An aerobic denitrifying bacterium and application thereof in denitrification in aquaculture water

Abstract

This invention discloses a heterotrophic-aerobic denitrifying bacterium C-3 and its applications. The heterotrophic-aerobic denitrifying bacterium C-3 is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified as *Brevundimonas naejangsanensis*, with accession number CGMCC No. 31729, and deposited on August 23, 2024. The heterotrophic-aerobic denitrifying vesicular *Brevundimonas naejangsanensis* strain C-3 provided by this invention exhibits excellent nitrate nitrogen removal efficiency (100%) under aerobic conditions using sodium acetate as a carbon source, a C / N mass ratio of 10, a pH of 7, a temperature of 30℃, and a rotation speed of 200 r / min. In actual aquaculture wastewater, it demonstrates excellent removal effects on nitrate nitrogen, nitrite nitrogen, ammonia nitrogen, and total nitrogen. Furthermore, this strain exhibits high safety and has significant application value in treating aquaculture wastewater.

Description

Technical Field

[0001] This invention relates to a microbial denitrification process for aquaculture water bodies, specifically a heterotrophic nitrifying-aerobic denitrifying bacterium C-3 and its application in denitrification in freshwater aquaculture ponds. Background Technology

[0002] To conserve fishery resources, aquaculture has generally adopted intensive, factory-style, and high-density farming models. These models often involve excessive fertilization and feeding, and the excessive discharge of aquatic animal waste exacerbates eutrophication, generating large amounts of nitrogen-containing polluting aquaculture wastewater. The discharge of this wastewater into the surrounding environment not only endangers human drinking water safety but also seriously affects the ecological balance of aquatic bodies. Therefore, how to reduce the nitrogen content in aquaculture wastewater to meet discharge standards has become a research hotspot for purifying aquaculture water bodies.

[0003] Currently, the main methods for nitrogen removal from wastewater in my country include physical, chemical, and microbial methods. Among them, microbial nitrogen removal technology is favored by aquaculture practitioners due to its advantages such as low cost, safety, high efficiency, and environmental friendliness. The principle of microbial nitrogen removal is mainly through reactions such as ammoniation, nitrification, and denitrification, converting organic and inorganic nitrogen in the water into nitrogen-containing gases that are discharged from the water body, thereby achieving nitrogen removal and purification of wastewater. Since the nitrification and denitrification processes of traditional nitrogen-removing microorganisms take place in two extreme environmental zones—aerobic and anaerobic—in practical applications, they often require a large land area, high energy consumption, and complex operation and management. Furthermore, aquaculture water bodies contain a certain concentration of dissolved oxygen, making it difficult to achieve an anaerobic environment, which inhibits the denitrification process of nitrogen-removing microorganisms and affects their nitrogen removal efficiency.

[0004] In recent years, to improve nitrogen removal efficiency, shorten the nitrogen removal process, and reduce wastewater treatment costs, many researchers have focused on the study of aerobic denitrification processes and related bacteria, and have isolated a large number of aerobic denitrifying bacteria, mainly including *Alcaligenes*, *Acinetobacter*, *Bacillus*, and *Pseudomonas*. These microorganisms can simultaneously complete nitrification and denitrification reactions under aerobic conditions, and they grow and reproduce rapidly. They can use the products of nitrification as a nitrogen source for denitrification, accelerating the reaction process. Furthermore, these microorganisms can produce alkali during denitrification, which can neutralize the acid produced by nitrification, maintaining pH stability during nitrogen removal. Aerobic denitrifying bacteria also have low requirements for dissolved oxygen concentration, making them very suitable for nitrogen removal treatment of aquaculture wastewater. Although aerobic denitrifying bacteria have many advantages in denitrification treatment of aquaculture wastewater, several problems remain in practical applications: For example, current research on aerobic denitrifying bacteria mainly focuses on the denitrification mechanisms and conditions of the strains, limiting their practical application; different species of aerobic denitrifying bacteria exhibit significant differences in growth characteristics and denitrification capacity, and not all aerobic denitrifying strains possess efficient denitrification capabilities; since aquaculture water comes into direct contact with aquatic animals, the microorganisms used in aquaculture must have high safety, but most of the currently screened and reported aerobic denitrifying strains have not undergone relevant safety verification; therefore, screening for efficient, safe bacteria capable of simultaneous nitrification and denitrification under aerobic conditions is of significant research importance and application value for denitrification of polluted aquaculture water. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a safe and efficient heterotrophic-aerobic denitrifying bacterium C-3 and its application for rapid denitrification in aquaculture where the nitrogen content of the water is relatively low compared to the wastewater, and where it is necessary to ensure that the denitrification operation does not affect the growth and survival of fish.

[0006] Technical solution: The heterotrophic nitrifying-aerobic denitrifying bacteria C-3 described in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] Heterotrophic-aerobic denitrifying bacteria C-3, namely Brevundimonas naejangsanensis C-3, with accession number CGMCC No.31729, was deposited at the China General Microbiological Culture Collection Center on August 23, 2024.

[0008] Application of heterotrophic-aerobic denitrifying bacteria C-3 in nitrogen removal treatment of aquaculture water.

[0009] Application of heterotrophic-aerobic denitrifying bacteria C-3 in the removal of nitrate nitrogen from aquaculture water.

[0010] The method for denitrifying nitrogen-containing water using the heterotrophic-aerobic denitrifying bacteria C-3 involves inoculating the heterotrophic-aerobic denitrifying bacteria C-3 into the nitrogen-containing water, adding a carbon source to achieve a C / N ratio of 8–12, and culturing the bacteria at a pH of 6–8, 25–35°C, and 150–250 r / min to remove different forms of nitrogen from the water.

[0011] The method wherein the carbon source is one of sodium acetate, sucrose, glucose, or sodium citrate, and the nitrogen is in the form of single nitrate nitrogen, single ammonia nitrogen, or a mixture of nitrate nitrogen and ammonia nitrogen.

[0012] The 16S rDNA gene sequence of the heterotrophic-aerobic denitrifying bacteria C-3 is shown in SEQ ID NO.1.

[0013] The bacterial agent or bacterial solution contains the heterotrophic-aerobic denitrifying bacteria C-3.

[0014] The application of the bacterial agent or bacterial solution in denitrification treatment of aquatic water.

[0015] The application of the bacterial agent or bacterial solution in the removal of nitrate nitrogen from aquaculture water.

[0016] The application of heterotrophic-aerobic denitrifying bacteria C-3 in combination with sodium acetate in water denitrification.

[0017] The method for safety assessment of heterotrophic-aerobic denitrifying bacteria C-3 in denitrification of aquaculture water includes testing the hemolytic activity of the heterotrophic-aerobic denitrifying bacteria C-3 and determining the survival rate of grass carp infected in vivo and in vitro.

[0018] The heterotrophic nitrifying-aerobic denitrifying bacterium C-3 of this invention was isolated from the bottom mud of a catfish farming pond in Dongtai City, Jiangsu Province. The colony characteristics on LB agar plates are as follows: round, opaque, milky white, with a raised and glossy surface, neat edges, short straight rod-shaped cells, grooved surface, non-clustered, exhibiting unipolar hair movement in a short wave, and Gram staining results are negative. After 16S rDNA identification, it was determined to be a strain of the genus Brevundimonas sp. and named Brevundimonas C-3.

[0019] This invention also relates to a method for denitrifying nitrogen-containing water bodies using the heterotrophic-aerobic denitrifying bacteria C-3, the method comprising: inoculating the heterotrophic-aerobic denitrifying bacteria C-3 into nitrogen-containing water bodies, adding a carbon source to achieve a C / N ratio of 10, wherein the carbon source is sodium acetate, and culturing the bacteria at a pH of 7, a temperature of 30°C, and a rotation speed of 200 r / min to remove nitrogen from the water body.

[0020] This invention also relates to the application of the heterotrophic-aerobic denitrifying bacteria C-3 in the denitrification treatment of nitrogen-containing aquaculture wastewater.

[0021] The present invention also relates to a method for safety assessment of the heterotrophic-aerobic denitrifying bacteria C-3 in aquaculture water, the method comprising: conducting hemolytic activity testing of the heterotrophic-aerobic denitrifying bacteria C-3 and inoculating the heterotrophic-aerobic denitrifying bacteria C-3 at low and high concentrations into the grass carp aquaculture water and the peritoneal cavity of the grass carp, respectively, conducting in vivo and in vitro infection tests, feeding the fish normally, and recording and observing the survival status of the grass carp in each tank at regular intervals every day for 7 days.

[0022] Beneficial effects: (1) The heterotrophic-aerobic denitrifying vesicular shortwave monoclonal bacteria C-3 (Brevundimonas sp.) of the present invention can utilize a variety of carbon sources, and has the best denitrification effect of 100% when cultured aerobicly for 36 h under the conditions of sodium acetate as the optimal carbon source, C / N ratio of 10, pH of 7, temperature of 30℃, and rotation speed of 200 r / min. (2) This strain has a good removal effect on both ammonia nitrogen and total nitrogen, and the bacteria are non-hemolytic, and can remove nitrogen at concentrations below 1×10⁻⁶. 8 Within the safe concentration range of Cfu / mL, it has no toxic effect on grass carp, which is of great practical significance for its application in the denitrification treatment of aquaculture tailwater. (3) When the nitrogen content in the water exceeds a certain value, it will cause fish to die. Therefore, the nitrogen content in the water where aquaculture is being carried out is usually not very high, much lower than that of sewage or wastewater. The strain of the present invention has been experimentally proven to achieve a good denitrification effect even under low nitrogen content conditions. Attached Figure Description

[0023] Figure 1 This is a colony characteristic diagram of the heterotrophic-aerobic denitrifying bacteria C-3 of the present invention;

[0024] Figure 2 This is a Gram staining result of the heterotrophic-aerobic denitrifying bacteria C-3 of this invention;

[0025] Figure 3 This is a scanning electron microscope image of the heterotrophic-aerobic denitrifying bacteria C-3 of this invention;

[0026] Figure 4 This is a phylogenetic tree diagram of the heterotrophic-aerobic denitrifying bacteria C-3 of this invention;

[0027] Figure 5 This is a schematic diagram illustrating the growth of the heterotrophic-aerobic denitrifying bacteria C-3 and its ability to degrade nitrate nitrogen according to the present invention.

[0028] Figure 6 This is a schematic diagram illustrating the growth of heterotrophic-aerobic denitrifying bacteria C-3 and its ability to degrade ammonia nitrogen according to the present invention.

[0029] Figure 7 This is a schematic diagram illustrating the growth of heterotrophic-aerobic denitrifying bacteria C-3 and its ability to degrade mixed nitrogen sources according to the present invention.

[0030] Figure 8 This is a schematic diagram illustrating the degradation of nitrate nitrogen by the heterotrophic-aerobic denitrifying bacteria C-3 under different carbon sources according to the present invention.

[0031] Figure 9 This is a schematic diagram illustrating the denitrification effect of the heterotrophic-aerobic denitrifying bacteria C-3 of the present invention in actual aquaculture wastewater applications;

[0032] Figure 10 This is a graph showing the hemolytic activity of C-3 heterotrophic-aerobic denitrifying bacteria according to the present invention. Detailed Implementation

[0033] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0034] Example 1

[0035] Screening and identification of a heterotrophic-aerobic denitrifying bacterium

[0036] In a clean bench, 10g of sediment sample was added to a 250mL Erlenmeyer flask containing 90mL of sterile deionized water, along with an appropriate amount of glass culture medium. The flask was shaken at 200 rpm and 30℃ for 3 hours, then removed and allowed to stand at room temperature for 30 minutes. 10mL of the supernatant was then transferred to 90mL of fresh DM medium, and enrichment culture was continued under the same conditions for 48 hours. This enrichment culture was repeated three times.

[0037] The above DM culture medium formula:

[0038]

[0039] Add 1 mL of the bacterial suspension obtained after three enrichments to 9 mL of sterile PBS buffer solution to dilute to 10⁻⁶. -1 Then from 10 -1 Take 1 mL of the solution and repeat this step until diluting to 10. -8 Each step of the dilution process was performed using a vortex mixer to ensure thorough mixing. 0.1 mL of each dilution was taken and evenly spread onto BTB solid agar plates, with three replicates per group.

[0040] The above BTB culture medium formula:

[0041]

[0042] The spread BTB agar plates were placed in a constant-temperature biochemical incubator and incubated upside down at 30°C in the dark until distinct single colonies were observed. Then, single colonies of different morphologies that changed the medium color from green to blue were picked using a sterile inoculation loop. The colonies were purified by streaking on BTB agar plates using the partitioning method. During purification, strains that no longer showed color change were discarded, while strains that did show color change were purified again using BTB medium, for at least three purification cycles.

[0043] The isolated and purified strains were picked up with a 10 μL sterile inoculation loop and inoculated into DM medium, and cultured for 3 days in a shaker at 30°C and 200 rpm. The shaker tubes were then centrifuged at 8000 rpm and 4°C for 5 min. 1 mL of the supernatant was then transferred to two sterile centrifuge tubes, and diphenylamine and Griess reagent were added to each tube for re-screening.

[0044] The re-screened strains were inoculated into 40 mL of sterile liquid LB medium and cultured for 24 h in a shaker at 30 °C and 200 rpm. Then, the culture was centrifuged at 8000 rpm and 4 °C for 5 min, the supernatant was discarded, and the culture was washed twice with sterile ultrapure water. An appropriate amount of sterile PBS buffer was added, and the culture was resuspended in a vortex mixer to allow the OD to adjust. 600 ≈1. After autoclaving the uniformly prepared liquid denitrification medium, dispense 100 mL into sterile Erlenmeyer flasks, then inoculate with bacterial culture at a 1% inoculum rate. Use an equal volume of sterile water as a blank control. Incubate at 30℃ and 200 rpm for 3 days. After 3 days, take 50 mL of culture medium and measure the OD value. 600 The value was then determined by centrifugation at 8000 r / min and 4℃ for 5 min, and the supernatant was collected for NO3 determination. - -N, NO2 - The concentrations of -N and TN were calculated, and NO3 was determined. - Removal rates of -N and TN. The optimal TN removal rate and NO2 removal rate were selected. - Strains with low or no accumulation of -N were identified, and subsequent research was conducted based on these strains.

[0045] Colony characteristics such as Figure 1 As shown, the colonies are round, opaque, milky-white, with a raised, glossy surface and neat edges. A small amount of C-3 from the slant culture was picked, Gram-stained, and the prepared thin sections were examined under an oil immersion microscope. The results are as follows. Figure 2 The results showed that C-3 bacteria stained red (Gram-negative), were short rod-shaped, non-spore-forming, and singly distributed. A small amount of strain C-3 was washed with water, fixed with glutaraldehyde, and dried before being observed under a scanning electron microscope. The results are as follows: Figure 3As shown, strain C-3 exhibits short, straight rod-like structures with grooved surfaces, does not cluster, and displays a short-wave unipolar hair movement.

[0046] Genomic DNA was extracted from the bacterial strain using the bacterial genomic DNA extraction kit (B518255) from Sangon Biotech (Shanghai) Co., Ltd. The 16S rDNA sequence of the strain was amplified by PCR using the upstream primer (27F): 5′-AGAGTTTGATCCTGGCTCAG-3′; and the downstream primer (1492R): 5′-GGCTACCTTGTTACGACTT-3′. After confirming the PCR results by electrophoresis on a 1×TAE gel at 150V, 100mA, and 1.5% agarose gel for 20 min, the product was sent to Sangon Biotech (Shanghai) Co., Ltd. for purification and sequencing.

[0047] PCR amplification. Reaction conditions:

[0048]

[0049] The 16S rDNA sequence of strain C-3 is 1398 bp in length, and its gene sequence is shown in SEQ ID NO.1.

[0050] Sequence Listing SEQ ID NO.1:

[0051]

[0052] The strain sequence was uploaded to the NCBI database and compared with existing bacterial 16S rDNA gene sequences. The results showed that the bacterium was *Brevundimonas naejangsanensis* strain. A phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.0 software to analyze the genetic characteristics of the strain, determine its species status and evolutionary position. The results are shown in […]. Figure 4 .

[0053] The strain C-3 was named *Brevundimonas naejangsanensis* strain C-3, with accession number CGMCC No. 31729. It was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on August 23, 2024.

[0054] Example 2

[0055] Identification of the growth and denitrification ability of strain C-3 in different nitrogen source media

[0056] Culture medium formulation:

[0057]

[0058] The treatment method was as follows: 1% of the strain seed culture was inoculated into 500 mL of DM-1, DM-2, and DM-3 liquid culture medium, respectively, and incubated at 30℃ and 200 rpm for 72 h. Then, every 12 h, 10 mL of culture medium was collected, centrifuged at 8000 rpm and 4℃ for 5 min, and the supernatant was used to determine the OD of strain C-3. 600 TN, NH4 + -N, NO2 - -N and NO3 - The concentration of -N. Results are shown in... Figures 5-7 .

[0059] Seed culture preparation: Inoculate 100 mL of LB liquid medium with a slant culture of strain C-3 and culture at 30℃ and 200 rpm for 24 h. Centrifuge the cultured bacterial solution at 8000 rpm for 2 min, discard the supernatant, wash the cells three times with sterile PBS solution, and finally resuspend in 10–20 mL of PBS solution to allow OD200 to adjust. 600 Approximately 1, which is the seed liquid.

[0060] Depend on Figure 5 It can be seen that when the culture conditions are 30℃ and 200r / min, strain C-3 reduced the nitrate nitrogen concentration from 78.10mg / L to 0.645mg / L within 36h, and then completely degraded it, with a degradation rate of 100%. OD 600 The maximum value was 1.27, indicating that strain C-3 has a good ability to degrade nitrate nitrogen. With the degradation of nitrate nitrogen, nitrite nitrogen and ammonia nitrogen accumulated, and the total nitrogen removal rate was not 100%, so it is speculated that some nitrate nitrogen was converted into nitrite nitrogen and ammonia nitrogen.

[0061] Depend on Figure 6 It can be seen that when the culture conditions are 30℃ and 200 r / min, strain C-3 can utilize ammonia nitrogen for growth and reproduction, and the OD value after 60 h is [data missing]. 600 Maintaining an pH of 1.48 also provides good degradation ability for ammonia nitrogen; after 72 hours, the degradation rate of ammonia nitrogen is 53.57%.

[0062] Depend on Figure 7 It can be seen that when the culture conditions are 30℃ and 200r / min, the OD of strain C-3 after 72h in a mixed nitrogen source is... 600The concentration reached 1.9496, indicating that strain C-3 exhibited rapid growth over a prolonged period, demonstrating its well-adaptability to environments where ammonia and nitrate nitrogen coexist. Under the simultaneous presence of both ammonia and nitrate nitrogen ions, strain C-3 preferentially utilized nitrate nitrogen. After rapid growth and reproduction, it then began to utilize ammonia nitrogen simultaneously. After 48 hours, the nitrate nitrogen degradation rate reached 95.21%, and the ammonia nitrogen degradation rate reached 68.04%. Subsequently, the ammonia nitrogen concentration showed a slight rebound, presumably due to the dissolution of ammonia nitrogen caused by bacterial cell death.

[0063] Example 3

[0064] Carbon source adaptability test of strain C-3

[0065] Sucrose (1.64 g / L), glucose (1.73 g / L), sodium acetate (2.36 g / L), and sodium citrate (2.0 g / L) were used as the sole carbon source, respectively, and KNO3 (0.5 g / L) was used as the nitrogen source, while other components of the basal culture medium remained unchanged. The cultures were incubated at 30℃ and 200 rpm for 60 h. Every 12 h, 10 mL of culture medium was collected, centrifuged at 8000 rpm and 4℃ for 5 min, and the supernatant was collected for the determination of strain C-3OD. 600 TN, NH4 + -N, NO2 - -N and NO3 - The concentration of -N was used to understand the growth and denitrification characteristics of the strains. The results are shown in […]. Figure 8 .

[0066] Depend on Figure 8 It can be seen that strain C-3 has a broad carbon source spectrum, and its response to NO3 varies among different carbon sources. - The removal rates of NO3- showed significant differences. After 60 hours, strain C-3 achieved a nitrate nitrogen removal rate of 60.72% with sucrose as the carbon source, with nitrite nitrogen accumulation reaching 5.98 mg / L and ammonia nitrogen accumulation reaching 9.274 mg / L. With glucose as the carbon source, the nitrate nitrogen removal rate was 82.25%, with ammonia nitrogen accumulation of 34.234 mg / L and nitrite nitrogen of 6.725 mg / L. With sodium citrate as the carbon source, the degradation of nitrate nitrogen was similar to that with glucose, but the accumulation of nitrite nitrogen and ammonia nitrogen was much lower. With sodium acetate as the carbon source, although the biomass of the strain was low, the removal rate of NO3- was high. - -N was removed most quickly. After 60 hours of cultivation, the removal rate reached 91.94%; the accumulation of nitrate nitrogen and nitrite nitrogen was also relatively low. Therefore, sodium acetate was the optimal carbon source for strain C-3.

[0067] Example 4

[0068] Application of a heterotrophic-aerobic denitrifying bacterium C-3 in aquaculture wastewater treatment

[0069] Aquaculture wastewater samples were collected on March 12, 2024, from a grass carp and crucian carp polyculture pond at the Large Freshwater Fish Promotion and Demonstration Base in Xinyi City, Jiangsu Province. A 30L mixed water sample was collected using the three-point sampling method and transported back to the laboratory under refrigeration. FiveL of the above aquaculture wastewater sample was then placed in two clean 10L plastic containers and inoculated with 1% of strain C-3 seed culture. The control group was inoculated with an equal volume of sterile water. The experiment was conducted continuously at room temperature for 7 days. The contents of nitrate nitrogen, nitrite nitrogen, ammonia nitrogen, and total nitrogen in the wastewater were measured daily. The results are shown in [Figure number missing]. Figure 9 .

[0070] Depend on Figure 9 It was found that the most abundant inorganic nitrogen in aquaculture wastewater was nitrate nitrogen (8.9551 mg / L), followed by nitrite nitrogen (1.28 mg / L), ammonia nitrogen (2.28 mg / L), and total nitrogen (14.98 mg / L). The experimental group with added strains showed a significant degradation effect on nitrate nitrogen. After 7 days, strain C-3 achieved a degradation rate of 89.75 ± 0.13% for nitrate nitrogen, 36.42 ± 0.20% for nitrite nitrogen, 57.62 ± 0.39% for ammonia nitrogen, and 74.27 ± 0.166% for total nitrogen.

[0071] Example 5

[0072] Safety testing of a heterotrophic-aerobic denitrifying bacterium C-3

[0073] Single colonies of strain C-3 were picked using a sterile inoculation loop and placed on fresh blood agar plates (purchased from Qingdao Rishui Biotechnology Co., Ltd., catalog number 31402). The plates were incubated upside down for 24 hours to determine hemolytic activity. Results are shown below. Figure 10 .

[0074] Depend on Figure 10 It can be seen that strain C-3 does not have hemolytic activity and can be used for subsequent tests.

[0075] Grass carp external infection: The infection solution was added to the aquaculture water in a specific ratio to maintain a bacterial concentration of approximately 1×10⁻⁶. 8 Cfu / mL and 1×10 11 Cfu / mL), and an equal volume of sterile water was added to the blank control group; grass carp were injected with infection: grass carp were intraperitoneally injected into a low-dose injection group (bacterial concentration approximately 1×10⁻⁶ Cfu / mL), and an equal volume of sterile water was added to the blank control group; grass carp were injected with infection: grass carp were divided into a low-dose injection group (bacterial concentration approximately 1×10⁻⁶ Cfu 8 Cfu / mL) and high-dose injection group (bacterial concentration approximately 1×10⁻⁶ Cfu / mL) 11The control group was injected with 0.6% sterile saline (Cfu / mL). The injection dose was 0.2 mL. Each group was replicated twice. Ten fish were kept in each tank. During the experiment, two replicates were performed. The fish were fed normally, and the number of surviving grass carp in each tank was recorded and observed at regular intervals every day for 7 days. The results are shown in Table 1.

[0076] Table 1. Infection results of strain C-3 on grass carp.

[0077]

[0078] Table 1 shows that strain C-3 caused mortality in both high-dose in vitro infection and high-dose intraperitoneal injection experiments, indicating that high-concentration in vitro infection with strain C-3 may be lethal to grass carp. However, when the concentration is not higher than 1×10⁻⁶, the mortality rate may be lower. 8 No deaths occurred at Cfu / mL. Furthermore, in the natural environment, the pathogenic dose of the microorganism is approximately 1×10⁻⁶. 4 CFU / mL, it is difficult for a single strain to reach a concentration of 1×10⁻⁶. 8 Cfu / mL, therefore, strain C-3 is non-toxic within the safe concentration range.

Claims

1. A heterotrophic-aerobic denitrifying bacterium C-3, characterized in that: It is *Shortwave vesicular* C-3, with accession number CGMCC No. 31729, and was deposited at the China General Microbiological Culture Collection Center on August 23, 2024.

2. The application of the heterotrophic-aerobic denitrifying bacteria C-3 as described in claim 1 in the denitrification treatment of aquaculture water.

3. The application of the heterotrophic-aerobic denitrifying bacteria C-3 as described in claim 1 in the removal of nitrate nitrogen from aquaculture water.

4. A method for denitrifying nitrogen-containing water using the heterotrophic-aerobic denitrifying bacteria C-3 as described in claim 1, characterized in that: The heterotrophic-aerobic denitrifying bacteria C-3 were inoculated into nitrogen-containing water bodies, and a carbon source was added to achieve a C / N ratio of 8–12. The bacteria were then cultured at a pH of 6–8, 25–35°C, and 150–250 r / min to remove different forms of nitrogen from the water.

5. The method as described in claim 4, characterized in that: The carbon source is a single sodium acetate, sucrose, glucose, or sodium citrate, and the nitrogen is in the form of single nitrate nitrogen, single ammonia nitrogen, or a mixture of nitrate nitrogen and ammonia nitrogen.

6. The heterotrophic-aerobic denitrifying bacteria C-3 as described in claim 1, characterized in that: The 16S rDNA gene sequence is shown in SEQ ID NO.

1.

7. A bacterial agent or bacterial solution, characterized in that: It includes the heterotrophic-aerobic denitrifying bacteria C-3 as described in claim 1.

8. The application of the bacterial agent or bacterial solution as described in claim 7 in the denitrification treatment of aquaculture water.

9. The application of the heterotrophic-aerobic denitrifying bacteria C-3 as described in claim 1 in combination with sodium acetate for denitrification in water bodies.

10. A method for safety assessment of the heterotrophic-aerobic denitrifying bacteria C-3 as described in claim 1 for denitrification in aquaculture water, characterized in that: The hemolytic activity of the heterotrophic-aerobic denitrifying bacteria C-3 was tested, and the survival rate of grass carp infected in vivo and in vitro was determined.

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