SALT-TOLERANT HETEROTROPHIC NITRIFICATION AEROBIC DENITRIFICATION PHOSPHORUS REMOVAL BACTERIAL STRAIN AND APPLICATION THEREOF

Abstract

The application relates to the field of microbiology and presents a salt-tolerant heterotrophic nitrification aerobic denitrifying and phosphorus removal bacterial strain and its application. The strain is Pseudomonas mendocina A4, deposited on Nov. 4, 2022, at the Guangdong Microbial Culture Collection Center, located at Building 59, 5th Floor, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with the deposit number GDMCC No: 62944. The Pseudomonas mendocina A4 strain provided by the present application possesses both heterotrophic nitrification and aerobic denitrification functions and can be applied in the field of wastewater treatment. It has a strong tolerance to high concentrations of organic carbon while utilizing various organic carbon sources in wastewater and exhibits excellent organic carbon removal capabilities in water. This strain is particularly suitable for treating nitrogen-containing wastewater with a high C/N ratio.

Claims

1. A salt-tolerant heterotrophic nitrification aerobic denitrification and phosphorus removal bacterial strain, comprising Pseudomonas mendocina A4, was deposited on Nov. 4, 2022, at the Guangdong Microbial Culture Collection Center, located at Building 59, 5th floor, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with the deposit number GDMCC No: 62944.

2. An application of a salt-tolerant heterotrophic nitrification aerobic denitrification and phosphorus removal bacterial strain as claimed in claim 1, wherein using the Pseudomonas mendocina A4 in a field of wastewater treatment.

3. The application of the salt-tolerant heterotrophic nitrification aerobic denitrification and phosphorus removal bacterial strain as claimed in claim 2, wherein a carbon source of the wastewater is at least one of sodium citrate, sodium succinate, sucrose, and glucose.

4. The application of the salt-tolerant heterotrophic nitrification aerobic denitrification and phosphorus removal bacterial strain as claimed in claim 3, wherein the carbon source of the wastewater is sodium succinate.

5. The application of the salt-tolerant heterotrophic nitrification aerobic denitrification and phosphorus removal bacterial strain as claimed in claim 2, wherein using the Pseudomonas mendocina A4 in the treatment of nitrogen and phosphorus-containing wastewater, with a C/N ratio of 0 to 15.

6. The application of the salt-tolerant heterotrophic nitrification aerobic denitrification and phosphorus removal bacterial strain as claimed in claim 2, wherein using the Pseudomonas mendocina A4 in the treatment of nitrogen and phosphorus-containing wastewater, with a C/N ratio of 10.

7. The application of the salt-tolerant heterotrophic nitrification aerobic denitrification and phosphorus removal bacterial strain as claimed in claim 2, wherein using the Pseudomonas mendocina A4 in the treatment of nitrogen and phosphorus-containing wastewater, with a P/N ratio of 0 to 1.

8. The application of the salt-tolerant heterotrophic nitrification aerobic denitrification and phosphorus removal bacterial strain as claimed in claim 2, wherein using the Pseudomonas mendocina A4 in the treatment of nitrogen and phosphorus-containing wastewater, with a pH value of 5 to 9.

9. The application of the salt-tolerant heterotrophic nitrification aerobic denitrification and phosphorus removal bacterial strain as claimed in claim 2, wherein using the Pseudomonas mendocina A4 in the treatment of nitrogen and phosphorus-containing wastewater, at a temperature of 250? C. to 40? C.

10. The application of the salt-tolerant heterotrophic nitrification aerobic denitrification and phosphorus removal bacterial strain as claimed in claim 2, wherein a salinity of the wastewater is 0 to 15%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings are used to further illustrate the present application, but the embodiments in the drawings do not constitute any limitation on the present application. Skilled persons in the art can obtain other drawings based on the following drawings without any creative work.

[0028] FIG. 1 is a colony morphology of Pseudomonas mendocina A4, an embodiment of the present application, on a nutrient agar plate.

[0029] FIG. 2 is a Gram stain image of Pseudomonas mendocina A4, an embodiment of the present application.

[0030] FIG. 3 is a comparison of the growth and denitrification effects of Pseudomonas mendocina A4, an embodiment of the present application, under different organic carbon sources and different inorganic nitrogen sources.

[0031] FIG. 4 is a comparison of the growth and denitrification effects of Pseudomonas mendocina A4, an embodiment of the present application, under different C/N ratios and different inorganic nitrogen sources.

[0032] FIG. 5 is a comparison of the growth and denitrification effects of Pseudomonas mendocina A4, an embodiment of the present application, under different P/N ratios and different inorganic nitrogen sources.

[0033] FIG. 6 is a comparison of the growth and denitrification effects of Pseudomonas mendocina A4, an embodiment of the present application, under different pH and different inorganic nitrogen sources.

[0034] FIG. 7 is a comparison of the growth and denitrification effects of Pseudomonas mendocina A4, an embodiment of the present application, under different temperatures and different inorganic nitrogen sources.

[0035] FIG. 8 is a comparison of the growth and denitrification effects of Pseudomonas mendocina A4, an embodiment of the present application, under different salinities and different inorganic nitrogen sources.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] To more clearly illustrate the present application, the technical features, objectives, and beneficial effects of the present application will be further understood in conjunction with FIGS. 1-8 and embodiments. The technical solution of the present application is now detailed below, but should not be construed as limiting the scope of the present application.

[0037] In the experiments of the present application, the determination and analysis methods for three nitrogen elements NH.sub.4.sup.+, NO.sub.3.sup.?, and NO.sub.2.sup.? referred to the national standards, including:

[0038] NH.sub.4.sup.+ determination and analysis according to the Water qualityDetermination of Ammonia NitrogenNessler's Reagent Spectrophotometry (GB HJ535-2009);

[0039] NO.sub.3.sup.? determination and analysis according to the Water qualityDetermination of Nitrate NitrogenUltraviolet Spectrophotometry (GB HJ/T346-2007);

[0040] NO.sub.2.sup.? determination and analysis according to the Water qualityDetermination of Nitrite NitrogenSpectrophotometry (GB 7493-87);

[0041] PO.sub.4.sup.3?P using the ammonium molybdate spectrophotometric method.

[0042] The embodiment provides a salt-tolerant heterotrophic nitrification aerobic denitrification and phosphorus-removing bacterial strain, the strain being Pseudomonas mendocina A4, deposited on Nov. 4, 2022, at the Guangdong Microbial Culture Collection Center, located at Building 59, 5th Floor, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with the deposit number GDMCCNo: 62944.

[0043] This strain is a Pseudomonas mendocina strain collected and bred by the inventors' team from aquaculture ponds in Guangdong Province, and has been endowed with the biological characteristics of efficient degradation of NH.sub.4.sup.+N, NO.sub.3.sup.?N, NO.sub.2.sup.?N, and PO.sub.4.sup.3?P under certain salinity conditions. It is gram-stain negative, with white opaque colonies on nutrient agar, raised surface, circular, smooth, moist, and shiny, with intact edges, rod-shaped cells, and no flagella. It has the function of heterotrophic nitrification and aerobic denitrification.

[0044] The adaptability and safety of the Pseudomonas mendocina A4 strain are good, therefore it has good application prospects in the biological denitrification and phosphorus removal treatment of aquaculture effluent or other high-nitrogen and phosphorus-containing high-salt sewage.

[0045] The raw materials, reagents, or devices used in the following embodiments can be obtained from conventional commercial sources, unless otherwise specified, or can be obtained by existing known methods.

[0046] The present application is further described with multiple specific implementation examples below.

Embodiment 1

[0047] This embodiment provides a strain of Pseudomonas mendocina A4 (GDMCC No: 62944) with both heterotrophic nitrification and aerobic denitrification functions. The breeding process includes the following steps:

Sample Collection

[0048] The original strain of Pseudomonas mendocina A4 of the present application was obtained by screening and isolating water and mud samples from the breeding ponds in Nanshui Area, Doumen District, Zhuhai City, Guangdong Province. The sample collection was conducted according to the Mixed Sample Collection Method in the Technical Specifications for Soil Environment Monitoring (HJ/T 166-2004), using the plum blossom point sampling method to collect surface, middle, and deep water and bottom sediment from the breeding ponds in sterile sampling bags, which were then transported and stored at 4? C.

2. Preparation of Culture Media and Solutions

[0049] (1) Salt Solution (g/L): NaCl 2.5 g, MgSO.sub.4.Math.7H.sub.2O 2.5 g, FeSO.sub.4.Math.7H.sub.2O 0.05 g, MnSO.sub.4.Math.4H.sub.2O 0.05 g; [0050] (2) Trace Element Solution (g/L): MgSO.sub.4.Math.7H.sub.2O 50.0 g, CaCl.sub.2) 5.5 g, CuSO.sub.4.Math.5H.sub.2O 1.57 g, ZnSO.sub.4.Math.7H.sub.2O 2.2 g, FeSO.sub.4, 5.0 g, MnCl.sub.2.Math.4H.sub.2O 5.06 g, CoCl.sub.2.Math.6H.sub.2O 1.60 g, Na.sub.2EDTA, 50.0 g; [0051] (3) Enrichment Culture Medium (g/L): 5.62 g sodium succinate, 0.087 g KH.sub.2PO.sub.4, 0.24 g NaNO.sub.3, 0.165 g NaNO.sub.2, 0.472 g (NH.sub.4).sub.2SO.sub.4, 50 mL salt solution; [0052] (4) BTB Medium (g/L): Sodium citrate dihydrate 6.45 g, 1% BTB ethanol solution 1 mL, KH.sub.2PO.sub.41.5 g, MgSO.sub.4.Math.7H.sub.2O 0.01 g, Na.sub.2HPO.sub.47.9 g, NaNO.sub.30.8415 g, NH.sub.4Cl 0.192 g, NaNO20.362 g, trace element solution 2 mL, agar 20 g, pH 7.0?7.5; [0053] (5) Single Nitrogen Source Fermentation Medium (DMI) (g/L): 5.62 g sodium succinate, 0.087 g KH.sub.2PO.sub.4, 0.472 g (NH.sub.4).sub.2SO.sub.4, trace element solution 2 mL, pH 7.0; [0054] (6) Single Nitrogen Source Fermentation Medium (DMII) (g/L): 5.62 g sodium succinate, 0.087 g KH.sub.2PO.sub.4, 0.607 g NaNO.sub.3, trace element solution 2 mL, pH 7.0; [0055] (7) Single Nitrogen Source Fermentation Medium (DMIII) (g/L): 5.62 g sodium succinate, 0.087 g KH.sub.2PO.sub.4, 0.4928 g NaNO.sub.2, trace element solution 2 mL, pH 7.0.

[0056] The basic culture media used in the experiments were sterilized at 121? C. for 20 minutes using high-pressure steam before use.

Embodiment 2

[0057] A method for selecting a strain of Pseudomonas mendocina A4 with both heterotrophic nitrification and aerobic denitrification functions, including enrichment, isolation, and screening of the original strain of Pseudomonas mendocina A4, specifically including the following steps: [0058] (1) Sample pretreatment: Take 10 g of pond mud, add it to a 300 mL wide-mouth triangular flask containing 90 mL of 0.9% sterile physiological saline solution on a super clean workbench, and add a few glass beads that have been sterilized by high-pressure steam at 121? C. for 15 minutes, and shake at 160 r/min for 1 h to disperse the mud sample, so that the microorganisms in the mud sample are fully suspended in the physiological saline solution; the water sample is treated similarly; [0059] (2) Enrichment culture: Take 22.2 mL of the above-mentioned mixed liquid (10.sup.?1 mud) and add it to a 500 mL conical flask containing 200 mL of enrichment culture medium, and shake at 30? C. and 160 r/min for 2?3 days. For the water sample, take 10 mL and inoculate it into a 300 mL wide-mouth triangular flask containing 90 mL of enrichment culture medium, and shake at 30? C. and 160 r/min for 1 h; [0060] (3) Sample plate coating: Take 1 mL of the pretreated water sample and mud sample from (1), add it to a test tube containing 9 mL of sterile physiological saline solution on a super clean workbench, and gently blow or shake with a pipette to mix. Take 1 mL of the liquid from the test tube and add it to a new test tube containing 9 mL of sterile physiological saline solution, repeat this operation, and dilute the 10.sup.?1 mud and water sample original solutions to 10.sup.?2?10.sup.?4 concentrations. Take 100 ?L?200 ?L of the original solution of sediment and water samples at concentrations of 10.sup.?1?10.sup.?4, and directly plate them on BTB agar medium, with 3 parallel groups set for each gradient, 1 blank control group, and incubate upside down at 30? C. in a constant temperature biochemical incubator for 2?3 days; [0061] (4) Sample enrichment liquid plate coating: dilute the gradient of the enriched culture suspension from (2) as follows: take 1 mL of the bacterial suspension from the conical flask in (2) and add it to a test tube containing 9 mL of sterile physiological saline solution, mix thoroughly, and dilute the concentration to 10.sup.?1. Then, take 1 mL of liquid from this test tube and add it to a new test tube containing 9 mL of sterile physiological saline solution, mix thoroughly, and repeat this step, sequentially diluting to reach concentrations of 10.sup.?2?10.sup.?8. Then take 100 ?L?200 ?L from each concentration gradient mixture and plate them on a pre-made BTB solid plate medium, indicating the dilution gradient and date, and incubate upside down at 30? C. in a constant temperature biochemical incubator for 2?3 days; [0062] (5) Separation and purification: Using an inoculation loop to pick different morphological colonies. Use the streaking method on BTB solid plate medium for isolation and purification. After streaking, the plates are placed in a laminar flow hood at room temperature for 5 minutes and then inverted and incubated at 30? C. in a constant temperature biochemical incubator for 2?3 days. Repeat this step, pick single colonies, and streak for purification 3?4 times. [0063] After observing no morphological abnormalities in the colonies, pick single colonies, perform a crystal violet stain, and examine under a microscope for purity (100? oil immersion lens); [0064] (6) Spot inoculation initial screening: Use an inoculation needle to spot inoculate the purified strains on BTB denitrification identification medium and culture for 2?3 days. Select strains with high denitrification capabilities based on colony growth and the size of the blue halo around the colonies on the BTB medium. Generally, the larger the blue halo, the higher the denitrification capability. After incubation at 30? C. on a slant for 2?3 days, the test tubes are stored at 4? C.; [0065] (7) Nitration and denitrification performance re-screening: Use an inoculation loop to take the obtained strains for activation and inoculate them on a slant in nutrient broth, shaking at 30? C., 160 r/min for 1 day, and measure their OD.sub.600. Then, inoculate with 1% inoculum into an enriched culture medium, shaking at 30? C., 160 r/min for 0 h, 12 h, 24 h, 36 h, 48 h, and measure the OD.sub.600 of the culture at each time point. After low-speed centrifugation at 12000 rpm, 4? C. for 5 min, measure the content of NH.sub.4.sup.+N, NO.sub.3.sup.?N, NO.sub.2.sup.?N, and PO.sub.4.sup.3?P in the supernatant, and select the strain with the best biological characteristics as Pseudomonas mendocina A4.

Embodiment 3

[0066] The biological characteristics identification steps for the strain Pseudomonas mendocina A4, which was re-screened and found to have both heterotrophic nitrification and aerobic denitrification functions, include: [0067] (1) Morphological identification: After the above screening and isolation, a strain of heterotrophic nitrification-aerobic denitrification bacterium A4 was obtained. This strain is gram-negative, rod-shaped, and non-flagellated. The colonies on nutrient agar are white, opaque, raised, circular, smooth, moist, and shiny, with a complete edge. [0068] (2) Molecular biology identification: The DNA extraction of strain A4 is carried out using Takara Lysis Buffer for Microorganism to Direct PCR. The 16S rDNA is amplified using a pair of universal primers: upstream primer (27F): 5-AGAGTTTGATCCTGGCTCAG-3; downstream primer (1492R): 5-GGCTACCTTGTTACGACTT-3. The universal primers are synthesized by Shanghai Bioengineering Co., Ltd. The PCR reaction system (25 ?L) consists of 2?Unique?Taq Master Mix (With Dye) 12.5 ?L, 1 ?L of each upstream and downstream primer, 1 ?L of DNA template, and 9.5 ?L of ddH.sub.2O. The PCR program is as follows: {circle around (1)} 94? C., 5 min; {circle around (2)}94? C. pre-denaturation, 1 min; {circle around (3)} 55? C. annealing, 1 min; {circle around (4)} 72? C. extension 1.5 min; {circle around (5)} 72? C., 10 min; cycles of {circle around (2)} ?{circle around (4)} are repeated 30 times. The results are analyzed by 1% agarose gel electrophoresis. The PCR product sequencing is completed by Shanghai Bioengineering Co., Ltd.

[0069] After the above screening, cultivation, and isolation, a strain with the best biological characteristics and stable properties of heterotrophic nitrification-aerobic denitrification, namely Pseudomonas mendocina A4, is obtained. The physiological and biochemical characteristics of this strain are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Re- Re- Re- Item sult Item sult Item sult Gelatin ? Starch hydrolysis ? Methyl red + liquefaction SIM ? Indole production + V-P test + Sucrose + Triple sugar ? Growth: + iron agar 41? C. Lactose + D-(+)-glucose + Glycerol ? D-lactose ? D-xylose + D-fructose + L-phenylalanine ? Mannitol ? Citrate + H.sub.2S production ? Note: + indicates positive; ? indicates negative.

[0070] After the above screening steps, a strain of heterotrophic nitrification-aerobic denitrification bacterium A4 was obtained. Based on its 16S rDNA, bacterial morphology, colony morphology, and physiological and biochemical characteristics, it is determined that strain A4 belongs to Pseudomonas mendocina.

Embodiment 4

[0071] The above-mentioned heterotrophic nitrifying-aerobic denitrifying bacterium Pseudomonas mendocina A4 (hereinafter referred to as strain A4), which simultaneously possesses the functions of heterotrophic nitrification and aerobic denitrification, is applied in the field of wastewater treatment. The testing and validation of its optimal growth and denitrification conditions include the following steps:

(1) Influence of Different Organic Carbon Sources on the Growth and Denitrification Performance of Strain A4

[0072] Four carbon sources, sucrose, sodium succinate, glucose, and sodium citrate, are selected. With a fixed C/N ratio of 10, the experiment is conducted at 30? C., 160 rpm/min, and pH=7.0. Based on the DM fermentation culture medium, the amount of each organic carbon source added per liter of culture medium is 5.00 g for sucrose, 5.62 g for sodium succinate, 2.50 g for glucose, and 4.08 g for sodium citrate. As a single inorganic nitrogen source, the amounts of (NH.sub.4).sub.2SO.sub.4, NaNO.sub.3, and NaNO.sub.2 added per liter of culture medium are 0.472 g, 0.607 g, and 0.493 g, respectively. The candidate strains are inoculated into nutrient broth culture medium and incubated at 30? C., 160 r/min for 1 day. Then, with a 1% inoculation amount, they are inoculated into the denitrification culture medium with different organic carbon sources, and the culture liquid is taken at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h to measure their OD.sub.600. After low-speed centrifugation at 5000 r/min, 4? C. for 5-10 min, the supernatant is taken to measure the content of NH.sub.4.sup.+N, NO.sub.3.sup.?N, NO.sub.2.sup.?N, and PO.sub.4.sup.3?P. The experiment sets up three technical replicates of the experimental group and one blank control group, with the control group receiving an equal amount of physiological saline. The analysis examines the effects of four different organic carbon sources-sucrose, sodium succinate, glucose, and sodium citrate-on the growth denitrification and phosphorus removal effects of strain A4.

[0073] FIG. 3 shows the nitrogen and phosphorus removal by Pseudomonas mendocina A4 under different carbon source conditions; wherein: (a) Ammonium nitrogen and phosphate removal rate; (b) Nitrate and phosphate removal rate; (c) Nitrite and phosphate removal rate.

(2) Influence of Different C/N Ratios on the Growth and Denitrification Performance of Strain A4

[0074] Sodium succinate is selected as the carbon source in the denitrification culture medium. Under fixed conditions of 30? C., 160 r/min, P/N=0.2, and pH=7.0, C/N gradients of 0, 2, 5, 10, 15 were set. The amount of sodium succinate added to the culture medium for each gradient is 0 g/L, 1.125 g/L, 2.812 g/L, 5.62 g/L, 8.44 g/L; as a single inorganic nitrogen source, the amounts of (NH.sub.4).sub.2SO.sub.4, NaNO.sub.3, and NaNO.sub.2 added per liter of culture medium are 0.472 g, 0.607 g, and 0.493 g, respectively. The candidate strains were inoculated into a nutrient broth medium and cultured at 30? C. and 160 r/min for one day. Then, 1% of the inoculum was added to the above-mentioned media at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h. The culture liquid was taken to measure its OD.sub.600, and after low-speed centrifugation at 5000 r/min and 4? C. for 5?10 minutes, the supernatant was taken to measure the content of NH.sub.4.sup.+N, NO.sub.3.sup.?N, NO.sub.2.sup.?N, and PO.sub.4.sup.3?P. The experiment sets up three technical replicates of the experimental group and one blank control group, with the control group receiving an equal amount of physiological saline. The analysis examines the effects of five different C/N ratios (0, 2, 5, 10, 15) on the growth and denitrification and phosphorus removal effects of strain A4.

[0075] FIG. 4 shows the nitrogen and phosphorus removal by Pseudomonas mendocina A4 under different C/N ratios; where: (a) Ammonium nitrogen and phosphate removal rate; (b) Nitrate and phosphate removal rate; (c) Nitrite and phosphate removal rate.

[0076] FIG. 5 shows the nitrogen and phosphorus removal by Pseudomonas mendocina A4 under different P/N ratios; where: (a) Ammonium nitrogen and phosphate removal rate; (b) Nitrate and phosphate removal rate; (c) Nitrite and phosphate removal rate.

(3) Influence of Different C/N Ratios on the Growth and Denitrification Performance of Strain A4

[0077] Sodium succinate is selected as the carbon source in the denitrification culture medium. Under fixed conditions of 30? C., 160 r/min, C/N=10, and pH=7.0, P/N gradients of 0, 0.1, 0.2, 0.5, and 1 were set. The amount of KH.sub.2PO.sub.4 added to the culture medium for each gradient was 0.0000 g/L, 0.0430 g/L, 0.0870 g/L, 0.2193 g/L, 0.4387 g/L; as a single inorganic nitrogen source, the amounts of (NH.sub.4).sub.2SO.sub.4, NaNO.sub.3, and NaNO.sub.2 added per liter of culture medium are 0.472 g, 0.607 g, and 0.493 g, respectively. The candidate strains were inoculated into a nutrient broth medium and cultured at 30? C. and 160 r/min for one day. Then, 1% of the inoculum was added to the above-mentioned media at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h. The culture liquid was taken to measure its OD.sub.600, and after low-speed centrifugation at 5000 r/min and 4? C. for 5?10 minutes, the supernatant was taken to measure the content of NH.sub.4.sup.+N, NO.sub.3.sup.?N, NO.sub.2.sup.?N, and PO.sub.4.sup.3?P. The experiment sets up three technical replicates of the experimental group and one blank control group, with the control group receiving an equal amount of physiological saline. The analysis examines the effects of five different P/N ratios (0, 0.1, 0.2, 0.5, 1) on the growth and denitrification and phosphorus removal effects of strain A4.

(4) Influence of Different pH on the Growth and Denitrification Performance of Strain A4

[0078] Under fixed conditions of C/N=10, P/N=0.2, 30? C., 160 r/min, and sodium succinate as the sole organic carbon source, pH gradients of 5, 6, 7, 8, and 9 were set. The amounts of (NH.sub.4).sub.2SO.sub.4, NaNO.sub.3, and NaNO.sub.2 added per liter of culture medium as single inorganic nitrogen sources were 0.472 g, 0.607 g, and 0.493 g, respectively. Candidate strains were inoculated into a nutrient broth medium and cultured at 30? C., 160 r/min for one day. Then, 1% of the inoculum is transferred to the above-mentioned culture media, and the culture liquid is sampled at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h to determine its OD.sub.600. After low-speed centrifugation at 5000 r/min and 4? C. for 5?10 minutes, the supernatant is collected to measure the content of NH.sub.4.sup.+N, NO.sub.3.sup.?N, NO.sub.2.sup.?N, and PO.sub.4.sup.3?P. The experiment includes three technical replicates and a blank control group with an equal amount of physiological saline added. The impact of five different pH levels (5, 6, 7, 8, 9) on the growth denitrification and phosphorus removal effects of strain A4 is analyzed.

[0079] FIG. 6 shows the nitrogen and phosphorus removal by Pseudomonas mendocina A4 under different pH conditions; where: (a) Ammonium nitrogen and phosphate removal rate; (b) Nitrate and phosphate removal rate; (c) Nitrite and phosphate removal rate.

(5) Influence of Different Temperatures on the Growth and Denitrification Performance of Strain A4

[0080] Under fixed conditions of C/N=10, pH=7.0, 160 r/min, and sodium citrate as the sole organic carbon source, temperature gradients of 25? C., 30? C., 35? C., 40? C. are set. The amounts of (NH.sub.4).sub.2SO.sub.4, NaNO.sub.3, and NaNO.sub.2 added per liter of culture medium as single inorganic nitrogen sources are 0.472 g, 0.607 g, and 0.493 g, respectively. Candidate strains are inoculated into a nutrient broth medium and cultured at 30? C., 160 r/min for one day. Then, 1% of the inoculum is transferred to the above-mentioned culture media, and the culture liquid is sampled at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h to determine its OD.sub.600. After low-speed centrifugation at 5000 r/min and 4? C. for 5?10 minutes, the supernatant is collected to measure the content of NH.sub.4.sup.+N, NO.sub.3.sup.?N, NO.sub.2.sup.?N, and PO.sub.4.sup.3?P. The experiment includes three technical replicates and a blank control group with an equal amount of physiological saline added. The impact of four different temperatures (25? C., 30? C., 35? C., 40? C.) on the growth and denitrification and phosphorus removal effects of strain A4 is analyzed.

[0081] FIG. 7 shows the nitrogen and phosphorus removal by Pseudomonas mendocina A4 under different temperature conditions; where: (a) Ammonium nitrogen and phosphate removal rate; (b) Nitrate and phosphate removal rate; (c) Nitrite and phosphate removal rate.

(6) Influence of Different Salinities on the Growth and Denitrification Performance of Strain A4.

[0082] Under fixed conditions of C/N=10, P/N=0.2, pH=7.0, 160 r/min, and sodium succinate as the sole organic carbon source, salinity gradients of 0%, 3%, 5%, 10%, and 15% are set. Salinity is controlled by adding NaCl, with corresponding amounts of 0 g, 3 g, 5 g, 10 g, and 15 g per 100 mL of culture medium. The amounts of (NH.sub.4).sub.2SO.sub.4, NaNO.sub.3, and NaNO.sub.2 added per liter of culture medium as single inorganic nitrogen sources are 0.472 g, 0.607 g, and 0.493 g, respectively. Candidate strains are inoculated into a nutrient broth medium and cultured at 30? C., 160 r/min for one day. Then, 1% of the inoculum is transferred to the above-mentioned culture media, and the culture liquid is sampled at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h to determine its OD.sub.600. After low-speed centrifugation at 5000 r/min and 4? C. for 5?10 minutes, the supernatant is collected to measure the content of NH.sub.4.sup.+N, NO.sub.3.sup.?N, NO.sub.2+-N, and PO.sub.4.sup.3?P. The experiment includes three technical replicates and a blank control group with an equal amount of physiological saline added. The impact of five different salinities (0%, 3%, 5%, 10%, 15%) on the growth and denitrification and phosphorus removal effects of strain A4 is analyzed.

[0083] FIG. 8 shows the nitrogen and phosphorus removal by Pseudomonas mendocina A4 under different salinity conditions: Ammonium nitrogen and phosphate removal rate (a); Nitrate and phosphate removal rate (b); Nitrite and phosphate removal rate (c).

[0084] From Embodiment 4 (FIGS. 3-8) and Embodiment 3 (Table 1), it can be seen that Pseudomonas mendocina A4 can grow using various organic carbon sources such as sodium citrate and sodium succinate. The best growth and nitrogen and phosphorus removal effects are observed when using sodium succinate as the carbon source. The strain can grow under C/N ratios of 0?15, P/N ratios of 0?1, pH of 6?9, and temperatures of 25? C.?40? C. Even under a C/N ratio of 0, the strain A4 can still grow, indicating its ability to aggregate and release phosphorus for energy metabolism. When the P/N ratio is 0, the nitrogen removal efficiency is significantly lower than when the P/N ratio is 0.1, indicating that phosphorus is essential for the growth metabolism of strain A4. The optimal denitrification conditions are using sodium succinate as the carbon source, C/N=10, P/N=0.2, pH=7, and T=30? C. The denitrification efficiency of strain A4 is 99.58%, 990.99%, and 99.83%, with corresponding phosphorus removal rates of 95.96%, 880.28%, and 94.69%. Under the optimal denitrification and phosphorus removal conditions, increasing salinity from 0% to 5% does not show a statistically significant difference in the corresponding nitrogen and phosphorus removal rates, indicating its good tolerance to salinity.

[0085] The Pseudomonas mendocina A4 bred in the present application can be applied to the treatment of nitrogen and phosphorus-containing wastewater in saltwater aquaculture, without adverse effects on aquaculture organisms, and has high aquatic biologic safety, suitable for application in most aquaculture waters. The strain has both heterotrophic nitrification and aerobic denitrification, as well as phosphorus removal functions. It can utilize multiple organic carbon sources while exhibiting strong tolerance to high concentrations of organic carbon and salinity, and has a good ability to remove organic carbon in water. The strain is particularly suitable for the treatment of nitrogen-containing wastewater with a high C/N ratio. Under fully aerobic conditions, the strain can use NH.sub.4.sup.+, NO.sub.3.sup.?, and NO.sub.2.sup.? as the sole inorganic nitrogen sources for aerobic nitrification and denitrification. The strain can overcome the incompatibility problem between nitrification and denitrification caused by different oxygen requirements, making it possible to synchronize nitrification and denitrification in the same aerobic reactor, with good economic and environmental benefits and broad application prospects.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application, and not to limit the scope of the present application. Although the best embodiments have been described in detail concerning the present application, those skilled in the art should understand that the technical solution of the present application can be modified or replaced with equivalent alternatives without departing from the essence and scope of the present application.