A highly efficient denitrifying strain with the function of dissimilatory reduction to ammonium and its application

Abstract

A highly efficient denitrifying strain with the function of dissimilatory reduction to ammonium and its application, which relates to the field of water treatment. The present invention aims to screen out functional strains with the function of efficiently removing nitrate nitrogen or nitrite nitrogen with natural growth advantages. The Aeromonas provided by the present invention has high nitrate and nitrite enzyme activities and rapid cell growth. The strain also has the function of dissimilatory reduction to ammonium, which can achieve the rapid removal of nitrate nitrogen in water and effectively reduce the generation of nitrogen oxide gases. The Aeromonas media L20, which is an aerobic denitrifying bacterium, is preserved in the China Center for Type Culture Collection. The preservation date is July 12, 2023, and the preservation number is CCTCC NO: M 20231191.

Description

Technical Field

[0001] The present invention belongs to the field of water treatment, and particularly relates to a highly efficient denitrifying strain with the function of dissimilatory reduction to ammonium and its application. Background Art

[0002] The concentration of nitrogen element is often used as an important standard to measure the balance and stability in the natural environment. When the ammonia nitrogen concentration in water is too high, it will cause water eutrophication; when the nitrate nitrogen concentration in water is too high, it will cause cancer and malformations in infant development. Therefore, removing nitrogen from water has always been a key issue in the sewage treatment process. At the same time, the main denitrification products of the current water denitrifying strains mainly concentrate on nitrogen gas and nitrogen oxide gases, and the emission of a large amount of greenhouse gases has a serious impact on the atmospheric environment. Therefore, it is very necessary to screen out a strain with strong denitrification ability and significantly reduced nitrogen oxide gas emissions, especially with strong nitrate or nitrite nitrogen treatment ability. Among the existing strains with the function of dissimilatory reduction to ammonium, there is no Aeromonas. For example, the patent publication number CN 111793573B, with the invention name "A Shewanella algae strain with the functions of heterotrophic and autotrophic nitrate dissimilatory reduction to ammonium, its cultivation method and application", discloses a Shewanella algae strain with the functions of heterotrophic and autotrophic nitrate dissimilatory reduction to ammonium, its cultivation method and application. Specifically, it is a Shewanella algae strain with the functions of heterotrophic nitrate dissimilatory reduction to ammonium and sulfur autotrophic nitrate dissimilatory reduction to ammonium, its cultivation method and its use in wastewater and waste gas treatment. This Shewanella algae strain is Shewanella algae, which can reduce nitrate, nitrite and NO in Fe(II)EDTANO produced by complex absorption flue gas denitrification in wastewater to ammonium through heterotrophic nitrate dissimilatory reduction to ammonium or sulfur autotrophic nitrate dissimilatory reduction to ammonium under anaerobic conditions. After ammonium is enriched in water, ammonia gas can be recovered by stripping. The above functions of this Shewanella algae strain can realize the resource utilization of nitrate, nitrite in wastewater and NO in flue gas, and have great uses in the fields of wastewater denitrification and waste gas denitrification. This patent is about Shewanella algae strain, not Aeromonas, and it cannot effectively remove high concentrations of nitrate nitrogen and nitrite nitrogen within 12 hours. Summary of the Invention

[0003] Aiming at the above problems, the object of the present invention is to screen out a highly efficient strain with the function of removing nitrate nitrogen or nitrite nitrogen with natural growth advantages. The Aeromonas provided by the present invention has high nitrate and nitrite enzyme system activities and rapid bacterial growth. The strain also has the function of dissimilatory reduction to ammonium, which can effectively reduce the generation of nitrogen oxide gas while quickly removing nitrate nitrogen in water.

[0004] The object of the present invention is achieved by the following technical solutions: An Aeromonas strain, namely Aeromonas media L20, which is an aerobic denitrifying bacterium, is preserved in the China Center for Type Culture Collection. The preservation date is July 12, 2023, and the preservation number is CCTCC NO: M 20231191.

[0005] Application of a highly efficient nitrogen-removing strain with the function of dissimilatory reduction to ammonium in the present invention, wherein the highly efficient nitrogen-removing strain is used for sewage treatment.

[0006] Further, the sewage treatment refers to the removal of nitrate nitrogen and / or nitrite nitrogen in water.

[0007] Further, the sewage treatment is to remove nitrate nitrogen with a concentration of 50 - 200 mg / L and / or nitrite nitrogen with a concentration of 50 - 200 mg / L in water.

[0008] Further, the sewage treatment refers to the complete removal of nitrate nitrogen with a concentration of 50 - 200 mg / L and / or nitrite nitrogen with a concentration of 50 - 200 mg / L in sewage within 12 hours.

[0009] Further, the sewage treatment refers to the removal of nitrate nitrogen and / or nitrite nitrogen in water, and at the same time, partially converting nitrate nitrogen and / or nitrite nitrogen into ammonium.

[0010] Application of a highly efficient nitrogen-removing strain with the function of dissimilatory reduction to ammonium in the present invention, wherein the highly efficient nitrogen-removing strain is used for preparing an aerobic denitrifying and dissimilatory reduction to ammonium bacterial agent.

[0011] The second object of the present invention is to provide a screening and identification method for the above-mentioned Aeromonas media L20, including the following steps:

[0012] 1. Enrichment: Collect activated aerobic sludge, break up the sludge and let it stand, then take the supernatant and inject it into the first enrichment medium for enrichment cultivation. Transfer the cultivated bacterial liquid into the second enrichment medium for enrichment cultivation, and so on.

[0013] 2. Isolation: Dilute and spread the bacterial liquid obtained by enrichment cultivation on the BTB medium, and select single colonies with blue halos from the cultivated colonies for purification by streaking on a plate to obtain Aeromonas.

[0014] The third object of the present invention is to provide the above-mentioned Aeromonas for treating nitrogen-containing sewage, and the nitrogen-containing sewage includes NO3 - or (and) NO2 - .

[0015] The third object of the present invention is to provide the application of the above-mentioned Aeromonas in the treatment of nitrogen-containing sewage.

[0016] The present invention has the following beneficial effects:

[0017] I. Using Aeromonas media L20 of the present invention to remove nitrate nitrogen or nitrite nitrogen in water, 100 mg / L of NO3 - -N can be completely removed without residue within 12 hours, and more than 95% of 50 mg / L of NO2 - -N can be removed; while in the prior art, no other strains that can completely remove 100 mg / L of NO3 — -N within 12 hours have been found.

[0018] II. Using Aeromonas media L20 of the present invention to remove nitrate nitrogen in water, ammonium nitrogen accumulation can be achieved through the metabolism of partial nitrate nitrogen by the dissimilatory reduction to ammonium pathway, reducing the complete gaseous loss of nitrogen; BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 are two dissimilatory reduction pathways of microbial nitrate nitrogen;

[0020] Figure 2 is the colony morphology diagram of strain L20;

[0021] Figure 3 is the phylogenetic tree of the strains of the present invention;

[0022] Figure 4 is the curve diagram of NO3 - -N removal and growth of strain L20 of the present invention over time;

[0023] Figure 5 is the curve diagram of NO2 - -N removal and growth of strain L20 of the present invention over time;

[0024] Figure 6 is the curve diagram of NH4 + -N production of strain L20 of the present invention over time;

[0025] Figure 7 is the process diagram of strain L20 of the present invention for removing nitrate nitrogen and nitrite nitrogen in artificially prepared sewage. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer and more understandable, the spirit of the content disclosed by the present invention will be described in detail below. After any person skilled in the art understands the embodiments of the content of the present invention, the techniques taught by the content of the present invention can be changed and modified without departing from the spirit and scope of the content of the present invention.

[0027] Schematic embodiments of the present invention and their descriptions are used to explain the present invention, but not to limit the present invention.

[0028] Example 1

[0029] (1) Enrichment of strains

[0030] Take 2 g of sediment collected from the sewage treatment plant and place it in a 250 mL conical flask containing 98 mL of sterile water. Incubate overnight at 30 °C with shaking at 150 r·min -1 Under sterile conditions, withdraw 5 mL of the suspension and inoculate it into a 250 mL conical flask containing 95 mL of LB liquid medium. Incubate with shaking at 30 °C and 150 r·min -1 for 12 h to achieve the purpose of strain enrichment.

[0031] (2) Isolation and purification of strains

[0032] In a laminar flow hood, use a pipette to aspirate 0.1 mL of the enriched culture solution and inoculate it into a centrifuge tube containing 0.9 mL of sterile water. Shake well to obtain a 10 -1 bacterial solution. Dilute the bacterial solution to 10 -2 -10 -6 and other gradient bacterial solutions according to the gradient dilution method. Respectively aspirate 10 -4 、10 -5 、10 -6 100 μL of the bacterial solutions of the three dilution gradients, spread them on the LB solid medium (each gradient is repeated 3 times), make marks and invert them in an incubator at 30 °C for 48 - 72 h until single colonies grow.

[0033] After single colonies grow, use an inoculation loop to select single colonies with good growth conditions and different morphological characteristics for streak plate isolation, and invert them in an incubator at 30 °C for 48 - 72 h. Repeat the above operation three times until the surface characteristics of the colonies are completely the same and the morphological characteristics of the bacteria are consistent under microscopic examination. Pick the purified single colonies, inoculate them into LB liquid medium for activation, then use an inoculation loop to dip a small amount of the bacterial solution and streak it on the BTB solid medium, and incubate it in an incubator at 30 °C for 72 h. Preliminary screening of strains with certain aerobic denitrification ability is carried out according to the color change of the BTB solid medium.

[0034] The formula of the BTB medium is: add 2% of technical agar and 1% of bromothymol blue ethanol solution on the basis of the enrichment medium.

[0035] The formula for the LB plate is: based on the LB broth medium, add 2% technical agar. The formula for the LB broth medium is (g / L): tryptone: 10, yeast extract: 5, sodium chloride: 10. Aliquot 50 ml into 150 ml Erlenmeyer flasks and sterilize by moist heat at 121 °C for 20 min, with the final pH being 7.0 ± 0.2.

[0036] (3) Preservation of strains

[0037] Under aseptic conditions, pick a single colony and inoculate it into LB liquid medium. Incubate it on a shaker at 30 °C and 150 r·min -1 for 12 h. Then pipette 75 μL of the bacterial solution and mix it with 125 μL of 80% glycerol, and store it in an ultra-low temperature freezer at -80 °C. Inoculate the strain onto the medium and culture it at 30 °C. The colonies are round, milky white, smooth on the surface, and opaque.

[0038] (4) Strain identification

[0039] After activating the screened bacteria, extract the genomic DNA of the bacteria according to the operation instructions of the bacterial genomic DNA extraction kit, and use each bacterial genome as a template for PCR amplification of 16S rDNA. The primer sequences are shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR amplification reaction conditions are set as shown in Table 3. After the amplified product is correctly detected by 1.0% agarose gel electrophoresis, use the agarose gel DNA recovery kit for recovery and send it to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results are submitted to the Genbank database. Blastn align the 16S rDNA sequences of the screened bacteria in the NCBI nucleic acid sequence database, and use the MEGA11 software to construct a phylogenetic tree using the NJ method (Neighbor-Joining Algorithm) (as Figure 2 shown). By alignment, several strains with high homology to it are all Aeromonas media. Therefore, this strain can be identified as Aeromonas media and named Aeromonas media L20.

[0040] The 16S rDNA sequence table described

[0041]

[0042] Table 1 16S rDNA primer sequences

[0043]

[0044] Table 2 16S rDNA PCR amplification reaction system

[0045]

[0046] Table 3 16S rDNA PCR amplification reaction conditions

[0047]

[0048] Example 2

[0049] The denitrification function of Aeromonas media L20 in Example 1 was tested to determine the nitrate nitrogen removal ability of the strain

[0050] Pick the purified single colony and inoculate it into an aerobic denitrification liquid medium with NaNO3 as the sole nitrogen source (nitrate nitrogen concentration is 100 mg / L). Incubate at 30 °C, 150 r·min -1 Cultivate until OD 600 Reaches 0.6. Use a pipette to aspirate 3 mL of the bacterial solution of Aeromonas media L20 in Example 1 and inoculate it into 100 mL of the medium with nitrate as the sole nitrogen source. Incubate at 30 °C, 150 r·min -1 Shake and cultivate. Take 1 mL of the culture every 2 h. After centrifuging at 8000 g for 10 min, take the supernatant to detect the concentration change of NO3 - -N, and determine the NO3 content at different time points according to the standard curve - -N content, the denitrification ability of the strain (as Figure 4 shown). The results show that the strain has a very strong ability to remove nitrate nitrogen and can achieve residue-free removal within 12 h

[0051] Example 3

[0052] The denitrification function of Aeromonas media L20 in Example 1 was tested to determine the nitrite nitrogen removal ability of the strain

[0053] Pick the purified single colony and inoculate it into an aerobic denitrification liquid medium with NaNO2 as the sole nitrogen source (nitrite nitrogen concentration is 50 mg / L). Incubate at 30 °C, 150 r·min -1Cultured until the OD600 reached 0.6, then 3 mL of the bacterial solution of Aeromonas media L20 from Example 1 was aspirated using a pipette and inoculated into 100 mL of a medium with nitrite as the sole nitrogen source, and cultured at 30 °C with 150 r·min -1 shaking culture. 1 mL of the culture was taken every 2 h. After high-speed centrifugation at 8000 g for 10 min, the supernatant was taken to detect the concentration change of NO2 - -N. According to the standard curve, the NO2 - -N content was determined, and the denitrification ability of the strain (as Figure 5 shown). The results showed that nitrite had some effects on the growth of the strain, but a removal rate of more than 90% could still be achieved within 12 h.

[0054] Example 4

[0055] While testing the denitrification function of Aeromonas media L20 in Example 1, the determination of the function of the strain to dissimilatory reduce to ammonium was determined.

[0056] A single purified colony was picked and inoculated into an aerobic denitrifying liquid medium with NaNO3 as the sole nitrogen source (the nitrate nitrogen concentration was 100 mg / L), and cultured at 30 °C with 150 r·min -1 cultured until the OD600 reached 0.6, then 3 mL of the bacterial solution of Aeromonas media L20 from Example 1 was aspirated using a pipette and inoculated into 100 mL of a medium with nitrate as the sole nitrogen source, and cultured at 30 °C with 150 r·min -1 shaking culture. 1 mL of the culture was taken every 2 h. After high-speed centrifugation at 8000 g for 10 min, while detecting the NO3 - -N content, the NH4 + -N content was detected. The detection of ammonia was based on the national standard detection method: 37.5 μL of the chromogenic reagent (salicylic acid-sodium potassium tartrate solution), 10 μL of sodium nitroferricyanide, 10 μL of sodium hypochlorite, and 20 μL of deionized water were added and mixed evenly. After color development for 60 min, the absorbance value at a wavelength of 697 nm was measured with a spectrophotometer. According to the standard curve, the ammonia content was determined, and the values were as Figure 6 shown. Since the culture system had only nitrite as the sole nitrogen source, the ability of the strain to dissimilatory reduce to ammonium during the removal of nitrate nitrogen or nitrite nitrogen could be proved by the production of NH4 + -N.

[0057] Example 5 The process of removing artificially prepared nitrate nitrogen and nitrite nitrogen sewage by the Aeromonas media L20 strain in Example 1

[0058] The strain screened in Example 1 was inoculated into a 500 mL conical flask containing 200 mL of LB medium and cultured at 30 °C and 150 rpm for 12 h. After centrifugation at 8000 rpm for 10 min, the cells were obtained, washed twice with sterile water, and then made into a cell suspension with an OD 600 of 0.900 - 1.000; then, they were respectively transferred to the artificially prepared sewage containing 200 mg / L of NaNO3 at an inoculation amount of 2% (V / V) (200 mL of the culture medium was filled in a 500 mL conical flask, formula: 0.2 g of NaNO3, 5 g of glucose, 1000 mL of H2O, pH 7), cultured at 30 °C and 150 rpm, and then centrifuged at 8000 rpm for 10 min, and then the NO2 - -N concentration and NO3 - -N concentration in the supernatant, as well as the amount of NH4+-N produced, were measured. As Figure 7 shown, the results showed that with the removal of nitrate nitrogen, NH4 + -N gradually accumulated, and at the same time, nitrite nitrogen did not accumulate significantly, indicating that the transformation occurred through the pathways of cyclic denitrification and dissimilatory reduction.

Claims

1. A denitrifying strain with the function of dissimilatory reduction to ammonium, characterized in that The denitrifying strain is Aeromonas media L20, which was deposited in the China Center for Type Culture Collection on July 12, 2023, with the deposit number CCTCC NO: M 20231191.

2. The application of a denitrifying strain with the function of dissimilatory reduction to ammonium according to claim 1, characterized in that The denitrifying strain described above is used for sewage treatment.

3. The application according to claim 2, characterized in that The sewage treatment described above refers to the removal of nitrate nitrogen and / or nitrite nitrogen in water.

4. The application according to claim 2 or 3, characterized in that The sewage treatment described above is to remove nitrate nitrogen with a concentration of 50 - 200 mg / L in water and / or nitrite nitrogen with a concentration of 50 - 200 mg / L.

5. The application according to claim 4, characterized in that The sewage treatment described above means that the complete removal of nitrate nitrogen with a concentration of 50 - 200 mg / L in sewage and / or nitrite nitrogen with a concentration of 50 - 200 mg / L is completed within 12 h.

6. The application according to claim 2 or 3, characterized in that The sewage treatment described above refers to the removal of nitrate nitrogen and / or nitrite nitrogen in water, and at the same time, partially converting nitrate nitrogen and / or nitrite nitrogen into ammonium.

7. The application of a denitrifying strain with the function of dissimilatory reduction to ammonium according to claim 1, characterized in that The denitrifying strain described above is used for preparing an aerobic denitrification and dissimilatory reduction to ammonium bacterial agent.

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