Pseudomonas fluorescens with benzo [a] anthracene degradation capability and application thereof

Abstract

The invention discloses pseudomonas fluorescens with benzo [a] anthracene degradation capacity and application of the pseudomonas fluorescens, and relates to the technical field of microbial degradation. The bacterial strain is Pseudomonas fluorescens SXU-ZW-01 and is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms, the preservation number of the bacterial strain is CGMCC NO.36511, and the preservation date of the bacterial strain is November 05, 2025. The strain is separated from an environmental sample of a coking plant, has a typical high-temperature adaptation characteristic and can efficiently degrade benzo [a] anthracene under the conditions that the temperature is 45 DEG C and the pH value is 7.0-8.0, and the degradation rate of benzo [a] anthracene within 96 hours can reach 95% or above. The invention provides core strain resources and technical support for green treatment of coking flue gas, and has good industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial degradation technology, and in particular relates to a fluorescent Pseudomonas strain with benzo[a]anthracene degradation ability and its application. Background Technology

[0002] Benzo[a]anthracene is a typical tetracyclic polycyclic aromatic hydrocarbon (PAH) compound, classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC). It exhibits strong teratogenic, carcinogenic, and mutagenic properties, posing a serious threat to the ecological environment and human health. This compound primarily originates from the high-temperature pyrolysis and incomplete combustion processes of organic matter such as coal, petroleum, and wood, and is widely present in industrial exhaust gases from coking, steel, power, and chemical industries, as well as in vehicle emissions. PAH pollutants are chemically stable, possess strong hydrophobicity and bioaccumulation properties, and can persist in soil, water, and atmospheric particulate matter after entering the environment. They accumulate and amplify through the food chain, ultimately harming human health. Therefore, the remediation of PAH pollution has become a critical issue urgently needing to be addressed in the field of environmental science.

[0003] In the coking industry, the emission problem of benzo[a]anthracene is particularly prominent. During coke oven production, large amounts of complex flue gas containing benzo[a]anthracene are emitted at various stages, including coal charging, coke pushing, coke quenching, and top-of-furnace emissions. This type of flue gas has an extremely complex composition, containing not only polycyclic aromatic hydrocarbons but also tar mist, particulate matter, water vapor, sulfur dioxide, nitrogen oxides, and other components. The temperature of coking flue gas is generally between 180-300℃, and the humidity is generally between 12%-18%. Currently, most of the Taiyuan coking plant uses wet desulfurization technology for cooling, resulting in low-temperature saturated wet flue gas with a temperature of 45-55 degrees Celsius and high dust and tar content, posing a severe challenge to the stable operation of pollution control technologies. The current "Emission Standard of Pollutants for Coking Chemical Industry" (GB16171-2012) imposes strict special emission limits on characteristic pollutants such as benzo[a]anthracene, necessitating the development of green and efficient treatment technologies suitable for high-temperature, high-humidity, and multi-pollutant operating conditions in coking enterprises.

[0004] Currently, the treatment of polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]anthracene mainly employs physicochemical methods, including activated carbon adsorption, catalytic oxidation, wet scrubbing, and photocatalytic degradation. However, these traditional technologies have many limitations when applied to coking flue gas treatment. While adsorption technology shows good initial removal efficiency, the adsorbent is easily saturated, regeneration is difficult, operating costs are high, and saturated adsorbents, as hazardous waste, require further disposal. Catalytic oxidation technology has high requirements for reaction conditions; tar and dust in coking flue gas easily lead to catalyst poisoning and deactivation, and catalytic bed clogging is a serious problem. Wet scrubbing technology generates large amounts of oily and dusty scrubbing wastewater, easily causing secondary pollution, while equipment corrosion and packing blockage are frequent problems, resulting in high operating and maintenance costs. Photocatalytic technology is affected by factors such as poor light transmittance and high moisture content of the flue gas, making it difficult to achieve stable results in actual flue gas treatment. These physicochemical methods generally suffer from poor operational stability, high energy consumption, and a high risk of secondary pollution when dealing with complex conditions such as high temperature, high humidity, and multiple pollutant coexistence, like coking flue gas, making it difficult to meet increasingly stringent environmental emission standards.

[0005] Microbial degradation technology has become a research hotspot for the in-depth treatment of polycyclic aromatic hydrocarbons (PAHs) due to its advantages such as thorough mineralization, low cost, mild operating conditions, and minimal secondary pollution. This technology utilizes the metabolism of microorganisms to completely mineralize organic pollutants such as benzo[a]anthracene into carbon dioxide and water, achieving the harmless treatment of pollutants. In recent years, researchers at home and abroad have isolated some microbial strains with PAH degradation capabilities from contaminated soil, river sediment, activated sludge, and other environments, such as *Pseudomonas*, *Bacillus*, *Rhodococcus*, and *Mycobacterium*. However, these reported degradation strains are mainly isolated from ambient temperature (25-35℃), low-salt, and low-dust environments. Their optimal growth temperatures are generally low, and the thermal stability of their degradation enzyme systems is poor, making it difficult to adapt to the high-temperature conditions of coking flue gas (45-60℃). When the flue gas temperature rises above 45°C, the cell membrane fluidity of conventional microbial strains changes, and conformational changes in enzymes lead to reduced activity or even inactivation. This significantly inhibits microbial metabolic activity, resulting in a sharp decline in the degradation efficiency of benzo[a]anthracene and a seasonal collapse in the removal performance of treatment facilities such as biofilters. Furthermore, the high concentrations of tar particles and SO2 in coking flue gas have strong toxic and inhibitory effects on microorganisms, making it difficult for conventional strains to survive and maintain metabolic activity in such harsh environments. The mismatch between the extreme operating conditions of coking flue gas and the physiological adaptability of existing degradation strains has become a key bottleneck restricting the widespread application of biological purification technology in the coking industry.

[0006] Therefore, screening and identifying a single dominant strain capable of maintaining high-efficiency degradation of benzo[a]anthracene at 45℃, systematically studying its high-temperature degradation characteristics, physiological and biochemical features, and environmental adaptability, and revealing its degradation behavior under high-concentration pollutants, high dust, and high humidity conditions, is crucial for constructing a biological purification process adapted to the characteristics of coking flue gas. This is an urgent need to achieve stable and compliant emissions of benzo[a]anthracene pollutants throughout the year in the coking industry and is also an important research direction in the field of environmental microbiology. Screening and domesticating indigenous microorganisms from environmental samples at coking plants is expected to yield excellent degradation strains adapted to local extreme working conditions, providing core microbial resources and technical support for the biological purification of coking flue gas. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes a fluorescent Pseudomonas strain with benzo[a]anthracene degradation ability and its application. This fluorescent Pseudomonas strain SXU-ZW-01 belongs to the genus Pseudomonas and has excellent benzo[a]anthracene degradation ability. In particular, it can maintain high efficiency degradation activity under high temperature conditions and has a high degradation rate.

[0008] To achieve the above objectives, the present invention provides a fluorescent Pseudomonas strain with benzo[a]anthracene degradation ability, wherein the strain is Fluorescent Pseudomonas (… Pseudomonas fluorescens SXU-ZW-01, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.36511, on November 5, 2025.

[0009] The present invention also provides the application of the above-mentioned fluorescent Pseudomonas strain with benzo[a]anthracene degradation ability in environmental remediation.

[0010] Furthermore, the application involves using the fluorescent Pseudomonas SXU-ZW-01 to degrade benzo[a]anthracene in the environment.

[0011] Furthermore, the environment is at least one of coking flue gas, coke oven escaping gas, industrial waste gas, polluted soil, or polluted water.

[0012] The present invention also provides a method for treating benzo[a]anthracene in coking flue gas using the above-mentioned fluorescent Pseudomonas bacteria, comprising the following steps: passing coking flue gas containing benzo[a]anthracene into a biofilter tower loaded with fluorescent Pseudomonas bacteria SXU-ZW-01, and carrying out a biodegradation reaction at 35-55℃ to achieve the removal of benzo[a]anthracene.

[0013] Furthermore, the temperature of the biodegradation reaction is 40-50℃; and the pH of the coking flue gas is 5.0-9.0.

[0014] Furthermore, the empty residence time of the biofiltration tower is 30-90 seconds.

[0015] Furthermore, the temperature of the biodegradation reaction is 45°C; the pH of the coking flue gas is 7.0-8.0; and the empty residence time of the biofiltration tower is 45-60 seconds.

[0016] The present invention also provides a microbial agent for degrading benzo[a]anthracene, wherein the microbial agent contains the above-mentioned fluorescent Pseudomonas SXU-ZW-01 as an active ingredient.

[0017] The present invention also provides a biological filtration device, the device comprising the above-mentioned microbial agent and a porous packing material for loading the microbial agent.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects: The *Pseudomonas fluorescens* SXU-ZW-01 strain obtained in this invention is a benzo[a]anthracene degrading strain with excellent high-temperature degradation performance. This strain can efficiently degrade benzo[a]anthracene at 45℃ and pH 7.0-8.0, achieving a degradation rate of over 95% in 96 hours. It also exhibits good tolerance to coexisting pollutants such as tar and dust, demonstrating long-term operational stability in simulated coking flue gas environments. SXU-ZW-01 shows significant advantages in degradation performance under high-temperature conditions. This strain provides core bacterial resources and technical support for the green biological treatment of benzo[a]anthracene in coking flue gas, and has broad prospects for industrial application. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The growth status of *Pseudomonas fluorescens* SXU-ZW-01 on solid culture medium; Figure 2 The degradation rate of benzo[a]anthracene by *Pseudomonas fluorescens* SXU-ZW-01 under different pH conditions; Figure 3 The degradation rate of benzo[a]anthracene by *Pseudomonas fluorescens* SXU-ZW-01 under different temperature conditions; Figure 4 The degradation rate of different concentrations of benzo[a]anthracene by *Pseudomonas fluorescens* SXU-ZW-01 is shown. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0023] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0024] Example 1: Screening and isolation of *Pseudomonas fluorescens* SXU-ZW-01 1. Sample Collection The bacterial strains used in the experiment were screened from total suspended particulate matter (TSP) samples from the escaping area of ​​the coke oven roof at a coking plant in Taiyuan City, Shanxi Province. The sampling point was located on the main working platform of the coke oven roof, an area constantly affected by coke oven escaping flue gas, with flue gas temperature fluctuations ranging from 45-55℃ and relative humidity >85%. A high-flow-rate air sampler was used with a quartz fiber filter membrane (Whatman, 90mm in diameter) as the carrier, at a standard flow rate of 1.0 m³ / h. 3 Continuous sampling was conducted for 24 hours at a rate of [number] min. After sampling, the filter membrane was removed with sterile forceps, wrapped in sterilized aluminum foil, and transported to the laboratory in an insulated box with ice packs. It was then frozen and stored at -20°C for later use.

[0025] 2. Preparation of bacterial suspension In a sterile operating room, the quartz fiber sampling membrane stored at -20℃ was removed and thawed at room temperature for 30 minutes. Approximately 1 / 6 of the adherent area of ​​the membrane was cut using sterile scissors and placed in a sterile centrifuge tube. 35 mL of pre-chilled sterile phosphate-buffered saline (PBS, 0.01 mol / L, pH 7.2) was added. The centrifuge tube was placed in an ice bath and ultrasonically cleaned at 150 W for 10 minutes to allow particles adhering to the membrane to detach and enter the buffer solution while maintaining the integrity of the bacterial cells. After ultrasonication, the centrifuge tube was centrifuged at 200 × g for 3 minutes at 4℃ to remove large particles and tar residue. The supernatant was collected and transferred to a new 50 mL sterile centrifuge tube. The above procedure was repeated once with the original membrane, and the supernatants from both treatments were combined to obtain approximately 70 mL of initial bacterial suspension.

[0026] A negative pressure filtration device was used to filter the initial bacterial suspension through a sterile polyethersulfone (PES) membrane (47 mm in diameter) with a pore size of 0.22 μm, trapping the bacteria on the membrane surface. After filtration, the membrane was removed with sterile forceps and placed face up into a 2 mL sterile EP tube. Immediately, 1 mL of sterile PBS buffer was added, and the membrane surface was repeatedly agitated 20 times with a pipette to fully elute the trapped bacteria, yielding approximately 1 mL of concentrated bacterial suspension. To maximize bacterial recovery, another 1 mL of sterile PBS was added for a second elution, and the two eluates were combined.

[0027] 3. Enrichment culture and separation / purification The above concentrated bacterial suspension was prepared at a ratio of 10 -1 10 -2 10 -3 Three serial dilutions were performed, with 100 μL of each dilution spread onto beef extract peptone agar plates, and three replicates were set up for each dilution. After spreading, the plates were incubated in a clean bench for 30 min to allow the bacterial culture to be fully absorbed by the medium, and then inverted in a 45℃ incubator for 2-7 days, observing colony growth daily. The beef extract peptone medium formula was: 5 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 20 g / L agar, pH 7.0±0.2.

[0028] After single colonies have grown on the plates, single colonies of different morphologies are selected based on their phenotypic characteristics such as morphology, size, color, edge features, and gloss. These colonies are then streaked onto new beef extract peptone agar plates for purification. The streaked plates are incubated at 45°C for 24-48 hours. Single colonies with consistent morphology are then selected, and the streaking purification process is repeated at least three times until a pure culture is obtained.

[0029] 4. Targeted screening of benzo[a]anthracene degrading bacteria To obtain strains with the ability to degrade benzo[a]anthracene, the purified single colonies were inoculated onto inorganic salt solid culture plates with benzo[a]anthracene as the sole carbon source for targeted screening.

[0030] The inorganic salt culture medium formula is as follows: 2.0 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2·2H2O, 0.001 g / L FeSO4·7H2O, 1.5 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO4, 0.004 g / L MnCl2·4H2O, 0.001 g / L ZnCl2, 0.001 g / L CoCl4·6H2O, 0.002 g / L NiCl2·6H2O, 0.0003 g / L CuSO4·5H2O, 18 g / L agar, pH 7.0±0.2.

[0031] After sterilization, the culture medium was cooled to about 50°C, and then benzo[a]anthracene-acetone stock solution that had been sterile filtered was added to make the final concentration of benzo[a]anthracene 50 mg / L. After thorough mixing, the mixture was poured into plates.

[0032] The purified bacterial culture was streaked onto inorganic salt solid medium plates containing benzo[a]anthracene using an inoculation loop, with three parallel plates inoculated for each strain. The plates were incubated upside down at 45°C for 5-7 days, and colony growth was observed daily. Strains that grew on benzo[a]anthracene and formed distinct colonies were selected and re-inoculated onto fresh inorganic salt solid medium plates containing benzo[a]anthracene for further screening. The streaking isolation and purification process was repeated until a single dominant strain with stable growth and consistent colony morphology on a medium using benzo[a]anthracene as the sole carbon source was obtained, named SXU-ZW-01.

[0033] 5. Preservation of microbial strains The purified SXU-ZW-01 strain was inoculated into beef extract peptone liquid medium and cultured with shaking at 45℃ and 180 r / min until the logarithmic growth phase (OD50). 600 ≈0.8). Mix 500 μL of bacterial culture with 500 μL of 50% sterile glycerol, dispense into 2 mL cryovials, and store at -80°C for long-term preservation. Simultaneously, inoculate the strain onto beef extract peptone agar slant medium, incubate at 45°C for 24 h, and then store at 4°C as the working strain.

[0034] This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) under the name SXU-ZW-01 and classified as *Pseudomonas fluorescens*. Pseudomonas fluorescens The collection has the accession number CGMCC NO.36511, the accession date is November 5, 2025, and the accession address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0035] Example 2: Identification of Fluorescent Pseudomonas SXU-ZW-01 1. Observation of morphological characteristics The SXU-ZW-01 strain was inoculated onto beef extract peptone agar plates, and single colonies were isolated using the streak plating method. Colony morphology was observed after incubation at 45°C for 24 hours. Figure 1 As shown, this strain forms circular colonies approximately 1 mm in diameter on solid culture medium, with neat edges, a smooth and moist surface, and a yellowish-green, slightly transparent, and somewhat glossy appearance. Single colonies were picked and Gram-stained. Under an optical microscope, the bacteria appeared coccobacillus-shaped, Gram-negative, and non-motile.

[0036] 2. 16S rDNA sequence analysis 16S rDNA sequencing was performed on strain SXU-ZW-01, and the obtained sequences were submitted to the NCBI database. The sequences were then compared with the 16S rDNA sequences of known strains in GenBank using the BLAST program. The results showed that strain SXU-ZW-01 is similar to *Pseudomonas fluorescens* (…). Pseudomonas fluorescens The type strain of SXU-ZW-01 showed a sequence similarity of 99.88%. Using MEGA 11.0 software, a phylogenetic tree was constructed using the Neighbor-Joining method. Further analysis showed that strain SXU-ZW-01... Pseudomonas fluorescens Clustered on the same branch, they are most closely related. Based on morphological characteristics and physiological and biochemical identification results, strain SXU-ZW-01 was identified as *Pseudomonas fluorescens* (…). Pseudomonas fluorescens ).

[0037] 3. Identification of physiological and biochemical characteristics The physiological and biochemical characteristics of strain SXU-ZW-01 were determined according to the methods described in the "Manual of Systematic Identification of Common Bacteria" and "Bergey's Manual of Bacterial Identification".

[0038] The results showed that this strain is a strict aerobic bacterium with an optimal growth temperature of 45℃, and can grow in the range of 35-50℃; the optimal growth pH is 7.0-8.0, and it can grow in the range of pH 6.0-9.0; it is catalase positive and oxidase positive, and can utilize carbon sources such as glucose, fructose, and sucrose, but cannot utilize lactose; it is negative for nitrate reduction test, gelatin liquefaction test, and starch hydrolysis test; it can tolerate 2.0% NaCl, and is sensitive to ampicillin, kanamycin, and streptomycin.

[0039] Example 3: Degradation characteristics of benzo[a]anthracene by *Pseudomonas fluorescens* SXU-ZW-01 under different pH conditions 1. Preparation of bacterial suspension The SXU-ZW-01 glycerol strain, stored at -80℃, was streaked onto beef extract peptone agar plates and activated by incubation at 45℃ for 12 h. Single colonies of activated bacteria were picked and inoculated into 100 mL of beef extract peptone liquid medium, and cultured at 45℃ with shaking at 180 rpm until the logarithmic growth phase (OD50) was reached. 600 ≈0.8). Take an appropriate amount of culture medium, centrifuge at 8000×g for 5 min at 4℃, discard the supernatant and collect the bacterial cells. Wash the bacterial cell pellet twice with sterile inorganic salt medium (without carbon source), resuspend it in the same medium, and adjust the OD. 600 Adjust the concentration to 0.20±0.02 to obtain the working bacterial suspension, store at 4℃ for later use, and use within 2 hours.

[0040] 2. Establishment of the degradation system Add 50 mL of sterile inorganic salt culture medium (formulation as in Part 4 of Example 1, excluding agar) to each 120 mL thermostable serum bottle. Add benzo[a]anthracene-acetone stock solution using a microsyringe, and remove the acetone solvent by nitrogen blowing to fix the initial concentration of benzo[a]anthracene at 50 mg / L. Adjust the pH of the system precisely to 5.0, 6.0, 7.0, 8.0, and 9.0 (accuracy ±0.1) using 1 mol / L HCl or 1 mol / L NaOH, with three replicates for each pH treatment. Inoculate each bottle with 2% (v / v) of working bacterial suspension (i.e., 1.0 mL) and immediately seal with a PTFE stopper and aluminum cap. A blank control group without inoculation is also set up to correct for abiotic losses of benzo[a]anthracene.

[0041] 3. Cultivation and Sampling The sealed serum bottles were placed in a constant temperature shaking incubator and incubated in the dark at 45℃ and 180 rpm. At 0, 12, 24, 48, 72, and 96 h of incubation, 1.0 mL of the reaction solution was aspirated through the stopper using a sterile syringe and immediately injected into a centrifuge tube containing 2.0 mL of chromatographically pure n-hexane to terminate the reaction. The centrifuge tube was placed in an ice bath and ultrasonically extracted for 15 min (40 kHz, 100 W) to ensure complete transfer of benzo[a]anthracene to the organic phase. The extract was centrifuged at 12000 × g for 5 min at 4℃. The upper organic phase was carefully aspirated, filtered through a 0.22 μm organic filter membrane, and transferred to a brown sample vial for HPLC-FLD analysis.

[0042] 4. HPLC-FLD determination conditions An Agilent 1260 Infinity II high-performance liquid chromatography system, equipped with a fluorescence detector and a ZORBAX Eclipse PAH column (4.6 mm × 250 mm, 5 μm), was used. The mobile phase was acetonitrile / water = 90 / 10 (v / v), with isocratic elution at a flow rate of 1.0 mL / min; the column temperature was 35 °C; the injection volume was 20 μL; the fluorescence detector had an excitation wavelength of 280 nm and an emission wavelength of 410 nm. Quantification was performed using the external standard method. A series of concentrations (0.05, 0.1, 0.5, 1.0, 5.0, 10.0 mg / L) of benzo[a]anthracene standard were prepared to plot a standard curve. The linear correlation coefficient R0 was calculated. 2 >0.999.

[0043] 5. Experimental Results The 96-h degradation rate of benzo[a]anthracene by *Pseudomonas fluorescens* SXU-ZW-01 under different pH conditions is as follows: Figure 2 As shown in the figure. The results (n=3) indicate that the degradation rate was fastest in the pH 7.0 group, reaching (95.3±1.2)% after 96 hours; the degradation effect of the pH 8.0 group was comparable to that of the pH 7.0 group, with a degradation rate of (95.7±1.8)% after 96 hours; the degradation rate of the pH 6.0 group decreased to (78.0±2.5)%; the degradation effect of the pH 5.0 group was the worst, at only (57.4±3.1)%; and the degradation rate of the pH 9.0 group was (82.1±2.9)%, slightly lower than that under neutral and weakly alkaline conditions. These results indicate that strain SXU-ZW-01 can maintain high efficiency in degrading benzo[a]anthracene in the neutral to weakly alkaline range (pH 7.0-8.0), and this pH range is highly consistent with the actual operating conditions of coking flue gas.

[0044] Example 4: Degradation characteristics of benzo[a]anthracene by *Pseudomonas fluorescens* SXU-ZW-01 under different temperature conditions 1. Preparation of bacterial suspension The working bacterial suspension was prepared according to the method described in step 1 of Example 3. OD 600 Adjust to 0.20±0.02 and store at 4℃ for later use.

[0045] 2. Temperature gradient setting Five temperature points were set in the constant temperature shaking incubator: 35℃, 40℃, 45℃, 50℃, and 55℃, with a temperature control accuracy of ±0.2℃ and a shaking amplitude of 25mm. Each temperature point was pre-run for 30 minutes before the experiment to ensure temperature stability.

[0046] 3. Establishment of the degradation system Add 50 mL of sterile inorganic salt culture medium to each 120 mL thermostable serum bottle, then add benzo[a]anthracene-acetone stock solution and remove the solvent by nitrogen evaporation to achieve an initial benzo[a]anthracene concentration of 50 mg / L. Adjust the pH to 7.0 ± 0.1 using 1 mol / L HCl / NaOH. Inoculate each bottle with 1.0 mL of working bacterial suspension (2% v / v) and seal immediately. Set up three replicates for each temperature and two blank control bottles without inoculation. Place the serum bottles in shaking incubators at different temperatures and incubate at 180 rpm in the dark.

[0047] 4. Sampling and Pretreatment At 0, 12, 24, 48, 72, and 96 hours of culture, 1.0 mL of sample was taken using a sterile syringe, and extraction and sample pretreatment were performed according to the method described in step 3 of Example 3.

[0048] 5. HPLC-FLD determination The measurement conditions are the same as step 4 of Example 3.

[0049] 6. Experimental Results The 96-h degradation rate of benzo[a]anthracene by *Pseudomonas fluorescens* SXU-ZW-01 under different temperature conditions is as follows: Figure 3 As shown in the figure. The results (n=3) indicate that the degradation effect was best in the 45℃ group, with a degradation rate of (95.3±1.5)% after 96 hours; the degradation rate in the 40℃ group was (74.7±1.8)%; the degradation rate in the 35℃ group was only (68.4±2.1)%, significantly lower than that in the 45℃ group; the degradation rate in the 50℃ group was (88.0±1.2)%, still maintaining high activity; and the degradation rate in the 55℃ group decreased to (82.1±2.7)%, but was still above 50%. This result shows that strain SXU-ZW-01 has typical high-temperature adaptation characteristics, and its optimal degradation temperature (45℃) matches the actual emission temperature of coking flue gas, while ordinary room-temperature strains often have difficulty surviving or experience a significant decrease in activity at this temperature.

[0050] Example 5: Degradation characteristics of benzo[a]anthracene by *Pseudomonas fluorescens* SXU-ZW-01 under different initial concentrations 1. Concentration gradient setting Using a 1 g / L benzo[a]anthracene-acetone stock solution as the mother liquor, the solution was aseptically filtered through a 0.22 μm organic filter membrane and then serially diluted with sterile inorganic salt medium to prepare benzo[a]anthracene degradation systems with initial concentrations of 30, 40, 50, 60, and 70 mg / L (accuracy ±0.5 mg / L). Each concentration gradient solution was stored at 4°C protected from light and used on the same day.

[0051] 2. Establishment of the degradation system For each concentration gradient, the pH was uniformly controlled at 7.0±0.1, the culture temperature at 45℃, the shaking speed at 180 r / min, and the inoculum size at 2% v / v. 50 mL of the corresponding concentration of benzo[a]anthracene-inorganic salt medium was added to a 120 mL serum bottle, and the bottle was immediately sealed after inoculation with the working bacterial suspension. Three replicates were prepared for each concentration, along with a blank control without inoculation.

[0052] 3. Cultivation and Sampling The serum bottles were placed in a 45°C constant temperature shaking incubator and incubated at 180 rpm in the dark for 96 h. Samples were taken at 0, 12, 24, 48, 72, and 96 h to determine the residual concentration of benzo[a]anthracene. The sampling and pretreatment methods were the same as in step 3 of Example 3.

[0053] 4. Experimental Results The 96-h degradation rate of benzo[a]anthracene by *Pseudomonas fluorescens* SXU-ZW-01 under different initial concentrations is as follows: Figure 4 As shown in the figure. The results (n=3) indicate that strain SXU-ZW-01 exhibited good degradation ability within the concentration range of 30-70 mg / L, with no obvious substrate inhibition. The highest degradation rate was observed in the 50 mg / L group (95.7±1.8)%, followed by the 30 mg / L group (92.1±1.0)% and the 70 mg / L group (90.5±2.2)%. It is noteworthy that strain SXU-ZW-01 maintained a degradation rate of over 85% within the above concentration range, meeting the adaptability requirements for pollutant concentration fluctuations under actual operating conditions.

[0054] Example 6: Long-term operational stability of *Pseudomonas fluorescens* SXU-ZW-01 in a simulated coking flue gas environment 1. Experimental Objective To verify the long-term operational stability of strain SXU-ZW-01 under actual operating conditions, a pilot-scale biofilter system simulating coking flue gas environment was constructed to investigate the variation of benzo[a]anthracene removal efficiency during 30 days of continuous operation.

[0055] 2. Construction of biological filtration tower The biological filtration tower is constructed using plexiglass columns (100mm inner diameter, 500mm packing layer height), with polyurethane foam as the packing material. The packing material has a porosity of approximately 92% and a specific surface area of ​​approximately 600m². 2 / m 3 The activated and expanded SXU-ZW-01 bacterial suspension (OD) 600 ≈1.0) is sprayed onto the surface of the packing material in a cycle, and the biofilm is formed in a cycle for 7 days, so that the bacteria can fully attach and grow, forming a stable biofilm.

[0056] 3. Simulated flue gas preparation Simulated coking flue gas composition: Benz[a]anthracene as the target pollutant, with a concentration of 80±5 mg / m³. 3 Add tar mist (50±10mg / m³) 3 ) and dust particles (30±5mg / m 3 The flue gas temperature was controlled at 45±2℃; the relative humidity was maintained at 90±5%; and the empty tower residence time was set to 60s.

[0057] 4. Operating conditions and monitoring The biofilter operated continuously for 30 days, with the concentrations of benzo[a]anthracene at the inlet and outlet measured daily to calculate the removal efficiency. Simultaneously, the pressure drop across the packing layer, biofilm growth, and the stability of the microbial community structure were monitored.

[0058] 5. Experimental Results Continuous monitoring over 30 days showed that strain SXU-ZW-01 exhibited excellent long-term operational stability in a simulated coking flue gas environment. During the start-up period (1-3 days), the removal efficiency gradually increased to over 90%; during the stable operation period (4-25 days), the removal efficiency remained between 92% and 96%, with an average removal rate of (93.8±1.5)%; in the later stage of operation (26-30 days), the removal efficiency decreased slightly but still remained above 88%. Throughout the entire operation, the pressure drop of the packing layer remained stable within the range of 80-120 Pa, with no significant blockage observed. Periodic collection of biofilm samples from the packing layer for microbial community analysis showed that SXU-ZW-01 consistently maintained its dominant microbial community status, with a relative abundance exceeding 75%. This result indicates that strain SXU-ZW-01 can maintain long-term stable benzo[a]anthracene degradation capacity in the high-temperature, high-humidity, and multi-pollutant coexistence environment of simulated coking flue gas, demonstrating promising prospects for industrial application.

[0059] Example 7: Experiment on the tolerance of *Pseudomonas fluorescens* SXU-ZW-01 to tar and dust interference 1. Experimental Objective Coking flue gas contains high concentrations of tar mist and dust particles, which have toxic and inhibitory effects on microorganisms. This experiment aims to investigate the degradation performance of strain SXU-ZW-01 under conditions of coexistence of tar and dust.

[0060] 2. Tar tolerance test In a basic degradation system (50 mg / L benzo[a]anthracene, pH 7.0, 45℃, 180 r / min), 0, 50, 100, 200, and 500 mg / L of coking tar (collected from a coking plant in Taiyuan and sterile filtered) were added to investigate the 96-h degradation rate of benzo[a]anthracene by strain SXU-ZW-01 under different concentrations of tar.

[0061] 3. Dust tolerance test In the basic degradation system, coking dust (collected from a bag filter of a coking plant in Taiyuan and sterilized at high temperature) was added at concentrations of 0, 30, 50, 100, and 200 mg / L to investigate the degradation performance of the strain under the presence of dust.

[0062] 4. Composite Interference Experiment The degradation performance of the strain was investigated under the condition of simultaneous addition of tar (100 mg / L) and dust (50 mg / L).

[0063] 5. Experimental Results Tar tolerance experiments showed that when the tar concentration was ≤100 mg / L, the degradation rate of benzo[a]anthracene by strain SXU-ZW-01 could still reach over 91.2%, which was slightly lower than the tar-free control group (95.3%) but the difference was not significant. When the tar concentration increased to 200 mg / L, the degradation rate decreased to 83.7%; when the tar concentration was 500 mg / L, the degradation rate could still be maintained at 70.5%. Dust tolerance experiments showed that when the dust concentration was ≤100 mg / L, the degradation rate remained above 88%; when the dust concentration was 200 mg / L, the degradation rate was 79.6%. Under the combined interference of tar (100 mg / L) and dust (50 mg / L), the 96-hour degradation rate of benzo[a]anthracene by the strain was 86.4%, still maintaining a high level of activity.

[0064] The above results indicate that strain SXU-ZW-01 has good tolerance to tar and dust, and can still maintain high degradation activity in the complex environment of coking flue gas with multiple pollutants, which is an important advantage for its application in coking flue gas treatment.

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fluorescent Pseudomonas strain capable of degrading benzo[a]anthracene, characterized in that, The strain is *Pseudomonas fluorescens* (…). Pseudomonas fluorescens SXU-ZW-01, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.36511, on November 5, 2025.

2. The application of the fluorescent Pseudomonas bacterium with benzo[a]anthracene degradation capability as described in claim 1 in environmental remediation.

3. The application according to claim 2, characterized in that, The application involves using the fluorescent Pseudomonas SXU-ZW-01 to degrade benzo[a]anthracene in the environment.

4. The application according to claim 3, characterized in that, The environment is at least one of coking flue gas, coke oven gas, industrial waste gas, polluted soil, or polluted water.

5. A method for treating benzo[a]anthracene in coking flue gas using the *Pseudomonas fluorescens* strain described in claim 1, characterized in that, Includes the following steps: Coking flue gas containing benzo[a]anthracene was passed into a biofilter loaded with Pseudomonas fluorescens SXU-ZW-01 and subjected to biodegradation at 35-55℃ to remove benzo[a]anthracene.

6. The method according to claim 5, characterized in that, The temperature of the biodegradation reaction is 40-50℃; the pH of the coking flue gas is 5.0-9.

0.

7. The method according to claim 5, characterized in that, The empty residence time of the biofiltration tower is 30-90s, preferably 45-60s.

8. The method according to claim 5, characterized in that, The temperature of the biodegradation reaction is 45℃; the pH of the coking flue gas is 7.0-8.0; and the empty residence time of the biofiltration tower is 45-60s.

9. A microbial inoculant for degrading benzo[a]anthracene, characterized in that, The microbial agent contains the fluorescent Pseudomonas SXU-ZW-01 as the active ingredient as described in claim 1.

10. A biological filtration device, characterized in that, The device comprises the microbial agent of claim 9, and a porous packing material for loading the microbial agent.

Images