Salt-resistant sulfonamide antibiotic-resistant synchronous nitrification and denitrification pseudomonas and application thereof

Abstract

The invention relates to the field of water treatment, and discloses a salt-resistant sulfonamide antibiotic-resistant synchronous nitrification and denitrification pseudomonas and application thereof. The novel pseudomonas Z-08 is obtained through screening, and the pseudomonas Z-08 can still realize efficient denitrification through a heterotrophic nitrification-aerobic denitrification way in a high-salt and high-concentration sulfonamide antibiotic environment, so that the pseudomonas Z-08 can be applied to treatment of wastewater containing high-concentration salt, high-concentration sulfonamide antibiotics and high-concentration nitrogen.

Description

Technical Field

[0001] The present invention relates to the field of water treatment, and particularly to a salt-tolerant and sulfonamide antibiotic-resistant simultaneous nitrification and denitrification Pseudomonas bacterium and its application. Background Art

[0002] With the development of the pharmaceutical and chemical industries and social economy, the environmental protection requirements for the pharmaceutical and chemical industries are getting higher and higher. In particular, the efficient and green treatment of pharmaceutical and chemical wastewater has become an urgent task. Pharmaceutical and chemical wastewater mainly includes process wastewater, flushing wastewater, circulating cooling water, experimental wastewater, domestic sewage, etc. Among them, the composition of process wastewater is the most complex, containing high concentrations of reactants, products, by-products, solvents, catalysts, etc. The water quality presents the following characteristics: (1) High salt content: Due to the use of a large amount of inorganic salts, acids and bases in the synthesis and separation and purification processes, the wastewater has a high and fluctuating salt content, and the salinity range is usually several thousand to several hundred thousand mg / L; (2) High nitrogen content: The synthesis reaction involves the use of a large amount of nitrogen-containing substances, and the concentrations of organic nitrogen and ammonia nitrogen in the wastewater are extremely high, generally reaching several hundred or even over a thousand mg / L; (3) Many new pollutants: Due to the use of organic solvents in the production process and the limitation of product separation efficiency, the wastewater usually contains various new pollutants represented by antibiotics. Among them, sulfamethoxazole (SMX), a widely used sulfonamide antibiotic, is commonly present in pharmaceutical and chemical wastewater and is also one of the antibiotics with the highest detection frequency in wastewater. Under normal circumstances, the presence of SMX and a large amount of inorganic salts will inhibit the normal growth and metabolism of bacteria, affect the structure and function of the indigenous microbial community and the biofilm in the artificially constructed sewage treatment system, impact the stable operation of the activated sludge system, and cause a decrease in the microbial denitrification efficiency, resulting in the difficulty of meeting the effluent quality standard of the sewage.

[0003] Excessive nitrogen will not only damage the water ecosystem, causing acidification and eutrophication problems in lakes, rivers, and coastal waters, but also pose a threat to human health and biological survival in severe cases. In traditional water body denitrification technologies, biological denitrification is widely used due to its advantages such as low cost, mild reaction conditions, and no secondary pollution. The traditional biological denitrification process includes the aerobic nitrification of autotrophic bacteria and the anaerobic denitrification of heterotrophic bacteria. However, since this process consists of two independent technological steps, it leads to problems such as complex operation, high energy consumption, and expensive operation cost. If simultaneous heterotrophic nitrification-aerobic denitrification can be achieved, it can not only reduce the number of sewage treatment system structures and floor area, but also shorten the hydraulic retention time in the reactor.

[0004] On the other hand, since nitrogen-containing wastewater generally also contains high concentrations of salts and high concentrations of antibiotics, this poses relatively high requirements for the salt and antibiotic tolerance of microorganisms. At present, there are few reports on strains that simultaneously possess the ability of simultaneous nitrification and denitrification and salt and SMX tolerance. Therefore, the development of functional microorganisms that can complete simultaneous nitrification and denitrification under high-salt and high-sulfonamide antibiotic conditions is of great significance for the green and efficient treatment of pharmaceutical and chemical wastewater. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a salt- and sulfonamide antibiotic-tolerant Pseudomonas aeruginosa with simultaneous nitrification and denitrification and its application. The present invention screened and obtained a new strain of Pseudomonas aeruginosa Z-08, and the present invention found that it can still achieve efficient nitrogen removal through the heterotrophic nitrification-aerobic denitrification pathway under high-salt and high-concentration sulfonamide antibiotic environments. Therefore, it can be applied to the treatment of wastewater containing high concentrations of salts, high concentrations of sulfonamide antibiotics, and high concentrations of nitrogen.

[0006] The specific technical solution of the present invention is as follows: First, the present invention provides a salt- and sulfonamide antibiotic-tolerant Pseudomonas aeruginosa with simultaneous nitrification and denitrification. This Pseudomonas aeruginosa is named Z-08 and was deposited at the China Center for Type Culture Collection on September 27, 2024, with the deposit number CCTCC M 20242106, and the microbial taxonomic name is Pseudomonas ( Pseudomonas sp . ) Z-08.

[0007] The Pseudomonas aeruginosa Z-08 of the present invention was screened from the bacteria in the sludge of a pharmaceutical and chemical wastewater treatment plant. After its isolation and cultivation, the morphological and physiological and biochemical characteristics of the obtained strain are as follows: Gram-negative bacteria, which need to grow under aerobic conditions; when cultured on a solid plate of the acclimation medium for 5 days, the colonies are 1.0 - 1.2 mm in size, with irregular edges, convex, and opaque (as Figure 1 shown); the temperature growth range is 25 - 45 °C (optimum 35 °C), and the pH growth range is 5.0 - 9.0 (optimum 7.5 - 8.0). The 16S rRNA nucleotide sequence of Pseudomonas aeruginosa Z-08 is shown in SEQ ID NO:1. According to the above morphological characteristics and 16S rRNA sequence analysis, it is shown that this bacterium is a new strain of Pseudomonas ( Pseudomonas sp . ).

[0008] The present invention further discovers through experiments that the Pseudomonas sp. Z-08 of the present invention can denitrify nitrogen-containing wastewater through the heterotrophic nitrification-aerobic denitrification pathway. Specifically, it can convert ammonia nitrogen substances in the wastewater into nitrite nitrogen, convert nitrite nitrogen into nitrate nitrogen, and convert nitrate nitrogen into nitrogen gas. Moreover, the more remarkable property of this strain is that during the process of converting ammonia nitrogen, nitrite nitrogen, or nitrate nitrogen respectively, there will be no accumulation of nitrite nitrogen or nitrate nitrogen in the wastewater. This integrated treatment process can significantly improve the denitrification efficiency, reduce the residence time of nitrogen in the water body, thereby reducing the risk of water eutrophication. At the same time, it can also reduce the risk of secondary pollution that these intermediate products may bring, which is of great significance for protecting the water ecological environment and human health.

[0009] On this basis, the present invention also discovers that the Pseudomonas sp. Z-08 has particularly excellent tolerance to sulfonamide antibiotics and salts. Therefore, it can denitrify nitrogen-containing wastewater with high salt and high-concentration sulfonamide antibiotics (ordinary strains have poor tolerance and poor denitrification activity or even cannot survive in an environment with high salt and high-concentration sulfonamide antibiotics).

[0010] In the second aspect, the present invention provides the application of the above-mentioned Pseudomonas sp. in denitrifying nitrogen-containing wastewater.

[0011] Furthermore, the nitrogen-containing substances in the nitrogen-containing wastewater are one or more of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, and organic nitrogen.

[0012] Furthermore, the nitrogen concentration in the nitrogen-containing wastewater is 100 - 1000 mg / L, more preferably 200 - 800 mg / L, even more preferably 300 - 600 mg / L, and most preferably 400 - 500 mg / L.

[0013] Furthermore, the nitrogen-containing wastewater also contains sulfonamide antibiotics and / or salts.

[0014] Still further, the concentration of sulfonamide antibiotics in the nitrogen-containing wastewater is 0 - 100 mg / L.

[0015] Still further, the salinity of the nitrogen-containing wastewater is 0 - 80 g / L.

[0016] Furthermore, the sulfonamide antibiotic is sulfamethoxazole.

[0017] In the third aspect, the present invention provides a microbial degrading agent for nitrogen-containing wastewater, which contains the above-mentioned Pseudomonas sp. or its cell culture.

[0018] In the fourth aspect, the present invention provides a biological denitrification method for nitrogen-containing wastewater, which is to put the above-mentioned Pseudomonas sp. or its cell culture into the nitrogen-containing wastewater to achieve denitrification through the heterotrophic nitrification-aerobic denitrification pathway.

[0019] Further, the temperature for denitrification is 25 - 40 °C.

[0020] Further, the nitrogen-containing wastewater contains sulfonamide antibiotics and / or salts.

[0021] Still further, the concentration of sulfonamide antibiotics in the nitrogen-containing wastewater is 0 - 100 mg / L.

[0022] Still further, the salinity of the nitrogen-containing wastewater is 0 - 80 g / L.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: A new strain of Pseudomonas Z-08 is screened in the present invention. It is found that the strain can still achieve efficient denitrification through the heterotrophic nitrification-aerobic denitrification pathway in an environment with high salt and high concentrations of sulfonamide antibiotics. Therefore, it can be applied to the treatment of wastewater containing high concentrations of salt, high concentrations of sulfonamide antibiotics, and high concentrations of nitrogen. Description of the Drawings

[0024] Figure 1 It is a photograph of the colony of Pseudomonas Z-08.

[0025] Figure 2 It is the effect diagram of Pseudomonas Z-08 removing ammonia nitrogen and nitrate nitrogen.

[0026] Figure 3 It is the effect diagram of Pseudomonas Z-08 removing ammonia nitrogen and nitrite nitrogen. Detailed Embodiments

[0027] The present invention will be further described below in conjunction with embodiments.

[0028] General Embodiment First, a salt-tolerant and sulfonamide antibiotic-tolerant Pseudomonas for synchronous nitrification and denitrification. This Pseudomonas is named Z-08 and was deposited at the China Center for Type Culture Collection on September 27, 2024, with the deposit number CCTCC M20242106. The microbial taxonomic name is Pseudomonas ( Pseudomonas sp . ) Z-08. The 16S rRNA nucleotide sequence of Pseudomonas Z-08 is shown in SEQ ID NO:1:

[0029] Second aspect, application of the above-mentioned Pseudomonas in denitrification of nitrogen-containing wastewater: the Pseudomonas denitrifies nitrogen-containing wastewater through the heterotrophic nitrification-aerobic denitrification pathway.

[0030] Furthermore, the nitrogen-containing substances in the nitrogen-containing wastewater are one or more of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, and organic nitrogen.

[0031] In some specific implementation cases, the nitrogen concentration in the nitrogen-containing wastewater is 100-1000 mg / L, more preferably 200-800 mg / L, still more preferably 300-600 mg / L, and most preferably 400-500 mg / L.

[0032] In some specific implementation cases, the nitrogen-containing wastewater also contains sulfonamide antibiotics and / or salts.

[0033] In some specific implementation cases, the concentration of sulfonamide antibiotics in the nitrogen-containing wastewater is 0-100 mg / L.

[0034] In some specific implementation cases, the salinity of the nitrogen-containing wastewater is 0-80 g / L.

[0035] In some specific implementation cases, the sulfonamide antibiotics are sulfamethoxazole, etc.

[0036] In some specific implementation cases, the salts are sodium chloride, etc.

[0037] Third aspect, a microbial degrader for nitrogen-containing wastewater, containing the above-mentioned Pseudomonas or its cell culture.

[0038] Fourth aspect, a biological denitrification method for nitrogen-containing wastewater, adding the above-mentioned Pseudomonas or its cell culture to the nitrogen-containing wastewater, and realizing denitrification through the heterotrophic nitrification-aerobic denitrification pathway.

[0039] In some preferred implementation cases, the temperature for denitrification is 25-40 °C.

[0040] In some specific implementation cases, the nitrogen-containing wastewater contains sulfonamide antibiotics and / or salts.

[0041] In some specific implementation cases, the concentration of sulfonamide antibiotics in the nitrogen-containing wastewater is 0-100 mg / L.

[0042] In some specific implementation cases, the salinity of the nitrogen-containing wastewater is 0-80 g / L.

[0043] In some specific implementation cases, the sulfonamide antibiotics are sulfamethoxazole, etc.

[0044] In some specific embodiments, the salts are sodium chloride and the like. Specific embodiments Example 1: Domestication, isolation and identification of strains (1) Preparation of the culture medium: Domestication medium (per liter of water): KH 2 HPO 4 •3H 2 O 1.50 g, NaNO 3 1.1 g, NH 4 Cl 0.7 g, KH 2 PO 4 0.5 g, NaCl 19.3 g, MgSO 4 •7H 2 O 0.2 g, sulfamethoxazole (SMX) 20.0 mg.

[0046] 1 / 5 LB medium (per liter of water): tryptone 2.0 g, yeast extract 1.0 g, NaCl 2.0 g, sulfamethoxazole (SMX) 20.0 mg.

[0047] The preparation method of the solid medium is to add 1.5% (w / v) agar to the corresponding liquid medium formula. The sterilization method is high-temperature high-pressure sterilization at 121 °C for 30 min.

[0048] (2) Domestication steps: Take the sludge from the pharmaceutical and chemical wastewater treatment plant into the domestication medium, shake and culture it at a certain temperature for one week to obtain the first-generation bacterial community. Then take part of the first-generation bacterial community into the fresh domestication medium and culture it for one week. Repeat the above subculture process multiple times at the same ratio to obtain a stable primary bacterial community.

[0049] (3) Isolation of single bacteria: After continuously diluting part of the primary bacterial community culture solution, take 0.1 mL of the diluted suspension and spread it on the solid domestication medium. After controlled-temperature culture, isolate single colonies with different morphologies and repeatedly purify them on the solid domestication medium to obtain the strain, named Pseudomonas Z-08.

[0050] (4) Strain identification: The morphological, physiological and biochemical characteristics of Pseudomonas Z-08 are as follows: Gram-negative bacteria, which need to grow under aerobic conditions. After culturing on the solid plate of the domestication medium for 5 days, the colonies are 1.0 - 1.2 mm in size, with irregular edges, convex, and opaque (as Figure 1 shown). The temperature growth range is 25 - 45 °C (optimum 35 °C), and the pH growth range is 5.0 - 9.0 (optimum 7.5 - 8.0).

[0051]

[0052] Based on the above morphological characteristics and 16S rDNA sequence analysis results, it is shown that this bacterium is a new Pseudomonas ( Pseudomonas sp.), and this strain was deposited at the China Center for Type Culture Collection on September 27, 2024, with the deposit number CCTCC M 20242106, and the microbial taxonomic name is Pseudomonas ( Pseudomonas sp.) Z-08.

[0053] Example 2: Heterotrophic nitrification-aerobic denitrification ability of Pseudomonas Z-08 under high-salt environment (1) Preparation of the culture medium: Heterotrophic nitrification-aerobic denitrification medium 1 (per liter of water): C 6 H 5 Na 3 O 7 4.74 g, NH 4 Cl 0.25 g, NaNO 3 1.1697 g, NaCl 20 g, MgSO 4 ·7H 2 O 0.1 g, KH 2 PO 4 1.5 g, K 2 HPO 4 ·3H 2 O 7.9 g, pH = 7.0, sulfamethoxazole (SMX) 20 mg.

[0054] Heterotrophic nitrification-aerobic denitrification medium 2 (per liter of water): C 6 H 5 Na 3 O 7 4.74 g, NH 4 Cl 0.25 g, NaNO 2 0.25 g, NaCl 20 g, MgSO 4 ·7H 2 O 0.1 g, KH 2 PO 4 1.5 g, K 2 HPO 4 ·3H 2 O 7.9 g, pH = 7.0, sulfamethoxazole (SMX) 20 mg.

[0055] The sterilization method is autoclaving at 121 °C for 30 min.

[0056] (2) Preparation of the seed solution: Pseudomonas sp. Z-08 was cultured in a solid domestication medium (same as in Example 1) at 30 °C for 5 d, and single colonies were picked. The single colonies were inoculated into 10 mL of 1 / 5 LB liquid medium with a salinity of 20 g / L and cultured to the logarithmic phase (cultured for 12 h) under the conditions of a rotation speed of 150 r / min and a temperature of 35 °C. The obtained bacterial liquid was transferred to 40 mL of 1 / 5 LB medium with a salinity of 20 g / L and cultured until OD 600 = 1.0, then centrifuged to collect the bacterial cells, washed twice with physiological saline, and resuspended with physiological saline to prepare a cell suspension, which was used as the seed liquid.

[0057] (3)Denitrification characteristics of Pseudomonas sp. Z-08 for removing ammonia nitrogen and nitrate nitrogen: The seed liquid was transferred to heterotrophic nitrification-aerobic denitrification medium 1 with a salinity of 20 g / L at an inoculation amount of 10% and cultured under the conditions of a temperature of 35 °C and a rotation speed of 150 rpm. Samples were taken to measure NH 4 + -N, NO 3 - -N, NO 2 - -N and OD 600 。

[0058] The results were as Figure 2 shown. From 8 h, Pseudomonas sp. Z-08 began to grow rapidly, and there was a synchronous downward trend in ammonia nitrogen and nitrate nitrogen. The final removal rate of ammonia nitrogen was close to 100% at 24 h, and 83.79 mg / L of nitrate nitrogen could be removed at 24 h, and there was no accumulation of nitrite nitrogen during the experiment. These results indicate that Pseudomonas sp. Z-08 can quickly adapt to a high-salt environment and remove ammonia nitrogen and nitrate nitrogen simultaneously under the stress of sulfamethoxazole.

[0059] (5)Denitrification characteristics of Pseudomonas sp. Z-08 for removing ammonia nitrogen and nitrite nitrogen: The seed liquid was transferred to heterotrophic nitrification-aerobic denitrification medium 2 with a salinity of 20 g / L at an inoculation amount of 10% and cultured under the conditions of a temperature of 35 °C and a rotation speed of 150 rpm. Samples were taken to measure NH 4 + -N, NO 3 - -N, NO 2 - -N and OD 600 。

[0060] The results were as Figure 3As shown, Pseudomonas sp. Z-08 began to grow rapidly after 6 h, and the ammonia nitrogen and nitrite nitrogen showed a synchronous downward trend. Moreover, the removal rate of ammonia nitrogen was faster than that of nitrite nitrogen. At 24 h, the final removal rate of ammonia nitrogen was close to 100%, and the removal rate of nitrite nitrogen was 4.10 mg / L / h. There was no accumulation of nitrate nitrogen during the experiment. These results indicate that Pseudomonas sp. Z-08 can quickly adapt to the high-salt environment and remove ammonia nitrogen and nitrite nitrogen simultaneously under the stress of sulfamethoxazole.

[0061] Through the above experiments, the present invention discovered a novel strain of Pseudomonas sp. Z-08, which can denitrify nitrogen-containing wastewater through the heterotrophic nitrification-aerobic denitrification pathway. Specifically, it can convert ammonia nitrogen in the wastewater into nitrite nitrogen, convert nitrite nitrogen into nitrate nitrogen, and convert nitrate nitrogen into gaseous nitrogen. Moreover, the more remarkable property of this strain is that during the process of converting ammonia nitrogen, nitrite nitrogen, or nitrate nitrogen respectively, there will be no accumulation of nitrite nitrogen or nitrate nitrogen in the wastewater. This integrated treatment process can significantly improve the denitrification efficiency, reduce the residence time of nitrogen in the water body, thereby reducing the risk of water eutrophication. At the same time, it can also reduce the risk of secondary pollution that these intermediate products may bring, which is of great significance for protecting the water ecological environment and human health.

[0062] On this basis, the present invention also found that Pseudomonas sp. Z-08 has particularly excellent tolerance to sulfonamide antibiotics and salts. Therefore, it can denitrify nitrogen-containing wastewater with high salt and high concentration of sulfonamide antibiotics (ordinary strains have poor tolerance and poor denitrification activity or even cannot survive in the environment of high salt and high concentration of sulfonamide antibiotics).

[0063] In the present invention, the raw materials and equipment used, unless otherwise specified, are all common raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0064] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modification, change, and equivalent transformation made to the above embodiments according to the technical essence of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A salt-resistant and sulfonamide-resistant simultaneous nitrifying and denitrifying Pseudomonas sp., characterized in that: The Pseudomonas was named Z-08, and was deposited in the China Center for Type Culture Collection on September 27, 2024, with a deposit number of CCTCC M20242106. The microbial classification was named Pseudomonas ( Pseudomonas sp.).

2. The use of Pseudomonas according to claim 1 in denitrification of nitrogen-containing wastewater, characterized in that: The Pseudomonas denitrifies nitrogen-containing wastewater through a heterotrophic nitrification-aerobic denitrification pathway.

3. The use according to claim 2, characterized in that: The nitrogen-containing substances in the nitrogen-containing wastewater are one or more of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and organic nitrogen.

4. The use according to claim 3, characterized in that: The nitrogen concentration in the nitrogen-containing wastewater is 100-1000 mg / L.

5. The use according to claim 2, characterized in that: The nitrogen-containing wastewater also contains sulfonamide antibiotics and / or salts.

6. The use according to claim 5, characterized in that: The concentration of sulfonamide antibiotics in the nitrogen-containing wastewater is 0-100 mg / L; The salinity of the nitrogen-containing wastewater is 0-80 g / L.

7. A nitrogen-containing wastewater microbial degradation agent, characterized in that: Containing the Pseudomonas or its bacterial culture according to claim 1.

8. A biological denitrification method for nitrogen-containing wastewater, characterized in that: Add nitrogen to wastewater The Pseudomonas or bacterial culture thereof according to claim 1 achieves denitrification through a heterotrophic nitrification-aerobic denitrification pathway.

9. The biological denitrification method for nitrogen-containing wastewater according to claim 8, characterized in that: The denitrification temperature is 25~40°C.

10. The biological denitrification method for nitrogen-containing wastewater according to claim 8, characterized in that: The nitrogen-containing wastewater contains sulfonamide antibiotics and / or salts; The concentration of sulfonamide antibiotics in the nitrogen-containing wastewater is 0 to 100 mg / L; The salinity of the nitrogen-containing wastewater is 0-80 g / L.

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