Preparation and application of heterotrophic nitrification and aerobic denitrification bacteria suitable for aquarium industry

Abstract

The invention discloses a nitrifying bacterium agent suitable for aquarium, fishpond and other aquatic industries, the strain of the nitrifying bacterium agent is Glutamicacter nicotianae TJ-X, the nitrifying bacterium agent is preserved in Guangdong Microbial Culture Collection Center on February 12, 2025, and the preservation number is GDMCC NO.65877. The invention further discloses a preparation method of the nitrifying bacterium agent. The nitrifying bacteria TJ-X strain suitable for the aquarium industry can perform heterotrophic nitrification and aerobic denitrification under the condition that the salt content is 5wt%, under the condition of laboratory pure culture, within 8 hours, the removal rate of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen is 99% or above, the COD degradation rate is 81%, the nitrifying bacteria TJ-X strain is suitable for the environment where organic matter, ammonia nitrogen, nitrite and nitrate coexist, and the nitrifying bacteria TJ-X strain can be applied to the aquarium industry. The microbial agent has the functions of degrading organic matters such as excrement of ornamental fish and fish food, converting organic nitrogen into inorganic nitrogen, and simultaneously degrading inorganic nitrogen such as ammonia nitrogen, nitrite nitrogen and nitrate nitrogen into nitrogen. And the strain is high in biological safety, high in environmental adaptability and high in growth density, can be applied to water industries with high organic matter content and inorganic nitrogen, such as the aquarium industry and aquaculture, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a multifunctional bacterial agent for heterotrophic nitrification and agronomical denitrification that can be used in the aquarium industry. Background Technology

[0002] In the aquarium industry, nitrifying bacteria play a crucial role, especially in maintaining water quality and promoting aquatic ecological balance. Nitrifying bacteria are microorganisms that purify water by converting harmful substances (such as ammonia nitrogen and nitrite) into safer nitrates. In aquariums, fish ponds, and other aquatic environments, the presence and function of nitrifying bacteria effectively reduce the concentration of toxic substances, maintain water quality stability, and ensure the healthy growth of aquatic organisms. In traditional denitrification systems, the nitrification process, dominated by nitrifying bacteria, is a biotransformation process primarily involving two types of bacteria: 1. Ammonia-oxidizing bacteria (AOBs).

[0003] Nitrifying bacteria convert ammonia (NH3) in water into nitrite (NO2⁻), a process known as ammonia oxidation. Nitrite-oxidizing bacteria (NOB) further convert nitrite into nitrate (NO3⁻), a process called nitrite oxidation. However, both types of nitrifying bacteria are typically autotrophic, with slow growth and reproduction rates. In newly established aquariums, water quality is often unstable, with high concentrations of ammonia nitrogen and nitrite. Nitrifying bacteria may need several weeks or even longer to fully multiply and begin to function. During this period, aquarium systems may face "ammonia nitrogen crisis" and "nitrite crisis." Without other interventions, aquatic organisms may suffer from poisoning. In addition, because autotrophic nitrifying bacteria do not have the function of decomposing organic matter, undecomposed fish food and feces often breed a large number of harmful saprophytic bacteria, leading to water quality deterioration, microbial imbalance in the aquarium, and other problems. Although traditional nitrifying bacteria can effectively treat ammonia nitrogen and nitrite in the water, due to their autotrophic growth characteristics, a high organic matter environment actually hinders their growth and reproduction. Therefore, it is impossible to completely solve the problem of high organic matter pollution in water caused by excessive stocking density and excessive fish food in the short term.

[0004] Compared to autotrophic nitrifying bacteria, heterotrophic nitrifying bacteria have advantages such as rapid growth rate, low dissolved oxygen requirement, completion of nitrification and denitrification in the same reactor, ability to metabolize various forms of nitrogen compounds, and improved COD removal rate. Therefore, heterotrophic nitrifying bacteria are increasingly being used in aquaculture water treatment. For example, patent number CN 111100811 A discloses a heterotrophic nitrifying compound bacterial agent and its application. This compound bacterial agent tolerates a C / N ratio range of 0-200, within which its NH4+... +The removal rate of -N is higher than 93.0%, it can adapt to high concentrations of organic matter, and this compound microbial agent can tolerate NH4+. + -N concentration 0-500 mg•L -1 While possessing strong ammonia nitrogen degradation capabilities, this composite bacterium primarily functions through heterotrophic nitrification, removing only ammonia nitrogen from water. It cannot perform denitrification in an aerobic environment to reduce nitrate accumulation. Patent CN110656066 A discloses a short-cutting nitrifying and denitrifying variant of Acinetobacter bacillus and its applications. This invented short-cutting nitrifying and denitrifying variant of Acinetobacter bacillus exhibits highly efficient removal capabilities for total nitrogen, ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen, and can be applied in nitrogen removal treatment of freshwater aquaculture water. Currently, these denitrifying bacteria mainly originate from freshwater environments. Novel denitrifying bacteria that have been reported to be isolated and purified include *Thiosphaera pantotropha*, *Alcaligenes* sp., *Acinetobacter* sp., and *Pseudomonas* sp., among others. Due to the problems and shortcomings of traditional nitrification processes, current nitrifying bacteria products on the market often add various functional bacterial groups, such as heterotrophic bacteria that decompose organic matter and convert organic nitrogen into ammonia, autotrophic nitrifying bacteria, and denitrifying bacteria. The bacteria added to these products contribute to the nitrogen cycle in the aquarium in steps, and each step of degradation requires a significant amount of time. This often leads to fish death due to ammonia and nitrite poisoning, necessitating manual intervention such as frequent water changes to dilute the toxins and prevent fish mortality. Furthermore, in the aquarium's nitrogen cycle, organic nitrogen ultimately decomposes into nitrates under aerobic conditions. Nitrates often require denitrification under strictly anaerobic conditions to be converted into nitrogen gas and removed. In aquarium systems with high dissolved oxygen requirements, nitrates cannot be converted into nitrogen gas by denitrifying bacteria and must be removed through manual water changes. Although nitrates are weakly toxic, long-term accumulation can still lead to problems such as weakened immunity in ornamental fish and algae blooms in the aquarium. Finally, the cultivation and production of heterotrophic decomposing bacteria such as Bacillus subtilis, lactic acid bacteria, and yeast, as well as the cultivation and production of autotrophic nitrifying bacteria, usually need to be carried out separately. This undoubtedly leads to a lengthy and complex production process, increased production costs, and uncontrollable product quality inspection. Summary of the Invention

[0005] This invention aims to overcome existing technical deficiencies and provides a heterotrophic nitrifying-aerobic denitrifying bacterial agent suitable for the aquarium industry. This agent decomposes organic waste in aquariums, degrades highly toxic substances such as ammonia nitrogen and nitrite, and performs aerobic denitrification, converting nitrates into nitrogen gas for removal. The heterotrophic effect accelerates growth and reproduction, enabling the rapid establishment of a mature nitrogen cycle system in the aquarium in a short period. It removes organic nitrogen into nitrogen gas in one step, reducing complex and wasteful steps such as water changes and water conditioning. The agent of this invention, with a single bacterial species, can achieve the functions of multiple bacterial species, replacing traditional compound bacterial agents currently on the market. It achieves multiple functions with one bacterial species and does not cause dysbiosis between heterotrophic and autotrophic denitrifying bacteria. The agent of this invention belongs to heterotrophic nitrifying-aerobic denitrifying bacteria, which can simultaneously degrade organic matter and inorganic nitrogen under suitable environmental conditions and quickly become the dominant bacteria, reducing the growth and reproduction of aerobic pathogens.

[0006] To achieve the above objectives, the technical solution adopted by this invention is as follows: One strain of nitrifying bacteria in this invention is derived from the activated sludge of a biological treatment unit in a municipal wastewater treatment plant. It grows and reproduces using ammonia nitrogen and nitrate nitrogen as nitrogen sources, and sodium citrate dihydrate as both a carbon source and energy source. It is enriched using heterotrophic nitrification and aerobic denitrification media. During the cultivation process, the salt concentration is gradually increased for gradient acclimatization, and the bacteria are obtained through streak plate separation and purification.

[0007] The strain *Glutamicibacternicotianae* TJ-X in the heterotrophic nitrifying aerobic denitrifying bacterial agent was deposited at the Guangdong Provincial Microbial Culture Collection Center on February 12, 2025, with accession number GDMCC NO.65877 and NCBI gene sequence number PQ524502.1. *Glutamicibacternicotianae* TJ-X is a Gram-positive bacterium. The bacteria are short rods, small and club-shaped, with blunt ends. Colonies are round, light yellow, and smooth, measuring 1.5-3.0 mm. Strain TJ-X possesses simultaneous nitrification and denitrification capabilities and can grow and reproduce in a medium with a salt content of 5 wt%. Physiological and biochemical tests showed positive results for the catalase test, glycolysis test, nitrate reduction test, citrate test, and methyl red test; negative results for the VP test, starch hydrolysis test, oil test, casein hydrolysis test, and gelatin liquefaction test.

[0008] The heterotrophic nitrifying aerobic denitrifying denitrifying bacteria agent is used in environments with high levels of organic matter, ammonia nitrogen, nitrite, nitrate, etc., such as in the aquarium industry, aquaculture industry, food processing wastewater, and pharmaceutical wastewater industry.

[0009] By adopting the above technical solution, the present invention has the following positive effects compared with the prior art.

[0010] The TJ-X strain of nitrifying bacteria, applicable to the aquarium industry, can perform heterotrophic nitrification and aerobic denitrification for nitrogen removal under a salt content of 5 wt%. Under pure laboratory culture conditions, within 8 hours, the removal rates of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen are over 99%, and the COD degradation rate is 81%. This bacterial agent can be used in the aquarium industry, aquaculture industry, and wastewater treatment industries such as food processing wastewater, pharmaceutical wastewater, and oil refining wastewater, with broad application prospects.

[0011] Therefore, the nitrifying bacteria provided by this invention, suitable for the aquarium industry, has a significant effect on biological denitrification of environments containing salt, high organic matter, ammonia nitrogen, nitrite, nitrate, etc. Attached Figure Description

[0012] Figure 1 The following is a phylogenetic tree of strain TJ-X.

[0013] Figure 2 The image shown is a colony diagram of strain TJ-X.

[0014] Figure 3 The image shown is a Gram staining diagram of strain TJ-X.

[0015] Figure 4 The figure shows the COD removal rate of strain TJ-X.

[0016] Figure 5 The figure shown is a graph of inorganic nitrogen removal capacity of strain TJ-X.

[0017] Figure 6 The graph shown is a trend chart of ammonia nitrogen content changes in a fishless circulating aquarium.

[0018] Figure 7 The graph shown is a trend chart of nitrite content changes in a fishless, circulating aquarium.

[0019] Figure 8 The graph shown illustrates the trend of nitrate content changes in a fishless, recirculating aquarium.

[0020] Figure 9 The graph shows the trend of ammonia nitrogen and nitrite content in the aquarium with added nitrifying bacteria.

[0021] Figure 10 The graph shown depicts the changing trends of ammonia nitrogen and nitrite levels in the aquarium without added bacteria.

[0022] The nucleotide sequence of strain TJ-X is shown in SEQ ID NO.1:

[0023] Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of protection thereof.

[0025] Example 1: Isolation and purification of strains

[0026] (1) Source of strains

[0027] The bacteria source was activated sludge from the biological treatment unit of the municipal wastewater treatment plant in Shenzhen, Guangdong Province.

[0028] (2) Isolation and purification of strains

[0029] The continuously acclimatized municipal sludge was inoculated into sterile water, cultured in a constant temperature shaker for 3 hours, allowed to stand, and the supernatant was used as a bacterial suspension. Five mL of bacterial suspension was inoculated into 100 mL of sterilized heterotrophic nitrifying-aerobic denitrifying bacteria enrichment culture medium with a salinity of 1%, and cultured for 24 h under the same conditions for the first round of enrichment. Five mL of the first round of enriched bacterial suspension was inoculated into heterotrophic nitrifying-aerobic denitrifying bacteria enrichment culture medium with a salinity of 3%, and cultured for 24 h under the same conditions for the second round of enrichment. Five mL of the second round of enriched bacterial suspension was inoculated into heterotrophic nitrifying-aerobic denitrifying bacteria enrichment culture medium with a salinity of 5%, and cultured for 24 h under the same conditions for the third round of enrichment. The third round of enriched bacterial suspension was serially diluted and inoculated into heterotrophic nitrifying-aerobic denitrifying bacteria separation solid medium with a salinity of 5% using the dilution plating method. After culturing for 72 h, the colony morphology was observed, and typical colonies were selected for streak isolation on the plate to purify the screened strains. Two streak isolations were performed. The isolated and purified single strain was inoculated into a 100 mL Erlenmeyer flask containing heterotrophic nitrifying-aerobic denitrifying bacteria medium and cultured on a shaker for 24 hours. The ammonia nitrogen concentration in the medium was measured to screen for the strain with the highest ammonia nitrogen and nitrate nitrogen removal rates for denitrification performance verification experiments. The strain TJ-X with the best overall performance was selected for preservation in slant agar and glycerol tubes.

[0030] The composition of the heterotrophic nitrifying-aerobic denitrifying bacteria enrichment medium is: Na3C6H5O7*2H2O 2.17 g, (NH4)2SO4 0.5 g, K2HPO4 0.8 g, MgSO4*7H2O 0.4 g, NaCl 0.4 g, FeSO4*7H2O 0.04 g, CaCl2*H2O 0.03 g, adjusted to 1 L, pH 7.0-7.2; add 20 g of agar to obtain the solid medium.

[0031] The heterotrophic nitrifying-aerobic denitrifying bacteria isolation medium consisted of: Na₃C₆H₅O₇·2H₂O 2.17 g, (NH₄)₂SO₄ 0.5 g, K₂HPO₄ 0.6 g, MgSO₄·7H₂O 0.2 g, NaCl 0.4 g, and FeSO₄·7H₂O 0.04 g, diluted to 1 L, with a pH of 7.0–7.2. Adding 20g of agar completed the solid medium. All media were sterilized at 121°C for 30 min before use. A certain concentration of NaCl was added to each of the above media, progressively increasing the salt content for acclimatization.

[0032] Example 2: Strain Identification

[0033] (1) Identification of 16S rDNA of strain TJ-X

[0034] Genomic DNA was extracted from strain TJ-X using a DNA extraction kit, followed by 16S rDNA cloning and sequencing. The 16S rDNA sequence is shown in the sequence listing. Blast alignment in GenBank revealed that strain TJ-X shared 99% sequence similarity with *Glutamicibacter nicotianae*. A phylogenetic tree was constructed using MEGA 11.0 software, and the results are as follows: Figure 1 As shown, this strain belongs to the same branch as *Bacillus nicotine* with a reliability of 93%.

[0035] (2) Physiological and biochemical identification of strain TJ-X

[0036] like Figure 2 As shown, the TJ-X(L2Y) strain is morphologically short rod-shaped, club-shaped with blunt, rounded ends. Colonies are round, light yellow, and smooth. Figure 3 As shown, Gram staining indicates that strain TJ-X is a Gram-positive bacterium. Physiological and biochemical tests showed that strain TJ-X was positive for catalase, glycolysis, nitrate reduction, citrate, and methyl red tests; and negative for VP, starch hydrolysis, lipid, casein hydrolysis, and gelatin liquefaction tests. Based on the 16S rDNA identification results of strain TJ-X, it was classified as *Glutamicibacternicotianae*.

[0037] Example 3: Heterotrophic nitrification, aerobic denitrification, and nitrogen removal characteristics of strain TJ-X

[0038] (1) Preparation of single-strain seed culture

[0039] A certain number of bacterial cells were scraped from the slant culture medium of strain TJ-X using a sterile inoculation loop and resuspended in 2 mL of sterile physiological saline. The resuspended bacterial cells were then inoculated into 200 mL of heterotrophic nitrification-aerobic denitrification medium and cultured in a constant temperature shaker at 30°C and 200 rpm / min for 15 hours.

[0040] (2) Scale-up production in 100L fermenters

[0041] After cleaning the 100L fermenter, sterilize it by emptying it. After sterilization, prepare 7.5L of the above-mentioned fermentation medium and sterilize it at 121℃ for 30 minutes. After cooling, inoculate the primary seed culture into the 100L fermenter, control the temperature at 30℃, stir at 350 rpm, and aerate at 1.7 M. 3 Cultured at / h for 72 h.

[0042] (3) Characteristics of heterotrophic nitrification and aerobic denitrification

[0043] Methods: The fermented inoculum was added at a rate of 3‰ to evaluation water containing 500 mg / L COD, 50 mg / L ammonia nitrogen, 50 mg / L nitrite, and 50 mg / L nitrate. The C / N ratio was 10, dissolved oxygen was 5 mg / L, the temperature was 30℃, the rotation speed was 200 r / min, and the mixture was incubated on a shaker for 8 hours. Initial samples and samples after 8 hours of incubation were taken for the above indicators to be detected.

[0044] The results showed that the COD removal rate of strain TJ-X was as follows: Figure 4 As shown, the experimental group with added bacterial agent TJ-X achieved a COD removal rate of 81% after 8 hours, while the blank control group without the bacterial agent of this invention had a COD removal rate of only 5%. COD (Chemical Oxygen Demand) refers to the amount of oxygen equivalent consumed by a chemical reaction to oxidize reducing inorganic and organic matter (generally organic matter) in a water sample. It is a very important indicator for judging whether the water environment is polluted. This indicator is also one of the comprehensive indicators of the relative content of organic matter. The higher the COD removal rate, the lower the organic matter content in the water. Therefore, this experiment directly proves that strain TJ-X has the corresponding degradation ability for organic matter in water, and has good application prospects in water purification in aquariums, aquaculture, and other industries containing organic water. The remaining inorganic nitrogen (ammonia nitrogen, nitrate nitrogen, nitrite) content in the water is evaluated as follows: Figure 5As shown, after 8 hours, the levels of inorganic nitrogen (ammonia nitrogen, nitrate nitrogen, and nitrite) in the evaluation system with strain TJ-X decreased from the initial 50 mg / L to 0.056, 0.53, and 0.15 mg / L, respectively, with removal rates of 99.9%, 99.0%, and 99.7%. In contrast, the levels of ammonia nitrogen, nitrate nitrogen, and nitrite in the blank control system without strain TJ-X decreased from the initial 50 mg / L to 48.5, 49.0, and 49.2 mg / L, respectively, with removal rates of 3%, 2%, and 1.6%. This evaluation system demonstrates that strain TJ-X exhibits heterotrophic nitrification-aerobic denitrification characteristics under pure culture and optimal growth conditions. These results indicate that strain TJ-X can remove organic matter, ammonia nitrogen, and nitrite, and can remove nitrate nitrogen under aerobic conditions. This indicates that strain TJ-X has broad application prospects in water bodies where organic matter and inorganic nitrogen coexist, such as aquarium fish farming water bodies, aquaculture water bodies, food processing wastewater, and pharmaceutical wastewater.

[0045] Example 4: Application Case of Nitrifying Bacteria for Denitrification in Aquariums in the Aquarium Industry

[0046] (1) Case 1: Verification of the denitrification effect of nitrogen circulation in aquariums without fish

[0047] The aquarium fishless nitrogen cycle originated in Europe and the United States as a method to establish a nitrification system instead of introducing starter fish. This method involves directly adding fish food or ammonium chloride solution to a new aquarium to accelerate or omit the conversion of organic nitrogen to inorganic ammonia. The ammonia stimulates the growth of autotrophic ammonia-oxidizing bacteria, which then convert the ammonia into nitrite, stimulating the formation of nitrifying bacteria. Compared to traditional methods, this process is more user-friendly, convenient, and simple. Combined with nitrifying bacteria, it can accelerate the establishment of a nitrification system. Therefore, this invention combines the fishless cycle method with grinding the fish food to accelerate the conversion of organic nitrogen to ammonia by heterotrophic bacteria, allowing for faster verification of the denitrification effect of nitrifying bacteria.

[0048] Specific implementation steps: Add the nitrifying bacteria agent at a dosage of 1 mL (‱) per 10L of water to each aquarium containing 10L of water. Turn on the air pump and control the dissolved oxygen to above 6 mg / L. Place 1cm of filter media at the bottom of each aquarium to settle feces and fish food, and add finely ground fish food to the water. This test plan consists of four test stages: Stage 1, organic nitrogen conversion stage (fish food decomposes to produce ammonia nitrogen); Stage 2, ammonia oxidation stage (ammonia nitrogen is converted into nitrite); Stage 3, nitrite conversion stage (nitrite is converted into nitrate); Stage 4, aerobic denitrification stage (nitrate is degraded in an aquarium without plants).

[0049] By detecting the ammonia nitrogen content in a new fish tank, the rate at which heterotrophic bacteria decompose organic matter can be observed. The results are as follows... Figure 6 As shown, the aquarium water with the added nitrifying bacteria agent of this invention showed an ammonia nitrogen level of 0.6 mg / L on the second day of the experiment, and a peak ammonia nitrogen level was detected on the fourth day. In contrast, the blank control group without the added bacteria agent showed an ammonia nitrogen level of 0.2 mg / L on the fourth day, and a peak ammonia nitrogen level was not detected until the eighth day. This indicates that adding this nitrifying bacteria agent can accelerate the decomposition of organic matter and speed up the appearance of the ammonia peak, which is beneficial for accelerating the establishment of the nitrification system. In addition, on the fourth day after adding the nitrifying bacteria agent, the ammonia nitrogen content in the aquarium began to decrease sharply, and the ammonia nitrogen content was 0 mg / L on the ninth day. This indicates that by the ninth day, the nitrogen cycle in the new aquarium had completed the first and second stages, namely the conversion of organic nitrogen and the ammonia oxidation stage, taking 9 days, which is half the time of the traditional ammonia oxidation process.

[0050] The results of the third stage are as follows: Figure 7 In aquariums treated with the nitrifying bacteria agent of this invention, a peak nitrite level was detected on day 8. The nitrite level began to decrease sharply on day 9, reaching 0 mg / L on day 13. In contrast, the sterile control group showed no significant decrease in nitrite level on day 13, and even exhibited an increasing trend. This indicates that by day 13, the third stage of nitrification—the conversion of nitrate by nitrifying bacteria—had been completed in the aquarium treated with the nitrifying bacteria agent of this invention, and the nitrification process was fully established. This reduces the time required for setting up and conditioning the aquarium by more than half compared to the traditional process without added bacteria.

[0051] From day 12 to day 17 of the entire nitrogen cycle without fish, the nitrate level in the aquarium was measured daily for a total of 6 days. The results are as follows: Figure 8As shown, the aquarium with added nitrifying bacteria exhibited a fluctuating trend in nitrate levels, decreasing until day 5, when the nitrate concentration dropped from a peak of 50 mg / L to 10 mg / L. In contrast, the control group showed a continuous increase in nitrate levels. This is because continuous aeration by the air pump in the aquarium environment ensures dissolved oxygen levels remain above 6 mg / L. However, current mainstream traditional denitrification systems (activated sludge) consider dissolved oxygen levels to be below 0.5 mg / L for denitrification. Some also believe that nitrification and denitrification occur simultaneously when dissolved oxygen levels are maintained between 0.5 and 1.0 mg / L. However, in ornamental fish farming, low dissolved oxygen hinders the growth and reproduction of autotrophic nitrifying bacteria. Ammonia nitrogen and nitrite cannot be decomposed by nitrifying bacteria in a timely manner, leading to water quality deterioration. Furthermore, when dissolved oxygen levels in the aquarium fall below 6 mg / L, ornamental fish will experience adverse reactions. The low-oxygen environment promotes the proliferation of harmful heterotrophic bacteria, leading to bacterial diseases in the fish. The nitrifying bacteria agent for the aquarium industry described in this invention has a unique aerobic denitrification ability, which can convert nitrates into nitrogen gas in a high dissolved oxygen environment, and establish a mature and complete nitrogen cycle system in the aquarium ecological environment.

[0052] (2) Case 2: Stability application of the nitrification system established by the microbial agent of the present invention

[0053] When aquariums are subjected to sudden improper handling, such as overfeeding, adding new fish at excessively high densities, or not changing the water for an extended period, heterotrophic bacteria can proliferate, leading to excessive ammonia and nitrogen levels. This can ultimately cause the nitrification system to collapse, resulting in cloudy water, sudden fish deaths, and acute poisoning. Therefore, establishing a rapid and stable nitrification system is a pressing issue in the aquarium industry. However, traditional denitrification technologies cannot solve this problem biologically and rely solely on frequent water changes.

[0054] The nitrifying bacteria agent of this invention was added to an aquarium with dimensions of 60*30*40cm, a water volume of 70L, and containing one goldfish 8-9cm long. Under stable water quality conditions, the feeding amount was increased from once a day (1g each time) to three times a day (1g each time). Three new 8-9cm long-tailed goldfish were added. The dissolved oxygen content in the aquarium was controlled at 6mg / L or higher, and the water temperature was maintained at 26-30℃. A filter was used to collect feces, and the bacteria agent of this invention was added to the filter media. An aquarium with the same configuration but without the bacteria agent was used as a control group for testing.

[0055] The results are as follows Figure 9The results showed that in a fish tank with previously stable water quality (ammonia nitrogen 0 mg / L, nitrite 0 mg / L), a surge in ammonia nitrogen occurred after a sudden overfeeding and increase in fish density. After adding the bacterial agent of this invention for 24 hours, the ammonia nitrogen in the tank dropped from a peak of 0.8 mg / L to 0 mg / L, and under the same feeding conditions, it remained at 0 mg / L on day 8. Nitrite levels fluctuated continuously between 0-0.2 mg / L under the sudden water quality change, and after continuous monitoring until day 8, the nitrite content approached 0. The results of the empty control group without the bacterial agent of this invention are as follows. Figure 10 As shown, within 1-8 days, ammonia nitrogen increased exponentially without any downward trend, and nitrite levels also increased, albeit slowly. This indicates that the nitrification system collapsed after sudden overfeeding and increased fish density, and the recovery time was slow. During this period, the reproduction rate of nitrite-producing bacteria in the nitrifying flora was slow, and ammonia nitrogen levels exceeding 0.2 mg / L at this stage could easily lead to the death of ornamental fish. Therefore, this case demonstrates that the heterotrophic nitrifying aerobic denitrifying bacteria agent of this invention can be used to accelerate the re-establishment of the nitrification system and maintain its stability in cases of nitrification system collapse and water quality deterioration in ornamental fish, thus preventing acute ammonia poisoning and nitrite poisoning in ornamental fish under deteriorating water conditions.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A heterotrophic nitrifying and agrotrophic denitrifying bacteria agent, characterized in that: A strain of *Glutamicibacter nicotianae* TJ-X was inoculated at a volume ratio of 1-2% into a heterotrophic nitrifying aerobic denitrification medium and cultured at 30°C, dissolved oxygen level of 2-5 mg / L, and rotation speed of 200-220 r / min for 15 h to obtain a culture solution. This *Glutamicibacter nicotianae* TJ-X strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on February 12, 2025, with accession number GDMCC NO. 65877.

2. The heterotrophic nitrifying and agrotrophic denitrifying bacteria agent according to claim 1, characterized in that: The bacterial culture was conducted at a temperature of 30°C, a dissolved oxygen level of 5 mg / L, a rotation speed of 200 r / min, and a culture time of 15 h.

3. The heterotrophic nitrifying and agrotrophic denitrifying bacteria agent according to claim 1, characterized in that: The heterotrophic nitrification-aerobic denitrification medium comprises 2.17 g of Na3C6H5O7*2H2O, 0.5 g of (NH4)2SO4, 0.6 g of K2HPO4, 0.2 g of MgSO4*7H2O, 0.4 g of NaCl, and 0.04 g of FeSO4*7H2O, diluted to 1 L, with a pH of 7.0–7.

2.

4. The application of a heterotrophic nitrifying agrotrophic denitrifying bacteria agent as described in any one of claims 1-3, characterized in that: This microbial agent was applied to reduce the levels of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in aquarium water used for raising ornamental fish.

5. The application of the heterotrophic nitrifying and agrotrophic denitrifying bacteria agent according to claim 4, characterized in that: The application of the heterotrophic nitrifying and aerobic denitrifying bacteria in reducing ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in aquaculture water includes the following steps: Under aerobic conditions, the bacteria carry out heterotrophic nitrification and aerobic denitrification nitrogen removal with a salt content of 5wt%. Under pure laboratory culture conditions, within 8 hours, the removal rate of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen is over 99%, and the COD (carbon source) degradation rate is 81%.

6. The application of the heterotrophic nitrifying and agrotrophic denitrifying bacteria agent according to claim 5, characterized in that: The aquarium used for ornamental fish farming uses organic carbon as a carbon source, including sodium citrate, fish feces, and / or fish feed.

7. The application of the heterotrophic nitrifying and agrotrophic denitrifying bacteria agent according to claim 5, characterized in that: The aerobic conditions refer to a dissolved oxygen level of 2-10 mg / L in the water during the cultivation process.

8. The application of the heterotrophic nitrifying and agrotrophic denitrifying bacteria agent according to claim 5, characterized in that: The aerobic conditions refer to a water temperature of 26-30 degrees Celsius during the cultivation process.

9. The application of the heterotrophic nitrifying and agrotrophic denitrifying bacteria agent according to claim 5, characterized in that: Add the bacterial agent to the ornamental fish aquaculture water at a dosage of 1 mL per 10 L of water (1‱). Before degradation, the ammonia nitrogen concentration, nitrite concentration, and nitrate content of the water are 0-50 mg / L, 0-50 mg / L, and 0-50 mg / L, respectively. The pH of the aquaculture water is 7.0-7.

2. The culture temperature is 30 degrees Celsius, and the C / N ratio is 10. After degradation, the water body has the following characteristics: residual ammonia nitrogen concentration of 0-0.02 mg / L, residual nitrite concentration of 0-0.01 mg / L, and residual nitrate concentration of 0-10 mg / L.

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