Pseudomonas monteilii and application thereof

Abstract

This invention discloses a strain of *Pseudomonas monteilii* and its applications, relating to the field of microbial technology. The *Pseudomonas monteilii* strain is named *Pseudomonas monteilii* XC-1, deposited at the Guangdong Provincial Microbial Culture Collection Center on January 9, 2023, with accession number GDMCC No: 63127. *Pseudomonas monteilii* XC-1 exhibits good adaptability to different environments, tolerating pH values ​​of 5–9, temperature ranges of 15–35°C, and salinity of 0.4%–5%. It can degrade various PAHs such as phenanthrene, carbazole, anthracene, and pyrene, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Pseudomonas montelukastii and its applications. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of aromatic compounds composed of two or more benzene rings, characterized by high melting points, high boiling points, low solubility, and strong hydrophobicity. Once in the environment, PAHs readily adsorb onto solid surfaces, making them difficult for microorganisms to access. Furthermore, due to their stable structure and low natural degradation rate, PAHs persist in environmental media for extended periods. PAHs can enter the human body through the respiratory tract, skin, and digestive tract, exhibiting teratogenic, carcinogenic, and mutagenic effects. In recent years, the incomplete combustion of fossil fuels such as coal and oil, as well as leaks and pollution during crude oil extraction, transportation, and production, have led to a surge in environmental PAH emissions, posing a growing threat to human health and the entire ecosystem.

[0003] To control the environmental pollution caused by PAHs, it is necessary to remediate the contaminated media. Early physicochemical remediation techniques, such as thermal desorption, solvent extraction, and electrochemical remediation, were employed. While these techniques yielded good results, they were costly and prone to causing secondary pollution. In recent years, PAH bioremediation technology has gained increasing attention due to its advantages of being environmentally friendly, cost-effective, and renewable. The bioremediation efficacy of PAHs is primarily influenced by the performance of the degrading strains. Therefore, screening for highly efficient PAH-degrading strains is crucial for the bioremediation of PAH pollution. Furthermore, multiple PAHs often coexist in contaminated media, which increases the difficulty of bioremediation.

[0004] Therefore, it is very important to provide a strain that can efficiently degrade PAHs. Summary of the Invention

[0005] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a strain of *Pseudomonas montelukastii* capable of effectively degrading polycyclic aromatic hydrocarbons (PAHs).

[0006] The present invention also provides a microbial agent.

[0007] The present invention also provides a product for degrading polycyclic aromatic hydrocarbons.

[0008] The present invention also provides the application of the above-mentioned Pseudomonas montelukastii or its agents or products in the degradation of polycyclic aromatic hydrocarbons.

[0009] The present invention also provides a method for degrading polycyclic aromatic hydrocarbons.

[0010] According to the first aspect of the present invention, a strain of Pseudomonas monteilii is named Pseudomonas monteilii XC-1. The depositary institution is Guangdong Provincial Center for Microbial Culture Collection, located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. The deposit date is January 9, 2023, and the deposit number is GDMCC No:63127.

[0011] The *Pseudomonas montelukastii* strain according to embodiments of the present invention has at least the following beneficial effects:

[0012] The *Pseudomonas montelukastella* XC-1 strain described in this example exhibits good adaptability to various environments, tolerating pH levels of 5–9, temperatures ranging from 15°C to 35°C, and salinity from 0.4% to 5%. It is capable of degrading various PAHs, including phenanthrene, carbazole, anthracene, and pyrene, and shows promising application prospects. Furthermore, high temperature, high salinity, slightly acidic, and slightly alkaline environments significantly promote the degradation of PAHs by *Pseudomonas montelukastella* XC-1, and it also demonstrates excellent tolerance to Tween 80.

[0013] According to a second aspect of the present invention, a bacterial agent contains the aforementioned *Pseudomonas montelukastiensis* XC-1. Since the bacterial agent employs all the technical solutions of the *Pseudomonas montelukastiensis* XC-1 bacterial cells described in the above embodiments, it possesses at least all the beneficial effects brought about by the technical solutions of the above embodiments.

[0014] According to some embodiments of the present invention, the microbial agent further includes a surfactant.

[0015] According to some embodiments of the present invention, the surfactant includes Tween 80.

[0016] According to some embodiments of the present invention, the bacterial agent contains live cells of Pseudomonas montmorillonii XC-1, freeze-dried bacterial cells, immobilized cells, liquid bacterial agent, solid bacterial agent, or Pseudomonas montmorillonii XC-1 strain existing in any other form.

[0017] According to some embodiments of the present invention, the microbial agent may also contain other bacteria or fungi.

[0018] According to some embodiments of the present invention, the active ingredient of the bacterial agent includes the above-mentioned Pseudomonas montelukastiensis XC-1.

[0019] According to some embodiments of the present invention, the microbial agent may further include at least one of a carrier, a surfactant, a stabilizer, and a pH adjuster.

[0020] According to some embodiments of the present invention, the formulation of the microbial agent can be of various forms, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0021] A product for degrading polycyclic aromatic hydrocarbons according to a third aspect embodiment of the present invention comprises the aforementioned *Pseudomonas montelukastiensis* XC-1 or the aforementioned bacterial agent. Since the product employs all the technical solutions of the *Pseudomonas montelukastiensis* XC-1 bacterial cells of the above embodiments, it possesses at least all the beneficial effects brought about by the technical solutions of the above embodiments.

[0022] According to some embodiments of the present invention, the polycyclic aromatic hydrocarbon includes at least one of phenanthrene, carbazole, anthracene, and pyrene.

[0023] Application of any one of A1) to A3) of the fourth aspect of the present invention in the degradation of polycyclic aromatic hydrocarbons,

[0024] A1) The *Pseudomonas montmorillonii* XC-1 described in the first aspect embodiment;

[0025] A2) The microbial agent described in the second aspect embodiment;

[0026] A3) The product described in the third aspect embodiment.

[0027] According to some embodiments of the present invention, the polycyclic aromatic hydrocarbon includes at least one of phenanthrene, carbazole, anthracene, and pyrene.

[0028] A method for degrading polycyclic aromatic hydrocarbons according to a fifth aspect embodiment of the present invention includes the following steps:

[0029] Use any one of A1) to A3) to contact polycyclic aromatic hydrocarbons;

[0030] A1) The *Pseudomonas montmorillonii* XC-1 described in the first aspect embodiment;

[0031] A2) The microbial agent described in the second aspect embodiment;

[0032] A3) The product described in the third aspect embodiment. Since the method adopts all the technical solutions of Pseudomonas montelukastiensis XC-1 bacterial cells of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments.

[0033] According to some embodiments of the present invention, the temperature conditions during the degradation are 15°C to 35°C. More specifically, they can be 20°C to 35°C. Even more specifically, they can be 30°C to 35°C.

[0034] According to some embodiments of the present invention, the salinity conditions during the degradation are 0.5% to 5%.

[0035] According to some embodiments of the present invention, the pH conditions during the degradation are 5 to 9.

[0036] According to some embodiments of the present invention, the degradation also includes a surfactant.

[0037] According to some embodiments of the present invention, the content of the surfactant is 0.5% or more. More specifically, it can be 0.5% to 6%.

[0038] According to some embodiments of the present invention, the surfactant is Tween 80.

[0039] According to some embodiments of the present invention, the contact may specifically be: inoculating the above-mentioned Pseudomonas montelukastiensis XC-1 or the above-mentioned bacterial agent or the above-mentioned product into water or soil containing polycyclic aromatic hydrocarbons.

[0040] According to some embodiments of the present invention, during the contact process, the inoculation amount of the bacterial suspension containing *Pseudomonas montelukastiensis* XC-1 is 1% to 20% (V / V). The OD600nm of the bacterial suspension is 0.75 to 1.25. The OD600nm of the bacterial suspension is approximately 1.

[0041] According to some embodiments of the present invention, the phenanthrene concentration in the water body is 5 mg / L to 120 mg / L. Preferably, it is 10 mg / L to 40 mg / L.

[0042] According to some embodiments of the present invention, the degradation time is 3 to 20 days, preferably 3 to 15 days.

[0043] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0044] Figure 1 Colony morphology (A) and Gram staining results (B) of Pseudomonas monteilii XC-1;

[0045] Figure 2 SEM image of Pseudomonas monteilii XC-1 (scale bar 5 μm);

[0046] Figure 3 A phylogenetic tree based on the 16S rDNA sequences of Pseudomonas monteilii XC-1 and related strains;

[0047] Figure 4 The growth status of Pseudomonas monteilii XC-1 under different pH (A), different temperatures (B), and different salinities (C);

[0048] Figure 5 The growth curve of Pseudomonas monteilii XC-1 in MSM medium with phenanthrene as the sole carbon source is shown.

[0049] Figure 6 The degradation curve of phenanthrene by Pseudomonas monteilii XC-1;

[0050] Figure 7 The results of detecting biofilm formation in Pseudomonas monteilii XC-1 using different concentrations of phenanthrene;

[0051] Figure 8 The results of biofilm formation by Pseudomonas monteilii XC-1 using different PAHs (A) and its degradation efficiency (B);

[0052] Figure 9 The growth of Pseudomonas monteilii XC-1 under different concentrations of Tween 80;

[0053] Figure 10 The degradation rate of phenanthrene by Pseudomonas monteilii XC-1 at different concentrations of Tween 80;

[0054] Figure 11 The removal rate of phenanthrene by Pseudomonas monteilii XC-1 at different concentrations in the presence of 1.0 g / L Tween 80;

[0055] Figure 12 The removal rate of phenanthrene by Pseudomonas monteilii XC-1 under different environmental conditions. Detailed Implementation

[0056] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0057] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0058] In the description of this invention, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0059] Example 1 (Isolation, Screening, and Identification of Strains)

[0060] 1. Screening and isolation of strains

[0061] (1) Activated sludge was collected from a municipal wastewater treatment plant in Shenzhen and stabilized for 7 days using artificial wastewater (500 mg / L glucose, 100 mg / L yeast extract, 236 mg / L (NH4)2SO4, 22.5 mg / L MgSO4·7H2O, 1.7 mg / L NH4Cl, 27.5 mg / L CaCl2·2H2O, 0.25 mg / L FeCl3·6H2O, 840 mg / L NaHCO3, 21.75 mg / L K2HPO4·12H2O, 8.5 mg / L KH2PO4, 44.6 mg / L Na2HPO4·12H2O).

[0062] (2) Prepare concentrated solutions of phenanthrene, pyrene, and benzo[a]pyrene (acetonitrile as solvent). Add the three PAHs concentrated solutions to a shake flask. After the solvent evaporates, add the basic salt medium (MSM medium, NaHPO4 2800 mg / L, KH2PO4 1000 mg / L, (NH4)2PO4 500 mg / L, MgCl2 53 mg / L, Ca(NO3)2 50 mg / L, EDTA-2Na 0.5 mg / L, FeSO4·7H2O 0.2 mg / L, ZnSO4 0.01 mg / L, MnCl2 0.003 mg / L, H3BO3 0.03 mg / L, CoCl2 0.02 mg / L, CuCl2·2H2O 0.001 mg / L, NiCl2·6H2O) 0.001 mg / L NaMoO4·2H2O 0.003 mg / L) were used to prepare MSM medium (containing 100 mg / L phenanthrene, 100 mg / L pyrene, and 50 mg / L benzo[a]pyrene) with PAHs as the carbon source. The stabilized activated sludge was inoculated into the PAHs-based MSM medium at a volume ratio of 10%. After 7 days of shake-flask culture, the sludge was transferred to fresh PAHs-based MSM medium at a volume ratio of 10%, and cultured for another 7 days. This process was repeated three times. PAHs-degrading strains were then enriched.

[0063] (3) The enriched sludge was diluted with sterile water and spread onto MSM agar plates containing PAHs (containing 100 mg / L phenanthrene, 100 mg / L pyrene and 50 mg / L benzo[a]pyrene) and incubated in a 30°C incubator for 3 days. 48 single colonies were randomly picked from the plate with a sterile needle and inoculated into two 24-well plates, each containing 2 mL of MSM medium with the same PAH concentration (containing 100 mg / L phenanthrene, 100 mg / L pyrene and 50 mg / L benzo[a]pyrene). After incubating the 24-well plate at 30℃ for 7 days, the bacterial suspension was transferred to a 96-well plate. The OD600nm value was analyzed using a microplate reader. The five wells with the best growth were selected, and the resulting bacterial suspension was again spread onto MSM agar plates containing PAHs (100 mg / L phenanthrene, 100 mg / L pyrene, and 50 mg / L benzo[a]pyrene) to obtain pure bacteria capable of degrading PAHs. Single colonies of the five obtained pure bacteria were picked and cultured in MSM agar containing PAHs (100 mg / L phenanthrene, 100 mg / L pyrene, and 50 mg / L benzo[a]pyrene) for 7 days. The OD600nm value was measured, and the strain with the highest OD600nm value was selected (denoted as: XC-1). The bacterial culture of this strain was spread onto LB agar plates (10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, pH 7.0) and stored at 4°C for subsequent studies after single cell formation.

[0064] 2. Identification of strains

[0065] (1) Morphological characteristics identification

[0066] 1) Plate colonies: Select a single colony of XC-1 and incubate it overnight in LB medium. Wash the bacterial cells twice with sterile physiological saline, then resuspend the bacterial cells with an equal volume of sterile physiological saline. Use an inoculation stick to streak the bacterial solution back and forth to isolate pure bacteria. After incubating at 30℃ for 24 hours, observe the growth status of the colonies.

[0067] 2) Gram staining: Take 20 μL of XC-1 bacterial culture onto a clean glass slide, slowly dry and fix the bacterial culture with the outer flame of an alcohol lamp, add crystal violet for staining for 2 min, wash, add iodine solution (1.0 g potassium iodide and 0.5 g iodine dissolved in 150 mL distilled water) for staining for 2 min, wash, add 95% alcohol for decolorization for 10 s, and finally add safranin (2.5% (w / v) safranin ethanol solution mixed with distilled water at a volume ratio of 1:4) for counterstaining for 1 min, rinse, and observe under a microscope.

[0068] 3) Scanning Electron Microscopy: Take 1 mL of bacterial culture, remove the supernatant, add 1 mL of 2.5% glutaraldehyde solution to resuspend the bacterial culture, and fix it overnight at 4℃. Wash the bacterial culture three times with sterile water, then resuspend it in 1 mL of sterile water. Transfer 50 μL to a coverslip, allow it to air dry, and then perform gradient dehydration with 50%, 70%, 80%, 90%, and 95% ethanol aqueous solutions and anhydrous ethanol for 20 min each time. Then, freeze-dry the sample for 48 h. Sputter the sample with gold in a magnetron sputtering apparatus and observe it under a scanning electron microscope.

[0069] Morphological identification results such as Figure 1 and Figure 2 As shown.

[0070] Morphological identification results showed that XC-1 colonies were milky white, round, raised, with clear and relatively moist edges; after Gram staining, the bacteria were red, indicating that XC-1 was a Gram-negative bacterium; the size of XC-1 bacteria was approximately 0.5 μm × 2.0 μm, and they were straight rods with rounded ends.

[0071] (2) Molecular biological identification

[0072] Genomic DNA was extracted from XC-1 according to the instructions of the Ezup column-based bacterial genomic DNA extraction kit. After verifying the purity, PCR amplification of the XC-1 genomic DNA was performed using universal amplification primers 7F (5'-CAGAGTTTGATCCTGGCT-3') and 1540R (5'-AGGAGGTGATCCAGCCGCA3'). The amplified fragments obtained by PCR were subjected to gel electrophoresis, and the bands of approximately 1500 bp were cut, purified, and recovered. The recovered products were stored in centrifuge tubes and sent to Shanghai Sangon Biotech Co., Ltd. for 16S rDNA sequencing.

[0073] The sequencing results are as follows:

[0074] >XC-1(5'→3')

[0075]

[0076] The 16S rDNA sequence of strain XC-1 was entered into the GenBank database. The 16S rDNA sequence of strain XC-1 was compared with known 16S rDNA sequences using the local comparison search tool BLAST, and a phylogenetic tree was constructed using the neighbor-joining method (NJ method).

[0077] The resulting evolutionary tree is as follows Figure 3 As shown.

[0078] Based on the combined morphological and molecular biological identification results, XC-1 is a straight bacillus with blunt, rounded ends, belonging to Gram-negative bacteria. The XC-1 strain has a sequence similarity of 99.93% with the Pseudomonas monteilii strain, and the bacterium is named Pseudomonas monteilii XC-1.

[0079] Pseudomonas monteilii XC-1 was deposited on January 9, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No:63127.

[0080] Example 2 (Growth conditions of Pseudomonas monteilii XC-1)

[0081] Single colonies of Pseudomonas monteilii XC-1 were picked and cultured overnight in LB medium. The bacterial culture was centrifuged at 4000 rpm for 15 min and the supernatant was discarded. The OD600nm was adjusted to 1.0 with fresh LB medium (pH 7.0) to obtain Pseudomonas monteilii XC-1 bacterial culture for subsequent experiments.

[0082] (1) Optimal growth pH

[0083] Prepare LB medium with initial pH values ​​of approximately 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 (adjust pH 4.0–5.0 with a 0.1 mol / L citrate-sodium citrate buffer system; adjust pH 6.0–8.0 with 0.1 mol / L KH₂PO₄ and NaOH; adjust pH 9.0–11.0 with 0.1 mol / L HCl and NaHCO₃), and sterilize for later use. Add 200 μL of the above-mentioned LB medium at different pH values ​​and 4 μL of Pseudomonas monteilii XC-1 bacterial suspension (OD600nm = 1.0) to each well of a 96-well plate. Perform three replicates for each pH condition, and continuously measure OD600nm values ​​for 24 hours using a microplate reader. Plot the growth curves of Pseudomonas monteilii XC-1 under different initial pH (30℃) culture conditions to determine the optimal growth pH.

[0084] (2) Optimal growth temperature

[0085] Add 10 mL of LB medium (pH 7.0) and 100 μL of Pseudomonasmonteilii XC-1 bacterial suspension (OD600nm = 1.0) to a 50 mL sterile centrifuge tube. Incubate for 24 h at 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, and 40 °C on a shaker (200 rpm). Then, take a 200 μL sample of the bacterial suspension and measure OD600nm in a 96-well plate to determine the optimal growth temperature. Perform three replicates for each temperature condition.

[0086] (3) Optimal growth salinity

[0087] Add NaCl to LB medium (originally containing 1.0% NaCl) to prepare LB medium with salinities (NaCl concentrations) of 2%, 3%, 4%, 5%, 6%, 7%, and 8%. Add 200 μL of the above-mentioned LB medium at different salinities and 4 μL of *Pseudomonas monteilii* XC-1 bacterial culture (OD600nm = 1.0) to each well of a 96-well plate sequentially. Perform three replicates for each salinity condition, and continuously measure the OD600nm value using a microplate reader for 24 hours to obtain the growth curves of *Pseudomonas monteilii* XC-1 under different salinity conditions (30℃, pH 7.0) to determine the optimal salinity for growth.

[0088] Test results are as follows Figure 4 As shown.

[0089] Pseudomonas monteilii XC-1 maintained good growth under a wide range of pH (6–9), temperature (15℃–35℃), and salinity (1%–5%) conditions. This indicates that Pseudomonas monteilii XC-1 has good adaptability to different environmental conditions and has the potential for application in environmental remediation.

[0090] Example 3 (Degradation of phenanthrene by Pseudomonas monteilii XC-1)

[0091] (1) Centrifuge the overnight cultured Pseudomonas monteilii XC-1 bacterial suspension at 4000 rpm for 10 min, remove the supernatant, wash twice with MSM medium (pH 7.0) and resuspend the bacterial suspension. Adjust the OD600nm of the bacterial suspension to 1.0 to obtain the XC-1 resuspension. Take three 50 mL sterile centrifuge tubes, add phenanthrene concentrate (final concentration 100 mg / L) to them, wait for the solvent to evaporate completely in a clean bench, add 5 mL of MSM medium and 50 μL of XC-1 resuspension, set up an experimental group without phenanthrene as a blank control, mix well and place in a constant temperature shaker at 30℃ and 200 rpm. Take samples every 24 h to analyze the OD600nm value of the bacterial suspension.

[0092] The results are as follows Figure 5 As shown.

[0093] In MSM medium with phenanthrene as the sole carbon source, the *Pseudomonas monteilii* XC- strain achieved massive proliferation within 2 days and maintained its bacterial count for a considerable period. This indicates that *Pseudomonas monteilii* XC-1 can utilize phenanthrene for its own growth, demonstrating good application potential.

[0094] (2) Add the phenanthrene concentrate to a 50 mL centrifuge tube. After the organic solvent is dried, add 4 mL of MSM medium and 1 mL of seudomonas monteilii XC-1 bacterial suspension (OD600nm=1.0). The final concentration of phenanthrene is 100 mg / L. The treatment group without bacterial suspension is used as the blank control group. Each group is repeated three times.

[0095] Test tubes from each treatment group were continuously incubated at 30℃ and 200 rpm in the dark for 15 days. On days 1, 3, 5, 7, 10, and 15, three test tubes were randomly selected, and internal standards (carbazole and benzo[a]anthracene) and 10 mL of dichloromethane were added. Extraction was carried out at 30℃ and 200 rpm for 2 hours. 1 mL of the organic phase was accurately pipetted into a brown chromatographic vial, the solvent was dried using a nitrogen evaporator, and 1 mL of chromatographically pure acetonitrile was added to reconstitute the sample. The reconstituted sample was filtered through a 0.22 μm nylon filter membrane, and the filtrate was transferred to a new chromatographic vial for quantitative analysis of phenanthrene using a high-performance liquid chromatograph (Ultimate 3000).

[0096] The chromatographic column was a ZORBAX Eclipse PAH (4.6*150mm*3.5μm); the operating parameters were as follows: mobile phase was acetonitrile and degassed deionized water, 0–0.75 min 62% acetonitrile, 0.75–5.25 min 62%→100% acetonitrile, 5.25–7.5 min 100% acetonitrile, 7.5–9.75 min 100%→62% acetonitrile, 9.75–11 min 62% acetonitrile; flow rate 2 mL / min, retention time 12 min, column temperature 25℃, injection volume 5 μL, detection wavelength 250 nm (phenanthrene) and 290 nm (internal standard); the recoveries of all samples were 90%–110%, and the limit of quantitation was 0.01 mg / L.

[0097] Test results as follows Figure 6 As shown.

[0098] Lower concentrations of Pseudomonas monteilii XC-1, using phenanthrene at an initial concentration of 100 mg / L as the sole carbon source, can degrade and remove approximately 50% of phenanthrene within 7 days and approximately 80% within 15 days.

[0099] (3) Select a single colony of *Pseudomonas monteilii* XC-1 and culture it overnight. Wash it twice with MSM medium and adjust the OD600nm of the bacterial solution to 1.0 to obtain a cleaned bacterial solution. Mix the MSM medium and the cleaned bacterial solution at a volume ratio of 4:1 to obtain a diluted bacterial solution. In a clean bench, add different volumes of phenanthrene concentrate to a 96-well plate. After the organic solvent evaporates, add 200 μL of diluted bacterial solution to each well. The final concentrations of phenanthrene are 0 mg / L (blank control), 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, 600 mg / L, 800 mg / L, and 1000 mg / L, with 8 replicates for each concentration. Add 200 μL of LB medium to the first and last columns to check for contamination during the culture process. Seal the 96-well plate with sealing film and incubate it in a 30°C incubator. After 3 days of incubation, the plate was gently inverted and the bacterial suspension was discarded. Each well was gently washed three times with phosphate-buffered saline (pH 7.3). The plate was then placed upright in a 60°C oven for 1 hour to fix the biofilm. Next, 1% crystal violet solution was added to each well for staining in the dark for 30 minutes. After thoroughly washing away any remaining crystal violet solution, the plate was allowed to air dry. The crystal violet fixed in the biofilm was reconstituted with 95% ethanol, and the OD595nm value was measured using a microplate reader. A higher OD595nm value indicates a higher biofilm formation rate of *Pseudomonas monteilii* XC-1.

[0100] Test results as follows Figure 7 As shown.

[0101] Compared with the blank control group, the amount of biofilm formed increased with increasing phenanthrene concentration. This indicates that *Pseudomonas monteilii* XC-1 can survive in a high phenanthrene concentration range and can utilize phenanthrene as a carbon source to synthesize biofilms.

[0102] Example 4 (Degradation effect of Pseudomonas monteilii XC-1 on different PAHs)

[0103] Select a single colony of Pseudomonas monteilii XC-1 and culture it overnight. Wash it twice with MSM medium and adjust the OD600nm of the bacterial solution to 1.0 to obtain the washed bacterial solution, which will be used for subsequent experiments.

[0104] (1) Prepare diluted bacterial suspension by mixing MSM medium with the washing bacterial suspension (OD600nm = 1.0) at a volume ratio of 4:1. Open a sterile 24-well plate in a laminar flow hood and add concentrated solutions of carbazole, anthracene, or pyrene sequentially. After the solvent evaporates, add 1 mL of diluted bacterial suspension. A control group without PAHs (carbazole, anthracene, and pyrene) was set up; the final concentration of the three PAHs in the experimental groups was 100 mg / L; both the experimental groups and the control group were repeated four times. Seal the 24-well plate with sealing film and incubate at 30°C. After 3 days of static incubation, analyze the amount of biofilm formed in each well (method as in Example 3).

[0105] (2) Prepare diluted bacterial solutions by mixing MSM medium and washing bacterial solution at a volume ratio of 4:1. Take 50 mL sterile plastic centrifuge tubes and add concentrated carbazole, anthracene, or pyrene solutions respectively. After the organic solvent is dried, add 5 mL of diluted bacterial solution and mix. The final concentration of carbazole, anthracene, or pyrene is 10 mg / L. Each group is set up in triplicate, and an experimental control without bacterial addition is also set up. After incubation at 30℃ and 200 rpm for 3 days, the residual PAHs are analyzed by HPLC to calculate the PAHs removal rate.

[0106] Test results as follows Figure 8 As shown.

[0107] Compared with the blank control, Pseudomonas monteilii XC-1 can utilize different PAHs (carbazole, anthracene, and pyrene) as carbon sources for biofilm synthesis; within 3 days, the degradation rates of carbazole, anthracene, and pyrene at an initial concentration of 10 mg / L reached 8.8% ± 1.8%, 15.8% ± 3.6%, and 30.1% ± 5.9%, respectively. This indicates that Pseudomonas monteilii XC-1 has the function of degrading multiple PAHs.

[0108] Example 5 (Effect of Tween 80 on the degradation of phenanthrene by Pseudomonas monteilii XC-1)

[0109] In practical soil remediation processes, surfactants are typically added to enhance the bioavailability of PAHs. Tween 80 is a representative nonionic surfactant commonly used in soil remediation processes for organic pollutants such as PAHs. However, when Tween 80 is used as a surfactant, it tends to inhibit the degradation performance of PAH-degrading bacteria.

[0110] Select a single colony of Pseudomonas monteilii XC-1 and culture it overnight. Wash it twice with MSM medium and adjust the OD600nm of the bacterial solution to 1.0 to obtain the washed bacterial solution, which will be used for subsequent experiments.

[0111] (1) Add 1 mL of washing bacterial solution and 4 mL of MSM medium containing Tween 80 to a 50 mL centrifuge tube. The final mass (g) of Tween 80 as a percentage (w / v) of liquid volume (mL) was 0 (blank control group), 0.50%, 1%, 2%, 3%, 4%, and 5%, respectively, with each group repeated 3 times. Incubate at 30 °C and 200 rpm for 14 days with constant temperature shaking. Analyze the OD600 nm using a microplate reader to evaluate the growth of Pseudomonas monteilii XC-1.

[0112] Test results as follows Figure 9 As shown.

[0113] During the first four days of cultivation, different concentrations of Tween 80 slightly inhibited the OD600nm process. From the fifth day onwards, the blank control group (without Tween 80) of *Pseudomonas monteilii* XC-1 entered the death phase, with the OD600nm gradually decreasing. In the experimental groups, the higher the Tween 80 content, the later *Pseudomonas monteilii* XC-1 entered the death phase. Notably, in the experimental group containing 5% Tween 80, the OD600nm continued to increase for 14 days, suggesting that *Pseudomonas monteilii* XC-1 can utilize Tween 80 as a carbon source for growth.

[0114] (2) Add phenanthrene concentrate to a 50 mL centrifuge tube. After the solvent evaporates, add 1 mL of washing bacterial solution and 4 mL of MSM medium containing Tween 80. The final concentrations of Tween 80 are 0%, 0.5%, 1%, 2%, 3%, 4%, and 5%, respectively, and the final concentration of phenanthrene is 40 mg / L. Each group is repeated 3 times. After incubation at 30℃ and 200 rpm for 3 days, the remaining PAHs are analyzed by HPLC.

[0115] Test results as follows Figure 10 As shown.

[0116] When 1%, 2%, 3%, 4%, and 5% Tween 80 were added, the average degradation rates of phenanthrene by Pseudomonas monteilii XC-1 were 27.4% ± 4.0%, 28.3% ± 2.5%, 26.0% ± 3.4%, 24.6% ± 1.4%, and 27.8% ± 4.1%, respectively, with no significant differences between groups (P-value: 1.0). Therefore, adding higher concentrations of Tween 80 surfactant did not have a significant negative impact on the degradation efficiency of phenanthrene by Pseudomonas monteilii XC-1.

[0117] (3) Add phenanthrene to 50 mL centrifuge tubes at final concentrations of 5 mg / L, 10 mg / L, 20 mg / L, 40 mg / L, 80 mg / L, and 120 mg / L, respectively. After the solvent evaporates, add 1 mL of washing bacterial solution and 4 mL of MSM medium containing Tween 80 (final concentration of Tween 80: 1.0 g / L). Each group has 3 replicates. After incubation at 30 °C and 200 rpm for 3 days with constant temperature shaking, analyze the remaining PAHs by HPLC.

[0118] Test results as follows Figure 11 As shown.

[0119] Pseudomonas monteilii XC-1 exhibits varying degradation efficiencies towards different initial concentrations of phenanthrene. When the initial concentration of phenanthrene is between 10 and 40 mg / L, the average degradation rate can exceed 30%; however, when the concentration is as low as 5 mg / L or increases to 80 and 120 mg / L, the degradation efficiency drops to around 20%. Therefore, both excessively low and excessively high initial PAH concentrations will affect the degradation efficiency of XC-1. In practical remediation applications, the amount of XC-1 added should be adjusted according to the actual PAH concentration to achieve good degradation and remediation results.

[0120] In practical soil remediation processes, surfactants are often added to promote the dissolution of PAHs and enhance their microbial availability. Some degrading bacteria have poor adaptability to surfactants, thus their PAH degradation performance is inhibited in the presence of surfactants. However, the *Pseudomonas monteilii* XC-1 of this invention exhibits stable degradation performance against PAHs in both the presence and absence of surfactants, indicating its good practical potential.

[0121] Example 6 (Effects of environmental conditions on the degradation of phenanthrene by Pseudomonas monteilii XC-1)

[0122] The physicochemical properties of contaminated soil can affect the effectiveness of soil remediation; therefore, it is necessary to investigate the impact of different environmental conditions on the strain. This experiment selected three main environmental factors—pH, temperature, and salinity—and used phenanthrene degradation rate as a metric to evaluate the impact of their critical conditions on the *Pseudomonas monteilii* XC-1 strain.

[0123] Single colonies of Pseudomonas monteilii XC-1 were cultured overnight, washed twice with MSM medium, and the OD600nm of the bacterial culture was adjusted to 1.0 to obtain the washed bacterial solution. Phenanthrene concentrate was added to a 50 mL centrifuge tube. After the solvent evaporated, 1 mL of the washed bacterial solution and 4 mL of MSM medium were added. The final concentration of phenanthrene was 40 mg / L. The mixture was mixed and cultured in a shaker at 200 rpm for 3 days. The remaining PAHs were analyzed by HPLC.

[0124] In this experiment, the pH of the MSM medium used was the pH condition corresponding to the specific experimental group. The pH settings for the investigation were 5.0 and 9.0 (the pH of the medium was adjusted using a 0.1 mol / L citrate-sodium citrate buffer system and a 0.1 mol / L HCl-NaHCO3 system, respectively); the salinity settings were 3% and 5% (the salinity of the medium was changed by adding NaCl); and the culture temperatures were 15℃, 20℃, and 35℃. The experimental group with experimental conditions of pH 7.0, culture temperature of 30℃, and salinity of 0.44% of the total basic salt concentration in the MSM was set as the blank control group. Each group was repeated 3 times.

[0125] Test results as follows Figure 12 As shown.

[0126] Compared with the control group, the degradation rate of phenanthrene did not decrease significantly under high temperature (35℃), high salinity (3% and 5%), slightly acidic (pH 5.0), and slightly alkaline (pH 9.0) conditions; in fact, it increased. Temperature had a significant impact on the degradation rate of phenanthrene, but even under low temperature (15℃) conditions, the 3-day degradation rate of phenanthrene remained at 14.5%. This indicates that *Pseudomonas monteilii* XC-1 has strong adaptability to different environmental critical conditions and has good application potential in practical soil remediation.

[0127] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A strain of *Pseudomonas montelukastii*, characterized in that, Named Pseudomonas montelukastii ( Pseudomonas monteilii XC-1, depositary institution: Guangdong Provincial Center for Microbial Culture Collection, deposit date: January 9, 2023, accession number: GDMCCNo: 63127.

2. A microbial agent, characterized in that, The bacterial agent contains Pseudomonas montelukastii XC-1 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent also includes surfactants.

4. A product for degrading polycyclic aromatic hydrocarbons, characterized in that, Includes Pseudomonas montelukastiensis XC-1 as described in claim 1 or the bacterial agent as described in any one of claims 2 to 3.

5. The application of any one of A1) to A3) in the degradation of polycyclic aromatic hydrocarbons, A1) The Pseudomonas montelukastii XC-1 as described in claim 1; A2) The microbial agent according to claim 2 or 3; A3) The product as described in claim 4; The polycyclic aromatic hydrocarbon is at least one of phenanthrene, carbazole, anthracene, and pyrene.

6. A method for degrading polycyclic aromatic hydrocarbons, characterized in that, Includes the following steps: Use any one of A1) to A3) to contact polycyclic aromatic hydrocarbons; A1) The Pseudomonas montelukastii XC-1 as described in claim 1; A2) The microbial agent according to claim 2 or 3; A3) The product as described in claim 4; The polycyclic aromatic hydrocarbon is at least one of phenanthrene, carbazole, anthracene, and pyrene.

7. The method according to claim 6, characterized in that, The temperature conditions for the degradation are 15℃~35℃.

8. The method according to claim 6, characterized in that, The salinity conditions during the degradation are 0.5% to 5%.

9. The method according to claim 7, characterized in that, The pH conditions for the degradation are 5 to 9.

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