A method for alleviating pharmaceutical stress in a short-cut nitrification process using a bacteriophage

Abstract

This invention discloses a method for alleviating drug stress in short-cut nitrification processes using bacteriophages, comprising: inoculating a reactor with a dominant bacterium... Nitrosomonas Activated sludge was introduced into the reactor at 30±1℃ under aerobic conditions, with influent containing ammonia nitrogen and ibuprofen at a pH of 8.0-8.5. Siphoviridae Bacteriophages enhance the denitrification performance of reactors. The method of this invention can promote the activity of ammonia-oxidizing bacteria, strengthen the denitrification effect, and has the advantage of low operating costs, thus broadening the application scenarios of the process.

Description

Technical Field

[0001] This invention relates to the field of biological denitrification technology in wastewater treatment, specifically to a method for alleviating chemical stress in short-cut nitrification processes using bacteriophages. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Short-cut nitrification processes are widely used in the treatment of various high-ammonia-nitrogen wastewaters due to their advantages of low energy consumption and low carbon source consumption. However, wastewater from industries such as pharmaceuticals, aquaculture, and fine chemicals generally contains toxic substances such as antibiotics, antibacterial drugs, and nonsteroidal anti-inflammatory drugs, which can exert continuous drug stress on the short-cut nitrification system and severely stress the metabolic activity of nitrifying microorganisms.

[0004] Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) used for pain relief and is the third most used drug globally. Ibuprofen is one of the active compounds detected in the influent and effluent of wastewater treatment plants, with concentrations in final effluent and surface water ranging from μg / L. -1 up to mg / L -1 This fact reflects the potential risks of ibuprofen in the environment. Ibuprofen has a significant inhibitory effect on the growth and reproduction of some fungi and bacteria. Studies have shown that 10 μg / L... -1 Ibuprofen exposure can lead to a slight decrease in the relative abundance of Proteobacteria and cause changes in the community structure of ammonia-oxidizing bacteria.

[0005] In recent years, researchers have been exploring the challenges of short-cut nitrification processes in maintaining nitrite accumulation. However, in the treatment of ibuprofen-containing wastewater, the toxicity of the influent leads to the inactivation of a large number of core functional bacteria (ammonia-oxidizing bacteria), hindering bacterial metabolism and causing a significant decrease in nitrite accumulation rate, a sharp drop in denitrification efficiency, and even problems such as sludge disintegration and process paralysis. This greatly limits the promotion and application of short-cut nitrification processes. Therefore, it is necessary to find effective strategies to simultaneously address these problems and maintain the stable operation of short-cut nitrification processes under drug stress.

[0006] Patent specification CN117699971B discloses a method for alleviating antibiotic stress in mainstream anaerobic ammonia oxidation processes. This method maintains a dissolved oxygen concentration of 0.3-1.2 mg·L⁻¹ in the aqueous layer of the mainstream anaerobic ammonia oxidation reactor. -1 The dissolved oxygen concentration in the sludge layer was 0.1-0.4 mg·L⁻¹. -1This invention effectively reduces the inhibitory effect of antibiotics on mainstream anaerobic ammonia oxidation. However, this method is not intended to alleviate the stress caused by nonsteroidal anti-inflammatory drugs (NSAIDs).

[0007] Patent specification CN105152325A discloses a method for cultivating oxytetracycline-tolerant anaerobic ammonia oxidation granular sludge cultures. The method employs an upflow anaerobic sludge bed reactor, using anaerobic ammonia oxidation granular sludge as the inoculum and simulated wastewater containing ammonia nitrogen and nitrite nitrogen as the influent. The culture is carried out under anaerobic, light-protected conditions at a temperature of 35±1℃, an influent pH of 8.10±0.14, and a hydraulic retention time of 0.8-2.5 h until the reactor stabilizes. Oxytetracycline is then gradually added to the influent in stages for continued cultivation. Subsequently, anaerobic ammonia oxidation granular sludge is added to the reactor, and cultivation continues until the microbial activity within the reactor is 2-3 times that of the initially inoculated anaerobic ammonia oxidation granular sludge, thus obtaining an oxytetracycline-tolerant anaerobic ammonia oxidation granular sludge culture. This method is not applicable to short-cut nitrification processes. Summary of the Invention

[0008] To address the aforementioned technical problems and shortcomings in the field, this invention provides a method for alleviating drug stress in short-cut nitrification processes using bacteriophages. By precisely regulating the nitrifying bacteria community through specific bacteriophages, the short-cut nitrification reaction can be stably maintained, improving the nitrogen removal performance of the process, reducing operating costs, and broadening the applicable scenarios of the process.

[0009] The specific technical solution is as follows: A method for alleviating pharmaceutical stress in a short-cut nitrification process using bacteriophages includes: The dominant bacteria inoculated in the reactor are Nitrosomonas Activated sludge was introduced into the reactor at 30±1℃ under aerobic conditions, with influent containing ammonia nitrogen and ibuprofen at a pH of 8.0-8.5. Siphoviridae Bacteriophages enhance the denitrification performance of reactors.

[0010] In some preferred embodiments, the method for alleviating drug stress in short-cut nitrification processes using bacteriophages employs a sequencing batch reactor (SBR). This reactor allows for flexible adjustment of parameters at each stage, enabling control of optimal conditions.

[0011] In some preferred embodiments, the method of using bacteriophages to alleviate drug stress in short-cut nitrification processes employs intermittent reactor operation, which is beneficial to the growth and metabolism of activated sludge.

[0012] In some preferred embodiments, the method for using bacteriophages to alleviate drug stress in short-cut nitrification processes involves the reactor operating in two cycles per day. Each cycle includes: 5 minutes of water inlet, stirring and aeration started simultaneously with water inlet and continued for 11 hours, 50 minutes of settling, and 5 minutes of drainage.

[0013] This invention's method is applicable to influent with high ammonia nitrogen content and has a wide range of applications. In some preferred embodiments, the method for using bacteriophages to alleviate chemical stress in short-cut nitrification processes involves an influent ammonia nitrogen concentration of 200 mg / L. -1 above.

[0014] The method for relieving drug pressure in short-cut nitrification process using bacteriophages can be described in which the ammonia nitrogen source in the influent can be (NH4)2SO4.

[0015] The method for relieving drug stress in short-cut nitrification processes using bacteriophages may also include trace elements and sodium bicarbonate (for pH adjustment) in the influent.

[0016] Furthermore, the trace elements may include EDTA-2Na, FeSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, H3BO3, MnCl2·4H2O, Na2MoO4·H2O, NiCl2·6H2O, and ZnSO4·7H2O.

[0017] Furthermore, the influent contains a trace element I solution and a trace element II solution, wherein the trace element I solution contains EDTA-2Na and FeSO4·7H2O, and the trace element II solution contains EDTA-2Na, CoCl2·6H2O, CuSO4·5H2O, H3BO3, MnCl2·4H2O, Na2MoO4·H2O, NiCl2·6H2O, and ZnSO4·7H2O.

[0018] Furthermore, the concentration of trace element I solution in the influent is 1.07 mL / L. -1 The concentration of trace element II solution was 1.07 mL / L. -1 The trace element I solution contains 5 g / L -1 EDTA-2Na and 9.14 g L -1 FeSO4·7H2O; Trace element II solution contains 15 g L -1 EDTA-2Na, 0.24 g / L -1 CoCl2·6H2O, 0.25 g L -1 CuSO4·5H2O, 0.014 g / L -1 H3BO3, 0.99 g / L -1 MnCl2·4H2O, 0.22 g L -1 Na2MoO4·H2O, 0.21 g L -1 NiCl2·6H2O and 0.43 g L-1 ZnSO4·7H2O.

[0019] In some preferred embodiments, the method for alleviating pharmaceutical stress in a short-cut nitrification process using bacteriophages, wherein the ibuprofen concentration in the influent does not exceed 0.5 mg / L. -1 .

[0020] In some preferred embodiments, the method for using bacteriophages to alleviate drug stress in short-cut nitrification processes operates in a volume of 70%-75% of the reactor volume. Exceeding this volume can easily lead to sludge overflow and sludge loss during the influent process.

[0021] In some preferred embodiments, the method for alleviating chemical stress in short-cut nitrification processes using bacteriophages, wherein the aerobic condition refers to a dissolved oxygen concentration of 0.5-0.8 mg / L in the reactor water. -1 For example, 0.6 mg L -1 The present invention has found that dissolved oxygen concentrations of 0.5-0.8 mg / L are suitable. -1 It satisfies the normal metabolism of ammonia-oxidizing bacteria and inhibits nitrite-oxidizing bacteria.

[0022] In some preferred embodiments, the method for alleviating drug stress in short-cut nitrification processes using bacteriophages does not involve sludge discharge during reactor operation. The method of this invention can operate long-term without sludge discharge. This sludge-free technical solution reduces sludge treatment costs in practical engineering applications, resulting in economic benefits.

[0023] In some preferred embodiments, the method for alleviating chemical stress in short-cut nitrification processes using bacteriophages involves adding bacteriophages to the influent at an abundance of 10. 8 PFU mL -1 above.

[0024] In some preferred embodiments, the method for alleviating pharmaceutical stress in short-cut nitrification processes using bacteriophages employs Klebsiella phage PZK-KV19, with accession number CCTCC NO: M 20261189. This bacteriophage is deposited at the China Center for Type Culture Collection (CCTCC) on June 4, 2026, at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0025] In some preferred embodiments, the method of using bacteriophages to alleviate chemical stress in short-cut nitrification processes involves inoculating activated sludge that has been naturally settled for more than 24 hours beforehand. The technical solution of this invention can obtain concentrated activated sludge solely through gravity settling, eliminating the need for centrifugation, flocculation, or purchased bacterial agents, thus simplifying on-site operations and reducing start-up costs.

[0026] Compared with the prior art, the beneficial effects of this invention are as follows: This invention activates the activity of ammonia-oxidizing bacteria and enhances denitrification by releasing substances released from the host through bacteriophage lysis. This invention relies on endogenous microbial ecological regulation, requires no chemical agents, causes no secondary pollution, and produces stable sludge. The technology has low preparation and operation costs, requires no equipment modification, and is suitable for various wastewater scenarios and reactors. Attached Figure Description

[0027] Figure 1 A schematic diagram of an experimental apparatus for alleviating drug pressure in a short-cut nitrification process. In the diagram: 1-Inlet tank; 2-Peristaltic pump; 3-Sequencing batch reactor; 4-Aeration device; 5-Agitator; 6-Dissolved oxygen online monitor; 7-Outlet tank.

[0028] Figure 2 The diagram shows the specific ammonia oxidation activity of reactors R0 and R1.

[0029] Figure 3 Phage abundance map retained for the reactor. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0031] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0032] This embodiment uses a sequencing batch reactor (SBR) device with an effective volume of 1.5 L, such as... Figure 1 As shown, the reactor includes an inlet tank 1, a peristaltic pump 2, a sequencing batch reactor (SBR) 3, an aeration device 4, a stirrer 5, an online dissolved oxygen monitor 6, and an outlet tank 7. The inlet tank 1, peristaltic pump 2, and SBR 3 are connected sequentially. Under the action of the peristaltic pump 2, water enters the SBR 3 from the inlet tank 1. The stirrer 5 is installed inside the SBR 3 for stirring. The aeration device 4 is connected to the SBR 3 to aerate and create aerobic conditions. The online dissolved oxygen monitor 6 is used to monitor the dissolved oxygen concentration in the water within the SBR 3. The outlet tank 7 is connected to the SBR 3 to receive the wastewater from the SBR 3.

[0033] Before inoculation, the remaining sludge was allowed to settle naturally for 24 hours. Then, the sludge was inoculated into sequencing batch reactor 3 with a concentration of 1068 ± 0.1 mg L⁻¹ from a membrane bioreactor in a municipal wastewater treatment plant. -1 Activated sludge (dominant bacteria are) NitrosomonasThe sample was placed in a constant temperature chamber at 30±1℃ and enriched under aerobic conditions. The influent pH was 8.0-8.5, and the influent matrix included ammonia nitrogen ((NH4)2SO4), trace elements, and sodium bicarbonate (used to adjust the pH), with an ammonia nitrogen concentration of 200 mg / L. -1 The dissolved oxygen concentration was controlled at 0.5-0.8 mg / L during the stirring and aeration process. -1 The trace elements include EDTA-2Na, FeSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, H3BO3, MnCl2·4H2O, Na2MoO4·H2O, NiCl2·6H2O, and ZnSO4·7H2O. Furthermore, the influent contains trace element I solution and trace element II solution, with trace element I solution comprising 1.07 mL / L. -1 The concentration of trace element II solution was 1.07 mL / L. -1 Furthermore, the trace element I solution contains 5 g / L -1 EDTA-2Na and 9.14 g L -1 FeSO4·7H2O; Trace element II solution contains 15 g L -1 EDTA-2Na, 0.24 g / L -1 CoCl2·6H2O, 0.25 g L -1 CuSO4·5H2O, 0.014 g / L -1 H3BO3, 0.99 g / L -1 MnCl2·4H2O, 0.22 g L -1 Na2MoO4·H2O, 0.21 g L -1 NiCl2·6H2O and 0.43 g L -1 ZnSO4·7H2O.

[0034] Sequencing batch reactor 3 operates intermittently, with an operating volume of 75% of the reactor's capacity. It operates for two cycles per day. Each cycle includes: 5 minutes of influent intake, followed by simultaneous agitation and aeration for 11 hours, 50 minutes of settling, and 5 minutes of effluent discharge. No sludge is discharged during the operation of sequencing batch reactor 3. At the end of each cycle, the specific ammonia oxidation activity of sequencing batch reactor 3 is monitored to examine the impact of bacteriophages on reactor activity.

[0035] Two sequencing batch reactors, R0 and R1, were set up in this experiment. For example... Figure 2 As shown, in the initial stage, the specific ammonia oxidation activity of reactors R0 and R1 is 32.1 mgN gVSS. -1 h -1and 29.1 mgN gVSS -1 h -1 Thirty days later, 0.5 mg L was added to reactor R1. -1 After ibuprofen administration, the specific ammonia oxidation activity of reactor R1 decreased to 26.4 mgN gVSS. -1 h -1 With reactor R0 unchanged, the specific ammonia oxidation activity remains at 31.9 mgN gVSS. -1 h -1 Then, bacteriophage Klebsiella phage PZK-KV19 (CCTCC NO: M 20261189) was added to reactor R1, while reactor R0 remained unchanged. The concentration and separation of the bacteriophage were as follows: 20 L of activated sludge mixture was allowed to stand for 0.5 h. The supernatant was filtered through two stages (0.45 μm and 0.22 μm) and then concentrated to 10 mL using a 100 kDa ultrafiltration membrane (Millipore, America) on ice. The concentrate was mixed with the host culture (host bacterium Klebsiella variicola (Ningbo Mingzhou Biotechnology Co., Ltd., catalog number BMZ147331)) at a volume ratio of 1:10 and cultured at 30℃ and 150 rpm for 24 h with shaking until lysis and clarification. The lysate was centrifuged at 12000 rpm for 5 min, and the supernatant was aseptically filtered through a 0.22 μm filter to obtain the original bacteriophage solution. Add to 5 mL of semi-solid culture medium, pour onto the bottom plate, and incubate overnight at 30°C with the plate inverted. Repeat single-spot purification three times to obtain pure phage. Phage identification method: Extract genomic DNA from the phage and perform whole-genome identification (SEQ ID NO: 1). Phage addition method: Pick a single phage from the overnight double-layer plate and inoculate it with Klebsiella variicola (product number BMZ147331), a logarithmic-phase host bacterium purchased from Ningbo Mingzhou Biotechnology Co., Ltd., and amplify by shaking at 30°C and 150 rpm for 3 h, followed by centrifugation at 12000 rpm for 5 min. After amplification, add the phage to the reactor until the abundance reaches 10. 8 PFU mL -1 Considering the potential loss of bacteriophages, new bacteriophages are added regularly each day to ensure that the abundance of bacteriophages in the influent remains stable at 10. 8 PFU mL -1 .

[0036] like Figure 3 As shown, after the addition of bacteriophages, the abundance of bacteriophages in the reactor effluent remained at 1.1 PFU / mL for the first four days. -1The phage removal rate in the reactor gradually increased from 0.11 LRV to 1.03 LRV from day 6 to day 24 after phage addition. Then, from day 26 to day 30, the phage abundance gradually stabilized, fluctuating around 0.80 LRV. This indicates that some phage was retained by the process. During this process, the specific ammonia oxidation activity of reactor R1 increased to 55.1 mgN / gVSS. -1 h -1 This demonstrates that the present invention has significant advantages in alleviating the stress on ibuprofen caused by short-path nitration processes.

[0037] In summary, the method of the present invention can promote the activity of ammonia-oxidizing bacteria, enhance the denitrification effect, has the advantage of low operating cost, and broadens the application scenarios of the process.

[0038] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for alleviating pharmaceutical stress in short-cut nitrification processes using bacteriophages, characterized in that, include: The dominant bacteria inoculated in the reactor are Nitrosomonas Activated sludge was introduced into the reactor at 30±1℃ under aerobic conditions, with influent containing ammonia nitrogen and ibuprofen at a pH of 8.0-8.

5. Siphoviridae Bacteriophages enhance the denitrification performance of reactors.

2. The method according to claim 1, characterized in that, The reactor is a sequencing batch bioreactor; The reactor operates intermittently. The reactor operates in two cycles per day. Each cycle includes: 5 minutes of water inlet, stirring and aeration starting simultaneously with water inlet and continuing for 11 hours, 50 minutes of settling, and 5 minutes of drainage.

3. The method according to claim 1, characterized in that, The ammonia nitrogen concentration in the influent is 200 mg / L. -1 above.

4. The method according to claim 1, characterized in that, The concentration of ibuprofen in the influent shall not exceed 0.5 mg / L. -1 .

5. The method according to claim 1, characterized in that, The operating volume is 70%-75% of the reactor volume.

6. The method according to claim 1, characterized in that, Aerobic conditions refer to a dissolved oxygen concentration of 0.5-0.8 mg / L in the reactor water. -1 .

7. The method according to claim 1, characterized in that, No sludge is discharged during reactor operation.

8. The method according to claim 1, characterized in that, Bacteriophages were added to the influent to an abundance of 10. 8 PFU mL -1 above.

9. The method according to claim 1, characterized in that, The bacteriophage is Klebsiella phage PZK-KV19, with accession number CCTCC NO: M 20261189.

10. The method according to claim 1, characterized in that, The inoculated material is activated sludge that has been naturally settled for more than 24 hours.

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