Method for evaluating response mechanism of activated sludge system under antibiotic impact and application thereof

Abstract

The invention provides a method for evaluating an activated sludge system response mechanism under antibiotic impact and application thereof, and belongs to the technical field of water environment risk evaluation and management. The method mainly relates to detection of water quality indexes (ammonia nitrogen, total nitrogen, total phosphorus and chemical oxygen demand) in the activated sludge and physicochemical characteristics (extracellular polymeric substance EPS, sludge settling performance and Zeta potential) of the activated sludge, and a response mechanism of an activated sludge system under antibiotic impact is analyzed by taking ciprofloxacin (CIP), ofloxacin (OFL) and enrofloxacin (ENR) as indication pollutants. Effective reference is provided for system regulation and control when a sewage plant faces antibiotic impact, and a new insight is provided for removing antibiotics and drug-resistant genes in wastewater by explaining the relation among functional genes, drug-resistant genes and microbial communities in an activated sludge system under antibiotic exposure.

Description

Technical Field

[0001] This invention belongs to the field of water environment risk assessment and management technology, specifically a method for assessing the response mechanism of activated sludge systems under antibiotic shock and its application. Background Technology

[0002] Wastewater treatment plants play a vital role as facilities for treating urban domestic sewage and industrial wastewater. With the development of healthcare, large amounts of antibiotics are entering wastewater treatment plants with the wastewater, leading to the deterioration of the performance of activated sludge within the plants.

[0003] Microorganisms are an important component of activated sludge systems, playing a crucial role in their sludge removal performance and are essential for solving aquatic environmental problems. Exposure to new pollutants such as antibiotics may alter the microbial community and related genes in activated sludge from wastewater treatment plants; however, the patterns and mechanisms of these changes require further investigation. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for evaluating the response mechanism of an activated sludge system under antibiotic shock. This method reveals the effects of antibiotics on the microbial community and carbon, nitrogen, and phosphorus-related functional genes in activated sludge, and clarifies the relationship between functional genes, resistance genes, and microbial communities in the activated sludge system under antibiotic exposure. This provides a theoretical basis and operable technical means for wastewater treatment plants to scientifically assess risks, optimize operating processes, precisely control sludge performance, and effectively curb the spread of resistance genes in the practice of treating new pollutants.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for evaluating the response mechanism of an activated sludge system under antibiotic shock, comprising the following steps: Reactor construction and acclimatization: Establish at least four parallel sequencing batch reactors and inoculate them with activated sludge. Acclimatize them with synthetic wastewater until the effluent quality is stable. Gradient concentration antibiotic exposure: One of ciprofloxacin, ofloxacin, and enrofloxacin was added to each reactor, and at least five concentration gradients were set up for long-term exposure experiments, while a control group without antibiotics was set up. System performance monitoring: Daily monitoring of the decontamination characteristics of the reactor influent and effluent samples; Activated sludge sample analysis and sludge system response mechanism assessment: Activated sludge samples were collected at the end of each concentration gradient stage, and metagenomic DNA of sludge microorganisms was extracted for metagenomic sequencing; based on metagenomic data, the microbial community structure, antibiotic resistance genes, and functional genes were analyzed; collinear network analysis was used to elucidate the correlation between resistance genes, functional genes, and the microbial community; and by integrating sludge removal performance, physicochemical properties, and metagenomic data, the response mechanism of the activated sludge system under antibiotic shock was obtained.

[0006] Preferably, the operating cycle of the sequencing batch reactor includes influent, anaerobic, aerobic, sedimentation, drainage, and settling.

[0007] Preferably, the pollution removal performance indicators include ammonia nitrogen concentration, total nitrogen concentration, total phosphorus concentration, and chemical oxygen demand.

[0008] Preferably, the physicochemical properties include extracellular polymeric substances (EPS), activated sludge morphology, sludge settling properties, and zeta potential.

[0009] More preferably, the analysis of the extracellular polymeric EPS includes extracting the extracellular polymeric EPS using a heating extraction method, determining the protein content using the Lowry method and the polysaccharide content using the phenol-sulfuric acid method, and simultaneously analyzing the component characteristics of the extracellular polymeric EPS using three-dimensional fluorescence spectroscopy.

[0010] Preferably, the activated sludge sample analysis further includes high-throughput sequencing analysis of the DNA of the activated sludge sample.

[0011] More preferably, ASV clustering analysis is performed on the data from the high-throughput sequencing analysis to obtain information on the species composition and relative abundance of the microbial community.

[0012] Preferably, the collinear network analysis uses Spearman correlation analysis to construct the association network between drug resistance genes, functional genes and key microbial groups, and visualizes it using Cytoscape software.

[0013] The present invention also provides an application of the method in the treatment of new pollutants in wastewater treatment plants.

[0014] The present invention also provides an application of the method in risk control of wastewater treatment plants.

[0015] Compared with the prior art, the present invention has the following advantages: This invention provides a method for evaluating the response mechanism of activated sludge systems under antibiotic shock. By simulating exposure scenarios of typical fluoroquinolone antibiotics (FQs) (ciprofloxacin CIP, ofloxacin OFL, and enrofloxacin ENR) in municipal, pharmaceutical, and hospital wastewater at environmentally relevant concentrations, this invention systematically studies the effects of FQs on the microbial community structure and C / N / P cycle functional genes in activated sludge systems. Through analysis of sequencing batch reactor (SBR) operating performance, changes in biofilm EPS (extracellular polymeric substances), and three-dimensional fluorescence spectroscopy, this invention reveals the self-protective mechanism of microorganisms under fluoroquinolone antibiotic exposure: regulating extracellular polymeric substances (EPS) secretion and enhancing protein metabolism to improve environmental adaptability. This invention elucidates the correlation between functional genes, antibiotic resistance genes, and microbial communities under fluoroquinolone antibiotic exposure at the genetic level, providing a theoretical basis for understanding the risk of antibiotic resistance spread. The multi-gradient concentration SBR reactor construction and metagenomic analysis strategy proposed in this invention provides an operable experimental framework and evaluation method for assessing the impact of antibiotic pollution on functional microorganisms and pollution control effectiveness in actual wastewater treatment plants (WWTPs). Attached Figure Description

[0016] Figure 1 The graph shows the changes in chemical oxygen demand (COD) of reactor influent and effluent under different concentrations of fluoroquinolone antibiotics. (A) is the control group, (B) is the CIP group, (C) is the OFL group, and (D) is the ENR group. Figure 2 The graph shows the changes in ammonia nitrogen concentration in the reactor influent and effluent under different concentrations of fluoroquinolone antibiotics. (A) is the control group, (B) is the CIP group, (C) is the OFL group, and (D) is the ENR group. Figure 3 The graph shows the changes in total phosphorus concentration in the reactor influent and effluent under different concentrations of fluoroquinolone antibiotics. (A) is the control group, (B) is the CIP group, (C) is the OFL group, and (D) is the ENR group. Figure 4 The graph shows the changes in the main components of extracellular polymers under different concentrations of fluoroquinolone antibiotics, where (A) is the control group, (B) is the CIP group, (C) is the OFL group, and (D) is the ENR group. Figure 5 Three-dimensional fluorescence spectra of extracellular polymers at different concentrations of fluoroquinolone antibiotics are shown. (A) is the stage I control group, (B) is the stage I CIP group, (C) is the stage I OFL group, (D) is the stage I ENR group, (E) is the stage III control group, (F) is the stage III CIP group, (G) is the stage III OFL group, (H) is the stage III ENR group, (I) is the stage V control group, (J) is the stage V CIP group, (K) is the stage V OFL group, and (L) is the stage V ENR group. Figure 6The graph shows the changes in Zeta potential at different concentrations of fluoroquinolone antibiotics, where (A) is the control group, (B) is the CIP group, (C) is the OFL group, and (D) is the ENR group. Figure 7 Abundance diagrams of bacterial community species composition under different concentrations of fluoroquinolone antibiotics, where (A) represents the abundance of species at the phylum level and (B) represents the abundance of species at the genus level; Figure 8 The graph shows the relative abundance of functional genes at different concentrations of fluoroquinolone antibiotics, where (A) represents the relative abundance of N-cycle functional genes and (B) represents the relative abundance of P-cycle functional genes. Figure 9 A graph showing the abundance and composition of resistance genes at different concentrations of fluoroquinolone antibiotics; Figure 10 This is a collinear network diagram of resistance genes, functional genes, and microbial communities at different concentrations of fluoroquinolone antibiotics. Detailed Implementation

[0017] This invention provides a method for evaluating the response mechanism of an activated sludge system under antibiotic shock, the method preferably comprising the following steps: Reactor construction and acclimatization: Establish at least four parallel sequencing batch reactors and inoculate them with activated sludge. Acclimatize them with synthetic wastewater until the effluent quality is stable. Gradient concentration antibiotic exposure: One of ciprofloxacin, ofloxacin, and enrofloxacin was added to each reactor, and at least five concentration gradients were set up for long-term exposure experiments, while a control group without antibiotics was set up. System performance monitoring: Daily monitoring of the decontamination characteristics of the reactor influent and effluent samples; Activated sludge sample analysis and sludge system response mechanism assessment: Activated sludge samples were collected at the end of each concentration gradient stage, and metagenomic DNA of sludge microorganisms was extracted for metagenomic sequencing; based on metagenomic data, the microbial community structure, antibiotic resistance genes, and functional genes were analyzed; collinear network analysis was used to elucidate the correlation between resistance genes, functional genes, and the microbial community; and by integrating sludge removal performance, physicochemical properties, and metagenomic data, the response mechanism of the activated sludge system under antibiotic shock was obtained.

[0018] In this invention, the operating cycle of the sequencing batch reactor preferably includes influent, anaerobic, aerobic, sedimentation, drainage, and settling; more preferably, it includes influent (20-30 min), anaerobic stage (30-40 min), aerobic stage (400-500 min), sedimentation (80-100 min), drainage (20-30 min), and settling (60-80 min); and even more preferably, it includes timed aeration influent (20 min), anaerobic stage (40 min), aerobic stage (480 min), sedimentation (90 min), drainage (20 min), and settling (70 min).

[0019] In this invention, the antibiotic used in the exposure test is preferably a fluoroquinolone antibiotic, which further includes one of ciprofloxacin, ofloxacin, and enrofloxacin; the antibiotic concentration gradient in the exposure test is set with at least five concentration gradients, which are preferably 10 μg / L, 100 μg / L, 500 μg / L, 1000 μg / L, and 2000 μg / L.

[0020] In this invention, the preferred formulation of the synthetic wastewater includes: CH3COONa•3H2O 240 mg / L; NH4Cl 20 mg / L; KH2PO4 4 mg / L; MgSO4•7H2O 25 mg / L; CaCl2 20 mg / L; NaHCO3 50 mg / L; CuSO4•5H2O 0.06 mg / L; CoCl2•6H2O 0.03 mg / L; KI 0.03 mg / L; Citric Acid 2.73 mg / L; FeCl3•6H2O 1.5 mg / L; H3BO3 0.25 mg / L; ZnSO4•7H2O 0.15 mg / L; MnCl2•4H2O 0.12 mg / L; Na2MoO4•2H2O 0.03 mg / L.

[0021] In this invention, the pollution removal characteristic indicators include ammonia nitrogen concentration, total nitrogen concentration, total phosphorus concentration, and chemical oxygen demand (COD). In this invention, conventional methods in the art can be used to detect these pollution removal characteristic indicators. In this invention, as a preferred embodiment, ammonia nitrogen concentration is detected using the salicylic acid-hypochlorite spectrophotometric method, total nitrogen concentration is detected using the alkaline potassium persulfate digestion-ultraviolet spectrophotometric method, and total phosphorus concentration is detected using the molybdenum blue spectrophotometric method.

[0022] In this invention, the physicochemical properties include extracellular polymeric substances (EPS), activated sludge morphology, sludge settling properties, and zeta potential. Preferably, the analysis of the EPS includes extracting the EPS using a heating extraction method, determining the protein content using the Lowry method, determining the polysaccharide content using the phenol-sulfuric acid method, and simultaneously using three-dimensional fluorescence spectroscopy (3D-EEM) to analyze the component characteristics of the EPS.

[0023] In this invention, the activated sludge sample analysis further includes high-throughput sequencing analysis of the activated sludge sample DNA. In this invention, ASV clustering analysis is performed on the data from the high-throughput sequencing analysis to obtain information on the species composition and relative abundance of the microbial community.

[0024] In this invention, the high-throughput sequencing analysis targets the V3-V4 region of the microbial 16S rRNA gene, using primers 341F and 805R for amplification. The metagenomic sequencing employs a sequencing platform for paired-end sequencing. As a preferred method, the sequencing platform is used, with a paired-end sequencing mode of 150 bp. The V3-V4 region of the bacterial 16S rRNA gene is amplified using forward primer 341F: 5'-CCTACGGGNGGCWGCAG-3' (SEQ ID NO.1) and reverse primer 805R: 5'-GACTACHVGGGTATCTAATCC-3' (SEQ ID NO.2). Based on the aforementioned universal primer sequences, long primers with sample-specific tags and sequencing adapters are synthesized for amplification. After amplification, the target product is obtained, a library is constructed, and sequencing is performed to obtain raw paired-end sequencing data. In this invention, N, W, H, and V in the universal primers are degenerate bases, wherein the normal bases corresponding to N are A / T / C / G; the normal bases corresponding to W are A / T; the normal bases corresponding to H are A / T / C; and the normal bases corresponding to V are G / A / C.

[0025] The present invention preferably processes the raw sequencing data as follows: FastP is used for quality control to remove adapter sequences, low-quality reads, sequences containing N, and excessively short fragments, resulting in clean, high-quality data. Subsequently, MEGAHIT assembly software is used to assemble the clean reads into contigs. The unassembled contigs are then aligned to species annotation databases using tools such as Kraken2 and MetaPhlAn3 to achieve species classification annotation, obtaining the taxonomic composition and relative abundance of microorganisms in twenty sludge samples across five stages.

[0026] In this invention, the annotation of antibiotic resistance genes (ARGs) is accomplished by aligning contigs obtained from metagenomic assembly with the Antibiotic Resistance Gene Database (ARDB). The alignment parameters require a similarity greater than 80% and a coverage greater than 70%. As a preferred method, contigs are aligned with relevant databases using MMseq2, while ARDB is used for resistance gene identification. Alignment parameter settings generally require high similarity (identity > 80%) and a relatively high coverage (coverage > 70%) to ensure annotation accuracy. After annotation, the relative abundance of each gene is calculated using the RPKM method, combining gene length and alignment coverage information. Ultimately, the types and abundances of different ARGs in twenty sludge samples across five stages can be obtained.

[0027] This invention also provides an application of the method in the treatment of new pollutants in wastewater treatment plants. In this invention, the method is preferably used to assess the potential risks of new antibiotic pollutants in the influent of actual wastewater treatment plants to treatment efficiency and sludge properties, and to provide a theoretical basis and operational strategies for optimizing process parameters, controlling sludge properties, and inhibiting the spread of drug resistance.

[0028] This invention also provides an application of the method in the risk control of wastewater treatment plants. In this invention, based on the critical inhibitory concentration of antibiotics and the microbial response patterns determined by the method, an early warning and control strategy for wastewater treatment plants to cope with antibiotic shocks is established. This invention's research found that free radical sludge (FQs) at concentrations of 500 μg / L and above significantly impact the decontamination characteristics of activated sludge, especially in nitrogen and phosphorus removal. Through the detection of physicochemical factors, it was found that FQs significantly interfere with the physicochemical properties of activated sludge, providing a valid reference for the risk control of wastewater treatment plants.

[0029] In a specific embodiment of the present invention, the metagenomic sequencing is outsourced to Shanghai Paisenno Co., Ltd.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0032] Example 1 A method for evaluating the response mechanism of an activated sludge system under antibiotic shock, the method comprising the following steps: Reactor construction and acclimatization: Four parallel sequencing batch reactors were built and inoculated with activated sludge. Synthetic wastewater was used for acclimatization until the effluent quality stabilized. Gradient concentration antibiotic exposure: One of ciprofloxacin, ofloxacin, and enrofloxacin was added to each reactor, and five concentration gradients of 10 μg / L, 100 μg / L, 500 μg / L, 1000 μg / L, and 2000 μg / L were set up for long-term exposure experiments. At the same time, a control group without antibiotics was set up. System performance monitoring: Daily monitoring of the influent and effluent water samples from the reactor to measure their pollution removal characteristics, including chemical oxygen demand (COD) and ammonia nitrogen (NH4). + Concentrations of total nitrogen (TN) and total phosphorus (TP); Activated sludge sample analysis and sludge system response mechanism assessment: Activated sludge samples were collected at the end of each concentration gradient stage for the following analysis: (1) Analyze the physicochemical properties of activated sludge, including the content and composition of extracellular polymeric substances (EPS), sludge settling performance, and zeta potential; (2) Extract metagenomic DNA from sludge microorganisms and perform high-throughput sequencing and metagenomic sequencing; (3) Based on metagenomic data, analyze the composition of microbial community structure, antibiotic resistance genes (ARGs), and functional genes; (4) Integrate the decontamination performance, physicochemical properties and metagenomic data, and use co-occurrence network analysis to clarify the relationship between microbial community structure, functional genes and drug resistance genes under antibiotic stress, thereby revealing the response mechanism of activated sludge system.

[0033] Example 2 Activated sludge was collected from the oxidation ditch of the Southeast Wastewater Treatment Plant in Changchun City, Jilin Province, and four cylindrical sequencing batch reactors (SBRs) (5 L each) were constructed in the laboratory for experiments. The four SBRs served as the control group (without antibiotics), the CIP group, the OFL group, and the ENR group, and the reactor operation was divided into five stages. For the first 20 days (Phase I), the synthetic wastewater (CH3COONa•3H2O 240mg / L; NH4Cl 20mg / L; KH2PO4 4mg / L; MgSO4•7H2O 25mg / L; CaCl2 20mg / L; NaHCO3 50mg / L; CuSO4•5H2O 0.06mg / L; CoCl2•6H2O 0.03mg / L; KI 0.03mg / L; Citric Acid 2.73mg / L; FeCl3•6H2O 1.5mg / L; H3BO3 0.25mg / L; ZnSO4•7H2O 0.15mg / L; MnCl2•4H2O 0.12mg / L; Na2MoO4•2H2O 0.03mg / L) was fed into four SBR reactors to adapt the activated sludge. After the effluent stabilized, three types of FQs were added to the influent, and the four reactors were run for another 40 days using different influent FQs concentrations: 10 μg / L for days 21-30 (Phase II); 100 μg / L for days 31-40 (Phase III); 500 μg / L for days 41-50 (Phase IV); and 2000 μg / L for days 51-60 (Phase V). Figures 1-3 The results show that basic water quality indicators were monitored daily, and ammonia nitrogen (NH4+) was measured using a DR3900 visible light spectrophotometer. + -N), total phosphorus (TP), total nitrogen (TN), nitrite (NO2) - -N) and nitrates (NO3) - Monitoring results showed that under different concentrations of FQs stress, the COD removal rate of the activated sludge system was basically unaffected in the five stages of FQs stress, maintaining a similar removal efficiency as the control group. FQs had no significant inhibitory effect on aerobic heterotrophic bacteria in the system. Nitrogen removal performance exhibited a concentration gradient effect; low concentrations of FQs had little effect on ammonia nitrogen oxidation but inhibited denitrification; at high concentrations, especially at 2000 μg / L, the total nitrogen removal rate decreased significantly, with the CIP group being most affected. Phosphorus removal performance was more sensitive to FQs; at 500 μg / L, the total phosphorus removal rate had already dropped below 85%, and at higher concentrations, the removal rates of all groups further decreased, with the CIP group showing the largest decrease. These results indicate that FQs have a significant impact on the nitrogen and phosphorus removal performance of activated sludge, and there are differences among different FQs compounds (mainly attributed to their chemical structural characteristics).

[0034] In the five phase IV stages, activated sludge samples were collected from four SBR reactors in triplicate at the end of each stage, and extracellular polymeric substances (EPS) were extracted using a heating method. The sludge samples were centrifuged at 3200 rpm for 15 minutes, the supernatant was discarded, and the precipitate was resuspended to its original volume using 0.05% NaCl solution. The mixture of each sample was heated at 60°C for 30 minutes, followed by centrifugation at 12000 rpm for 15 minutes. Finally, the supernatant obtained from centrifugation was filtered through a membrane filter with a pore size of 0.22 µm to obtain EPS. Figure 4 The results showed that the Lowry method was used to determine proteins, the phenol-sulfuric acid method to determine polysaccharides, and a fluorescence spectrophotometer was used to measure the three-dimensional fluorescence spectra at five different time points to analyze the changes in EPS (experiments per unit volume) of activated sludge under antibiotic shock. With increasing FQ (free radical scavenging) concentration, the total EPS showed a significant upward trend. Compared with polysaccharides, tryptophan-containing proteins were the main components resisting FQs. Furthermore, according to... Figure 5 Three-dimensional fluorescence spectroscopy showed that humic acid and fulvic acid-like substances were clearly present in EPS at the beginning of stage V, at which the bacteria began to die and lyse.

[0035] In the five stages of Phase IV, at the end of each stage, activated sludge samples were collected from four SBR reactors in a triple-replication manner. The activated sludge samples were examined under an inverted microscope, and the settling properties of the sludge samples were tested to investigate the effects of antibiotics on the morphology and settling properties of activated sludge.

[0036] according to Figure 6 The results showed that the zeta potential of the sludge samples collected above was measured using Nano ZS to help determine the adsorption capacity of the sludge for antibiotics. The increase in zeta potential indicates that the removal of antibiotics by the activated sludge is mainly through adsorption, and that the antibiotics adsorbed on the activated sludge are difficult to biodegrade.

[0037] Example 3 The response of the microbial community was further evaluated using the method described in Example 2.

[0038] 1. Sample collection and metagenomic sequencing In the five phase IV trials, activated sludge samples were collected from four SBR reactors in triplicate at the end of each phase. The samples were centrifuged at 12,000 rpm, 4°C, for 5 min, and the precipitates were retained and stored at -80°C for later use. Sequencing was performed using a paired-end 150 bp sequencing strategy. Quality control was performed using FASTP to remove adapter sequences, low-quality reads, sequences containing nitrogen (N), and excessively short fragments, obtaining clean, high-quality data. The clean reads were then assembled using MEGAHIT assembly software to obtain contigs. The assembled contigs were aligned to species annotation databases using tools such as Kraken2 and MetaPhlAn3 to achieve species classification annotation, obtaining omics information on the microorganisms in twenty sludge samples across the five phases.

[0039] 2. Based on metagenomic sequencing, the species diversity of activated sludge microorganisms under different antibiotics in stages I, III, and V was analyzed. As shown in Table 1, the Chao 1, Shannon, and OTU indices of activated sludge all showed a decreasing trend after antibiotic addition, while the Simpson index increased, indicating that the addition of antibiotics led to a decrease in species richness and diversity in activated sludge. The CIP group showed the most significant changes, which may be due to its stronger antibacterial activity, stronger selectivity for microorganisms, and a more concentrated dominant species.

[0040] Table 1. Analysis of microbial species diversity in activated sludge under different antibiotics.

[0041] 3. Metagenomic sequencing was performed on activated sludge samples. Figure 7 Figure (A) illustrates the phylum-level community composition of microorganisms at each stage of the SBR system. In all systems, Proteobacteria (… Proteobacteria Bacteroidetes () hold an absolute dominant position. Bacteroidota Secondly, it is worth noting that with the addition of fluoroquinolone antibiotics, the number of planktonic fungi ( Planctomycetota The relative abundance of *Ploveromycetes* increased significantly (by 4.16%-14.05%), making it the third most abundant phylum. *Ploveromycetes* is widely distributed in environments such as wastewater treatment plants and aquaculture, and is often associated with antibiotic resistance genes; antibiotics can promote its abundance increase. Meanwhile, the abundance of *Actinomycetes*... Actinobacteria ) and acidobacteria ( Acidobacteria The relative abundance of these bacteria decreased after antibiotic addition, indicating that their growth was inhibited. Actinobacteria often participate in synergistic effects to promote biological phosphorus removal, while acidobacteria play an important role in phosphorus fixation. Therefore, the suppression of these two functional phyla provides a reasonable explanation for the decline in phosphorus removal performance of sludge systems under antibiotic exposure. Nitrifying Spirulina (… NitrospiraeThe abundance of this phylum decreased synchronously (this phylum is related to nitrite nitrogen (NO2)). - (closely related to the transformation of -N), further corroborating the adverse effects of antibiotics on phosphorus removal function.

[0042] At the genus level ( Figure 7 (B) ), Microphytes genus ( Hyphomicrobium The relative abundance of this genus increased significantly after antibiotic addition (by 3.36%-8.78%), making it the dominant genus. This genus is known to degrade polycyclic aromatic hydrocarbons and fluorinated compounds and is often enriched in co-metabolism systems involving multiple pollutants. This phenomenon suggests that antibiotic exposure may promote the growth of microorganisms with potential antibiotic-degrading capabilities (such as...). Hyphomicrobium The proliferation of denitrifying bacteria (often found in wastewater treatment systems) has been observed, although their ability to directly degrade antibiotics has not been reported. In the antibiotic treatment group, denitrifying bacteria—common in wastewater treatment systems—[…]. Thauera The abundance of this genus showed a decreasing trend, consistent with the changes in the Acidobacteria phylum. This may be one of the reasons for the decline in the denitrification performance of sludge systems under antibiotic exposure. Furthermore, Amaricoccus The relative abundance of this genus increased from 0.11% in the control group to a maximum of 2.82% (control group). Overgrowth of this genus is often associated with viscous (non-filamentous) bulking of activated sludge, as it stimulates the secretion of polysaccharide-rich extracellular polymers (EPS). This also explains why antibiotic exposure still leads to a significant increase in the sludge settling index (SV) of activated sludge (AS) even with low filamentous abundance.

[0043] 4. Perform metagenomic sequencing on activated sludge samples to obtain functional gene data, such as... Figure 8 As shown in (A), denitrification-related functional genes include nirK, nirS, nosZ and narG ; Nitrification-related functional genes include nxrA, nxrB, amoA, amoB and amoC .like Figure 8 As shown in (A), in stage III, nitrification genes amoB and denitrification genes narG The increased relative abundance suggests that low concentrations of fluoroquinolone antibiotics may have promoted partial nitrification and denitrification. However, in phase V, the abundance of all detected functional genes showed a decreasing trend. This was particularly evident in the ciprofloxacin (CIP) treatment group. nirS and nirK The most significant decrease was in gene abundance, with reductions of 55.0% and 55.1%, respectively. nirS and nirK Genetically encoded nitrite reductase plays a crucial rate-limiting role in denitrification, responsible for catalyzing the formation of nitrite (NO2). -It is reduced to nitric oxide (NO). Because this enzyme is located in the periplasm of bacteria, it is more susceptible to external environmental factors (such as antibiotic exposure).

[0044] Figure 8 As shown in (B), phnK This gene is crucial for the growth and reproduction of *E. coli* under conditions where phosphonic acid is the sole phosphorus source. The ATP-binding protein encoded by this gene binds and recruits ATP, participating in nutrient absorption and cellular metabolic processes, playing a vital role in phosphorus, nitrogen, and carbon cycles. Furthermore, active cellular metabolism has a high demand for phosphorus. As shown in the illustration, the presence of fluoroquinolone antibiotics is associated with… phoD and phoX Downregulation of the gene (encoding alkaline phosphatase) further interfered with phosphorus metabolism.

[0045] Metagenomic sequencing was performed on activated sludge samples to obtain drug resistance gene data, such as... Figure 9 This study showcases the top 20 most abundant antibiotic resistance genes (ARGs) after antibiotic exposure in stages I, III, and V. The relative abundance of ARGs increases with increasing antibiotic concentration. Based on their resistance mechanisms, ARGs can be categorized into four types: antibiotic efflux, antibiotic inactivation, antibiotic target substitution, and antibiotic target protection. Figure 9 As shown, antibiotic efflux-related genes (such as...) bcr, tetQ, macB, MexE and MexW These constitute the majority of ARGs, indicating that antibiotic efflux is the most significant resistance mechanism after FQ exposure. Furthermore, some dominant ARGs possess antibiotic target substitution functions, including... VanB and pbp Interestingly, the relative abundance of these genes remained largely unchanged in stage III, but a sharp increase was observed in stage V. This phenomenon suggests that efflux mechanisms are insufficient to resist toxic stress under high-dose FQs exposure; microorganisms begin to further enhance resistance by altering antibiotic targets (such as peptidoglycan synthase). To further elucidate the intrinsic connections between various elements under antibiotic shock, such as... Figure 10 As shown, this invention constructs a co-occurrence network among antibiotic resistance genes, functional genes, and key microbial taxa. This network diagram clearly reveals the synergistic adaptation mechanism of microbial communities under antibiotic stress. Core ammonia-oxidizing bacteria and denitrifying bacteria communicate through key functional genes (such as ammonia-oxidizing genes). amoA Denitrification genes nirK / nirS and narG The synergistic expression of multiple drug resistance genes (mdr) and efflux pump genes (mdr) maintains the nitrogen cycle compensation function; at the same time, the synergistic expression of multiple drug resistance genes (mdr) and efflux pump genes (mdr) maintains the nitrogen cycle compensation function. MexC / MexE The widespread activation of ) reflects a key survival strategy for microorganisms in response to antibiotic stress.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for evaluating the response mechanism of an activated sludge system under antibiotic shock, characterized in that, Includes the following steps: Reactor construction and acclimatization: Establish at least four parallel sequencing batch reactors and inoculate them with activated sludge. Acclimatize them with synthetic wastewater until the effluent quality is stable. Gradient concentration antibiotic exposure: One of ciprofloxacin, ofloxacin, and enrofloxacin was added to each reactor, and at least five concentration gradients were set up for long-term exposure experiments, while a control group without antibiotics was set up. System performance monitoring: Daily monitoring of the decontamination characteristics of the reactor influent and effluent samples; Activated sludge sample analysis and sludge system response mechanism assessment: Activated sludge samples were collected at the end of each concentration gradient stage, and metagenomic DNA of sludge microorganisms was extracted for metagenomic sequencing. Based on metagenomic data, we analyzed the microbial community structure, antibiotic resistance genes, and functional genes; collinear network analysis was used to elucidate the correlation between resistance genes, functional genes, and the microbial community; and by integrating decontamination performance, physicochemical properties, and metagenomic data, we obtained the response mechanism of the activated sludge system under antibiotic shock.

2. The method according to claim 1, characterized in that, The operating cycle of the sequencing batch reactor includes influent, anaerobic, aerobic, sedimentation, drainage, and settling.

3. The method according to claim 1, characterized in that, The pollution removal performance indicators include ammonia nitrogen concentration, total nitrogen concentration, total phosphorus concentration, and chemical oxygen demand.

4. The method according to claim 1, characterized in that, The physicochemical properties are measured by indicators including extracellular polymeric substances (EPS), activated sludge morphology, sludge settling properties, and zeta potential.

5. The method according to claim 4, characterized in that, The analysis of the extracellular polymeric substances (EPS) included extracting the EPS using a heating extraction method, determining the protein content using the Lowry method and the polysaccharide content using the phenol-sulfuric acid method, and simultaneously analyzing the component characteristics of the EPS using three-dimensional fluorescence spectroscopy.

6. The method according to claim 1, characterized in that, The analysis of activated sludge samples also includes high-throughput sequencing analysis of the DNA in the activated sludge samples.

7. The method according to claim 6, characterized in that, ASV clustering analysis was performed on the high-throughput sequencing data to obtain information on the species composition and relative abundance of the microbial community.

8. The method according to claim 1, characterized in that, The collinear network analysis employed Spearman correlation analysis to construct the association network between drug resistance genes, functional genes, and key microbial taxa, and visualized it using Cytoscape software.

9. The application of the method according to any one of claims 1-8 in the treatment of new pollutants in wastewater treatment plants.

10. The application of the method according to any one of claims 1-8 in risk control of wastewater treatment plants.

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