Method for purifying antibiotic-containing wastewater by functional microorganisms

Abstract

The application discloses a method for purifying antibiotic-containing wastewater by using functional microorganisms and relates to the technical field of wastewater treatment. The method comprises the following steps: expanding culture of Thiobacillus denitrificans, Propionibacterium acidipropionici, Bradyrhizobium and Thiobacillus ferroxidans to obtain bacterial liquid; inoculating the Thiobacillus denitrificans, the Propionibacterium acidipropionici and Bradyrhizobium into an anaerobic tank, and inoculating the Thiobacillus ferroxidans group into an aerobic tank to perform A 2 / O treatment process. After the treatment, the content of antibiotics and the abundance of antibiotic resistance genes in the wastewater are significantly reduced, and the purification of the antibiotic-containing wastewater is effectively realized.

Description

Technical Field

[0001] This invention relates to the field of wastewater purification technology, specifically to a method for purifying antibiotic-containing wastewater using functional microorganisms. Background Technology

[0002] Antibiotics are widely used in various fields such as medicine and animal husbandry, resulting in a large amount of antibiotic-containing wastewater, such as hospital wastewater, livestock and poultry farm wastewater, and antibiotic production wastewater. This wastewater often contains high concentrations of antibiotic residues. Once discharged into the environment, it not only affects the safety of organisms and disrupts the ecological balance, but also induces microorganisms to develop drug-resistant genes and bacteria, rendering previously effective antibiotics ineffective and leading to difficulties in treating human and animal diseases. Therefore, the efficient removal of antibiotics and the spread of antibiotic resistance genes (ARGs) from wastewater is crucial for human health and safety.

[0003] While current wastewater treatment methods can effectively reduce organic pollutants in wastewater, they often fail to simultaneously reduce the abundance of antibiotic resistance genes (ARGs). Some studies have attempted to treat antibiotic-containing wastewater using specific microorganisms, but most focus only on degrading specific antibiotics without addressing how to simultaneously reduce the abundance of antibiotic resistance genes. In some cases, the addition of antibiotic-resistant microorganisms or microorganisms that promote the growth of antibiotic-resistant microorganisms in the water may even increase the abundance of antibiotic resistance genes, exacerbating the problem of residual antibiotic resistance genes in the wastewater. Therefore, developing a treatment method that can effectively reduce both antibiotic content and ARG abundance in wastewater is of significant practical importance. Summary of the Invention

[0004] In view of the shortcomings of existing technologies, this invention aims to provide a method for purifying antibiotic-containing wastewater using functional microorganisms. By treating wastewater with functional microorganisms, the residual amount of antibiotics and the abundance of antibiotic resistance genes in the wastewater are effectively reduced, solving the problem that existing wastewater treatment technologies cannot simultaneously and effectively reduce antibiotics and antibiotic resistance genes in organic wastewater, thus achieving the goal of highly efficient purification of antibiotic-containing wastewater.

[0005] After organic wastewater is treated in anaerobic, anoxic, aerobic, and sedimentation tanks, a large number of different types of microorganisms remain in the wastewater treatment ponds. Through extensive preliminary research and analysis, the inventors discovered that regardless of the bacterial species present in the wastewater or related commercially available strains, *Thiobacillus denitrificationis* (…) is the most prevalent. Thiobacillus denitrificans Propionibacterium propionitum ( Propionibacterium acidipropionici ATCC 25562, Slow-growing rhizobia ( Bradyrhizobium sp. CCTCC AB 2012237 and Thiobacillus ferrooxidans ( Thiobacillus ferrooxidans The dominant bacterial flora not only removes antibiotics but also carries fewer antibiotic resistance genes. Adding them to wastewater does not introduce large amounts of resistance genes. Furthermore, these bacteria can inhibit the growth of microorganisms carrying abundant antibiotic resistance genes in the raw water through metabolic activity, thus purifying the wastewater of antibiotics and antibiotic resistance genes. During their growth, they may inhibit the growth of microorganisms containing antibiotic resistance genes such as quinolones (ARGs) by competing with microorganisms rich in antibiotic resistance genes for nutrients and living space, or by secreting antibacterial active substances (after microbial death, the genes they carry are usually gradually degraded due to their own enzymatic degradation, environmental microbial decomposition, and physicochemical factors), thereby reducing the abundance of antibiotic resistance genes and achieving comprehensive purification of organic wastewater.

[0006] Based on previous research findings, this application proposes the following technical solutions: A method for purifying antibiotic-containing wastewater using functional microorganisms includes the following steps: (1) The bacterial culture was expanded by denitrifying thiobacillus, propionate-producing propionate bacillus, slow-growing rhizobium and ferrooxidizing thiobacillus to obtain bacterial solution; (2) Inoculate the anaerobic tank with bacterial solutions of *Thiobacillus denitrifyingus*, *Propionibacterium propionitum*, and *Slow-growing Rhizobium*, and inoculate the aerobic tank with bacterial solution of *Thiobacillus ferrooxidans* for A. 2 / O processing technology; Preferably, in step (1), the concentration of the bacterial solution is 10. 9 cfu / mL.

[0007] Preferably, in step (2), the inoculation amount of the bacterial solution is at least 1% (v / v).

[0008] Preferably, in step (2), the treatment time is at least 6 hours. Sufficient treatment time allows the microorganisms to fully exert their effects.

[0009] Preferably, the wastewater includes wastewater from antibiotic pharmaceutical factories, hospital wastewater, livestock and poultry farming wastewater, or domestic sewage. These types of wastewater typically contain antibiotics and microorganisms carrying antibiotic resistance genes, and can be treated using this method.

[0010] Preferably, the wastewater contains antibiotics and microorganisms, and the microorganisms contain antibiotic resistance genes.

[0011] Preferably, the antibiotics include quinolone antibiotics, tetracycline antibiotics, or sulfonamide antibiotics.

[0012] Preferably, the antibiotic resistance gene includes quinolone, tetracycline, or sulfonamide antibiotic resistance genes.

[0013] Preferably, the antibiotic resistance gene includes qepA , qnrB Gene, Su1I Gene, TetA Gene.

[0014] Preferably, the antibiotics include levofloxacin, sulfamethoxazole, and erythromycin.

[0015] The antibiotics or antibiotic resistance genes mentioned above are only a partial list and do not represent all antibiotics or antibiotic resistance genes that this method can handle. This method can also be used to treat other antibiotics and antibiotic resistance genes known in the art.

[0016] Compared with the prior art, the beneficial results of the present invention are as follows: The method of this invention effectively degrades organic matter in water, enabling wastewater to meet better discharge standards and thus contributing to the protection of water resources and the ecological environment. Antibiotic removal rate reaches 96%, significantly reducing the potential harm of antibiotics in wastewater to the environment and human health, and minimizing the spread and accumulation of antibiotics in the environment.

[0017] The method of this invention can also reduce the abundance of antibiotic resistance genes in wastewater by more than 78%, effectively inhibiting the spread and diffusion of antibiotic resistance genes in the environment.

[0018] This method is simple to operate and easy to implement. At the same time, it is based on screening microorganisms in raw water, which is relatively low in cost and has good environmental and economic benefits, making it conducive to promotion and application in practical engineering. Detailed Implementation

[0019] To better understand the technical content of this invention, the invention will be further described below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the experimental detection methods are conventional methods. Unless otherwise specified, the reagents and bacterial strains are commercially available products and can be routinely obtained through commercial channels.

[0020] Experimental methods: The CODcr determination method adopted was the conventional microwave digestion titration method.

[0021] Antibiotic concentrations were determined using high performance liquid chromatography-tandem mass spectrometry.

[0022] Resistance gene detection: For water samples, microorganisms were obtained by filtering through a 0.22 μm filter membrane. DNA samples were extracted using a DNA kit according to the instructions. The abundance of resistance genes such as qepA and qnrB was detected by quantitative PCR (qPCR). Three parallel experiments were set up for each sample, and the average value was taken.

[0023] Example 1: (1) Organic wastewater discharged from antibiotic pharmaceutical plants is treated using traditional A 2 After treatment using the / O process (the wastewater is sequentially fed into an anaerobic tank, an anoxic tank, an aerobic tank, and a sedimentation tank for treatment, with a wastewater retention time of 6 hours), the antibiotic content and resistance gene abundance in the sedimentation tank effluent are measured. (2) Activate and expand culture to a concentration of 10 at an inoculum amount of 1% (v / v). 9 Thiobacillus denitrification strain DSM 12475, at a concentration of 10 cfu / mL. 9 Propionibacterium propionitum ATCC 25562, at a concentration of 10 cfu / mL. 9 A solution of slow-growing rhizobium CCTCC AB 2012237 (cfu / mL) was inoculated into the anaerobic tank, activated, and cultured to a concentration of 10. 9 A CFU / mL solution of *Thiobacillus ferrooxidans* ATCC 23270 was inoculated into an aerobic tank to treat the raw water (organic wastewater from an antibiotic pharmaceutical factory) underwent A... 2 / O treatment process (sewage retention time is 6h), detect antibiotic content and resistance gene abundance in sedimentation tank effluent.

[0024] Table 1

[0025] The results showed that in the treated water samples, the content of sulfamethoxazole decreased from 106.0 μg / L to 5.6 μg / L, levofloxacin decreased from 625.4 μg / L to 37.1 μg / L, and erythromycin decreased from 50.5 μg / L to 4.3 μg / L, indicating a significant reduction in antibiotic residues with a removal rate exceeding 91%. The abundance of antibiotic resistance genes, including quinolone resistance genes, tetracycline resistance genes, and sulfonamide resistance genes, was also significantly reduced, with a reduction rate exceeding 77%.

[0026] Example 2 (1) Medical wastewater is treated using traditional A 2 After treatment by the / O process (the wastewater is sequentially fed into an anaerobic tank, an anoxic tank, an aerobic tank, and a sedimentation tank for treatment, with a wastewater retention time of 6 hours in the anaerobic, anoxic, and aerobic tanks), the antibiotic content and resistance gene abundance in the effluent from the sedimentation tank are tested. (2) Activate and expand culture to a concentration of 10 at an inoculum amount of 1% (v / v). 9 Thiobacillus denitrification strain DSM 12475, at a concentration of 10 cfu / mL. 9 Propionibacterium propionitum ATCC 25562, at a concentration of 10 cfu / mL. 9 A solution of slow-growing rhizobium CCTCC AB 2012237 (cfu / mL) was inoculated into the anaerobic tank, activated, and cultured to a concentration of 10. 9A CFU / mL solution of *Thiobacillus ferrooxidans* ATCC 23270 was inoculated into the aerobic tank, and the raw water (medical wastewater) was subjected to A... 2 / O treatment process (sewage retention time is 6h), detect antibiotic content and resistance gene abundance in sedimentation tank effluent.

[0027] Test results: Table 2

[0028] The treated water samples showed a significant reduction in antibiotic residues, with a removal rate exceeding 95%. The abundance of antibiotic resistance genes also decreased significantly, with a reduction rate exceeding 80%.

[0029] Example 3: (1) Domestic sewage is treated by traditional A 2 After treatment by the / O process (the wastewater is sequentially fed into an anaerobic tank, an anoxic tank, an aerobic tank, and a sedimentation tank for treatment, with a wastewater retention time of 6 hours in the anaerobic, anoxic, and aerobic tanks), the antibiotic content and resistance gene abundance in the effluent from the sedimentation tank are tested. (2) Activate and expand culture to a concentration of 10 at an inoculum amount of 1% (v / v). 9 Thiobacillus denitrification strain DSM 12475, at a concentration of 10 cfu / mL. 9 Propionibacterium propionitum ATCC 25562, at a concentration of 10 cfu / mL. 9 A solution of slow-growing rhizobium CCTCC AB 2012237 (cfu / mL) was inoculated into the anaerobic tank, activated, and cultured to a concentration of 10. 9 A CFU / mL solution of *Thiobacillus ferrooxidans* ATCC 23270 was inoculated into the aerobic tank, and the raw water (domestic sewage) was subjected to A... 2 / O treatment process (sewage retention time is 6h), detect antibiotic content and resistance gene abundance in sedimentation tank effluent.

[0030] Test results: Table 3

[0031] The treated water samples showed a significant reduction in antibiotic residues, with a removal rate exceeding 94%. Antibiotic resistance genes were reduced by over 85%, and their abundance was significantly decreased.

[0032] Comparative Example 1: (1) Organic wastewater discharged from antibiotic pharmaceutical plants is treated using traditional A 2After treatment by the / O process (the wastewater is sequentially fed into an anaerobic tank, an anoxic tank, an aerobic tank, and a sedimentation tank for treatment, with a wastewater retention time of 6 hours in the anaerobic, anoxic, and aerobic tanks), the antibiotic content and resistance gene abundance in the effluent from the sedimentation tank are tested. (2) At an inoculum rate of 1% (v / v), activate and expand the culture to a concentration of 10. 9 CFU / mL of denitrifying thiobacillus DSM 12475 was inoculated into the anaerobic tank, and the raw water (organic wastewater discharged from an antibiotic pharmaceutical factory) was subjected to A... 2 / O treatment process (sewage retention time is 6h), detect antibiotic content and resistance gene abundance in sedimentation tank effluent.

[0033] Test results: Table 4

[0034] In the treated water sample, the antibiotic removal rate was approximately 76%. The resistance gene was reduced by about 51%, but the effect was significantly less than in Example 1.

[0035] Comparative Example 2: Organic wastewater discharged from antibiotic pharmaceutical plants is treated using traditional A 2 After treatment by the / O process (the wastewater is sequentially fed into an anaerobic tank, an anoxic tank, an aerobic tank, and a sedimentation tank for treatment, with a wastewater retention time of 1 hour in the anaerobic, anoxic, and aerobic tanks), the antibiotic content and resistance gene abundance in the effluent from the sedimentation tank are tested. (2) At an inoculum rate of 1% (v / v), activate and expand the culture to a concentration of 10. 9 A 1% (v / v) inoculum of *Thiobacillus ferrooxidans* ATCC 23270 was inoculated into the aerobic tank, and the raw water (organic wastewater from an antibiotic pharmaceutical factory) was treated with A... 2 / O treatment process (sewage retention time is 6h), detect antibiotic content and resistance gene abundance in sedimentation tank effluent.

[0036] Test results: Table 5

[0037] In the treated water samples, the antibiotic removal rate was 67%–70%, and the resistance genes were reduced by 39%–58%.

[0038] The wastewater treatment effects of Comparative Example 1 and Comparative Example 2 were not as good as those of Example 1, suggesting that the simultaneous addition of multiple Thiobacillus strains is more conducive to the removal of antibiotics and antibiotic resistance genes.

[0039] Comparative Example 3: (1) Organic wastewater discharged from antibiotic pharmaceutical plants is treated using traditional A 2After treatment by the / O process (the wastewater is sequentially fed into an anaerobic tank, an anoxic tank, an aerobic tank, and a sedimentation tank for treatment, with a wastewater retention time of 1 hour in the anaerobic, anoxic, and aerobic tanks), the antibiotic content and resistance gene abundance in the effluent from the sedimentation tank are tested. (2) Activate and expand culture to a concentration of 10 at an inoculum amount of 1% (v / v). 9 Thiobacillus denitrification strain DSM 12475, at a concentration of 10 cfu / mL. 9 Propionibacterium propionitum ATCC 25562, at a concentration of 10 cfu / mL. 9 A 10 CFU / mL solution of the slow-growing rhizobium CCTCC AB 2012237 was obtained. 9 A CFU / mL solution of *Thiobacillus ferrooxidans* ATCC 23270 was inoculated into an aerobic tank to treat the raw water (organic wastewater from an antibiotic pharmaceutical factory) underwent A... 2 / O treatment process (sewage retention time is 6h), detect antibiotic content and resistance gene abundance in sedimentation tank effluent.

[0040] Test results: Table 6

[0041] In this comparative example, the bacterial community was inoculated into the aerobic tank. The treated water sample showed an antibiotic removal rate of approximately 72% and a reduction in antibiotic resistance genes of approximately 61%–64%. The removal efficiency of antibiotics and antibiotic resistance genes was not as good as in Example 1.

[0042] Comparative Example 4: (1) Organic wastewater discharged from antibiotic pharmaceutical plants is treated using traditional A 2 After treatment using the / O process (the wastewater is sequentially fed into an anaerobic tank, an anoxic tank, an aerobic tank, and a sedimentation tank for treatment, with a wastewater retention time of 6 hours), the antibiotic content and resistance gene abundance in the sedimentation tank effluent are measured. (2) Activate and expand culture to a concentration of 10 at an inoculum amount of 1% (v / v). 9 Thiobacillus denitrification strain DSM 12475, at a concentration of 10 cfu / mL. 9 CFU / mL of methanolophilic bacteria ( Methanolinea mesophila NBRC 105659, concentration 10 9 A solution of slow-growing rhizobium CCTCC AB 2012237 (cfu / mL) was inoculated into the anaerobic tank, activated, and cultured to a concentration of 10. 9 CFU / mL of *Thiopyroximate* (CFU / mL) Acidithiobacillus caldus CCTCC ZB 2008153 bacterial solution was inoculated into the aerobic tank to treat the raw water (organic wastewater discharged from an antibiotic pharmaceutical factory) under A treatment. 2 / O treatment process (sewage retention time is 6h), detect antibiotic content and resistance gene abundance in sedimentation tank effluent.

[0043] Table 7

[0044] The results showed that the removal rate of antibiotics in the treated water samples was relatively high, but the removal rate of antibiotic resistance genes was as low as 39%. Compared with Example 1, the removal effect of resistance genes in this comparative example was poor. It can be seen that the specific functional bacterial strain combination screened and grouped for inoculation in this application plays a key role in the removal of antibiotics and their resistance genes, and has a better removal effect than other microbial combinations with similar functions.

[0045] The above description is only a part of the embodiments 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 shall fall within the protection scope of the present invention.

Claims

1. A method for purifying antibiotic-containing wastewater using functional microorganisms, characterized in that, Includes the following steps: (1) The bacterial culture was expanded by denitrifying thiobacillus, propionate-producing propionate bacillus, slow-growing rhizobium and ferrooxidizing thiobacillus to obtain bacterial solution; (2) Inoculate *Thiobacillus denitrifyingus*, *Propionibacterium propionate*, and *Slow-growing rhizobium* into the anaerobic tank, and inoculate *Thiobacillus ferrooxidans* into the aerobic tank to carry out new A... 2 / O processing technology.

2. The method according to claim 1, characterized in that, In step (2), the inoculation amount of the bacterial solution is at least 1% (v / v).

3. The method according to claim 1, characterized in that, In step (2), the processing time is at least 6 hours.

4. The method according to claim 1, characterized in that, The antibiotic-containing wastewater includes wastewater from antibiotic pharmaceutical factories, hospital wastewater, livestock and poultry farming wastewater, or domestic wastewater.

5. The method according to claim 1, characterized in that, The wastewater contains antibiotics and microorganisms, and the microorganisms contain antibiotic resistance genes.

6. The method according to claim 5, characterized in that, The antibiotics include at least one of quinolone antibiotics, tetracycline antibiotics, or sulfonamide antibiotics.

7. The method according to claim 5, characterized in that, The antibiotic resistance gene includes at least one of the following: quinolone, tetracycline, or sulfonamide antibiotic resistance genes.

8. The method according to claim 5, characterized in that, The antibiotic resistance gene includes qepA , qnrB Gene, Su1I Gene, TetA At least one of the genes.

9. The method according to claim 5, characterized in that, The antibiotics include at least one of levofloxacin, sulfamethoxazole, and erythromycin.

10. The method according to claim 1, characterized in that, The concentration of the bacterial solution is 10. 9 cfu / mL or higher.

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