Method for realizing efficient denitrification through directional enrichment of antibiotic-resistant denitrification functional flora

Abstract

The invention relates to the technical field of biological denitrification of sewage, in particular to a method for realizing efficient denitrification through directional enrichment of antibiotic-resistant denitrification functional flora. According to the invention, an activated sludge stress system of'high-concentration antibiotic stress effect-antibiotic resistance gene multi-protection-denitrification function gene co-expression 'and an activated sludge strengthening system of'high-concentration antibiotic remaining effect-resistant denitrification function strengthening-resistant denitrification flora proliferation' are constructed; through a two-stage screening reinforcement system of antibiotic-resistant denitrification functional florae, the antibiotic-resistant denitrification functional florae of activated sludge are increased, and the denitrification efficiency of high-concentration antibiotic wastewater is remarkably improved. Compared with other denitrification methods for wastewater containing high-concentration antibiotics, the method has the advantages of simplicity, economy, high efficiency and the like. Due to high operation stability and efficient biological nitrogen removal efficiency, the method has development and engineering application prospects, and a new technical path is provided for efficient nitrogen removal of wastewater containing high-concentration antibiotics.

Description

Technical Field

[0001] The present invention relates to the technical field of biological denitrification of sewage, and in particular to a method for achieving efficient denitrification through the targeted enrichment of antibiotic-resistant denitrifying functional bacteria. The method is suitable for the treatment of high-concentration antibiotic wastewater such as pharmaceutical wastewater, livestock breeding wastewater and aquaculture wastewater. Background Art

[0002] Antibiotics, as effective drugs for preventing and treating diseases and as growth promoters for animals, are widely used in industries such as human medicine, animal husbandry, and aquaculture. However, most antibiotics are difficult to fully absorb and degrade by organisms. Approximately 80% of these antibiotics are excreted into the environment in the form of parent antibiotics or metabolites through feces or urine, ultimately contaminating wastewater treatment plants, where they inevitably impact the denitrification efficiency of activated sludge systems.

[0003] Biological denitrification is an effective treatment technology for nitrogen pollution in water. Traditional biological denitrification processes primarily rely on the synergistic action of nitrifying and denitrifying bacteria in activated sludge to remove total nitrogen. However, these processes present technical challenges when exposed to high antibiotic concentrations. First, nitrifying bacteria are highly sensitive to environmental changes. When antibiotic concentrations exceed 5 mg / L, they severely inhibit the activity of nitrifying bacteria, thereby reducing ammonia oxidation rates. Second, the denitrification process is affected by toxic inhibition, leading to nitrite accumulation and unstable denitrification efficiency. Existing research has shown that direct addition of denitrifying agents or adjustments to system operation strategies can improve denitrification performance to a certain extent. However, the added denitrifying bacteria often suffer from poor adaptability and easily inhibited metabolic activity. Therefore, how to adjust system operation strategies to achieve targeted enrichment of activated sludge-resistant denitrifying bacteria, thereby achieving efficient denitrification in activated sludge under high antibiotic concentrations, has become an urgent issue.

[0004] Related studies have found that antibiotic-resistant denitrifying bacteria (such as Thauera and Pseudomonas) carry antibiotic resistance genes that undergo horizontal gene transfer (HGT) under antibiotic stress, activating multiple protective mechanisms of antibiotic resistance genes and achieving coordinated expression of denitrification and antibiotic resistance genes. Therefore, under the stress and selection effect of high-concentration antibiotics, the differences in antibiotic resistance among different bacterial genera in the activated sludge of sewage treatment plants promote differential responses among different bacterial genera. Simultaneously, the antibiotic resistance genes carried by activated sludge microorganisms in sewage treatment plants activate multiple protective mechanisms, enabling them to maintain normal metabolism and reproduction under high-concentration antibiotic stress, thereby achieving the goal of enriching antibiotic-resistant denitrifying bacteria. After completing the stress screening and enrichment of antibiotic-resistant denitrifying bacteria, the pressure of high-concentration antibiotic stress is promptly released, and the role of high-concentration antibiotic carryover (i.e., the persistent impact of residual antibiotics on subsequent microbial communities) in the directional regulation of bacterial communities is utilized to achieve enhanced enrichment of antibiotic-resistant denitrifying bacteria.

[0005] Based on the above background, the present invention intends to achieve the effect of targeted enrichment of antibiotic-resistant denitrifying functional bacteria to achieve efficient denitrification by constructing an activated sludge stress system of "high-concentration antibiotic stress effect-multiple protection of antibiotic resistance genes-co-expression of denitrification functional genes" and an activated sludge strengthening system of "high-concentration antibiotic residual effect-enhancement of resistance denitrification function-proliferation of resistant denitrifying bacteria". Summary of the Invention

[0006] The present invention aims to provide a method for achieving efficient denitrification through the targeted enrichment of antibiotic-resistant denitrifying functional bacteria, thereby addressing the aforementioned problems of the prior art. The present invention proposes a two-stage implementation strategy for the enrichment of antibiotic-resistant denitrifying functional bacteria, which reduces the operational difficulty of enriching the antibiotic-resistant denitrifying functional bacteria and improves the feasibility of engineering applications.

[0007] The present invention identifies high-concentration enrofloxacin (ENR) stress, multiple protections by antibiotic resistance genes, and a high-concentration ENR residual effect as screening conditions for the targeted enrichment of resistant denitrifying functional bacterial communities. Activated sludge with excellent nitrification performance is used as the inoculum sludge. The inoculum sludge is first subjected to stress-enrichment of antibiotic-resistant denitrifying functional bacterial communities. By leveraging the differential responses of activated sludge microorganisms under high-concentration ENR stress, the activation of protective mechanisms by denitrifying functional bacterial genera carrying multiple antibiotic resistance genes, and the coordinated expression of antibiotic resistance and denitrifying functional genes, a "high-concentration ENR stress effect - multiple protections by antibiotic resistance genes - co-expression of denitrifying functional genes" activated sludge stress system is successfully constructed. This system regulates the microbial community during this stage, increases the relative abundance of denitrifying functional bacterial genera, and achieves stress-enrichment of antibiotic-resistant denitrifying functional bacterial communities. At the end of the stress enrichment phase, the relative abundance of ammonia oxidizing bacteria (AOB) in the nitrifying bacterial community reached 0.021%, the relative abundance of complete ammonia oxidizing bacteria (Comammox) reached 0.016%, a 50.0% increase, and the relative abundance of nitrite oxidizing bacteria (NOB) reached 0.177%. Among the denitrifying bacteria, the relative abundance of denitrifying bacteria (DNB) reached 29.540%, a 27.8% increase, and the relative abundance of simultaneous nitrifying and denitrifying bacteria (SND) reached 20.578%, a 65.4% increase. The presence of antibiotic resistance genes increased by 9.1%. A total nitrogen removal rate exceeding 80% was achieved in just five cycles during this phase and remained stable throughout the stress phase. The next step was to enhance the enrichment of antibiotic-resistant denitrifying bacteria. By eliminating the stress caused by the residual effects of high ENR concentrations, the antibiotic-resistant denitrifying bacteria further proliferated. This resulted in a successful activated sludge enhancement system combining the "residual effects of high ENR concentrations, enhanced denitrification, and increased resistant denitrification bacteria," achieving enhanced enrichment of antibiotic-resistant denitrifying bacteria. At the end of the enhanced enrichment phase, the relative abundance of ammonia-oxidizing bacteria in the nitrifying bacterial population reached 0.063%, a 200% increase compared to the previous phase, and the relative abundance of complete ammonia-oxidizing bacteria reached 0.021%, a 30.3% increase compared to the previous phase. The relative abundance of denitrifying bacteria remained high, with a relative abundance of 22.826% for denitrifiers and 14.291% for simultaneous nitrifiers and denitrifiers. The presence of antibiotic resistance genes also increased by 2.4% compared to the inoculated sludge. Total nitrogen removal efficiency remained above 75% throughout this phase.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a method for achieving efficient denitrification by targeted enrichment of antibiotic-resistant denitrifying functional bacteria, comprising the following steps:

[0010] a. Use activated sludge with good nitrification performance as inoculated sludge;

[0011] b. The inoculated sludge and wastewater containing a high concentration of enrofloxacin were mixed evenly and aerated to obtain an antibiotic-resistant denitrification functional bacterial community stress enriched activated sludge;

[0012] c. The activated sludge enriched by the antibiotic-resistant denitrification functional bacteria and the wastewater without enrofloxacin are evenly mixed and aerated to obtain the activated sludge enriched by the antibiotic-resistant denitrification functional bacteria, thereby completing the efficient denitrification treatment of the wastewater.

[0013] In the present invention, activated sludge with good nitrification performance is used as inoculum sludge, and the inoculum sludge and artificial synthetic wastewater are evenly mixed according to a certain volume ratio. Through the high-concentration enrofloxacin (ENR) stress effect and the high-concentration ENR legacy effect, the targeted enrichment of antibiotic-resistant denitrifying functional bacteria is achieved, thereby achieving the goal of efficient denitrification of high-concentration antibiotic wastewater, as follows:

[0014] High-concentration enrofloxacin (ENR) stress effect stage: activated sludge with good nitrification performance is used as inoculum sludge, and artificial synthetic wastewater containing high-concentration ENR is added to screen for stress effects, thereby obtaining activated sludge enriched with antibiotic-resistant denitrification functional bacteria under stress;

[0015] High-concentration ENR legacy effect stage: wastewater without high-concentration ENR is added to the activated sludge enriched with antibiotic-resistant denitrification functional bacteria to enhance the legacy effect, thereby obtaining activated sludge enriched with antibiotic-resistant denitrification functional bacteria, and finally completing the efficient denitrification treatment of high-concentration antibiotic wastewater.

[0016] Preferably, in step a, the activated sludge has an ammonia nitrogen removal rate higher than 98%; the activated sludge is derived from flocculent sludge from a sewage treatment plant or excess sludge activated by air exposure.

[0017] Preferably, in step b, the concentration of enrofloxacin in the enrofloxacin-containing wastewater is 20±1 mg / L.

[0018] Preferably, in step b, the volume ratio of the activated sludge to the wastewater is 1:1; the MLSS of the material obtained after the activated sludge and the wastewater are evenly mixed is maintained between 4500 and 5000 mg / L; the temperature of the aeration treatment is 23-25°C, the rotation speed is 90-95 rad / min, and the pH is 7.5-8.0; the concentration of ammonia nitrogen in the wastewater is 30-35 mg / L, the concentration of COD is 300±10 mg / L, the concentration of trace elements is 1 mL / L, and the drainage ratio of each cycle is 50%;

[0019] In step c, the volume ratio of the activated sludge enriched by the antibiotic-resistant denitrifying functional bacteria and the wastewater is 1:1; the MLSS of the material obtained after the activated sludge enriched by the antibiotic-resistant denitrifying functional bacteria and the wastewater are evenly mixed is maintained between 4500 and 5000 mg / L; the temperature of the aeration treatment is 23-25°C, the rotation speed is 90-95 rad / min, and the pH is 7.5-8.0; the concentration of ammonia nitrogen in the wastewater is 30-35 mg / L, the concentration of COD is 300±10 mg / L, the concentration of trace elements is 1 mL / L, and the drainage ratio of each cycle is 50%.

[0020] Preferably, in step b, the aeration treatment operation cycle is 9 cycles; each cycle starts with the start of aeration treatment and ends when the ammonia nitrogen degradation degree reaches 98% or the ammonia nitrogen concentration remains stable;

[0021] In step c, the aeration treatment is operated for 6 cycles; each cycle starts with the start of aeration treatment and ends when the ammonia nitrogen degradation degree reaches 98% or the ammonia nitrogen concentration remains stable.

[0022] Further preferably, the aeration treatment of step b and step c is performed for a total of 15 cycles, and the concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen are sampled and determined every 1 hour in the 1st cycle, the 7th cycle and the 14th cycle, and only the concentrations of the three nitrogens in the inlet and outlet water are determined in the remaining cycles.

[0023] Preferably, the trace elements include components with the following concentrations:

[0024] 4.29g / LNa2EDTA, 1.99g / LFeCl2·4H2O, 0.08g / LMnCl2·2H2O, 0.02 / L g CuCl2·2H2O, 0.07g / LZnCl2, 0.02g / LNa2MoO4·2H2O, 0.03g / LNa2WoO4·2H2O and 0.06g / LH3BO3.

[0025] The present invention provides application of the above method in efficient denitrification of high-concentration antibiotic wastewater.

[0026] Preferably, the high-concentration antibiotic wastewater includes pharmaceutical wastewater, livestock breeding wastewater or aquaculture wastewater.

[0027] The present invention discloses the following technical effects:

[0028] The present invention uses a two-stage enrichment system of antibiotic-resistant denitrifying functional bacteria to increase the antibiotic-resistant denitrifying functional bacteria of activated sludge and significantly improve the denitrification efficiency. After the stress enrichment is completed, the relative abundance of ammonia oxidizing bacteria in the nitrifying functional bacteria is 0.021%, the relative abundance of complete ammonia oxidizing bacteria is 0.016%, an increase of 50.0%, the relative abundance of nitrite oxidizing bacteria is 0.177%, the relative abundance of denitrifying bacteria in the denitrifying functional bacteria is 29.540%, an increase of 27.8%, the relative abundance of synchronous nitrification and denitrification bacteria is 20.578%, an increase of 65.4%, and the antibiotic resistance gene increases by 9.1%. After five cycles of operation in this stage, a total nitrogen removal rate of more than 80% is achieved, and the relative abundance of nitrifying bacteria in the denitrifying functional bacteria is 0.021%, an increase of 50.0%, and the relative abundance of nitrite oxidizing bacteria is 0.177%. The collection stage remained stable; at the end of the enhanced enrichment, the relative abundance of ammonia oxidizing bacteria in the nitrifying functional bacteria was 0.063%, an increase of 200% compared with the previous stage, and the relative abundance of complete ammonia oxidizing bacteria was 0.021%, an increase of 30.3% compared with the previous stage; the relative abundance of denitrifying functional bacteria still maintained a high level, among which the relative abundance of denitrifying bacteria was 22.826%, and the relative abundance of synchronous nitrification and denitrifying bacteria was 14.291%. The antibiotic resistance gene still increased by 2.4% compared with the inoculated sludge, and the total nitrogen removal rate was maintained at more than 75% during this stage. Compared with other methods for denitrification of wastewater containing high-concentration antibiotics, the present invention has the advantages of simplicity, economy, and high efficiency. Because of its high operational stability and efficient biological denitrification efficiency, the present invention has simple development and engineering application prospects, and provides a new technical path for the efficient denitrification of wastewater containing high-concentration antibiotics. 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. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 NH4 for the control group in cycles 1-15 + -N, NO2 - -N and NO3 - -N and total nitrogen removal rate change curve; among them, NH4 + -N inf is the ammonia nitrogen content in the influent, NH4 + -N eff is the ammonia nitrogen content in the effluent, NO2 - -N inf is the nitrite nitrogen content of the influent, NO2 - -N eff is the nitrite nitrogen content in the effluent, NO3-N inf is the nitrate nitrogen content of the influent, NO3- -N eff is the nitrate nitrogen content in the effluent, TN removal rate is the total nitrogen removal rate;

[0031] Figure 2 NH4 in the experimental group from the 1st to the 15th cycle + -N, NO2 - -N and NO3 - -N and total nitrogen removal rate change curve; among them, NH4 + -N inf is the ammonia nitrogen content in the influent, NH4 + -N eff is the ammonia nitrogen content in the effluent, NO2 - -N inf is the nitrite nitrogen content of the influent, NO2 - -N eff is the nitrite nitrogen content in the effluent, NO3-N inf is the nitrate nitrogen content of the influent, NO3 - -N eff is the nitrate nitrogen content in the effluent, TN removal rate is the total nitrogen removal rate;

[0032] Figure 3 NH4 for the 1st, 7th and 14th full cycles of the experimental group + -N, NO2 - -N, NO3 - -N and TN change curves;

[0033] Figure 4 The relative abundance and composition distribution of denitrification functional bacteria in the control group and the experimental group at the 7th and 14th cycles;

[0034] Figure 5 represents the absolute copy number and relative abundance of antibiotic resistance genes in the control group and the experimental group at the 7th and 14th cycles; Tetracycline is tetracycline, Sulfonamide is sulfonamide, MLSB is macrolide-lincosamide-streptogramin B, FCA is fluoroquinolone, Beta_Lactamase is β-lactamase, and Aminoglycoside is aminoglycoside. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Example 1

[0041] A method for achieving efficient denitrification by targeted enrichment of antibiotic-resistant denitrifying functional bacteria, comprising the following steps:

[0042] Preparation of inoculum sludge: Activated sludge (or excess sludge activated by air exposure) taken from the aeration tank of a municipal sewage treatment plant in Beijing was added to the reactor, and artificial synthetic wastewater was added at a volume ratio of 1:1 (the concentration of ammonia nitrogen was controlled at 35 mg / L, the concentration of COD was controlled at 300 mg / L, the concentration of NaHCO3 was 840 mg / L, the concentration of KH2PO4 was 50 mg / L, the concentration of NaCl was 590 mg / L, the concentration of KCl was 80 mg / L, the concentration of trace elements was 1 mL / L, and the trace element composition was as follows: 4.29 g / L Na2EDTA, 1.99 g / L FeCl2·4H2O, 0.08 g / L MnCl2·2H2O, 0.02 g / L CuCl2·2H2O, 0.07g / LZnCl2, 0.02g / LNa2MoO4·2H2O, 0.03g / LNa2WoO4·2H2O and 0.06g / LH3BO3) were aerated. The ammonia nitrogen concentration was measured at the end of each cycle. One cycle was defined as when the ammonia nitrogen degradation reached 98% or above, and the cycle was continued for 3 cycles. Each cycle started with the start of aeration treatment and ended when the ammonia nitrogen degradation reached 98% or the ammonia nitrogen concentration remained stable. The activated sludge system reached stability and the activated sludge was diluted with ultrapure water to a MLSS (mixed liquor suspended solids concentration) between 9000 and 10000 mg / L. In this embodiment, the MLSS was controlled at 10000 mg / L to serve as inoculum sludge.

[0043] Stress enrichment of antibiotic-resistant denitrifying functional bacteria (high-concentration ENR stress screening): The inoculated sludge and synthetic wastewater (ammonia nitrogen concentration was controlled at 35 mg / L, COD concentration was controlled at 300 mg / L, NaHCO3 concentration was 840 mg / L, KH2PO4 concentration was 50 mg / L, NaCl concentration was 590 mg / L, KCl concentration was 80 mg / L, enrofloxacin concentration was 20 mg / L, trace element concentration was 1 mL / L, and the trace element composition was as follows: 4.29 g / L Na2EDTA, 1.99 g / LFeCl2·4H2O, 0.08 g / L MnCl2·2H2O, 0.02 g / L CuCl2·2H2O, 0.07 g / L ZnCl2, 0.02 g / LNa2MoO4·2H2O, 0.03 g / LNa2WoO4·2H2O and 0.06 g / LH3BO3) were mixed uniformly in a volume ratio of 1:1 and added to a 500 mL conical flask to 250 mL, and placed in a constant temperature shaking box (rotation speed of 95 rad / min), pH was controlled at 7.5-8.0, temperature was controlled at 25°C, MLSS was maintained between 4500 and 5000 mg / L, and 9 cycles were continuously operated with a drainage ratio of 50% in each cycle to stress and enrich the antibiotic-resistant denitrifying functional bacterial community. During the first and seventh cycles, samples were taken every hour to measure ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen concentrations. During the remaining cycles, only the concentrations of the three nitrogens (ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen) in the inlet and outlet water were measured. One cycle was run daily, and the specific cycle duration was based on the ammonia nitrogen degradation rate reaching 98% or the ammonia nitrogen concentration being continuously stable. Each cycle duration was the same or different (4-24 hours). That is, each cycle started with the start of aeration treatment and ended when the ammonia nitrogen degradation rate reached 98% or the ammonia nitrogen concentration was continuously stable. The ninth cycle ended, indicating the completion of the stress enrichment of the antibiotic-resistant denitrifying functional bacterial community.

[0044] Enhanced enrichment of antibiotic-resistant denitrifying functional bacteria (legacy effect): The activated sludge at the end of the 9th cycle was allowed to settle until the mud and water were completely separated, and 50% of the supernatant was discharged to obtain the stationary sludge; the stationary sludge was mixed with artificial synthetic wastewater (the concentration of ammonia nitrogen was controlled at 35 mg / L, the concentration of COD was controlled at 300 mg / L, the concentration of NaHCO3 was 840 mg / L, the concentration of KH2PO4 was 50 mg / L, the concentration of NaCl was 590 mg / L, the concentration of KCl was 80 mg / L, the concentration of enrofloxacin was 0 mg / L, the concentration of trace elements was 1 mL / L, and the trace element composition was as follows: 4.29 g / L Na2EDTA, 1.99 g / L FeCl2·4H2O, 0.08 g / LMnCl2·2H2O, 0.02 g / L CuCl2·2H2O, 0.07 g / L ZnCl2, 0.02 g / L Na2MoO4·2H2O, 0.03g / LNa2WoO4·2H2O and 0.06g / LH3BO3) were mixed in a volume ratio of 1:1. Specifically, the stationary sludge and synthetic wastewater were mixed evenly and then added to a 500mL conical flask to 250mL. The environmental conditions were basically the same as those in the 9 cycles of the stress enrichment of antibiotic-resistant denitrification functional bacteria, and the MLSS was also maintained between 4500 and 5000mg / L. The only difference was that enrofloxacin was no longer added from the beginning of this stage, that is, the synthetic wastewater did not contain enrofloxacin. The operation was continuous for 6 cycles, and the drainage ratio of each cycle was 50%. In the 14th cycle (stress enrichment cycle + enhanced enrichment cycle, a total of 15 cycles, the 14th cycle here refers to the 14th cycle after the two stages are added together), samples are taken every 1 hour to measure the concentrations of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen. In the remaining cycles, only the three nitrogen concentrations (ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen) of the inlet and outlet water are measured. One cycle is run every day. The specific cycle duration is based on the ammonia nitrogen degradation degree reaching 98% or the ammonia nitrogen concentration being continuously stable. The cycle duration is 4-24 hours, and the duration of each cycle is the same or different, that is, the time of each cycle starts with the start of aeration treatment and ends with the ammonia nitrogen degradation degree reaching 98% or the ammonia nitrogen concentration being continuously stable. The experimental group experiment is completed at the end of the 14th cycle (the enhanced enrichment of the antibiotic-resistant denitrification functional bacteria is completed), and activated sludge containing antibiotic-resistant denitrification functional bacteria is obtained, which is recorded as the experimental group.

[0045] Comparative Example 1

[0046] In this example, the inoculated sludge was used to directly treat synthetic wastewater without antibiotics for 15 consecutive cycles to obtain control sludge. The specific steps are as follows:

[0047] Preparation of inoculum sludge: Activated sludge (or excess sludge activated by aeration) from an aeration tank of a municipal sewage treatment plant in Beijing was added to the reactor, and artificial synthetic wastewater was added at a volume ratio of 1:1 (the concentration of ammonia nitrogen was controlled at 35 mg / L, the concentration of COD was controlled at 300 mg / L, the concentration of NaHCO3 was 840 mg / L, the concentration of KH2PO4 was 50 mg / L, the concentration of NaCl was 590 mg / L, the concentration of KCl was 80 mg / L, the concentration of trace elements was 1 mL / L, and the trace element composition was as follows: 4.29 g / L Na2EDTA, 1.99 g / L FeCl2·4H2O, 0.08 g / L MnCl2·2H2O, 0.02 g / L CuCl2·2H2O, 0.07g / LZnCl2, 0.02g / LNa2MoO4·2H2O, 0.03g / LNa2WoO4·2H2O and 0.06g / L H3BO3) were aerated, and the ammonia nitrogen concentration was measured at the end of each cycle. When the degree of ammonia nitrogen degradation reached more than 98%, it was considered one cycle. This cycle was continued for three cycles. When the activated sludge system reached stability, the activated sludge was diluted with ultrapure water to make its MLSS reach 10000mg / L, which was used as inoculum sludge.

[0048] Preparation of control sludge: inoculated sludge and artificial synthetic wastewater (the concentration of ammonia nitrogen was controlled at 35 mg / L, the concentration of COD was controlled at 300 mg / L, the concentration of NaHCO3 was 840 mg / L, the concentration of KH2PO4 was 50 mg / L, the concentration of NaCl was 590 mg / L, the concentration of KCl was 80 mg / L, the concentration of trace elements was 1 mL / L, and the trace element composition was as follows: 4.29 g / LNa2EDTA, 1.99 g / LFeCl2·4H2O, 0.08 g / LMnCl2·2H2O, 0.02 g / L CuCl2·2H2O, 0.07 g / LZnCl2, 0.02 g / LNa2MoO4·2H2O, 0.03 g / LNa2WoO4·2H2O and 0.06 g / L g H3BO3) were mixed in a volume ratio of 1:1, and specifically, the mixture was evenly added to a 500 mL conical flask to 250 mL, placed in a constant temperature shaking box (rotation speed of 90-95 rad / min), pH was controlled at 7.5-8.0, temperature was controlled at 23-25°C, and 15 cycles were continuously operated. The drainage ratio of each cycle was 50%, and the concentrations of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the inlet and outlet water were measured in each cycle. One cycle was operated per day for a total of 15 cycles. After the 15th cycle, the control group experiment was completed, and a control activated sludge was obtained, which was recorded as the control group.

[0049] Effect Example 1

[0050] The effects of Example 1 and Comparative Example 1 were investigated, and the results were as follows: Figure 1-Figure 5 shown.

[0051] Figure 1 It can be seen that the total nitrogen removal rate of the control group was maintained at 30%-50% in 15 cycles, with an average total nitrogen removal rate of 36.44%. During the reaction process, the concentrations of nitrite nitrogen and nitrate nitrogen were maintained at a low level (below 5 mg / L).

[0052] Figure 2 It can be seen that after the addition of high concentration of enrofloxacin, the total nitrogen removal rate of the experimental group increased from 49.1% to above 80% in only 5 cycles, and remained stable for 4 cycles. After the addition of enrofloxacin was stopped, it still basically maintained at a level above 75%.

[0053] Figure 3 It can be seen that the concentration of ammonia nitrogen in the first cycle of the experimental group decreased to 0 mg / L at 3 hours, but the nitrate nitrogen concentration in the effluent was 16.27 mg / L and the nitrite nitrogen concentration was 0.05 mg / L; the reaction time of the seventh cycle was 24 hours, and the concentrations of ammonia nitrogen and total nitrogen decreased in the same trend, with the final concentration of ammonia nitrogen being 3.04 mg / L and the final concentration of total nitrogen being 5.70 mg / L. There was no accumulation of nitrite nitrogen during this period, and the final effluent nitrate nitrogen concentration was only 2.66 mg / L; the reaction time of the fourteenth cycle was 15 hours, with the final ammonia nitrogen concentration being 0.59 mg / L, the nitrate nitrogen concentration being 8.59 mg / L, and the total nitrogen concentration being 11.47 mg / L. There was also no accumulation of nitrite nitrogen during this period.

[0054] Figure 4 It can be seen that compared with the experimental group, the relative abundance of ammonia oxidizing bacteria (AOB) in the control group (Control) decreased to 0.021% in the stress stage (ENR) and increased to 0.063% in the enhanced stage (ENR-P); the relative abundance of complete ammonia oxidizing bacteria (Comammox) increased to 0.016% in the stress stage and further increased to 0.021% in the enhanced stage; the relative abundance of nitrite oxidizing bacteria (NOB) decreased to 0.177% in the stress stage and increased to 0.275% in the enhanced stage; the relative abundance of denitrifying bacteria (DNB) increased to 29.540% in the stress stage and decreased to 22.825% in the enhanced stage; the relative abundance of simultaneous nitrifying and denitrifying bacteria (SND) increased to 20.578% in the stress stage and decreased to 14.291% in the enhanced stage. Through the stress pressure screening effect of high concentration enrofloxacin and the strengthening effect of the residual effect after the release of stress pressure, the above-mentioned changes in the denitrification functional bacteria are regulated, the targeted enrichment of antibiotic-resistant denitrification functional bacteria is achieved, and the goal of efficient denitrification is achieved.

[0055] Figure 5It can be seen that after the two stages of high-concentration ENR stress screening and residual effect, the absolute copy number of antibiotic resistance genes in the experimental group increased by 9.1% and 2.4% respectively compared with the control group, and the relative proportions of different types of antibiotic resistance genes changed. For example, the relative proportion of sulfonamide antibiotic resistance genes in the control group was 26.729%, the relative proportion in the high-concentration ENR stress stage was 23.348%, and the relative proportion in the high-concentration ENR residual effect stage reached 53.151%.

[0056] 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 method for achieving efficient denitrification by targeted enrichment of antibiotic-resistant denitrifying functional bacteria, characterized in that: The following steps are involved: a. Use activated sludge with good nitrification performance as inoculated sludge; b. The inoculated sludge and wastewater containing a high concentration of enrofloxacin were mixed evenly and aerated to obtain an antibiotic-resistant denitrification functional bacterial community stress enriched activated sludge; c. The activated sludge enriched by the antibiotic-resistant denitrification functional bacteria and the wastewater without enrofloxacin are evenly mixed and aerated to obtain the activated sludge enriched by the antibiotic-resistant denitrification functional bacteria, thereby completing the efficient denitrification treatment of the wastewater.

2. The method according to claim 1, characterized in that In step a, the activated sludge has an ammonia nitrogen removal rate higher than 98%; the activated sludge is derived from flocculent sludge from a sewage treatment plant or excess sludge activated by air exposure.

3. The method according to claim 1, characterized in that In step b, the concentration of enrofloxacin in the enrofloxacin-containing wastewater is 20±1 mg / L.

4. The method according to claim 1, wherein In step b, the volume ratio of the activated sludge to the wastewater is 1:1; the MLSS of the material obtained after the activated sludge and the wastewater are evenly mixed is maintained between 4500 and 5000 mg / L; the temperature of the aeration treatment is 23-25°C, the rotation speed is 90-95 rad / min, and the pH is 7.5-8.0; the concentration of ammonia nitrogen in the wastewater is 30-35 mg / L, the concentration of COD is 300±10 mg / L, the concentration of trace elements is 1 mL / L, and the drainage ratio of each cycle is 50%; In step c, the volume ratio of the activated sludge enriched by the antibiotic-resistant denitrifying functional bacteria and the wastewater is 1:1; the MLSS of the material obtained after the activated sludge enriched by the antibiotic-resistant denitrifying functional bacteria and the wastewater are evenly mixed is maintained between 4500 and 5000 mg / L; the temperature of the aeration treatment is 23-25°C, the rotation speed is 90-95 rad / min, and the pH is 7.5-8.0; the concentration of ammonia nitrogen in the wastewater is 30-35 mg / L, the concentration of COD is 300±10 mg / L, the concentration of trace elements is 1 mL / L, and the drainage ratio of each cycle is 50%.

5. The method according to claim 1, wherein In step b, the aeration treatment is operated for 9 cycles; each cycle starts with the start of the aeration treatment and ends when the ammonia nitrogen degradation degree reaches 98% or the ammonia nitrogen concentration remains stable; In step c, the aeration treatment is operated for 6 cycles; each cycle starts with the start of aeration treatment and ends when the ammonia nitrogen degradation degree reaches 98% or the ammonia nitrogen concentration remains stable.

6. The method according to claim 4, characterized in that The trace elements include the following components at the following concentrations: 4.29g / LNa2EDTA, 1.99g / LFeCl2·4H2O, 0.08g / LMnCl2·2H2O, 0.02 / L g CuCl2·2H2O, 0.07g / LZnCl2, 0.02g / LNa2MoO4·2H2O, 0.03g / LNa2WoO4·2H2O and 0.06g / LH3BO3.

7. Use of the method according to any one of claims 1 to 6 in the efficient denitrification of high-concentration antibiotic wastewater.

8. The use according to claim 7, characterized in that The high-concentration antibiotic wastewater includes pharmaceutical wastewater, livestock breeding wastewater or aquaculture wastewater.

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