Application of pseudomonas parayellow in biological denitrification and sewage treatment

Abstract

The invention discloses an application of pseudomonas paraxanthohuensis in biological nitrogen removal and sewage treatment. The invention belongs to the technical field of biology, and particularly relates to application of pseudomonas paraxanthoceras in biological denitrification and sewage treatment. The pseudomonas paraxanthomonas disclosed by the invention can be applied to the following aspects: (a1) biological nitrogen removal; (a2) preparing a biological denitrification product; (a3) sewage treatment; and (a4) preparing a product for sewage treatment. As a heterotrophic bacterium, the strain 1.15632 disclosed by the invention has the capability of converting ammonia into nitrogen under an aerobic condition, and also has good ammonia nitrogen and total nitrogen removal capability. The pseudomonas paraxanthoceras shows great application potential in the aspects of improving the sewage treatment effect, reducing the cost and energy consumption and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a strain of *Pseudomonas paraxanthizobium* in biological denitrification and wastewater treatment. Background Technology

[0002] Nitrogen is an essential nutrient for biological growth and reproduction, playing a vital role in Earth's ecosystems. The complex nitrogen cycle supports diverse life forms, enabling the Earth's ecosystems to maintain biodiversity. With increasing human activity, nitrogen pollution in water bodies has become a serious problem, posing a significant challenge to environmental protection. Excessive nitrogen emissions lead to a series of environmental problems, such as eutrophication, algal blooms, and the death of aquatic organisms due to elevated ammonia nitrogen concentrations. Therefore, exploring the mechanisms of microbial denitrification and developing effective denitrifying microbial strains are crucial for the remediation of aquatic environments.

[0003] Currently, wastewater treatment in China mainly relies on microbial methods, including nitrification-denitrification, anaerobic ammonium oxidation (ANAMMOX), and heterotrophic nitrification-aerobic denitrification (HN-AD). Nitrification-denitrification is a common nitrogen removal method, satisfying the needs of nitrifying and denitrifying bacteria respectively by carrying out nitrification and denitrification under different oxygen conditions. However, traditional nitrification-denitrification processes suffer from high energy consumption, the need for additional carbon sources, large equipment size, and N2O emissions. While anaerobic ammonium oxidation (ANAMMOX) has a high nitrogen removal capacity, its microbial growth is slow and its environmental requirements are stringent, limiting its application. In contrast, heterotrophic nitrification-aerobic denitrification simultaneously achieves nitrification and denitrification in a single reactor, efficiently removing COD and demonstrating advantages in rapid growth and wide adaptability. Despite the great potential shown by heterotrophic nitrification-aerobic denitrification, it is currently mostly in the laboratory stage, and its actual industrial application remains limited.

[0004] In recent years, scientists have discovered a novel denitrification pathway in heterotrophic bacteria—direct ammonia oxidation (Dirammox)—opening up new possibilities for biological denitrification. Dirammox, primarily occurring in Alcaligenes, is mediated by a specific dnf gene cluster and directly converts ammonia into nitrogen gas. Bioinformatics analysis indicates that the dnf gene and its homologs are widely distributed in heterotrophic bacteria, suggesting that direct ammonia oxidation may also occur in other bacteria. This new pathway significantly simplifies traditional denitrification steps and effectively improves denitrification efficiency. Summary of the Invention

[0005] The main problem this invention aims to solve is biological denitrification, wastewater treatment, and reduction of nitrogen pollution in water bodies. To address this problem, this invention provides applications of *Pseudomonas paraxanthioides* and its metabolites in the following:

[0006] 1. Application of Pseudomonas paraxanthomonas and its metabolites in biological denitrification.

[0007] 2. Application of Pseudomonas paraxanthizobium and its metabolites in the preparation of biological denitrification products.

[0008] 3. Application of Pseudomonas paraxanthomonas and its metabolites in wastewater treatment.

[0009] 4. Application of Pseudomonas paraxanthomonas and its metabolites in the preparation of wastewater treatment products.

[0010] In the above application, the *Pseudomonas paraxanthii* is *Pseudomonas paraxanthii* CGMCC 1.15632.

[0011] The *Pseudomonas paraxanthizobium* CGMCC 1.15632 of this invention contains a direct ammonia oxidation homologous gene cluster, including seven genes: dnfI, dnfH, dnfR, dnfG, dnfA, dnfB, and dnfC. Among these, dnfR, dnfA, dnfB, and dnfC are key functional genes in the Dirammox process, while dnfH, dnfG, and dnfI are non-essential genes. The dnfR is 1497 bp in length, with the nucleotide sequence being sequence 2 in the sequence listing, and is predicted to encode a PLP-dependent transaminase family protein; the dnfA is 963 bp in length, with the nucleotide sequence being sequence 3 in the sequence listing, and is predicted to encode a diferrooxygenase; the dnfB is 1062 bp in length, with the nucleotide sequence being sequence 4 in the sequence listing, and is predicted to encode a 2Fe-2S iron-sulfur cluster-binding domain protein; the dnfC is 696 bp in length, with the nucleotide sequence being sequence 5 in the sequence listing, and is predicted to encode a glutamine aminotransferase. At the genomic level, strain 1.15632 demonstrates the ability to denitrify via the direct ammonia oxidation (Dirammox) pathway.

[0012] The *Pseudomonas paraxanthii* can be prepared into a bacterial agent by the following method: *Pseudomonas paraxanthii* is inoculated at 1% (v / v) into 3 mL of LB liquid medium for activation. After 12 h at 30°C and 200 rpm, the bacterial solution is inoculated at 1% (v / v) into LB liquid medium and cultured at 30°C and 200 rpm for 24 h to obtain a bacterial concentration of 10. 8 CFU / mL of Pseudomonas paraxanthizobium CGMCC1.15632 inoculum.

[0013] The active ingredient of the above-mentioned microbial agent may be the above-mentioned Pseudomonas paraxanthii and / or the metabolites of the above-mentioned Pseudomonas paraxanthii. The active ingredient of the above-mentioned microbial agent may also contain other biological or non-biological components.

[0014] In the above text, the metabolites can be obtained from the fermentation broth of *Pseudomonas paraxanthifolius*. The metabolites can be sterile metabolites of *P. paraxanthifolius* or bacterial metabolites of *P. paraxanthifolius*. Specifically, the sterile metabolites of *P. paraxanthifolius* (sterile fermentation filtrate) can be prepared by culturing *P. paraxanthifolius* in a liquid culture medium and filtering to remove *P. paraxanthifolius* from the liquid culture (fermentation broth). Specifically, the bacterial metabolites of *P. paraxanthifolius* can be prepared by culturing *P. paraxanthifolius* in a liquid fermentation medium and collecting the fermentation broth, which is the bacterial metabolites of *P. paraxanthifolius*.

[0015] The term "culture" refers to a liquid or solid product (all substances within the culture container) that has grown a microbial community after artificial inoculation and cultivation. It is a product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0016] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0017] Depending on the needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the above-mentioned microbial agents.

[0018] In the above applications, the wastewater treatment refers to the treatment of ammonia nitrogen, nitrate nitrogen, and / or total nitrogen in wastewater.

[0019] The present invention also provides a wastewater treatment method, wherein the method comprises inoculating the aforementioned Pseudomonas paraxanthii and its metabolites or their culture medium into the wastewater for treatment.

[0020] In the above method, the inoculation medium for *Pseudomonas paraxanthizobium* is LB liquid medium or HNM medium.

[0021] Furthermore, the HNM culture medium is prepared at a temperature of 35°C, a pH of 11, a NaCl concentration of 0 g / L, a carbon-to-nitrogen ratio (C / N) of 10, and an ammonia nitrogen concentration (C(NH4)2) of [missing value]. + Under the condition of 140 mg / L, the total nitrogen (TN) and ammonia nitrogen (NH4+) of *Pseudomonas paraxanthizobium* CMGCC 1.15632 were... + It exhibits the best degradation ability.

[0022] The composition of HNM is as follows: 6.74g sodium succinate hexahydrate, 0.66g ammonium sulfate, 0.5g potassium dihydrogen phosphate, 0.2g magnesium sulfate heptahydrate, 1.25g disodium hydrogen phosphate dodecahydrate, 2mL trace elements (Trace), 1L ddH2O, pH 7.0.

[0023] Trace elements included: EDTA.Na 257.1 g, FeSO4·7H2O 5 g, ZnSO4·7H2O 3.9 g, MnCl2·4H2O 1 g, CoCl2·6H2O 1.6 g, CuSO4·5H2O 1.6 g, (NH4)6Mo7O24·4H2O 1.1 g, CaCl2·2H2O 7 g, ddH2O 1 L, pH 6.0.

[0024] In the above treatment method, the wastewater treatment conditions are 15-40℃.

[0025] The present invention also provides a method for preparing a wastewater treatment agent, wherein the method comprises inoculating the aforementioned *Pseudomonas paraxanthii* culture medium to obtain the wastewater treatment agent.

[0026] In the above method, the amount of wastewater treatment agent added is: 10 mg / L of agent per 1L of wastewater. 8 CFU / mL Pseudomonas paraxanthizobium CGMCC 1.15632.

[0027] The *Pseudomonas paraxanthizobium* CGMCC No. 1.15632 of this invention contains a direct ammonia oxidation homologous gene cluster, and it has been demonstrated that this strain has direct ammonia oxidation function. When ammonia nitrogen (140 mg / L) is used as the sole nitrogen source, 3.19% of the ammonia nitrogen can be converted into nitrogen gas, with an ammonia nitrogen removal rate of 79% and a total nitrogen removal rate of 51.45%. Strain 1.15632 exhibits good pH adaptability, growing well within a pH range of 5-11 and possessing direct ammonia oxidation capability. The removal efficiency of ammonia nitrogen and total nitrogen gradually increases with increasing pH, reaching 100% for ammonia nitrogen and 71.12% for total nitrogen. Strain 1.15632 grows well at NaCl concentrations less than or equal to 20 g / L, achieving ammonia nitrogen removal rates of 66-81% and total nitrogen removal rates of 43-38%. Strain 1.15632 has broad C / N adaptability, growing well within a C / N ratio range of 3-20, and can efficiently remove ammonia nitrogen and total nitrogen. Strain 1.15632 can maintain good growth and efficient nitrogen removal even under high ammonia nitrogen concentrations. Even at an ammonia nitrogen concentration of 1000 mg / L, it still grows well, achieving an ammonia nitrogen removal rate of 64.88% and a total nitrogen removal rate of 44.06%. This *Pseudomonas paraxanthum* has shown great application potential in improving wastewater treatment efficiency and reducing costs and energy consumption. Attached Figure Description

[0028] Figure 1 The comparison is between strain 1.15632 and the dnf gene cluster of Alcaligenes.

[0029] Figure 2 The sequence of the DnfR protein of strain 1.15632 was compared with that of Alcaligenes.

[0030] Figure 3 The sequence of DnfA protein of strain 1.15632 was compared with that of Alcaligenes.

[0031] Figure 4 The sequence of the DnfB protein of strain 1.15632 was compared with that of Alcaligenes.

[0032] Figure 5 The sequence of the DnfC protein of strain 1.15632 was compared with that of Alcaligenes.

[0033] Figure 6 The growth and direct ammonia oxidation capacity of strain 1.15632 with ammonia nitrogen as the sole nitrogen source were investigated.

[0034] Figure 7 Nitrogen balance of nitrogen-containing gaseous products of strain 1.15632 with ammonia nitrogen as the sole nitrogen source.

[0035] Figure 8 The total nitrogen removal rate of strain 1.15632 with ammonia nitrogen as the sole nitrogen source.

[0036] Figure 9 The effects of different pH conditions on the growth and direct ammonia oxidation capacity of strain 1.15632.

[0037] Figure 10 The effects of different NaCl concentrations on the growth and direct ammonia oxidation capacity of strain 1.15632 were investigated.

[0038] Figure 11 The effects of different C / N ratios on the growth and direct ammonia oxidation capacity of strain 1.15632.

[0039] Figure 12 The effects of different initial ammonia nitrogen concentrations on the growth and direct ammonia oxidation capacity of strain 1.15632 were investigated.

[0040] Figure 13 The effects of different dissolved oxygen (rotation speed) on the growth and direct ammonia oxidation capacity of strain 1.15632 were investigated.

[0041] Figure 14 The effects of different temperatures on the growth and direct ammonia oxidation capacity of strain 1.15632.

[0042] Figure 15The effects of different carbon sources on the growth and direct ammonia oxidation capacity of strain 1.15632. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0045] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0046] The *Pseudomonas paraxanthii* CGMCC No. 1.15632 in the following examples has been deposited at the China General Microbiological Culture Collection Center (CGMCC). The public can purchase this biological material from https: / / cgmcc.net / directory / detail?cgmccid=1.15632&number=1.15632&genus=&s pecies=&yiming=&page=1.

[0047] The culture medium preparation methods involved in the following examples are as follows:

[0048] 1) LB medium: 10g tryptone, 5g yeast extract, 10g NaCl, 1L ddH2O, pH 7.0.

[0049] 2) Heterotrophic Nitrification Media (HNM): Sodium succinate hexahydrate 6.74g, ammonium sulfate 0.66g, potassium dihydrogen phosphate 0.5g, magnesium sulfate heptahydrate 0.2g, disodium hydrogen phosphate dodecahydrate 1.25g, trace elements (Trace) 2mL, made up to 1L with ddH2O, pH 7.0. Trace elements (Trace): EDTA·Na 257.1g, FeSO4·7H2O 5g, ZnSO4·7H2O 3.9g, MnCl2·4H2O 1g, CoCl2·6H2O 1.6g, CuSO4·5H2O 1.6g, (NH4)6Mo7O 24· 4H2O 1.1 g, CaCl2·2H2O 7 g, add ddH2O to make up to 1 L, pH 6.0.

[0050] The following examples used GraphPad Prism 9 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0051] Example 1: Identification of the dnf gene cluster in *Pseudomonas paraxanthizobium* 1.15632

[0052] *Pseudomonas paraxanthum* 1.15632 is a Gram-negative bacterium. Colonies are yellow, round, smooth, moist, opaque, and have relatively regular edges. After culturing on LB agar at 30°C for 2 days, the colony diameter is approximately 2-3 mm. Cells are straight or slightly curved short rods. Its 16S rRNA gene sequence is sequence 1 in the sequence listing.

[0053] *Pseudomonas paraxanthomonas* possesses a direct ammonia oxidation homologous gene cluster, including seven genes: dnfI, dnfH, dnfR, dnfG, dnfA, dnfB, and dnfC. A comparison of the dnf gene cluster of strain 1.15632 with that of *Alcaligenes aeruginosa* is shown below. Figure 1 As shown in the figure. Among them, dnfR, dnfA, dnfB, and dnfC genes are key functional genes in the Dirammox process, while dnfH, dnfG, and dnfI are non-essential genes. dnfR is 1497 bp in length, with its nucleotide sequence being sequence 2 in the sequence listing. It is predicted to encode a PLP-dependent transaminase family protein. The sequence alignment of strain 1.15632 with that of *Alcaligenes aeruginosa* is shown in the figure. Figure 2 As shown, the protein identity is 75.5%; the dnfA protein is 963 bp in length, and its nucleotide sequence is sequence 3 in the sequence listing. It is predicted to encode diferrooxygenase. The DnfA protein sequence of strain 1.15632 is compared with that of Alcaligenes as follows. Figure 3 As shown, the protein identity was 71.9%; dnfB was 1062 bp in length, with the nucleotide sequence being sequence 4 in the sequence listing, and was predicted to encode a 2Fe-2S iron-sulfur cluster-binding domain protein. The DnfB protein sequence of strain 1.15632 was compared with that of *Alcaligenes aeruginosa* as shown below. Figure 4 As shown, the protein identity is 51.7%; dnfC is 696 bp in length, and its nucleotide sequence is sequence 5 in the sequence listing. It is predicted to encode glutamine aminotransferase. The sequence alignment of strain 1.15632 with the DnfB protein of Alcaligenes is shown below. Figure 5As shown, the protein identity was 62.8%. At the genomic level, strain 1.15632 was demonstrated to have the potential to complete denitrification via the direct ammonia oxidation (Dirammox) pathway.

[0054] Example 2: Denitrification capacity analysis of Pseudomonas paraxanthizobium 1.15632

[0055] 1. Determination of growth ability and direct ammonia oxidation ability of strain 1.15632 with ammonia nitrogen as the sole nitrogen source.

[0056] Strain 1.15632 was pre-cultured in LB medium to complete strain activation. Then, 200 μL of strain 1.15632 seed culture (inoculation concentration 10) was added. 8 CFU / mL was inoculated into 50 mL Erlenmeyer flasks containing 20 mL of HNM medium (C / N = 7). The HNM medium contained 140 mg / L of initial ammonia nitrogen, with (NH4)2SO4 as the nitrogen source. The flasks were incubated on a rotating shaker at 30 °C and 160 rpm. Bacterial growth (OD) was measured every 6 hours during the 54-hour incubation period. 600 ), NH4 + -N, NH2OH-N, NO2 - -N content.

[0057] The determination methods for ammonia nitrogen are as follows: Determination of ammonia nitrogen in water quality by salicylic acid spectrophotometry HJ 536-2009; the determination methods for nitrite nitrogen are as follows: Determination of nitrite nitrogen in water quality by spectrophotometry GB 7493-87; the determination methods for hydroxylamine nitrogen are as follows: the paper "Frear, DS, & Burrel l, RC (1955). SPECTROPHOTOMETRIC METHOD FOR DETERMINING HYDROXYLAMINE REDUCTASE ACTIVITY IN HIGHER PLANTS. Analytical Chemistry, 27(10), 1664-1665." The determination principle is that in the presence of ethanol and sodium carbonate, hydroxylamine reacts quantitatively with excess 8-hydroxyquinoline to generate a stable green condensate 5,8-quinolinequinone-5-(8-hydroxy-5-quinolyl imide). The hydroxylamine content in the sample can be quantified by measuring the photometric value of its absorption peak at a wavelength of 705 nm.

[0058] Tests on its ammonia oxidation and denitrification capabilities in HNM medium revealed that ammonia consumption accompanied cell growth during ammonia conversion. Figure 6 As shown, hydroxylamine accumulates to a certain extent within 6 hours, and is then rapidly consumed, with a small amount of nitrite produced throughout the process.

[0059] After 6 hours of growth, strain 1.15632 cells entered the logarithmic growth phase, and the OD value reached 18 hours after culture. 600 It reached a peak (0.762), then decreased slightly. Before 6 hours, it increased with NH4... + -N decreases, and NH2OH-N gradually accumulates, reaching a maximum of 0.9 mg / L at 6 h, and becoming almost undetectable after 12 h. Small amounts of nitrite (0.5 mg / L) and nitrate (0.5 mg / L) were detected throughout the reaction process.

[0060] 2. Detection of nitrogen-containing gaseous products from strain 1.15632, which uses ammonia nitrogen as the sole nitrogen source.

[0061] Strain 1.15632 was pre-cultured in LB medium, and then 40 μL of 1.15632 bacterial culture was inoculated into a medium containing 4 mL of HNM (containing 5 mM ( 15 The culture medium (NH4)2SO4 was placed in a 50 mL anaerobic flask. The headspace of the flask was filled with He / O2 at a ratio of 4:1 as described above. The flask was incubated on a rotating shaker at 30°C and 160 rpm for 72 h. N2-N and N2O-N were measured, followed by bacterial growth (OD). 600 ), NH4 + -N, NH2OH-N, NO2 - -N, NO3 - -N.

[0062] The results showed that strain 1.15632 produced nitrogen gas during the aerobic conversion of ammonia. Based on nitrogen balance analysis, strain 1.15632 could ultimately convert most of the 10 mM nitrogen gas into nitrogen. 15 NH4-N consumption, and can consume 3.19% of it. 15 NH4 + Transform into 15 N2, with a production of 0.01% 15 N2O ( Figure 7 Meanwhile, trace amounts of nitrite (0.06%) and nitrate (0.16%) were detected. In addition, most of the consumed ammonia was converted into cellular nitrogen and organic nitrogen, with a total nitrogen removal rate of 51.45%.

[0063] 3. Detection of total nitrogen removal rate of strain 1.15632 using ammonia nitrogen as the sole nitrogen source.

[0064] The removal rate of total nitrogen in water by strain 1.15632 was determined by culturing strain 1.15632 in HNM medium until the bacterial concentration reached 10⁻⁶. 8CFU / mL, centrifuged fermentation broth. Total nitrogen was determined according to the method described in "Determination of Total Nitrogen in Water Quality - Alkaline Potassium Persulfate Digestion - Ultraviolet Spectrophotometry GB 11894-89". Total nitrogen was converted to nitrate nitrogen through high-temperature digestion before spectrophotometric determination. Biomass nitrogen (biomass-N) was determined using the same method as total nitrogen, calculated via the differential method. The total nitrogen removal rate of strain 1.15632 with ammonia nitrogen as the sole nitrogen source was as follows: Figure 8 As shown, at 30°C and 160 rpm, strain 1.15632 can remove approximately 51.45% of the total nitrogen (TN) from HNM medium.

[0065] Example 3: Effects of different HNM culture medium conditions on the growth and direct ammonia oxidation capacity of strain 1.15632

[0066] 1. Optimization of pH conditions in HNM medium

[0067] The initial pH of the HNM medium was adjusted to 5, 6, 7, 8, 9, 10, and 11. Sodium succinate was used as the sole carbon source, and the initial ammonia nitrogen concentration was set at 140 mg / L with a C / N ratio of 7. The culture was carried out with shaking at 30°C and 160 rpm. The growth and direct ammonia oxidation capacity of strain 1.15632 were measured. The growth assay, direct ammonia oxidation capacity assay, and total nitrogen (TN) removal rate assay were performed using the same methods as in Example 2.

[0068] The results are as follows Figure 9 As shown, strain 1.15632 grows well in the pH range of 5-11 and exhibits good direct ammonia oxidation capacity. There was no significant difference in growth among strains within the pH range of 5-11; however, at pH 5, a high level of hydroxylamine accumulation was detected, approximately 2.5 mg / L (approximately 178.57 μM); the maximum detectable accumulation of nitrite nitrogen was 0.35 mg / L (approximately 25 μM).

[0069] The removal capacities of ammonia nitrogen and total nitrogen are shown in Table 1 below. Within the pH range of 5-11, strain 1.15632 achieved an ammonia nitrogen removal rate of 68.95-97.77% and a total nitrogen removal rate of 32.66-71.14%.

[0070] Table 1. Removal capacity of ammonia nitrogen and total nitrogen from fermentation broth under different pH conditions

[0071] pH Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 5 68.95±1,91 32.66±0.74 6 78.53±2.42 39.15±2.10 7 81.21±0.43 48.71±0.58 8 85.08±0.61 53.91±1.14 9 89.92±1.83 58.67±1.07 10 96.35±0.36 65.73±0.42 11 97.77±0.32 71.14±2.59

[0072] 2. Optimization of different NaCl concentrations in HNM medium

[0073] The NaCl concentration in HNM medium was adjusted to 0 g / L, 20 g / L, 40 g / L, 60 g / L, and 80 g / L, respectively. Sodium succinate was used as the sole carbon source. The initial ammonia nitrogen concentration was 140 mg / L, the C / N ratio was 7, and the initial pH was 7.0. The culture was carried out with shaking at 30°C and 160 rpm. The growth and direct ammonia oxidation capacity of strain 1.15632 were measured. The growth determination, direct ammonia oxidation capacity determination, and total nitrogen (TN) removal rate detection were performed using the same methods as in Example 2.

[0074] The results are as follows Figure 10 As shown, strain 1.15632 grows well and exhibits direct ammonia oxidation ability in the NaCl concentration range of 0-20 g / L. However, the growth of the strain is significantly inhibited at NaCl concentrations of 40-80 g / L.

[0075] The removal capacity of ammonia nitrogen and total nitrogen is shown in Table 2 below. The strain can grow normally in the range of NaCl concentration of 0-20 g / L. At this time, the removal rate of ammonia nitrogen is 66.1-81.21% and the removal rate of total nitrogen is 43.08-48.71%. When the NaCl concentration is greater than 40 g / L, the growth of the strain is significantly inhibited.

[0076] Table 2. Removal capacity of ammonia nitrogen and total nitrogen in fermentation broth under different NaCl concentrations

[0077] NaCl (g / L) Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 0 81.21±0.43 48.71±0.58 20 66.15±3.44 43.08±0.45 40 8.15±1.87 9.99±0.87 60 8.12±1.10 5.37±1.1 80 4.89±1.60 6.89±1.04

[0078] 3. Optimization of different C / N conditions in HNM culture medium

[0079] Sodium succinate was used as the sole carbon source, and the C / N ratios were adjusted to 3, 7, 10, 15, and 20, respectively. The initial ammonia nitrogen concentration was set to 140 mg / L, and the initial pH of the culture medium was adjusted to 7.0. The culture was then incubated at 30°C and 160 rpm with shaking. The growth and direct ammonia oxidation capacity of strain 1.15632 were determined. The growth assay, direct ammonia oxidation capacity assay, and total nitrogen (TN) removal rate assay were performed using the same methods as in Example 2.

[0080] The results are as follows Figure 11 As shown, strain 1.15632 grows well in the C / N ratio range of 3-20 and exhibits good direct ammonia oxidation ability. Within the C / N range of 3-20, the growth rate and amount of strain 1.15632 increase with increasing C / N ratio, while the hydroxylamine accumulation shows no significant difference within the C / N range of 3-20.

[0081] The removal capacities of ammonia nitrogen and total nitrogen are shown in Table 3 below. Within the C / N ratio range of 3-20, strain 1.15632 achieved an ammonia nitrogen removal rate of 65.79%-99.9% and a total nitrogen removal rate of 40.5%-54.8%.

[0082] Table 3. Removal capacity of ammonia nitrogen and total nitrogen in fermentation broth under different C / N ratios

[0083] C / N Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 3 65.79±3.71 40.5±2.2 7 81.21±0.43 54.8±2.2 10 99.02±0.23 54.3±1.9 15 99.22±0.08 51.9±1.8 20 99.90±0.02 40.5±2.2

[0084] 4. Different NH4+ in HNM culture medium + -N concentration conditions optimization

[0085] NH4 in HNM medium + The -N concentration was adjusted to 35, 70, 140, 500, 750, and 1000 mg / L, respectively. Sodium succinate was used as the sole carbon source with an initial carbon source concentration of 980 mg / L and an initial pH of 7.0. The strain was cultured at 30°C and 160 rpm with shaking. The growth and direct ammonia oxidation capacity of strain 1.15632 were measured. The growth assay, direct ammonia oxidation capacity assay, and total nitrogen (TN) removal rate assay were performed using the same methods as in Example 2.

[0086] The results are as follows Figure 12 As shown, strain 1.15632 grew well and consistently within an ammonia nitrogen concentration range of 500-1000 mg / L. Growth was good at ammonia nitrogen concentrations of 35-140 mg / L, increasing with increasing concentration. Hydroxylamine accumulation was strongest at 6 hours with an initial ammonia nitrogen concentration of 140 mg / L. The strain produced small amounts of nitrite and nitrate at ammonia nitrogen concentrations of 35-140 mg / L, and a large amount of nitrate (3.14-3.48 mg / L) at ammonia nitrogen concentrations of 500-1000 mg / L, while nitrite production was relatively low at this concentration.

[0087] The removal capacities of ammonia nitrogen and total nitrogen are shown in Table 4 below. Within the ammonia nitrogen concentration range of 35-1000 mg / L, the ammonia nitrogen removal rate of strain 1.15632 is 64.87-94.80%, and the total nitrogen removal rate is between 52.19-71.43%.

[0088] Table 4. Different NH4 4+ Ammonia and total nitrogen removal capacity in fermentation broth under -N concentration conditions

[0089] Ammonia nitrogen concentration (mg / L) Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 35 86.39±2.94 14.44±4.31 70 87.34±2.37 40.14±9.20 140 94.80±0.82 42.09±5.19 500 82.78±0.82 53.45±3.11 750 74.29±1.11 41.70±4.66 1000 64.87±0.32 44.06±4.45

[0090] 5. Optimization of different dissolved oxygen conditions in HNM medium

[0091] Sodium succinate was used as the sole carbon source. The shaking speed was adjusted to 0, 80, 160, and 200 rpm, the initial ammonia nitrogen concentration was set to 140 mg / L, the initial pH of the culture medium was adjusted to 7.0, C / N = 7, and the culture was carried out at 30℃. The growth, direct ammonia oxidation capacity, and total nitrogen (TN) removal rate of strain 1.15632 were measured.

[0092] The results are as follows Figure 13 As shown, strain 1.15632 grows well within a rotation speed range of 0-200 rpm. Within a C / N ratio range of 0-200 rpm, the time for strain 1.15632 to enter the stationary phase increases with increasing dissolved oxygen content (rotation speed); hydroxylamine production is best at 160 rpm.

[0093] The ammonia nitrogen and total nitrogen removal capabilities of strain 1.15632 are shown in Table 5. Within the rotation speed range of 0-200 rpm, the ammonia nitrogen removal rate of strain 1.15632 is 94.84-95.65%, and the total nitrogen removal rate is between 49.3-54.61%.

[0094] Table 5. Removal capacity of ammonia nitrogen and total nitrogen in fermentation broth under different dissolved oxygen conditions

[0095] Rotational speed (rpm) Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 0 94.84±0.19 50.82±5.06 80 95.15±0.18 49.30±3.82 160 95.64±0.12 53.66±8.38 200 95.57±0.16 54.61±4.12

[0096] 6. Optimization of different culture temperature conditions in HNM medium

[0097] Sodium succinate was used as the sole carbon source. The temperature was adjusted to 15, 20, 30, 35 and 40 °C, the initial ammonia nitrogen concentration was set to 140 mg / L, the initial pH of the culture medium was adjusted to 7.0, C / N = 7, and the culture was shaken at 160 rpm. The growth, direct ammonia oxidation capacity and total nitrogen (TN) removal rate of strain 1.15632 were measured.

[0098] The results are as follows Figure 14 As shown, strain 1.15632 grows well in the temperature range of 10-40℃ and has good direct ammonia oxidation ability. In the temperature range of 15-35℃, the time for strain 1.15632 to enter the stationary phase decreases with increasing temperature, and the growth of the strain is significantly inhibited at 40℃.

[0099] The ammonia nitrogen and total nitrogen removal capabilities of strain 1.15632 are shown in Table 6. Within the temperature range of 10-40℃, the ammonia nitrogen removal rate of strain 1.15632 ranges from 23.01% to 94.69%, and the total nitrogen removal rate ranges from 24.82% to 50.34%.

[0100] Table 6. Removal capacity of ammonia nitrogen and total nitrogen in fermentation broth under different culture temperatures

[0101] Temperature (°C) Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 15 76.63±0.45 28.43±5.85 20 78.32±0.34 32.79±7.23 30 89.42±1.13 49.01±4.27 35 94.69±1.09 50.34±4.15 40 23.01±1.40 24.82±7.89

[0102] 7. Optimization of different carbon source conditions in HNM medium

[0103] Using ammonium sulfate as the sole nitrogen source, different carbon sources (citric acid, L-(-)-malic acid, fumaric acid, α-ketoglutarate, and sodium pyruvate) were added. The initial pH was 7.0, and the strain was cultured with shaking at 30°C and 160 rpm. The growth and total nitrogen removal capacity of strain 1.15632 were determined. The growth assay, direct ammonia oxidation capacity assay, and total nitrogen (TN) removal rate assay were performed using the same methods as in Example 2.

[0104] The results are as follows Figure 15 As shown, strain 1.15632 grew well in citric acid, L-(-)-malic acid, fumaric acid, α-ketoglutarate, and sodium pyruvate. Under conditions where sodium pyruvate was the sole carbon source, strain 1.15632 achieved the highest hydroxylamine accumulation of 4.17 mg / L (226 μM).

[0105] The ammonia nitrogen and total nitrogen removal capabilities of strain 1.15632 are shown in Table 7. Under different carbon source conditions, the ammonia nitrogen removal rate of strain 1.15632 was 97.7-99.52%, and the total nitrogen removal rate was between 54.35-61.19%.

[0106] Table 7. Removal capacity of ammonia nitrogen and total nitrogen in fermentation broth under different carbon source conditions

[0107] carbon source Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) Citric acid 97.70±0.08 54.35±1.85 L-(-)-malic acid 99.52±0.12 61.19±3.62 fumaric acid 98.78±0.20 55.91±1.02 α-Ketoglutarate 98.01±0.17 58.52±5.32 Sodium pyruvate 99.47±0.04 51.61±2.69

[0108] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of Pseudomonas paraxanthomonas and its metabolites in biological denitrification.

2. Application of Pseudomonas paraxanthomonas and its metabolites in the preparation of biological denitrification products.

3. Application of Pseudomonas paraxanthomonas and its metabolites in wastewater treatment.

4. Application of Pseudomonas paraxanthomonas and its metabolites in the preparation of wastewater treatment products.

5. The application according to any one of claims 1-4, characterized in that, The *Pseudomonas paraxanthii* was *Pseudomonas paraxanthii* CGMCC 1.15632.

6. The application according to claim 3 or 4, characterized in that, The wastewater treatment involves removing ammonia nitrogen, nitrate nitrogen, and / or total nitrogen from wastewater.

7. A wastewater treatment method, characterized in that, The method involves inoculating wastewater with the *Pseudomonas paraxanthum* and its metabolites or their culture medium as described in claims 1-5.

8. The method according to claim 7, characterized in that: The inoculation medium for *Pseudomonas paraxanthizobium* was HNM medium.

9. The method according to claim 8, characterized in that: The HNM culture medium consisted of 4.72 g sodium succinate hexahydrate, 0.66 g ammonium sulfate, 0.5 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 1.25 g disodium hydrogen phosphate dodecahydrate, 2 mL trace elements, 1 L ddH2O, and pH 7.

0. The trace elements were: EDTA·Na 257.1 g, FeSO4·7H2O 5 g, ZnSO4·7H2O 3.9 g, MnCl2·4H2O 1 g, CoCl2·6H2O 1.6 g, CuSO4·5H2O 1.6 g, (NH4)6Mo7O24·4H2O 1.1 g, CaCl2·2H2O 7 g, 1 L ddH2O, and pH 6.

0.

10. A method for preparing a wastewater treatment agent, characterized in that, The method involves inoculating the culture medium with *Pseudomonas paraxanthii* as described in any one of claims 1-5 to obtain the wastewater treatment agent.

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