Heterotrophic nitrification-aerobic denitrification bacteria and application thereof in wastewater denitrification treatment

Abstract

The application discloses a heterotrophic nitrification-aerobic denitrification bacterium and application thereof in sewage denitrification treatment, and belongs to the technical field of microorganisms. Enterobacter asburiae The heterotrophic nitrification-aerobic denitrification bacterium is Enterobacter asburiae WZCH (WZCH), which was preserved in the China Center for Type Culture Collection on June 16, 2025, and the preservation number is CCTCC NO: M 2025272. The strain has high reduction capacity, can convert ammonia nitrogen, nitrate nitrogen and the like in sewage into gaseous nitrogen, has no nitro nitrogen accumulation, is green and environment-friendly, and has high practical application value.

Description

Technical Field

[0001] This invention belongs to the field of wastewater microbial denitrification technology, and more specifically, relates to a heterotrophic nitrifying-aerobic denitrifying bacterium and its application in wastewater denitrification treatment. Background Technology

[0002] In recent years, with the increasing severity of pollution, the nitrogen cycle in most water bodies has been disrupted. To address nitrogen pollution, the methods currently used are often physical and chemical nitrogen removal. However, the toxic intermediate products generated during the nitrogen removal process, low nitrogen removal efficiency, high cost, and harsh nitrogen removal environments have become major challenges.

[0003] Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria can oxidize and decompose organic matter under aerobic conditions, simultaneously reducing nitrates to nitrogen gas or nitrous oxide, thus achieving nitrogen removal. Under aerobic conditions, they can also reduce nitrates to nitrogen gas, and the different valence states of nitrogen produced in the process can meet the needs of different aquatic plants and animals, facilitating the nitrogen cycle in the water. This method is environmentally friendly, does not rely on chemical agents, reduces the risk of secondary pollution, can recover nitrogen gas and other valuable byproducts, achieving resource recycling, and is economical and efficient, utilizing organic matter in wastewater as a carbon source, reducing treatment costs. Therefore, utilizing HN-AD bacteria to degrade nitrogen is a crucial step in the treatment of water pollution.

[0004] Currently, HN-AD bacteria are mostly Klebsiella, Pseudomonas, Zobelil, Aeromonas, Acinetobacter, Marinebacter, Rhodococcus, Paracoccus, and Achromobacter. Research on Enterobacteriaceae as HN-AD bacteria is relatively limited; patent application CN117050895A reports a species of Enterobacter aestivum (…). Enterobacter asburiae DNW01, this strain is used in short-cut denitrification technology, but it will produce a large accumulation of nitrite. Excessive nitrite will lead to eutrophication, which will cause algae to proliferate and form harmful algal blooms. In addition, nitrite is also toxic to aquatic organisms, especially sensitive species such as fish and shrimp, causing environmental damage.

[0005] This invention screens out a highly efficient heterotrophic nitrifying-aerobic denitrifying bacterium for nitrogen removal. It can not only effectively degrade nitrogen, but also promote the growth and recycling of various aquatic plants and animals through its byproducts. This provides an important reference for its application in degrading nitrogen pollution in water bodies and for the research and development of highly efficient biodegradable agents and nitrogen-containing products for nitrogen in water bodies. Summary of the Invention

[0006] The application aims to provide a heterotrophic nitrification-aerobic denitrification bacteria and its application in wastewater denitrification treatment, and solve the problem of nitrite accumulation during the denitrification of Enterobacter asburiae in the prior art.

[0007] To achieve the above-mentioned purposes, the application adopts the following technical solutions.

[0008] A heterotrophic nitrification-aerobic denitrification bacteria, which is Enterobacter asburiae WZCH, was preserved in the China Center for Type Culture Collection on February 21, 2025, and the preservation number is CCTCC NO: M 2025272.

[0009] Preferably, the 16S rDNA sequence of the Enterobacter asburiae WZCH is shown in SEQ ID NO: 1.

[0010] The application further provides an application of the heterotrophic nitrification-aerobic denitrification bacteria in wastewater denitrification, and the bacteria is Enterobacter asburiae WZCH.

[0011] Preferably, the Enterobacter asburiae WZCH converts the ammonia nitrogen and nitrate nitrogen in the wastewater into gaseous nitrogen.

[0012] The application also provides a wastewater denitrification method, wherein the heterotrophic nitrification-aerobic denitrification bacteria (i.e., Enterobacter asburiae WZCH) is cultured to obtain a seed liquid; the seed liquid is inoculated into wastewater for fermentation, and the ammonia nitrogen and nitrate nitrogen in the wastewater are converted into gaseous nitrogen.

[0013] Preferably, the content of the heterotrophic nitrification-aerobic denitrification bacteria in the seed liquid is 0.1-10 wt%.

[0014] Preferably, the fermentation medium formula is as follows:

[0015] Manganese chloride tetrahydrate 0.008 ~ 0.015 g / L, potassium dihydrogen phosphate anhydrous 0.22 ~ 0.28 g / L, dipotassium hydrogen phosphate anhydrous 0.70 ~ 0.80 g / L, magnesium sulfate heptahydrate 0.025 ~ 0.040 g / L, trisodium citrate dihydrate 5.5 ~ 7.0 g / L, ammonium chloride 0.45 ~ 0.55 g / L.

[0016] Preferably, the fermentation time is 60 ~ 84 h.

[0017] Preferably, the pH of the fermentation is 7.2 to 7.8.

[0018] Preferably, the fermentation temperature is 18 to 22°C.

[0019] Biological Preservation Information

[0020] Enterobacter asburiae WZCH was deposited at the China Center for Type Culture Collection (CCTCC) on February 21, 2025, with accession number CCTCC NO: M 2025272, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention isolated a strain of Enterobacter aeruginosa with highly efficient heterotrophic nitrification-aerobic denitrification capabilities—Enterobacter argentiformis WZCH—from samples collected from multiple wastewater treatment plants. Enterobacter asburiae After 72 hours of treatment with the WZCH strain, the final removal rate of TN in the water reached 91.87%, and NO3... — The final removal rate of nitrogen (N) reached 95.73%, and NH4... + -N was ultimately removed at a rate of 90.62%, and NO2 was also removed. - The nitrogen (N) accumulation decreased from an initial 0.0003 mg / L to a final 0.00273 mg / L, with no significant accumulation observed. The strain of this invention exhibits high reducing activity, effectively degrading nitrogen and converting ammonia and nitrate nitrogen into gaseous nitrogen, thereby altering water quality, maintaining the nitrogen cycle in the water, and possessing high practical application value. Attached Figure Description

[0023] Figure 1 Scanning electron microscope image (left) and microscope image (right) of strain WZCH.

[0024] Figure 2 A phylogenetic tree of strain WZCH constructed based on the 16S rRNA gene sequence.

[0025] Figure 3 The effects of different carbon sources on the growth of the strain;

[0026] Among them, A. changes in total nitrogen concentration; B. changes in nitrate nitrogen concentration; C. changes in nitrite nitrogen concentration; D. changes in ammonia nitrogen concentration.

[0027] Figure 4 The effects of different nitrogen sources on the growth of bacterial strains;

[0028] Wherein, A, total nitrogen concentration change; B, nitrate nitrogen concentration change; C, nitrite nitrogen concentration change; D, ammonia nitrogen concentration change.

[0029] Figure 5 Effect of different carbon nitrogen ratio on strain growth;

[0030] Wherein, A, total nitrogen concentration change; B, nitrate nitrogen concentration change; C, nitrite nitrogen concentration change; D, ammonia nitrogen concentration change.

[0031] Figure 6 Effect of different pH value on strain growth;

[0032] Wherein, A, total nitrogen concentration change; B, nitrate nitrogen concentration change; C, nitrite nitrogen concentration change; D, ammonia nitrogen concentration change.

[0033] Figure 7 Effect of different temperature on strain growth;

[0034] Wherein, A, total nitrogen concentration change; B, nitrate nitrogen concentration change; C, nitrite nitrogen concentration change; D, ammonia nitrogen concentration change.

[0035] Figure 8 Effect of different rotating speed on strain growth.

[0036] Wherein, A, total nitrogen concentration change; B, nitrate nitrogen concentration change; C, nitrite nitrogen concentration change; D, ammonia nitrogen concentration change. DETAILED DESCRIPTION

[0037] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without the specific details set forth in this description. In other instances, well-known methods have not been described in detail in order to avoid obscuring the present application.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. It is to be understood that the use of the singular herein includes the plural unless specifically stated otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. It is to be understood that the use of the singular herein includes the plural unless specifically stated otherwise. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0039] Example 1 Isolation and screening of strains

[0040] (1) Randomly selected sampling sites from different treatment units (such as aeration tank, sedimentation tank, sludge return system, etc.) of sewage treatment plants in Xiangtan City, and used a shovel to dig 4 cm x 4 cm x 4 cm sediment from the surface into a 1000 mL blue cap bottle, added sterile water to cover the soil sample, and stored the sample in a 4°C refrigerator.

[0041] (2) 10 mL sludge sample was inoculated into a 250 mL conical flask containing 90 mL sterilized HNM1 medium, and cultured at 30°C, 120 rpm for 3 days for enrichment in a shaking bed, and the process was repeated for 3 times to ensure that the dominant bacteria were obtained.

[0042] (3) The enriched bacterial solution was diluted with sterile water to obtain cell dilutions with gradients of 10 -2 , 10 -4 , 10 -6 , and 10 -8 , and 100 uL of the original solution and the cell dilutions were uniformly spread on HNM1 solid medium plates, which were placed in a constant temperature incubator and cultured at 30°C until visible colonies were observed. Different morphological single colonies were picked up with a loop and further purified by streaking on solid medium until pure strains were obtained.

[0043] (4) The obtained pure single colonies were picked up on BTB solid medium with nitrous acid nitrogen as the only nitrogen source or potassium nitrate as the only nitrogen source, treated by five-zone streaking method, and placed in a constant temperature incubator and cultured at 30°C. The color change of the medium was observed regularly. If the medium changed from green to blue, the screening was successful.

[0044] (5) The strain was subjected to gram staining, and its cell morphology was observed under a microscope, as shown in Figure 1 (right), and recorded its cell morphology characteristics. Scanning electron microscope samples were prepared, and the individual morphology of the strain was further observed by scanning electron microscope, as shown in Figure 1 (left).

[0045] (6) The genomic DNA of the screened strain was extracted using a universal DNA extraction kit (BBI Life Science Co., Ltd.), and the extracted genomic DNA was used as a template to amplify the 16S rDNA gene sequence by PCR using universal primers 27F (5'-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The 16S rDNA gene sequence of the selected high-homology model strain was sequenced by Shengong Bioengineering (Shanghai) Co., Ltd., and the sequence is shown in SEQ ID No. 1.

[0046] SEQ ID No. 1:

[0047]

[0048] (7) Finally, the phylogenetic tree is constructed using the adjacency join method in MEGA11 software, such as... Figure 2 As shown, the bacterial species has been classified as Enterobacter ( ). Enterobacter asburiae ), named WZCH.

[0049] Example 2: Fermentation treatment of simulated laboratory wastewater by strain WZCH (condition optimization)

[0050] (1) The effects of carbon source, nitrogen source, carbon-to-nitrogen ratio, pH, temperature, and initial shaking speed on the denitrification efficiency of the strain were investigated through single-factor variable experiments. The fixed laboratory simulated wastewater baseline conditions were: sodium acetate 5.125 g / L carbon source, ammonium sulfate 0.2357 g / L nitrogen source, carbon-to-nitrogen ratio 20, pH 7.0, temperature 30℃, and shaking speed 180 r / min. The influencing factors were as follows: carbon sources included glucose, sodium citrate, sodium acetate, and glycerol; nitrogen sources included urea, sodium nitrite, sodium nitrate, ammonium chloride, and potassium nitrate; carbon-to-nitrogen ratio (C / N) was 5, 10, 15, 20, and 25; pH was 6.5, 7, 7.5, 8, and 8.5; temperature was 20, 25, 30, 35, and 40℃; and shaking speed was 100, 125, 150, 175, and 200 r / min.

[0051] (2) Prepare culture media with different carbon sources (glucose, sodium citrate, sodium acetate, glycerol), inoculate with the strain seed liquid and shake for 72 h, take samples periodically (12 h), and measure the changes in water quality indicators such as ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and chemical oxygen demand in different culture media to screen out the optimal culture medium. Using the above optimal culture medium, set different nitrogen sources (urea, sodium nitrite, sodium nitrate, ammonium chloride, potassium nitrate), carbon-nitrogen ratios (5, 10, 15, 20, 25), pH (6.5, 7, 7.5, 8, 8.5), temperatures (20, 25, 30, 35, 40℃), and shaking speeds (100, 125, 150, 175, 200 r / min) and other culture conditions to further measure the denitrification effect of bacteria under different conditions, systematically analyze the key factors affecting the denitrification performance of strain WZCH, and determine the conditions for the highest denitrification efficiency of strain WZCH, such as Figures 3-8 The optimal fermentation medium for the strain to achieve the highest denitrification efficiency was determined to be: manganese chloride tetrahydrate 0.008–0.015 g / L, anhydrous potassium dihydrogen phosphate 0.22–0.28 g / L, anhydrous dipotassium hydrogen phosphate 0.70–0.80 g / L, magnesium sulfate heptahydrate 0.025–0.040 g / L, trisodium citrate dihydrate 5.5–7.0 g / L, and ammonium chloride 0.45–0.55 g / L; the fermentation time was 60–84 h; the initial pH was 7.2–7.8; and the fermentation speed was 180–200 r / min.

[0052] Example 3 Fermentation treatment of aquaculture wastewater by strain WZCH

[0053] The isolated heterotrophic nitrification-aerobic denitrification bacteria WZCH was added to the aquaculture wastewater (pH = 7.1), and the nitrogen removal effect was studied under the experimental conditions of 20℃ culture for 72 h. As shown in Table 1, the total nitrogen concentration of the water sample without strain WZCH treatment was 0.04 mg / mL or more; after treatment, the total nitrogen concentration was reduced to 0.02 mg / mL or less, and the denitrification effect was significant, and there was no obvious nitrite accumulation, which would not cause secondary pollution.

[0054] Table 1 Composition of sewage

[0055]

[0056] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A heterotrophic nitrification-aerobic denitrifying bacterium, which is Enterobius arthi (Enterobacter arthi) Enterobacter asburiae ) WZCH, deposited on February 21, 2025 in China Center for Type Culture Collection, with the accession number CCTCC NO: M2025272.

2. The application of the heterotrophic nitrifying-aerobic denitrifying bacteria according to claim 1 in wastewater denitrification, wherein the bacteria is Enterobacter aeruginosa (… Enterobacter asburiae WZCH; The specific application is to convert ammonia nitrogen and nitrate nitrogen in wastewater into gaseous nitrogen.

3. A wastewater denitrification method characterized by, The heterotrophic nitrification-aerobic denitrification bacteria in claim 1 are cultured to obtain a seed liquid; the seed liquid is inoculated into wastewater to perform fermentation, so that ammonia nitrogen and nitrate nitrogen in the wastewater are converted into gaseous nitrogen.

4. The method of claim 3, wherein the method is a method of denitrification of sewage. The content of the heterotrophic nitrification-aerobic denitrification bacteria in the seed liquid is 0.1-10wt%.

5. The method of claim 3, wherein the method is a method of denitrification of sewage. The fermentation time is 60-84 h.

6. The method of claim 3, wherein the method is a method of denitrification of sewage. The initial pH of the fermentation is 7.2-7.8.

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