Mixed polycyclic aromatic hydrocarbon degrading bacterium G3 suitable for variable environment and application thereof

Abstract

The invention relates to the technical field of environmental remediation microorganisms, and discloses a mixed polycyclic aromatic hydrocarbon degrading bacterium G3 suitable for variable environments and application thereof. The bacterial strain is pseudoxanthomonas sp. G3, and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC M 20251828. The strain G3 is enriched and separated from soil of a certain coking plant in Beijing, can utilize polycyclic aromatic hydrocarbons such as acenaphthene, fluorene, phenanthrene, fluoranthene and pyrene as a single carbon source, and can simultaneously degrade acenaphthene, fluorene, phenanthrene, fluoranthene and pyrene mixtures within 14 days, so that the degradation rate of each component is not lower than 70%; good growth and degradation activity are maintained under the conditions that the temperature is 20-37 DEG C, the salinity is 0-4% NaCl and the pH value is 5.0-9.0, and the degradation rates can reach 100% and 84.15% respectively after the strain is cultured in a single carbon source culture solution with the initial concentration of phenanthrene and pyrene being 50 mg.L <-1 > for 7 days; after the polycyclic aromatic hydrocarbon aging polluted soil is cultured for 28 days, the mixed polycyclic aromatic hydrocarbon in the soil can be effectively removed. According to the invention, the technical bottlenecks of poor environmental adaptability and low mixed pollutant degradation efficiency of the existing degrading bacteria are solved. Therefore, the strain has good application potential in the aspect of bioremediation of the polycyclic aromatic hydrocarbon polluted environment.

Description

[0001] This invention relates to the field of environmental remediation microbial technology, and in particular to a mixed polycyclic aromatic hydrocarbon degrading bacterium G3 suitable for variable environments and its applications. Background Technology

[0002] Cities, as centers of socio-economic activity, face severe environmental pollution problems due to resource and energy consumption. Industrial emissions, fossil fuel combustion, transportation activities, and waste disposal processes generate large amounts of pollutants, including petroleum hydrocarbons, benzene compounds, and polycyclic aromatic hydrocarbons (PAHs). PAHs are highly hydrophobic, persistent, and resilient, originating from the incomplete combustion of organic matter. They possess carcinogenic, teratogenic, and mutagenic effects (the "three-cause" effects) and bioaccumulation, threatening ecological security and human health through the food chain. The U.S. Environmental Protection Agency (EPA) has listed 16 PAHs as priority pollutants. In the environment, PAHs easily accumulate in soil through adsorption, deposition, and migration, making soil a significant PAH pollution accumulation zone. Therefore, the remediation and treatment of polycyclic aromatic hydrocarbon pollution in soil is receiving increasing attention.

[0003] Soil PAH pollution remediation technologies mainly include physical, chemical, and biological methods. Among them, bioremediation, especially microbial remediation, is gaining increasing attention due to its significant advantages such as low cost, high effectiveness, green and low-carbon operation, and adherence to natural decay patterns. Microbial remediation utilizes the metabolic activities of indigenous or exogenous microorganisms to degrade pollutants, and its core lies in the activity regulation of highly efficient degrading strains. Therefore, screening for highly efficient degrading bacteria that can adapt to environmental changes and maximizing microbial activity has become crucial to this technology.

[0004] Existing research has reported various PAH-degrading bacteria, such as *Mycobacterium*, *Rhodococcus*, *Pseudomonas*, and *Sphingomonas*, providing strain resources for the development of microbial remediation technologies. However, the complexity of polluted environments limits their practical application. On the one hand, fluctuations in environmental factors (such as temperature, pH, and salinity) limit the survival ability of most strains, necessitating the screening of strains adapted to variable environmental factors. On the other hand, existing studies have mostly explored the degradation ability of strains under single PAH conditions, while multiple PAHs often coexist in real-world polluted environments, making it difficult to directly apply research results to complex polluted sites. These dual limitations—the variability of environmental factors and the ability to degrade mixed pollutants—constitute the main challenges facing the practical application of PAH-degrading bacteria. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a highly efficient polycyclic aromatic hydrocarbon (PAH) degrading bacterium G3 suitable for variable environments and its application, in order to solve the problem of strain environmental adaptability and thus meet the technical needs of PAH degradation under variable environmental conditions.

[0006] The first objective of this invention is to provide a highly efficient polycyclic aromatic hydrocarbon degrading bacterium, G3, suitable for variable environments. This strain belongs to the genus *Pseudoxanthomonas* sp. After preliminary identification, the strain was named *Pseudoxanthomonas* sp. G3, with the accession number CCTCC M 20251828. It was deposited on August 15, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, China.

[0007] The degrading bacterium Pseudoxanthomonas sp.G3 provided by this invention was isolated from aging soil in a coking plant. After being cultured on Luria-Bertany (LB) solid medium plates at 37°C for 2-3 days, the colonies of this strain were yellow, round, with a raised, viscous, moist and opaque surface.

[0008] The morphological characteristics of the Pseudoxanthomonas sp. G3 strain provided by this invention are: Gram-negative, rod-shaped cells, about 2.0-3.0 μm long and about 0.3-0.5 μm in diameter.

[0009] The physiological and biochemical characteristics of the Pseudoxanthomonas sp. G3 strain provided by this invention are as follows: it can utilize α-D-glucose, sucrose, D-mannitol, and salicylic acid; it cannot utilize D-fructose and α-lactose; it is positive for urease test, positive for catalase test, and negative for methyl red test.

[0010] The 16S rDNA gene sequence characteristics of Pseudoxanthomonas sp. G3 provided by this invention are shown in the sequence listing.

[0011] The aforementioned Pseudoxanthomonas sp. G3 can degrade polycyclic aromatic hydrocarbon pollutants; the polycyclic aromatic hydrocarbon pollutants are acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, and pyrene.

[0012] The aforementioned Pseudoxanthomonas sp. G3 exhibits good degradation activity for single polycyclic aromatic hydrocarbon substrates such as acenaphthene, fluorene, phenanthrene, fluoranthene, and pyrene.

[0013] The aforementioned Pseudoxanthomonas sp.G3 exhibits good degradation activity for mixed polycyclic aromatic hydrocarbons, not only degrading acenaphthene, fluorene, phenanthrene, fluoranthene, and pyrene, but also degrading anthracene through cometabolism.

[0014] A second objective of this invention is to provide applications of the aforementioned Pseudoxanthomonas sp. G3 in degrading mixed polycyclic aromatic hydrocarbon pollutants under different environmental conditions.

[0015] The polycyclic aromatic hydrocarbon pollutants are acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Preferably, the mixed polycyclic aromatic hydrocarbons include phenanthrene and pyrene, and the different environmental factors include temperature (20-37°C), salinity (0-4% NaCl), and pH (5.0-9.0).

[0016] Preferably, pyrene in mixed polycyclic aromatic hydrocarbons is efficiently degraded under the conditions of 37°C, salinity 0-3% NaCl, and pH 5.0-8.0.

[0017] Preferably, phenanthrene in mixed polycyclic aromatic hydrocarbons is efficiently degraded under the conditions of temperature 28-37℃, salinity 0-2% NaCl, and pH 5.0-9.0.

[0018] A third objective of this invention is to provide the application of the aforementioned Pseudoxanthomonas sp. G3 in the bioremediation of environments contaminated with mixed polycyclic aromatic hydrocarbons.

[0019] Preferably, the polycyclic aromatic hydrocarbon (PAH) pollution of the environment includes PAH-polluted water bodies and / or soil. The mixed PAHs include acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, and pyrene.

[0020] Furthermore, the present invention also claims the application of the aforementioned Pseudoxanthomonas sp. G3 in degrading mixed polycyclic aromatic hydrocarbons in aging soil, characterized in that the Pseudoxanthomonas sp. G3 bacterial solution is sprayed onto aging soil contaminated with polycyclic aromatic hydrocarbons to degrade the mixed polycyclic aromatic hydrocarbons.

[0021] Preferably, the concentration of the *Pseudoxanthomonas* sp. G3 bacterial suspension applied is not less than 1 × 10⁻⁶. 5 CFU / g soil.

[0022] Technical features and beneficial effects of the present invention:

[0023] 1. This invention describes the enrichment and isolation of a highly efficient polycyclic aromatic hydrocarbon (PAH) degrading bacterium, G3, from contaminated soil samples from a coking plant in Beijing. Based on the strain's morphological and physiological characteristics, 16S rDNA gene sequencing analysis, and phylogenetic analysis, the strain was identified as *Pseudoxanthomonas* sp. G3. Strain G3 was isolated from the initial concentration of each component of the mixed PAHs at 10 mg / L. -1 After culturing in an inorganic salt culture medium for 14 days, the degradation rates of the mixed substrates acenaphthene, fluorene, phenanthrene, fluoranthene, and pyrene reached 89.69%, 80.43%, 95.61%, 77.93%, and 77.51%, respectively. It can also degrade 37.79% of anthracene through cometabolism.

[0024] 2. G3 can grow and rapidly degrade a mixture of phenanthrene and pyrene under conditions of temperature (20-37℃), salinity (0-4% NaCl), and pH (pH 5.0-9.0), with an initial mass concentration of 50 mg·L⁻¹ for both phenanthrene and pyrene. -1 After 7 days of cultivation, the degradation rate of phenanthrene was no less than 60%, and the degradation rate of pyrene was no less than 40%. Under conditions of 37℃, salinity 0-3% NaCl, and pH 5.0-8.0, after 7 days of cultivation, the degradation rate of mixed polycyclic aromatic hydrocarbons with an initial concentration of 50 mg·L⁻¹ was significantly reduced. -1 The degradation rate of pyrene was no less than 60%. Under conditions of 28-37℃, salinity 0-2% NaCl, and pH 5.0-9.0, after 7 days of cultivation, the degradation rate of pyrene in a mixed polycyclic aromatic hydrocarbon mixture with an initial concentration of 50 mg·L⁻¹ was significantly improved. -1 The degradation rate of phenanthrene is no less than 90%.

[0025] 3. The strain G3 was subjected to a concentration of not less than 1×10⁻⁶. 5 When CFU / g soil was applied to contaminated soil aged over 10 years, it significantly degraded various polycyclic aromatic hydrocarbons within 28 days. At a concentration of 5 × 10⁻⁶ CFU / g soil, [the following parameters were observed]: 7 At an inoculum concentration of CFU / g soil, the removal rates of phenanthrene and pyrene reached 29.28% and 29.69%, respectively; at a concentration of 1×10⁻⁶ CFU / g soil, the removal rates of phenanthrene and pyrene reached 29.28% and 29.69%, respectively. 7 At an inoculum concentration of CFU / g soil, the removal rates of anthracene and fluoranthene reached 60.23% and 27.17%, respectively; even at low inoculum concentrations (e.g., 5 × 10⁻⁶ CFU / g soil), the removal rates of anthracene and fluoranthene reached 60.23% and 27.17%, respectively; 6 The removal rates of fluorene and acenaphthene (CFU / g soil) reached 57.37% and 11.02%, respectively.

[0026] This invention effectively solves the technical bottlenecks of existing bioremediation technologies, such as single-function strains and poor environmental adaptability. It provides a highly efficient, stable, and directly applicable microbial resource and application method, thus having great environmental benefits and application prospects in the bioremediation of polycyclic aromatic hydrocarbons in actual contaminated soil. Attached Figure Description

[0027] Figure 1 The images show (a) a plate colony morphology of strain G3, (b) a scanning electron microscope image, and (c) a phylogenetic tree of the strain.

[0028] Figure 2 This is a graph showing the substrate broad-spectrum test results of strain G3 in degrading (a) single polycyclic aromatic hydrocarbons and (b) mixed polycyclic aromatic hydrocarbons.

[0029] Figure 3 The degradation characteristics of strain G3 (a) for phenanthrene, (b) for pyrene, (c) for a mixture of phenanthrene and pyrene, and (d) for its growth characteristics under polycyclic aromatic hydrocarbon pressure.

[0030] Figure 4 These are the growth curves of strain G3 at (a) different temperatures, (b) different salinities, and (c) different pH levels.

[0031] Figure 5 The degradation rate of phenanthrene mixture by strain G3 at (a) different temperatures, (b) different salinities, and (c) different pH values.

[0032] Figure 6 The removal efficiency of different concentrations of strain G3 on mixed polycyclic aromatic hydrocarbons (a) pyrene, (b) fluoranthene, (c) phenanthrene, (d) anthracene, (e) acenaphthene, and (f) fluorene in aging soil. Detailed Implementation

[0033] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0034] The culture medium formulation used in the following examples is as follows: The LB medium for Pseudoxanthomonas sp. G3 consists of: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0035] The LB medium solid plate composition is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L purified agar powder.

[0036] The composition of the MSM culture medium is as follows: Inorganic salt medium (MSM) contains the following components: NH4Cl 1.1g, K2HPO4 1.0g, NaCl 0.5g, MgSO4·7H2O 0.2g, KCl 0.2g, CaCl2 0.01g, and FeSO4 0.001g, 1mL of trace metal salt solution, 1000mL of distilled water, and a pH of 7.0. The formula for the trace metal salt solution is as follows: N(CH2COOH)3 1.5 g / L, MnSO4·H2O 0.5 g / L, NaCl 1.0 g / L, FeSO4·7H2O 0.1 g / L, CoSO4·7H2O 0.18 g / L, CaCl2·2H2O 0.1 g / L, ZnSO4·7H2O 0.18 g / L, CuSO4·5H2O 0.01 g / L, KAl(SO4)2·12H2O 0.02 g / L, H3BO3 0.01 g / L, Na2MoO4·2H2O 0.01 g / L, NiCl2·6H2O 0.025 g / L, Na2SeO3·5H2O 0.3 g / L.

[0037] The composition of the MSM culture medium solid plate is: the above-mentioned MSM culture medium components plus 15 g / L purified agar powder.

[0038] The strain of the present invention used in the following examples (Pseudoxanthomonas sp. G3) is abbreviated as G3.

[0039] Example 1: Isolation and identification of Pseudoxanthomonas sp. G3

[0040] Enrichment: Using polycyclic aromatic hydrocarbon (PAH) contaminated soil from the Beijing Coking Plant as the inoculum, 10g of the contaminated soil sample was weighed and added to a 250mL Erlenmeyer flask containing 90mL of sterile water and 0.5g of glass beads. The flask was incubated at 37℃ and 180rpm with shaking for 3 hours. After standing for 30 minutes, 5mL of the supernatant was transferred to 45mL of inorganic salt medium (MSM) containing 25mg / L of phenanthrene and pyrene, and incubated for 7 days. The culture was repeatedly subcultured four times, with the concentrations of phenanthrene and pyrene gradually increased to 50mg / L, 75mg / L, 100mg / L, and 125mg / L during the subculture. Enrichment of PAH-degrading bacteria was completed after five subcultures.

[0041] Screening: A certain amount of the final enrichment culture was spread onto an MSM solid plate, which was then inverted into a beaker containing pyrene. A pyrene film was deposited on the MSM solid plate using the sublimation method. The plate coated with the pyrene film was placed in a 30°C humidity incubator and incubated. During the incubation period, the colonies that showed a clear zone were identified as pyrene-degrading bacteria.

[0042] Isolation: Select colonies with clear zones and isolate them by repeated streak plating to obtain a pure strain of degrading bacteria, which is named G3.

[0043] Morphological observation: Strains G3 can grow single colonies after 3 days of incubation on LB medium at 37°C. The single colonies are yellow, round, with a raised, viscous, moist, and opaque surface (see appendix). Figure 1 a) Simultaneously, Gram staining was performed on the cultured bacteria. The stained cells were observed under a light microscope; the cells showed Gram-negative staining. Scanning electron microscopy revealed the cells to be rod-shaped, approximately 2.0-3.0 μm long and 0.3-0.5 μm in diameter (see appendix). Figure 1 b).

[0044] Physiological and biochemical characteristics: Strain G3 can utilize α-D-glucose, sucrose, D-mannitol, and salicylic acid as carbon sources, but cannot utilize D-fructose and α-lactose; it is positive for urease and catalase tests, and negative for methyl red test.

[0045] Molecular biological characteristics: Genomic DNA was extracted from *Pseudoxanthomonas* sp. G3 using a bacterial genomic DNA extraction kit (Tiangen Biotech Co., Ltd.). PCR amplification was performed using the upstream primer 5'-AGAGTTTGATCCTGGCTCAG-3' and the downstream primer 5'GGTTACCTTGTTACGACTT-3' to obtain its 16S rDNA gene fragment. The amplified product was sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing. Homologous sequences were analyzed using BLAST on the NCBI website, and a phylogenetic tree of the strain was constructed using MEGA 11 software. Sequencing results showed that the 16S rDNA gene sequence of this strain was 1452 bp in length. NCBI BLAST alignment showed the highest similarity (99%) to the 16S rDNA sequence of known *Pseudoxanthomonas* strains. A phylogenetic tree was constructed (see appendix). Figure 1 c) It can be preliminarily determined that this strain belongs to the genus Pseudoxanthomonas, and is named Pseudoxanthomonas sp.G3.

[0046] Example 2: Broad-spectrum substrate assay for the degradation of polycyclic aromatic hydrocarbons by the strain

[0047] Degradation ability of single polycyclic aromatic hydrocarbons (PAHs): Different PAHs (acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, and pyrene) dissolved in acetone were added to sterilized 250 mL Erlenmeyer flasks. After the acetone evaporated, MSM culture medium was added to make the concentration of each of the above six PAHs 10 mg / L (all PAHs below were added using this method). 0D was inoculated at an inoculum size of 10%. 600=0.8% G3 bacterial culture (obtained in Example 1, the same applies below). All Erlenmeyer flasks were placed in a shaker at 37°C and 180 rpm for incubation. Samples were taken on day 7 to determine the residual amount of each polycyclic aromatic hydrocarbon in the Erlenmeyer flasks. The experimental results showed that the strain could not effectively degrade anthracene in the above-mentioned PAHs, but showed effective degradation of the other 5 single polycyclic aromatic hydrocarbons. The degradation results are shown in Table 1 and Appendix. Figure 2 a.

[0048] Table 1. Degradation rate of single polycyclic aromatic hydrocarbons by Pseudoxanthomonas sp. G3 strain

[0049]

[0050] Degradation capacity of mixed PAHs: Acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, and pyrene dissolved in acetone were simultaneously added to a sterilized 250 mL Erlenmeyer flask, ensuring all six PAHs were present in the same flask. After the acetone evaporated, MSM culture medium was added, with each of the six PAHs at a concentration of 10 mg / L. OD was inoculated at a 10% inoculum. 600 =0.8% G3 bacterial culture. All Erlenmeyer flasks were incubated at 37℃ with shaking at 180 rpm. Samples were taken on day 14 to determine the residual amounts of each polycyclic aromatic hydrocarbon (PAH). The experimental results showed that strain G3 could effectively degrade six PAHs simultaneously and exhibited a cometabolism effect. The degradation results are shown in Table 2 and Appendix. Figure 2 b.

[0051] Table 2. Degradation rate of mixed polycyclic aromatic hydrocarbons by Pseudoxanthomonas sp. G3 strain

[0052]

[0053] Example 3: Degradation kinetics and growth characteristics of strains of phenanthrene, pyrene and their mixtures

[0054] Add phenanthrene, pyrene, and a mixture of phenanthrene and pyrene dissolved in acetone to sterilized 250 mL Erlenmeyer flasks. After the acetone evaporates, add MSM culture medium to ensure that the concentration of each component of the single or mixed polycyclic aromatic hydrocarbons in the flask is 50 mg / L. Inoculate OD at a 10% inoculum. 600 =0.8% G3 bacterial culture. Erlenmeyer flasks were placed in a shaker at 37°C and 180 rpm for incubation. Destructive sampling was performed on days 1, 3, 5, and 7 to determine the residual amount of polycyclic aromatic hydrocarbons in the flasks.

[0055] Each culture sample was added to 50 mL of methanol and ultrasonically cleaned for half an hour using a Shumei KQ-3000DE ultrasonic cleaner at 3000W. After shaking, 2 mL of the supernatant was filtered through a 0.22 μm polytetrafluoroethylene filter into a 2 mL brown chromatographic vial and stored at 4℃. The content of polycyclic aromatic hydrocarbons (PAHs) in each treated sample was determined using an Elite P1201 high-performance liquid chromatograph. The chromatographic column was a SinChromODS-BP column (5 μm, 4.6 × 200 mm), the mobile phase was methanol / water (v / v, 90:10), the detection wavelength was 254 nm, the flow rate was 1.0 mL / min, the injection volume was 20 μL, and the column temperature was set to 30℃. The obtained data were processed using Kromstation high-performance chromatography data management software, and PAH quantification was performed using a 6-point calibration curve.

[0056] Simultaneously, bacterial culture was collected at the corresponding time points, and OD was measured using a spectrophotometer. 600 The absorbance at that location.

[0057] Degradation kinetics of phenanthrene: Results are attached. Figure 3 a. The strain achieved a degradation rate of 40.80% for phenanthrene at an initial concentration of 50 mg / L on day 1, 86.51% on day 3, 99.93% on day 5, and 100% on day 7.

[0058] Degradation kinetics of pyrene alone: ​​results are attached. Figure 3 b. The strain achieved a degradation rate of 25.51% for pyrene at an initial concentration of 50 mg / L on day 1, 55.42% on day 3, 70.69% on day 5, and 84.15% on day 7.

[0059] Degradation kinetics of phenanthrene mixtures: results are attached. Figure 3 c. The strain achieved the following degradation rates for an initial concentration of 50 mg / L of phenanthrene and pyrene: on day 1, phenanthrene degradation reached 39.68% and pyrene degradation reached 19.01%; on day 3, phenanthrene degradation reached 85.98% and pyrene degradation reached 39.24%; on day 5, phenanthrene degradation reached 88.44% and pyrene degradation reached 53.43%; and on day 7, phenanthrene degradation reached 94.58% and pyrene degradation reached 64.81%.

[0060] Strain G3 grew well in systems with initial concentrations of 50 mg / L of single phenanthrene, single pyrene, and a mixture of phenanthrene and pyrene (see Appendix). Figure 3 d). In particular, in the phenanthrene-pyrene mixed system, the growth rate of strain G3 was significantly higher than that in the single phenanthrene or single pyrene system.

[0061] Example 4: Evaluation of the growth adaptability of strain G3 under different environmental factors

[0062] Temperature adaptability test: LB broth was used as the solvent and autoclaved at 121℃ for 25 min. 100 μL of OD was inoculated into 100 mL of LB broth. 600 Seed culture of G3 with a concentration of 0.6 was prepared and incubated in constant temperature shakers at 20℃, 28℃, and 37℃ in the dark. Then, 200 μL of the inoculated solution was transferred to each well of a 96-well plate and cultured, with the OD value monitored. 600 Changes in value.

[0063] Salinity adaptability test: Solutions with NaCl concentration gradients of 0%, 1%, 2%, 3%, and 4% were prepared using LB broth as solvent and autoclaved at 121℃ for 25 min. 100 μL LOD was then inoculated into 100 mL of LB broth at each salinity gradient. 600 =0.6% G3 seed culture. Then, take 200 μL of the inoculated solution and incubate in 96-well plates, monitoring the OD of the solution. 600 Changes in value.

[0064] pH adaptability test: Solutions with pH gradients of 5, 6, 7, 8, and 9 were prepared using LB culture medium as solvent and autoclaved at 121℃ for 25 min. 100 μL of OD was then inoculated into 100 mL of LB culture medium at each pH gradient. 600 =0.6% G3 seed culture. Then, take 200 μL of the inoculated solution and incubate in 96-well plates, monitoring the OD of the solution. 600 Changes in value.

[0065] As attached Figure 4 As shown, strain G3 can grow in LB medium at temperatures ranging from 20 to 37°C, with good growth observed at both 28°C and 37°C. This bacterium has relatively low salt tolerance, growing at salt concentrations from 0% to 4%, with the best growth observed at 1% NaCl. It can also grow at pH values ​​ranging from 5.0 to 9.0, reaching its highest growth levels at pH 6.0 to 8.0.

[0066] Example 5: Analysis of the mixed polycyclic aromatic hydrocarbon degradation activity of strain G3 under different environmental factors

[0067] Degradation efficiency determination under different temperature conditions: Phenanthrene and pyrene dissolved in acetone were added to a sterilized 250 mL Erlenmeyer flask. After the acetone evaporated, MSM culture medium (pH 7.0) was added to bring the initial concentration of phenanthrene and pyrene to 50 mg / L. 0D was inoculated at a 10% inoculum size. 600 =0.8% G3 bacterial culture. Erlenmeyer flasks were placed in shakers at 20℃, 28℃, and 37℃ at 180 rpm for incubation. On day 7, destructive sampling was performed to determine the residual amounts of phenanthrene and pyrene in the Erlenmeyer flasks.

[0068] Degradation efficiency determination under different salinity conditions: MSM medium was supplemented with NaCl at weights of 0%, 1%, 2%, 3%, and 4% (w / v), respectively, at pH 7.0. Phenanthrene and pyrene dissolved in acetone were added to sterilized 250 mL Erlenmeyer flasks. After the acetone evaporated, MSM culture medium at different salinities was added to achieve an initial concentration of 50 mg / L for phenanthrene and pyrene. OD was inoculated at a 10% inoculum. 600 =0.8% G3 bacterial culture. Erlenmeyer flasks were placed in a shaker at 37°C and 180 rpm for incubation. On day 7, destructive sampling was performed to determine the residual amounts of phenanthrene and pyrene in the Erlenmeyer flasks.

[0069] Degradation efficiency determination under different pH conditions: MSM culture media (1% NaCl salinity) with pH gradients of 5, 6, 7, 8, and 9 were prepared. Phenanthrene and pyrene dissolved in acetone were added to sterilized 250 mL Erlenmeyer flasks. After the acetone evaporated, MSM culture media at different pH values ​​were added to achieve an initial concentration of 50 mg / L for phenanthrene and pyrene. 0D was inoculated at a 10% inoculum size. 600 =0.8% G3 bacterial culture. Erlenmeyer flasks were placed in a shaker at 37°C and 180 rpm for incubation. On day 7, destructive sampling was performed to determine the residual amounts of phenanthrene and pyrene in the Erlenmeyer flasks.

[0070] The degradation effects of strain G3 under different temperature conditions are shown in the attached figure. Figure 5 As shown in Figure a, strain G3 achieved the following degradation rates on day 7 for an initial concentration of 50 mg / L of phenanthrene and pyrene: at 20°C, phenanthrene degradation reached 80.53% and pyrene degradation reached 40.00%; at 28°C, phenanthrene degradation reached 93.17% and pyrene degradation reached 57.00%; and at 37°C, phenanthrene degradation reached 95.29% and pyrene degradation reached 71.00%.

[0071] The degradation effects of strain G3 under different salinity conditions are shown in the attached figure. Figure 5As shown in b, for a mixture of phenanthrene and pyrene with an initial concentration of 50 mg / L, strain G3 achieved the following degradation rates on day 7: 94.64% for phenanthrene and 60.00% for pyrene with 0% NaCl added by weight; 94.98% for phenanthrene and 82.00% for pyrene with 1% NaCl added by weight; 90.49% for phenanthrene and 75.00% for pyrene with 2% NaCl added by weight; 71.56% for phenanthrene and 64.00% for pyrene with 3% NaCl added by weight; and 65.04% for phenanthrene and 43.00% for pyrene with 4% NaCl added by weight.

[0072] The degradation effects of strain G3 under different pH conditions are shown in the attached figure. Figure 5 As shown in c. For a mixture of phenanthrene and pyrene with an initial concentration of 50 mg / L, strain G3 achieved the following degradation rates on day 7: at pH 5, phenanthrene degradation reached 94.64% and pyrene degradation reached 60.00%; at pH 6, the degradation rates reached 96.11% and 68.00%; at pH 7, the degradation rates reached 95.29% and 71.00%; at pH 8, the degradation rates reached 93.71% and 70.00%; and at pH 9, the degradation rates reached 95.26% and 54.00%.

[0073] The results above show that strain G3 can effectively degrade mixed polycyclic aromatic hydrocarbons under different environmental conditions, with a degradation efficiency of >60% for phenanthrene and >40% for pyrene. This indicates that strain G3 has good application potential in bioremediation under different environmental conditions.

[0074] Example 6: Evaluation of the removal effect of strain G3 with different inoculum amounts on phenanthrene and pyrene in aged contaminated soil

[0075] Preparation of reconstituted soil: Weigh 50g of aged soil contaminated with polycyclic aromatic hydrocarbons from Beijing Coking Plant and place it in a sterile 250mL Erlenmeyer flask. Add 15mL of sterile water, stir well, and let it stand for one day for soil reconstitution.

[0076] Add 1×10 to the revived soil suspension. 5 1×10 6 5×10 6 1×10 7 5×10 7The G3 bacterial culture of this invention was inoculated at an inoculum amount of CFU / g soil and then placed in a constant temperature incubator and cultured at 30°C for 28 days.

[0077] Methods for Determination of Polycyclic Aromatic Hydrocarbons (PAHs) in Solid Phase: The methods for determining PAHs in solid soil are based on HJ 805-2016. The methods for determining PAHs in aqueous phase are based on HJ 478-2009.

[0078] Removal rate = {1 - [(PAHs content in the solid phase of soil suspension after 28 days + PAHs content in the aqueous phase) / (PAHs content in the solid phase of soil suspension on day 0 + PAHs content in the aqueous phase)]} × 100%.

[0079] The results are shown in Table 3 and Appendix. Figure 6 As shown. In polycyclic aromatic hydrocarbon (PAH) aged soil, the addition of strain G3 of this invention removed phenanthrene and pyrene. Under the experimental conditions, after a short-term treatment of PAH-aged soil for 28 days, 1×10⁻⁶ PbS was added. 5 In the CFU / g strain G3 treatment group, the removal rates of acenaphthene, fluorene, anthracene, phenanthrene, fluoranthene, and pyrene were 7.10%, 57.37%, 46.30%, 15.69%, 14.39%, and 4.64%, respectively; the addition of 1×10 6 In the CFU / g strain G3 treatment group, the removal rates of acenaphthene, fluorene, anthracene, phenanthrene, fluoranthene, and pyrene were 6.22%, 63.32%, 43.82%, 16.68%, 16.31%, and 9.86%, respectively; with the addition of 5×10 6 In the CFU / g strain G3 treatment group, the removal rates of acenaphthene, fluorene, anthracene, phenanthrene, fluoranthene, and pyrene were 11.02%, 57.75%, 52.16%, 20.71%, 21.07%, and 23.88%, respectively; the addition of 1×10 7 In the CFU / g strain G3 treatment group, the removal rates of acenaphthene, fluorene, anthracene, phenanthrene, fluoranthene, and pyrene were 6.66%, 39.47%, 60.23%, 23.89%, 23.26%, and 27.17%, respectively; with the addition of 5×10 7 In the treatment group of strain G3 with CFU / g, the removal rates of acenaphthene, fluorene, anthracene, phenanthrene, fluoranthene, and pyrene were 7.64%, 29.81%, 44.39%, 29.28%, 29.69%, and 23.58%, respectively; that is, the strain of the present invention can significantly remove mixed polycyclic aromatic hydrocarbons in contaminated soil.

[0080] The results above indicate that strain G3 is a strain capable of degrading persistent organic pollutants in polycyclic aromatic hydrocarbon (PAH)-aged soil, and has good application potential in in-situ bioremediation of PAH-contaminated sites.

[0081] Table 3. Polycyclic aromatic hydrocarbon content in aged soil after 28 days with different concentrations of G3 bacterial solution.

[0082]

[0083] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A highly efficient polycyclic aromatic hydrocarbon degrading bacterium G3 adapted to variable environments, characterized in that, This strain belongs to the genus Pseudoxanthomonas sp. and is named Pseudoxanthomonas sp.G3. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20251828.

2. The mixed polycyclic aromatic hydrocarbon high-efficiency degrading bacteria G3 according to claim 1, characterized in that, The 16S rDNA sequence is shown in SEQ ID NO:

1.

3. The mixed polycyclic aromatic hydrocarbon high-efficiency degrading bacteria G3 according to claim 1, characterized in that, After being cultured for 7 days at a temperature of 20-37℃, a salinity of 0-4% NaCl, and a pH of 5.0-9.0, the strain exhibits a degradation rate of no less than 60% for phenanthrene and no less than 40% for pyrene in mixed polycyclic aromatic hydrocarbons containing phenanthrene and pyrene.

4. The mixed polycyclic aromatic hydrocarbon high-efficiency degrading bacteria G3 according to claim 1, characterized in that, Under conditions of 37℃, 0-3% NaCl salinity, and pH 5.0-8.0, the degradation rate of pyrene in mixed polycyclic aromatic hydrocarbons was no less than 60% after 7 days of cultivation.

5. The mixed polycyclic aromatic hydrocarbon high-efficiency degrading bacteria G3 according to claim 1, characterized in that, Under conditions of 28-37℃, 0-2% NaCl salinity, and pH 5.0-9.0, the degradation rate of phenanthrene in mixed polycyclic aromatic hydrocarbons is no less than 90% after 7 days of cultivation.

6. Application of G3, a highly efficient polycyclic aromatic hydrocarbon (PAH) degrading bacterium suitable for variable environments, in the degradation of mixed PAH pollution.

7. The application according to claim 6, characterized in that, The mixed polycyclic aromatic hydrocarbons include one or more of acenaphthene, fluorene, anthracene, fluoranthene, phenanthrene, and pyrene.

8. The application according to claim 6, characterized in that, The aforementioned degradation of mixed polycyclic aromatic hydrocarbon pollution refers to the degradation of mixed polycyclic aromatic hydrocarbon pollution in water bodies or soil.

9. The application according to claim 6, characterized in that, The G3 high-efficiency polycyclic aromatic hydrocarbon degrading bacteria of claim 1 is applied to a polycyclic aromatic hydrocarbon contaminated environment.

10. The application according to claim 6, characterized in that, The effective viable count of the mixed polycyclic aromatic hydrocarbon high-efficiency degrading bacteria G3 is ≥1×10⁻⁶. 5 CFU / g soil can degrade mixed polycyclic aromatic hydrocarbons in the soil.

11. The application according to claim 6, characterized in that, The highly efficient polycyclic aromatic hydrocarbon (PAH) degrading bacteria G3 degrades PAH-contaminated soil that has been aged for more than 10 years.

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