Efficient nitrogen and phenol removal strain and application thereof

Abstract

The invention belongs to the technical field of environmental microorganisms, and discloses an efficient nitrogen and phenol removal strain and application thereof. The efficient nitrogen and phenol removal bacterial strain is citronellol pseudomonas, and the citronellol pseudomonas is named as citronellol pseudomonas BY-1. The strain is preserved in the China Center for Type Culture Collection, and the preservation number of the strain is CCTCC No.M 2025818. The 16S rDNA sequence of the gene is as shown in SEQ ID No: 1. The strain citronellol pseudomonas BY-1 provided by the invention can obviously reduce the nitrogen content of wastewater in high C / N ratio wastewater treatment, and has the effect of enhancing nitrogen and carbon removal. The nitrogen content and the phenol content of the wastewater can be remarkably reduced in the phenol-containing wastewater, and the effect of enhancing nitrogen and phenol removal is achieved.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, and specifically relates to a highly efficient nitrogen- and phenol-removing bacterial strain and its applications. More specifically, it relates to a heterotrophic nitrifying-aerobic denitrifying bacterium, BY-1, and its nitrogen- and phenol-removing applications. Background Technology

[0002] Biological nitrogen removal is widely used in wastewater treatment processes due to its low cost and environmental friendliness. Traditional biological nitrogen removal processes achieve nitrogen removal through the coupling of two continuous reactions: nitrification and denitrification. These processes are completed by aerobic nitrifying bacteria and anoxic denitrifying bacteria under different environmental conditions, respectively, to remove NH4+ from wastewater. + -N and COD are converted into N2 and CO2, respectively, thus achieving the simultaneous removal of carbon and nitrogen. However, in order to meet the growth requirements of different functional microorganisms, this process requires the configuration of multi-stage treatment structures, which leads to problems such as complex process design, high construction costs, and unstable performance.

[0003] Heterotrophic nitrification-aerobic denitrification (HN-AD) is the process by which a single microorganism (HN-AD bacteria) removes NH4 under aerobic and heterotrophic conditions through simultaneous heterotrophic nitrification and aerobic denitrification. + -N, NO3 - -N and NO2 - -N. As aerobic heterotrophic bacteria, HN-AD strains exhibit higher growth rates and nitrogen removal efficiencies than traditional nitrifying and denitrifying bacteria. Furthermore, because simultaneous nitrogen and carbon removal can be achieved within a single structure, this process effectively alleviates operational complexity and significantly reduces land requirements and construction costs. HN-AD strains are widely sourced, isolated from environments such as lakes, soils, wetlands, and sludge. Currently, over 20 functional genera of HN-AD bacteria have been identified, including *Pseudomonas*, *Alcaligenes*, *Acinetobacter*, *Flavobacterium*, *Bacillus*, and *Trichomonas*, demonstrating significant potential for treating various types of wastewater, such as swine wastewater, marine aquaculture wastewater, and saline wastewater.

[0004] Phenolic wastewater is a typical type of industrial wastewater, mainly originating from industries such as petrochemicals, pharmaceuticals, coking, pesticides, and dyes. It is characterized by high toxicity and poor biodegradability, posing significant challenges in biological treatment. Studies have shown that phenolic compounds can serve as an organic carbon source for nitrogen metabolism during the HN-AD process, thereby achieving the simultaneous removal of nitrogen and phenolic pollutants from phenolic wastewater. This demonstrates the great potential of HN-AD technology in the treatment of phenolic wastewater. However, currently, the reported microbial resources capable of simultaneously removing nitrogen and phenols are limited, and related research is still in its early stages.

[0005] Therefore, screening high-performance strains capable of simultaneously removing nitrogen and phenols is of significant research importance. This not only helps enrich the HN-AD functional strain library but also determines the adaptability and operational stability of these strains in practical wastewater treatment systems. More importantly, it provides a crucial scientific foundation for the practical application of simultaneous nitrogen-phenol removal technology. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a highly efficient nitrogen-removing and phenol-removing strain and its application. The strain *Pseudomonas citronellolis* BY-1 provided by this invention can significantly reduce the nitrogen content of wastewater with a high C / N ratio, demonstrating enhanced nitrogen and carbon removal effects. In phenol-containing wastewater, it can significantly reduce both nitrogen and phenol content, exhibiting enhanced nitrogen and phenol removal effects.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] The first objective of this invention is to provide a highly efficient nitrogen- and phenol-removing strain, which is a heterotrophic nitrifying, aerobic, and denitrifying strain that simultaneously removes nitrogen and phenols. This highly efficient nitrogen- and phenol-removing strain is *Pseudomonas citronellolis*, named *Pseudomonas citronellolis* BY-1. It has been deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC No. M 2025818. The deposit address is Wuhan University, Wuhan, Hubei Province, China, and the deposit date is April 18, 2025.

[0009] The biological characteristics of the above-mentioned *Pseudomonas citronellolis* BY-1 are as follows:

[0010] (1) It can grow on liquid culture media with (NH4)2SO4 as the sole nitrogen source or with a mixture of (NH4)2SO4 and NaNO3 as nitrogen sources.

[0011] (2) It can grow on liquid culture media with sodium succinate, sodium citrate, sodium acetate, phenol and other carbon sources.

[0012] (3) The colonies are round, smooth and moist, milky white and opaque. Scanning electron microscopy (SEM) results show that the cells of strain BY-1 are rod-shaped with elliptical ends and wavy cell walls, which makes the surface of the bacteria slightly rough.

[0013] (4) The full-length 16S rDNA sequence of strain Pseudomonas citronellolis BY-1 contains 1421 base pairs.

[0014] The second objective of this invention is to request protection for a screening method for the above-mentioned strain *Pseudomonas citronellol* BY-1, comprising the following steps:

[0015] Take 100 mL of activated sludge (washed with physiological saline) from the aerobic tank of a coking wastewater treatment plant of Anshan Iron and Steel Group in Anshan City, Liaoning Province, and place it in a 250 mL Erlenmeyer flask. Add a few glass beads and shake in a shaker (30±0.5℃, 150 r / min) for 48 h. After standing, use the supernatant as the inoculum. Take 5 mL of the supernatant and incubate it in 100 mL of fresh basal culture medium. Repeat this process three times. Then, perform serial dilutions of the inoculum, diluting it to 10⁻⁶ each time. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 and 10 -8 Gradient. Under aseptic conditions, 10 -4 10 -6 and 10 -8 Take 200 μL of bacterial suspension from each gradient and spread it evenly on the surface of solid basal medium. Incubate at 30℃ for 1-3 days. Select single colonies with good growth and purify them further on new solid basal medium. Repeat this process three times to obtain a single strain. Store the single strain in a -80℃ freezer.

[0016] Furthermore, the formulation of the basal culture medium (g / L) is as follows:

[0017] (NH4)2SO4 0.472, C6H5OH 1.0, Na2HPO4 4.26, KH2PO4 2.65, MgSO4·7H2O 0.2, NaCl 0.5 and 1 mL of trace elements, pH 7.2.

[0018] Furthermore, the formulation of the solid basal culture medium (g / L) is as follows:

[0019] (NH4)2SO4 0.472, C6H5OH 1.0, Na2HPO4 4.26, KH2PO4 2.65, MgSO4·7H2O 0.2, NaCl 0.5, agar powder 18 and 1 mL trace elements, pH 7.2.

[0020] A third objective of this invention is to request protection for the application of the above-mentioned strain *Citronellol Pseudomonas* BY-1 in simultaneous denitrification and phenol removal.

[0021] Application method one specifically refers to its application in biological denitrification of aquatic bodies. The biological denitrification of aquatic bodies is an enhanced denitrification process for wastewater with a high carbon-to-nitrogen ratio. The bacterial culture of *Pseudomonas citronellolis* BY-1 is inoculated into the wastewater with a high carbon-to-nitrogen ratio at an inoculum rate of 0.8-1.2%, and cultured at 25-35℃ and 150-250 rpm for 9-24 hours.

[0022] The preparation method of bacterial suspension is as follows: the slant culture of Pseudomonas citronellolis BY-1 strain is inoculated into LB liquid medium and cultured at 25-35℃ and 150-250rpm for 12-24h. Then, the precipitate is obtained by centrifugation and the precipitate is resuspended in PBS to obtain bacterial suspension.

[0023] Application method two specifically involves the simultaneous denitrification and phenol removal of phenol-containing wastewater. The aforementioned *Pseudomonas citronellolis* BY-1 is inoculated into the phenol-containing wastewater and cultured. The bacterial culture of *Pseudomonas citronellolis* BY-1 is inoculated into the phenol-containing wastewater at an inoculum rate of 1.0-10.0%, and cultured at 25-35℃ and 150-250 rpm for 16-144 hours.

[0024] The preparation method of bacterial suspension is as follows: the slant culture of Pseudomonas citronellolis BY-1 strain is inoculated into LB liquid medium and cultured at 25-35℃ and 150-250rpm for 12-24h. Then, the precipitate is obtained by centrifugation and the precipitate is resuspended in PBS to obtain the bacterial suspension.

[0025] The formulation of LB medium (g / L) is as follows:

[0026] NaCl 10, peptone 10, yeast extract 5.

[0027] The third application method involves biological removal of combined nitrogen and degradation of phenols in water. It contains the active ingredient *Pseudomonas citronellolis* BY-1.

[0028] The advantages of this invention compared to the prior art are:

[0029] 1. The *Pseudomonas citronellolis* BY-1 provided by this invention can significantly reduce the content of COD and total nitrogen (TN) in wastewater with a high C / N ratio, thus enhancing the denitrification and carbon removal effect.

[0030] 2. The *Pseudomonas citronellolis* BY-1 provided by this invention can simultaneously perform heterotrophic nitrification and aerobic denitrification, and can treat bacteria containing NH4+. + -N, NO2 - -N and NO3 - -N wastewater. In the form of NH4+ + When -N is the nitrogen source, after 16 hours of cultivation, NH4 + -N removal rate reached 100%, and NO2 was eliminated. - -N and NO3 - -N accumulation; with NH4 + -N and NO3 - When -N is a mixed nitrogen source, after 16 hours of cultivation, NH4 + -N, NO3 - The removal rates of -N and TN reached 100%, 86.60%, and 65.06%, respectively.

[0031] 3. The citronellolis Pseudomonas BY-1 provided by this invention can tolerate high concentrations of phenol and has a high phenol degradation rate.

[0032] 4. The *Pseudomonas citronellolis* BY-1 provided by this invention can significantly reduce the nitrogen and phenol content in phenol-containing wastewater during wastewater treatment, demonstrating enhanced denitrification and phenol removal effects. (Using NH4+) + When -N is the nitrogen source, phenol and NH4 + The removal ratio of -N is 12:1. Attached Figure Description

[0033] Figure 1 This is a colony characteristic diagram of Pseudomonas BY-1 in this invention.

[0034] Figure 2 This is a microscopic image of the cell characteristics of BY-1 bacteria as described in this invention.

[0035] Figure 3 This is the phylogenetic tree of BY-1 in this invention, constructed using the neighbor-joining method based on the 16S rDNA sequence.

[0036] Figure 4Different carbon sources were used to simulate wastewater NH4 in BY-1 in Example 2 of this invention. + -N denitrification results diagram.

[0037] Figure 5 The different C / N ratios of BY-1 used in Example 3 of this invention simulate wastewater NH4 + -N denitrification results diagram.

[0038] Figure 6 This is a graph showing the results of nitrogen and phenol removal from simulated wastewater using different nitrogen sources in Example 4 of the present invention, BY-1. (A) represents the results using NH4+. + -N represents the nitrogen source, and (B) represents NO3. - -N represents the nitrogen source, and (C) represents NH4. + -N and NO3 - -N represents a mixed nitrogen source diagram.

[0039] Figure 7 This is a graph showing the effect of BY-1 in Example 7 of the present invention on the removal of nitrogen, phenols, and COD from actual coking wastewater. (A) shows the change in nitrogen pollutant concentration, and (B) shows the change in phenolic pollutant and COD concentration. Detailed Implementation

[0040] The present invention is described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0041] Example 1

[0042] Isolation, screening and identification of Pseudomonas citronellolis BY-1

[0043] Take 100 mL of activated sludge (washed with physiological saline) from the aerobic tank of a coking wastewater treatment plant of Anshan Iron and Steel Group in Anshan City, Liaoning Province, and place it in a 250 mL Erlenmeyer flask. Add a few glass beads and shake in a shaker (30±0.5℃, 150 r / min) for 48 h. After standing, use the supernatant as the inoculum. Take 5 mL of the supernatant and culture it in 100 mL of fresh basal culture medium (formula: (NH4)2SO4 0.472 g / L, C6H5OH 1.0 g / L, Na2HPO4 4.26 g / L, KH2PO4 2.65 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.5 g / L and trace elements 1 mL, pH 7.2). Repeat this process three times. Then, perform serial dilutions on the bacterial culture, diluting each solution to 10⁻⁶. -1 10 -2 10 -3 10-4 10 -5 10 -6 10 -7 and 10 -8 Isogradient. Under aseptic conditions, 10 -4 10 -6 and 10 -8 Take 200 μL of bacterial suspension at each of the same gradients and spread it evenly on the surface of a solid basal medium (formula: (NH4)2SO4 0.472 g / L, C6H5OH 1.0 g / L, Na2HPO4 4.26 g / L, KH2PO4 2.65 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.5 g / L, agar powder 18 g and trace elements 1 mL, pH 7.2). Incubate at 30℃ for 1-3 days. Select single colonies with good growth and purify them further on a new solid basal medium. Repeat this process three times to obtain a single strain. Store the single strain in a -80℃ freezer.

[0044] Morphological identification: Colonies are round, smooth and moist, milky white, and opaque. Figure 1 Scanning electron microscopy (SEM) results showed that the cells of strain BY-1 were rod-shaped with elliptical ends, and the cell wall had a wavy structure, making the cell surface appear slightly rough. Figure 2 ).

[0045] Molecular biological identification: DNA was extracted from the screened strains using a bacterial genome extraction kit. PCR amplification of the conserved target fragment was performed using universal primers 27F and 1492R for the 16S rDNA gene. The amplified products were then recovered from the gel and sequenced. PCR amplification, sequencing, and BLAST alignment showed that the 16S rDNA sequence of strain BY-1 had a high similarity to *Pseudomonas citronellolis*, with a similarity exceeding 99% to *Pseudomonas citronellolis* strain NBRC 103043. Figure 3 Therefore, strain BY-1 was identified as *Pseudomonas citronellolis* BY-1.

[0046] Example 2

[0047] Carbon source adaptation study of Pseudomonas citronellolis BY-1

[0048] The slant seed culture of *Pseudomonas citronellolis* BY-1 was transferred to 100 mL of LB liquid medium and cultured at 30 °C and 150 rpm for 12 h. The cultured bacterial suspension was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the cells were washed three times with sterile PBS solution. Finally, the suspension was resuspended in 10 mL of PBS solution to obtain the *Pseudomonas citronellolis* BY-1 bacterial suspension.

[0049] Transfer 1.0 mL of bacterial culture to 100 mL of NH4+. + -N simulated wastewater (100mg / LNH4) + -N, the substrate general-purpose culture medium contains Na2HPO4 4.26 g / L, KH2PO4 2.65 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.5 g / L, and 1 mL / L trace elements, pH 7.2. The trace element solution formula is: 100 mg / L EDTA, 4.4 mg / L ZnSO4, 11 mg / L CaCl2, 10.2 mg / L MnCl2, 10 mg / L FeSO4·7H2O, 2.2 mg / L (NH4)6Mo7O 24 ·4H2O, 3.2 mg / L CuSO4·5H2O, 3.2 mg / L COCl2·6H2O), sodium succinate, sodium citrate, sodium acetate, and sucrose were selected as carbon sources and added to NH4. + In simulated wastewater containing -N, the optimal carbon source available to *Pseudomonas citronellolis* BY-1 was investigated. The samples were cultured at 30℃ and 150 rpm for 9 h, with 5 mL samples taken every 3 h and stored at 4℃. NH4+ levels in the samples were determined using Nessler's reagent spectrophotometry. + -N concentration. Results are as follows: Figure 4 As shown, when sodium succinate and sodium citrate were used as carbon sources, strain *Pseudomonas citronellolis* BY-1 exhibited high ammonia removal performance and growth rate. After 9 hours, the ammonia removal concentrations were 81.86 ± 2.62 mg / L and 73.34 ± 0.58 mg / L, respectively. In contrast, when sodium acetate was used as the carbon source, the ammonia removal concentration was only 39.57 ± 2.73 mg / L, and strain *Pseudomonas citronellolis* BY-1 was almost unable to utilize sucrose for metabolism. This indicates that sodium succinate is the optimal carbon source for the growth of strain BY-1.

[0050] Example 3

[0051] A study on the C / N ratio adaptation of Pseudomonas citronellolis BY-1.

[0052] The *Pseudomonas citronellolis* BY-1 bacterial suspension prepared in Example 2 was inoculated at a ratio of 1% into simulated domestic sewage containing different C / N ratios (100 mg / L NH4). + -N, C / N = 10, 15, 20, 25, 30, carbon source is sodium succinate, substrate general culture medium Na2HPO4 4.26 g / L, KH2PO4 2.65 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.5 g / L and 1 mL / L trace elements, pH 7.2, trace element solution formula is: 100 mg / L EDTA, 4.4 mg / L ZnSO4, 11 mg / L CaCl2, 10.2 mg / L MnCl2, 10 mg / L FeSO4·7H2O, 2.2 mg / L (NH4)6Mo7O 24 The medium was incubated at 30℃ and 150 rpm for 9 h using a solution of 3.2 mg / L CuSO4·5H2O, 3.2 mg / L COCl2·6H2O, and 4H2O. After incubation, the NH4+ concentration in each culture medium was determined using Nessler's reagent spectrophotometry. + -N content, and calculate NH4+ for each C / N ratio. + -N removal amount. The effect is as follows: Figure 5 As shown, when the C / N ratio exceeds 15.0, the ammonia removal rate and OD... 600 There were no significant differences in growth rates. However, when the C / N ratio was 5.0 or 10.0, the ammonia removal concentration and OD... 600 The fold changes were all lower than those at a C / N ratio of 15. This indicates that the optimal C / N ratio for strain Pseudomonas citronellolis BY-1 is approximately 15.0, and this strain has potential applications in treating wastewater with high C / N ratios.

[0053] Example 4

[0054] Phenol tolerance study of Pseudomonas citronellolis BY-1

[0055] The *Pseudomonas citronellolis* BY-1 bacterial suspension prepared in Example 2 was inoculated at a ratio of 5% into simulated phenol-containing wastewater (50 mg / L NH4) containing different concentrations of phenol. + -N, 500 mg / L, 800 mg / L, 1000 mg / L, 1300 mg / L phenol; general-purpose substrate medium: Na2HPO4 4.26 g / L, KH2PO4 2.65 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.5 g / L, and 1 mL / L trace elements; pH 7.2; trace element solution formulation: 100 mg / L EDTA, 4.4 mg / L ZnSO4, 11 mg / L CaCl2, 10.2 mg / L MnCl2, 10 mg / L FeSO4·7H2O, 2.2 mg / L (NH4)6Mo7O 24 The *Pseudomonas citronellolis* BY-1 was cultured in a solution of 3.2 mg / L CuSO4·5H2O, 3.2 mg / L COCl2·6H2O at 30 °C and 150 rpm for several hours to detect its tolerance and degradation ability to phenol, and to simultaneously detect NH4+. + -N concentration changes. As shown in Table 1, with increasing phenol concentration, the hysteresis loop of *Pseudomonas citronellolis* BY-1 prolonged, and the degradation rate of phenol gradually decreased. Specifically, 500 mg / L phenol was completely degraded within 16 hours, with an ammonia removal concentration of 44.05 ± 7.28 mg / L. Phenol at concentrations of 800 mg / L, 1000 mg / L, and 1300 mg / L required 24 hours, 48 ​​hours, and 120 hours for complete degradation, respectively, with ammonia removal concentrations of 68.61 ± 3.27 mg / L, 79.90 ± 2.93 mg / L, and 112.54 ± 5.93 mg / L. This indicates that strain *Pseudomonas citronellolis* BY-1 exhibits excellent performance in both phenol tolerance and phenol removal rate. It should be noted that the ratio of phenol degradation to ammonia removal remains stable at approximately 12:1.

[0056] Table 1. Degradation efficiency of Pseudomonas citronellolis BY-1 strain for different phenol concentrations.

[0057]

[0058] Example 5

[0059] The study on the adaptability of *Pseudomonas citronellolis* BY-1 to different nitrogen sources involved using *Pseudomonas citronellolis* BY-1 bacterial suspension prepared in Example 2, which was inoculated at a ratio of 5% into simulated phenol-containing wastewater (50 mg / L NH4) containing different nitrogen sources. + -N, 500 mg / L phenol; 50 mg / L NO3 - -N, 500 mg / L phenol; 25 mg / L NH4 + -N+25mg / LNO3 - -N, 500 mg / L phenol, general-purpose substrate medium: Na₂HPO₄ 4.26 g / L, KH₂PO₄ 2.65 g / L, MgSO₄·7H₂O 0.2 g / L, NaCl 0.5 g / L, and 1 mL / L trace elements, pH 7.2. Trace element solution formulation: 100 mg / L EDTA, 4.4 mg / L ZnSO₄, 11 mg / L CaCl₂, 10.2 mg / L MnCl₂, 10 mg / L FeSO₄·7H₂O, 2.2 mg / L (NH₄)₆Mo₇O 24 The medium was incubated with 3.2 mg / L CuSO4·5H2O, 3.2 mg / L COCl2·6H2O at 30 °C and 150 rpm for 16 h. After incubation, the NH4+ content in each culture medium was measured. + -N, NO2 - -N, NO3 - -N and TN contents were analyzed, and TN removal was calculated under different nitrogen source conditions. The results are as follows: Figure 6 As shown, when *Pseudomonas citronellolis* BY-1 was in NH4... + -N medium (NH4) + When cultured in an environment with a nitrogen concentration of 45.52 ± 0.75 mg / L and a phenol concentration of 520.17 ± 17.17 mg / L, NH4+ was completely removed within 16 hours. + -N, the phenol concentration decreased to 26.92±10.34 mg / L, NH4 + NO3 was not detected during the degradation of -N. - -N or NO2 - -N accumulation ( Figure 6 (Figure A)). When Pseudomonas citronellolis BY-1 was in NO3 - When cultured in -N medium, simultaneous removal of NO3 was not observed.- The ability of -N and phenol ( Figure 6 (Figure B) in the diagram. However, in the mixed nitrogen source (NH4) + -N and NO3 - When cultured under conditions of NO3-N coexistence, *Pseudomonas citronellolis* BY-1 exhibited NO3-... - -N removal capacity, NH4+ removal capacity during 8-12 hours + -N concentration decreased from 22.34±0.23 mg / L to 8.83±0.40 mg / L, NO3 - -N removal was not significant; residual NH4+ remained within 12-16 hours. + -N is completely removed, NO3 - The -N concentration decreased from 26.13±1.42 mg / L to 3.50±1.10 mg / L, and NO2 was detected at the 16th hour. - -N accumulation (14.29±1.04 mg / L). Within 16 hours, the phenol concentration decreased from 489.50±26.06 mg / L to 7.25±8.04 mg / L, OD... 600 The value increased from 0.081±0.001 to 0.431±0.001, and the TN removal efficiency was 65.06±2.07%. Figure 6 (Figure C) in the middle.

[0060] Example 6

[0061] Application of Pseudomonas citronellolis BY-1 in actual coking wastewater

[0062] The *Pseudomonas citronellolis* BY-1 bacterial culture prepared in Example 2 was inoculated into actual coking wastewater at a ratio of 5%, and cultured at 30°C and 150 rpm for 30 hours. NH4 levels were measured before and after inoculation. + -N, NO2 - -N, NO3 - The study investigated the changes in N, phenolic compounds, and COD concentrations to explore the treatment performance of Pseudomonas citronellolis BY-1 on actual coking wastewater. Figure 7The performance of strain *Pseudomonas citronellolis* BY-1 in removing ammonia and phenolic compounds from actual coking wastewater was demonstrated. Within 36 hours, the ammonia concentration significantly decreased from 44.90 ± 4.30 mg / L to 14.29 ± 3.68 mg / L, and NO3 was not detected. - -N and NO2 - -N. For example... Figure 7 As shown in (B), phenolic pollutants were completely removed. Furthermore, the chemical oxygen demand (COD) concentration decreased from 2628.94 ± 95.8 mg / L to 1271.21 ± 75.95 mg / L. These results collectively demonstrate that strain *Pseudomonas citronellolis* BY-1 exhibits significant capacity for treating nitrogenous and phenolic pollutants in actual coking wastewater.

[0063] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A highly efficient nitrogen-degrading and phenol-removing strain, characterized in that it is A heterotrophic nitrifying, aerobic, and denitrifying strain that simultaneously removes nitrogen and phenols is *Pseudomonas citronellol*, named *Pseudomonas citronellol* BY-1. It has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M 2025818. The deposit address is Wuhan University, Wuhan, Hubei Province, China, and the deposit date is April 18, 2025.

2. The application of the highly efficient denitrifying and phenol-removing strain *Citronellol Pseudomonas* BY-1 as described in claim 1 in simultaneous denitrification and phenol removal.

3. The application as described in claim 2, characterized in that, Application in biological denitrification of aquatic bodies.

4. The application as described in claim 2, characterized in that, Application of simultaneous denitrification and phenol removal in phenol-containing wastewater.

5. The application as described in claim 3, characterized in that, The biological denitrification of the water body is an enhanced denitrification process for wastewater with a high carbon-to-nitrogen ratio.

6. The application as described in claim 3, characterized in that the application... Specifically, the bacterial suspension of Citronellol Pseudomonas BY-1 was inoculated into wastewater with a high carbon-to-nitrogen ratio at an inoculation rate of 0.8-1.2%, and cultured at 25-35℃ and 150-250 rpm for 9-24 hours.

7. The application as described in claim 4, characterized in that, The specific application involves inoculating Citronellol Pseudomonas BY-1 into phenol-containing wastewater for cultivation.

8. The application as described in claim 4, characterized in that, The specific application is as follows: the above-mentioned citronellol Pseudomonas BY-1 bacterial suspension is inoculated into phenol-containing wastewater at an inoculation rate of 1.0-10.0%, and cultured at 25-35℃ and 150-250rpm for 16-144h.

9. The application as described in claim 6 or claim 8, characterized in that, The preparation method of bacterial suspension is as follows: the slant culture of Pseudomonas citronellol BY-1 strain is inoculated into LB liquid medium and cultured at 25-35℃ and 150-250rpm for 12-24h. Then, the precipitate is obtained by centrifugation, and the precipitate is resuspended in PBS to obtain bacterial suspension.

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