Denitrifying bacteria and their applications

Abstract

This invention relates to the field of microbiology and discloses a denitrifying bacterium and its application. The denitrifying bacterium is *Pseudomonas peli*, with the preservation number CGMCC No. 30426. The denitrifying bacterium provided by this invention can effectively reduce nitrate nitrogen in water bodies, and this strain exhibits good ecological safety and is non-toxic to fish.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a denitrifying bacterium and its applications. Background Technology

[0002] my country is the world's largest aquaculture nation, with a freshwater aquaculture output of 32.9 million tons in 2022. The feed conversion ratio for aquatic animals is 1-2, meaning that over 32.9 million tons of feed are used annually in various freshwater aquaculture models. However, only 25% of the nutrients in this feed are converted into nutrients for the animals. The majority of nutrients remain in the water in organic or inorganic forms. Intensive aquaculture generates various forms of pollutants, including nitrogen and phosphorus, which are discharged into receiving water bodies, leading to the deterioration of the surrounding aquatic environment. Excessive nitrogen levels are a key indicator of pollution load.

[0003] With increasing emphasis on the ecological environment and the green and sustainable development of aquaculture, freshwater aquaculture wastewater treatment facilities have been further strengthened. Currently, most major aquaculture provinces and cities across the country have issued aquaculture wastewater discharge standards, requiring the treatment of aquaculture wastewater to meet relevant standards before discharge. There are currently two main methods for treating aquaculture wastewater: centralized treatment and in-situ treatment. Of these two methods, compared to chemical methods, microbial denitrification processes, primarily based on denitrification, have attracted widespread attention from researchers due to their low operating costs and lack of residue. Unlike domestic sewage biological treatment ponds, aquaculture ponds and wastewater treatment facilities contain farmed animals, resulting in relatively high dissolved oxygen levels, making anaerobic denitrification difficult to achieve in freshwater aquaculture. The lack of denitrifying bacteria used in aquaculture and wastewater treatment makes nitrogen removal from aquaculture wastewater a persistent challenge. Summary of the Invention

[0004] The purpose of this invention is to overcome the problem that the existing technology lacks denitrifying bacteria used in aquaculture and aquaculture wastewater treatment, which makes it difficult to remove nitrogen from aquaculture wastewater, and to provide a denitrifying bacteria and its application in freshwater aquaculture water.

[0005] The inventors of this invention isolated a strain from the intestine of the channel catfish, which was identified as *Pseudomonas peli*. This strain has good removal capabilities for both nitrate and nitrite nitrogen and can be used for nitrate removal in aquaculture. Therefore, to achieve the above objective, the first aspect of this invention provides a denitrifying bacterium, *Pseudomonas peli*, with accession number CGMCC No. 30426.

[0006] The second aspect of the present invention provides the application of the denitrifying bacteria described in the first aspect of the present invention in water treatment.

[0007] A third aspect of the present invention provides a method for treating water, characterized in that the method includes contacting the denitrifying bacteria as described above with the water to be treated.

[0008] Through the above technical solution, the present invention has at least the following beneficial effects:

[0009] (1) The denitrifying bacteria provided by the present invention were isolated from the intestine of spotted catfish. It is a strain of Pseudomonas rotundifolia obtained from the intestine of farmed fish. It has good environmental and ecological safety and is non-pathogenic to fish.

[0010] (2) The denitrifying bacteria provided by the present invention have a good function of removing nitrate nitrogen and nitrite nitrogen in freshwater aquaculture water, and can be effectively used for the removal of nitrate nitrogen in aquaculture, and the treatment cycle is relatively short.

[0011] Biological Preservation

[0012] The *Pseudomonas peli* strain of this invention, with accession number H46-1, was deposited on April 24, 2024, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) (abbreviation of depositary institution: CGMCC), with accession number CGMCC No. 30426. Attached Figure Description

[0013] Figure 1 The hemolytic activity of Staphylococcus aureus (left) and Pseudomonas sphaeroides (right) of the present invention;

[0014] Figure 2 This describes the growth of *Pseudomonas septica* in nitrate nitrogen medium according to the present invention.

[0015] Figure 3 This is a diagram illustrating the effect of Pseudomonas sphaeroides in removing nitrate nitrogen according to the present invention;

[0016] Figure 4 This describes the growth of *Pseudomonas septica* in a nitrite-rich medium according to the present invention.

[0017] Figure 5 This is an image showing the effect of Pseudomonas sphaeroides in removing nitrite from the present invention;

[0018] Figure 6 This is a diagram showing the effect of Pseudomonas sphaeroides of the present invention on the removal of nitrite nitrogen in a circulating water system (C / N=15);

[0019] Figure 7 This is a diagram showing the effect of Pseudomonas sphaeroides of the present invention on the removal of nitrate nitrogen in a circulating water system (C / N=15);

[0020] Figure 8 This is a diagram showing the effect of Pseudomonas sphaeroides of the present invention on the removal of nitrite nitrogen in a circulating water system (C / N=10);

[0021] Figure 9 This is a diagram showing the effect of Pseudomonas sphaeroides of the present invention on the removal of nitrate nitrogen in a circulating water system (C / N = 10). Detailed Implementation

[0022] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0023] During their research, the inventors of this invention isolated a denitrifying bacterium from the intestines of the channel catfish. 16S RNA sequencing identified it as *Pseudomonas peli*, with the preservation number CGMCC No. 30426. In this invention, it is designated as H46-1.

[0024] The 16S rRNA gene sequence of denitrifying bacteria provided by the present invention is shown in SEQ ID NO:1.

[0025] SEQ ID NO:1

[0026]

[0027] The morphological characteristics of the denitrifying bacteria of this invention are as follows: After incubation on denitrification agar plates at 28°C for 48 hours, the colonies are round with neat edges, approximately 1 mm in diameter, and slightly raised on the surface. Under a microscope, the cells are rod-shaped, motile, and show no spores; Gram staining is negative. The denitrification agar plate composition is: sodium citrate 5 g / L, K₂HPO₄ 1 g / L, KH₂PO₄ 1 g / L, MgSO₄·7H₂O 0.2 g / L, NaNO₃ 1 g / L, trace element solution 2 mL / L, bromophenol blue 0.05 g / L. The trace element solution contains: CaCl₂ 0.2 g / L, FeSO₄·7H₂O 1 g / L, CoCl₂·6H₂O 0.35 g / L, CuSO₄·5H₂O 0.4 g / L, MnCl₂·7H₂O 0.8 g / L, (NH₄)₆Mo₇O₇ 24 ·4H2O 0.65g / L, ZnSO4 0.4g / L.

[0028] The denitrifying bacteria of the present invention were tested using API 20NE, and their physiological and biochemical characteristics are shown in Table 1 below.

[0029] Table 1. Physiological and biochemical characteristics of *Pseudomonas sphaeroides*

[0030]

[0031] The second aspect of the present invention provides the application of the denitrifying bacteria described in the first aspect of the present invention in water treatment.

[0032] In this invention, the water body can be a common water body that requires nitrate nitrogen treatment, preferably a freshwater aquaculture water body, such as a freshwater fish farming water body.

[0033] In this invention, the method can process water with a C / N ratio of 5-20, preferably 10-15.

[0034] In this invention, the method can treat water bodies with nitrite nitrogen content of 0-20 mg / L and nitrate nitrogen content of 0-50 mg / L. Preferably, the water body has nitrite nitrogen content of 0.2-5 mg / L and nitrate nitrogen content of 5-30 mg / L.

[0035] A third aspect of the present invention provides a water treatment method, the method comprising contacting denitrifying bacteria as described above with water to be treated. The water may be as described above.

[0036] In this invention, the contact methods between denitrifying bacteria and water include direct addition and / or loading them onto a carrier before adding them to the water to be treated.

[0037] In this invention, to achieve better nitrate nitrogen treatment, the concentration of denitrifying bacteria in the water is 1×10⁻⁶. 2 -1×10 8 CFU / mL, preferably 1×10⁻⁶ 3 -1×10 6 CFU / mL.

[0038] In this invention, the contact conditions may include: a temperature of 25-30°C and a contact time of 1-30 days.

[0039] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0040] Unless otherwise specified, all reagents and materials used in the following examples were purchased from reputable chemical reagent suppliers and were of analytical purity.

[0041] The *Pseudomonas sphaeroides* culture medium formulation used in the following examples is as follows: K₂HPO₄ 1 g / L, KH₂PO₄ 1 g / L, MgSO₄·7H₂O 0.2 g / L, trace element solution 2 mL / L, NaNO₃ 0.085 g / L, and trisodium citrate dihydrate 0.856 g / L. The trace element solution includes CaCl₂ 0.2 g / L, FeSO₄·7H₂O 1 g / L, CoCl₂·6H₂O 0.35 g / L, CuSO₄·5H₂O 0.4 g / L, MnCl₂·7H₂O 0.8 g / L, and (NH₄)₆Mo₇O₇. 24 The solution was sterilized by filtration through a 0.22μm filter membrane with 0.65g / L 4H2O and 0.4g / L ZnSO4.

[0042] In the following examples, the nitrite nitrogen concentration was determined using the spectrophotometric method for the determination of nitrite nitrogen in water quality (GB 7493-87). The nitrate nitrogen concentration was determined using the ultraviolet spectrophotometric method for the determination of nitrate nitrogen in water quality (HJ / T 346-2007).

[0043] Example 1

[0044] This embodiment is used to illustrate the safety of the denitrifying bacteria provided by the present invention.

[0045] The densities of *Pseudomonas septicemia* were set at 0 and 1×10⁻⁶. 5 1×10 6 and 1×10 7Four groups of experiments were conducted at CFU / mL, with three parallel experiments in each group. Fifteen zebrafish were used in each group. After anesthesia, approximately 20% of the scales on the tail surface were scraped off, and the fish were immersed in water containing the bacteria for infection testing. The fish were observed for 14 days, with daily mortality rates and the presence of infection symptoms recorded. The *Pseudomonas aeruginosa* population density was 1×10⁻⁶. 5 1×10 6 and 1×10 7 In the experimental groups at three CFU / mL concentrations and the control group without bacteria, no zebrafish died within 14 days, and no obvious abnormalities were observed, indicating that *Pseudomonas sphaeroides* is not pathogenic to zebrafish.

[0046] Example 2

[0047] This embodiment is used to illustrate the safety of the denitrifying bacteria provided by the present invention.

[0048] Activate and culture *Pseudomonas septica* for 24 hours, then inoculate 2 μL of the bacterial solution onto blood agar plates and incubate at 28°C for 48 hours. Observe whether a hemolytic zone is formed. *Staphylococcus aureus* is used as a positive control.

[0049] like Figure 1 As shown, on blood agar plates, the positive control Staphylococcus aureus produced a clear hemolytic zone, while Pseudomonas septicemia did not.

[0050] Example 3

[0051] This embodiment is used to illustrate the denitrification performance of the denitrifying bacteria provided by the present invention for nitrate nitrogen.

[0052] Nitrate nitrogen removal test medium: sodium citrate dihydrate 5 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.2 g / L, trace element solution 2 mL / L, NaNO3 1 g / L.

[0053] Prepare a medium for testing the denitrification capacity of nitrates, with three replicates per group. Inoculate the logarithmic growth phase bacterial culture into the test medium, ensuring an initial OD600 of 0.1-0.2, and incubate at 28°C with shaking at 180 rpm. Sample and measure OD600 and nitrate nitrogen concentration every 12 hours until 72 hours.

[0054] like Figure 2 As shown, *Pseudomonas aeruginosa* grew well in the nitrate denitrification capacity test medium, reaching its maximum bacterial density after 24 hours, at which point the nitrate removal rate was greater than 95.3%. Figure 3 During the 36-72h period, the average level of nitrate nitrogen was below 10mg / L.

[0055] Example 4

[0056] This embodiment is used to illustrate the denitrification performance of the denitrifying bacteria provided by the present invention for nitrite nitrogen.

[0057] Nitrite removal test medium: sodium citrate dihydrate 5 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.2 g / L, trace element solution 2 mL / L, KNO2 0.5 g / L.

[0058] Prepare a test medium for the denitrification capacity of nitrite, with three replicates per group. Inoculate the logarithmic growth phase bacterial culture into the test medium, ensuring an initial OD600 of 0.1-0.2, and incubate at 28℃ with shaking at 180 rpm. Sample and measure OD600 and nitrite concentration every 12 hours until 72 hours.

[0059] like Figure 4 As shown, *Pseudomonas sphaeroides* grew well in the nitrite denitrification capacity test medium, reaching its maximum bacterial density after 24 hours, at which point the nitrite removal rate reached 95.28%. Figure 5 The concentration of nitrite nitrogen ranged from 3.7 to 7.7 mg / L within 36-72 hours.

[0060] Example 5

[0061] This embodiment illustrates the performance of the denitrifying bacteria provided by the present invention in removing nitrite and nitrate nitrogen in a circulating water system.

[0062] The experimental circulating water system consists of aquaculture facilities, a circulating pump, and a filtration device. 60L plastic basins are used as aquaculture facilities, each filled with 30L of tap water. A filtration device is installed above the plastic basins, consisting of a filter box and filter media, including volcanic rock, gravel, and filter cotton.

[0063] The entire circulating water system was disinfected with trichlorfon and aerated for two days. The water temperature was 26±2℃. Each system was stocked with 15 zebrafish, each weighing 0.25±0.1g. The fish were fed once daily, with a feed amount approximately 3% of their body weight. The circulating water was shut off for 30 minutes during feeding. Feeding continued for four days. Day 0 was observed after four days of feeding.

[0064] Filter cotton was soaked in 1.8L of fresh *Pseudomonas aeruginosa* culture medium, and 200mL of *Pseudomonas aeruginosa* was inoculated. Aeration was maintained, and the mixture was incubated at 28℃ for 12 hours, at which point the bacterial density was 2×10⁻⁶. 8 CFU / mL. Remove the bacterial-containing filter cotton from Day 0 and place it into the filtration device. Turn on the circulation pump; the bacterial density in the aquaculture water is approximately 1×10⁻⁶. 6 CFU / mL.

[0065] Six independent circulating water systems were used, with three parallel experiments set up for both the control group (without bacteria) and the treatment group (with bacteria). Sodium nitrite was added to both the control and treatment groups until nitrite nitrogen reached 4 mg / L, and sodium nitrate was added until nitrate nitrogen in the aquaculture facility water reached 41 mg / L, with a C / N ratio of 15. Filter cotton containing *Pseudomonas aeruginosa* and sodium citrate dihydrate at a final concentration of 2.756 g / L were added. Nitrite and nitrate nitrogen levels were measured on Days 0, 1, 3, 5, 7, 9, and 11.

[0066] like Figure 6 As shown, in the circulating water system, the nitrite nitrogen level in the control group ranged from 3.62 to 4.10 mg / L over 11 days. On Day 0, the nitrite nitrogen level in the control group was 3.96 ± 0.09 mg / L, while that in the treatment group was 4.04 ± 0.10 mg / L. After one day, the nitrite nitrogen level in the treatment group significantly increased to 20.07 ± 1.39 mg / L, significantly higher than that in the control group (p < 0.05). On Day 3, the nitrite nitrogen level in the treatment group decreased to 3.97 ± 0.28 mg / L. After Day 5, the nitrite nitrogen concentration in the treatment group was below the detection limit (0.003 mg / L), and the nitrite nitrogen level was below the detection limit on Days 5, 7, 9, and 11.

[0067] like Figure 7 As shown, in the circulating water system, the nitrate nitrogen level in the control group ranged from 35.11 to 42.84 mg / L over 11 days. On Day 0, the nitrate nitrogen level in the control group was 40.11 ± 1.72 mg / L, while that in the treatment group was 40.00 ± 2.36 mg / L. After one day, the nitrate nitrogen level in the treatment group was significantly lower than that in the control group (p < 0.05), reaching 12.61 ± 2.49 mg / L. After three days, the nitrate nitrogen concentration in the treatment group decreased to 3.66 ± 0.92 mg / L, a significant difference from the control group (p < 0.05). On days 5, 7, 9, and 11, the nitrate nitrogen concentration in the treatment group decreased to below 3 mg / L.

[0068] Example 6

[0069] This embodiment illustrates the performance of the denitrifying bacteria provided by the present invention in removing nitrite and nitrate nitrogen in a circulating water system.

[0070] The experimental circulating water system consists of aquaculture facilities, a circulating pump, and a filtration device. 60L plastic basins are used as aquaculture facilities, each filled with 30L of tap water. A filtration device is installed above the plastic basins, consisting of a filter box and filter media, including volcanic rock, gravel, and filter cotton.

[0071] The entire circulating water system was disinfected with trichlorfon and aerated for two days. The water temperature was 26±2℃. 15 zebrafish (0.25±0.1g / fish) were placed in each container. They were fed once daily, with a food intake of approximately 3% of their body weight. The circulating water was turned off for 30 minutes during feeding. Feeding continued for 4 days. Day 0 was observed after 4 days of feeding.

[0072] Filter cotton was soaked in 1.8L of fresh *Pseudomonas aeruginosa* culture medium, and 200mL of *Pseudomonas aeruginosa* was inoculated. Aeration was maintained, and the mixture was incubated at 28℃ for 12 hours, at which point the bacterial density was 2×10⁻⁶. 8 CFU / mL. Remove the bacterial-containing filter cotton from Day 0 and place it into the filtration device. Turn on the circulation pump; the bacterial density in the aquaculture water is approximately 1×10⁻⁶. 6 CFU / mL.

[0073] Six independent circulating water systems were used, with three parallel experiments set up for both the control group (without bacteria) and the treatment group (with bacteria). Sodium nitrite was added to both the control and treatment groups until nitrite nitrogen reached 4 mg / L, and sodium nitrate was added until nitrate nitrogen in the aquaculture facility water reached 41 mg / L, with a C / N ratio of 10. Filter cotton containing *Pseudomonas aeruginosa* and sodium citrate dihydrate at a final concentration of 1.837 g / L were added. Nitrite and nitrate nitrogen levels were measured on Days 0, 1, 3, 5, 7, 9, and 11.

[0074] like Figure 8 As shown, in the circulating water system, the nitrite nitrogen level in the control group ranged from 3.62 to 4.10 mg / L over 11 days. On Day 0, the nitrite nitrogen level in the control group was 3.96 ± 0.09 mg / L, while that in the treatment group was 3.95 ± 0.12 mg / L. After one day, the nitrite nitrogen level in the treatment group significantly increased to 19.34 ± 1.27 mg / L, significantly higher than that in the control group (p < 0.05). On Day 3, the nitrite nitrogen level in the treatment group decreased to 9.57 ± 1.25 mg / L. On Day 5, the nitrite nitrogen concentration in the treatment group was 4.87 ± 1.41 mg / L, similar to that in the control group. On Day 7, it was 1.05 ± 0.17 mg / L; on Day 9, it was 0.76 ± 0.23 mg / L; and on Day 11, it was 0.76 ± 0.14 mg / L.

[0075] like Figure 9 As shown, in the circulating water system, the nitrate nitrogen level in the control group ranged from 35.11 to 42.84 mg / L over 11 days. On Day 0, the nitrate nitrogen level in the control group was 40.11 ± 1.72 mg / L, while that in the treatment group was 40.91 ± 3.43 mg / L. After one day, the nitrate nitrogen level in the treatment group was significantly lower than that in the control group (p < 0.05), reaching 21.42 ± 7.98 mg / L. After three days, the nitrate nitrogen concentration in the treatment group decreased to 3.43 ± 1.47 mg / L, a significant difference from the control group (p < 0.05). On days 5, 7, 9, and 11, the nitrate nitrogen concentration in the treatment group all decreased to below 3 mg / L.

[0076] Example 7

[0077] This embodiment illustrates the performance of the denitrifying bacteria provided by the present invention in removing nitrite and nitrate nitrogen in ponds.

[0078] The bacterial strain was tested for its effects on nitrite and nitrate nitrogen in ponds at a fish farm in Beijing. The experiment was conducted in early September. Details are as follows:

[0079] Pond #2 had high nitrite nitrogen levels (0.56 mg / L) and nitrate nitrogen levels (12.63 mg / L). Pond #5 had nitrite nitrogen levels (0.38 mg / L) and nitrate nitrogen levels (10.82 mg / L). Pond #2 was used as the treatment group, and the entire pond was treated with *Pseudomonas aeruginosa* culture solution. Pond #5 served as the control group and received no treatment. Pond #2 had an area of ​​4.6 mu (approximately 0.24 hectares), and Pond #5 had a surface area of ​​7 mu (approximately 0.47 hectares). Both ponds #2 and #5 primarily cultured channel catfish, with silver carp, bighead carp, and common carp as intercropping, and the stocking density, size, and timing were similar.

[0080] After culturing with *Pseudomonas sphaeroides* culture medium for 24 hours under laboratory conditions, the bacterial concentration was 2 × 10⁻⁶. 8 CFU / mL, 10L of bacterial culture was added to *Pseudomonas aeruginosa* culture medium and 5kg of brown sugar in a 100L clean tank at the breeding farm for further expansion. After 12 hours of incubation, the bacterial density on the plate was 1×10⁻⁶. 8 CFU / mL, 100L of the expansion broth was poured into the treated pond, and the final concentration of *Pseudomonas aeruginosa* in the pond water was approximately 1×10⁻⁶. 3 CFU / mL.

[0081] The control group ponds and the treatment group ponds were managed with the same feeding and aeration measures. The pond water temperature, dissolved oxygen, nitrite nitrogen and nitrate levels were monitored at 0d, 1d, 2d, 4d, 8d, 10d and 12d.

[0082] *Pseudomonas sphaeroides* significantly reduced nitrite levels in pond #2. After 8 days, nitrite levels decreased to 0.24 mg / L, a 57.14% reduction compared to day 0. At 10 and 12 days, the levels were 0.3 and 0.31 mg / L, respectively. In contrast, the control pond showed a slight increase (see Table 2 for details).

[0083] Table 2. Changes in nitrite concentration in aquaculture ponds over 12 days after treatment with *Pseudomonas sphaeroides*.

[0084]

[0085] *Pseudomonas sphaeroides* significantly reduced nitrate levels in two ponds. After 8 days, in treatment group 2, nitrate nitrogen levels decreased to 6.27 mg / L, a 50.35% reduction compared to day 0. Nitrate nitrogen levels at days 10 and 12 were 6.57 and 6.42 mg / L, respectively. In contrast, the nitrate nitrogen level in control pond #5 remained similar to that on day 0, showing no change.

[0086] Table 3. Changes in nitrate concentration in aquaculture ponds over 12 days after treatment with *Pseudomonas sphaeroides*.

[0087]

[0088] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of denitrifying bacteria, characterized in that, The denitrifying bacteria is *Pseudomonas aeruginosa* (…). Pseudomonas peli (The accession number is CGMCC No.30426).

2. The application of the denitrifying bacteria as described in claim 1 in water treatment.

3. The application according to claim 2, wherein, The water body in question is a freshwater aquaculture water body.

4. The application according to claim 2, wherein, The C / N ratio of the water body is 5-20.

5. The application according to claim 4, wherein, The C / N ratio of the water body is 10-15.

6. The application according to claim 2, wherein, The nitrite nitrogen content in the water body is 0-20 mg / L, and the nitrate nitrogen content is 0-50 mg / L.

7. The application according to claim 6, wherein, The nitrite nitrogen content in the water body is 0.2-5 mg / L, and the nitrate nitrogen content is 5-30 mg / L.

8. A method for treating water bodies, characterized in that, The treatment method includes: contacting the denitrifying bacteria of claim 1 with the water to be treated.

9. The method according to claim 8, wherein, The contact method involves directly adding denitrifying bacteria or loading them onto a carrier before adding them to the water body to be treated.

10. The method according to claim 8, wherein, The dosage of the denitrifying bacteria is 1×10⁻⁶. 2 -1×10 8 CFU / mL water.

11. The method according to claim 10, wherein, The dosage of the denitrifying bacteria is 1×10⁻⁶. 3 -1×10 6 CFU / mL water.

12. The method according to claim 8, wherein, The water body is a freshwater aquaculture water body; And / or, the C / N ratio of the water body is 5-20; And / or, the nitrite nitrogen content in the water body is 0-20 mg / L and the nitrate nitrogen content is 0-50 mg / L.

13. The method according to claim 12, wherein, The C / N ratio of the water body is 10-15; And / or, the nitrite nitrogen content in the water body is 0.2-5 mg / L and the nitrate nitrogen content is 5-30 mg / L.

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