A method for improving sewage treatment effect under ciprofloxacin stress

Abstract

The present invention belongs to the field of sewage treatment technology, and specifically relates to a method for improving sewage treatment efficiency under ciprofloxacin stress. The method involves adding polyamide to sewage containing ciprofloxacin, followed by aerobic denitrification treatment. Experiments have shown that while ciprofloxacin can severely inhibit sludge performance and affect the metabolic activity of aerobic denitrifying bacteria, polyamide can significantly mitigate the effects of ciprofloxacin on aerobic denitrifying bacteria under short-term and long-term stress, promoting the denitrification metabolic process, enhancing nitrate reduction, and improving the aerobic denitrification treatment efficiency of sewage.

Description

Technical Field

[0001] The invention belongs to the technical field of sewage treatment, and particularly relates to a method for improving sewage treatment effect under ciprofloxacin stress. Background Art

[0002] Water is a precious resource essential to human survival. As water consumption increases, water quality faces severe challenges. Industrialization, agricultural production, and urban life lead to environmental degradation and pollution, adversely affecting water bodies (rivers and oceans) essential for life, ultimately impacting human health and sustainable social development. Factors contributing to water pollution are primarily industrialization, agricultural activities, urban life, and sewage treatment facilities.

[0003] Compared to traditional physicochemical and biological methods, biological denitrification processes are gaining popularity due to their cost-effectiveness, minimal byproduct generation, reliable operation, and environmental compatibility in water pollution treatment. Traditional biological denitrification processes include the A / A / O process, oxidation ditch process, and SBR process. While these technologies require simple equipment and are easy to operate, they also suffer from numerous drawbacks, such as long cycle times, high energy consumption, and large footprints. With the continued exploration and study of biological denitrification technologies in the academic research community, researchers have begun to focus on new, high-efficiency, low-energy biological denitrification technologies, such as aerobic denitrification, anaerobic ammonium oxidation, and short-term nitrification and denitrification. Among these, aerobic denitrification, as a novel biological denitrification system, offers structural integration, reduced footprint, and simplified operational management. Furthermore, the higher growth rate and denitrification capacity of aerobic denitrifying bacteria give aerobic denitrification processes advantages such as rapid startup, stable operation, and flexibility with oxygen and organic substrates. In the field of water pollution treatment, aerobic denitrification processes and the stress effects of pollutants on aerobic denitrification have become a focus of research.

[0004] Antibiotics, a class of drugs that can combat pathogenic microorganisms, are primarily derived from compounds with antipathogenic activity produced during the metabolism of microorganisms such as bacteria and actinomycetes. At low concentrations, antibiotics can specifically kill or inhibit specific pathogens. Ciprofloxacin (CIP) is a highly representative broad-spectrum antibiotic. CIP and its biotransformation products (TPs) pose a serious threat to ecosystems and human health. Subinhibitory concentrations of CIP can promote the emergence and proliferation of drug-resistant genes. Furthermore, CIP and its TPs can inhibit the growth of plants (such as duckweed and algae), are cytotoxic to bacteria (Escherichiacoli K12), and are genotoxic to animals (such as mice and fathead minnows). Numerous studies have indicated that commonly used antibiotics, such as ciprofloxacin, tetracycline, and amoxicillin, have varying degrees of inhibitory effects on denitrification performance.

[0005] In water pollution, pollutants such as antibiotics, heavy metals, and microplastics often coexist. Microplastics, as carriers, can affect the migration and transformation of pollutants such as metal ions, antibiotics, and plasticizers. Their interactions with pollutants may even alter their toxicity. For example, Ma et al. found that 50-nanometer polystyrene particles, when combined with the organic compound phenanthrene, produced additive toxic effects on Daphnia. Guilhermino et al. also reported that 1-5 micrometer microplastics synergistically interacted with the antibiotic florfenicol, exacerbating neurotoxicity and oxidative stress in bivalves. Furthermore, Masud et al. found that combined exposure to 0.3-mm polypropylene microplastics and glyphosate herbicide resulted in a 73% mortality rate in wild fish, demonstrating a synergistic toxic effect between the two. However, research by Zhang et al. demonstrated that amino-modified 200-nanometer polystyrene particles strongly adsorbed glyphosate, reducing its toxicity to Microcystis aeruginosa, demonstrating an antagonistic effect between the two. Zhu et al. studied the combined toxicity of copper nanoparticles and 1μm polyvinyl chloride microplastics on microalgae and found that microplastics reduced the toxicity of nanocopper and copper ions by adsorbing a certain amount of copper ions. However, the impact of PA on aerobic denitrification wastewater treatment processes under ciprofloxacin stress has not been reported. Summary of the Invention

[0006] In view of the above technical problems, the present invention provides a method for improving the sewage treatment effect under ciprofloxacin stress, which uses polyamide to reduce the impact of ciprofloxacin on aerobic denitrifying bacteria in an aerobic denitrification sewage treatment process.

[0007] The specific technical solutions provided by the present invention are as follows:

[0008] The present invention provides a method for improving the sewage treatment effect under ciprofloxacin stress, which comprises adding polyamide to the sewage containing ciprofloxacin and then performing aerobic denitrification treatment;

[0009] When the content of ciprofloxacin in sewage is 0.1 mg / L to 1.0 mg / L, the amount of polyamide added is 5 mg / L to 500 mg / L.

[0010] As a preferred embodiment of the present invention, the polyamide is used to reduce the inhibitory effect of ciprofloxacin on the activity of aerobic denitrifying bacteria.

[0011] As a preferred embodiment of the present invention, the polyamide is used to improve the degradation rate of NO3--N, TN, COD and the conversion efficiency of NO2--N in the presence of ciprofloxacin.

[0012] As a preferred embodiment of the present invention, the polyamide is used to improve the stability and sedimentation rate of sludge in water to be treated in the presence of ciprofloxacin.

[0013] As a preferred embodiment of the present invention, the polyamide is used to improve the denitrification performance of sludge in water to be treated in the presence of ciprofloxacin.

[0014] As a preferred embodiment of the present invention, the polyamide is used to improve the denitrification activity in the presence of ciprofloxacin.

[0015] As a preferred embodiment of the present invention, the polyamide is used to improve the stability of aerobic denitrifying bacteria communities in sewage in the presence of ciprofloxacin and their tolerance to adverse environments.

[0016] The present invention also provides a sewage treatment agent, which includes polyamide and is used to reduce the influence of ciprofloxacin on the treatment effect in an aerobic denitrification treatment process.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] The present invention provides a method for improving wastewater treatment efficiency under ciprofloxacin stress. The method comprises adding a specific concentration of polyamide to wastewater containing ciprofloxacin, followed by aerobic denitrification treatment. Experiments have demonstrated that ciprofloxacin (CIP) severely inhibits sludge performance and affects the metabolic activity of aerobic denitrifying bacteria, while polyamide (PA) significantly mitigates the effects of ciprofloxacin on aerobic denitrifying bacteria under short-term stress, promoting the denitrification metabolic process and enhancing nitrate reduction. In long-term stress tests, CIP resulted in a greater decrease in sludge concentration than PA, and the addition of PA under combined stress conditions can mitigate this effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The carbon and nitrogen metabolism performance of aerobic denitrifying bacteria under short-term stress of PA and CIP; a, NO3--N, b, NO2--N, c, TN, d, COD;

[0020] Figure 2 is the 3D-EEM spectrum under short-term stress of PA and CIP;

[0021] Figure 3 It is a parallel factor analysis based on the PARAFAC model; C1: humic acid-like C2: fulvic acid-like);

[0022] Figure 4 These are the fluorescent components under different concentrations of PA and CIP stress; C1: humic acid-like C2: fulvic acid-like;

[0023] Figure 5 is the PN, PS, and EPS contents of reactor sludge under short-term stress and their relative changes;

[0024] Figure 6Denitrification enzyme activity of aerobic denitrifying bacteria under different concentrations of PA and CIP stress; a: NAR enzyme activity; b: NIR enzyme activity

[0025] Figure 7 is the Pearson correlation analysis of related indicators of aerobic denitrifying bacteria under short-term stress; *, p < 0.05; **, p < 0.01;

[0026] Figure 8 The carbon and nitrogen metabolism performance of aerobic denitrifying bacteria under long-term PA and CIP stress; a, NO3--N, b, nitrogen source removal rate, c, NO2--N, d, COD;

[0027] Figure 9 is the 3D-EEM spectrum under long-term PA and CIP stress (first stage);

[0028] Figure 10 is the 3D-EEM spectrum under long-term PA and CIP stress (second stage);

[0029] Figure 11 It is a parallel factor analysis based on the PARAFAC model; C1: tryptophan-like C2: humic acid-like;

[0030] Figure 12 These are the fluorescent components under different concentrations of PA and CIP stress; C1: tryptophan-like C2: humic acid-like;

[0031] Figure 13 is the change in reactor sludge concentration under long-term stress;

[0032] Figure 14 is the EPS content and its relative changes in the reactor sludge under the two stages of long-term stress;

[0033] Figure 15 are the EPS-PN (a) and EPS-PS (b) contents and their relative changes in the reactor sludge under the two stages of long-term stress;

[0034] Figure 16 The denitrification enzyme activities of aerobic denitrifying bacteria under different concentrations of PA and CIP stress; a: NAR enzyme activity; b: NIR enzyme activity;

[0035] Figure 17 The phylum level of the microbial community structure of aerobic denitrifying sludge under long-term stress of different concentrations of PA and CIP;

[0036] Figure 18 It is the genus level of the aerobic denitrifying sludge microbial community structure under long-term stress of different concentrations of PA and CIP. DETAILED DESCRIPTION

[0037] The following description is merely a preferred embodiment of the present invention and does not limit the scope of protection of the present invention.

[0038] In order to verify the effect of PA on the aerobic denitrification water treatment process under ciprofloxacin stress, the present invention is described in detail through short-term stress tests and long-term stress tests.

[0039] It should be noted that in actual engineering applications, due to the rapid metabolic rate and high proliferation rate of aerobic denitrification sludge, periodic sludge discharge is required. This process removes ciprofloxacin and polyamide PA, which are enriched within the sludge. This removes pollutants from the water while ensuring the normal operation of the sludge system. Furthermore, the discharged sludge is in the form of flocs, which have a large volume and specific surface area, capable of absorbing large amounts of ciprofloxacin and polyamide. Further dehydration of the discharged sludge significantly reduces its volume and increases its carbon content, allowing it to be incinerated, further reducing the amount of ciprofloxacin and polyamide remaining in the wastewater.

[0040] The aerobic denitrification treatment process is carried out with reference to the existing technology, such as “THIRD KA, GIBBS B, NEWLAND M, et al. Long-term aeration management for improved N-removal via SND in a sequencing batch reactor [J]. Water Research, 2005, 39 (15): 3523-3530.”, “MASOUDI SMA, HEDAYATI MOGHADDAM A, SARGOLZAEIJ, et al. Investigation and optimization of the SND-SBR system for organic matter and ammonium nitrogen removal using the central composite design [J]. Environmental Progress & Sustainable Energy, 2018, 37 (5): 1638-1646.”. However, those skilled in the art should know that this is not a special limitation on the aerobic denitrification process in the present invention.

[0041] 1. Method

[0042] 1.1 Short-term effect test

[0043] In the short-term effect experiment, 200-400 mesh polyamide (PA) produced by Shanghai Yuanye Biotechnology Co., Ltd. was used to simulate microplastic pollutants. The CIP required for the experiment was purchased from Aladdin Biochemical Technology Co., Ltd. with a purity of 98%. CIP was pre-prepared into a mother liquor with a concentration of 10g / L and properly stored in a refrigerator at 4°C until use. As for the source of activated sludge, 1ml of a sludge-water mixture was taken from a stably operating aerobic denitrification reactor and added to LB culture medium. The mixture was activated in a shaker and inoculated into an inorganic salt culture medium within 48 hours of activation and cultured on a shaker.

[0044] The experiment was conducted in a 250ml conical flask, with a shaker temperature of 30 degrees Celsius and a rotation speed of 150r / min. The basic components were 150ml of inorganic salt culture medium, 1ml of enriched bacterial liquid cultured in LB medium, and appropriate amounts of PA and configured CIP mother liquor were added to achieve the corresponding stress concentration. After careful review of relevant data, the following experimental plan was designed: it included 1 group of blank control experiments, 2 groups of experiments containing only PA, 2 groups of experiments containing only CIP, and 4 groups of composite experiments containing both PA and CIP. In the composite experiments, the present invention specifically selected low concentrations (50mg / L) and high concentrations (500mg / L) of PA, as well as low concentrations (0.1mg / L) and high concentrations (1mg / L) of CIP, to comprehensively explore the effects of different concentration combinations on the experimental subjects.

[0045] 1.2. Long-term effect experiment

[0046] The long-term effect experiment used the same PA and CIP sources as the short-term effect experiment. Four aerobic denitrification reactors, already operating stably, were used for the experiment. The four reactors were R0, R1, R2, and R3. R0 served as a control group without the addition of additional pollutants, R1 was subjected to continuous PA stress, R2 to continuous CIP stress, and R3 to a continuous combined PA and CIP stress. The experiment lasted a total of 70 days. The low-concentration stress phase lasted from day 0 to 35, with PA concentrations of 5 mg / L and CIP concentrations of 0.1 mg / L; the high-concentration stress phase lasted from day 36 to 70, with PA concentrations of 50 mg / L and CIP concentrations of 1 mg / L. All four reactors had been operating stably for a period of time before the stress treatment. The synthetic wastewater used in the four reactors was primarily KNO₃ as the nitrogen source. The inlet pH was approximately 7.0-7.5. The reactors had an effective volume of approximately 1.5 L and were operated in the dark. The agitator speed in all four reactors was 110 rpm. During daily water changes, appropriate amounts of PA and prepared CIP stock solution were added to achieve the desired stress concentration. PA and CIP were added continuously during the stress phase. During the one-day cycle, aeration and stirring were performed for 23 hours, followed by a 30-minute standing period, and a 30-minute water exchange period for simulated wastewater treatment plant influent and effluent. The ambient temperature was maintained at approximately 20-25°C throughout the long-term stress experiment.

[0047] 2. The source of bacterial flora and culture medium are as follows:

[0048] The inoculum sludge used in the experiment was taken from denitrification sludge that had been stored frozen in the laboratory for a long time. Through careful adjustment of the rotation speed and aeration operation in a custom-made SBR reactor, and after a long period of activation and cultivation, aerobic denitrification sludge with excellent nitrogen and phosphorus removal performance was successfully cultivated.

[0049] Culture medium components: CH3COONa 4.698 g·L -1 , KNO31g·L -1 , KH2PO40.65g·L -1 ,MgSO4·7H2O0.1g·L -1 , CuSO40.1mg·L -1 , CaCl20.01g·L -1 , FeSO4·7H2O 0.006g·L -1 Dissolved in deionized water, C / N=10, pH range of 7.0-7.5, containing approximately 140 mg·L -1 of nitrate nitrogen.

[0050] 3. Main analysis items and measurement methods

[0051] 3.1 Water quality testing methods

[0052] Before testing water samples, a pretreatment step involving centrifugation and filtration is required. Centrifugation is performed at 8000 rpm for 5 minutes, followed by filtration through a 0.22 μm filter membrane. Basic water quality indicators are determined using the corresponding national standard methods.

[0053] The present invention adopts the gravimetric method to determine the mixed liquor suspended solids concentration (MLSS) (g / L) and the mixed liquor volatile suspended solids concentration (MLVSS) (g / L). The specific steps are as follows:

[0054] (1) First, dry the quantitative filter paper in an oven at 105°C for 2 hours, then take it out and place it in a desiccator to cool to constant weight, which is recorded as m0 (g).

[0055] (2) Next, filter 30 mL of the sample using the dried quantitative filter paper. Place the filter paper in an oven at 105°C and dry for 2 hours. After drying, place the sample in a desiccator and cool to a constant weight, which is recorded as m1 (g).

[0056] (3) At the same time, place the clean crucible in an oven at 105°C and dry for 2 hours. After taking it out, place it in a desiccator and cool it to constant weight. Record it as m2 (g).

[0057] (4) Finally, place the quantitative filter paper from step (2) into the crucible from step (3) and place them together in a muffle furnace. After the temperature of the muffle furnace rises to 600°C, calcine for 1 hour. After calcination, remove the crucible and filter paper, cool naturally at room temperature, and then place them back in a desiccator until a constant weight is reached. Record this as m³ (g).

[0058] According to the above steps, the present invention can be calculated according to the following formula:

[0059] MLSS(g / L)=[(m1~m0) / V]*1000

[0060] MLVSS(g / L)={[(m1+m2~m0)~m3] / V}*1000

[0061] 3.2 EEM spectrum analysis

[0062] The EEM spectra of the extracted EPS were measured using a 3D-EEM spectrometer (F-4500 fluorophotometer). Before testing, the sample was diluted to an absorbance of <0.05 at 200 nm. The sample was scanned using the F-4500 fluorophotometer, using a deuterium lamp as the excitation light source, a PMT voltage of 700 V, an excitation wavelength of 200 nm to 500 nm (with 5 nm intervals), and an emission wavelength of 220 nm to 600 nm (with 5 nm intervals). The scan speed was 12,000 nm / min.

[0063] 3.3 PARAFAC analysis

[0064] The Parallel Factor Analysis (PARAFAC) algorithm uses alternating least squares to decompose a trilinear model. This algorithm can reduce the interference of overlapping fluorophores between various compounds in the EEM and mathematically decompose each EEM spectrum into individual fluorescent components and quantify their relative amounts.

[0065]

[0066] Where:

[0067] f—a component in the model; F—the number of model components;

[0068] x ijk — represents the fluorescence intensity of the i-th sample at the j-th emission wavelength and the k-th excitation wavelength;

[0069] e ijk — represents the residual sum of squares in the model;

[0070] a if—The score of the component in the i-th sample model is proportional to the concentration of the f-th component in sample i;

[0071] b jf —Estimated value of the emission spectrum of the fth component;

[0072] c kf —Estimated value of the excitation spectrum of the fth component.

[0073] 3.4 Quantification and Extraction of EPS

[0074] EPS was extracted using a heat extraction method. First, the sample was thoroughly washed three times with saline and then transferred to a 50-ml centrifuge tube. Next, 30 ml of a 0.05% NaCl solution was added to the centrifuge tube and heated at 60°C for 10 minutes. Subsequently, the tube was centrifuged at 4000 rpm for 15 minutes. After centrifugation, the supernatant was aspirated and filtered through a 0.45-μm cellulose acetate membrane. The filtrate obtained was the EPS extract and stored at ~20°C until use.

[0075] To determine the volatile suspended solids (VLS) in the sludge mixture, the present invention uses the standard gravimetric method. The protein and polysaccharide contents in EPS are calculated based on the PN and PS contents per gram of MLVSS. PN is determined using Coomassie blue staining, and PS is determined using the anthrone-sulfuric acid method.

[0076] 3.5 Denitrification enzyme activity: NAR, NIR

[0077] The biological denitrification effect of aerobic denitrifying bacteria mainly depends on the denitrification process, which is regulated by nitrate reductase (NAR) and nitrite reductase (NIR). Therefore, in order to study the effects of different concentrations of microplastic PA and antibiotic CIP on aerobic denitrifying bacteria, the present invention can explore their biochemical response by detecting the activities of nitrate reductase and nitrite reductase.

[0078] After denitrification, the bacterial suspension was removed and washed three times with 0.01 M PBS (pH 7.4). The resuspended bacterial cells were then disrupted using ultrasonic probes (300 W, 5 min) in an ice bath. The crude crystals in the supernatant were collected after centrifugation (12,000 g, 10 min). Enzyme activity was determined based on protein content using the Bradford method with bovine serum albumin (BSA) as the standard.

[0079] NAR and NIR activities were evaluated using sodium bisulfite (Na2S2O4), methyl vinoxyl (MV), and sodium as electron donors. Typically, 500 μL of 100 mM Na2S2O4, 500 μL of 4 mM MV, 500 μL of 0.04 M PBS (pH 7.4), 200 μL of NaNO3 or NaNO2, and 300 μL of the crude product were added to a 2 mL tube and incubated at 30°C in a shaker (150 rpm) for 30 min. The reduced NaNO3 and NaNO2 were then measured. NAR and NIR activities can be expressed as μmol NaNO3 / (min·mg protein) and μmol NaNO2 / (min·mg protein).

[0080] 3.6 Microbial diversity testing

[0081] Sludge samples were collected from each reactor on days 35 and 70 to analyze community changes after the addition of different pollutants. High-throughput 16S rRNA gene sequencing was performed by Magigene (Guangdong, China). The 16S rRNA gene V4 region was amplified using bacterial primers 515F and 806R. Sequences were compared with microbial data from the Silva database. High-quality sequences with a similarity threshold greater than 97% were obtained and clustered into operational taxonomic units.

[0082] To study microbial diversity, the present invention can use single-sample diversity analysis (i.e., Alpha diversity) to reveal the richness and diversity characteristics of microbial communities. The following are several commonly used community distribution diversity indices and their calculation methods:

[0083] When drawing a relative abundance distribution map, the present invention first needs to count the sequence information of different classification levels (such as phylum, class, order, family, genus, species and OTU, etc.) in OTU_table and calculate the relative abundance of each taxonomic group. Then, the present invention can select taxonomic groups with a relative abundance exceeding 1% (default value) and the top 15 taxonomic groups with the highest abundance (default value), and draw the corresponding relative abundance distribution map based on the sample or group. The present invention selects the top 10 taxonomic groups at the phylum level of community structure and the top 15 taxonomic groups at the genus level of community structure to draw relative abundance distribution maps.

[0084] 3.7 Pearson Correlation Analysis

[0085] Pearson correlation analysis is a statistical method used to quantify the strength and direction of the linear relationship between two variables. The core of this analysis is to calculate the Pearson correlation coefficient, which is obtained by dividing the covariance of the two variables by the product of their respective standard deviations, and its value range is limited to between -1 and 1. If the correlation coefficient is positive, it indicates that the two variables are positively correlated; if it is negative, they are negatively correlated; if the coefficient is zero, it means that there is no linear association between the two variables. In addition, the closer the absolute value of the correlation coefficient is to 1, the more significant the linear relationship between the two variables; conversely, the closer the absolute value is to 0, the weaker the linear relationship. In order to more intuitively display the distribution of the Pearson correlation coefficient, a heat map is usually used for visualization.

[0086] The present invention uses SPSS (v.26.0) to perform Pearson correlation tests to find correlations between key parameters. P < 0.05 is considered a significant correlation, and P < 0.01 is considered a highly significant correlation. All figures were drawn using the CorrelationPlot plug-in in Origin (v.2021).

[0087] 4. Results

[0088] 4.1 Nitrogen and carbon removal under short-term stress

[0089] The changes in the concentrations of basic water quality indicators under different concentrations of microplastics and antibiotic stress are as follows: Figure 1 As shown, over the five-day reactor reaction period, NO₃-N, NO₂-N, TN, and COD concentrations remained relatively stable. The control group achieved higher removal rates for NO₃-N, TN, and COD than the group treated with microplastics and antibiotics. The accumulation rate of NO₂-N was also lower than that of the group treated with microplastics and antibiotics. This indicates that 50 and 500 mg / L PA and 0.1 and 1 mg / L CIP exerted acute stress on aerobic denitrifying bacteria.

[0090] from Figure 1 It can be seen that under the condition of single pollutant stress, with the increase of PA and CIP concentrations, the removal rates of NO3--N, TN, and COD all decreased to varying degrees, and the acute stress effect of CIP on aerobic denitrifying bacteria was stronger than that of PA. At the beginning of the reaction, there was no NO2--N in the culture medium. As the bacteria reduced NO3--N, NO2--N gradually began to accumulate, and the accumulation of nitrite in the microorganisms had a certain inhibitory effect on their activity. Figure 1 The data in (b) prove this point, and CIP has a stronger inhibitory effect on the conversion of NO2--N than PA.

[0091] Antibiotics, especially high concentrations of antibiotics, can effectively block the pores and material exchange channels on the surface of high-molecular sludge. This may be an important reason that affects the transfer of substances during biological metabolism. However, after the addition of the pollutant microplastic PA, the degradation rates of NO3--N, TN, and COD were significantly reduced by the antibiotic CIP, and the conversion efficiency of NO2--N was enhanced. It can be inferred that the presence of CIP inhibits the aerobic denitrification process and reduces the denitrification capacity, while PA may reduce the negative impact of CIP. In addition, the larger specific surface area of PA can provide more attachment sites for aerobic denitrification sludge, which helps the sludge to flocculate in water. Larger sludge particles can adapt to high concentrations of CIP for a period of time and exhibit higher biological activity.

[0092] 4.2 Changes in DOM Fluorescence Groups Under Short-term Stress

[0093] Three-dimensional fluorescence can analyze more abundant UV-active substances in organic matter, such as Figure 2 The following table shows the EEM spectra of organic components in reactor samples under different concentrations of PA and CIP stress to explore the impact on the metabolic capacity of aerobic denitrifying microbial communities. Based on the different regions of the EEM spectra, organic matter can be divided into five categories, as shown in Table 1.

[0094] Table 1 Three-dimensional fluorescent material partitioning

[0095]

[0096] exist Figure 2 There are two obvious fluorescence peaks, among which SpeakA (Ex / Em250~400nm / 380~500nm) is a humic acid-like fluorescence peak, and SpeakB (Ex / Em200~250nm / 380~500nm) is a fulvic acid-like fluorescence peak. Humic acid substances are related to the growth of certain bacteria, the decomposition of macromolecular organic matter (such as PS and PN), and dead cells. Fulvic acid substances are a low molecular weight substance with biological activity and are the ultimate microstructure form after active biological decomposition. Figure 2As can be seen, under single pollutant stress, both humic and fulvic acid-like fluorescence peaks are present, with the difference being that at a CIP concentration of 1 mg / L, the hues of these peaks are darker. Under combined pollutant stress, due to the presence of CIP, both humic and fulvic acid-like fluorescence peaks are more pronounced than under any other single stress except 1 mg / L CIP. The above data analysis indicates that the antibiotic CIP causes the accumulation of large humic acid-like substances, especially at high CIP concentrations. However, varying concentrations of microplastics (PA) have little effect on the organic components of water samples. When PA and CIP are present simultaneously, PA mitigates the impact of CIP, while changes in fulvic acid-like substances are not evident in the EEM spectra. Therefore, this experiment further analyzed the PARAFAC model based on the three-dimensional fluorescence spectral data.

[0097] 4.3 Analysis of EEM spectra based on the PARAFAC model

[0098] The PARAFAC model was used to perform parallel factor analysis on the three-dimensional fluorescence data of all samples, as shown in Figure 3 The results showed that two groups of fluorescent components could be resolved, namely C1: (Ex / Em270 / 320nm~415nm): humic acid-like; C2: (Ex / Em250nm~430nm): fulvic acid-like.

[0099] Figure 4 Two fluorescent components, C1 humic acid and C2 fulvic acid, were quantitatively characterized in nine sample groups. 3D-EEM fluorescence spectroscopy revealed that the concentrations of C1 and C2 in the control group were 0 and 1551, respectively. Under single-pollutant stress, the concentrations of C1 and C2 in the 50 and 500 mg / L PA and 0.1 and 1 mg / L CIP stress groups were 0, 0, 427, and 11099, and 1548, 1357, 1201, and 0, respectively. Under combined stress, the concentrations of C1 and C2 in the PA1+CIP1, PA1+CIP2, PA2+CIP1, and PA2+CIP2 stress groups were 1947, 2238, 199, and 2704, and 1023, 901, 1345, and 835, respectively. Humic acid content varied significantly under single and combined stresses with different concentrations of PA and CIP. The antibiotic CIP causes the accumulation of large-molecule humic acids, while smaller-molecule fulvic acids decrease with the addition of CIP. The addition of PA slightly reduces the accumulation of fulvic acids but does not lead to the accumulation of humic acids, and it also reduces the impact of CIP. These data demonstrate that the addition of CIP inhibits the humification of DOM, and this inhibitory effect increases with increasing CIP concentration. This suggests that the addition of CIP strongly inhibits the activity of aerobic denitrifying bacteria, while the addition of PA reduces this inhibitory effect.

[0100] 4.4 Biochemical responses of microbial communities to short-term combined stress

[0101] 4.4.1 Changes in EPS Content under Short-term Stress

[0102] EPS is a complex mixture of various substances including PN and PS, which accounts for about 80% of the sludge. This substance not only helps to aggregate and protect microorganisms, but also provides effective protection for denitrification sludge under harsh environmental conditions. Previous studies have confirmed that there is a positive correlation between the concentration of EPS and the stability of sludge, that is, an increase in EPS concentration will improve the stability of sludge. After a 5-day short-term stress experiment, the present invention tested the EPS content of the sludge in the reactor system, and the results are as follows: Figure 5 shown.

[0103] After adding different concentrations of PA and CIP to the nine reactors, the PN and PS content of EPS changed significantly. The PN and PS concentrations of the control group were 464.33 and 67.98 mg / (g·MLVSS), respectively. Under single pollutant stress, the PN and PS concentrations in the 50 and 500 mg / L PA and 0.1 and 1 mg / L CIP stress groups were 367.06, 370.95, 5.01, and 426.72 mg / (g·MLVSS) and 49.03, 55.45, 110.20, and 20.61 mg / (g·MLVSS), respectively. Under combined stress, the PN and PS concentrations in the PA1+CIP1, PA1+CIP2, PA2+CIP1, and PA2+CIP2 stress groups were 359.27, 243.84, 208.82, and 307.39 mg / (g·MLVSS) and 101.37, 65.57, 89.97, and 56.42 mg / (g·MLVSS), respectively. Polysaccharides (PS) and proteins (PN) are the main components of EPS, and their hydrophilic and hydrophobic groups show significant differences. The lower the PS / PN ratio, the better the biomass flocculation and sedimentation effect. After adding CIP, the PS content increased significantly and the PN content decreased, and the relationship was linear with the increase of CIP concentration. However, the reactor with PA was less affected. Figure 5 Analysis suggests that the abnormally high EPS concentrations originate from damaged bacterial cells. The above data demonstrate that PA improves sludge stability and accelerates sedimentation. Overall, the addition of PA not only mitigates CIP-induced sludge damage but also enhances aerobic denitrification sludge denitrification performance.

[0104] 4.4.2 Changes in key denitrase activities under short-term stress

[0105] The good biological denitrification of aerobic denitrifiers depends on the denitrification process, which is controlled by nitrate reductase (NAR) and nitrite reductase (NIR). Figure 6 shown.

[0106] from Figure 6 (a) It can be seen that the NAR enzyme activity of the aerobic denitrifying bacteria was significantly inhibited under the stress of different concentrations of PA and CIP. The NAR enzyme activity of the control group was 0.341 μmol / (min / mg protein). Under single pollutant stress, the enzyme activities of the 50 and 500 mg / L PA and 0.1 and 1 mg / L CIP stress groups were 0.334, 0.319, 0.268, and 0.244 μmol / (min / mg protein), respectively. Under combined stress, the enzyme activities of the PA1+CIP1, PA1+CIP2, PA2+CIP1, and PA2+CIP2 stress groups were 0.295, 0.285, 0.294, and 0.282 μmol / (min / mg protein), respectively. From the above experimental results, it can be seen that under single stress, the NAR activity of the bacterial community decreases with the increase of pollutant concentration, and CIP has a greater impact on aerobic denitrification than PA; under combined stress, the NAR activity of the bacterial community also decreases with the increase of pollutant concentration. From the comparison of the two sets of data, it can be seen that the addition of PA reduces the damage of CIP to enzyme activity.

[0107] Depend on Figure 6 (b) The changes in bacterial NIR enzyme activity showed similar trends to those in NAR enzyme activity. Under single pollutant stress, the NIR enzyme activity of the control group was 0.0112 μmol / (min / mg protein), while the enzyme activities of the 50 and 500 mg / L PA and 0.1 and 1 mg / L CIP stress groups were 0.0098, 0.0096, 0.0069, and 0.0061 μmol / (min / mg protein), respectively. Under combined stress, the enzyme activities of the PA1+CIP1, PA1+CIP2, PA2+CIP1, and PA2+CIP2 stress groups were 0.0097, 0.0089, 0.0095, and 0.0087 μmol / (min / mg protein), respectively. It can be seen that the NIR enzyme activity of the bacterial community showed a downward trend with increasing stress concentrations. Under combined stress, the damage of CIP to enzyme activity decreased with the addition of PA.

[0108] 4.5 Correlation between Physiological and Biochemical Responses

[0109] In order to analyze the significant effects of various physiological responses and biochemical responses of aerobic denitrifying bacteria under short-term combined stress, the present invention conducted a Pearson correlation analysis and analyzed the response mechanism of aerobic denitrification by combining the correlation between physiological and biochemical responses and literature.

[0110] The correlation between PA, CIP and physiological and biochemical responses is the key point that needs to be explored. CIP is negatively correlated with denitrification indicators (NO3--N, NO2--N, COD removal rate) in physiological responses and fulvic acid-like substances in DOM, and positively correlated with humic acid-like substances in DOM. In addition, it is also negatively correlated with all biochemical response indicators. CIP has been reported to effectively inhibit the biological activity of aerobic denitrifying bacteria and the denitrification process, which can be mutually confirmed by the correlation analysis. The accumulation of humic acid-like substances can represent the deepening of the degree of biological corruption in the water body, which may be the reason for the significant difference between humic acid-like substances and other indicators. However, when comparing PA and CIP, the present invention found that the significance of PA to bacteria is almost completely opposite to that of CIP. This shows that under short-term stress, PA can weaken the negative impact of CIP on bacteria and improve denitrification performance. Figure 7 .

[0111] In order to further analyze the state of aerobic denitrifying bacteria after short-term combined stress of PA and CIP and to further study the aerobic denitrification system, the present invention discussed the correlation between physiological response and biochemical response. Denitrification-related indicators showed significant differences with other indicators, among which COD had the most significant correlation (6 pairs, p < 0.05), followed by NO3--N (4 pairs, p < 0.05). COD and NO3--N were significantly positively correlated with fulvic acid-like substances, key denitrification enzyme activities and ETSA activity, and significantly negatively correlated with humic acid-like substances. The results showed that systems with good denitrification performance usually had lower humic acid-like substances and higher accumulation of fulvic acid-like substances, key denitrification enzyme activities and ETSA activity. However, EPS-related indicators only had a strong correlation with NO2--N, which was significantly negatively correlated with EPS-PN and positively correlated with EPS-PS. According to the correlation between EPS and NO2--N, it can be calculated that the accumulation of NO2--N is positively correlated with the PS / PN status, which means that the accumulation of NO2--N will inhibit the aerobic denitrification system by affecting the EPS status.

[0112] Overall, PA significantly alleviated the effects of CIP on aerobic denitrifiers under short-term stress, promoted the denitrification metabolic process, and enhanced nitrate reduction.

[0113] 4.6 Nitrogen and carbon removal under long-term stress

[0114] During the 75-day operation period, there were two different time periods: 5 mg / LPA and 0.1 mg / LCIP (days 1 to 35, stage 1) and 50 mg / LPA and 1 mg / LCIP (days 36 to 75, stage 2). Different concentrations of microplastics and antibiotics, alone or in combination, had varying degrees of impact on the aerobic denitrification process. Adding microplastics to the combined stress condition reduced the impact.

[0115] from Figure 8 It can be seen that compared with the control group, the NO3--N, nitrogen source removal rate, NO2--N, and COD concentrations in the effluent of the PA group were relatively stable. In the first stage, the NO3--N and NO2--N in the effluent of the PA group were lower than those of the control group, and the nitrogen source removal rate was higher. This shows that low concentrations of PA can enhance the carbon removal and denitrification performance of the aerobic denitrifying bacteria sludge. However, the performance of COD was completely different, with a slight increase in concentration, indicating that low-concentration microplastic PA reduced the COD consumption of the system. In the second stage, the concentration of PA increased, and the NO3--N, NO2--N, and COD in the effluent of the PA group were higher than those of the control group, with a lower nitrogen source removal rate. This shows that the long-term stress of 50 mg / L PA has been able to inhibit the performance of the aerobic denitrifying bacteria sludge.

[0116] In the CIP group and the PA+CIP group, the trends of effluent NO3--N, nitrogen source removal rate, NO2--N and COD concentrations were similar ( Figure 8 As the influent antibiotic CIP concentration increased, the effluent nitrogen concentration and COD in each reactor increased to varying degrees. After a period of unstable operation, they gradually decreased and stabilized. This may be because the antibiotic CIP affects the state and biological characteristics of the sludge. Higher CIP concentrations have a greater impact on aerobic denitrifying bacteria. This leads to the accumulation of NO₃-N, NO₂-N, and COD, and a decrease in nitrogen source removal efficiency.

[0117] from Figure 8 Long-term experimental data show that bacterial activity gradually recovers as they acquire drug resistance, but increasing CIP concentrations can lead to slow or irreversible recovery of carbon and nitrogen removal capabilities. However, after adding microplastic PA, nitrogen concentration and COD were less affected by CIP in the early stages of the two phases, and the recovery time was also shorter than that of the CIP group.

[0118] From this, it can be inferred that the presence of CIP inhibits aerobic denitrification and reduces denitrification capacity, while PA may reduce the negative impact of CIP. In addition, on the one hand, PA helps sludge agglomeration, and larger sludge agglomerates can adapt to high concentrations of CIP for a period of time and show higher biological activity. On the other hand, microplastic PA has a large specific surface area and can adsorb a certain amount of CIP, thereby reducing the impact of CIP on the reactor.

[0119] 4.7 Changes in DOM Fluorescence Groups Under Long-term Stress

[0120] Three-dimensional fluorescence spectroscopy can effectively infer the metabolic capacity of aerobic denitrifying microbial communities by analyzing the dissolved organic matter in the reactor effluent. Water samples were collected at 35 and 75 days of reaction time and analyzed for three-dimensional fluorescence spectroscopy to analyze the sources of organic components in the effluent samples under different concentrations of PA and long-term CIP stress.

[0121] After 35 days of stress in the first stage, according to the EEM spectrum Figure 9 As shown, in all four reactors, C1 and C2 components were detected within the Ex / Em ranges of 240-250 / 300-330 nm and 250-350 / 450-475 nm, respectively. C1 is believed to be a tryptophan-like substance, and C2 is a humic acid-like substance. It can be seen that humic acid substances increased significantly after the addition of CIP, a trend similar to that observed in the short-term stress analysis, further demonstrating that CIP can affect the humification level of DOM.

[0122] At the end of the second phase, the water samples were subjected to three-dimensional fluorescence spectroscopy. Figure 10 It can be seen that after adding higher concentrations of PA and CIP, the three-dimensional fluorescence spectra of the four reactors showed similar characteristics to those in the first stage. The prominent C1 (Ex / Em 200-250 / 330-380 nm) represents the tryptophan fluorescence peak, while the C2 (Ex / Em 250-400 / 380-500 nm) represents the humic substance fluorescence peak. With the addition of CIP, the abundance of humic acid substances in the three-dimensional fluorescence spectra increased.

[0123] In summary, under EEM spectral analysis, the two stages of the long-term stress experiment have similarities. Tryptophan proteins and humic acid proteins are important components of soluble organic matter, and tryptophan fluorescence peaks usually contain peaks related to microbial activity. Humic acid is involved in the growth of certain bacteria, macromolecular organic matter and the decomposition of dead cells. The present invention noted that the fluorescence peaks of different concentrations of PA were similar to those of the control group, while under the stress of different concentrations of CIP, the content of tryptophan proteins remained stable, while the content of humic acid proteins was significantly higher than that of other samples, indicating that microbial decay intensified and metabolic activity decreased, leading to the accumulation of humic substances. When CIP was not present, humic acid proteins could hardly be observed in the sample, but in the presence of CIP, their proportion was significantly increased.

[0124] 4.8 Analysis of EEM spectra based on the PARAFAC model

[0125] according to Figure 11 As shown in the figure, a PARAFAC model was established to perform parallel factor analysis on the existing three-dimensional fluorescence data. The results revealed two groups of fluorescence components: C1 (Ex / Em 230nm-335nm): tryptophan-like; C2 (Ex / Em 255nm-460nm): humic acid-like.

[0126] In order to further analyze the main components of DOM in the water body where aerobic denitrifying bacteria are located under long-term combined stress, the EEM-PARAFAC model was established to quantitatively describe the fluorescent components C1 tryptophan substances and C2 humic acid substances in four groups of reactors in two stages. Figure 12As shown, in the first phase, the tryptophan and humic acid contents in the control, PA, CIP, and PA+CIP groups were 1010, 1163, 1080, and 1148, respectively, and 345, 355, 532, and 481, respectively. In the second phase, the tryptophan and humic acid contents in the control, PA, CIP, and PA+CIP groups were 1095, 951, 1303, and 1218, respectively, and 263, 213, 1070, and 1122, respectively. Comparison of tryptophan contents between the two phases reveals that the amount of tryptophan remains stable. The close correlation between tryptophan content and microbial activity demonstrates the strong adaptability of the aerobic denitrifying bacterial community. The humic acid content varied significantly with the phase. At the same phase, the humic concentration in the reactor with CIP addition was significantly higher than that in the control and PA groups. The addition of PA reduced the accumulation of humic acid to a certain extent. When the CIP concentration was increased from 0.1 mg / L to 1 mg / L, the accumulation of humic acid substances in the 75-day CIP group was twice that of the 35-day CIP group. This also shows that long-term stress caused by high-concentration CIP can lead to increased microbial decay and reduced metabolic activity, which in turn leads to the accumulation of humic substances.

[0127] 4.9 Changes in sludge concentration and EPS content under long-term stress

[0128] The aerobic denitrification sludge system is affected to a certain extent by the sludge concentration. The changes in the sludge concentration in the aerobic denitrification reactor operation system during the long-term stress experiment are shown in the figure below. Figure 13 As shown, observe Figure 13 It was found that in the early stages of each phase, the sludge concentrations in the PA, CIP, and PA+CIP groups all showed a downward trend compared to the control group. The downward trend was more pronounced in the CIP group with ciprofloxacin added, and the sludge concentration in the group containing both PA and CIP was higher than that in the CIP group. These results are similar to those of the basic water quality indicators. The addition of CIP inhibits the sludge concentration of aerobic denitrifying bacteria and has an inhibitory effect on the denitrification process, while the addition of PA can mitigate this effect.

[0129] Extracellular polymeric substances (EPS) serve as the first barrier to toxic external compounds from penetrating bacterial cells and play a crucial role in determining the performance of activated sludge. EPS, composed of substances such as PN and PS, makes up the vast majority of sludge weight. Therefore, EPS analysis can effectively help understand the state and structural stability of sludge.

[0130] The effects of EPS content (PS and PN) on sludge structure under long-term stress of different concentrations of PA and CIP were investigated. Figure 14). In the first stage, there were significant differences among the reactors. The EPS content of the PA group, CIP group, and PA+CIP group began to increase significantly in the early stage of the first stage, with the maximum increases being 616.475 mg.g-1MLVSS, 625.557 mg.g-1MLVSS, and 659.079 mg.g-1MLVSS, respectively. Studies have shown that under adverse environmental or toxic stress, microorganisms often cause cell lysis and release a large amount of EPS into the water body in the early stage, and the more unfavorable the growth conditions, the more EPS is produced. However, in the middle and late stages of the first stage, the EPS in the PA group, CIP group, and PA+CIP group began to recover, indicating that under long-term stress of lower concentrations of PA and CIP, the sludge state will gradually recover after being damaged. However, the addition of PA alleviated the effect of CIP on the sludge state to a certain extent.

[0131] After adding different concentrations of PA and CIP to each reactor at different times, the changes of PN and PS in EPS ( Figure 15 ). Protein (PN) and polysaccharide (PS) are the main components of EPS, and there are obvious differences in their hydrophilic and hydrophobic groups. In comparison, the lower the PS / PN, the more conducive it is to biomass flocculation and the better the sedimentation effect. In the present invention, regardless of whether PA and CIP are added, the change trends of PN and PS are similar. In the early stage of each stage, the PN and PS contents will increase significantly, and then gradually decrease. In order to more clearly show the change levels of PN and PS in the EPS of the four reactors, the average PS / PN values of different reactors in two stages were taken. Among them, in the first stage, the PS / PN of the control group, PA group, CIP group and PA+CIP group were 21%, 31%, 44% and 35% respectively; in the second stage, the PS / PN of the control group, PA group, CIP group and PA+CIP group were 15%, 28%, 20% and 18% respectively. This shows that although long-term PA stress will inhibit the rate of biomass flocculation, PA will also make the sludge have better stability and faster sedimentation rate under the stress of CIP. In general, long-term PA stress itself will cause certain damage to the sludge, but it can also reduce the damage of CIP to the sludge and enable aerobic denitrification sludge to maintain a higher denitrification performance.

[0132] 4.10 Changes in Key Denitrase Activities Under Long-term Stress

[0133] The experiment tested the activities of nitrate reductase (NAR) and nitrite reductase (NIR) to explore the biochemical response of aerobic denitrifying bacteria under long-term stress of different concentrations of microplastic PA and antibiotic CIP. The experimental results are as follows: Figure 16 shown.

[0134] from Figure 16(a) It can be seen that under the long-term stress of PA and CIP at different concentrations, the NAR enzyme activity of the aerobic denitrifying bacteria was significantly affected by the stress. At 18 days, the NAR enzyme activity of the control group was 0.204 μmol / (minmgprotein), and the enzyme activities of the PA group, CIP group and combined stress group were 0.190, 0.144 and 0.177 μmol / (minmgprotein), respectively; at 36 days, the NAR enzyme activity of the control group was 0.230 μmol / (minmgprotein), and the enzyme activities of the PA group, CIP group and combined stress group were 0.219, 0.203 and 0.225 μmol / (minmgprotein), respectively; On day 54, the NAR enzyme activity of the control group was 0.199 μmol / min / mg protein, while that of the PA, CIP, and combined stress groups was 0.165, 0.135, and 0.158 μmol / min / mg protein, respectively. Finally, on day 72, the NAR enzyme activity of the control group was 0.215 μmol / min / mg protein, while that of the PA, CIP, and combined stress groups was 0.190, 0.137, and 0.165 μmol / min / mg protein, respectively. Analysis of the experimental results showed that, based on the duration of stress, the NAR activity of the bacterial communities showed a trend of first decreasing and then increasing. Under CIP stress conditions at the same time point and stage, the NAR enzyme activity of the bacterial communities was lower than that of the PA group and the combined pollution group combined with PA. This result suggests that CIP has a more significant inhibitory effect on bacterial denitrification.

[0135] Depend on Figure 16 (b) It can be seen that the change trend of bacterial NIR enzyme activity is similar to that of NAR enzyme activity. Under the long-term stress of different concentrations of PA and CIP, the NAR enzyme activity of the aerobic denitrifying bacteria was significantly affected by the stress. At 18 days, the NIR enzyme activity of the control group was 0.0095 μmol / (minmgprotein), while the enzyme activities of the PA group, CIP group and combined stress group were 0.0077, 0.0049 and 0.0063, respectively.

[0136] 0.0068μmol / (minmgprotein); at 36 days, the NIR enzyme activity of the control group was 0.0101μmol / (minmgprotein), and the enzyme activities of the PA group, CIP group and combined stress group were 0.0088, 0.0069 and 0.0087μmol / (minmgprotein), respectively; at 54 days, the NIR enzyme activity of the control group was 0.0088μmol / (minmgprotein), and the enzyme activities of the PA group, CIP group and combined stress group were 0.0065, 0.0032 and 0.0050μmol / (minmgprotein), respectively; finally, at 72 days, the NIR enzyme activity of the control group was 0.0099μmol / (minmgprotein), and the enzyme activities of the PA group, CIP group and combined stress group were 0.0082, 0.0041 and 0.0061μmol / (minmgprotein), respectively. Observations revealed that with increasing stress duration, bacterial NIR enzyme activity initially decreased and then increased. Under the same CIP stress concentration, enzyme activity in the PA-added group was generally lower compared to the CIP-only group. This finding further explains why, at the same time point and CIP concentration, the CIP group showed higher NO₂-N accumulation than the PA-added group.

[0137] From the above data, it can be seen that under CIP stress at the same stage and time, the NAR and NIR enzyme activities of the aerobic denitrifying bacteria group were lower than those in the PA group, CIP group, and combined stress group, indicating that CIP has a stronger inhibitory effect on the key enzyme activities of bacterial denitrification. This is due to the toxicity of the antibiotic CIP itself to the activated sludge. The key enzyme activity of the PA group also decreased slightly compared with the control group, which shows that PA will also have a slight inhibitory effect on the denitrification capacity of aerobic denitrification. However, when PA was added to the CIP group, the effect of CIP on the key enzyme activities of aerobic denitrifying bacteria was reduced to a certain extent. This is due to the adsorption of the microplastic PA itself and the stimulation of sludge flocculation to improve the sludge's resistance to pollutants.

[0138] 4.11. Adaptation of microbial communities under long-term combined stress

[0139] This study employed high-throughput 16S rRNA sequencing technology to conduct an in-depth study of the microbial communities within the reaction system. The results of the alpha diversity analysis are detailed in Table 2. As can be seen, the coverage of all samples exceeded 99.9%, demonstrating that the sequencing depth was sufficient to cover virtually all microorganisms within the samples, ensuring that the sequencing results accurately reflect the actual microbial populations within the samples.

[0140] In addition, the present invention uses the Shannon index to characterize the diversity of the microbial community. Generally speaking, as the Shannon index increases, the diversity of the microbial community will also increase accordingly. At the same time, the Simpson index is also an important reference index. The smaller its value, the greater the diversity of the community usually means. The richness of the microbial community can be reflected by the ACE index and the Chao1 index. The comprehensive analysis of these indices provides a comprehensive and in-depth understanding of the microbial community diversity for the present invention. As shown in the results of Table 2, the abundances and biodiversity of the communities are CIP < PA + CIP < 0 < PA in sequence. It shows that CIP has a significant adverse effect on the diversity and stability of the microbial community, while PA enhances the stability of the community structure and the tolerance to adverse environments. Therefore, from the perspective of system stability, the aerobic denitrification community in the reactor after adding PA may obtain better environmental tolerance.

[0141] Table 2 Microbial diversity indices of aerobic denitrifying bacteria under long-term stress of different concentrations of PA and CIP

[0142]

[0143] Analyze the dominant phyla and genera at each sampling stage to explore the effects of PA and CIP on the community structure composition. The community dynamics at the phylum level are as Figure 17 shown. The dominant phyla are Proteobacteria, Bacteroidota, Chloroflexi, Verrucomicrobiota, Planctomycetota, and Actinobacteriota, accounting for more than 80% in each reactor. The results show that during the whole experiment, the dominant phyla in each reactor basically did not change, but the relative abundances changed dynamically. Proteobacteria is the dominant phylum in all samples, proving its important role in participating in the nitrogen cycle and playing an important role in the denitrification process. From Figure 17It can be seen that under long-term pollutant stress, the relative abundances of Proteobacteria in the four reactors in the two stages were 0 < PA < PA+CIP < CIP, indicating that Proteobacteria can better adapt to long-term pollutant stress and obtain better activity after adapting to the stress. Compared with the 0 group, the PA group and the combined stress group, the relative abundance of Bacteroidota in the CIP group was lower on the 35th day. Bacteroidota is sensitive to the influence of adverse external environments due to its thin cell wall and low peptidoglycan content. Therefore, the decrease in Bacteroidota may be due to its inability to tolerate CIP, or due to interspecies competition. The increase in Proteobacteria may lead to its decrease. Chloroflexi can form a network structure with other bacteria, promote microbial flocculation, and can use dead bacterial cells as substrates for metabolism. The relative abundance of Chloroflexi in the PA group was much higher than that in the 0 group, the PA group and the combined stress group, which indirectly proves that PA can reduce the impact of CIP stress and make aerobic denitrifying sludge bind more tightly. The proportions of the other three bacterial communities related to denitrification, Verrucomicrobiota, Planctomycetota, and Actinobacteriota, were all < 4%, indicating that there is also competition among denitrifying bacteria.

[0144] At the genus level, the changes in the relative abundances of the bacterial communities in each sample are as Figure 18As shown. The dominant genera include Thauera, Azoarcus, and Taibaiella. To clearly show the changes in the dominant genera under single and combined stress of PA and CIP, the average relative abundances of the dominant genera in the reactor at different stages were taken. The relative abundance of Thauera in the control group was 4%, and the relative abundances of Thauera in the PA group, CIP group, and combined stress group were 16%, 21%, and 19% respectively; the relative abundance of Azoarcus in the control group was 24%, and the relative abundances of Azoarcus in the PA group, CIP group, and combined stress group were 25%, 37%, and 34% respectively; while the relative abundance of Taibaiella in the control group was 14%, and the relative abundances of Taibaiella in the PA group, CIP group, and combined stress group were 11%, 5%, and 9% respectively. The above-mentioned dominant genera all have denitrification performance and show high CIP tolerance in this community, but there are still certain differences among these dominant genera. The genus with the strongest competitiveness is Azoarcus, which is an aerobic genus dependent on nitrate, and its relative abundances are 0 < PA < PA + CIP < CIP in sequence. This relative abundance trend is similar to that of Thauera and opposite to that of Taibaiella. This indicates that Azoarcus and Thauera can show stronger resistance to the long-term stress of PA and CIP, and enable the aerobic denitrifying bacterial community to maintain a high denitrification efficiency under the long-term stress of PA and CIP.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving sewage treatment effect under ciprofloxacin stress, characterized in that, The method is to add polyamide to sewage containing ciprofloxacin and then perform aerobic denitrification treatment; when the content of ciprofloxacin in the sewage is 0.1mg / L to 1.0mg / L, the amount of polyamide added is 5mg / L to 500mg / L; The polyamide is used to reduce the inhibitory effect of ciprofloxacin on the activity of aerobic denitrifying bacteria; Under the condition of single pollutant stress, with the increase of PA and CIP concentrations, the removal rates of NO3--N, TN, and COD all decreased to varying degrees, and CIP had a stronger acute stress effect on aerobic denitrifying bacteria than PA. At the beginning of the reaction, there was no NO2--N in the culture medium. As the bacteria reduced NO3--N, NO2--N gradually began to accumulate. The accumulation of nitrite in the microorganisms had a certain inhibitory effect on their activity. Compared with PA, CIP had a stronger inhibitory effect on the conversion of NO2--N. After the addition of the pollutant microplastic PA, the degradation rates of NO3--N, TN, and COD were significantly reduced by the antibiotic CIP, and the conversion efficiency of NO2--N was enhanced. The presence of CIP inhibited the aerobic denitrification process and reduced the denitrification capacity, while PA reduced the negative impact of CIP. The larger specific surface area of PA provides more attachment sites for aerobic denitrification sludge, which helps the sludge flocculate in the water. Larger sludge particles can better adapt to high concentrations of CIP over a period of time and show higher biological activity. The polyamide is used for improving the degradation rate of NO3--N, TN and COD and the conversion efficiency of NO2--N in the presence of ciprofloxacin.

2. The method for improving sewage treatment effect under ciprofloxacin stress according to claim 1, wherein The polyamide is used for improving the stability and sedimentation rate of sludge in water to be treated in the presence of ciprofloxacin.

3. The method for improving sewage treatment effect under ciprofloxacin stress according to claim 1, wherein The polyamide is used for improving the denitrification performance of sludge in water to be treated in the presence of ciprofloxacin.

4. The method for improving sewage treatment effect under ciprofloxacin stress according to claim 1, wherein The polyamide is used to improve the denitrification activity in the presence of ciprofloxacin.

5. The method for improving sewage treatment effect under ciprofloxacin stress according to claim 1, wherein The polyamide is used for improving the stability of aerobic denitrifying bacteria communities in sewage in the presence of ciprofloxacin and their tolerance to adverse environments.

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