A method for inhibiting the spread of resistance genes using ammonia-oxidized sludge

Abstract

This invention discloses a method for inhibiting the spread of resistance genes using ammonia-oxidized sludge, belonging to the field of wastewater treatment technology. The invention proposes a method for inhibiting the spread of resistance genes using ammonia-oxidized sludge. Experiments were conducted using synthetic wastewater as the influent to a reactor enriched with ammonia-oxidized sludge. As the antibiotic concentration increased, the abundance of highly abundant resistance genes decreased. Simultaneously, under long-term operating conditions, the biological denitrification effect was not inhibited. This method not only removes conventional pollutants from wastewater but also effectively inhibits the spread of resistance genes, reducing the spread and damage of pathogens in nature, thereby protecting biodiversity and ecosystem stability.

Description

Technical Field

[0001] This invention relates to a method for inhibiting the spread of resistance genes using ammonia-oxidized sludge, belonging to the field of wastewater treatment technology. Background Technology

[0002] The release of antibiotics into the aquatic environment has numerous adverse effects on humans and the environment, such as chronic toxicity, the emergence and spread of drug-resistant bacteria and resistance genes, which have attracted increasing attention. Antibiotics are widely used to treat various infectious diseases caused by pathogenic microorganisms and also to promote the growth of plants and animals. In recent years, resistance genes have been frequently detected in wastewater treatment plants. Common resistance genes include macrolide resistance genes, sulfonamide resistance genes, quinolone resistance genes, tetracycline resistance genes, and β-lactam resistance genes. Resistance genes can spread and diffuse between bacteria in the environment and between different environmental media, and can continuously replicate within organisms, leading to antibiotic resistance and reduced or even ineffective antibiotic efficacy. Resistance genes can spread between bacteria through mutation, horizontal gene transfer, and vertical gene transfer. Given these harmful effects, the suppression of resistance genes is essential.

[0003] Wastewater treatment typically focuses on removing pollutants such as ammonia nitrogen, phosphates, COD, and antibiotics. However, commonly used wastewater treatment methods do not consider how to inhibit the spread of resistance genes. Biological treatment, the most common method in wastewater treatment plants, due to its high biomass, not only fails to inhibit resistance genes but may actually promote their spread. Therefore, there is an urgent need to develop technologies that can effectively inhibit resistance genes. Summary of the Invention

[0004] To address one or more of the problems mentioned above, this invention provides a method for inhibiting the spread of resistance genes using ammonia-oxidized sludge. The technical solution is as follows:

[0005] This invention proposes a method for inhibiting the spread of resistance genes using ammonia-oxidized sludge, the method being as follows:

[0006] (1) Enriching ammonia oxidation sludge in an ammonia oxidation sludge enrichment reactor;

[0007] (2) Add nitrogen and phosphorus sources to the influent of the reactor and control the pH to be maintained at 7.5-8.0; set the operating cycle of the reactor;

[0008] (3) Antibiotics were added to the reactor feed water in six stages, with the concentration gradually increasing.

[0009] In one embodiment, the enrichment of ammonia oxidation sludge in step (1) is carried out using the method described in patent document CN115849574A.

[0010] In one embodiment, in step (2), the nitrogen source is NH4HCO3 with a content of 2-2.5 g / L.

[0011] In one embodiment, in step (2), the phosphorus source is one or more of K2HPO4 and KH2PO4, with a content of 0.06-0.07 g / L.

[0012] In one implementation, in step (2), pH is controlled by adding NaHCO3.

[0013] In one embodiment, the antibiotic in step (3) is sulfadiazine (SDZ).

[0014] In one implementation, in step (3), the concentration of SDZ in the influent of the six stages is 0 mg / L in the first stage, 0.1 mg / L in the second stage, 0.5 mg / L in the third stage, 2.5 mg / L in the fourth stage, 10.0 mg / L in the fifth stage, and 0 mg / L in the sixth stage. Each stage is operated for 10-13 days.

[0015] In one embodiment, the reactor operates four times a day, with each cycle lasting 360 minutes, including 60 minutes of influent, 260 minutes of aeration, 30 minutes of sedimentation, 5 minutes of drainage, and 5 minutes of settling; the drainage ratio is 25%.

[0016] The method is applied to suppressing the spread of resistance genes.

[0017] The beneficial effects of this invention are:

[0018] Since traditional biological treatment technologies, such as activated sludge processes, cannot effectively suppress resistance genes, this invention proposes a method to inhibit the spread of resistance genes using ammonia-oxidized sludge. This invention involves enriching ammonia-oxidized sludge and using synthetic wastewater as the influent to an ammonia-oxidized sludge enrichment reactor. Experiments showed that as antibiotic concentration increased, the abundance of highly abundant resistance genes decreased. Simultaneously, under long-term operating conditions, the biological denitrification effect was not inhibited. This method not only removes conventional pollutants from wastewater but also effectively inhibits the spread of resistance genes, reducing the spread and damage of pathogens in nature, thereby protecting biodiversity and ecosystem stability. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The effect of enriched ammonia oxidation sludge on the removal of conventional pollutants under different sulfadiazine concentration conditions is shown in the figure.

[0021] Figure 2 The removal rates of conventional pollutants and the specific ammonia oxidation rates of ammonia-oxidized sludge enriched under different sulfadiazine concentration conditions are plotted.

[0022] Figure 3 The effect of enriched ammonia-oxidized sludge on the removal of sulfadiazine under different sulfadiazine concentration conditions is shown in the figure.

[0023] Figure 4 A graph showing the changes in the abundance of sulfonamide resistance genes in ammonia-oxidized sludge under different sulfadiazine concentrations.

[0024] Figure 5 A graph showing the changes in the abundance of sulfonamide resistance genes in traditional activated sludge under different sulfadiazine concentrations. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0026] Test methods

[0027] Ammonia nitrogen concentration: determined by Nessler's reagent spectrophotometry.

[0028] Nitrite concentration: determined by N-(1-naphthyl)-ethylenediamine spectrophotometry.

[0029] Nitrate concentration: determined by ultraviolet spectrophotometry.

[0030] Specific ammonia oxidation rate (SAOR): This is obtained by calculating the ratio of the ammonia oxidation rate (AOR) to the volatile suspended solids concentration (MLVSS) of the mixed liquor. Water samples were taken from the reactor every 10 minutes over 1 hour, and the ammonia nitrogen concentration at each time point was measured. The slope of the linearly fitted line is then used as the AOR. MLVSS was determined by gravimetric method.

[0031] SDZ concentration: determined by high-performance liquid chromatography (HPLC). The HPLC column was a C18 column (250×4.6mm, 5μm; Agilent Technologies, Germany). Mobile phase A was acetonitrile, mobile phase B was 0.1% formic acid solution, the liquid flow rate was 1mL / min, the column temperature was 25℃, the injection volume was 20μL, and the detection wavelength was 370nm.

[0032] Example 1: Enrichment of ammonia oxidation sludge

[0033] A method for inhibiting the spread of resistance genes using ammonia-oxidized sludge, the method being as follows:

[0034] (1) Enriching ammonia oxidation sludge in a reactor using the method described in patent document CN115849574A;

[0035] (2) NH4HCO3 was added as a nitrogen source to the influent of the reactor at a concentration of 2.2571 g / L, KH2PO4 was added as a phosphorus source at a concentration of 0.064 g / L, and NaHCO3 (4 g / L) was added to control the pH at 7.8 ± 0.2.

[0036] The SBR reactor is set to operate for 4 cycles per day, each cycle lasting 360 minutes, including 60 minutes of influent, 260 minutes of aeration, 30 minutes of sedimentation, 5 minutes of effluent discharge, and 5 minutes of settling; the effluent ratio is 25%.

[0037] Antibiotics were added to the reactor feed water in six stages, with the concentration gradually increasing. The concentration of SDZ in the feed water was 0 mg / L in the first stage, 0.1 mg / L in the second stage, 0.5 mg / L in the third stage, 2.5 mg / L in the fourth stage, 10.0 mg / L in the fifth stage, and 0 mg / L in the sixth stage. Each stage lasted for 13 days.

[0038] The effect of enriching ammonia oxidation sludge with different sulfadiazine concentrations on pollutant removal is as follows: Figure 1-4 As shown, where Figure 1 This is a diagram showing the removal effect of conventional pollutants; Figure 2 The graph shows the removal rate of conventional pollutants and the specific ammonia oxidation rate. Figure 3 The image shows the effect of sulfadiazine removal. Figure 4 This is a graph showing the changes in the abundance of sulfonamide resistance genes.

[0039] In the ammonia oxidation sludge reactor, the ammonia nitrogen removal rate was over 96% without the addition of sulfadiazine (SDZ), and the average SAOR reached 24.77 mgN·(gVSS·h)⁻¹. When the concentration of SDZ was 0.1 mg / L, the SAOR value in the ammonia oxidation sludge decreased to 10.88 mgN·(gVSS·h)⁻¹ in the initial stage of addition, and the ammonia nitrogen removal rate significantly decreased to 45%. After stable operation, the ammonia nitrogen removal rate recovered to over 96%, and the SAOR recovered to 17.53 mgN·(gVSS·h)⁻¹, indicating that the microorganisms in the ammonia oxidation sludge can adapt to the environment containing 0.1 mg / L of SDZ. When the concentration of SDZ was further increased to 0.5 mg / L, the SAOR of the enriched ammonia oxidation sludge initially decreased to 4.31 mg N·(gVSS·h)⁻¹, gradually recovering to 12.57 mg N·(gVSS·h)⁻¹ after 40 days of operation. However, this was significantly lower than the activity without SDZ and with 0.1 mg / L SDZ, indicating that 0.5 mg / L SDZ had a significant inhibitory effect on microbial activity. When the SDZ concentration was increased to 2.5 mg / L, the SAOR of the enriched ammonia oxidation sludge continued to decrease to 9.57 mg N·(gVSS·h)⁻¹. When the SDZ concentration was further increased to 10 mg / L, the SAOR did not decrease significantly and stabilized at 11.19 mgN·(gVSS·h)-1. At this point, the average ammonia nitrogen removal rate reached over 95%, indicating that although the ammonia oxidation activity of the ammonia-oxidizing sludge microorganisms was lower than in the first three stages under the stress of high concentrations of SDZ (2.5 mg / L and 10 mg / L), they were able to recover high ammonia nitrogen removal performance after adapting to the environment.

[0040] When the SDZ concentration was 0.1 mg / L, the average removal rate of SDZ in the ammonia-enriched sludge reactor was 66%. Increasing the SDZ concentration to 0.5 mg / L rapidly increased the removal rate from 83% to over 95%, indicating that in the initial stage of SDZ addition, the microorganisms in the ammonia-enriched sludge adapted quickly to the stimulation of SDZ, rapidly activating the enzymes responsible for biodegradation, thereby quickly removing SDZ through biodegradation to resist its stress. When the SDZ concentration in the reactor was further increased to 2.5 mg / L, the removal rate stabilized at over 95%.

[0041] Comparative Example 1: Conventional Activated Sludge Process

[0042] The conventional activated sludge process for removing sulfadiazine is as follows:

[0043] (1) Set the SBR reactor to work 3 cycles per day, each cycle is 480 min, including 5 min of influent, 150 min of anaerobic, 270 min of aerobic, 1 min of sludge discharge, 30 min of sedimentation, 5 min of drainage and 20 min of settling, with a drainage ratio of 25%.

[0044] (2) Add 50 mg N / L NH4Cl as a nitrogen source, 10 mg P / L KH2PO4 as a phosphorus source, 500 mg COD / L sodium acetate as an organic carbon source, and 125 mg / L NaHCO3 to the influent of the reactor to control the pH.

[0045] (3) Sulfadiazine (SDZ) was gradually added to the influent in six stages in order of increasing concentration. The concentration of SDZ in the influent was 0 mg / L in the first stage, 0.1 mg / L in the second stage, 0.5 mg / L in the third stage, 2.5 mg / L in the fourth stage, 10.0 mg / L in the fifth stage, and 0 mg / L in the sixth stage. Each stage was run for 13 days to acclimate activated sludge that could stably and efficiently remove sulfadiazine.

[0046] Changes in the abundance of sulfonamide resistance genes in traditional activated sludge under different sulfadiazine concentrations are as follows: Figure 5 As shown.

[0047] Example 2: Analysis of the characteristics of resistance gene occurrence in Example 1 and Comparative Example 1

[0048] Analysis of the characteristics of sulfonamide resistance genes:

[0049] Sludge samples were taken on the last day of each operating phase of the two reactors in Example 1 and Comparative Example 1 for metagenomic analysis, including COG functional annotation analysis, KEGG functional annotation analysis, and CARD resistance gene functional annotation. This experiment was conducted by Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0050] (1) COG functional annotation analysis was performed using the eggNOG database (Evolutionary genealogy of genes: Non-supervised Orthologous Groups). http: / / eggnog.embl.de / The non-redundant gene set sequences were aligned with the eggNOG database using DIAMOND software (parameters: blastp; E-value ≤ 1e-5) to obtain the corresponding orthologous protein clusters (COGs). The relative abundance of the COG was then calculated using the sum of the gene abundances corresponding to the COG.

[0051] (2) KEGG functional annotation analysis was performed using the KEGG database (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ), a large knowledge base that systematically analyzes gene function and connects genomic and functional information. The KEGG GENES database provides gene and protein sequence information; the KEGG PATHWAY database includes various metabolic pathways, synthetic pathways, membrane transport, signal transduction, cell cycle, and disease-related pathways; KEGG Pathology (KO) is an orthologous classification system that can group genes with similar sequences and functions into one category, using the functions of known genes as the functions of this KO for cross-species annotation. The DIAMOND software was used to align the non-redundant gene set sequences with the KEGG gene database (parameters: blastp; E-value ≤ 1e-5), and the relative abundance of the functional category was calculated based on the sum of gene abundance corresponding to KO, Pathway, and EC.

[0052] (3) CARD resistance gene functional annotation utilizes the comprehensive ARGs database (http: / / arpcard.Mcmaster.ca), which contains reference genes related to antibiotic resistance from various organisms, genomes, and plasmids. This database can be used to guide research on antibiotic resistance groups and mechanisms in the environment, humans, and animal microbiota. DIAMOND (https: / / github.com / bbuchfink / diamond) is used to align the non-redundant gene set with the CARD database (parameters: blastp; E-value ≤ 1e-5). The target gene is then combined with its resistance functional annotation information to obtain the annotation results.

[0053] In the distribution of ARG subtypes of resistance genes in each sample in Example 1, four sulfonamide resistance genes were annotated in the CARD database, namely sul1, sul2, sul3, and sul4. When the concentration of SDZ in the reactor increased to 0.5 mg / L, the four sulfonamide resistance genes in Example 1 all showed a decreasing trend, with their relative abundance decreasing from 0.417%, 0.736%, 0.040%, and 0.763% to 0.349%, 0.733%, 0.016%, and 0.728%, respectively. It is speculated that ammonia-oxidizing bacteria can inhibit the enrichment and spread of sulfonamide resistance genes in low concentrations of SDZ. When the SDZ concentration in the reactor increased to 10 mg / L, the relative abundance of sul1 increased significantly, from the initial 0.349% to 1.538%, while the relative abundance of sul2 decreased significantly, from the initial 0.733% to 0.160%. The relative abundances of sul3 and sul4 also showed a decreasing trend, decreasing from the initial 0.016% and 0.728% to 0.013% and 0.640%, respectively. This indicates that high concentrations of SDZ may promote the enrichment of sul1 while inhibiting the enrichment of sul2, sul3, and sul4. In Comparative Example 1, when the SDZ concentration increased to 10 mg / L, the relative abundances of the sulfonamide resistance genes sul1 and sul2 both showed an increasing trend, increasing from the initial 0.017% and 0.077% to 0.142% and 0.090%, respectively.

[0054] Analysis of the characteristics of other types of resistance genes:

[0055] In Example 1, the five ARGs with relatively high abundance were macrolide resistance gene macB, tetracycline resistance gene tetA (58), polyamino acid resistance gene bcrA, aminocoumarin resistance gene novA, and macrolide resistance gene oleC. As the concentration of SDZ in the reactor increased, the abundance of these ARGs decreased. The relative abundance of tetracycline resistance gene tetA (58) decreased from 4.962% to 4.633%, the relative abundance of macrolide resistance gene macB decreased from 7.080% to 5.564%, and the relative abundance of polyamino acid resistance gene bcrA decreased from 7.080% to 5.564%. In the reactor of Comparative Example 1, the relative abundance of these AGRs increased significantly. The relative abundance of the tetracycline resistance gene tetA(58) increased from 4.149% to 5.162%, the relative abundance of the macrolide resistance gene macB increased from 8.349% to 8.448%, and the relative abundance of the polyamino acid resistance gene bcrA increased from 3.108% to 3.523%. Compared with traditional activated sludge, enriched ammonia oxidation sludge may inhibit the enrichment and spread of resistance genes for non-corresponding antibiotics, possibly because the high concentration of SDZ inhibits the enrichment of bacteria capable of carrying ARGs.

[0056] The removal rates of various resistance genes in Example 1 and Comparative Example 1 are shown in Table 1:

[0057] Table 1

[0058]

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for inhibiting the spread of resistance genes using ammonia-oxidized sludge, characterized in that, Includes the following steps: (1) Enriching ammonia oxidation sludge in an ammonia oxidation sludge enrichment reactor; (2) Add nitrogen and phosphorus sources to the influent of the reactor and control the pH to be maintained at 7.5-8.0; set the operating cycle of the reactor; (3) Sulfadiazine was added to the reactor feed water in six stages with gradually increasing concentration. The concentration of sulfadiazine in the feed water was 0 mg / L in the first stage, 0.1 mg / L in the second stage, 0.5 mg / L in the third stage, 2.5 mg / L in the fourth stage, 10.0 mg / L in the fifth stage, and 0 mg / L in the sixth stage. Each stage was run for 10-13 days.

2. The method according to claim 1, characterized in that, In step (2), the nitrogen source is NH4HCO3, and the content is 2-2.5 g / L.

3. The method according to claim 1, characterized in that, In step (2), the phosphorus source is one or more of K2HPO4 and KH2PO4, with a content of 0.06-0.07 g / L.

4. The method according to claim 1, characterized in that, In step (2), pH is controlled by adding NaHCO3.

5. The method according to claim 1, characterized in that, In step (2), the reactor is set to operate for 4 cycles per day, with each cycle lasting 360 minutes, including 60 minutes of water inlet, 260 minutes of aeration, 30 minutes of sedimentation, 5 minutes of drainage, and 5 minutes of settling; the drainage ratio is 25%.

6. The application of any one of the methods described in claims 1-5 in suppressing the spread of resistance genes.

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