A method for adsorbing and degrading high concentrations of tetracycline in high-salt wastewater using activated sludge.

Abstract

This invention discloses a method for adsorbing and degrading high-concentration tetracycline in high-salt wastewater using activated sludge. The method comprises two steps. The first step uses a continuous influent process in a completely mixed reactor to selectively acclimate heterotrophic activated sludge with sulfate-reducing bacteria (SRB) as the primary functional bacteria. Adding influent cultured with low tetracycline concentrations enhances the tetracycline tolerance of the microorganisms and promotes the secretion of extracellular polymeric substances (EPS) by the bacteria. The EPS adsorbs and degrades the tetracycline. By adjusting the pH value in stages, the horizontal transfer of antibiotic resistance genes is effectively inhibited. The second step utilizes the successfully acclimatized anaerobic activated sludge to efficiently degrade the high-concentration tetracycline. This invention successfully achieves effective treatment of high-concentration tetracycline wastewater generated by industries such as marine aquaculture, while simultaneously curbing the spread of resistance genes, resulting in significant environmental and economic benefits.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment technology, specifically to a method for degrading high concentrations of tetracycline in high-salt wastewater by adsorbing activated sludge. Background Technology

[0002] With the rapid development of aquaculture and the medical industry, the frequency and scope of antibiotic use are increasing daily. Large quantities of antibiotics enter the aquatic environment through various pathways, including medical wastewater, livestock farm discharge, and domestic sewage. Wastewater containing antibiotics is often discharged into urban sewage systems without effective treatment, ultimately flowing directly or indirectly into the ocean through sewage outlets or runoff, causing persistent antibiotic pollution, especially in marine aquaculture areas and waters near sewage treatment plant outlets. Tetracycline is the most commonly detected antibiotic, and its potential risks to aquatic ecosystems and public health cannot be ignored.

[0003] Currently, the activated sludge process is the mainstream technology for wastewater treatment, demonstrating good results in treating conventional organic pollutants. Its mechanism of action mainly includes microbial adsorption and biodegradation. However, traditional activated sludge systems face severe challenges when dealing with high-salinity wastewater containing antibiotics. The high salinity of seawater severely inhibits microbial activity, reducing its ability to adsorb and degrade tetracyclines, and significantly lowering treatment efficiency. Traditional sludge is easily deactivated under the dual stress of high salinity and tetracyclines, resulting in limited treatment effectiveness. Therefore, developing a biological treatment technology that can maintain microbial activity in a high-salinity environment, effectively degrade tetracycline antibiotics, and inhibit the spread of resistance genes has become a key issue that urgently needs to be addressed in the field of wastewater treatment. Summary of the Invention

[0004] This invention addresses the above-mentioned problems by researching and designing a method for adsorbing and degrading high concentrations of tetracycline in high-salt wastewater using activated sludge. The technical means employed in this invention are as follows:

[0005] A method for adsorbing and degrading high concentrations of tetracycline in high-salt wastewater using activated sludge includes the following steps:

[0006] S1: Targeted acclimatization stage of sludge: Activated sludge is placed in a completely mixed reactor, maintaining an anaerobic environment, using a continuous influent method, with a hydraulic retention time of 24 hours. The reactor temperature is set at 25-40℃. Acclimatization water is introduced into the reactor, and the pH value is adjusted from 7 to 7.2. Once the sludge has initially formed a flocculated state, the pH value is then adjusted to 7.5. The concentration of SO42- (S) in the acclimatization water is 850-900 mg / L, and the TOC (C) concentration is 700-750 mg / L. When the removal rates of TOC and SO42- continue to rise and stabilize above 95%, and the S2- generation rate rises to a stable level, tetracycline at a concentration of 80-120 μg / L is added to slowly raise the pH value to 7.5-8. The activated sludge forms brown settling flocs. Once the system treatment efficiency fluctuates within ±10%, the pH value is raised to 8-8.5 to inhibit the transfer of resistance genes until the removal rates of TOC, SO42- and S2- generation rate stabilize, thus completing the acclimatization of the activated sludge.

[0007] S2: Wastewater treatment stage: Wastewater is treated using a fully mixed reactor containing activated sludge acclimated in step S1. Continuous influent is used, with a hydraulic retention time of 24 hours. The temperature inside the reactor is set at 25-40℃, and the pH value is maintained at 8-8.5.

[0008] Furthermore, in step S1, the activated sludge is aerobic activated sludge.

[0009] Furthermore, in step S1, the acclimatization water is artificially prepared water, the source of SO42- in the artificially prepared water is natural clean seawater, the carbon source is peptone, and it also includes NaHCO3 and tetracycline.

[0010] Furthermore, in step S1, the inoculation concentration (MLSS) of the activated sludge is 38-40 g / L.

[0011] Furthermore, in step S1, the functional microbial community in the acclimated activated sludge is sulfate-reducing bacteria, with dominant genera including Dethiosulfatibacter, Desulfatiglans, and Desulfobacter.

[0012] Furthermore, in step S2, the influent treatment of wastewater is carried out in two stages. In the first stage, the concentration of tetracycline in the influent is controlled at 1-1.5 mg / L, and in the second stage, the concentration of tetracycline in the influent is controlled at 1-5 mg / L.

[0013] Compared with existing technologies, the method of using activated sludge to adsorb and degrade high-concentration tetracycline in high-salt wastewater described in this invention enhances the combined tolerance of sulfate-reducing bacteria (SRB) to salinity and tetracycline by domesticating and improving the tolerance of SRB to salinity and tetracycline, and inhibits the transmission of resistance genes by adjusting pH value in stages. This has important practical significance for enhancing the ability of activated sludge to degrade tetracycline in high-salt environments, and can provide a feasible biological treatment solution for the treatment of high-salt tetracycline wastewater. It achieves effective treatment of high-concentration tetracycline wastewater generated by industries such as marine aquaculture, and has significant environmental and economic benefits. Attached Figure Description

[0014] Figure 1 This is a graph showing the relationship between the removal rates of SO42-, TOC, and tetracycline, as well as the formation rate of S2-, and the number of treatment days during the sludge-oriented acclimatization stage in step one of the embodiments of the present invention.

[0015] Figure 2 This is a graph showing the relationship between the removal rates of SO42-, TOC, and tetracycline, as well as the formation rate of S2-, and the number of treatment days in step two of the wastewater treatment stage of this embodiment of the invention.

[0016] Figure 3 This is a diagram showing the distribution pattern of functional bacteria in the wastewater treatment system according to their genus classification in an embodiment of the present invention.

[0017] Figure 4 This is a graph showing the relationship between pH level regulation and the abundance of resistance genes in an embodiment of the present invention. Detailed Implementation

[0018] This embodiment provides a method for degrading high concentrations of tetracycline in high-salt wastewater using activated sludge adsorption, including the following steps:

[0019] Step 1, Targeted acclimatization of sludge:

[0020] Activated sludge was acclimated in two identical completely mixed reactors using the following method: maintaining an anaerobic environment, continuous influent, and a hydraulic retention time of 24 hours. The reactor temperature was set at 35±0.5℃, and the pH was adjusted from 7 to 7.2 until initial sludge flocculation was achieved. The pH was then adjusted to 7.5, and high-salt organic wastewater containing SO42- was introduced into the reactors. The SO42- concentration (S) in the influent was 850-900 mg / L, provided entirely by seawater without the addition of additional chemical agents. The organic matter (C) concentration was 700 mg / L, with peptone as the carbon source. When the TOC and SO42- removal rates continued to rise and eventually remained above 95%, and the S2- formation rate stabilized at around 15%, 100 μg / L of tetracycline was added to the influent to cultivate microbial tolerance to tetracycline. The pH of reactor 1 was slowly increased to 7.5-8, causing the activated sludge to form brown settling flocs. Once the system treatment efficiency fluctuation was within ±10%, the pH was increased to 8.5 to inhibit the transfer of resistance genes. The pH of reactor 2 was maintained at 7.5. When the tetracycline removal rate reached 90%, and the TOC, SO42- removal rates, and S2- formation rates stabilized, the heterotrophic activated sludge acclimation, mainly composed of SRB, was completed, and the first step of the system startup was successful.

[0021] Step Two, Wastewater Treatment Stage:

[0022] After the first stage of system startup was successful, high concentrations of tetracycline were added to the reactor influent in two stages, at 1 mg / L and 3 mg / L respectively, using a continuous influent method with a hydraulic retention time of 24 hours. The reactor temperature was set at 35 ± 0.5℃, and the pH values ​​of both reactors remained constant. High-salt organic wastewater containing SO42- was introduced into the reactors. The SO42- concentration (S) in the influent was 850-900 mg / L, provided entirely by seawater without the addition of additional chemical agents. The organic matter (C) concentration was 700 mg / L, with peptone as the carbon source. The concentrations of TOC, SO42-, S2-, and tetracycline in the water were detected. When the system was running stably and the tetracycline removal rate reached over 80%, the next stage began. If the tetracycline removal rate remained stable above 80% in both stages, the second stage of system startup was successful.

[0023] When treating high-salt wastewater containing tetracycline, different tetracycline concentrations will have varying degrees of inhibitory effect on SRB and affect its degradation capacity. This embodiment utilizes acclimated sludge to treat high-concentration tetracycline wastewater of 1-3 mg / L, and simultaneously evaluates the system's operating status, tetracycline removal efficiency, microbial community evolution, and abundance of resistance genes, providing a feasible biological treatment solution for the treatment of high-salt tetracycline wastewater.

[0024] The activated sludge acclimated in this embodiment was taken from the residual aerobic activated sludge in the secondary sedimentation tank of a municipal wastewater treatment plant.

[0025] In step one of this embodiment, the activated sludge acclimatization uses artificially prepared water, which consists of natural clean seawater, peptone, NaHCO3, and tetracycline.

[0026] In this embodiment, the inoculation concentration (MLSS) of the activated sludge is 38-40 g / L.

[0027] The main functional bacterial group acclimated by the system in this embodiment for adsorbing and degrading tetracycline is sulfate-reducing bacteria, with dominant genera including Dethiosulfatibacter, Desulfatiglans, and Desulfobacter.

[0028] In this embodiment, during the sludge-oriented acclimatization stage (Step 1), activated sludge is acclimatized through the synergistic stress of low-concentration tetracycline (100 μg / L) and a high-salt environment, forming a salt-tolerant and tetracycline-resistant functional bacterial community with SRB as the dominant species. In the wastewater treatment stage (Step 2), under high-salt conditions, the acclimatized activated sludge efficiently degrades tetracycline, achieving highly efficient removal of high concentrations of 1 mg / L and 3 mg / L tetracycline.

[0029] In this embodiment, during the sludge acclimatization stage in step one, the pH value was adjusted from 7 to 7.2 until the sludge initially formed a flocculated state. Then, the pH value was adjusted to 7.5, and after adding 100 μg / L tetracycline, the pH value of reactor 1 was slowly increased to 7.5-8. The activated sludge formed brown settling flocs. Once the system treatment efficiency fluctuation was within ±10%, the pH value was increased from 8 to 8.5. During the wastewater treatment stage, the pH value was also set at 8.5 to achieve the goal of inhibiting the transfer of resistance genes while efficiently removing tetracycline. Reactor 2, with the pH value consistently maintained at 7.5, was set up as a control group to demonstrate the inhibitory effect of pH value on the transfer of resistance genes.

[0030] In this implementation plan, SO42-, tetracycline, S2-, and total organic carbon (TOC) in the wastewater after treatment by the two reactors were measured:

[0031] Step 1: The entire acclimatization phase of the device lasted 45 days, and the results are shown below. Figure 1 , Figure 1 The graph shows the relationship between the removal rates of SO42-, tetracycline, and S2-, as well as the S2- formation rate and the number of treatment days. In the completely mixed reactor, the removal rates of SO42- and TOC both reached over 95%, and the S2- formation rate stabilized at around 15%. After adding 100 μg / L of tetracycline to acclimate its tolerance and degradation ability to tetracycline, the removal rates of SO42-, TOC, and tetracycline all reached over 95%, and the S2- formation rate stabilized at around 15%, indicating that the first step of the SRB acclimatization stage was completed, and the acclimatization results of the two reactors were basically consistent.

[0032] Step 2: The wastewater treatment process for the two reactors was divided into two stages, A and B, with tetracycline concentrations of 1 mg / L and 3 mg / L, respectively. Each stage lasted 6 days. Results are shown below. Figure 2 , Figure 2 The graph shows the relationship between the removal rates of SO42-, TOC, and tetracycline, as well as the formation rate of S2-, and the number of treatment days. In stage A, the removal rate of tetracycline steadily increased to 85%, and the system operated stably. In stage B, when the tetracycline concentration increased, the removal rate of tetracycline decreased and then gradually recovered to 80%, and the system operated stably. In both stages, the removal rate of tetracycline remained stable above 80%, indicating that the reaction system had a strong ability to degrade tetracycline, and the operating efficiency of the two reactors was similar.

[0033] according to Figure 3 As shown, in order to gain a deeper understanding of the metabolic functions of the system's microorganisms, the composition of the microbial community at the genus level in reactor 1 was analyzed to analyze in detail the distribution of functional bacteria in the device under high salt and high concentration tetracycline environment.

[0034] Dethiosulfatibacter is a dominant SRB genus in the system with high salt tolerance. It can utilize carbon sources to perform sulfate reduction reactions, reducing SO42- or S2O32- to S2-. Dethiosulfatibacter can survive in high-salt environments and is the dominant genus in the system. At pH 7.5-8.5, it can secrete a large amount of EPS, which is beneficial for antibiotic adsorption, and it also has strong resistance to antibiotics.

[0035] Desulfatiglans is a salt-tolerant sulfate-reducing bacterium that can adapt to a wide range of temperatures. It can reduce S2O32- or SO42- to H2S. It is a dominant bacterium in the system, with a strong ability to secrete EPS and is resistant to antibiotics.

[0036] Desulfobacter can utilize organic acids and glucose for growth and metabolism, converting SO42- into S2-. It is tolerant to the salinity of seawater, and its relative abundance does not fluctuate much with increasing tetracycline concentration, indicating that it has strong tolerance and is a dominant genus in the system.

[0037] Thermovirga, a genus of *Thermobacterium*, can drive the conversion of thiosulfate to hydrogen sulfide in anaerobic environments and can also utilize amino acids, organic acids, and proteins for metabolism. *Thermovirga* can adapt to high-salinity environments, but its thermophilicity has not become dominant under normal seawater conditions. Its relative abundance increases slightly with increasing tetracycline concentration, showing relatively little influence from tetracycline concentration and exhibiting good tetracycline tolerance.

[0038] Peptoclostridium is an acid-producing bacterium that decomposes large organic molecules to generate volatile fatty acids, providing readily available small-molecule carbon sources for SRB (sulfate-reducing bacteria), thereby activating the sulfate-reduction metabolic pathway. The rapid decrease in its relative abundance due to increased tetracycline concentration indicates that Peptoclostridium plays a role in the adsorption and degradation of tetracycline at lower concentrations, but its adsorption and degradation capacity is inhibited at higher concentrations.

[0039] Soehngenia is an acid-producing bacterium that decomposes large organic molecules. Its relative abundance increases with increasing tetracycline concentration. Both Soehngenia and Peptoclostridium promote sulfate reduction, while key intermediate metabolites produced during SRB accelerate the decomposition of large organic molecules. However, Soehngenia is less affected by tetracycline concentration.

[0040] Lentimicrobiaceae, as a typical saccharophilic Lactobacillus genus, can utilize simple organic substances such as glucose, but has difficulty utilizing small-molecule organic acids such as acetic acid, propionic acid, and butyric acid. Dethiosulfatibacter competes with Lentimicrobiaceae for organic substrates, which also leads to a decrease in the abundance of Lentimicrobiaceae.

[0041] according to Figure 4 As shown, to demonstrate the inhibitory effect of pH regulation on resistance genes, qPCR was used to detect typical resistance genes (tetB, tetX, and tetM) in the activated sludge of the two reactors at the end of the wastewater treatment stage. The results showed that the abundance of resistance genes in the reactor was greater at pH 7.5 than at pH 8.5, indicating that the pH 8.5 environment inhibits the transfer of resistance genes.

[0042] This example shows high concentrations of sulfate (SO4) in seawater. 2 -) Providing conditions for the proliferation of sulfate-reducing bacteria (SRB). To better remove tetracycline from high-salinity wastewater, the abundance of SRB in the environment is increased through targeted domestication of activated sludge. As a dominant bacterial group, SRB has the natural ability to reduce sulfate and degrade organic matter, providing a new pathway for the treatment of high-salinity wastewater. However, tetracycline significantly inhibits SRB activity. Therefore, improving the combined tolerance of SRB to salinity and tetracycline through domestication, and periodically adjusting the pH value to inhibit the transmission of resistance genes, enhances the ability of activated sludge to degrade tetracycline in high-salinity environments. This has important practical significance.

[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for degrading high-concentration tetracycline in high-salinity wastewater by using activated sludge adsorption, characterized in that, The method comprises the following steps: S1: sludge directional acclimation stage: the activated sludge is placed in a complete mixing reactor, an anaerobic environment is maintained, a continuous water feeding mode is adopted, the hydraulic retention time is 24 h, the temperature in the reactor is set to 25-40°C, acclimation water is introduced into the reactor, the pH value is adjusted from 7 to 7.2, the sludge flocculation state is preliminarily formed, then the pH value is adjusted to 7.5, the SO4 2- concentration in the acclimation water is 850-900 mg / L in terms of S, and the TOC is 700-750 mg / L in terms of C, the TOC and SO4 2- removal rates continuously increase and stabilize at above 95%, the S 2- when the generation rate is increased and stabilized, tetracycline with a concentration of 80-120 μg / L is added, the pH value is slowly increased to 7.5-8, the activated sludge forms brown settleability zooglea, the system processing efficiency fluctuation amplitude is within ±10%, and then the pH value is increased to 8- 8.5, inhibition of transfer of resistance genes, up to TOC, SO4 2- Removal and S 2- Generation rate stable, completion of acclimation of activated sludge; S2: sewage treatment stage: using a complete mixing reactor containing activated sludge domesticated in step S1 to treat sewage, using continuous water feeding mode, the hydraulic retention time is 24 h, the temperature in the reactor is set to 25-40℃, and the pH value is maintained at 8-8.

5.

2. The method for adsorbing and degrading high-concentration tetracycline in high-salinity wastewater by using activated sludge according to claim 1, characterized in that, In step S1, the activated sludge is aerobic activated sludge. 3.The method according to claim 1, wherein, The domesticated water is artificial water, and a source of SO4 2- in the artificial water is natural clean seawater, a carbon source is peptone, and the artificial water further comprises NaHCO3 and tetracycline.

4. The method for adsorbing and degrading high-concentration tetracycline in high-salinity wastewater by using activated sludge according to claim 1, characterized in that, In step S1, the inoculation concentration MLSS of the activated sludge is 38-40 g / L. 5.The method for adsorbing and degrading high-concentration tetracycline in high-salinity wastewater by using activated sludge according to claim 1, characterized in that, In step S1, the functional flora in the domesticated activated sludge is sulfate-reducing bacteria, and the dominant genera thereof include Dethiosulfatibacter, Desulfatiglans and Desulfobacter. 6.The method for adsorbing and degrading high-concentration tetracycline in high-salinity wastewater by using activated sludge according to claim 1, characterized in that, In step S2, the water feeding mode of the sewage is divided into two stages, the concentration of tetracycline in the first stage is controlled to be 1-1.5 mg / L, and the concentration of tetracycline in the second stage is controlled to be 1-5 mg / L.

Images