A strain of direct ammonia-oxidizing coprologenic bacterium AP3-14 and its application

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of direct ammonia-oxidizing fecal alkalogen AP3-14 and its applications. The fecal alkalogen provided by this invention (… Alcaligenes faecalis AP3-14 was deposited on November 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36650, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This strain not only possesses direct ammonia oxidation capabilities, achieving efficient denitrification under aerobic conditions through this pathway, but also simultaneously degrades ammonia nitrogen and phenol in wastewater or sewage. Furthermore, this strain can tolerate a wide range of ammonia nitrogen concentrations, phenol concentrations, and pH levels.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of direct ammonia-oxidizing alkali-producing bacteria AP3-14 and its applications. Background Technology

[0002] The production processes of industries such as petrochemicals, pharmaceuticals, and papermaking generate large amounts of wastewater. Wastewater containing phenol and ammonia nitrogen is discharged into sewage treatment systems and the natural environment, posing a serious threat to ecosystems and human health. Ammonia nitrogen, a major pollutant in industrial wastewater, not only causes a series of problems such as decreased dissolved oxygen, eutrophication, and water quality deterioration when discharged in excess, but also poses a serious threat to human health and the growth of aquatic plants and animals due to the toxicity of some nitrogenous substances, disrupting the balance of ecosystems and increasing water treatment costs. Phenolic wastewater is also listed as a key hazardous wastewater to be addressed. Phenol, as the most toxic and polluting phenolic compound, has a serious toxic effect on the growth and reproduction of biological populations, severely disrupting the natural ecological balance. Phenol can also damage cell membranes, enter cells and damage intracellular structures, affecting cell activity. Furthermore, phenol is carcinogenic; after contact, it can penetrate the human body, causing serious damage or even death to the brain, digestive system, and respiratory system.

[0003] Currently, various methods exist for removing ammonia nitrogen and phenol from wastewater or sewage. Among these, biological treatment methods are widely used due to their advantages such as simple operation, mild process conditions, and environmental friendliness. However, biological treatment methods still have certain limitations. On the one hand, biological treatment technology mainly consists of two stages: autotrophic nitrification and heterotrophic denitrification. However, because these two stages have different environmental requirements, they need to be completed in two separate reaction systems, resulting in disadvantages such as long process flow and high energy consumption. Furthermore, nitrifying bacteria are autotrophic, and their slow growth and reproduction rate leads to slow reactor start-up. Denitrifying bacteria, as heterotrophic bacteria, require external organic matter for growth and reproduction, but nitrifying bacteria cannot tolerate organic matter, making the treatment process even more complex. On the other hand, for phenol-containing wastewater, the recalcitrant nature of phenol and its biotoxicity to microorganisms (especially autotrophic nitrifying bacteria) often lead to low treatment efficiency or even system collapse in traditional processes.

[0004] In recent years, a novel microbial nitrogen transformation pathway known as "direct ammonia oxidation" has gradually attracted attention. Researchers have isolated direct ammonia-oxidizing bacteria and found that these bacteria can directly oxidize ammonia to hydroxylamine under aerobic conditions, which is then further converted into nitrogen gas without the intermediate step of traditional nitrification. Furthermore, these strains exhibit advantages such as rapid growth, strong environmental adaptability, and low greenhouse gas emission risk. However, existing ammonia-oxidizing bacteria often suffer from limited functionality; for wastewater containing multiple pollutants, several different bacterial agents are required to achieve the desired degradation effect. Summary of the Invention

[0005] In view of this, the present invention provides a direct ammonia oxidation alkaloid-producing bacterium AP3-14 and its application. This alkaloid-producing bacterium can not only efficiently denitrify through direct ammonia oxidation, but also simultaneously degrade ammonia nitrogen and phenol in polluted water bodies, and has a wide adaptability to ammonia nitrogen concentration, phenol concentration and pH range.

[0006] To solve the above technical problems, the first aspect of the present invention provides a strain of fecal alkali-producing bacteria that directly oxidizes ammonia ( Alcaligenes faecalis AP3-14, this strain was deposited on November 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36650, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The *Alkaliformis* AP3-14 strain provided by this invention can directly oxidize ammonia nitrogen in water into nitrogen gas, which is harmless to the environment. Furthermore, the N2O conversion rate is extremely low during the nitrogen conversion process, preventing air pollution during application. Simultaneously, this strain can also degrade ammonia nitrogen and phenol in water. When water contains both ammonia nitrogen and phenol, this strain alone is sufficient for effective degradation, eliminating the need to consider antagonistic effects between different strains when added simultaneously. This improves degradation efficiency while reducing costs, which is of great significance for water treatment. In addition, this strain can use phenol as a carbon source for denitrification, simultaneously utilizing and degrading phenol. It exhibits good denitrification and phenol removal effects over a wide range of ammonia nitrogen concentrations, phenol concentrations, and pH levels, allowing for wastewater or sewage treatment under extreme environmental conditions (such as strong alkalinity). This strain also has broad adaptability to tetracycline antibiotics, enabling its application in the treatment of wastewater or sewage containing tetracycline antibiotics. Furthermore, this strain is not hemolytic and shows sensitivity to most antibiotics, so it will not cause environmental impact due to its own excessive reproduction, which greatly reduces potential ecological risks.

[0008] The second aspect of the present invention provides an application of the above-mentioned fecal alkaloid-producing bacteria AP3-14 in the denitrification and dephenolization treatment of sewage or wastewater under aerobic conditions.

[0009] In conjunction with the second aspect, the sewage or wastewater includes landfill leachate, municipal sewage, aquaculture sewage, pharmaceutical wastewater, kitchen waste wastewater, coal chemical wastewater, fine chemical wastewater, textile printing and dyeing wastewater, and papermaking wastewater. Other sewage or wastewater that meets the degradation conditions of strain AP3-14 may also be included.

[0010] In conjunction with the second aspect, the bacterial solution of the fecal alkali-producing bacteria AP3-14 is inoculated into sewage or wastewater at a volume ratio of 4%-8%, mixed evenly, and then decomposed at 25-35℃.

[0011] In conjunction with the second aspect, the pH of the sewage or wastewater is 6-11, and the carbon-to-nitrogen ratio is 2-16.

[0012] Generally, when treating actual water samples, the water quality is analyzed beforehand. Once the water quality conditions meet the degradation conditions of strain AP3-14, the strain is inoculated for degradation. If the raw sewage or wastewater (i.e., untreated) or the water quality conditions (including carbon-to-nitrogen ratio, ammonia-to-nitrogen concentration, phenol concentration, and pH) still do not meet the degradation conditions of the strain, the water quality can be adjusted to suitable degradation conditions before degradation.

[0013] Ammonia nitrogen and phenol are two completely different pollutants. Experimental results show that when the ammonia nitrogen concentration is 100 mg / L and sodium acetate (sodium acetate is a small molecule organic compound and an easily utilized carbon source) is used as the carbon source, a carbon-to-nitrogen ratio (C / N ratio) of 2-16 can effectively remove nitrogen, while a C / N ratio of 10-16 shows highly efficient nitrogen removal. When the ammonia nitrogen concentration is 100 mg / L and sodium acetate is used as the carbon source while 1000 mg / L of phenol is added, a C / N ratio of 2-10 can effectively remove nitrogen and efficiently degrade phenol, with a C / N ratio of 4 showing the best denitrification and phenol removal effects. When phenol is used as the carbon source, a C / N ratio of 2-8 can effectively remove nitrogen and efficiently degrade phenol, with a C / N ratio of 8 showing the best simultaneous denitrification and phenol removal effects.

[0014] In conjunction with the second aspect, the ammonia nitrogen content in the sewage or wastewater is 0-1000 mg / L, and the phenol content is 0-1100 mg / L.

[0015] When the ammonia nitrogen content is 0-1000 mg / L and the phenol content is 0-1100 mg / L, strain AP3-14 can effectively degrade ammonia nitrogen and phenol.

[0016] A third aspect of the present invention provides a microbial agent comprising the above-mentioned strain Alcaligenes faecalis AP3-14.

[0017] The fourth aspect of this invention provides the application of a microbial agent in the denitrification or dephenolization treatment of sewage or wastewater. Attached Figure Description

[0018] Figure 1 This is a diagram showing the growth morphology of AP3-14, a bacterium that produces alkaloids in feces. Figure 2 Scanning electron microscope image of AP3-14, a fecal alkaloid bacterium; Figure 3The figure shows the denitrification characteristics of strain AP3-14 using sodium acetate as the carbon source under different carbon-to-nitrogen ratios. (a) shows the changes in ammonia nitrogen concentration and the removal rates of ammonia nitrogen and total dissolved nitrogen before and after degradation at different carbon-to-nitrogen ratios. (b) shows the changes in the concentrations of nitrate nitrogen, nitrite nitrogen, and hydroxylamine nitrogen after 6 days of degradation under different carbon-to-nitrogen ratios, and the OD of the strain. 600 value; Figure 4 The denitrification and phenol removal characteristics of strain AP3-14 under different carbon-to-nitrogen ratios using sodium acetate as the carbon source and 1000 mg / L phenol as the additive are shown in the figure. (a) shows the changes in ammonia nitrogen concentration and the removal rates of ammonia nitrogen, total dissolved nitrogen, and phenol before and after degradation under different carbon-to-nitrogen ratios. (b) shows the changes in the concentrations of nitrate nitrogen, nitrite nitrogen, and hydroxylamine nitrogen and the OD of the strain after 6 days of degradation under different carbon-to-nitrogen ratios. 600 value; Figure 5 The graph shows the denitrification and phenol removal characteristics of strain AP3-14 using phenol as a carbon source under different carbon-to-nitrogen ratios. (a) shows the changes in ammonia nitrogen concentration and the removal rates of ammonia nitrogen, total dissolved nitrogen, and phenol before and after degradation at different carbon-to-nitrogen ratios. (b) shows the changes in the concentrations of nitrate nitrogen, nitrite nitrogen, and hydroxylamine nitrogen after 6 days of degradation under different carbon-to-nitrogen ratios, and the OD of the strain. 600 value; Figure 6 The denitrification characteristics of strain AP3-14 at different initial ammonia nitrogen concentrations are shown in the figure. (a) shows the changes in ammonia nitrogen concentration before and after degradation at different initial ammonia nitrogen concentrations, as well as the removal rates of ammonia nitrogen and total dissolved nitrogen. (b) shows the changes in the concentrations of nitrate nitrogen, nitrite nitrogen, and hydroxylamine nitrogen after 6 days of degradation under different initial ammonia nitrogen concentrations, as well as the OD of the strain. 600 value; Figure 7 The denitrification characteristics of strain AP3-14 at different initial pH values ​​are shown in the figure. (a) shows the changes in ammonia nitrogen concentration and the removal rates of ammonia nitrogen, total dissolved nitrogen, and phenol before and after degradation at different initial pH values. (b) shows the changes in the concentrations of nitrate nitrogen, nitrite nitrogen, and hydroxylamine nitrogen and the OD values ​​of the strain after 6 days of degradation under different initial pH conditions. 600 value; Figure 8 This is a diagram of the hemolysis experiment of strain AP3-14; Figure 9 The image shows the results of the drug susceptibility test for strain AP3-14. Figure 10 The denitrification characteristics of strain AP3-14 under different antibiotic concentrations are shown in the figure. (a) shows the changes in ammonia nitrogen concentration and the removal rates of ammonia nitrogen and total dissolved nitrogen before and after degradation at different antibiotic concentrations. (b) shows the changes in the concentrations of nitrate nitrogen, nitrite nitrogen, and hydroxylamine nitrogen and the OD of the strain after 6 days of degradation under different antibiotic concentrations.600 value; Figure 11 The nitrogen balance distribution diagrams for strain AP3-14 in different reaction systems in Example 8 are shown, where (a) is the nitrogen balance distribution diagram in reaction system (1) and (b) is the nitrogen balance distribution diagram in reaction system (2). Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed embodiments are provided. For example, the present invention will be described in further detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods in the art. Unless otherwise specified, the raw materials and reagents used were obtained commercially.

[0021] Example 1 Isolation and screening of fecal alkaloid-producing bacteria AP3-14 Fresh activated sludge was obtained from the aerobic tank of a pharmaceutical wastewater treatment plant in Shijiazhuang City. 10 mL of fresh activated sludge was placed in a 250 mL Erlenmeyer flask containing 100 mL of LB medium and cultured at 30℃ and 120 rpm on a shaker. Every 2 days, the sludge was transferred to fresh LB medium at a 5% (v / v) ratio for enrichment twice. After enrichment, a 5% (v / v) bacterial suspension was transferred to a 250 mL Erlenmeyer flask containing 150 mL of nitrification medium and cultured at 30℃ and 120 rpm on a shaker for acclimatization. This process was repeated 10 times, transferring the suspension to fresh nitrification medium every 2 days. All media were subjected to continuous digestion at 121℃ for 30 min. After the bacteria were domesticated, they were serially diluted. Different concentrations of bacteria were spread onto solid nitrification medium, sealed, and inverted in a 30°C constant temperature incubator. When visible colonies were observed, single bacteria were isolated by streaking on the solid medium. The streaking was repeated 4 times to obtain purified single bacteria. The purified single bacteria were then transferred to a 4°C refrigerator for screening.

[0022] The purified single strains were sequentially inoculated into nitrification medium to verify their ammonia nitrogen degradation efficiency. Strains with an ammonia nitrogen removal efficiency of ≥90% were preserved, and the expanded bacterial culture was thoroughly mixed with sterile glycerol at a 6:4 ratio and then frozen. AP3-14 was among the single strains isolated with excellent performance. The culture media and formulations used in the above separation and screening steps include: LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH adjusted to 7.2, autoclaved at 121°C for 30 min.

[0023] Nitrification medium: CH3COONa 1.3667 g / L, C6H6O 1 g / L, (NH4)2SO4 0.472 g / L, Vickers salt solution (including K2HPO4·3H2O 0.75 g / L, NaH2PO4·2H2O 0.25 g / L, NaCl 0.12 g / L, MnSO4·H2O 0.01 g / L, MgSO4·7H2O 0.05 g / L, FeSO4·7H2O 0.01 g / L), autoclaved at 121℃ for 30 min.

[0024] Solid nitrification medium: CH3COONa 1.3667 g / L, C6H6O 1 g / L, (NH4)2SO4 0.472 g / L, Vickers salt solution (including K2HPO4·3H2O 0.75 g / L, NaH2PO4·2H2O 0.25 g / L, NaCl 0.12 g / L, MnSO4·H2O 0.01 g / L, MgSO4·7H2O 0.05 g / L, FeSO4·7H2O 0.01 g / L), agar 18 g / L, autoclaved at 121℃ for 30 min.

[0025] Buffer solution: NaCl 8 g / L, KCl 0.2 g / L, Na2HPO4·12H2O 3.63 g / L, KH2PO4 0.24 g / L, adjust pH to 7.4.

[0026] Example 2 Identification and whole-genome analysis of AP3-14, a fecal alkaloid-producing bacterium. Observe the morphology of strain AP3-14 obtained by isolation and screening in Example 1, and record its morphological characteristics: the colony diameter is approximately 0.5~1.5 mm (e.g., ...). Figure 1 As shown in the image, the colonies are opaque, white, slightly raised, with a moist and smooth surface and regular edges. They have a uniform texture, no special odor, and are easy to pick up.

[0027] After enrichment with strain AP3-14, the bacterial cells were centrifuged to obtain a precipitate. This precipitate was then fixed, dehydrated, replaced, and freeze-dried before scanning electron microscopy (SEM) experiments to observe the morphology and size of the bacteria. (Specific details are as follows...) Figure 2 As shown, the AP3-14 strain is short rod-shaped.

[0028] Genomic DNA was extracted from this strain, and PCR products were amplified using the universal primers 27F / 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3') for bacterial 16S rRNA gene amplification. The resulting PCR products were sent to Shanghai Meiji Biotechnology Co., Ltd. for sequence sequencing, and their species relationship was identified through comparison analysis in the NCBI database. The strain AP3-14 was found to be related to... Alcaligenes faecalis The strain was closest in distance and had the highest similarity (99.93%). Based on the above results, the strain of this invention was preliminarily identified as *Alcaligenes faecalis*, and its taxonomic name is *Alcaligenes faecalis*. Alcaligenes faecalis AP3-14. The 16S rRNA gene sequence of strain AP3-14 is shown in SEQ No. 1.

[0029] Whole-genome analysis of strain AP3-14 revealed the presence of the key gene cluster DnfABC (a key gene cluster for direct ammonia oxidation in the model strain Alcaligenes ammonioxydans HO-1). Specifically, DnfA exhibited 100% coverage and 96.85% homology, DnfB 100% coverage and 95.71% homology, and DnfC 100% coverage and 97.92% homology. Based on the combined physicochemical and genetic results, this strain was identified as a novel biological denitrification microorganism—a "direct ammonia oxidizer." The genetic information of the DnfABC gene cluster is shown in Table 1. The sequence of DnfA is shown in SEQ No. 2, the sequence of DnfB is shown in SEQ No. 3, and the sequence of DnfC is shown in SEQ No. 4.

[0030] Table 1. DnfABC information table for strain AP3-14

[0031] This strain AP3-14 was deposited on November 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36650, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its taxonomic name is *Alcaligenes faecalis*. Alcaligenes faecalis .

[0032] Example 3 This example studies the growth and denitrification characteristics of strain AP3-14 under different carbon-nitrogen ratios. (1) Sodium acetate is the carbon source The activated strain AP3-14 bacterial suspension was inoculated into NH4 at a ratio of 5% (v / v). +In fresh nitrified medium with -N=100mg / L and C / N ratios of 2, 4, 6, 8, 10, 12, 14 and 16 (NH4+ fixation) + (The N-N concentration was kept constant, and the carbon-to-nitrogen ratio was controlled by changing the sodium acetate content). The mixture was incubated at 30℃ and 120 rpm for 6 days with constant temperature shaking. OD values ​​were measured at the beginning and end of the incubation period. 600 Nitrate nitrogen (NO3) - -N), ammonia nitrogen (NH4) + -N), nitrite nitrogen (NO2) - -N), hydroxylamine nitrogen (NH2OH-N), dissolved total nitrogen (TDN), and total nitrogen (TN).

[0033] For example, with a carbon-to-nitrogen ratio of 12, the nitrification medium formula is as follows: CH3COONa 4.1 g / L, (NH4)2SO4 0.472 g / L, Vickers salt solution (including K2HPO4·3H2O 0.75 g / L, NaH2PO4·2H2O 0.25 g / L, NaCl 0.12 g / L, MnSO4·H2O 0.01 g / L, MgSO4·7H2O 0.05 g / L, FeSO4·7H2O 0.01 g / L), autoclaved at 121℃ for 30 min.

[0034] The results are as follows Figure 3 As shown, the growth of the strain is directly proportional to the carbon-nitrogen ratio of the culture medium; the higher the carbon-nitrogen ratio, the stronger the strain's growth ability. When the carbon-to-nitrogen ratios were 2, 4, 6, 8, 10, 12, 14, and 16, the ammonia nitrogen removal rates of strain AP3-14 were 43.22±3.13%, 62.81±2.52%, 73.82±2.21%, 79.37±0.49%, 87.12±0.35%, 91.65±1.77%, 92.48±0.80%, and 95.14±0.08%, respectively; the total dissolved nitrogen removal rates were 36.23±4.38%, 51.31±2.27%, 59.79±2.83%, 67.33±0.52%, 73.04±1.27%, 74.17±1.15%, 77.88±83.34%, and 83.34±0.76%, respectively. A small amount of hydroxylamine nitrogen and nitrite nitrogen were also detected during the ammonia nitrogen removal process. Furthermore, the ammonia nitrogen removal rate of the strains reached over 90% when the carbon-to-nitrogen ratio was 12, 14, and 16.

[0035] (2) Sodium acetate was used as the carbon source and 1000 mg / L phenol was added. The activated strain AP3-14 was inoculated into NH4 at a ratio of 5% (v / v). +-N=100mg / L, with sodium acetate as the carbon source and carbon-to-nitrogen ratios of 2, 4, 6, 8, and 10, and all supplemented with 1000mg / L phenol (fixing NH4). + (The N-N concentration was kept constant, and the carbon-to-nitrogen ratio was controlled by changing the sodium acetate content). The mixture was incubated at 30℃ and 120 rpm for 6 days with constant temperature shaking. OD values ​​were measured at the beginning and end of the incubation period. 600 Nitrate nitrogen (NO3) - -N), ammonia nitrogen (NH4) + -N), nitrite nitrogen (NO2) - The concentrations of hydroxylamine nitrogen (NH2OH-N), total dissolved nitrogen (TDN), total nitrogen (TN), and phenol (C6H6O) were measured.

[0036] For example, with a carbon-to-nitrogen ratio of 4, the nitrification medium formula is as follows: CH3COONa 1.3667 g / L, C6H6O 1 g / L, (NH4)2SO4 0.472 g / L, Vickers salt solution (including K2HPO4·3H2O 0.75 g / L, NaH2PO4·2H2O 0.25 g / L, NaCl 0.12 g / L, MnSO4·H2O 0.01 g / L, MgSO4·7H2O 0.05 g / L, FeSO4·7H2O 0.01 g / L), autoclaved at 121℃ for 30 min.

[0037] The results are as follows Figure 4 As shown, in the culture medium supplemented with phenol, the growth of the strain is directly proportional to the carbon-to-nitrogen ratio of the medium, and the growth capacity of the strain generally increases with the increase of the carbon-to-nitrogen ratio. When the carbon-to-nitrogen ratios are 2, 4, 6, 8, and 10, the removal rates of ammonia nitrogen by strain AP3-14 are 82.60±0.76%, 96.54±0.07%, 91.62±0.85%, 96.97±0.45%, and 88.96±2.06%, respectively; the removal rates of dissolved total nitrogen are 67.94±1.01%, 87.32±1.00%, 85.61±0.36%, 87.51±0.95%, and 86.54±2.12%, respectively. Furthermore, a small amount of hydroxylamine nitrogen and nitrite nitrogen were detected to accumulate during the ammonia nitrogen removal process.

[0038] Furthermore, when the carbon-to-nitrogen ratio was between 2 and 8, strain AP3-14 exhibited a higher phenol removal rate at 2 and 4 days, indicating that the strain could tolerate phenol toxicity and utilize phenol as a carbon source for denitrification after utilizing sodium acetate. At 6 days, the phenol removal rates for different carbon-to-nitrogen ratios were 100.00±0.00%, 100.00±0.00%, 100.00±0.00%, 74.46±9.05%, and 95.07±3.60%, respectively. This demonstrates that strain AP3-14 can utilize phenol as a carbon source for growth and denitrification, achieving simultaneous degradation of phenol and ammonia nitrogen. However, when a large amount of sodium acetate was added, the strain's utilization of phenol was delayed, indicating that when a mixed carbon source was added, the strain utilized the carbon source in the order of sodium acetate followed by phenol.

[0039] (3) Phenol is the carbon source The activated strain AP3-14 bacterial suspension was inoculated into NH4 at a ratio of 5% (v / v). + -N=100mg / L, in fresh nitrified media with carbon-to-nitrogen ratios of 2, 4, 6, 8 and 10 using phenol as the carbon source (fixed NH4) + (The -N concentration remained constant, and the carbon-to-nitrogen ratio was controlled by changing the phenol content). The mixture was incubated at 30℃ and 120 rpm for 6 days with constant temperature shaking. OD values ​​were measured at the beginning and end of the incubation period. 600 Nitrate nitrogen (NO3) - -N), ammonia nitrogen (NH4) + -N), nitrite nitrogen (NO2) - The concentrations of hydroxylamine nitrogen (NH2OH-N), total dissolved nitrogen (TDN), total nitrogen (TN), and phenol (C6H6O) were measured.

[0040] For example, with a carbon-to-nitrogen ratio of 8, the nitrification medium formula is as follows: C6H6O 1.044 g / L, (NH4)2SO4 0.472 g / L, Vickers salt solution (including K2HPO4·3H2O 0.75 g / L, NaH2PO4·2H2O 0.25 g / L, NaCl 0.12 g / L, MnSO4·H2O 0.01 g / L, MgSO4·7H2O 0.05 g / L, FeSO4·7H2O 0.01 g / L), autoclaved at 121℃ for 30 min.

[0041] The results are as follows Figure 5As shown, the growth of the strain is directly proportional to the carbon-to-nitrogen ratio (except when the carbon-to-nitrogen ratio is 10, the strain shows no significant growth). The higher the carbon-to-nitrogen ratio, the stronger the strain's growth ability. When the carbon-to-nitrogen ratios are 2, 4, 6, 8, and 10, the removal rates of ammonia nitrogen by strain AP3-14 are 25.95±1.91%, 44.52±2.15%, 52.72±1.04%, 58.86±1.20%, and 6.29±1.48%, respectively; the removal rates of dissolved total nitrogen are 25.23±2.22%, 40.21±1.43%, 45.06±1.63%, 55.42±1.61%, and 5.25±1.45%, respectively. Furthermore, a small amount of hydroxylamine nitrogen and nitrite nitrogen were detected during the ammonia nitrogen removal process. The removal rates of phenol by the strain at carbon-to-nitrogen ratios of 2–10 were 100.00±0.00%, 99.99±0.03%, 100.00±0.00%, 100.00±0.00%, and 6.87±1.36%, respectively. This indicates that strain AP3-14 not only tolerates the biotoxicity of phenol but can also utilize phenol as the sole carbon source for denitrification, further verifying that strain AP3-14 can achieve simultaneous degradation of phenol and ammonia nitrogen. At a carbon-to-nitrogen ratio of 10, the strain showed no significant growth and no significant removal of ammonia nitrogen, total dissolved nitrogen, or phenol. This is because the toxicity of phenol has a detrimental effect on strain AP3-14, leading to its death and preventing the strain from utilizing phenol as an energy source and thus hindering the denitrification reaction.

[0042] Example 4 This study investigated the growth and nitrogen removal of strain AP3-14 under different initial ammonia nitrogen concentrations. The activated AP3-14 bacterial culture was transferred at a ratio of 5% to fresh nitrification medium with a carbon-to-nitrogen ratio of 12 and different ammonia nitrogen concentrations (20, 50, 100, 250, 500, and 1000 mg / L). The medium was incubated at 30°C with shaking at 120 rpm for 6 days. OD values ​​were measured at the beginning and end of the incubation period. 600 Nitrate nitrogen (NO3) - -N), ammonia nitrogen (NH4) + -N), nitrite nitrogen (NO2) - -N), hydroxylamine nitrogen (NH2OH-N), dissolved total nitrogen (TDN), and total nitrogen (TN). The fresh nitrification medium used was the same as that used in Section (1) of Example 2 (carbon-nitrogen ratio of 12, NH4+). + -N=100mg / L).

[0043] The results are as follows Figure 6As shown, the growth of strain AP3-14 is basically proportional to the ammonia nitrogen concentration; the higher the ammonia nitrogen concentration, the stronger the strain's growth ability. When the carbon-to-nitrogen ratio is 12 and the ammonia nitrogen concentrations are 20, 50, 100, 250, 500, and 1000 mg / L, the ammonia nitrogen removal rates of strain AP3-14 are 92.04±1.63%, 91.07±0.61%, 88.93±2.47%, 60.77±4.07%, 45.28±1.77%, and 27.35±1.56%, respectively; the removal rates of dissolved total nitrogen are 63.86±2.22%, 57.52±2.86%, 75.11±1.22%, 57.29±3.51%, 39.60±2.21%, and 15.66±2.27%. This indicates that strain AP3-14 has a wide tolerance range for ammonia nitrogen concentration and can achieve denitrification in environments with high concentrations of ammonia nitrogen.

[0044] Example 5 This study investigated the growth and nitrogen removal of strain AP3-14 under different initial pH values. The activated AP3-14 bacterial culture was transferred at a ratio of 5% to fresh nitrification medium with a carbon-to-nitrogen ratio of 4 and 1000 mg / L phenol added (initial pH values ​​were 6, 7, 8, 9, 10, 11, and 12). The medium was incubated at 30°C with shaking at 120 rpm for 6 days. OD values ​​were measured at the beginning and end of the incubation period. 600 Nitrate nitrogen (NO3) - -N), ammonia nitrogen (NH4) + -N), nitrite nitrogen (NO2) - -N), hydroxylamine nitrogen (NH2OH-N), dissolved total nitrogen (TDN), total nitrogen (TN), and phenol (C6H6O). The fresh nitrification medium used was the same as that used in Section (2) of Example 3 (C / N ratio of 4).

[0045] The results are as follows Figure 7As shown, strain AP3-14 can grow well and achieve efficient nitrogen removal at pH 6 to 11. Although its growth is poor at pH 12, it can still remove most of the nitrogen. When the carbon-to-nitrogen ratio was 4 and 1000 mg / L phenol was added, and the pH range was 6–12, the removal rates of ammonia nitrogen by strain AP3-14 were 95.11±1.91%, 92.50±1.25%, 94.42±0.31%, 97.84±0.14%, 96.68±0.35%, 97.97±0.61%, and 77.40±2.09%, respectively; and the removal rates of phenol were 100.00±0.00%, 100.00±0.00%, 100.00±0.00%, 82.75±4.60%, 30.94±4.62%, 9.95±3.13%, and 0.58±0.88%, respectively. This shows that when the carbon source concentration is fixed, the removal rate of ammonia nitrogen by the strain reaches more than 90% in the pH range of 6-11, while the removal rate of phenol decreases with the increase of pH. This indicates that strain AP3-14 has a wide pH adaptation range and requires less carbon source in a weakly alkaline environment, that is, it is more suitable for growth and denitrification in a weakly alkaline environment.

[0046] Example 6 This example demonstrates the safety assessment of strain AP3-14. (1) Inoculate strain AP3-14 onto BA (blood agar) plates. Pick strain AP3-14 from the preserved plates and incubate at 30℃ for 48 hours using the streak method. The hemolytic activity of the strain is determined by observing the hemolytic area around the colonies. The hemolytic activity assessment results are as follows: Figure 8 As shown, strain AP3-14 can grow normally on BA plates and no hemolytic zone was observed around the bacteria, thus confirming that strain AP3-14 does not have hemolytic activity.

[0047] The BA culture medium comprises: 1.5 g / L soluble starch, 6 g / L beef extract, 17.5 g / L acid-hydrolyzed casein, 25 ml / L defibrinated sheep blood, and 18 g / L agar.

[0048] (2) Antimicrobial susceptibility testing of strain AP3-14 against 31 different antibiotics was conducted using the disc diffusion method. 0.2 mL of a mixed bacterial culture that had been cultured in nitrified medium for 3 days was evenly spread onto the surface of LB solid medium. The plate was placed on a clean bench and allowed to stand for 2-3 minutes until the bacterial culture dried. Antimicrobial susceptibility discs were then placed on the plate surface using sterile forceps and gently pressed to ensure tight adhesion. The plate was then incubated at 35°C for 18-24 hours. Three replicates were set up for each experiment. After incubation, the diameter of the inhibition zone was measured with calipers. Results were judged according to the antibiotic susceptibility criteria of the Clinical and Laboratory Standards Institute (CLSI). If the diameter of the inhibition zone was ≥20 mm, the strain was classified as highly sensitive (S); if the diameter was between 14 and 20 mm, it was considered moderately sensitive (I); and if the diameter was ≤14 mm, it was classified as resistant (R). The results of the antimicrobial susceptibility test are as follows: Figure 9 As shown in Table 2, the results of the antibiotic resistance test show that strain AP3-14 exhibited high sensitivity to 14 of the 31 tested antibiotics, especially β-lactams (ceftriaxone), and moderate sensitivity to 9 other antibiotics. This indicates that it has good overall antibacterial sensitivity characteristics in potential environmental applications, and its use will not damage the original ecological environment, thus greatly reducing potential ecological risks.

[0049] Table 2. Antibiotic resistance results of strain AP3-14

[0050] Example 7 This example studies the denitrification characteristics of strain AP3-14 under a tetracycline antibiotic background. The activated seed culture of strain AP3-14 was inoculated at a ratio of 5% into fresh nitrification medium containing 1, 5, and 10 mg / L tetracycline hydrochloride (TC) and 1, 5, and 10 mg / L oxytetracycline hydrochloride (OTC), respectively, with shaking at 30°C and 120 rpm for 6 days. OD values ​​were measured at the beginning and end of the incubation period. 600 Nitrate nitrogen (NO3) - -N), ammonia nitrogen (NH4) + -N), nitrite nitrogen (NO2) - The nitrification medium used was the same as that used in Section (1) of Example 2.

[0051] The results are as follows Figure 10As shown, when the reaction time was 6 days, compared with no antibiotics, the addition of 1, 5, and 10 mg / L tetracycline hydrochloride and oxytetracycline hydrochloride had no significant effect on the growth of strain AP3-14 and its ammonia nitrogen removal efficiency. At 6 days, the addition of different concentrations of antibiotics (TC and OTC) all resulted in ammonia nitrogen removal rates exceeding 90%. This indicates that strain AP3-14 can tolerate the toxicity of tetracycline antibiotics and perform denitrification, meaning that this strain has a broad tolerance to tetracycline antibiotics and can be applied to the treatment of wastewater containing tetracycline antibiotics.

[0052] Example 8 This example studies the gaseous products of strain AP3-14. Using isotope labeling, the activated strain AP3-14 was centrifuged and resuspended in PBS buffer, and inoculated into two reaction systems at a ratio of 5% respectively: (1) phenol as carbon source, carbon-nitrogen ratio of 8 15 In N-nitrified medium; (2) sodium acetate as carbon source, carbon-nitrogen ratio of 1:2, 10 mg / L tetracycline hydrochloride added. 15 In N-nitrification medium, the reaction was carried out in a 2.5L sealed anaerobic flask with the headspace replaced with 99.99% high-purity oxygen. The mixture was incubated at 30°C with constant temperature shaking at 120 rpm. Nitrogen balance (including NO3-) was measured at the beginning and end of the reaction. - -N,NH4 + -N, NO2 - -N, NH2OH-N, TDN, TN), phenol (C6H6O), and collect the headspace gas at the end of the reaction into a 100mL gas bag, and determine the N2O content at the end of the reaction using GC-MS. Among them, the fresh nitrification medium used in reaction system (1) is the same as the medium used in section (3) of Example 3 (nitrogen ratio of 8); the fresh nitrification medium used in reaction system (2) is the same as the medium used in Example 7 (with 10mg / L tetracycline hydrochloride added), wherein (NH4)2SO4 is replaced with ( 15 NH4)2SO4.

[0053] The results showed that, after 12 days of reaction, the nitrogen transformation of strain AP3-14 in reaction system (1) was as follows: Figure 11 As shown in (a), it can be seen that during the nitrogen transformation process, hydroxylamine nitrogen accumulated, accounting for 0.70±0.05%, and nitrogen loss accounted for 14.66±0.83%, with the N2O conversion rate measured as 0% (as shown in Table 3); the nitrogen transformation results of strain AP3-14 in reaction system (2) after 6 days of reaction are as follows Figure 11As shown in (b), similarly, hydroxylamine nitrogen accumulated during nitrogen transformation, with a nitrogen loss rate of 26.35 ± 0.87%, and the measured N2O conversion rate was only 0.19%. These results confirm that the nitrogen transformation process of the strain includes direct ammonia oxidation (NH4+). + The results also indicate that the conversion rate of greenhouse gas N2O during nitrogen transformation by strain AP3-14 is extremely low, with almost all nitrogen loss being converted into N2 and emitted into the air. This confirms that strain AP3-14 will not cause air pollution in practical applications.

[0054] Table 3. N2O determination results for different reaction systems

[0055] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A strain of fecal alkali-producing bacteria that directly oxidizes ammonia ( Alcaligenes faecalis AP3-14, characterized in that, It was deposited on November 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36650, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of the fecal alkaloid-producing bacteria AP3-14 as described in claim 1 in the denitrification and phenol removal treatment of sewage or wastewater under aerobic conditions.

3. The application of the fecal alkaloid-producing bacteria AP3-14 as described in claim 2 in the denitrification or phenol removal treatment of sewage or wastewater, characterized in that, The wastewater or sewage includes landfill leachate, municipal sewage, livestock wastewater, pharmaceutical wastewater, kitchen wastewater, coal chemical wastewater, fine chemical wastewater, textile dyeing and printing wastewater, and papermaking wastewater.

4. The application of the fecal alkaloid-producing bacteria AP3-14 as described in claim 2 in the denitrification or phenol removal treatment of sewage or wastewater, characterized in that, The bacterial solution of the fecal alkaline bacteria AP3-14 was inoculated into sewage or wastewater at a volume ratio of 4%-8%, mixed evenly, and decomposed at 25-35℃.

5. The application of the fecal alkaloid-producing bacteria AP3-14 as described in claim 2 in the denitrification or phenol removal treatment of sewage or wastewater, characterized in that, The pH of the wastewater or sewage is 6-11, and the carbon-to-nitrogen ratio is 2-16.

6. The application of the fecal alkaloid-producing bacteria AP3-14 as described in claim 2 in the denitrification or phenol removal treatment of sewage or wastewater, characterized in that, The ammonia nitrogen content in the wastewater is 0-1000 mg / L, and the phenol content is 0-1100 mg / L.

7. A microbial inoculant, characterized in that, Includes the fecal alkaloid bacterium AP3-14 as described in claim 1.

8. The application of the microbial agent according to claim 7 in the denitrification or dephenolization treatment of sewage or wastewater.

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