Heterotrophic nitrification-aerobic denitrification bacterium and application thereof in sewage denitrification treatment

Abstract

The invention discloses a heterotrophic nitrification-aerobic denitrification bacterium and application thereof in sewage denitrification treatment, and belongs to the technical field of microorganisms. The heterotrophic nitrification-aerobic denitrification bacterium is Enterobacter asbriae WZCH, is preserved in the China Center for Type Culture Collection on June 16, 2025, and has a preservation number of CCTCC (China Center for Type Culture Collection) NO: M 2025272. The invention further discloses a preparation method of the heterotrophic nitrification-aerobic denitrification bacterium. The strain has relatively high reducing capacity, can convert ammoniacal nitrogen, nitrate nitrogen and the like in sewage into gaseous nitrogen, is free of nitro nitrogen accumulation, is green and environment-friendly, and has relatively 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 purpose of this invention is to provide a heterotrophic nitrifying-aerobic denitrifying bacterium and its application in wastewater denitrification treatment, using Enterobacter aeruginosa as the fermentation strain, to solve the technical problem of nitrite accumulation during denitrification by Enterobacter aeruginosa in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A heterotrophic nitrifying-aerobic denitrifying bacterium, namely Enterobacterasburiae WZCH, was deposited at the China Center for Type Culture Collection on February 21, 2025, with accession number CCTCCNO: M 2025272.

[0008] Preferably, the 16S rDNA sequence of Enterobacter asburiae WZCH is shown in SEQ ID NO:1.

[0009] The present invention further provides an application of the heterotrophic nitrifying-aerobic denitrifying bacteria in wastewater denitrification, wherein the bacteria is Enterobacter asburiae WZCH.

[0010] Preferably, the Enterobacter asburiae WZCH converts ammonia nitrogen and nitrate nitrogen in wastewater into gaseous nitrogen.

[0011] The present invention also provides a wastewater denitrification method, wherein the heterotrophic nitrifying-aerobic denitrifying bacteria (i.e., Enterobacter aeruginosa WZCH) are cultured to obtain a seed culture; the seed culture is inoculated into wastewater and fermented to convert ammonia nitrogen and nitrate nitrogen in the wastewater into gaseous nitrogen.

[0012] Preferably, the content of heterotrophic nitrifying-aerobic denitrifying bacteria in the seed solution is 0.1-10 wt%.

[0013] Preferably, the fermentation culture medium formula is as follows: 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, ammonium chloride 0.45 ~ 0.55 g / L.

[0014] Preferably, the fermentation time is 60 to 84 hours.

[0015] Preferably, the pH of the fermentation is 7.2 to 7.8.

[0016] Preferably, the fermentation temperature is 18 to 22°C.

[0017] Biological Preservation Information

[0018] 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.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 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

[0020] Figure 1 Scanning electron microscope image (left) and microscope image (right) of strain WZCH.

[0021] Figure 2 A phylogenetic tree of strain WZCH constructed based on the 16S rRNA gene sequence.

[0022] Figure 3 The effects of different carbon sources on the growth of the strain; 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.

[0023] Figure 4 The effects of different nitrogen sources on the growth of bacterial strains; 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.

[0024] Figure 5 The effect of different carbon-nitrogen ratios on the growth of strains; 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.

[0025] Figure 6 The effect of different pH values ​​on the growth of bacterial strains; 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.

[0026] Figure 7 The effect of different temperatures on the growth of bacterial strains; 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 8 The effect of different rotation speeds on the growth of the strain.

[0028] 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. Detailed Implementation

[0029] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways than those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0030] 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 invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. It should be noted that the reagents and other materials used in these embodiments are all commercially available products.

[0031] Example 1: Isolation and Screening of Strains (1) Sampling locations were randomly selected from the drainage outlets of different treatment units (such as aeration tanks, sedimentation tanks, sludge return systems, etc.) of multiple sewage treatment plants in Xiangtan City. A 4cm×4cm×4cm bottom mud was dug from the ground with an iron shovel, placed in a 1000mL blue cap bottle, and sterile water was added to cover the soil sample. The sample was then stored in a 4℃ refrigerator.

[0032] (2) Inoculate 10 mL of sludge sample into a 250 mL Erlenmeyer flask containing 90 mL of sterile HNM1 medium, and incubate in a shaker at 30 °C and 120 rpm for 3 days for enrichment. Repeat the process 3 times to ensure that the dominant bacterial species are obtained.

[0033] (3) Dilute the enriched bacterial solution with sterile water until a gradient of 10 is obtained.-2 10 -4 10 -6 10 -8 The cell dilution buffer was prepared by evenly spreading 100 μL each of the stock solution and the cell dilution buffer onto HNM1 solid medium plates and incubating at 30°C until visible colonies were observed. Single colonies of different morphologies were picked up with an inoculation loop and streaked onto solid medium for further purification until a pure strain was obtained.

[0034] (4) Pick the obtained pure single colonies onto BTB solid medium with nitrite nitrogen as the sole nitrogen source or potassium nitrate as the sole nitrogen source, treat them with the five-zone streak method, and incubate them in a constant temperature incubator at 30°C. Observe the color change of the medium periodically. If the medium changes from green to blue, the screening is successful.

[0035] (5) Gram staining of the strain, such as... Figure 1 As shown on the right, the cell morphology was observed under a microscope, and its morphological characteristics were recorded. Scanning electron microscopy (SEM) samples were prepared, and the individual morphology of the strain was further observed using an SEM, as shown below. Figure 1 As shown on the left.

[0036] (6) Genomic DNA of the selected strains was extracted using a universal DNA extraction kit (BBI Life Sciences Co., Ltd.). Using the extracted genomic DNA as a template, the 16S rDNA gene sequence was amplified by PCR using universal primers 27F (5'-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The 16S rDNA gene sequence of the model strain with high homology was selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequence is shown in SEQ ID No. 1.

[0037] SEQ ID No. 1: (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.

[0038] Example 2: Fermentation treatment of simulated laboratory wastewater by strain WZCH (condition optimization) (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.

[0039] (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.

[0040] Example 3: Fermentation treatment of aquaculture wastewater using strain WZCH The isolated heterotrophic nitrifying-aerobic denitrifying bacteria WZCH were added to aquaculture wastewater (pH=7.1) and cultured at 20℃ for 72 h to study its nitrogen removal effect. The results are shown in Table 1. The total nitrogen concentration in the untreated water sample was above 0.04 mg / mL; after treatment with WZCH, the total nitrogen concentration was reduced to below 0.02 mg / mL, showing a significant denitrification effect and no obvious nitrite accumulation, thus avoiding secondary pollution.

[0041] Table 1. Components of wastewater

[0042] The above description is only a preferred embodiment 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 should be included within the protection scope of the present invention.

Claims

1. A heterotrophic nitrifying-aerobic denitrifying bacterium, wherein the bacterium is Enterobacterasburiae WZCH, which was deposited at the China Center for Type Culture Collection on February 21, 2025, with accession number CCTCCNO: M 2025272.

2. The heterotrophic nitrifying-aerobic denitrifying bacteria according to claim 1, characterized in that, Its 16S rDNA sequence is shown in SEQ ID NO:

1.

3. The application of the heterotrophic nitrifying-aerobic denitrifying bacteria according to claim 1 or 2 in wastewater denitrification, wherein the bacteria is Enterobacter asburiae WZCH.

4. The application according to claim 3, characterized in that, The Enterobacter bacillus WZCH converts ammonia nitrogen and nitrate nitrogen in wastewater into gaseous nitrogen.

5. A wastewater denitrification method, characterized in that, The heterotrophic nitrifying-aerobic denitrifying bacteria described in claim 1 or 2 are cultured to obtain a seed culture; the seed culture is inoculated into wastewater and fermented to convert ammonia nitrogen and nitrate nitrogen in the wastewater into gaseous nitrogen.

6. The wastewater denitrification method according to claim 5, characterized in that, The content of heterotrophic nitrifying-aerobic denitrifying bacteria in the seed solution is 0.1-10 wt%.

7. The wastewater denitrification method according to claim 5, characterized in that, The culture medium formula for fermentation is as follows: 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, ammonium chloride 0.45 ~ 0.55 g / L.

8. The wastewater denitrification method according to claim 5, characterized in that, The fermentation time is 60 to 84 hours.

9. The wastewater denitrification method according to claim 5, characterized in that, The initial pH of the fermentation is 7.2 to 7.8.

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