A salt-tolerant strain capable of degrading tetracycline antibiotics and its application

Abstract

The present invention relates to a salt-tolerant strain capable of degrading tetracycline antibiotics and its application. The strain has been deposited in the Guangdong Provincial Microbial Culture Collection Center on February 24, 2022, with a deposit number of GDMCC No: 61970. The present invention shows that strain FD-08 has a good degradation effect on tetracycline, oxytetracycline, chlortetracycline and doxycycline, with degradation rates of 93.20%, 52.34%, 66.07% and 38.36%, respectively; among them, the degradation effect on tetracycline is the best. At the same time, the FD-08 strain can tolerate higher salinity, so that it also has a good ability to remove tetracycline in the soil under high salinity conditions, and can be widely used in the tetracycline pollution remediation and soil pollution remediation fields of coastal saline soil.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and more specifically, relates to a salt-tolerant bacterial strain capable of degrading tetracycline antibiotics and its application. Background Art

[0002] Since the discovery of penicillin in 1928, thousands of antibiotics have been identified and used as drugs and food additives to prevent diseases and infections in humans and livestock. Among them, tetracycline is one of the most widely used antibiotics worldwide due to its low price and broad-spectrum antibacterial activity. As the world's second largest and China's largest producer and user of tetracycline, China produces 1.2×10 4 ttetracycline, of which about 70% is used to prevent livestock and poultry diseases, promote animal growth and development, and improve the quality of livestock and poultry products. Since only a small part of tetracycline is absorbed and utilized, it is frequently overused or misused in animal husbandry, resulting in high residue rates in animal feces (40% to 90%) and urine. Animal feces produced by animal husbandry are often directly applied to farmland as organic fertilizer. Its insufficient biodegradation leads to increased concentrations of tetracycline residues in the soil. It contains phytotoxic components and can cause environmental damage. Therefore, soil is an inevitable source of tetracycline from fertilizers, direct excrement of grazing livestock, or wastewater irrigation. The concentration of tetracycline in the soil in most coastal areas of my country, such as the Yangtze River Delta and the Pearl River Delta, is 10 -3 ~300mg / kg, which has seriously exceeded the environmental ecotoxicity trigger value of 100μg / kg. Tetracycline pollution can inhibit the growth and activity of soil microorganisms and seriously affect the ecological function of the soil. In addition, therefore, it has become a top priority to develop an efficient and economical method to eliminate tetracycline pollution.

[0003] At present, tetracycline can be degraded in the environment through both abiotic and biotic pathways. The former includes photolysis and hydrolysis, while the latter is mainly microbial degradation. In most cases, the degradation rate of the former is much lower than that of the latter. Therefore, microbial degradation is the main degradation pathway of tetracycline in the natural environment. However, the broad-spectrum antibacterial activity of tetracycline and the particularity of its structure limit their microbial degradation. It is also related to the fact that tetracycline has an inhibitory effect on most Gram-positive and Gram-negative bacteria. High concentrations of tetracycline have a strong bactericidal effect, inhibiting the growth of microorganisms, resulting in its low bioavailability. At the same time, microbial degradation also faces application limitations such as "low environmental adaptability of strains, specificity of degradation substrates, and competition with indigenous microorganisms". According to the unique environmental conditions of the contaminated site, the selection of suitable high-quality strains is the key to determining whether microbial remediation technology can be successfully applied.

[0004] In recent years, some tetracycline antibiotic degradation strains have been discovered and applied in pesticide contaminated sites around the world. For example, Acinetobacter, Bacillus, Pseudomonas and Sphingomonas have been successfully used for soil tetracycline antibiotic pollution remediation. However, there are relatively few tetracycline degradation strains found so far, and the mechanism of using microorganisms to degrade tetracycline antibiotics is relatively complicated. In addition, most studies only focus on tetracycline degradation under normal conditions, and there are few studies on tetracycline degradation under high salinity conditions. However, high salinity can inhibit the growth of microorganisms, thereby affecting the degradation efficiency of tetracycline. Therefore, screening for efficient tetracycline degradation bacteria under high salinity conditions is a major breakthrough.

[0005] In view of this, in order to meet the growing demand for microbial remediation of tetracycline antibiotic pollution, provide more bacterial strain resources for microbial remediation of antibiotic pollution, carry out research on microbial remediation technology of tetracycline pollution, screen salt-tolerant tetracycline-efficient degrading microbial strains, and expand the antibiotic-degrading microbial strain library. It is very necessary to provide an effective means for remediating tetracycline-contaminated coastal saline soil, which has important economic value and practical significance for controlling environmental pollution. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the above-mentioned problems and provide a salt-tolerant strain capable of degrading tetracycline antibiotics and its application.

[0007] The purpose of the invention is to provide a salt-resistant tetracycline antibiotic-degrading bacterium.

[0008] Another object of the present invention is to provide new applications of the strain.

[0009] Another object of the present invention is to provide a tetracycline antibiotic-degrading bacterial agent.

[0010] Another object of the present invention is to provide a method for degrading tetracycline.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention screened out a Sphingobacterium multivorum FD-08 strain that efficiently degrades tetracycline from the soil of the coastal tidal flat pasture in Suixi County, Zhanjiang City, Guangdong Province, and deposited it in the Guangdong Provincial Microbiological Culture Collection Center on February 24, 2022, with a deposit number of GDMCC No: 61970. The nucleotide sequence of 16s rDNA of the FD-08 strain is shown in SEQ ID NO: 1. The FD-08 strain is a Gram-negative bacterium, and the colony is turbid and translucent, shiny, with round protrusions, a smooth and moist surface, neat edges, and a diameter of 0.5 to 0.8 μm; it is positive for catalase activity, oxidase activity, starch hydrolase activity, and sucrose and maltose hydrolysis; it is negative for Gram staining, gelatin liquefaction, indole reaction, and urease reaction. The suitable growth conditions for FD-08 strain are: pH range of 5-8, temperature range of 30-40°C, salinity range of 0-6%, inoculum range of 1%-7%, and tetracycline concentration range of 0-50 mg / L.

[0013] The research of the present invention shows that the FD-08 strain can degrade a variety of tetracycline antibiotics. At the same time, the strain FD-08 can effectively degrade tetracycline antibiotics under high salinity, among which tetracycline, oxytetracycline, chlortetracycline and doxycycline have a good degradation effect, and the degradation rates are 93.20%, 52.34%, 66.07% and 38.36% respectively; it can be applied to the restoration and treatment of coastal saline soil contaminated by tetracycline.

[0014] Therefore, the present invention provides the use of the Sphingobacterium multivorum FD-08 strain in degrading tetracycline antibiotics and / or in preparing tetracycline antibiotic-degrading bacterial agents, in repairing an environment contaminated by tetracycline antibiotics, or in repairing an environment contaminated by tetracycline antibiotics under high salt concentration.

[0015] Furthermore, the environment contaminated by the tetracycline antibiotics is soil or water.

[0016] Preferably, the environment contaminated by tetracycline antibiotics under high salt concentration is coastal saline soil.

[0017] Furthermore, the tetracycline antibiotic is one or more of tetracycline, oxytetracycline, chlortetracycline or doxycycline.

[0018] The invention provides a tetracycline antibiotic degradation bacterial agent, which contains the Sphingobacterium multivorum FD-08 strain or its bacterial liquid.

[0019] Preferably, the concentration of the strain is not less than 2.0×10 7 CFU / mL.

[0020] The invention provides a method for degrading tetracycline, which uses the Sphingobacillus multivorans FD-08 strain to treat soil or samples contaminated by tetracycline.

[0021] Preferably, the inoculation amount of the strain is 1% to 7%.

[0022] More preferably, the inoculum concentration of the strain is 5.69%.

[0023] Preferably, the treatment conditions are: pH 5-8, temperature 30-40° C., salinity 0-5%, and tetracycline concentration range 0-50 mg / L.

[0024] More preferably, pH: 7.15, temperature: 34.18°C, salinity: 4.77%.

[0025] The present invention has the following beneficial effects:

[0026] The Sphingobacterium multivorum FD-08 provided by the present invention can effectively degrade multiple tetracycline antibiotics (tetracycline, oxytetracycline, chlortetracycline and doxycycline) under high salinity conditions, wherein the degradation rate of tetracycline is as high as 93.20%, which has important practical significance and value for the degradation of tetracycline antibiotics and the repair of the environment polluted by tetracycline antibiotics.

[0027] The Sphingobacillus multivorans FD-08 provided by the present invention has good tolerance to salinity, can effectively remove tetracycline in coastal saline soil, can be widely used in the field of tetracycline pollution remediation and soil pollution remediation of coastal saline soil, and has good economic value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the morphological characteristics of strain FD-08;

[0029] Figure 2 is the phylogenetic tree of strain FD-08;

[0030] Figure 3 This is the growth curve of strain FD-08 and the performance test of degrading tetracycline;

[0031] Figure 4 is the tetracycline degradation ability of strain FD-08 under different growth conditions;

[0032] Figure 5 This is the 3D response surface diagram of tetracycline degradation by strain FD-08 under different growth conditions;

[0033] Figure 6 This is a test of the degradation ability of strain FD-08 on different tetracycline antibiotics. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0035] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0036] The inorganic salt culture medium used in the present invention is composed of: 1.0 g / L (NH4)2SO3, 0.5 g / L NaCl, 1.0 g / L NH4NO3, 0.5 g / L KH2PO4, 0.01 g / L CaCl2, 1.5 g / L K2HPO4·12H2O and 0.005 g / L yeast extract, pH = 7.0 ± 0.2.

[0037] The composition of LB medium was as follows: 10.0 g / L peptone, 5.0 g / L NaCl, 10.0 g / L yeast extract, pH = 7.0 ± 0.2.

[0038] The solid culture medium for the plate is the corresponding culture medium with 20 g of agar added.

[0039] The nucleotide sequence of 16S rDNA of FD-08 strain is as follows:

[0040]

[0041] Example 1 Strain enrichment, separation and purification, and characterization

[0042] (1) Enrichment of strains

[0043] Soil samples were collected from the coastal mudflat pastures of Suixi County, Zhanjiang City, Guangdong Province. Under aseptic operation, 10g of soil sample was accurately weighed and placed in a 250mL triangular flask containing 90mL of sterile water (with glass beads), and shaken on a shaker for 30min to fully mix the sample with water, disperse the cells, and form a uniform bacterial suspension. After standing for 20-30s, 1mL of supernatant was drawn into an inorganic salt medium containing 10mg / L tetracycline (with tetracycline as the only carbon source), and cultured at 30℃ and 180rpm in the dark. After 5 days of culture, it was inoculated again in an inorganic salt medium containing 20mg / L tetracycline with a 1% inoculum, and cultured under the same conditions, and so on, until the tetracycline concentration in the inorganic salt medium was 50mg / L. Each enrichment culture had a blank control without adding bacterial solution.

[0044] (2) Isolation and purification of strains

[0045] The enrichment culture with a tetracycline acclimation concentration of 50 mg / L was diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Through preliminary experiments, it was determined that each treatment would take 10 -4 , 10 -5 , 10 -6 100 μL of the dilution was spread on a solid culture medium plate with the corresponding tetracycline concentration (50 mg / L) and placed in a 30°C constant temperature incubator in the dark. This was repeated three times until a purified and highly efficient tetracycline pesticide-degrading bacterium was isolated and named FD-08.

[0046] (3) Characteristic identification

[0047] 3.1 Morphological characteristics of strains

[0048] The FD-08 strain is a Gram-negative bacterium. After culturing on LB medium for 24 hours, the colonies formed are as follows: Figure 1 As shown, it is turbid and translucent, shiny, round and convex, with a smooth and moist surface, neat edges, and a diameter of 0.5-0.8μm.

[0049] 3.2 Physiological and biochemical characteristics of strains

[0050] The FD-08 strain was positive for catalase activity, oxidase activity, starch hydrolase activity and sucrose and maltose hydrolysis; the strain was negative for Gram staining, gelatin liquefaction, indole reaction and urease reaction.

[0051] 3.3 Molecular biological characteristics

[0052] The total bacterial DNA was extracted using a kit. The bacterial 16S rDNA universal primers 27F and 1492R were used to PCR amplify the bacterial 16S rDNA, and an obvious band appeared near 1400 bp. The PCR amplification product was recovered and sequenced. The nucleotide sequence of the 16s rDNA of the FD-08 strain is shown in SEQ ID NO: 1. The obtained DNA sequence was subjected to Blast comparison analysis on the NCBI website (http: / / www.ncbi.nlm.nih.gov), and a phylogenetic tree was constructed using MEGA software (version: 7.0). Figure 2 As shown, it was found that the 16S rDNA sequence of the strain of the present invention has a high homology with Sphingobacterium multivorum of the genus Sphingobacterium in GenBank. Combined with the above-mentioned morphological characteristics and the results of the 16S rDNA sequence, the strain isolated by the present invention is classified as Sphingobacterium multivorum, named FD-08, and preserved in the Guangdong Provincial Microbiological Culture Collection Center on February 24, 2022, with a preservation number of GDMCC No: 61970, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0053] Example 2 Growth conditions of FD-08 and its degradation performance on tetracycline

[0054] (1) Growth curve of strain FD-08

[0055] The seed solution (OD 600 =0.8) was inoculated into an inorganic salt medium containing 50 mg / L tetracycline at a 3% inoculation rate, and cultured in a constant temperature shaking incubator at 30°C and 180 rpm in the dark for 72 h. Samples were taken every 6 h to determine the growth of the strain and the concentration of tetracycline.

[0056] The results are as follows Figure 3 As shown, strain FD-08 can significantly degrade tetracycline, and the growth rate of FD-08 strain and tetracycline degradation rate are significantly negatively correlated. After 72 hours of the experiment, the tetracycline degradation rate in the treatment with FD-08 strain added reached 96.20%.

[0057] (2) Tetracycline degradation experiment of strain FD-08 under different conditions

[0058] The tetracycline degradation experiment under different conditions mainly studied the effects of temperature, pH, salinity, inoculation size and tetracycline concentration on the ability of the strain to degrade tetracycline. The treatment groups of each influencing factor were: temperature (20℃, 25℃, 30℃, 35℃, 40℃); pH (5, 6, 7, 8, 9, 10); salinity (0%, 2%, 4%, 6%, 8%, 10%); inoculation size (1%, 3%, 5%, 7%, 9%); tetracycline concentration (10mg / L, 25mg / L, 50mg / L, 75mg / L, 100mg / L). After culturing in a constant temperature shaking incubator at 180rpm for 72 hours under the above different influencing factors, the growth of the strain (OD 600 ) and tetracycline concentration.

[0059] The results of the ability of the strain to degrade tetracycline under different temperature, pH, salinity, inoculum size and tetracycline concentration are shown in Figure 4 The results showed that the suitable temperature range of strain FD-08 was 30-40℃, and the tetracycline degradation rates were 96.85% and 94.20%, respectively. When the temperature was less than 25℃ or greater than 40℃, the strain grew slowly, especially at 20℃, the degradation ability of tetracycline was poor.

[0060] The suitable pH range of strain FD-08 is 5-8, and the tetracycline degradation rates are 91.76%, 98.19%, 96.20% and 90.21% respectively. When the pH is greater than 8, the growth of the strain is inhibited and the degradation ability of tetracycline is seriously reduced. Therefore, strain FD-08 can efficiently degrade tetracycline under weak acid or neutral conditions.

[0061] The suitable salinity range of strain FD-08 is 0-6%, and the tetracycline degradation rates are 96.20%, 97.83%, 98.55%, and 84.50%, respectively. When the salinity is greater than 6%, the strain's ability to degrade tetracycline decreases rapidly. Therefore, strain FD-08 can effectively degrade tetracycline under conditions of high salt concentration.

[0062] The effect of different inoculation amounts of strain FD-08 on the degradation of tetracycline was not obvious. The degradation rate of tetracycline was the best when the inoculation amount was 5%, up to 98.00%, and the degradation rate of tetracycline was poor when the inoculation amount was 9%, which was 86.74%. When the concentration of tetracycline was 10, 25, and 50 mg / L, the degradation rate of tetracycline by strain FD-08 was 96.04%, 95.44%, and 96.99%, respectively. When the concentration of tetracycline was greater than 50 mg / L, the degradation ability of the strain to tetracycline decreased rapidly.

[0063] In summary, the strain is suitable for a pH range of 5-8, a temperature range of 30-40°C, a salinity range of 0-6%, an inoculum range of 0%-6%, and a tetracycline concentration range of 0-50 mg / L.

[0064] Example 3 Response surface optimization design of FD-08 growth conditions

[0065] In order to clarify the degradation potential of strain FD-08 on tetracycline, the response surface method was applied to optimize the effects of temperature, pH, salinity and inoculum size and their interactions. Based on the Box-Behnken design, the above four main influencing factors were used as independent variables, the degradation rate was the response value, and the initial concentration of tetracycline was 50 mg / L. The Box-Behnken experiment was designed using Design Expert 13.0 software, with a total of 30 groups of treatments, each with 3 replicates. After 72 hours of culture, the residual tetracycline concentration in the bacterial solution was measured and the degradation rate was calculated. The specific treatments are shown in Table 1.

[0066] Table 1 Box-Behnken design and its resulting response values

[0067]

[0068]

[0069] In the above quadratic polynomial equation, Yi is the predicted response value, and X1, X2, X3, and X4 are the coded values ​​corresponding to temperature, pH, salinity, and inoculum size, respectively.

[0070] Polynomial regression analysis was performed on the experimental data, and a quadratic response model was established using Design Expert 13.0 software.

[0071] The fitted and derived quadratic polynomial equation is as follows:

[0072] Yi=98.18+8.82*X1+10.51*X1-9.65*X3+4.57*X4+4.24*X1X2+1.23*X1X3-3.26*X1X4-5.96*X2X3+0.5386*X2X4-0.1215*X3X4-3.38*X1 2 -25.12*X2 2 -7.83*X3 2 -2.99*X4 2

[0073] Table 2 Equation analysis of the fitted model (ANOVA)

[0074]

[0075]

[0076] In the above Table 2, Sum of Squares is the sum of the sequence, DF is the degree of freedom, Mean Square is the mean square, and a P value less than 0.05 indicates that the model item is significant, otherwise it is not significant.

[0077] The statistical analysis of Design Expert 13.0 software shows that the model has a very high significance (p<0.05). At the same time, the correlation coefficient R 2 is 0.9712, and the predicted correlation coefficient R 2 is 0.8345, and the difference between the two does not exceed 0.2, indicating that there is a high correlation between the measured value and the predicted value, and the model is suitable for the theoretical prediction of tetracycline degradation rate. In the regression equation, the response coefficients of temperature, pH, salinity and inoculum size all reached a significant level (p<0.05), which shows that the above single factors have a significant effect on the degradation results. In addition, the interaction coefficients between temperature and pH, temperature and inoculum size, and pH and salinity are significant (p<0.05), indicating that the above interactions can significantly enhance the degradation of tetracycline by the strain. At the same time, the secondary response coefficients of pH and salinity also significantly affect the degradation of tetracycline by the strain.

[0078] 3D response surface diagram can effectively and intuitively reflect the various factors and their interactions in the test results, such as Figure 5 As shown. In this study, the quadratic polynomial model was successfully fitted, which reduced the number of treatments and resource inputs, and achieved the purpose of optimizing the degradation conditions. The maximum degradation rate at the theoretical optimal point was 99.88%, and the coding values ​​of X1, X2, X3, and X4 were 0.418, -0.140, -0.046, and 0.1725, respectively; the actual values ​​were: temperature was 34.18°C, pH was 7.15, salinity was 4.77%, and inoculation size was 5.69%. In order to predict the prediction accuracy of the above model, the degradation experiment of tetracycline was carried out using the above optimized conditions. The results showed that after 72 hours of cultivation, the residual amount of tetracycline was only 0.62 mg / L, and the degradation rate was 98.76%, which was less than 0.01 different from the model prediction value. Therefore, the optimization of degradation conditions by this model is feasible and reliable.

[0079] Example 4 Degradation performance of FD-08 on different tetracycline antibiotics

[0080] In this example, tetracycline, oxytetracycline, chlortetracycline, and doxycycline were used to prepare an inorganic salt medium with a concentration of 50 mg / L. The salinity was set to 6%, and the seed liquid of strain FD-08 was inoculated at a 5% inoculation rate (OD 600 =0.8). After culturing in a constant temperature shaking incubator at 30°C and 180 rpm in the dark for 72 h, the residual concentrations of different tetracycline antibiotics were determined.

[0081] The results are as follows Figure 6 As shown, the FD-08 strain has a good degradation effect on tetracycline, oxytetracycline and chlortetracycline, with degradation rates of 93.20%, 52.34% and 66.07% respectively, and a poor degradation effect on doxycycline, with a degradation rate of only 38.36%.

[0082] Example 5FD-08 Removal of soil tetracycline in coastal saline soil

[0083] In order to observe the repair ability of strain FD-08 on tetracycline pollution in coastal saline soil, the soil used in this embodiment was collected from a ranch on the coastal beach of Suixi County, Zhanjiang City, Guangdong Province, with a soil salinity of 4.52%, a pH of 6.36, and a tetracycline concentration of 44.96 mg / kg. Weigh 100g of dry soil and place it in a conical flask, add seawater of the same salinity to keep it at 60% field moisture content. And access strain FD-08 with a 5% inoculum. The soil without bacteria was used as a blank control group, and 3 replicates were treated in each group and placed in a 30°C constant temperature incubator. Samples were collected at 0d, 1d, 3d, 5d, 7d and 9d during the culture stage, and the residual concentration of tetracycline in the soil was detected and its degradation rate was calculated.

[0084] The results are shown in Table 3. After 9 days, in the blank treatment, the degradation rate of tetracycline in the natural environment was poor, only 13.63%. On the contrary, in the treatment after the strain FD-08 was added, the residual concentration of tetracycline in the contaminated soil was only 5.07 mg / kg, and the degradation rate was 88.17%. Therefore, strain FD-08 can effectively repair tetracycline-contaminated soil in coastal saline soil.

[0085] Table 3 Degradation effect of strain FD-08 on tetracycline in coastal saline soil

[0086]

[0087] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A salt-tolerant Sphingobacterium multivorum FD-08 strain capable of degrading tetracycline antibiotics, characterized in that: The strain shown was deposited in Guangdong Provincial Microbiological Culture Collection Center on February 24, 2022, with the accession number GDMCC No: 61970.

2. Use of the Sphingobacterium multivorum FD-08 strain according to claim 1 in degrading tetracycline antibiotics and / or in preparing a tetracycline antibiotic degrading bacterial agent, characterized in that: The tetracycline antibiotic is one or more of tetracycline, oxytetracycline, chlortetracycline or doxycycline.

3. Use of the Sphingobacterium multivorum FD-08 strain according to claim 1 in repairing an environment contaminated by tetracycline antibiotics or repairing an environment contaminated by tetracycline antibiotics under high salt concentration, characterized in that: The tetracycline antibiotic is one or more of tetracycline, oxytetracycline, chlortetracycline or doxycycline.

4. The use according to claim 3, characterized in that: The environment contaminated by the tetracycline antibiotics is soil or water.

5. A tetracycline antibiotic degradation bacterial agent, characterized in that: Containing the Sphingobacterium multivorum FD-08 strain and / or its bacterial liquid as described in claim 1.

6. The degradation bacterial agent according to claim 5, characterized in that: The concentration of the bacterial solution is not less than 2.0×10 7 CFU / mL.

7. A method for degrading tetracycline, characterized in that: The Sphingobacterium multivorum FD-08 strain described in claim 1 is used to treat soil or samples contaminated with tetracycline.

8. The method according to claim 7, characterized in that: The inoculation amount of the strain is 1% to 7%.

9. The method according to claim 7, characterized in that: The treatment conditions are: pH 5-8, temperature 30-40°C, salinity 0-5%, and tetracycline concentration range 0-50 mg / L.

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