Synthetic bacterial community for degrading polycyclic aromatic hydrocarbons and application thereof

Abstract

This invention relates to a synthetic microbial community for degrading polycyclic aromatic hydrocarbons (PAHs) and its applications, belonging to the field of environmental remediation microbial technology. First, a strain of *Bacillus polymyxa* C9-5 is provided, deposited on June 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39269. Then, a synthetic microbial community for degrading PAHs is provided, comprising *Bacillus polymyxa* C9-5 and *Acinetobacter* B2-4. Finally, a composite microbial agent for degrading PAHs is provided, comprising a suspension of *Bacillus polymyxa* C9-5 and a suspension of *Acinetobacter* B2-4. This invention is the first to combine *Bacillus polymyxa* C9-5 and *Acinetobacter* B2-4 together to synergistically degrade PAHs in soil or wastewater. The degradation effect on PAHs is significantly better than that of any single strain, showing excellent efficacy in degrading high-ring PAHs and possessing broad application prospects.

Description

Technical Field

[0001] This invention relates to a synthetic microbial community that degrades polycyclic aromatic hydrocarbons and its applications, belonging to the fields of synthetic biology and environmental remediation microbial technology. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants widely distributed in the environment. They not only have carcinogenic, teratogenic, and mutagenic effects but also neurotoxicity, posing a significant threat to the ecological environment and public health. They mainly originate from fossil fuel combustion and industrial production processes, such as coal coking, oil extraction and refining, incomplete combustion of motor vehicle fuels, and forest fires. Due to the relatively good stability and inertness of the multiple benzene rings in their structure, and their natural decay rate being much lower than the input rate, PAHs cause persistent harm to the environment. PAHs can persist widely in various environments, such as the atmosphere, soil, water bodies, and sediments. They can first enter the atmosphere, existing in a gaseous state or bound to particulate matter, then settle onto the ground and into water bodies, eventually migrating and accumulating in deep anaerobic environments.

[0003] Remediation technologies for polycyclic aromatic hydrocarbon (PAH) contaminated soil mainly include physical remediation, chemical remediation, phytoremediation, and microbial remediation. Currently, there are 16 types of PAHs that are prioritized for control both domestically and internationally. Among them, low-ring PAHs (such as phenanthrene and anthracene) are more reactive than medium- and high-ring PAHs, making them more volatile and degradable. Existing technologies mainly focus on the biodegradation of medium- and low-ring PAHs, while biodegradation technologies for high-ring pyrene are rare, and those that exist have long degradation times and low degradation rates.

[0004] In view of this, there is an urgent need in the field to find strains or synthetic microbial communities with better degradation effects as microbial agents to better treat polycyclic aromatic hydrocarbons. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a synthetic microbial community for degrading polycyclic aromatic hydrocarbons and its applications.

[0006] The technical solution of the present invention is as follows:

[0007] A strain of Paenibacillus polymyxa C9-5 was deposited on June 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39269.

[0008] According to a preferred embodiment of the present invention, the gene sequence of the 16S rDNA of Paenibacillus polymyxa C9-5 is shown in SEQ ID NO.1.

[0009] A synthetic microbial community that degrades polycyclic aromatic hydrocarbons includes Paenibacillus polymyxa C9-5 and Acinetobacter sp. B2-4.

[0010] According to a preferred embodiment of the present invention, the Acinetobacter sp. B2-4 was deposited on March 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33689.

[0011] A compound bacterial agent for degrading polycyclic aromatic hydrocarbons, comprising a suspension of Bacillus polymyxa C9-5 and a suspension of Acinetobacter B2-4.

[0012] According to a preferred embodiment of the present invention, the *Bacillus polymyxa* C9-5 bacterial suspension and the *Acinetobacter* B2-4 bacterial suspension are prepared by centrifuging the fermentation broth cultured to the stationary phase, collecting the bacterial cells, resuspending them in sterile physiological saline, and adjusting the OD value to [value missing]. 600 =1.0, which generally corresponds to a viable count of 10. 8 ~10 9 CFU / mL.

[0013] According to a preferred embodiment of the present invention, the volume ratio of the Bacillus polymyxa C9-5 suspension to the Acinetobacter B2-4 suspension is 1:(1~1.2).

[0014] The preparation method of the above-mentioned composite bacterial agent for degrading polycyclic aromatic hydrocarbons includes the following steps:

[0015] (1) Polymyxin Bacillus C9-5 and Acinetobacter B2-4 were inoculated into LB liquid medium and activated and cultured at 25-30℃ and 150-200rpm for 20-30h. After centrifugation, the bacterial cells were collected. The bacterial cells were then resuspended and inoculated into inorganic salt medium and cultured at 25-30℃ and 150-200rpm for 20-30h to obtain polymyxin Bacillus C9-5 bacterial suspension and Acinetobacter B2-4 bacterial suspension.

[0016] (2) Mix the suspension of Bacillus polymyxa C9-5 and Acinetobacter B2-4 according to the ratio to obtain a composite bacterial agent for degrading polycyclic aromatic hydrocarbons.

[0017] The application of the aforementioned polymyxa Bacillus C9-5 in the degradation of polycyclic aromatic hydrocarbons.

[0018] According to a preferred embodiment of the present invention, the polycyclic aromatic hydrocarbon is pyrene.

[0019] The application of the above-mentioned synthetic microbial groups or compound microbial agents in the degradation of polycyclic aromatic hydrocarbons.

[0020] According to a preferred embodiment of the present invention, the polycyclic aromatic hydrocarbon is pyrene.

[0021] The application of the aforementioned Bacillus polymyxa C9-5, synthetic microbial groups, or compound microbial agents in the bioremediation of polycyclic aromatic hydrocarbon (PAH) contaminated environments.

[0022] According to a preferred embodiment of the present invention, the polycyclic aromatic hydrocarbon is pyrene.

[0023] Where this invention is not detailed, existing technologies may be used.

[0024] Technical features and beneficial effects of the present invention:

[0025] 1. This invention obtained a polycyclic aromatic hydrocarbon (PAH) degrading strain C9-5 through screening and isolation. Based on the strain's morphology, physiological characteristics, 16S rDNA gene sequencing analysis, and phylogenetic analysis, the strain was identified as a polymyxa bacillus with the function of degrading PAHs. It can be used for PAH pollution remediation and provides strain resources for PAH pollution remediation in the environment.

[0026] 2. The *Paenibacillus polymyxa* C9-5 of this invention is a facultative anaerobic bacterium that can grow under both aerobic and anaerobic conditions. It can survive and undergo metabolic transformation in a culture medium with polycyclic aromatic hydrocarbons as the sole carbon source. After 15 days of cultivation, the degradation rate of pyrene reached 39.8%.

[0027] 3. This invention is the first to combine *Bacillus polymyxa* C9-5 and *Acinetobacter* B2-4 together to synergistically degrade polycyclic aromatic hydrocarbons (PAHs) in soil or wastewater. This synthetic bacterial consortium, with *Acinetobacter* B2-4 as the degrading bacterium and *Bacillus polymyxa* C9-5 as the solubilizing bacterium, exhibits significantly better PAH degradation effects than either single strain, demonstrating excellent efficacy in degrading high-ring PAHs and showing broad application prospects. Attached Figure Description

[0028] Figure 1 This is an electron microscope image of the cell morphology of Paenibacillus polymyxa C9-5.

[0029] Figure 2 This is a colony morphology diagram of Paenibacillus polymyxa C9-5 on solid culture medium.

[0030] Figure 3 This is a phylogenetic tree analysis of Paenibacillus polymyxa C9-5.

[0031] Figure 4 The degradation curve of pyrene at an initial concentration of 100 mg / L is shown in the figure.

[0032] Figure 5 The concentrations of nitrate and nitrite in the C9-5 single-strain system.

[0033] Figure 6 Electron micrographs of the compound bacterial agent after cultivation in the pyrene degradation system;

[0034] In the figure, A is an electron microscope image of B2-4 single bacteria after culturing in the pyrene degradation system, B is an electron microscope image of C9-5 single bacteria after culturing in the pyrene degradation system, C is an electron microscope image of the synthetic bacteria in the early stage (t1) of culturing in the pyrene degradation system, and D is an electron microscope image of the synthetic bacteria in the later stage (t2) of culturing in the pyrene degradation system.

[0035] Figure 7 The degradation curve of the compound bacterial agent on pyrene at an initial concentration of 100 mg / L is shown.

[0036] Figure 8 The concentrations of nitrate and nitrite in the compound microbial agent system. Detailed Implementation

[0037] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0038] Microbial source: Paenibacillus polymyxa C9-5, deposited on June 5, 2026 at the China General Microbiological Culture Collection Center, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number CGMCC No. 39269.

[0039] Acinetobacter sp. B2-4 was deposited on March 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33689. This strain is an existing strain and has been disclosed in Chinese patent document CN120060083A.

[0040] The inorganic salt culture medium has the following formula: NaNO3 0.85 g / L, K2HPO4 4 g / L, KH2PO4 6 g / L, MgSO4 0.2 g / L, CaCl2 0.02 g / L, FeCl3 0.05 g / L, NH4Cl 1 g / L, trace element solution 1 mL, and pH adjusted to 7.0.

[0041] The formula for the trace element solution is as follows: EDTA 15g / L, H3BO3 0.014g / L, MnCl2·4H2O 0.99g / L, CuSO4·5H2O 0.25g / L, ZnSO4·7H2O 0.43g / L, NiCl2·6H2O 0.19g / L, Na2MoO4·2H2O 0.22g / L, CoCl2·6H2O 0.24g / L, and NaSeO4·10H2O 0.21g / L.

[0042] All raw materials and equipment used in the examples are conventional and commercially available.

[0043] Example 1: Screening and isolation of strain C9-5

[0044] 1. Sample Source

[0045] Samples were collected from oil-bearing soils in Shengli Oilfield, Dongying City, Shandong Province, and subjected to long-term acclimation using high concentrations of phenanthrene or pyrene as carbon sources to obtain sludge acclimated with polycyclic aromatic hydrocarbons.

[0046] 2. Screening and isolation of strains

[0047] (1) Take 80 mL of polycyclic aromatic hydrocarbon-acclimated sludge sample and place it in a 250 mL serum bottle. Add PBS buffer and sonicate to clean it 3 times.

[0048] (2) Dilute the sludge sample after cleaning in step (1) with inorganic salt culture medium to OD. 600 The value is 0.5~1;

[0049] (3) Add a gradient concentration of polycyclic aromatic hydrocarbon acetone solution (0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L) to a sterile 3 mL anaerobic bottle. After the acetone evaporates, add it to the sterilized inorganic salt culture medium.

[0050] (4) Inoculate the suspension from step (2) into the inorganic salt culture medium prepared in step (3) with polycyclic aromatic hydrocarbons as the sole carbon source at an inoculation rate of 10% (v / v), and culture it under constant temperature and shaking. Take samples at fixed intervals to test the microbial growth status of the culture medium.

[0051] (5) The anaerobic culture medium with the best microbial growth was serially diluted with sterile physiological saline, and 10 μL of each solution was taken. -4 10 -5 10 -6 and 10 -7 100 μL of bacterial suspension was spread onto an inorganic salt culture medium with polycyclic aromatic hydrocarbons as the sole carbon source and incubated statically at 30°C under anaerobic conditions.

[0052] (6) Once colonies appear, use an inoculation loop to select a single colony and streak it on a plate. Repeat the streaking process twice. Use an inoculation loop to pick a single colony and transfer it to a serum bottle containing 50 ml of liquid culture medium. Incubate at 30°C and 130 rpm for 24 hours with shaking. Add a 1:1 ratio of 50% glycerol solution and bacterial culture to the cryovial, mix well, and store in a -80°C ultra-low temperature freezer. The strain is designated C9-5.

[0053] Example 2: Identification of strain C9-5

[0054] 1. The morphology of strain C9-5 was observed using field emission scanning electron microscopy. The colony characteristics are as follows: Figure 1 and Figure 2 As shown.

[0055] Depend on Figure 1 and Figure 2 It can be seen that the biological characteristics of this strain are: cells are short rod-shaped, single, and 1.4μm~1.7μm×0.4μm~0.5μm in size (see [link to strain name]). Figure 1 Furthermore, the colony surface is smooth, raised, and moist, with a diameter of 2-3 mm (see [reference]). Figure 2 ).

[0056] 2. DNA was extracted from strain C9-5 obtained in Example 1 using the T5 Direct PCR Kit (Plant) bacterial genome extraction kit. The specific extraction method was as described in the kit's instructions. The 16S rDNA gene of strain C9-5 was amplified by PCR using primers 27F and 1492R, and the amplified specific fragment was sequenced.

[0057] Universal primers were used for PCR:

[0058] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 2),

[0059] 1492R: 5'-GGTTACCTTGTTACGACTTC-3' (SEQ ID NO. 3).

[0060] Sequencing results showed that the 16S rDNA sequence of C9-5 was 1382 bp in length (see SEQ ID NO.1). Comparison of this result with NCBI data, and BLAST alignment, revealed that strain C9-5 is most closely related to *Paenibacillus polymyxa*. A phylogenetic tree was constructed using the MEGA7 neighbor-joining method, and the results are as follows. Figure 3 As shown.

[0061] Based on the 16S rDNA sequence alignment results and combined with the biological characteristics of the strain, the strain was identified as Paenibacillus polymyxa and named Paenibacillus polymyxa C9-5.

[0062] Example 3: Strain C9-5 degrades pyrene

[0063] 1. A method for preparing a suspension of Paenibacillus polymyxa C9-5 bacteria, comprising the following steps:

[0064] (1) Inoculate Paenibacillus polymyxa C9-5 into LB liquid medium, activate and culture at 30℃ and 150rpm for 24h, centrifuge and collect the bacterial cells;

[0065] (2) After resuspending Paenibacillus polymyxa C9-5, the cells were inoculated into an inorganic salt medium and cultured at 30℃ and 150 rpm for 24 h. The cells were collected by centrifugation and resuspended in sterile physiological saline to obtain OD. 600 =1.0 Paenibacillus polymyxa C9-5 bacterial suspension.

[0066] OD was prepared using the same method. 600 =1.0 Acinetobacter B2-4 bacterial suspension.

[0067] 2. Preparation of degradation culture medium

[0068] Preparation method of pyrene degradation medium: Add 50 mL of inorganic salt medium to a 100 mL serum bottle, then add pyrene acetone solution to make the concentration of pyrene in the inorganic salt medium 100 mg / L. Place it in an aseptic chamber for 24 hours to allow the acetone to evaporate, thus obtaining the pyrene degradation medium.

[0069] 3. Degradation rate detection of polycyclic aromatic hydrocarbons

[0070] At an inoculum rate of 5% (v / v), a suspension of *Paenibacillus polymyxa* C9-5 was inoculated into pyrene degradation medium. The medium was sealed with a butyl rubber stopper, purged with sterile high-purity nitrogen for 30 min, and then transferred to a constant-temperature shaking incubator. Using nitrate (10 mM) as the electron acceptor, the culture was incubated at 30°C and 130 rpm in the dark for 15 days to obtain the C9-5 culture medium. Three parallel groups and sterile culture media were also included as blank controls.

[0071] Then, samples were taken at set time points, and the culture vials were ultrasonically extracted with an equal volume of cyclohexane. The pyrene content was determined by high-performance liquid chromatography (HPLC). The degradation rate, degradation rate constant, half-life, and nitrate and nitrite concentrations of pyrene were calculated. The results of the pyrene degradation rate are shown below. Figure 4 As shown, the concentrations of nitrate and nitrite are as follows: Figure 5 As shown.

[0072] Depend on Figure 4 It was found that strain C9-5 exhibited a degradation effect on pyrene under anaerobic conditions. After 15 days of culture, the degradation rate of pyrene by *Paenibacillus polymyxa* C9-5 was 39.8%, with a degradation rate constant of 0.018 d. −1 It has a half-life of 38.5 days.

[0073] Depend on Figure 5 It can be seen that after 72 h of culture of the C9-5 single-strain system, the nitrate (NO3) in the culture medium... - The concentration was 0.1 mmol / L, and the nitrite (NO2) concentration was... - The concentration was 0.5 mmol / L.

[0074] Simultaneously, B2-4 culture medium was prepared using the same method, and the degradation rate of pyrene by Acinetobacter B2-4 was determined to be 58.2%, with a degradation rate constant of 0.121 d. -1 It has a half-life of 15.73 days.

[0075] Example 4: Degradation of pyrene by a compound bacterial agent of strains C9-5 and B2-4

[0076] 1. A method for preparing a composite microbial agent for degrading polycyclic aromatic hydrocarbons, comprising the following steps:

[0077] The suspensions of Bacillus polymyxa C9-5 and Acinetobacter B2-4 were mixed evenly at a volume ratio of 1:1 to obtain a composite bacterial agent for degrading polycyclic aromatic hydrocarbons.

[0078] 2. Degradation rate detection of polycyclic aromatic hydrocarbons

[0079] The compound bacterial agent was inoculated into the pyrene degradation medium at a 5% (v / v) inoculation rate. After sealing with a butyl rubber stopper, the medium was purged with sterile high-purity nitrogen for 30 min and then transferred to a constant-temperature shaking incubator. Using nitrate (10 mM) as the electron acceptor, the medium was incubated at 30℃ and 130 rpm in the dark for 15 days to obtain the synthetic bacterial culture. Three parallel and sterile culture media were used as blank controls. Scanning electron microscopy was then used to observe the bacterial interactions, morphology, and distribution characteristics at different time points (early stage t1 and mid-to-late stage t2) in the pyrene degradation system. Single-cell culture characteristics were used as controls. The results are as follows: Figure 6 As shown.

[0080] Then, samples were taken at set time points, and the culture vials were ultrasonically extracted with an equal volume of cyclohexane. The pyrene content was determined by high-performance liquid chromatography (HPLC). The degradation rate, degradation rate constant, half-life, and nitrate and nitrite concentrations of pyrene were calculated. The results of the pyrene degradation rate are shown below. Figure 7 As shown, the concentrations of nitrate and nitrite are as follows: Figure 8 As shown.

[0081] Depend on Figure 6 It was observed that the bacterial cells in the pyrene degradation system exhibited a distinct aggregated state, with numerous bacterial cells tightly embedded within abundant spongy extracellular matrix-like material, forming a relatively dense cell-matrix complex structure, making it difficult to clearly distinguish the outline and boundaries of individual cells. These morphological characteristics suggest that *Bacillus polymyxa* C9-5 and *Acinetobacter* B2-4 can form stable cell aggregates under co-culture conditions, and the extracellular matrix-like material may help maintain the spatial proximity between bacterial cells and the stability of the synthetic bacterial community structure.

[0082] Depend on Figure 7 It can be seen that the compound bacterial agent has a significant degradation effect on pyrene under anaerobic conditions. After 15 days of cultivation, the degradation rate of pyrene by the compound bacterial agent was 80.3%, and the degradation rate constant was 0.158 d. −1 It has a half-life of 12.39 days.

[0083] Depend on Figure 8 It can be seen that after culturing for 72 hours in the compound bacterial agent system, the nitrate (NO3) in the culture medium... - The concentration was 0.1 mmol / L, and the nitrite (NO2) concentration was... - The concentration was 0.1 mmol / L.

[0084] Comparative analysis of the data revealed that the composite microbial agent increased the removal rate of pyrene to 80.3%, significantly higher than the 58.2% of the B2-4 single-strain system and the 39.8% of the C9-5 single-strain system. Furthermore, the degradation rate constant increased and the half-life shortened, indicating that the synergistic construction strategy of "degrading bacteria-solubilizing bacteria" effectively overcomes the bottlenecks of low bioavailability of high-cyclic PAHs and limited metabolic capacity of single bacteria, achieving a significant improvement in pyrene removal efficiency. Simultaneously, the peak nitrite accumulation in the composite microbial agent system decreased, suggesting that the synthetic microbial community, while enhancing pollutant degradation, also optimized the electron acceptor utilization efficiency of the nitrate reduction process.

[0085] Example 5: Determination of the solubilizing properties of Bacillus polymyxa C9-5 and its synthetic flora.

[0086] The C9-5 culture medium and the synthetic microbial culture medium obtained after 15 days of light-protected culture for the polycyclic aromatic hydrocarbon degradation rate detection experiment in Example 3 and Example 4 (point 3) were centrifuged at 4℃ and 9000 rpm for 10 min, respectively. The supernatant was collected and filtered through a 0.22 μm filter membrane to remove residual bacteria, yielding the cell-free fermentation supernatant of C9-5 and the cell-free fermentation supernatant of the synthetic microbial community. The cell-free fermentation supernatant of B2-4 was prepared using the same method, and a sterile inorganic salt medium that was not inoculated but underwent the same culture, centrifugation, and filtration treatment was used as a blank control. Each treatment was performed in triplicate.

[0087] Then, the surface tension of the cell-free fermentation supernatant of C9-5, the cell-free fermentation supernatant of the synthetic microbial community, the cell-free fermentation supernatant of B2-4, and the blank control was determined using a surface tension meter via the ring method. Excess pyrene was then added to each of the cell-free fermentation supernatant of C9-5, the cell-free fermentation supernatant of the synthetic microbial community, and the cell-free fermentation supernatant of B2-4. After equilibration, the supernatant was extracted with an equal volume of cyclohexane, and the pyrene content was determined by high-performance liquid chromatography (HPLC). The apparent solubility of pyrene was calculated, and the results are shown in Table 1.

[0088]

[0089] Table 1 shows that the surface tension of the blank culture medium was 72.32 mN / m, and the apparent solubility of pyrene was 0.14 mg / L. The surface tension of the B2-4 cell-free fermentation supernatant was 59.56 mN / m, and the apparent solubility of pyrene was 6.03 mg / L. The surface tension of the C9-5 cell-free fermentation supernatant was 47.92 mN / m, and the apparent solubility of pyrene was 27.45 mg / L. The surface tension of the synthetic microbial cell-free fermentation supernatant was 32.60 mN / m, and the apparent solubility of pyrene was 16.08 mg / L. The comparison shows that the surface tension of the C9-5 cell-free fermentation supernatant was 34% lower than that of the blank culture medium, and the apparent solubility of pyrene was 196 times that of the blank culture medium. Furthermore, compared with the blank culture medium and the B2-4 cell-free fermentation supernatant, the C9-5 cell-free fermentation supernatant exhibited stronger surface tension reduction and pyrene solubilization capabilities.

[0090] This indicates that *Bacillus polymyxa* C9-5 can produce surface-active extracellular metabolites during cultivation, improving the dispersion and apparent solubility of pyrene in the aqueous phase, thus enhancing its bioavailability. Therefore, *Bacillus polymyxa* C9-5 can be used as a solubilizing bacterium in combination with pyrene-degrading bacteria B2-4 to construct a synthetic microbial community. Furthermore, the strong surface activity and pyrene solubilizing ability of *Bacillus polymyxa* C9-5 are fully preserved in the synthetic microbial community culture system, demonstrating that *Bacillus polymyxa* C9-5 is beneficial in increasing the contact opportunities and bioavailability of hydrophobic pyrene with the bacterial cells, thereby supporting the synthetic microbial community in improving pyrene removal efficiency.

Claims

1. A synthetic microbial community that degrades polycyclic aromatic hydrocarbons, characterized in that, This includes Polymyxin Bacillus C9-5 and Acinetobacter B2-4.

2. The synthetic microbial community for degrading polycyclic aromatic hydrocarbons as described in claim 1, characterized in that, The polymyxa Bacillus C9-5 was deposited on June 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39269.

3. The synthetic microbial community for degrading polycyclic aromatic hydrocarbons as described in claim 1, characterized in that, The Acinetobacter B2-4 was deposited on March 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33689.

4. A composite microbial agent for degrading polycyclic aromatic hydrocarbons, characterized in that, This includes the aforementioned Bacillus polymyxa C9-5 bacterial suspension and Acinetobacter B2-4 bacterial suspension.

5. The composite microbial agent for degrading polycyclic aromatic hydrocarbons as described in claim 4, characterized in that, Both the *Bacillus polymyxa* C9-5 bacterial suspension and the *Acinetobacter* B2-4 bacterial suspension were prepared by centrifuging the fermentation broth to the stationary phase, collecting the bacterial cells, resuspending them in sterile physiological saline, and adjusting the OD value to [value missing]. 600 =1.0, which generally corresponds to a viable count of 10. 8 ~10 9 CFU / mL.

6. The composite microbial agent for degrading polycyclic aromatic hydrocarbons as described in claim 4, characterized in that, The volume ratio of the *Bacillus polymyxa* C9-5 suspension to the *Acinetobacter baumannii* B2-4 suspension is 1:(1~1.2).

7. The method for preparing the composite microbial agent for degrading polycyclic aromatic hydrocarbons according to claim 4, characterized in that, The steps include the following: (1) Polymyxin Bacillus C9-5 and Acinetobacter B2-4 were inoculated into LB liquid medium and activated and cultured at 25-30℃ and 150-200rpm for 20-30h. After centrifugation, the bacterial cells were collected. The bacterial cells were then resuspended and inoculated into inorganic salt medium and cultured at 25-30℃ and 150-200rpm for 20-30h to obtain polymyxin Bacillus C9-5 bacterial suspension and Acinetobacter B2-4 bacterial suspension. (2) Mix the suspension of Bacillus polymyxa C9-5 and Acinetobacter B2-4 according to the ratio to obtain a composite bacterial agent for degrading polycyclic aromatic hydrocarbons.

8. The application of the polymyxa C9-5 according to claim 2 in the degradation of polycyclic aromatic hydrocarbons.

9. The application of the synthetic microbial community of claim 1 or the composite microbial agent of claim 4 in the degradation of polycyclic aromatic hydrocarbons.

10. The application of the synthetic microbial community of claim 1, the Bacillus polymyxa C9-5 of claim 2, or the composite microbial agent of claim 4 in the bioremediation of polycyclic aromatic hydrocarbon contaminated environments.

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