Method for improving resistance of denitrification strain to new pollutant perfluorinated compound

Abstract

The invention relates to a method for improving resistance of a denitrification strain to a new pollutant perfluorinated compound, and belongs to the field of biological denitrification of sewage. The method comprises the following steps: under an aerobic condition, culturing a denitrification strain by using a sterilized TSB culture medium to obtain OD600 = 1.8; resuspending with a sterile PBS buffer solution to obtain a bacterial suspension; (2) preparing a synchronous nitrification and denitrification culture medium, and sterilizing for later use; (3) respectively adding a perfluorinated compound into the synchronous nitrification and denitrification culture medium, and respectively inoculating the bacterial suspension in the step (1) into the synchronous nitrification and denitrification culture medium containing the perfluorinated compound according to the inoculum size of 1% (v / v); and (4) placing the inoculated synchronous nitrification and denitrification culture medium in an aerobic environment, culturing for 2 hours in a shaking table at the temperature of 30 DEG C and the rotating speed of 150rpm, and carrying out weak ultrasonic treatment on the strain by using an ultrasonic generator. According to the method, the denitrification strain is treated by weak ultrasound, so that the inhibition effect of the perfluorinated compound on the denitrification strain is slowed down, and the resistance of the denitrification strain on the perfluorinated compound is improved.

Description

Technical Field

[0001] The invention belongs to the field of biological denitrification of sewage, and particularly relates to a method for improving the resistance of denitrification strains to new pollutant perfluorinated compounds. Background Art

[0002] With the continuous development of industrialization, the total amount of nitrogen-containing substances discharged into the aquatic environment is increasing, and the removal of nitrogen from water bodies has become urgent. At the same time, the large amount of wastewater containing perfluorinated compounds discharged into the water during the production of chemical products will inevitably pose a serious threat to the environment and human health. Due to the toxic effects of perfluorinated compounds, they will inevitably have a certain impact on the denitrification ability of microorganisms. Traditional biological denitrification technologies mainly rely on the nitrification and denitrification of microorganisms to convert ammonia nitrogen in wastewater into nitrogen gas, thereby achieving nitrogen removal. However, new biological denitrification technologies have effectively overcome the shortcomings of traditional biological denitrification technologies and have attracted widespread attention due to their energy-saving, environmentally friendly, and easy-to-operate denitrification advantages. Among them, heterotrophic nitrification and aerobic denitrification biological denitrification technology stands out among many new biological denitrification technologies due to its advantages such as reduced floor space, cost savings, and efficient nitrogen removal. Summary of the Invention

[0003] Based on this, the present invention proposes a method for improving the resistance of denitrifying strains to new pollutant perfluorinated compounds. The present invention uses weak ultrasound to treat the denitrifying strains to slow down the inhibitory effect of new pollutant perfluorinated compounds on the denitrifying strains, thereby improving the resistance of the denitrifying strains to new pollutant perfluorinated compounds.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] A method for improving the resistance of denitrifying strains to new pollutant perfluorinated compounds comprises the following steps:

[0006] (1) Under aerobic conditions, the denitrification strain was cultured in a sterilized TSB medium at 30°C and 150 rpm in a shaker for 18 hours to obtain the OD 600 =1.8; then wash with sterile PBS buffer 2 to 3 times and resuspend to obtain a bacterial suspension;

[0007] (2) Prepare the simultaneous nitrification and denitrification culture medium and sterilize it for later use;

[0008] (3) adding perfluorinated compounds to the simultaneous nitrification and denitrification culture medium respectively, with the concentrations of perfluorinated compounds being 0 mg / L, 1 mg / L, and 20 mg / L, respectively, and inoculating the bacterial suspension in step (1) into the simultaneous nitrification and denitrification culture medium containing perfluorinated compounds at an inoculum volume of 1% (v / v);

[0009] (4) The inoculated synchronous nitrification and denitrification culture medium was placed in an aerobic environment, cultured in a shaker at 30°C and 150 rpm for 2 h, and then the strain was subjected to weak ultrasonic treatment using an ultrasonic generator.

[0010] Furthermore, in step (1), the denitrification strain is Paracoccus denitrificans.

[0011] Furthermore, in step (1), the formula of the TSB culture medium used is: 15.0 g / L trypsin, 5.0 g / L soy protein, 5.0 g / L sodium chloride, 2.5 g / L glucose, 2.5 g / L Na2HPO4, the pH is adjusted to 7.0-7.5 using 1 M sodium hydroxide solution or hydrochloric acid solution, and the sterilization temperature is set to 121°C for 20 min.

[0012] Furthermore, in step (2), the simultaneous nitrification and denitrification culture medium includes inorganic salts, carbon sources and trace element solutions, wherein:

[0013] Inorganic salts include: ammonium chloride 0.382g / L, potassium nitrate 0.722g / L, potassium dihydrogen phosphate 1.5g / L, disodium hydrogen phosphate 0.42g / L, magnesium sulfate 1.0g / L;

[0014] The carbon source was sodium succinate 9.44 g / L;

[0015] Trace element solution 2mL / L;

[0016] Use 1M sodium hydroxide solution or hydrochloric acid solution to adjust the pH to 7.0-7.5 and set the sterilization temperature to 121°C for 20 minutes.

[0017] Furthermore, in step (2), the trace element solution is composed of: 10.0 g / L ethylenediaminetetraacetic acid, 1.0 g / L ferric sulfate, 0.5 g / L manganese sulfate, 0.05 g / L copper sulfate, 0.1 g / L zinc sulfate, 0.025 g / L ammonium molybdate, 0.025 g / L cobalt chloride, and 0.1 g / L calcium chloride.

[0018] Furthermore, the perfluorinated compound in step (3) is one of PFOA, PFOS, and PFBS.

[0019] Furthermore, in step (3), the initial inoculum size is OD 600 =1.0.

[0020] Furthermore, in step (4), the specific process of weak ultrasonic treatment is as follows: placing the probe of the ultrasonic generator 2 cm below the surface of the culture medium, the ultrasonic power is 10 to 25 W, and the ultrasonic time is 6 to 14 minutes.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention uses weak ultrasound to treat denitrification strains and conducts denitrification culture under the stress of perfluorochemicals. Compared with strains that have not been treated with weak ultrasound, strains treated with weak ultrasound can cause membrane deformation and pore expansion, which increases membrane permeability, promotes the absorption of external substances, and enhances strain metabolism and growth; secondly, ultrasound increases the release of extracellular polymers, which enhances the formation of protective cell layers or biofilms, thereby reducing the adverse effects of perfluorochemicals. In addition, ultrasound enhances the ATPase activity of the strain, accelerates the supply and metabolism of energy, and promotes the growth and activity of the strain. It also improves the removal rates of ammonia nitrogen and nitrate nitrogen, and reduces the accumulation of nitrite nitrogen; and it can simultaneously and efficiently denitrify in a single reactor. Compared with other physical and chemical denitrification methods, it has low cost and no secondary pollution.

[0023] 2. This invention aims to develop a biological denitrification method that is resistant to perfluorinated compound inhibition by subjecting denitrifying bacterial strains to weak ultrasound treatment. This method can improve the denitrification performance of microorganisms in perfluorinated compound-contaminated environments. This invention provides a new solution for the biological treatment of high-concentration perfluorinated compound-contaminated wastewater and has broad application prospects. DETAILED DESCRIPTION

[0024] The technical solutions and effects of the present invention are further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0025] Perfluorinated compounds, which are widely present in the environment and difficult to degrade, significantly affect the metabolic activity and denitrification efficiency of microorganisms. To ensure the effectiveness of denitrifying strains in removing nitrogen from wastewater, the present invention provides a method for improving the resistance of denitrifying strains to new pollutants, perfluorinated compounds. This method can increase the resistance of denitrifying strains to perfluorinated compounds, improve the removal rate of ammonia nitrogen and nitrate nitrogen by denitrifying strains, reduce the accumulation of nitrite nitrogen, and enable simultaneous and efficient denitrification in a single reactor. Compared with other physical and chemical denitrification methods, it is low-cost and does not cause secondary pollution.

[0026] Example 1

[0027] This example uses the denitrification strain Paracoccus denitrificans as an example to provide a method for improving the resistance of the denitrification strain Paracoccus denitrificans to new pollutants, perfluorinated compounds. The denitrification strain Paracoccus denitrificans used in this example was purchased from Wuhan Huizao Biotechnology Co., Ltd., and the perfluorinated compound used is PFOA.

[0028] The method of the present invention for improving the resistance of the denitrifying strain Paracoccus denitrificans to the new pollutant PFOA comprises the following steps:

[0029] (1) Under aerobic conditions, the denitrification strain Paracoccus denitrificans was cultured in a sterilized TSB medium at 30°C and 150 rpm in a shaker for 18 hours to obtain the OD 600 =1.8; then wash it 2 to 3 times with sterile PBS buffer and resuspend it to obtain a Paracoccus denitrificans bacterial suspension.

[0030] In this step, the formula of the TSB medium used is: 15.0 g / L trypsin, 5.0 g / L soy protein, 5.0 g / L sodium chloride, 2.5 g / L glucose, and 2.5 g / L Na2HPO4. Use 1 M sodium hydroxide solution or hydrochloric acid solution to adjust the pH to 7.0-7.5, and set the sterilization temperature to 121°C for 20 min.

[0031] (2) Prepare the simultaneous nitrification and denitrification culture medium and sterilize it for later use.

[0032] In this step, the simultaneous nitrification and denitrification culture medium includes inorganic salts, carbon sources and trace element solutions, wherein:

[0033] Inorganic salts include: ammonium chloride 0.382g / L, potassium nitrate 0.722g / L, potassium dihydrogen phosphate 1.5g / L, disodium hydrogen phosphate 0.42g / L, magnesium sulfate 1.0g / L;

[0034] The carbon source was sodium succinate 9.44 g / L;

[0035] Trace element solution 2mL / L;

[0036] Use 1M sodium hydroxide solution or hydrochloric acid solution to adjust the pH to 7.0-7.5 and set the sterilization temperature to 121°C for 20 minutes.

[0037] Among them, the trace element solution composition is: ethylenediaminetetraacetic acid 10.0g / L, ferric sulfate 1.0g / L, manganese sulfate 0.5g / L, copper sulfate 0.05g / L, zinc sulfate 0.1g / L, ammonium molybdate 0.025g / L, cobalt chloride 0.025g / L, and calcium chloride 0.1g / L.

[0038] (3) PFOA was added to the simultaneous nitrification and denitrification culture medium at concentrations of 0 mg / L, 1 mg / L, and 20 mg / L, respectively. The Paracoccus denitrificans suspension prepared in step 1 was inoculated into the simultaneous nitrification and denitrification culture medium containing PFOA at an inoculum volume of 1% (v / v) of the culture medium volume. The initial inoculum volume was OD 600 =1.0.

[0039] (4) The inoculated simultaneous nitrification and denitrification culture medium was placed in an aerobic environment at 30°C and a shaking incubator at 150 rpm for 2 h, and then the strain was subjected to weak ultrasonic treatment using an ultrasonic generator. The specific process was as follows: the probe of the ultrasonic generator was placed 2 cm below the liquid surface of the culture medium, and ultrasonic treatment was performed at a power of 15 W for 10 min. In this embodiment, the ultrasonic frequency was selected to be 40 kHz.

[0040] The control group was not subjected to weak ultrasound treatment.

[0041] The simultaneous nitrification and denitrification medium configured above simulates domestic sewage, and the reaction system in this experiment is the culture medium. Therefore, the specific denitrification steps are: The activated strains in the TSB culture medium are prepared into a bacterial suspension, which is then added to the simultaneous nitrification and denitrification medium to carry out the denitrification reaction (to simulate the denitrification effect of actual domestic sewage). In other words, domestic sewage is actually the simultaneous nitrification and denitrification medium. The parameters of the simultaneous nitrification and denitrification medium can be calculated based on the above formula: nitrate nitrogen and ammonia nitrogen concentrations are both 100 mg / L, COD concentration is 2500 mg / L, and total nitrogen concentration is 220 mg / L.

[0042] Sampling and Measurement. During the reaction, samples were collected periodically using a sterile disposable syringe in a clean bench. After centrifugation at 5000 rpm for 5 minutes, the supernatant was collected for nitrate nitrogen and ammonia nitrogen determination. The results are shown in Tables 1 and 2. Nitrate nitrogen was determined using the phenol disulfonic acid spectrophotometric method, and ammonia nitrogen was determined using Nessler's reagent spectrophotometric method. The measurements were performed using a 721G UV-Vis spectrophotometer.

[0043] Table 1 Determination results of nitrate nitrogen

[0044]

[0045] Table 2 Determination results of ammonia nitrogen

[0046]

[0047] The results showed that under PFOA stress, the sonicated strain improved both nitrate and ammonia nitrogen removal compared to the unsonicated strain. Under 1 mg / L PFOA stress, after 9 hours of culture, the nitrate nitrogen removal rate of the unsonicated strain and the sonicated strain increased from 47.96% to 68.77%. Under 20 mg / L PFOA stress, after 9 hours of culture, the nitrate nitrogen removal rate of the unsonicated strain and the sonicated strain increased from 37.09% to 55.79%. Under 1 mg / L PFOA stress, after 9 hours of culture, the ammonia nitrogen removal rate of the unsonicated strain and the sonicated strain increased from 33.88% to 40%. Under 20 mg / L PFOA stress, after 9 hours of culture, the ammonia nitrogen removal rate of the unsonicated strain and the sonicated strain increased from 31.84% to 35.6%.

[0048] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving the resistance of denitrifying strains to new pollutants, perfluorinated compounds, characterized in that: The following steps are involved: (1) Under aerobic conditions, the denitrification strain was cultured in a sterilized TSB medium at 30°C and 150 rpm in a shaker for 18 hours to obtain the OD 600 =1.8; then wash with sterile PBS buffer 2 to 3 times and resuspend to obtain a bacterial suspension; (2) Prepare the simultaneous nitrification and denitrification culture medium and sterilize it for later use; (3) adding perfluorinated compounds to the simultaneous nitrification and denitrification culture medium respectively, with the concentrations of perfluorinated compounds being 0 mg / L, 1 mg / L, and 20 mg / L, respectively, and inoculating the bacterial suspension in step (1) into the simultaneous nitrification and denitrification culture medium containing perfluorinated compounds at an inoculum volume of 1% (v / v); (4) The inoculated synchronous nitrification and denitrification culture medium was placed in an aerobic environment, cultured in a shaker at 30°C and 150 rpm for 2 h, and then the strain was subjected to weak ultrasonic treatment using an ultrasonic generator.

2. The method for improving the resistance of denitrifying strains to new pollutants, perfluorinated compounds, according to claim 1, characterized in that: In the step (1), the denitrification strain is paracoccus denitrificans .

3. The method for improving the resistance of denitrifying strains to new pollutants, perfluorinated compounds, according to claim 2, characterized in that: In step (1), the formula of the TSB culture medium used is: 15.0 g / L trypsin, 5.0 g / L soy protein, 5.0 g / L sodium chloride, 2.5 g / L glucose, 2.5 g / L Na2HPO4, and the pH is adjusted to 7.0-7.5 using 1 M sodium hydroxide solution or hydrochloric acid solution. The sterilization temperature is set at 121°C for 20 min.

4. The method for improving the resistance of denitrifying strains to new pollutants, perfluorinated compounds, according to claim 1, characterized in that: In the step (2), the simultaneous nitrification and denitrification culture medium includes inorganic salts, carbon sources and trace element solutions, wherein: Inorganic salts include: ammonium chloride 0.382g / L, potassium nitrate 0.722g / L, potassium dihydrogen phosphate 1.5g / L, disodium hydrogen phosphate 0.42g / L, magnesium sulfate 1.0g / L; The carbon source was sodium succinate 9.44 g / L; Trace element solution 2mL / L; Use 1M sodium hydroxide solution or hydrochloric acid solution to adjust the pH to 7.0-7.5 and set the sterilization temperature to 121°C for 20 minutes.

5. The method for improving the resistance of denitrifying strains to new pollutants, perfluorinated compounds, according to claim 4, characterized in that: In step (2), the trace element solution is composed of: 10.0 g / L of ethylenediaminetetraacetic acid, 1.0 g / L of ferric sulfate, 0.5 g / L of manganese sulfate, 0.05 g / L of copper sulfate, 0.1 g / L of zinc sulfate, 0.025 g / L of ammonium molybdate, 0.025 g / L of cobalt chloride, and 0.1 g / L of calcium chloride.

6. The method for improving the resistance of denitrifying strains to new pollutants, perfluorinated compounds, according to claim 1, characterized in that: The perfluorinated compound in step (3) is one of PFOA, PFOS and PFBS.

7. The method for improving the resistance of denitrifying strains to new pollutants, perfluorinated compounds, according to claim 1, characterized in that: In step (3), the initial inoculum size is OD 600 =1.

0.

8. The method for improving the resistance of denitrifying strains to new pollutants, perfluorinated compounds, according to claim 1, characterized in that: In step (4), the specific process of weak ultrasonic treatment is as follows: placing the probe of the ultrasonic generator 2 cm below the liquid surface of the culture medium, the ultrasonic power is 10-25 W, and the ultrasonic time is 6-14 min.

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