Method for rapidly recovering performance of anaerobic ammonia oxidation system under stress of bisphenol A
By adding riboflavin and Shinella yambaruensis supernatant to enhance microbial quorum sensing and electron transfer, the performance degradation of the anaerobic ammonia oxidation system under bisphenol A stress was resolved, achieving rapid recovery and stable operation.
Patent Information
- Application Number
- CN202511286081.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-10
- Publication Date
- 2025-12-12
AI Technical Summary
Anaerobic ammonia oxidation systems experience performance degradation and slow recovery under bisphenol A stress. Existing recovery methods are unstable and difficult to respond to sudden pollution events.
Adding riboflavin and the supernatant of Shinella yambaruensis enhances the quorum sensing effect and electron transport of microorganisms. By continuously adding 0.5 mg/L of riboflavin, the supernatant of Shinella yambaruensis contains acyl homoserine lactone signaling molecules, which promote the rapid recovery of anaerobic ammonia-oxidizing bacteria.
It enables rapid recovery of anaerobic ammonia oxidation systems under bisphenol A stress, improves nitrogen removal rate, shortens recovery cycle, enhances system stability and resistance, and is suitable for the treatment of urban sewage and high-concentration toxic wastewater.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of wastewater biological treatment technology, specifically relating to a method for rapid performance recovery of an anaerobic ammonia oxidation system under bisphenol A stress. Background Technology
[0002] Anaerobic ammonia oxidation (AAO) is a highly efficient nitrogen removal process with advantages such as no need for organic carbon sources, low sludge production, and low energy consumption, and has been widely used in the treatment of high ammonia nitrogen wastewater. However, AAO bacteria are sensitive to environmental changes, especially under stress from endocrine disruptors such as bisphenol A (BPA). In such cases, system performance is significantly inhibited, manifesting as decreased nitrogen removal rate, reduced AAO bacterial activity, slow system recovery, and even system collapse. Studies have shown that when the BPA concentration reaches 10 mg / L, it leads to a significant decrease in the nitrogen removal efficiency of the AAO system, resulting in problems such as suppressed expression of key functional genes, reduced enzyme activity, and the death of some microorganisms.
[0003] Currently, there are limited methods for restoring anaerobic ammonia oxidation systems after being subjected to toxic pollutants such as bisphenol A. These methods mainly focus on controlling hydraulic retention time, diluting the concentration of toxic substances, and adjusting environmental parameters (such as pH and temperature). However, the recovery cycle is long, the effect is unstable, and it is difficult to cope with sudden pollution events.
[0004] In recent years, advancements in microbial communication have provided new insights into the regulation of microbial life activities. Quorum sensing is a crucial mechanism by which microorganisms coordinate collective behavior through the secretion and perception of signaling molecules. Signaling molecules, particularly acylhomoserine lactones (AHLs), have been shown to regulate microbial metabolism, proliferation, stress response, and extracellular polymer synthesis by activating quorum sensing mechanisms. In various environmental microbial systems, exogenous supplementation of signaling molecules has been found to significantly enhance microbial adaptability to environmental stresses, improving system stability and recovery efficiency. Current research indicates that quorum sensing has significant potential in regulating the metabolic activity, biofilm formation, and stress resistance of anaerobic ammonia-oxidizing bacteria, providing new insights into addressing the inhibition issues encountered in the practical application of anaerobic ammonia oxidation processes. Furthermore, riboflavin, as an electron shuttle, exhibits excellent biocompatibility and stability. It not only plays a vital role in extracellular electron transport but also acts as an intermediate carrier to promote electron flow between cells or between cells and electron acceptors, thereby enhancing the electron transport efficiency and energy metabolism capacity of microorganisms. Previous studies have shown that riboflavin can significantly promote the growth and metabolic activity of key functional microorganisms in certain anaerobic systems. It is speculated that it can also alleviate the damage caused by toxic stress in anaerobic ammonia oxidation systems by improving extracellular electron transfer efficiency and maintaining a reducing environment.
[0005] However, efficient and sustainable biofortification methods are still lacking for the regulation and recovery of anaerobic ammonia oxidation systems under bisphenol A (BPA) stress. Therefore, there is an urgent need to develop a regulatory strategy based on enhancing quorum sensing and electron transport to achieve rapid recovery of anaerobic ammonia oxidation systems after BPA stress and ensure their stable operation in practical applications. Due to the high cost of signaling molecules, cost-effective signaling molecule regulation strategies are also urgently needed. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention proposes a method for the rapid recovery of the performance of an anaerobic ammonia oxidation system under bisphenol A stress. By adding riboflavin and the supernatant of Shinella yambaruensis, the microbial quorum sensing effect and electron transfer between microorganisms are enhanced, thereby achieving a rapid recovery of the denitrification performance of the anaerobic ammonia oxidation system inhibited by bisphenol A.
[0007] To solve the above problems, the present invention adopts the following technical solution:
[0008] A method for rapid performance recovery of an anaerobic ammonia oxidation system under bisphenol A stress, the method comprising the following steps:
[0009] S1: Construct an anaerobic ammonia oxidation reaction system, introduce wastewater containing bisphenol A and run it continuously, and monitor the total nitrogen removal rate;
[0010] S2: When the total nitrogen removal rate drops to the warning value, it is determined that the anaerobic ammonia oxidation reaction system is inhibited;
[0011] S3: Add riboflavin and Shinellayambaruensis supernatant to the inhibited anaerobic ammonia oxidation system;
[0012] S4: Continuously add riboflavin and Shinella yambaruensis supernatant until the total nitrogen removal rate of the anaerobic ammonia oxidation system recovers to the standard value, and the anaerobic ammonia oxidation system is deemed to be functioning normally.
[0013] Furthermore, in S3, the riboflavin and the supernatant of Shinella yambaruensis are added continuously, the concentration of the riboflavin is 0.5 mg / L, and the riboflavin used is sodium riboflavin or riboflavin in bulk with a purity of ≥95%.
[0014] Furthermore, in S3, the supernatant of the Shinella yambaruensis contains a complex signaling molecule, which contains acyl homoserine lactones (AHLs).
[0015] Furthermore, the composite signal molecule is derived from the supernatant of Shinella yambaruensis bacterial culture and is obtained by organic solvent extraction, concentration, and purification. The specific extraction steps are as follows:
[0016] a) Culture the strain Shinella yambaruensis to the end of the logarithmic growth phase;
[0017] b) Obtain the supernatant of Shinella yambaruensis by centrifugation;
[0018] c) Concentrate the complex signal molecules in the supernatant using solid-phase extraction.
[0019] Furthermore, the optimal culture temperature for Shinella yambaruensis is 28°C, the pH is 7.0, the elution solvent for the solid-phase extraction method is methanol, the final concentration factor is 1000 times, and the total concentration range of the composite signal molecules is 0-5.0 mg / L.
[0020] Furthermore, in S2, the warning value is 60%, and in S4, the standard value is 85%.
[0021] Furthermore, the inhibitory concentration of bisphenol A on the anaerobic ammonia oxidation system is 20–50 mg / L.
[0022] Furthermore, after adding the supernatant of riboflavin and Shinella yambaruensis to the anaerobic ammonia oxidation system to restore its normal function, the activities of enzymes related to anaerobic ammonia oxidation bacteria all recovered to above the initial level, and the relative abundance of the dominant anaerobic ammonia oxidation bacteria Candidatus Jettenia increased to over 75%.
[0023] Advantages of this invention:
[0024] 1. By adding riboflavin and the supernatant of Shinella yambaruensis to the anaerobic ammonia oxidation system, the system performance under bisphenol A stress was rapidly restored, which has significant technical advantages.
[0025] 2. This method is simple to operate, requires no changes to the existing reactor structure and operating parameters, and is suitable for emergency response needs in actual engineering projects.
[0026] 3. The complex signaling molecules extracted from Shinella yambaruensis and added to the anaerobic ammonia oxidation system under bisphenol A stress effectively activated the quorum sensing effect of anaerobic ammonia oxidizing bacteria, enhanced the resistance of the anaerobic ammonia oxidation system to bisphenol A stress, and significantly improved the recovery rate of nitrogen removal performance of the system after being subjected to toxic interference.
[0027] 4. Riboflavin, as a natural electron shuttle, can promote extracellular electron transfer in microorganisms, enhance the metabolic activity of anaerobic ammonia-oxidizing bacteria, improve their stress resistance, and accelerate their functional recovery, thereby significantly shortening the system recovery cycle and improving nitrogen removal efficiency and operational stability.
[0028] 5. Compared with traditional methods such as physical dilution or adjustment of process parameters, this invention has the advantages of rapid response, strong adaptability, economy and environmental protection. It is suitable for promotion and application in urban sewage or high-concentration toxic wastewater treatment systems and has good engineering applicability and promotion value. Attached Figure Description
[0029] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0030] Figure 1 The graph shows the changes in denitrification performance of the anaerobic ammonia oxidation system under bisphenol A stress in Example 1 and the changes in denitrification performance after enhancement.
[0031] in, Figure 1 (a) is a graph showing the change in R1 (control group). Figure 1 (b) is a graph showing the changes in R2 (supernatant group with added riboflavin and Shinellayambaruensis);
[0032] Figure 2 This is a graph showing the changes in the concentration of extracellular polymers (proteins, polysaccharides, and extracellular DNA) of microorganisms in the anaerobic ammonia oxidation system during bisphenol A stress and recovery in Example 1.
[0033] in, Figure 2 (a) is a graph showing the change in R1 (control group). Figure 2 (b) is a graph showing the changes in R2 (supernatant after addition of riboflavin and Shinellayambaruensis);
[0034] Figure 3 To compare the performance of different measures in enhancing the activity of anaerobic ammonia oxidizing bacteria under bisphenol A stress in Example 1. Detailed Implementation
[0035] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, the main variations are obvious within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.
[0036] It should be noted that the technical terms used in this invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.
[0037] This solution provides a method for rapid performance recovery of an anaerobic ammonia oxidation system under bisphenol A stress, which specifically includes the following steps:
[0038] S1: Construct an anaerobic ammonia oxidation reaction system, introduce wastewater containing bisphenol A and run it continuously. The inhibitory concentration of bisphenol A on the anaerobic ammonia oxidation system is 20-50 mg / L, and monitor the total nitrogen removal rate.
[0039] S2: When the total nitrogen removal rate of the system drops below 60%, it is determined that the anaerobic ammonia oxidation reaction system is inhibited;
[0040] S3: Add riboflavin and the supernatant of Shinella yambaruensis to the inhibited anaerobic ammonia oxidation system;
[0041] Riboflavin and the supernatant of Shinella yambaruensis were added continuously. The concentration of riboflavin was 0.5 mg / L. The riboflavin used was sodium riboflavin or riboflavin in bulk with a purity of ≥95%. Riboflavin promotes the abundance of key enzyme genes (hzsA, hzo) in anaerobic ammonia oxidizing bacteria by enhancing electron transfer, thereby achieving rapid recovery of the system's denitrification performance.
[0042] The supernatant of *Shinella yambaruensis* contains a complex signaling molecule containing acyl homoserine lactones (AHLs). This complex signaling molecule was obtained from the supernatant of *Shinella yambaruensis* bacterial culture through organic solvent extraction, concentration, and purification. The specific extraction steps are as follows:
[0043] a) Culture the strain Shinella yambaruensis to the end of the logarithmic growth phase;
[0044] b) Obtain the supernatant of Shinella yambaruensis by centrifugation;
[0045] c) Concentrate the complex signal molecules in the supernatant using solid-phase extraction.
[0046] During the AHLs extraction process, the optimal culture temperature of Shinella yambaruensis was 28℃, the pH was 7.0, the elution solvent for solid-phase extraction was methanol, the final concentration factor was 1000 times, and the total concentration of the complex signaling molecules ranged from 0 to 5.0 mg / L. The signaling molecules are used to activate the signal transduction pathway of anaerobic ammonia-oxidizing bacteria, enhance their stress resistance, promote their activity recovery and extracellular polymer secretion.
[0047] S4: Continuously add riboflavin and the supernatant of Shinella yambaruensis until the total nitrogen removal rate of the anaerobic ammonia oxidation system recovers to more than 85%, at which point the anaerobic ammonia oxidation system is considered to be functioning normally.
[0048] During the recovery process, the components of extracellular polymers all increased. After the anaerobic ammonia oxidation system was restored to normal function by adding riboflavin and the supernatant of Shinella yambaruensis, the activities of enzymes related to anaerobic ammonia oxidation bacteria all recovered to above the initial level, and the relative abundance of the dominant anaerobic ammonia oxidation bacterium Candidatus Jettenia increased to over 75%.
[0049] Riboflavin enhances the anaerobic ammonia oxidation system under bisphenol A stress, and the system performance recovery rate is more than 90% of the total nitrogen removal rate within 7 days. The key to the method of signaling molecules to restore the anaerobic ammonia oxidation system under bisphenol A stress is (1) strengthening microbial cell repair, that is, stimulating the secretion of extracellular polymers (especially proteins and extracellular DNA), which is conducive to microorganisms resisting the impact of adverse factors; (2) enhancing metabolic activity, improving the activity of key enzymes (HZS, HZO), and strengthening the expression of functional genes (hzs, hdh); (3) regulating the microbial community structure and enriching dominant anaerobic ammonia oxidation bacteria.
[0050] Example 1
[0051] As a preferred embodiment of the present invention, the method specifically includes the following steps:
[0052] Two anaerobic ammonia oxidation biofilm reactors were used (R1 was the control group, and R2 was the riboflavin group). The hydraulic retention time was set to 24 hours, and the temperature of the anaerobic ammonia oxidation system was controlled at 36℃ using a constant temperature bath. The influent was artificial wastewater containing ammonium chloride (NH4Cl) 100 mgN / L, sodium nitrite (NaNO2) 132 mgN / L, magnesium sulfate heptahydrate (MgSO4·7H2O) 100 mg / L, calcium chloride dihydrate (CaCl2·2H2O) 180 mg / L, potassium dihydrogen phosphate (KH2PO4) 27 mg / L, sodium bicarbonate (NaHCO3) 500 mg / L, and trace elements. The influent pH was 7.6-7.8.
[0053] The experiment was conducted in three phases, with the following parameters for each phase: Phase I (days 1-37): Initial phase; Phase II (days 38-64): 10 mg / L bisphenol A was added; Phase III (days 65-98): 20 mg / L bisphenol A was added; Phase IV (days 99-171): 30 mg / L bisphenol A was added; Phase V (days 164-203): Recovery phase, with 30 mg / L bisphenol A continuously added in the anaerobic ammonia oxidation system. R1 was the blank control group with no added substances, and R2 was treated with 0.5 mg / L riboflavin and the supernatant of Shinella yambaruensis.
[0054] Enhancement effect: After enhancement with riboflavin and Shinella yambaruensis supernatant, the anaerobic ammonia oxidation system inhibited by bisphenol A was rapidly restored. The specific effects are as follows:
[0055] (1) As Figure 1 As shown, the total nitrogen removal rate of R2 was significantly improved: by day 6 after the addition of riboflavin and Shinellayambaruensis supernatant, the removal rate increased from 48% to over 90%;
[0056] (2) Figure 2 As shown, the activity of the key enzyme HZO in R2 increased to 141.25% of the initial level; the protein in the extracellular polymeric material increased by 162.67%; and the relative abundance of anaerobic ammonia oxidation-related functional genes (such as hzs and hdh) recovered to more than 112% of the initial level.
[0057] Comparative Example 1
[0058] To verify the effectiveness of the method of the present invention, comparative experiments with other enhancement measures were set up.
[0059] Four stable anaerobic ammonia oxidation systems were set up (influent nitrogen load 0.2 g N / L·d, operating temperature 30℃). Bisphenol A (BPA) at 30 mg / L was added to all four reactors until the total nitrogen removal rate decreased to 60%. One reactor served as a control group, with no substances added. The other three anaerobic ammonia oxidation reactors were added with 0.5 mg / L riboflavin, Shinella yambaruensis supernatant, and 0.5 mg / L riboflavin + Shinella yambaruensis supernatant, respectively. After one week of operation, the activity of anaerobic ammonia oxidizing microorganisms in the anaerobic ammonia oxidation systems was measured. Figure 3 As shown, the results indicate that the anaerobic ammonia oxidizing bacteria activity was highest in the supernatant group containing 0.5 mg / L riboflavin and Shinella yambaruensis.
[0060] The above examples have provided a detailed description of this embodiment, but the content is only a preferred embodiment and should not be considered as limiting the scope of implementation of this embodiment; all equivalent changes and improvements made in accordance with the scope of this embodiment should still fall within the patent coverage of this embodiment.
Claims
1. A method for rapid performance recovery of an anaerobic ammonia oxidation system under bisphenol A stress, characterized in that: The method includes the following steps: S1: Construct an anaerobic ammonia oxidation reaction system, introduce wastewater containing bisphenol A and run it continuously, and monitor the total nitrogen removal rate; S2: When the total nitrogen removal rate drops to the warning value, it is determined that the anaerobic ammonia oxidation reaction system is inhibited; S3: Add riboflavin and the supernatant of Shinella yambaruensis to the inhibited anaerobic ammonia oxidation system; S4: Continuously add riboflavin and Shinella yambaruensis supernatant until the total nitrogen removal rate of the anaerobic ammonia oxidation system recovers to the standard value, and the anaerobic ammonia oxidation system is deemed to be functioning normally.
2. The method for rapid performance recovery of an anaerobic ammonia oxidation system under bisphenol A stress according to claim 1, characterized in that: In S3, the riboflavin and the supernatant of Shinella yambaruensis are added continuously. The concentration of the riboflavin is 0.5 mg / L, and the riboflavin used is sodium riboflavin or riboflavin in bulk with a purity of ≥95%.
3. The method for rapid performance recovery of an anaerobic ammonia oxidation system under bisphenol A stress according to claim 1, characterized in that: In S3, the supernatant of the Shinella yambaruensis contains a complex signal molecule, which contains an acyl homoserine lactone.
4. The method for rapid performance recovery of an anaerobic ammonia oxidation system under bisphenol A stress according to claim 3, characterized in that: The composite signal molecule was derived from the supernatant of Shinella yambaruensis bacterial culture and obtained by organic solvent extraction, concentration and purification. The specific extraction steps are as follows: a) Culture the strain Shinella yambaruensis to the end of the logarithmic growth phase; b) Obtain the supernatant of Shinella yambaruensis by centrifugation; c) Concentrate the complex signal molecules in the supernatant using solid-phase extraction.
5. The method for rapid performance recovery of an anaerobic ammonia oxidation system under bisphenol A stress according to claim 4, characterized in that: The optimal culture temperature for *Shinella yambaruensis* is 28°C, the pH is 7.0, the elution solvent for the solid-phase extraction method is methanol, the final concentration factor is 1000 times, and the total concentration range of the composite signal molecules is 0-5.0 mg / L.
6. A method for rapid performance recovery of an anaerobic ammonia oxidation system under bisphenol A stress according to any one of claims 1-5, characterized in that: In S2, the warning value is 60%, and in S4, the standard value is 85%.
7. A method for rapid performance recovery of an anaerobic ammonia oxidation system under bisphenol A stress according to any one of claims 1-5, characterized in that: The inhibitory concentration of bisphenol A on the anaerobic ammonia oxidation system is 20–50 mg / L.
8. A method for rapid performance recovery of an anaerobic ammonia oxidation system under bisphenol A stress according to any one of claims 1-5, characterized in that: After the anaerobic ammonia oxidation system was restored to normal function by adding the supernatant of the riboflavin and Shinella yambaruensis, the activity of enzymes related to anaerobic ammonia oxidation bacteria all returned to above the initial level, and the relative abundance of the dominant anaerobic ammonia oxidation bacteria Candidatus Jettenia increased to over 75%.