Pseudomonas putida capable of removing p-xylene and nitrogen under anaerobic condition, microbial inoculum and method and application of pseudomonas putida

Abstract

The invention provides pseudomonas putida capable of removing p-xylene and nitrogen under an anaerobic condition, a microbial agent and a method and application of the pseudomonas putida, and relates to the field of microbial fermentation. The invention provides Pseudomonas putida capable of removing xylene and nitrogen under an anaerobic condition, the preservation number of the Pseudomonas putida is CCTCC (China Center for Type Culture Collection) NO: M20241686, and the Pseudomonas putida is preserved in the China Center for Type Culture Collection on July 25, 2024. When the strain takes NH4 < + >-N, NO3 <->-N and NO2 <->-N as single nitrogen sources (15mg / L), the removal rates of the strain on NH4 < + >-N, NO3 <->-N and NO2 <->-N respectively reach 59.08%, 96.90% and 100%; when p-xylene is used as a unique carbon source, the 72h removal rate of p-xylene of the strain can reach 55.54%. The optimum denitrification conditions of the strain are as follows: the optimum carbon source sodium succinate hexahydrate, the optimum C / N is 3, the optimum culture temperature is 30 DEG C, the optimum pH is 7, and meanwhile, the strain can tolerate a weak alkaline environment and a non-acid environment.

Description

Technical Field

[0001] The present invention belongs to the field of microbial fermentation, and relates to Pseudomonas putida, in particular to Pseudomonas putida with the ability to remove p-xylene and nitrogen under anaerobic conditions, a bacterial agent thereof, and methods and applications thereof. Background Art

[0002] High concentrations of NO 3 - -N in water bodies can lead to a series of human health and environmental risks. High concentrations of NO 3 - -N sewage entering rivers, lakes, etc. can stimulate the overgrowth of algae, resulting in water quality deterioration, reduced biodiversity, and ecosystem degradation. NO 3 - The toxicity of NO 3 - -N to livestock has been widely concerned. If the concentration of NO 3 - -N in feed or groundwater is too high, it will lead to an increase in livestock mortality. In ruminants, NO 2 - -N is reduced to NO 3 - -N, which can cause methemoglobinemia; the manifestations are cyanosis and hypoxia, which will immediately threaten the life of ruminants, and hypoxia can cause pregnant ruminants to abort. Therefore, the groundwater quality standard stipulates that the content of NO 3 - -N cannot exceed 20 mg / L. NO 3 - -N can be used by algae as a nutrient. When the content of NO

[0003] Aromatic VOCs are usually present in coal tar, petroleum products, and various organic chemical product formulations. They are a typical type of pollutant in chemical industrial polluted sites. Among them, refineries, pesticide factories, chemical plants, gas stations, and compost plants are the main pollution sources. Data shows that most of the volatile organic compounds in the environment are trace organic compounds with very low concentrations. Nearly half of the VOCs concentrations are between 0.01 and 1.0 μg / L, and some are below the detection limit. Although the concentration of VOCs in the environment is not high, due to the irritancy and toxicity of volatile organic compounds, they belong to the pollutants that are preferentially controlled in water quality monitoring in China.

[0004] There are many types of aromatic VOCs, including benzene, toluene, naphthalene, nitrobenzene, etc. Among them, p-xylene, as a typical VOC, is widely used in the production of plastics, rubber, paints and synthetic fibers. It is toxic, volatile, insoluble in water, and harmful to the skin, eyes, respiratory system and various organs. p-Xylene is an important chemical raw material in the petrochemical and pharmaceutical industries, and is used to produce many polymers, plastics, solvents and fuels. The International Oil Tanker Owners Pollution Federation has identified p-xylene as one of the 20 chemicals with the highest risk among hazardous and toxic substances. p-Xylene is widely used as an industrial solvent and has a high degree of mobility in the environment in gaseous, liquid or solid phases. p-Xylene is widely used as a solvent and can be distributed as a pollutant in the environments of various industries, such as leather, paint, rubber and printing, gasoline, aviation fuel, etc. According to the "Integrated Emission Standard of Air Pollutants in China" (GB 16297-1996), the maximum allowable emission concentration in the atmosphere is 90 mg / m 3 . Since p-xylene is a liquid at room temperature, it may enter surface water and soil. The limit value of p-xylene in the centralized drinking water surface water source of the "Surface Water Environment Quality Standard" (GB 3838-2002) is 0.5 mg / L. p-Xylene is very easy to evaporate, and high concentrations of p-xylene are rarely found in surface water or topsoil, but any p-xylene that does not evaporate from the topsoil can enter the groundwater downward. Therefore, eliminating p-xylene is crucial for the ecosystem and public safety and health.

[0005] Compared with the physical and chemical denitrification method, the commonly used biological denitrification method in the sewage treatment system is more green, economical and efficient. Conventional biological denitrification involves two processes: aerobic nitrification and anaerobic denitrification, which are carried out by nitrifying bacteria and denitrifying bacteria respectively. Denitrification is the key to biological denitrification technology. The denitrification reaction can be divided into three types according to the nutritional type and oxygen demand: heterotrophic anoxic denitrification, heterotrophic aerobic denitrification and autotrophic denitrification.

[0006] Due to water eutrophication, bottom inorganic nutrients will be regenerated to consume oxygen, increasing the primary productivity of water bodies. The growth and decay processes of primary water producers will form water organic matter, and the rapid degradation of this substance will consume dissolved oxygen (DO), leading to the existence of an anaerobic environment. Facultative anaerobes and anaerobes can survive in anaerobic conditions and form stable populations. The biggest characteristic of facultative anaerobic bacteria is their ability to survive and reproduce in an oxygen-free environment without being threatened by oxygen in the environment. The natural environments of sites contaminated by hydrocarbons, such as soil, groundwater aquifers, fresh and marine sediments, and oil reservoirs, etc., all share the commonality of being anaerobic, and facultative anaerobic bacteria can survive and reproduce in such environments. At the same time, facultative anaerobes can also use hydrocarbon pollutants as carbon and energy sources to achieve the purpose of removing pollutants. Therefore, it is possible to consider developing a stable population of microorganisms that can remove p-xylene under anaerobic denitrification conditions (Tucci M, et al. Toluene-driven anaerobic biodegradation of chloroform in a continuous-flow bioelectrochemical reactor[J]. Chemosphere, 2023.).

[0007] The prior art CN101892178A discloses a facultative anaerobic degradation bacterium for benzene compounds, which is Microbacterium schleiferi HBSD-C. It was deposited at the China Center for Type Culture Collection on November 27, 2009, with the deposit number: CCTCC No. M209284. Colony morphology: light yellow, round, semi-transparent, neat edges, sunken, smooth surface; cells are straight rod-shaped, Gram-positive, non-motile; main biochemical characteristics: oxidase positive, catalase positive, utilize glucose, fructose, sucrose, do not utilize maltose, denitrification positive, gelatin hydrolysis negative, facultative anaerobic, grow at 4°C - 41°C. This strain can degrade benzene, toluene, xylene, and mesitylene under facultative aerobic conditions, and the degradation rate of benzene compounds with a concentration of about 175.8 mg / L by this strain within 3 days is between 25.9% and 41.2%. The disadvantage of this technology is that the denitrification ability of the strain was not measured, and the degradation rate of p-xylene is low. Summary of the Invention

[0008] In view of the problem that there is a lack of strains capable of efficiently co-removing p-xylene and nitrogen under anaerobic conditions, the present invention provides a Pseudomonas putida with the ability to remove both p-xylene and nitrogen, as well as its culture solution, microbial agent, and application. The present invention provides a Pseudomonas putida capable of removing both p-xylene and nitrogen under anaerobic conditions, with a preservation number of CCTCC NO: M20241686, which was preserved at the China Center for Type Culture Collection on July 25, 2024. When this strain uses NH 4 + -N, NO 3 - -N, and NO 2 - -N as the sole nitrogen source (15 mg / L), the removal rates of NH 4 + -N, NO 3 - -N, and NO 2 - -N reach 59.08%, 96.90%, and 100% respectively; when using p-xylene as the sole carbon source, the 72-hour removal rate of p-xylene by this bacterium can reach 55.54%. The optimal conditions for nitrogen removal by this bacterium are as follows: the optimal carbon source is sodium succinate hexahydrate, the optimal C / N is 3, the optimal culture temperature is 30 °C, the optimal pH is 7, and it can tolerate a weakly alkaline environment but not an acidic environment.

[0009] To achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0010] On the one hand, the present invention provides a Pseudomonas putida with the ability to remove both p-xylene and nitrogen, with a preservation number of CCTCC NO: M20241686, which was preserved at the China Center for Type Culture Collection on July 25, 2024, and is named Pseudomonas putida strain 19, abbreviated as strain 19, and its 16S rDNA sequence is the sequence shown in SEQ ID NO.1.

[0011] On the other hand, the present invention provides a microbial agent, which contains the above-mentioned Pseudomonas putida or a preparation of the above-mentioned Pseudomonas putida.

[0012] Preferably, the preparation of the Pseudomonas putida includes a culture of the above-mentioned Pseudomonas putida, a culture extract, a freeze-dried powder, a fermentation broth, a fermentation broth precipitate, a fermentation broth supernatant, or a fermentation broth extract.

[0013] Preferably, the bacterial agent is inoculated into a culture medium with the above-mentioned Pseudomonas putida for fermentation. After filtration of the fermentation broth, a fermentation broth precipitate is obtained, which is then prepared by freeze-drying.

[0014] On the other hand, the present invention provides the application of the above-mentioned Pseudomonas putida or the above-mentioned bacterial agent, and the application includes denitrifying and removing nitrogen in water bodies or removing p-xylene in water bodies.

[0015] Preferably, the application includes denitrifying and removing nitrogen in water bodies and synergistically removing p-xylene.

[0016] On the other hand, the present invention provides a method for denitrifying and removing nitrogen in water bodies and synergistically removing p-xylene. The method is to add the above-mentioned Pseudomonas putida or the above-mentioned bacterial agent into a nitrogen-containing water body for cultivation.

[0017] Preferably, the method is to add the above-mentioned Pseudomonas putida or the above-mentioned bacterial agent into a nitrogen-containing water body for cultivation, which can achieve denitrifying and removing nitrogen and denitrifying and synergistically removing p-xylene in the water body under an anaerobic environment.

[0018] Preferably, the nitrogen-containing water body includes wastewater or artificial culture medium, and the wastewater or artificial culture medium contains the necessary nutrients for the growth of the above-mentioned Pseudomonas putida and has suitable pH conditions and temperature conditions.

[0019] Preferably, the nitrogen-containing water body includes a nitrogen-containing culture medium, and the components of the nitrogen-containing culture medium include: a carbon source, a nitrogen source, KH 2 PO 4 , Na 2 HPO 4 ·12H 2 O, MgSO 4 ·7H 2 O and trace elements, and the pH of the nitrogen-containing culture medium is 5-9.

[0020] Preferably, the carbon source includes sodium potassium tartrate, sodium succinate hexahydrate, sodium citrate, sodium acetate or glucose. The carbon source mainly provides energy for the growth of the strain and serves as an electron donor in the denitrification process. Different carbon sources have a greater impact on the denitrification ability of the strain.

[0021] Preferably, the carbon source is sodium succinate hexahydrate.

[0022] Preferably, the nitrogen source includes nitrate, nitrite or ammonium salt.

[0023] Preferably, the nitrogen source is nitrate.

[0024] Preferably, the carbon-nitrogen molar ratio of the carbon source and the nitrogen source is 1-60.

[0025] Preferably, the carbon-nitrogen molar ratio is 15.

[0026] Preferably, the pH of the nitrogen-containing medium is 7.0. Since pH can cause changes in the cell membrane charge, thereby affecting the absorption of nutrients by microorganisms and also affecting the activity of related enzymes within the cells, an appropriate pH has a great impact on the metabolic capacity of the strain.

[0027] Preferably, the cultivation temperature of the nitrogen-containing medium is 25 - 35 °C.

[0028] Preferably, the temperature of the nitrogen-containing medium is 30 °C.

[0029] Preferably, the components of the nitrogen-containing medium include KNO 3 0.100 - 0.120 g / L, sodium succinate hexahydrate 1.0000 - 1.2000 / L, KH 2 PO 4 0.200 - 0.250 g / L, Na 2 HPO 4 ·12H 2 O 0.780 - 0.800 g / L, MgSO 4 ·7H 2 O 0.008 - 0.012 g / L and trace elements 1.5 - 2.5 mL / L, and the trace elements include FeCl 2 ·4H 2 O 1.6 - 2.0 g / L, CoCl 2 ·6H 2 O 0.23 - 0.27 g / L, NiCl 2 ·6H 2 O 0.008 - 0.012 g / L, CuCl 2 ·2H 2 O 0.008 - 0.012 g / L, MnCl 2 ·4H 2 O 0.60 -

[0030] 0.80 g / L, ZnCl 2 0.08 - 0.12 g / L, H 3 BO 3 0.4 - 0.6 g / L, Na 2 MoO 4 ·2H 2 O 0.02 - 0.04 g / L and NaSeO 3 ·5H 2 O 0.008 - 0.012 g / L.

[0031] Preferably, the components of the nitrogen-containing medium include KNO3 0.108 g / L, sodium succinate hexahydrate 1.1562 g / L, KH 2 PO 4 0.226 g / L, Na 2 HPO 4 ·12H 2 O 0.792 g / L, MgSO 4 ·7H 2 O 0.01 g / L and trace elements 2.0 mL / L. The trace elements include FeCl 2 ·4H 2 O 1.8 g / L, CoCl 2 ·6H 2 O 0.25 g / L, NiCl 2 ·6H 2 O 0.01 g / L, CuCl 2 ·2H 2 O 0.01 g / L, MnCl 2 ·4H 2 O 0.70 g / L, ZnCl 2 0.1 g / L, H 3 BO 3 0.5 g / L, Na 2 MoO 4 ·2H 2 O 0.03 g / L and NaSeO 3 ·5H 2 O 0.01 g / L.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The anaerobic nitrogen-rich wastewater treated with the Pseudomonas putida 19 provided by the present invention has good anaerobic denitrification ability under the condition of low carbon-nitrogen ratio. At the same time, in the case of using p-xylene as the sole carbon source, it can effectively remove p-xylene and nitrogen in water. Strain 19 shows excellent anaerobic denitrification and nitrogen removal ability at low carbon-nitrogen ratio and good ability of synergistic denitrification to remove p-xylene. The removal rate of p-xylene by this strain can reach 55.54% in 72 h.

[0034] 2. The anaerobic Pseudomonas strain 19 provided by the present invention can simultaneously achieve the functions of nitrogen removal and p-xylene removal under anaerobic conditions, which is convenient to use and simple to operate, reducing the sewage treatment cost. This strain can be made into a new type of microecological bactericide and has good application prospects for treating composite wastewater.

[0035] Preservation description

[0036] Name of the strain: Pseudomonas putida;

[0037] Latin name: Pseudomonas putida;

[0038] Strain number: Pseudomonas putida strain 19;

[0039] Depositary institution: China Center for Type Culture Collection;

[0040] Abbreviation of the depositary institution: CCTCC;

[0041] Address: Inside Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0042] Date of deposit: July 25, 2024;

[0043] Deposit number: CCTCC NO: M20241686. Description of the drawings

[0044] Figure 1 Electron micrograph and Gram stain picture of strain 19, where A is the electron micrograph and B is the Gram stain picture.

[0045] Figure 2 Phylogenetic tree of strain 19.

[0046] Figure 3 PCR amplification of denitrifying enzyme-related genes (M is Marker: DL 2000DNA).

[0047] Figure 4 Growth curve of strain 19 when nitrate is the sole nitrogen source, NH 4 + -N concentration, NO 3 - -N concentration, NO 2 - -N concentration and TN concentration result graph.

[0048] Figure 5 Growth curve of strain 19 when ammonium salt is the sole nitrogen source, NH 4 + -N concentration, NO 3 - -N concentration, NO 2 - -N concentration and TN concentration result graph.

[0049] Figure 6 Growth curve of strain 19 when nitrite is the sole nitrogen source, NH 4 + -N concentration, NO 3- -N concentration, NO 2 - -N concentration and TN concentration result graph.

[0050] Figure 7 Effect of different carbon sources on the growth and denitrification of strain 19.

[0051] Figure 8 Effect of different C / N ratios on the growth and denitrification of strain 19.

[0052] Figure 9 Effect of different pH values on the growth and denitrification of strain 19.

[0053] Figure 10 Effect of different temperatures on the growth and denitrification of strain 19.

[0054] Figure 11 Growth curve of strain 19, NH 4 + -N concentration, NO 3 - -N concentration, NO 2 - -N concentration and TN concentration result graph.

[0055] Figure 12 p-Xylene removal by strain 19 at different culture times. Specific implementation manner

[0056] It should be noted that the raw materials used in the present invention are all ordinary commercially available products, and their sources are not specifically limited.

[0057] Setting of the culture medium:

[0058] DBM (enrichment medium): KNO 3 5.0 g / L, succinic acid hexahydrate 11.1 g / L, KH 2 PO 4 1.0 g / L, Na 2 HPO 4 ·12H 2 O 7.03 g / L, MgSO 4 ·7H 2 O 0.13 g / L, trace elements 2 mL / L, adjust pH to 7.0.

[0059] BTB (bromothymol blue solid medium): KNO 3 5.0 g / L, succinic acid hexahydrate 11.1 g / L, KH 2 PO 4

[0060] 1.0 g / L, Na 2 HPO 4 ·12H 2 O 7.03 g / L, MgSO 4 ·7H 2 O 0.13 g / L, trace elements 2 mL / L, 1% bromothymol blue, 2% agar powder, adjust pH to 7.0.

[0061] Nitrate nitrogen medium (C / N = 15:1): KNO 3 0.108 g / L, sodium succinate hexahydrate 1.1562 g / L, KH 2 PO 4 0.226 g / L, Na 2 HPO 4 ·12H 2 O 0.792 g / L, MgSO 4 ·7H 2 O 0.01 g / L, trace elements 2 mL / L, adjust pH to 7.0.

[0062] Nitrite nitrogen medium (C / N = 15:1): NaNO 2 0.0738 g / L, sodium succinate hexahydrate 1.1566 g / L, KH 2 PO 4 0.226 g / L, Na 2 HPO 4 ·12H 2 O 0.792 g / L, MgSO 4 ·7H 2 O 0.01 g / L, trace elements 2 mL / L, adjust pH to 7.0.

[0063] Ammonium nitrogen medium (C / N = 15:1): NH 4 Cl 0.058 g / L, sodium succinate hexahydrate 1.1723 g / L, KH 2 PO 4 0.226 g / L, Na 2 HPO 4 ·12H 2 O 0.792 g / L, MgSO 4 ·7H 2 O 0.01 g / L, trace elements 2 mL / L, adjust pH to 7.0.

[0064] Test medium for removing p - xylene as the sole carbon source (C / N = 15:1): KNO 3 0.108 g / L, KH 2 PO 4

[0065] 0.226 g / L, Na 2 HPO 4 ·12H 2 O 0.792 g / L, MgSO 4 ·7H 2 O 0.01 g / L, trace elements 2 mL / L, adjust pH to 7.0.

[0066] Trace elements: FeCl 2 ·4H 2 O 1.8 g / L, CoCl 2 ·6H 2 O 0.25 g / L, NiCl 2 ·6H 2 O 0.01 g / L, CuCl 2 ·2H 2 O 0.01 g / L, MnCl 2 ·4H 2 O 0.70 g / L, ZnCl 2 0.1 g / L, H 3 BO 3 0.5 g / L, Na 2 MoO 4 ·2H 2 O 0.03 g / L, NaSeO 3 ·5H 2 O 0.01 g / L.

[0067] Medium sterilization operation: The medium and required materials are sterilized in an autoclave at 121 °C for 20 minutes.

[0068] Detection method and data analysis:

[0069] OD 600 (indicating biomass), TN, NO 3 - -N, NO 2 - -N and NH 4 + -N contents are all determined using a spectrophotometer. The OD 600 value is directly measured at a wavelength of 600 nm. TN is determined using the alkaline potassium persulfate oxidation-ultraviolet spectrophotometry method. NO 3 - -N is determined using the sulfamic acid-ultraviolet spectrophotometry method. NO 2 - -N adopts the sulfanilamide-naphthyl ethylenediamine dihydrochloride method, and NH 4 + -N adopts the Nessler's reagent method for determination.

[0070] The determination of p-xylene refers to the method for determining the concentration of benzene series residues in the solution in the national environmental protection standard "Determination of Volatile Organic Compounds in Water - Headspace / Gas Chromatography-Mass Spectrometry" (HJ 810-2016); Chromatographic grade methanol is used to dilute the p-xylene pure standard into stock solutions with concentrations of 100 mg·L -1 and 1000 mg·L -1 The toluene-D8 pure standard is diluted into a stock solution of 1000 mg·L -1 for standby. Use a microsyringe to transfer the p-xylene stock solution into a headspace vial containing 10 mL of matrix modification solution to prepare solutions with p-xylene series concentrations of 50, 100, 160, 200, 250, 300 μg·L -1 The concentration of the internal standard toluene-D8 is fixed at 200 μg·L -1 , and then instrumental analysis is carried out by GC-MS. The results show that p-xylene has a good linearity in the concentration range of 0-300 μg·L -1 (R 2 > 0.99) and the method detection limit is less than 1 μg·L -1 . Using a GC-MS system, the parameters of the headspace injection system are: furnace temperature 80 °C, sampling needle temperature 95 °C, transfer line temperature 110 °C, carrier gas pressure (He) 19.0 psi, equilibration time 30 min, pressurization time 1 min, injection time 0.04 min, needle withdrawal time 0.5 min, and the GC cycle time is set to 33 min. The chromatographic column model is a DB-1 column (60 m × 0.32 mm × 1.0 μm), and the carrier gas is high-purity helium. The GC temperature program is: starting temperature 60 °C, holding for 2 min; then rising to 120 °C at a rate of 5 °C per minute and holding for 3 min; then rising to 230 °C at a rate of 20 °C per minute and holding for 3 min, with a total program of 25 min. The MS conditions are: electron impact (EI) ion source; ion source temperature 230 °C, ionization energy 70 eV, interface temperature 280 °C. Scanning mode: full scan (Scan); scanning range: 35 amu - 300 amu.

[0071] Example 1: Identification of strains

[0072] (1) Enrichment: Take 5 g of sediment and add 100 mL of sterile DBM, place it in an anaerobic bag, and statically culture it in an incubator at 30 °C. Take 5 mL of the culture solution and transfer it to a fresh sterile DBM medium, and repeat the enrichment 3 times.

[0073] (2) Isolation: Dilute the enrichment solution successively in gradients (10 -1 ~10 -7) Take 10 mL and spread it on the BTB solid medium. Place it in an anaerobic bag and culture it in an incubator at 30 °C until obvious single colonies appear. Pick the blue-changing single colonies, purify them by streaking multiple times, and store them in a refrigerator at 4 °C.

[0074] (3) Screening: Inoculate the isolated and purified strains into the nitrate nitrogen medium respectively, place them in an anaerobic bag, and statically culture them in an incubator at 30 °C. After 24 h, measure the change in nitrate nitrogen concentration, and screen to obtain strain No. 19 with the highest nitrate nitrogen removal rate.

[0075] (4) Gram staining: Inoculate strain No. 19 into the nitrate nitrogen medium, statically culture it in an incubator at 30 °C for 24 h, and monitor the OD 600 value. When the bacteria grow to the logarithmic phase, perform Gram staining on it and determine it as a Gram-negative strain (the staining results are shown in Figure 1 B).

[0076] (5) Biological identification:

[0077] After activating strain No. 19 by streaking on a plate, dilute and spread it on a BTB plate, and observe the colony morphological characteristics. The colonies are round, transparent, moist, and smooth on the surface; use a scanning electron microscope to observe the cell morphology of strain No. 19, and the results are as shown in Figure 1 A. The cells are rod-shaped, and the strain size is (0.9 - 0.92 μm) × 3.92 μm. Use a DNA extraction kit to extract bacterial DNA, use the 16S rRNA universal primer to perform PCR to obtain the 16S rRNA fragment and send it for sequencing. The length of the 16S rDNA sequence of strain No. 19 is 1431 bp, and the specific sequence is as shown in SEQ ID NO.1. Compare and analyze the sequencing results through Blast on NCBI, and find that the homology with Pseudomonas putida strain BBAL5-01 (Gene Bank number: FJ217182.1) is 99%. Phylogenetic tree analysis shows that strain No. 19 and Pseudomonas putida BBAL5 01 cluster into one branch, as shown in Figure 2 . According to the morphological characteristics, physiological and biochemical properties, and molecular biological identification characteristics, identify strain No. 19 as Pseudomonas putida. This strain was deposited in the China Center for Type Culture Collection on July 25, 2024, and the deposit number is CCTCC NO: M20241686, and it is named Pseudomonas putida strain 19, abbreviated as strain 19.

[0078] (6) PCR amplification of denitrifying enzyme-related genes:

[0079] PCR verification was performed on the narG, nirS, norR, and nosZ genes of strain 19, and the specific amplification primers and amplification conditions are shown in Table 1 and Table 2 respectively. The PCR products were electrophoresed on 1% agarose gel and detected by EB staining. The results are shown in Figure 3 , indicating that the genome of strain 19 contains the denitrifying enzyme-related genes narG, nirS, norR, and nosZ.

[0080] The amplification system was as follows: The total reaction system was 25 μL (10 μL ddH 2 O, 12.5 μL 2×PCR Mix, 1 μL upstream primer, 1 μL downstream primer, 1 μL bacterial liquid template DNA).

[0081] Table 1 Amplification primers for denitrifying enzyme-related genes

[0082]

[0083]

[0084] Table 2 Amplification program for denitrifying enzyme-related genes

[0085]

[0086] Example 2: Denitrification characteristics of the strain

[0087] Activation of the strain: The cryopreserved strain was inoculated into a 50 mL centrifuge tube containing 50 mL of nitrate nitrogen medium, and the bottle mouth was sealed with a sealing film and statically cultured in an incubator at 30 °C. Monitor the OD of the bacterial liquid 600 , and when the bacterial liquid grew to the logarithmic phase, then pour 10 mL of the bacterial liquid into a 50 mL centrifuge tube, add 40 mL of nitrate nitrogen medium, tighten the cap, seal it with a sealing film, and culture it again until the logarithmic phase for use.

[0088] The activated strain 19 was inoculated into nitrate nitrogen medium (50 mL, with NO 3 - -N as the nitrogen source), nitrite nitrogen medium (50 mL, with NO 2 - -N as the nitrogen source), and ammonia nitrogen medium (50 mL, with NH 4 + -N as the nitrogen source) in 50 mL centrifuge tubes respectively. The bottle mouth was wrapped with a sealing film and statically cultured in an incubator at 30 °C for 48 h. Samples were taken every 6 h to measure the OD 600 value, NH 4 + -N concentration, NO 3 - -N concentration, NO 2 --N concentration and TN concentration.

[0089] By selecting different inorganic nitrogen sources NO 3 - -N, NH 4 + -N and NO 2 - -N were used as the sole nitrogen sources respectively to study the nitrogen removal ability of strain 19. When NO 3 - -N was used as the single nitrogen source (see Figure 4 ), the growth curve of the strain increased first within 0 - 48 h, then gradually leveled off, and reached the maximum value of 0.031 at 36 h; meanwhile, within 18 h, the concentration of NO 3 - -N decreased from the initial 15 mg / L to 0.48 mg / L, the removal rate was 0.81 mg / (L·h), and the removal rate reached 96.90%. During this process, there was no accumulation of NO 2 - -N and NH 4 + -N. The maximum amount of NO 2 - -N was 6.51 mg / L, but it was completely degraded at the next time point, and the TN degradation rate was 92.48%. When NH 4 + -N (see Figure 5 ) and NO 2 - -N (see Figure 6 ) were used as the sole nitrogen sources respectively, the removal rates of NH 4 + -N and NO 2 - -N were 59.08% and 100% respectively, the removal rates reached 0.48 mg / (L·h) and 0.83 mg / (L·h), and there was almost no accumulation of nitrite nitrogen during the incubation process with NH 4 + -N as the single nitrogen source. The TN degradation rates were 20.17% and 88.97% respectively. These results indicate that strain 19 exhibits high-efficient inorganic nitrogen removal ability and has the potential for enhanced application in high-nitrogen wastewater. At the same time, the amplification of narG, nirS, norR, and nosZ genes in Example 1 further proves that this strain has anaerobic denitrification potential.

[0090] Example 3: Effects of carbon source, C / N, pH, and temperature on anaerobic denitrification of the strain

[0091] The strain was activated in the same way as in Example 2.

[0092] The activated bacterial solution was inoculated into a 50 mL centrifuge tube containing 50 mL of nitrate nitrogen medium at an inoculation amount of 2% v / v. Based on the nitrate nitrogen medium, single-factor optimization was carried out according to the following settings: In the optimization of carbon source, different carbon sources were set as sodium potassium tartrate, sodium succinate hexahydrate, sodium citrate, sodium acetate, and glucose, with a concentration of 225 mg / L each; in the optimization of C / N ratio, the concentration of KNO 3 remained unchanged, and different C / N ratios were set as 1, 3, 5, 10, 15, 30, and 60; in the optimization of pH, different pH values were set as 5, 7, and 9; in the optimization of culture temperature, different temperatures were set as 25 °C, 30 °C, and 35 °C; three parallel experiments were set for each group. After static culture in an incubator for 48 h, it was taken out to measure OD 600 and NO 3 - -N concentrations, and the nitrogen removal abilities of the four factors of carbon source, C / N ratio, temperature, and pH on the strain were investigated respectively.

[0093] The effects of different carbon sources on the growth and denitrification of strain 19 are shown in Figure 7 . The strain could grow under these five carbon sources. When glucose was the sole carbon source, the growth amount of strain 19 reached the highest value of 0.1278. However, at this time, the nitrogen removal rate of strain 19 was 96.01%. When sodium succinate hexahydrate was the carbon source, the nitrate nitrogen removal rate reached the maximum of 97.85%. The effects of different C / N ratios on the nitrogen removal ability of strain 19 are as shown in Figure 8 . As the C / N ratio increased, the OD 600 of the strain also increased continuously. When the C / N reached 15, OD 600 reached the maximum value of 0.036, and then showed a downward trend. As the C / N ratio increased, the denitrification efficiency of the strain first increased and then decreased, reaching the highest of 99.11% when the C / N was 3, and the nitrate nitrogen removal rate of this strain was higher than 80% in the range of C / N from 1 to 60. Especially when the C / N was 1, the strain still had a high nitrate nitrogen removal rate (96.53%). Given that strain 19 had the highest denitrification efficiency under the condition of C / N being 3, this could effectively solve the problems of insufficient carbon source and low denitrification efficiency of anaerobic denitrifying bacteria when treating sewage with a low C / N ratio. The nitrogen removal ability of strain 19 at different pH values is as shown in Figure 9 . The strain basically did not grow at pH 5, but could grow at pH 7 - 9. It grew best at pH 7, and the nitrogen removal ability basically reached 90%, indicating that this strain could only tolerate neutral and weakly alkaline environments and could not tolerate acidic environments. Different culture temperatures led to different nitrate nitrogen removal abilities of the strain. The results are as shown in Figure 10 . Strain 19 could grow under the culture conditions of 25 - 35 °C. It grew best at 35 °C, and OD 600It was 0.048, but at this time the lowest nitrite nitrogen removal rate was 82.67%. Strain 19 maintained a high removal rate when the temperature was between 25°C and 35°C, all reaching above 80%, and the highest could reach 96%. This indicates that this strain can tolerate a relatively wide temperature range.

[0094] Example 4: Removal characteristics of the strain for p-xylene

[0095] The strain activation was carried out in the manner of Example 2.

[0096] The activated strain was inoculated into a 100 mL anaerobic bottle containing 50 mL of test medium for removing p-xylene as the sole carbon source at an inoculation amount of 5% v / v. Argon was passed through the bottle for 4 minutes to create an anaerobic environment. After the inflation was completed, the bottle mouth was sealed with a sealing film. 10 μL of p-xylene solution was injected into it with a microsyringe to make its final concentration 160 mg / L, and it was placed in an incubator at 30°C and cultured at 160 rpm / min for 72 h. Three parallel experiments were set up; samples were taken every 12 h to measure the OD 600 value, NH 4 + -N concentration, NO 3 - -N concentration, NO 2 - -N concentration, TN concentration, and p-xylene concentration.

[0097] During the removal of p-xylene by Strain 19, the concentration changes of each component were as Figure 11 and Figure 12 shown. When p-xylene was used as the sole carbon source, the growth curve of the strain gradually decreased within 0 - 72 h and reached the minimum value of 0.025 at 72 h. At the same time, within 72 h, the concentration of NO 3 - -N gradually decreased with time, from the initial 17 mg / L to 2.40 mg / L, the removal rate was 0.20 mg / (L·h), and the removal rate reached 89.70%. The content of NO 2 - -N increased with time and reached the highest accumulation at 72 h, which was 12.29 mg / L. There was no NH 4 + -N accumulation during this process, and the TN removal rate was 10.93% (see Figure 11 ). The content of p-xylene also gradually decreased with time, from the initial 160 mg / L to 70.96 mg / L, the removal rate was 1.24 mg / (L·h), and the removal rate was 55.54% (see Figure 12 ).

[0098] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A Pseudomonas putida capable of removing both p-xylene and nitrogen, characterized in that: Its deposit number is CCTCC NO: M20241686, and it was deposited in the China Center for Type Culture Collection on July 25, 2024.

2. A bacterial agent, characterized in that The bacterial agent comprises the Pseudomonas putida according to claim 1 or a preparation of the Pseudomonas putida according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The preparation of Pseudomonas putida includes culture, culture extract, lyophilized powder, fermentation broth, fermentation broth precipitate, fermentation broth supernatant or fermentation broth extract of the Pseudomonas putida.

4. Use of the Pseudomonas putida according to claim 1 or the bacterial agent according to any one of claims 2 to 3, characterized in that: The application includes denitrification and nitrogen removal in water or removal of paraxylene in water.

5. A method for denitrification and synergistic removal of p-xylene in water, characterized in that: The method comprises adding the Pseudomonas putida described in claim 1 or the bacterial agent described in any one of claims 2 to 3 into a nitrogen-containing water body for culturing.

6. The method according to claim 5, characterized in that The nitrogen-containing water body comprises a nitrogen-containing culture medium, the components of which include: a carbon source, a nitrogen source, KH2PO4, Na2HPO4·12H2O, MgSO4·7H2O and trace elements, and the pH of the nitrogen-containing culture medium is 5-9.

7. The method according to claim 6, characterized in that The carbon source includes potassium sodium tartrate, sodium succinate hexahydrate, sodium citrate, sodium acetate or glucose.

8. The method according to claim 7, characterized in that The carbon source is sodium succinate hexahydrate.

9. The method according to claim 6, characterized in that The nitrogen source includes nitrate, nitrite or ammonium salt.

10. The method according to claim 9, characterized in that The nitrogen source is nitrate.

11. The method according to claim 6, characterized in that The carbon-nitrogen molar ratio of the carbon source to the nitrogen source is 1-60.

12. The method according to claim 11, characterized in that The carbon-nitrogen molar ratio is 15.

13. The method according to claim 6, characterized in that The pH was 7.

0.

14. The method according to claim 6, characterized in that The culture temperature of the nitrogen-containing culture medium is 25-35°C.

15. The method according to claim 14, characterized in that The culture temperature is 30°C.

16. The method according to claim 6, characterized in that The components of the nitrogen-containing culture medium include KNO3 0.100-0.120 g / L, sodium succinate hexahydrate 1.0000-1.2000 / L, KH2PO4 0.200-0.250 g / L, Na2HPO4·12H2O 0.780-0.800 g / L, MgSO4·7H2O 0.008-0.012 g / L and trace elements 1.5-2.5 mL / L, wherein the trace elements include FeCl2·4H2O 1.6-2.0 g / L, CoCl2·6H2O 0.23-0.27 g / L, NiCl2·6H2O 0.008-0.012 g / L, CuCl2·2H2O0.008-0.012 g / L, MnCl2·4H2O 0.60-0.80 g / L, ZnCl2 0.08-0.12 g / L, H3BO3 0.4-0.6 g / L, Na2MoO4·2H2O 0.02-0.04 g / L and NaSeO3·5H2O 0.008-0.012g / L.

17. The method according to claim 6, characterized in that The components of the nitrogen-containing culture medium include KNO3 0.108 g / L, sodium succinate hexahydrate 1.1562 g / L, KH2PO4 0.226 g / L, Na2HPO4·12H2O 0.792 g / L, MgSO4·7H2O 0.01 g / L and trace elements 2.0 mL / L, wherein the trace elements include FeCl2·4H2O 1.8 g / L, CoCl2·6H2O 0.25 g / L, NiCl2·6H2O 0.01 g / L, CuCl2·2H2O 0.01 g / L, MnCl2·4H2O 0.70 g / L, ZnCl2 0.1 g / L, H3BO3 0.5 g / L, Na2MoO4·2H2O 0.03 g / L and NaSeO3·5H2O 0.01g / L.

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