EPS; Accumulibacter was significantly negatively correlated with the two-component system, and was positively correlated with polysaccharides, tetO, and strB in LB-EPS, tetQ, tetS, and intl3 in LB-EPS, and intl3 in S-EPS. As a bacillus, Accumulibacter may play a mediating role in the photocatalytic coupled bioelectrochemical system, allowing environmental factors to affect the expression of EPS-related ARGs. In summary, ICPBES 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.The biofilm culture reactor used in the step of forming a biofilm inside the pores of the Kg-C3N4 photocatalyst nickel foam loaded in the present application, namely the BES reactor, is composed of two identical 216mL organic glass cubes (6 cm×6 cm×6 cm), which serve as the cathode chamber and the anode chamber of the BES reactor respectively and use a proton exchange membrane as a diaphragm, and the reaction conditions are: the anode uses a nickel foam electrode loaded with 400mg Kg-C3N4, the cathode is inserted with a carbon brush pretreated with an acetone solution, the electrodes are connected with a titanium wire with a diameter of 0.3mm, an external load of 100Ω resistance, and a voltage of 14V; when removing antibiotic resistance genes in the sludge mixed liquor to be treated by a photocatalytic coupled bioelectrochemical system, use the following. Figure 1 The pilot device shown in the figure is used for treatment, i.e., a single-chamber reactor. The pilot device includes a photocatalytic anode 1, a modified air cathode 2, an external resistor 3, a xenon lamp light source 4, and a reaction chamber 5. When treating sludge, the sludge is diluted with a 0.1 mpl / L glucose solution and then injected into the reaction chamber. After the voltage is stabilized, a photocatalytic experiment is performed. The illumination group uses a xenon lamp to irradiate the top of the photocatalytic anode with a light intensity of 4500 Lux. After nine hours, the sludge sample is taken out from the reaction chamber of the BES. The reaction is carried out at room temperature.

[0051] The sludge treated in this application is the sludge after ARGs in sewage are adsorbed. The activated sludge process is to continuously introduce air into wastewater, and after a certain period of time, aerobic microorganisms will reproduce to form sludge-like flocs. The treatment object of the technology of this application is the sludge in the sewage treatment plant, so the sludge in this application is the activated sludge flocs after ARGs in sewage are adsorbed.

[0052] Embodiment 1:

[0053] A method for removing antibiotic resistance genes from sludge by photocatalytic coupled bioelectrochemical system, denoted as ICPBES, comprises the following steps:

[0054] S1: Preparation of Kg-C3N4 photocatalyst:

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

[0056] S12: Preparation of Kg-C3N4 photocatalyst: weigh 2g of the g-C3N4 catalyst ground in step S11, add 0.2g of KCl, mix and grind for 30min, and after fully grinding, put it into a muffle furnace and calcine it at 500℃ for 3h with a heating rate of 2.5℃ / min to obtain Kg-C3N4 photocatalyst;

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

[0058] S21: Preparation of anode: coating the prepared Kg-C3N4 photocatalyst onto the nickel foam anode: cutting the nickel foam into required sizes, ultrasonically cleaning with acetone, alcohol and ultrapure water for ten minutes respectively, and drying in an oven to obtain a Kg-C3N4 photocatalyst loaded nickel foam electrode for standby use;

[0059] S21: Preparation of nickel foam electrode loaded with Kg-C3N4 photocatalyst:

[0060] Weigh 400 mg of Kg-C3N4 photocatalyst and put it into a beaker, add 2 ml of 5% Nafion solution and 1 mL of isopropanol, ultrasonicate for ten minutes, mix evenly, coat it on the cut nickel foam, and dry it in an oven to obtain a Kg-C3N4 photocatalyst loaded nickel foam electrode;

[0061] S22: Preparation of modified air cathode:

[0062] Carbon felt is used as the material for the air cathode. 10g of 60% PTFE is weighed, water is added to 120g and coated on one side of the carbon felt as a carbon-based layer. After drying at room temperature for two hours, it is placed in a 370°C oven and heated for 30 minutes. Four layers of 60% PTFE emulsion are then applied on the carbon-based layer as an air diffusion layer. Each layer is dried in a 370°C oven for 10-15 minutes. Then 318mg of activated carbon and 13.25mg of conductive carbon black are weighed, 1.48ml of isopropanol and 1ml of Nafion reagent are added, mixed evenly, and coated on the other side of the carbon felt (the side in contact with the electrolyte) as a catalytic layer, and dried in a 60°C oven for 6h.

[0063] S23: Acclimation of photocatalytic anode biofilm:

[0064] A double-chamber reactor configuration was used to construct a reactor dedicated to anode biofilm cultivation. The anode used a prepared nickel foam electrode, and the cathode was inserted with a carbon brush pretreated with acetone solution (the size of the carbon brush was 3 cm × 5 cm × 15 cm; the treatment method of the carbon brush was: put the carbon brush in a beaker containing acetone solution (13.5 mol / L), cover the beaker mouth with tin foil, and ultrasonicate for 10 min); the electrodes were connected by titanium wire with a diameter of 0.3 mm, and an external load of 100 Ω resistance; the anode chamber was inoculated with a mixture of sludge from a sewage treatment plant and sodium acetate solution (2.0 g / L), and the cathode solution was a mixed solution of 0.2 mol / L potassium dihydrogen phosphate and 0.1 mol / L potassium ferrocyanide as an electron acceptor. The output voltage value was recorded every day with a digital multimeter. The anode sludge and cathode liquid were replaced every three days. After about one month, the maximum voltage reached 0.6-0.7V, at which time the biofilm acclimation was completed, and a biofilm was formed inside the pores of the Kg-C3N4 photocatalyst nickel foam, thus preparing a photocatalytic anode of a photocatalytic coupled bioelectrochemical system.

[0065] S3: Removal of antibiotic resistance genes from the treated sludge mixed liquor by photocatalytic coupled bioelectrochemical system:

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

[0067] Embodiment 2:

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

[0069] A dual-chamber bioelectrochemical system (dual-chamber reactor) was used. The anode was the nickel foam anode without catalyst coating prepared in Example 1, and the cathode was the modified air cathode same as that in Example 1. After the anode biofilm was tamed according to Example 1, the sludge was diluted with 0.1 mpl / L glucose solution and injected into the anode reaction chamber. After the voltage was stabilized again, the sludge sample was taken out.

[0070] Embodiment 3:

[0071] A method for removing antibiotic resistance genes from sludge by a catalyst-coupled bioelectrochemical system, also named ICPBES-Dark, comprises the following steps:

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

[0073] Embodiment 4:

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

[0075] A dual-chamber catalyst-coupled bioelectrochemical system (dual-chamber reactor) was used, and the anode was the photocatalytic anode prepared in Example 1. After the anode biofilm was domesticated according to Example 1, the domesticated reactor was changed to a single-chamber catalyst-coupled bioelectrochemical system (single-chamber reactor), and the original cathode was replaced with a modified air cathode. The sludge was diluted with a 0.1 mpl / L glucose solution and injected into the reaction chamber, and the external circuit was disconnected to conduct the experiment. The reactor was placed on a magnetic stirrer, and a xenon lamp was used to irradiate the upper side of the photocatalytic anode. After nine hours, the sludge sample was taken out from the reaction chamber.

[0076] Example 5

[0077] A photocatalytic coupled bioelectrochemical system without biofilm connection for repairing antibiotic resistance genes in sludge, also named PES in this embodiment, comprises the following steps:

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

[0079] Performance Test:

[0080] (1) The Kg-C3N4 photocatalytic material prepared in the example was subjected to UV / visible diffuse reflectance (UV-Vis DRS) test, XRD (X-ray diffraction) test and EPR spectroscopy characterization, and pure g-C3N4 was used as a comparison. The results are as follows: Figure 3 As shown:

[0081] like Figure 3 a is the UV / visible diffuse reflectance spectra of g-C3N4 and Kg-C3N4. The DRS conversion Tacu diagram found that the UV-vis adsorption of Kg-C3N4 was red-shifted compared with pure g-C3N4. The band gap of Kg-C3N4 was 2.78eV, which was lower than the band gap of pure g-C3N4 (2.79eV). Therefore, the visible light absorption range of Kg-C3N4 was wider, and Kg-C3N4 had higher visible light absorption efficiency, improved light utilization, and higher photocatalytic activity than g-C3N4. Moreover, the luminescence intensity of Kg-C3N4 was also lower, so the recombination rate of electrons and holes was slowed down, the carrier lifetime was prolonged, and the interlayer spacing of Kg-C3N4 was increased, which was conducive to the transfer of charges. Therefore, the photocatalytic efficiency was improved. Under the irradiation of the xenon lamp, Kg-C3N4 was excited to generate electrons and holes. The holes reacted with water to generate ·OH, and the electrons were transferred to the cathode through the external circuit of the BES system, and O2 was further generated at the cathode. - and H2O2, these oxidative free radicals act directly on extracellular ARGs or on host cells.

[0082] In addition, the XRD spectra of g-C3N4 and Kg-C3N4 were characterized (e.g. Figure 3 b), an obvious diffraction peak was found at 27° in pure g-C3N4, and the diffraction peak in Kg-C3N4 shifted slightly (from 27.900 to 27.618) with the addition of K, indicating that the interlayer distance of g-C3N4 increased, which is beneficial to improve the charge transfer in the photocatalytic reaction. Figure 3 c is the photoluminescence PL spectra of Kg-C3N4 and g-C3N4 excited at 400nm at room temperature. The luminescence intensity of pure g-C3N4 is higher than that of Kg-C3N4, while the position of the luminescence peak is the same. Because the lower peak intensity indicates a lower photobiorecombination rate, Kg-C3N4 can effectively hinder the recombination rate of photogenerated electron-hole pairs relative to pure g-C3N4; Figure 3 d is the XPS spectrum of g-C3N4 and Kg-C3N4. It can be seen from the figure that K-gC3N4 has a K peak, indicating that K is successfully doped.

[0083] In order to further verify the degradation mechanism of ARGs by kg-C3N4 in the photocatalytic degradation system, EPR spectroscopy characterization was used to explore and verify that O2 -With DMPO as the scavenger, EPR spectra were performed in aqueous solution and methanol solution to detect the presence of DMPO-·OH and DMPO-·O2 - Signal peak. Figure 3 As shown in e and 3f, no signal peaks were detected under dark conditions, but after 10 min of visible light irradiation, obvious DMPO-·OH and DMPO-·O2 were detected. - The characteristic signal peak indicates that the catalytic effect of light stimulates Kg-C3N4 to produce a large number of oxidative free radicals.

[0084] (2) Investigating the effects of the methods of Examples 1-4 on polysaccharides and proteins in extracellular polymers (EPS) of sludge:

[0085] The sludge samples treated by the methods of Examples 1-4 were centrifuged to extract S-EPS, LB-EPS (TB-EPS (tightly bound cell aggregates)) in layers, as follows: 25 mL of sludge was taken and centrifuged for 15 min (4 ° C, 4000 r / min -1 ), the supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was S-EPS; the remaining sediment was supplemented with 0.9% NaCl to 25 mL, and centrifuged for 20 min (4 ° C 8000 r min -1 ), the supernatant was filtered through a 0.45 μm filter membrane to extract LB-EPS; the sediment was reused and supplemented with 0.9% NaCl solution to 25 ml, and then the mixture was stirred at 4000 r / min -1 , centrifuged at 4°C for 25 min, and the supernatant was filtered through a 0.45 μm filter membrane to extract TB-EPS. The sludge sample after EPS extraction was then centrifuged at 10,000 rpm for 10 min at 4°C, and iARGs were extracted from the residual particles.

[0086] The effect of the photocatalytic coupled bioelectrochemical system on the EPS characteristics of biofilm was investigated by detecting the content and composition of EPS. The EPS content is equal to the sum of protein and polysaccharide. The polysaccharide content was determined by the phenol-concentrated sulfuric acid method, and the protein content was determined by the Coomassie brilliant blue method.

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

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

[0089] In the PES group of Example 5, the content of polysaccharides and proteins also increased compared with the original mud group. In S-EPS, the polysaccharides and proteins increased by 209.55% and 25.25%, respectively, and in LB-EPS, they increased by 11.90% and 45.16%, respectively. There was no obvious change in TB-EPS, indicating that the free radicals produced 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 relatively small.

[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%. In LB-EPS, both polysaccharide and protein contents increased by 37.42% and 20.81% respectively compared with the original mud group. In TB-EPS, the polysaccharide content increased by 17.29% and the protein content did not change significantly. This indicates that the catalyst-coupled bioelectrochemical system can effectively destroy the EPS structure and even degrade the polysaccharides in the outer S-EPS.

[0091] In the ICPBES group in Example 1, the concentrations of polysaccharides and proteins reached 150.06 mg / L and 15.71 mg / L, respectively. In S-EPS, polysaccharides and proteins increased significantly by 804.18% and 303.03%, respectively, in LB-EPS, by 45.16% and 172.62%, respectively, while in TB-EPS, they only increased by 35.39% and 50.56%, respectively. This may be because the changes in the substances in the inner EPS are different from those in the outer EPS. The distance between TB-EPS and cells is closer, while SEPS and LB-EPS are farther away from cells, and the changes in substances are more affected by the reaction mechanism.

[0092] These results indicate that the photocatalytic coupled bioelectrochemical treatment is effective in destroying the EPS in each layer of sludge, and the effect is most significant on the outer EPS layer, with the effect of each part being ICPBES>PES>ICPBES-dark>BES. In general, the effective destruction of EPS in sludge by the photocatalytic coupled bioelectrochemical system treatment exposes the bacterial cells previously protected by EPS, making them more vulnerable 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] ① Changes in the abundance of total ARGs and MGEs

[0095] In order to explore the role of each part in the photocatalytic coupled bioelectrochemical system, the relative abundance of ARGs in each sample treated by the method provided in Examples 1-5 was compared. The specific method was as follows: the sludge samples treated by the methods of Examples 1-5 were taken and DNA was extracted using the Power Soil DNA Isolation Kit (MoBio, USA). A high-throughput quantitative polymerase chain reaction (HT-qPCR) was performed using the SmartChip real-time PCR system (Clontech, China), and a total of 48 genes were detected, including 40 major ARGs, 7 MGEs, including 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 different systems, the relative abundance of ARGs in sludge decreased compared with RS. Among them, the relative abundance of ARGs after treatment with BES and ICPBES systems was 0.09 and 0.07, respectively, and the removal rates of tetracycline, sulfonamide, macrolide, and β-lactam resistance genes by ICPBES reached 84.78%, 71.40%, 82.57%, and 84.28%, respectively; the removal rate of MGEs reached 69.65%, indicating that the BES system combined with photocatalysis promoted the reduction of ARGs. In ICPBES-Dark, in ICPBES-OC, the relative abundance of sludge ARGs was 0.18 and 0.17, respectively. Comparing these values ​​with the relative abundance of sludge ARGs after ICPBES treatment, it was shown that photocatalysis and extracellular electron transfer in ICPBES contributed 0.11 and 0.10, respectively. Comparing the relative abundance of ARGs in sludge after treatment with PES and ICPBES systems, the contribution of ICPB was calculated to be 0.15. These results indicate that the photocatalytic, bioelectrochemical processes, and ICPB in the ICPBES system all promote the attenuation of ARGs.

[0097] The abundance of ARGs in the sludge treated with BES 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 1.66×10 -2 5.00×10 -3, 2.85×10 -2 and 7.63×10 -3 sul1 and sul2 (sulfonamides) decreased by 1.27×10 -2 and 4.19×10 -2 Among tetracycline resistance genes, except for tetC which increased slightly, the others decreased, among which tetM decreased most significantly, decreasing by 1.29×10 -2 ( Figure 5 a). In addition, the relative abundances of blaOXY-1, blaOXY-1, and cphA (β-lactams) decreased by 2.59×10 -3 , 1.69×10 -3 and 1.54×10 -3 The relative abundances of aacA / aphD, aadA2-1, aadA5, and ampC (aminoglycosides) also decreased slightly. The relative abundances of all subtypes of MGEs decreased, with IS26 and tnpA-5 decreasing by 2.26×10 -2 and 2.00×10 -3 ( Figure 5 b).

[0098] Further research on the difference in the relative abundance of ARGs in the treated BES and ICPBES systems, as well as the effect of photocatalytic elimination of ARGs, showed that the relative abundance of aminoglycosides, β-lactams, and macrolide ARGs decreased by 4.46×10 -3 , 2.85×10 -4 and 8.41×10 -5 In addition, the total relative abundance of MGEs decreased by 7.31×10 3 Among them, ermF and mefA (macrolides) decreased by 1.59×10 -2 and 1.53×10 -3 strB and sul1 (sulfonamide) decreased by 1.01×10 -3 and 4.24×10 -3 tetQ and tetS (tetracyclines) decreased by 1.03×10 -5 and 1.49×10 -5 Among β-lactams, except for blaTEM, cphA and ampC which were slightly enriched, the other subtypes decreased, and blaCMY, blaCTX-M and blaMOX / blaCMY disappeared in the ICPBES system. The relative abundance of aac(6')-Ib and aadA2-1 (aminoglycosides) decreased significantly by 1.83×10 -3 Among the MGEs, only IS26 and intl2 increased by 2.03×10 -3and 2.27×10 -5 , other isoforms decreased, int1 and tnpA-7 decreased by 1.36×10 -3 and 1.20×10 -3 This indicates that the synergistic effect of photocatalysis and bioelectrochemical reactions has a significant elimination effect on both ARGs and MGEs, especially targeting macrolides, sulfonamides, and tetracycline resistance genes ( Figure 5 b).

[0099] ② Changes in EPS-related and intracellular ARGs abundance

[0100] like Figure 6 a, In ICPBES, the abundances of both i-ARGs and EPS-ARGs were significantly reduced (1 order of magnitude) compared with the original mud (RS). The total absolute abundance of EPS-ARGs ranged from 3.48×10 7 -9.91×10 7 copies / mL, and the total absolute abundance of i-ARGs was 7.25×10 7 copies / mL. Among them, LB-ARGs was the lowest (95.05% lower than the original mud), while TB-ARGs was the highest (74.12% lower than the original mud), and i-ARGs only decreased by 41.03%, indicating that the photocatalytic coupled bioelectrochemical system mainly destroyed LB-ARGs and caused ARGs to migrate and transform in the cells and EPS. The reason may be that the photocatalytic coupled bioelectrochemical system first destroyed the outer layer S-ARGs (ARGs in S-EPS), degraded them and reduced the absolute abundance of S-ARGs; then destroyed LB-ARGs and TB-ARGs from the outside to the inside, directly degraded them or migrated to the outer layer and then degraded; finally destroyed i-ARGs and released them from the cells to EPS for further migration and transformation. However, due to the limited capacity of the photocatalytic coupled bioelectrochemical system, the innermost i-ARGs were less damaged, and the damaged i-ARGs were first released into the TB-EPS. At the same time, the photocatalytic coupled bioelectrochemical system had a 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 fact that the rate at which the inner LB-ARGs and TB-ARGs were transferred to S-ARGs was greater than their own degradation rate. For example, the total absolute abundance of aminoglycoside ARGs decreased in all layers, but the decrease in S-ARGs was the least, and aac(6')-II was no longer detectable in LB-EPS and TB-EPS, while the absolute abundance in S-EPS was 8.38×10 4copies / mL; in addition, the total absolute abundance of tetracycline ARGs in S-EPS, LB-EPS, TB-EPS and cells decreased by 19.16%, 99.55%, 95.60% and 38.71%, respectively. Among them, tetM and tetT in S-EPS were significantly higher than those in other layers. The absolute abundance of tetM in S-EPS, LB-EPS, TB-EPS and cells 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. tetT was 1.14×10 6 copies / mL, 5.54×10 3 copies / mL, 5.90×10 4 copies / mL, 0 copies / mL. In addition, under the action of the photocatalytic coupled bioelectrochemical system, the degradation rates of aacA / aphD, blaCTX-M-1,3,15, ermB, and ermX in each layer of EPS exceeded 90%, and the degradation rate in the cell was 70.49%-90.38%. In summary, the photocatalytic coupled bioelectrochemical system has a destructive effect on ARGs in each layer, but the migration and transformation of LB-ARGs is most significant. And the removal effect of aacA / aphD, blaCTX-M-1,3,15, ermB, and 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, respectively, while i-ARGs and S-ARGs increased, indicating that the bioelectrochemical system alone can act on the ARGs in EPS, but is not enough to destroy i-ARGs, and the oxidation capacity of S-ARGs converted by migration in the inner layer of EPS is also limited. Therefore, the absolute abundance of S-ARGs increased compared with the original mud. For example, the absolute abundance of tetracycline ARGs increased in S-EPS, and decreased by 88.27%, 7.01% and 9.39% in LB-EPS, TB-EPS and cells, respectively. Among them, tetM, tetQ and tetX increased slightly in S-EPS and decreased in other layers. tetS increased slightly in S-EPS and cells, and also decreased in other layers. strB, sul1, sul2 (sulfonamides) increased slightly in S-EPS and cells, but decreased in other layers ( Figure 6b). In summary, the bioelectrochemical system had no significant effect on i-ARGs, but could cause the migration and transformation of ARGs in EPS, and showed good removal effects 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-lactams 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 increased slightly in cells and decreased in all layers of EPS, cphA increased slightly in S-EPS and decreased in other layers. Among sulfonamides, only strB decreased in S-EPS and cells, sul2 increased slightly in cells, and decreased in other layers ( Figure 6 b) In summary, photocatalysis is not enough to destroy i-ARGs, but it can cause the ARGs in EPS to migrate, causing LB-ARGs and TB-ARGs to migrate to S-ARGs, increasing the absolute abundance of S-ARGs. In addition, the intensity of photocatalysis on EPS-related ARGs is greater 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, the absolute abundance of LB-ARGs and S-ARGs increased because the migration rate of some ARGs from the inner layer was greater than the degradation rate. For example, the absolute abundance of aac(6')-Ib, aac(6')-II, and aacA / aphD in aminoglycosides decreased in TB-EPS and cells, but increased in S-EPS and LB-EPS. The absolute abundance of blaPSE and cphA in β-lactams also decreased in TB-EPS and cells, but increased in S-EPS and LB-EPS. Among them, blaPSE was almost undetectable in cells ( Figure 6 b). In summary, the catalyst-coupled bioelectrochemical system can destroy i-ARGs and TB-ARGs, but is not sufficient 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 outside the cells.

[0105] ③ Changes in EPS-related and intracellular MGEs abundance

[0106] like Figure 7 As shown in the figure, compared with RS, in the BES group, except for TB-MGEs, the abundance decreased by 9.93%, while the abundance in other layers increased, tnpA-5 and tnpA-7 decreased by 82.82% and 48.39%, respectively. ISCR1 decreased by 71.22% in LB-EPS, while the others increased. The above results indicate that BES is not enough to destroy i-MGEs, and the destroyed TB-MGEs may migrate to LB-EPS and S-EPS, 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 an absolute abundance reduction of 37.14%, a slight increase in TB-MGEs, and a significant increase in i-MGEs and S-MGEs, indicating that the photocatalytic reaction was not enough to destroy i-MGEs, and mainly destroyed LB-MGEs. And it had a significant effect on IS26, whose absolute abundance decreased by 60.74% and 8.96% in LB-EPS and TB-EPS, respectively, intl3 decreased by 46.62% in LB-EPS, disappeared in TB-EPS, and ISCR1 disappeared in all layers. It shows that the photocatalytic reaction has no obvious effect on i-MGEs and S-MGEs, but can make LB-MGEs and some TB-MGEs migrate and transform.

[0108] In the ICPBES-dark group, the absolute abundance of TB-MGEs and i-MGEs decreased by 85.58% and 11.51% respectively compared with the original mud, but LB-MGEs and S-MGEs increased, among which the effects on int1, tnpA-7 and ISCR1 were more obvious. The absolute abundance of int1 decreased by 84.91% and 16.27% in TB-MGEs and i-MGEs, tnpA-7 decreased by 95.19% and 22.56% respectively, and ISCR1 decreased by 19.38%, 83.67% and 7.91% in LB-MGEs, TB-MGEs and i-MGEs respectively. This indicates that the catalyst-coupled bioelectrochemical system can destroy i-ARGs and TB-ARGs, but is not enough to completely degrade LB-ARGs and S-ARGs that migrated to the outer layer.

[0109] Therefore, the photocatalytic coupled bioelectrochemical system also has obvious effects 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). Quantitative Insights Into Microbial Ecology (QIIME, version 1.9.1) was then used to perform quality control procedures (including trimming barcodes and primers, filtering low-quality reads and chimeras). Operational taxonomic units (OTUs) were defined based on 97% identity using Uparse (version 7.0.1001). Mothur software was used to classify and quantify OTUs for the Silva SSU rRNA database. The Shannon index was calculated to quantify the microbial alpha diversity of each sample. As shown in Table 1, the lowest level of the Coverag index of the five groups of samples reached 0.99, indicating that the data of the microbial samples determined this time were reliable.

[0113] Table 1 Alpha diversity index of microorganisms in sludge after treatment by different systems

[0114]

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

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

[0117] like 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 potential host of some ARGs (tetM, tetQ, tetX, sul1), and its relative abundance in ICPBES, BES, PES, and ICPBES-dark is 31.75%, 33.96%, 14.12%, and 7.96% lower than that in RS, respectively; Firmicutes is also one of the key hosts of ARGs and is considered to be the source of class I integrons. In ICPBES, BES, and PES, the relative abundances of Actinobacteriota, Acidobacteriota, Nitrospirota, and Planctomycetota decreased by 30.30%, 40.98%, and 14.65%, respectively. The relative abundances of Actinobacteriota, Acidobacteriota, Nitrospirota, and Planctomycetota decreased significantly by 65.28%, 21.19%, 61.30%, and 11.94% in ICPBES. However, Proteobacteria and Bacteroidota increased by 42.71% and 47.35%, respectively. In summary, the photocatalytic coupled bioelectrochemical system significantly changed the microbial community structure at the phylum level, with an inhibitory effect on Chloroflexi, Firmicutes, Actinobacteriota, Acidobacteriota, Nitrospirota, and Planctomycetota, and a promoting effect on Proteobacteria and Bacteroidota.

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

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

[0120] (5) Effects on metabolism

[0121] Related studies have shown that the spread of ARGs is highly dependent on related biological pathways. In order to understand the different effects of different treatments on functional modules, the functions of gene KO and KEGG database were predicted by PICRUSt 2. The expression levels of genes related to type IV secretion system (T4SS), two-component system (TCs) and bacterial secretion system were evaluated by KEGGorthologs. The metabolic function results based on KEGG were as follows: Fig. 9 As shown in b. Fig. 9 As shown in a, 21 metabolic pathways in each treatment group were significantly different from those in RS. Compared with RS, except for the BES group, the abundance of membrane transport, signal transduction, and infectious diseases: bacterial was reduced in each group. In addition, carbohydrate metabolism, other amino acid metabolism, and translation were reduced in each treatment group. This indicates that the photocatalytic coupled bioelectrochemical system may exert pressure on microorganisms, causing them to shift in these reactors. The following further studies the changes in genes related to membrane transport, signal transduction, and infectious diseases: bacterial.

[0122] ① Two-component control 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 each group, which was most obvious in the ICPBES group. Fig. 9 As shown in c, the relative abundance of rstA in ICPBES-dark, ICPBES, BES, and PES groups decreased by 22.22%, 55.56%, 11.11%, and 33.33%, respectively; chpB, chpC, and chpE involved in the synthesis of pili and flagella 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; degU and desR related to 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 reproduction of ARGs.

[0124] ②Bacterial secretion system

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

[0126] ③ Type IV secretion system

[0127] The type IV secretion system is considered to be an important mechanism for the transfer of genes and materials between microorganisms, which enables the genetic material to be transferred 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 ( Fig. 9 c). VirD4, a gene that plays a key role in substrate binding; VirB1 that promotes DNA release; VirB2, VirB3, and VirB5 that are responsible for the exchange of genetic material; VirB4 and VirB11 that participate in energy synthesis; VirB6, VirB8, and VirB10 that promote the horizontal transfer of ARGs; 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. This shows that ICPBES has an inhibitory effect on the genes of each module of T4SS, thereby weakening the dissemination and proliferation of ARGs in the system.

[0128] Apparently, the photocatalytically coupled 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) Analysis of microorganisms, metabolic pathways, EPS structure, MGEs and ARGs

[0130] Fig.10 The network analysis shown describes the process by which microorganisms affect EPS structure through metabolic pathways, such as Fig.10 As shown in the results, in ICPBES, EPS protein and polysaccharide significantly affected the movement of ARGs and MGEs, and ARGs and MGEs were 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, which was significantly correlated with polysaccharides in S-EPS, LB-EPS, and TB-EPS, and proteins in S-EPS and TB-EPS. For example, OLB14 was significantly negatively correlated with proteins in S-EPS and LB-EPS, and polysaccharides in S-EPS and TB-EPS (P<0.05). Rubrivivax and UBA12294 were also correlated with proteins and polysaccharides in EPS. At the same time, EPS-ARGs were also related to OLB14, Accumulibacter, Sulfuritalea, Azonexus, and Rubrivivax.

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

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

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

[0135] As mentioned above, changes in multiple parameters such as EPS structure, microbial status, bacterial diversity, dominant microbial community, metabolic pathways, and MGEs can affect the changes in ARGs in ICPBES. Therefore, partial mantel test was used to show the relationship between ARGs and diversity parameters ( Fig.11 ).like Fig.11 As shown, ARGs were significantly correlated with proteins and polysaccharides, and two-component systems, and were associated with Chao1, Simpson, ACE index, dominant microbial communities, and two-component systems.

[0136] In addition, there are intrinsic correlations among the factors. Proteins and polysaccharides are positively correlated with MGEs, Simpson index, and T4SS. The bacterial secretion system is extremely significantly correlated with Chao1, Shannon, ACE index, and two-component system (r>0.6). The dominant microbial community is correlated with Chao1, Shannon, and ACE index. Therefore, these influencing factors work together to achieve the removal of ARGs.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for removing antibiotic resistance genes from sludge by 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, KCl was added for mixed grinding, the mass ratio of the g-C3N4 catalyst to KCl was 10:1, the two were mixed, high-temperature calcination and grinding were performed to obtain Kg-C3N4 photocatalyst; S2: Preparation of photocatalytic anode for photocatalytic coupled bioelectrochemical system: Take Kg-C3N4 photocatalyst, add 5% Nafion solution and 1mL isopropanol, apply it on the nickel foam carrier after ultrasonic mixing, and obtain the nickel foam electrode loaded with Kg-C3N4 photocatalyst after drying; the mass ratio of Kg-C3N4 photocatalyst to Nafion solution and isopropanol is: 400mg:2mL:1mL; A nickel foam loaded with Kg-C3N4 photocatalyst 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 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 the cathode are electrically connected to form a loop, and electrical energy is generated through the chemical potential difference of the system itself. A biofilm is formed inside the pores of the nickel foam loaded with Kg-C3N4 photocatalyst. After the maximum voltage measured is stable, the domestication of the biofilm is completed, and a photocatalytic anode of a photocatalytic coupled bioelectrochemical system is prepared; S3: Removal of antibiotic resistance genes from the treated sludge mixed liquor by photocatalytic coupled 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 to remove the antibiotic resistance genes in the sludge mixture to be treated through the photocatalytic coupled bioelectrochemical system.

2. The method for removing antibiotic resistance genes in sludge by a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: 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 lid, and the crucible is placed in a muffle furnace and calcined at 550° C. for 3 h at a heating rate of 5° C. / min. After the product is naturally cooled, it is 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 by a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: In S2, the mixed solution containing anode sludge is a mixed solution 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 by 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 mpl / L glucose solution, wherein the pH value of the sludge to be treated is 7.58±0.01, the water 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 by a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: In S1, the high temperature calcination conditions are as follows: after the g-C3N4 catalyst is mixed with KCl, it is placed in a muffle furnace and calcined at 500°C for 3h with a heating rate of 2.5°C / min.

6. The method for removing antibiotic resistance genes in sludge by 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 by 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 by 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 to 0.7 V.

9. The method for removing antibiotic resistance genes in sludge by a photocatalytic coupled bioelectrochemical system according to claim 1, characterized in that: In S3, the preparation method of the modified air cathode is: use carbon felt as the material of the air cathode, weigh 10g of 60% PTFE, add water to 120g, and apply it on one side of the carbon felt as a carbon base layer, dry it at room temperature for two hours, and then put it in a 370°C oven to heat for 30 minutes, and then apply four layers of 60% PTFE emulsion on the carbon base layer as an air diffusion layer, and each layer is dried in a 370°C oven for 10-15 minutes; then weigh 318mg of activated carbon and 13.25mg of conductive carbon black, add 1.48ml of isopropanol and 1ml of Nafion reagent, mix well, and apply it on the other side of the carbon felt (the side in contact with the electrolyte) as a catalytic layer, and dry it in an oven at 60°C for 6h.

10. The method for removing antibiotic resistance genes in sludge by 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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