A method for degrading sulfamethoxazole using QXT-31-Mn

Abstract

The present invention discloses a method for degrading sulfamethoxazole using QXT-31-Mn, which belongs to the field of sewage treatment technology. QXT-31-Mn was deposited with the General Microbiology Center of the China Culture Collection Administration on December 3, 2024, with the deposit number CGMCC No. 32889 and the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The taxonomic name is Arthrobacter sp. The present invention utilizes QXT-31-Mn to produce a safer and more environmentally friendly cathode material that can efficiently degrade SMX.

Description

Technical Field

[0001] The invention belongs to the technical field of sewage treatment, and in particular relates to a method for degrading sulfamethoxazole by utilizing QXT-31-Mn. Background Art

[0002] Due to its excellent antibacterial effect, sulfamethoxazole (SMX) has been widely used in the treatment of bacterial infections in humans, livestock and aquaculture. However, after use, SMX enters sewage treatment plants through various pathways, but it is difficult for sewage treatment plants to completely remove the antibiotic, causing it to become a point source of river pollution. Currently, sewage treatment plants mainly rely on activated sludge systems to remove antibiotics. In traditional sewage treatment, the removal of SMX mainly depends on the adsorption and biodegradation processes of sludge, but these processes are often slow.

[0003] In recent years, bioelectrochemical systems (BESs) have attracted widespread attention due to their ability to utilize electroactive microorganisms to oxidize or reduce antibiotics at the anode or cathode, thereby effectively removing them. Existing BES research has mostly focused on the anode, while research on the cathode compartment is relatively insufficient. In most bioelectrochemical systems, the cathode reaction is typically abiotic, with the reduction of oxygen or ferricyanide being common. Ferricyanide has ideal properties as a cathode reactant, being unrestricted by solubility and independent of oxygen concentration. However, its toxicity to microorganisms limits its application in BESs. Furthermore, the slow reduction kinetics of oxygen at solid electrodes and its low solubility in water pose challenges to the practical application of oxygen as an oxidant. To address these limitations, many abiotic cathode BES studies are dedicated to improving the efficiency of oxygen reduction at solid electrodes to promote the development of microbial fuel cells.

[0004] Against this backdrop, several studies have attempted to use biomineralized manganese oxides as an oxygen substitute. In this alternative, oxygen serves as the final electron acceptor and acts as a redox mediator in the cathode reaction via the redox couple of manganese ions and manganese oxides.

[0005] The cathodes in existing bioelectrochemical systems (BES) mostly rely on abiotic reactions (such as the reduction of oxygen or ferrocyanide), which have limitations in performance and microbial compatibility. Furthermore, it is crucial to ensure the stability of the designed bioelectrochemical system over long periods of operation and verify its sustainability in the antibiotic degradation process to enable practical applications. Therefore, a biomanganese cathode composed of manganese-oxidizing bacteria and manganese oxides was developed, and its structure and performance were optimized to achieve stable electron transfer and efficient SMX degradation. Summary of the Invention

[0006] The present invention provides an Arthrobacter sp. QXT-31-Mn with a preservation number of CGMCC NO.32889. The taxonomic name of the QXT-31-Mn is Arthrobacter sp.

[0007] The present invention also provides a cathode prepared using the above-mentioned Arthrobacter QXT-31-Mn, and the preparation method of the cathode comprises the following steps:

[0008] (1) Cultivating QXT-31-Mn to obtain a QXT-31-Mn bacterial solution;

[0009] (2) Place the cathode in the container and use a solution containing 0.2-1 mM Mn 2+ QXT-31-Mn was cultured in a culture medium to promote its manganese oxidation to generate manganese oxides deposited on it.

[0010] Preferably, the culture medium used for culturing QXT-31-Mn in step (1) is PYG culture medium.

[0011] More preferably, HEPES buffer is added to the PYG medium when culturing QXT-31-Mn in step (1).

[0012] More preferably, in step (2), Mn 2+ The concentration is 0.4 mM.

[0013] More preferably, in step (2), Mn 2+ From MnCl2.

[0014] The present invention also provides a method for degrading sulfamethoxazole, which comprises the following steps:

[0015] (1) Activated sludge is inoculated into the anode chamber of the dual-chamber microbial electrochemical system and acclimated. The acclimated biofilm will adhere to the anode;

[0016] (2) using the cathode as the cathode of a dual-chamber microbial electrochemical system;

[0017] (3) adding cathode and anolyte to the cathode and anode chambers of the dual-chamber microbial electrochemical system, respectively;

[0018] (4) Apply an external voltage of -0.5 to -1 V to degrade SMX.

[0019] Preferably, the cathode electrolyte in step (3) is PYG culture medium.

[0020] More preferably, the external voltage in step (4) is -0.8V.

[0021] The present invention also provides the use of the above-mentioned Arthrobacter QXT-31-Mn in preparing a product for degrading sulfamethoxazole.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) By replacing traditional toxic cathode reactants such as ferrocyanide with a bio-manganese cathode, the present invention provides a safer and more environmentally friendly cathode material, reducing the toxic effects on microorganisms.

[0024] (2) The use of a biomanganese cathode that combines electroactive microorganisms with manganese oxides significantly enhances the efficiency of electron transfer, allowing the system to remain stable during long-term operation and ensuring continuous and efficient antibiotic degradation.

[0025] (3) By controlling the external resistance and moderate external voltage to adjust the system performance, the efficiency of electron transfer and redox reaction was significantly improved, and the overall performance of the bioelectrochemical system was optimized.

[0026] Biodeposit description of QXT-31-Mn:

[0027] Depository: General Microbiology Center of China Culture Collection Administration of Microorganisms;

[0028] Deposit number: CGMCC NO.32889;

[0029] Deposit date: December 3, 2024;

[0030] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0031] Taxonomic name: Arthrobacter sp. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 (a) OD600 changes and (b) Mn 2+ Concentration changes.

[0033] Figure 2 This is the cyclic voltammetry curve of manganese oxide in Example 2.

[0034] Figure 3 (a) The current when 1000Ω is applied, (b) The current at a constant potential of -0.8V, (c) The manganese reduction concentration at the biomanganese cathode, and (d) The electrochemical synergistic SMX degradation effect at the biomanganese cathode in Example 3.

[0035] Figure 4 This is a graph showing changes in manganese ion concentration in the dual-chamber electrochemical system in Example 4. DETAILED DESCRIPTION

[0036] In the following examples, Mn 2+ Both refer to MnCl2.

[0037] Example 1 Study on the Growth Characteristics of Manganese Oxidizing Bacteria

[0038] Cultivation of manganese oxidizing bacteria: scrape the manganese oxidizing bacteria from the solid culture medium and inoculate into 50 mL of Mn-free 2+ The culture was placed in a conical flask with autoclaved PYG medium (formula: 0.5 g MgSO₄·7H₂O, 0.06 g CaCl₂·2H₂O, 0.25 g glucose, 0.25 g yeast extract, 0.25 g tryptone, 1 L distilled water, pH 7.0-7.2) and shaken at 30°C and 170 rpm for 12 hours. The cultured bacterial suspension was inoculated into fresh PYG medium at a 3% volume ratio. HEPES buffer was then filtered through a 0.22 μm sterile filter membrane and added to the PYG medium to a final HEPES concentration of 0.5 M (pH 7.2), yielding a manganese-containing bacterial suspension.

[0039] In order to investigate the effect of micro-electric field stimulation on the growth of manganese oxidizing bacteria and manganese oxidation, a single-chamber electrochemical system was used to apply 0.1, 0.3, and 0.5 V applied voltages to QXT-31-Mn bacteria. Both the working electrode and the counter electrode were platinum mesh. First, the effect of different applied voltages on the growth of QXT-31-Mn was investigated ( Figure 1 a) The electrolyte was PYG medium supplemented with MnCl2, the addition amount of QXT-31-Mn bacterial solution was 10% (45 ml PYG medium, 5 ml bacterial solution), the initial MnCl2 concentrations were 0.1 V: 41.13 mg / L, 0.3 V: 44.50 mg / L, 0.5 V: 36.25 mg / L, and open circuit (OC): 37.83 mg / L.

[0040] The results showed that the growth curves under the conditions of 0.1V and 0.3V voltage were relatively similar. The bacterial growth rate at 0.3V was slightly lower than that at 0.1V, but both showed rapid initial growth, followed by a gradual stabilization of the growth rate. Under the condition of 0.5V voltage stimulation, the bacterial growth density was initially low, but began to rise rapidly from the 6th day. By the 10th day, the OD 600 However, there was no significant difference in OD between the different applied voltages and open circuit (OC) groups.

[0041] Secondly, the effect of different applied voltages on the oxidation of manganese in QXT-31-Mn was investigated ( Figure 1 b) All conditions are the same as above, Mn 2+The concentration of (MnCl2) remained relatively stable in the early stage of the experiment (about the first 4 days). Starting from the 4th day, the concentration of manganese ions under all conditions began to decrease significantly, but the rate of decrease did not differ significantly. By the 10th day of the experiment, the concentration of manganese ions under all conditions was close to or reached 0 mg / L, and all of them generated BioMnOx and attached to the electrodes. These changes may indicate that the existence and size of voltage have an impact on the effect of Mn 2+ In conclusion, micro-electric field stimulation has no inhibitory effect on the growth of manganese-oxidizing bacteria and manganese oxidation, and can be used as a cathode.

[0042] Example 2 Electrochemical Characterization of Biomanganese Oxide

[0043] In order to evaluate the redox ability of biogenic manganese oxides, cyclic voltammetry (CV) was used to characterize the biogenic manganese oxides ( Figure 2 ). Manganese oxidizing bacteria and biological manganese oxides were deposited (QXT-31-Mn was cultured in PYG medium containing 0.75mM MnCl2) on indium tin oxide (ITO) conductive glass as the working electrode, platinum mesh as the counter electrode, Ag / AgCl as the reference electrode, and the electrolyte was 0.1M saturated Na2SO4 solution. Cyclic voltammetry scanning was performed to observe the electrochemical state of biological manganese oxides. The magnitude of the current generated by biological manganese oxides is closely related to the amount of Mn present on the surface of Mn oxides. Based on the peak shape, we infer that the reduction peaks at 0.28V and -0.46V are attributed to the reduction of Mn(IV) to Mn(II) and Mn(IV) to Mn(III), respectively. The reduction of Mn(IV) to Mn(II) may be to promote bacterial utilization. In addition, the peaks at 0.92 and 0.01V are consistent with the dissolved Mn 2+ The CV curves provide information on the conversion of manganese in different electrochemical states, which helps to understand the electron transfer mechanism in manganese-based electrochemical systems, especially the redox kinetics of manganese.

[0044] Example 3: Feasibility of biomanganese cathode and degradation effect of bioanode on SMX

[0045] To explore the feasibility of biogenic manganese oxide as a cathode material, this example constructed a biogenic manganese cathode containing manganese oxide and the manganese-oxidizing bacterium QXT-31-Mn. First, a carbon felt electrode was placed in a shake flask filled with PYG medium containing 0.75 mM MnCl₂. QXT-31-Mn was inoculated and cultured to induce manganese oxidation, resulting in manganese oxide deposition on the carbon felt electrode. After 48 hours of incubation, the carbon felt electrode served as the cathode in a dual-chamber electrochemical system, with the PYG medium in the shake flask serving as the catholyte.

[0046] At the same time, potassium ferrocyanide was used to domesticate activated sludge and cultivate a microbial community dominated by electroactive bacteria to construct a biological anode for the biological manganese cathode.

[0047] Acclimation step: The anode chamber was inoculated with activated sludge from a sewage treatment plant and an anode solution (2.77 g / L NaH2PO4·2H2O, 11.55 g / L Na2HPO4·12H2O, 0.31 g / L NH4Cl, 0.13 g / L KCl, 1 mL / L vitamin solution (the vitamin solution (per liter of distilled water) contains: 2.0 mg biotin, 2.0 mg folic acid, 10.0 mg pyridoxine hydrochloride, 5.0 mg thiamine hydrochloride, 5.0 mg riboflavin, 5.0 mg niacin, 5.0 mg D-(+)-calcium pantothenate, 0.5 mg vitamin B12). 12 , 5 mg p-aminobenzoic acid and 5 mg lipoic acid), 1 mL / L of a mixture of mineral elements (the vitamin solution (per liter of distilled water) contains: 1.5 g N(CH2COOH)3, 3 g MgSO4·7H2O, 0.5 g MnSO4·H2O, 1 g NaCl, 0.1 g FeSO4·7H2O, 0.1 g CoCl2·6H2O, 0.1 g CaCL2, 0.1 g ZnSO4·7H2O, 0.01 g CuSO4·5H2O, 0.01 g AlK(SO4)2·12H2O, 0.01 g boric acid, 0.01 g NaMoO4·2H2O), 0.5 g / L sodium acetate solution), mixed in a ratio of 1:3 (v / v), 0.1 mM potassium ferricyanide solution was added to the cathode chamber, and an external resistor of 510 Ω was connected. The anode solution and cathode solution were replaced simultaneously every four days until a periodic current that first increased and then decreased appeared, indicating successful acclimation. New anode solution was replaced and 1 mg / L of SMX was added for subsequent experiments.

[0048] As shown in Table 1, a total of five operating modes were set for the reactor: when the external resistance was 1000Ω, the cathode was only QXT-31-Mn (denoted as MFC), and the cathode contained both QXT-31-Mn and manganese oxide (denoted as BioMnOx-MFC); when the external voltage was -0.8 V, the cathode was only QXT-31-Mn (denoted as -0.8 V), and the cathode contained both QXT-31-Mn and manganese oxide (denoted as -0.8 V-BioMnOx), as well as open circuit operation control.

[0049] The cathode with only QXT-31-Mn (denoted as MFC) is compared with the cathode containing manganese oxidizing bacteria and manganese oxides (denoted as BioMnOx-MFC). The difference is that no Mn is added during the culture. 2+ , only add bacterial solution.

[0050] Cathode chamber solution:

[0051] (1) External resistor 1000 ohms

[0052] ①MFC, PYG solution (biofree manganese oxide)

[0053] ②BioMnOx-MFC, PYG solution (with biological manganese oxide)

[0054] (2) External voltage -0.8V

[0055] ③-0.8V, PYG solution (no biological manganese oxide)

[0056] ④-0.8V-BioMnOx, PYG solution (with biological manganese oxide)

[0057] (3) Open circuit

[0058] ⑤OC: PYG solution (with biological manganese oxide)

[0059] Table 1

[0060]

[0061]

[0062] The current monitoring results of different groups showed that ( Figure 3 (a and 3b) When an external resistor was applied, the current of the cathode containing bioMnOx (BioMnOx-MFC) first increased and then decreased, reaching a maximum current of 0.03 mA in the first cycle and slightly increasing to 0.05 mA in the second cycle. The current of the cathode without bioMnOx (MFC) remained zero, indicating that bioMnOx effectively accepted electrons from the anode and participated in the reduction reaction. This demonstrates that bioMnOx has excellent electron acceptance and transfer capabilities, enabling the current to form a closed circuit. When an external voltage was applied, the current of the cathode containing bioMnOx (-0.8V-BioMnOx) rapidly increased to 0.14 mA and remained stable, while the current of the cathode without bioMnOx (-0.8V) also remained zero. This phenomenon further emphasizes the efficiency of the bioMnOx cathode in providing stable current output. This rapid and stable current increase indicates that the bioMnOx cathode can more efficiently transfer electrons and output energy when driven by voltage. In addition, the efficiency of current generation in the applied voltage operation mode is significantly higher than that in the mode using only an external resistor, which indicates that the electrochemical activity of biomanganese oxide is more fully utilized under higher potential driving.

[0063] In addition, in order to explore the reduction reaction of biological manganese cathode, this example also carried out 2+ Detection ( Figure 3c) The results of the study found that Mn can be detected 2+ The dissolution of Mn 2+ The dissolution rate of Mn was 1.20 mg / (L·h), which was significantly higher than that of the external resistor and open circuit groups. 2+ The dissolution rates of the biomanganese cathode were 0.45 and 0.22 mg / (L·h). This finding indicates that under the condition of applying an external voltage of -0.8 V, the reduction reaction of the biomanganese cathode is more active, thereby promoting more Mn 2+ In contrast, under external resistance and open circuit conditions, Mn 2+ The release rate of Mn is low, which may be due to the fact that the increase in current significantly accelerates the electrochemical reduction process of manganese. The higher applied voltage provides a greater driving force, prompting the rapid reduction of manganese ions at the biological manganese cathode. This rapid electrochemical activity not only increases the Mn 2+ The generation rate also shows that the application of voltage can effectively control and accelerate the reduction process.

[0064] On the premise of verifying the feasibility of the biological manganese cathode, the bioanode provides 1 mg / L SMX (1 mg / L SMX is added to the activated sludge after acclimation) for removal ( Figure 3 d). The results showed that SMX could be completely removed within four days. When the external voltage was -0.8V, the removal rate of SMX was 0.78mg / (L·d), which was significantly higher than that of the external resistor and open circuit group Mn 2+ The removal rates of SMX and Mn were 0.53 and 0.42 mg / (L·h). This indicates that the applied voltage can effectively improve the ability of the bioanode to treat SMX. It is worth noting that the efficient degradation of SMX and Mn 2+ The rapid reduction of manganese and the increase in current, which increase simultaneously under applied voltage, indicate that electrochemical driving forces play a key role in enhancing both manganese reduction and SMX biodegradation. This electrochemical activation likely leads to more efficient reduction of manganese oxides and enhanced electron-accepting capacity, thereby improving the overall system's pollutant treatment performance.

[0065] Example 4 Sustainability of Biomanganese Cathode

[0066] In this example, the QXT-31-Mn bacteria were first provided with Mn for solubility. 2+ , place a carbon felt electrode in a shake flask and use 0.4 mM Mn 2+ QXT-31-Mn was cultured to promote its oxidation of manganese to form manganese oxides deposited on it. QXT-31-Mn bacteria could completely oxidize manganese in 36 hours, with a manganese oxidation rate of 0.83 mg / (L·h). This result shows that QXT-31-Mn bacteria has efficient manganese oxidation ability and can quickly convert Mn 2+When these biologically generated manganese oxides are used as cathodes in bioelectrochemical systems, they can be efficiently reduced back to Mn 2+ , and has the same rate as oxidation 0.83 mg / (L·h). The other experimental conditions are the same as those in Example 3 (④-0.8V-BioMnOx, PYG solution (with biological manganese oxide), except that Mn 2+ The concentrations are different ( Figure 4 This symmetrical oxidation and reduction rate not only indicates the balanced nature of the process but also highlights the high efficiency and controllability of the system. The reversible redox properties of manganese oxide endow the system with remarkable reproducibility.

[0067] The cathode reaction mechanism involves the initial deposition of BioMnOx on the electrode surface and the subsequent reduction of electrons from the anodic reaction, resulting in the release of manganese ions. Since the release of divalent manganese occurs close to the electrode surface where the manganese-oxidizing bacteria are attached, the divalent manganese is immediately reoxidized to BioMnOx by the bacteria, thus completing the cycle. The anodic reaction mechanism primarily involves electroactive bacteria utilizing sodium acetate and SMX as a co-carbon source to degrade SMX and continuously provide electrons to the cathode. This mode of operation achieves the dual benefits of pollutant removal and resource recovery, demonstrating the enormous potential of bioelectrochemical technology in environmental management and resource recovery.

[0068] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A strain of Arthrobacter QXT-31-Mn with a deposit number of CGMCC NO.32889, characterized in that: The taxonomic designation of QXT-31-Mn is Arthrobacter sp.

2. A cathode prepared using the Arthrobacter QXT-31-Mn according to claim 1, characterized in that The preparation method of the cathode comprises the following steps: (1) Cultivating QXT-31-Mn to obtain a QXT-31-Mn bacterial solution; (2) Place the cathode in the container and use a solution containing 0.2-1 mM Mn 2+ QXT-31-Mn was cultured in a culture medium to promote its manganese oxidation to generate manganese oxides deposited on it.

3. The cathode according to claim 2, characterized in that The culture medium used for culturing QXT-31-Mn in step (1) is PYG culture medium.

4. The cathode according to claim 3, characterized in that When culturing QXT-31-Mn in step (1), HEPES buffer is added to the PYG culture medium.

5. The cathode according to claim 4, characterized in that In the step (2), Mn 2+ The concentration is 0.4 mM.

6. The cathode according to claim 5, characterized in that In the step (2), Mn 2+ From MnCl2.

7. A method for degrading sulfamethoxazole, characterized in that: The method comprises the following steps: (1) Activated sludge is inoculated into the anode chamber of the dual-chamber microbial electrochemical system and acclimated. The acclimated biofilm will adhere to the anode; (2) Using the cathode described in any one of claims 2 to 6 as the cathode of a dual-chamber microbial electrochemical system; (3) adding cathode and anolyte to the cathode and anode chambers of the dual-chamber microbial electrochemical system, respectively; (4) Apply an external voltage of -0.5 to -1 V to degrade SMX.

8. The method according to claim 7, characterized in that The cathode electrolyte in the step (3) is PYG culture medium.

9. The method according to claim 8, characterized in that In step (4), the external voltage is -0.8V.

10. Use of the Arthrobacter QXT-31-Mn according to claim 1 in preparing a product for degrading sulfamethoxazole.

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