Acinetobacter BJ03 and application thereof in degradation of polycyclic aromatic hydrocarbon

Abstract

The invention provides acinetobacter BJ03 and application thereof in degradation of polycyclic aromatic hydrocarbon, and belongs to the technical field of microorganisms. The acinetobacter sp. BJ03 is preserved in the China General Microbiological Culture Collection Center on May 26, 2025, and the preservation number of the acinetobacter sp. BJ03 is CGMCC No.34668. The microbial agent prepared by adopting the acinetobacter sp. BJ03 can be used for degrading polycyclic aromatic hydrocarbons.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to Acinetobacter BJ03 and its application in degrading polycyclic aromatic hydrocarbons. BACKGROUND

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a kind of refractory organic pollutants containing multiple benzene rings, which have genetic toxicity, mutagenicity and carcinogenicity. Farmland soil is one of the main enrichment places of PAHs in the environment, and the enrichment of PAHs in farmland soil can be adsorbed by organic matter, making it difficult to eliminate, and thus causing farmland soil to be polluted by PAHs, threatening the quality of agricultural products and human health in farmland soil.

[0003] Therefore, how to efficiently degrade PAHs in farmland soil is a problem to be solved. SUMMARY

[0004] The present application provides Acinetobacter BJ03 and its application in degrading polycyclic aromatic hydrocarbons, which can realize effective degradation of polycyclic aromatic hydrocarbons by a microbial inoculum constructed by Acinetobacter BJ03.

[0005] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0006] In a first aspect, the present application provides Acinetobacter BJ03, which was deposited with the China General Microbiological Culture Collection Center on May 26, 2025, and the deposit number is CGMCC No. 34668.

[0007] In a second aspect, the present application provides the application of Acinetobacter BJ03 provided in the first aspect in degrading polycyclic aromatic hydrocarbons.

[0008] In a third aspect, the present application provides a microbial inoculum for degrading polycyclic aromatic hydrocarbons, which comprises Acinetobacter BJ03 provided in the first aspect.

[0009] In an implementation manner of the third aspect, the microbial inoculum is Acinetobacter BJ03 loaded on a biochar carrier, and the biochar carrier is obtained by pyrolysis of plant material.

[0010] In a fourth aspect, the present application provides a carbon fixation and pollution removal microbial population, which comprises Acinetobacter BJ03 provided in the first aspect.

[0011] In an implementation form of the fourth aspect, the carbon fixation and pollution removal bacterial population comprises polycyclic aromatic hydrocarbon degrading bacteria and carbon fixation bacteria; the polycyclic aromatic hydrocarbon degrading bacteria comprises Pseudomonas putida CICC23685, Acinetobacter BJ03, Kocuria BJ05 and Paenibacillus PHE-2; and the carbon fixation bacteria is Nitrosogloea viennensis EN76.

[0012] In a fifth aspect, the present application provides application of the carbon fixation and pollution removal bacterial population provided in the fourth aspect to carbon fixation and pollution removal of organic contaminated soil.

[0013] In an implementation form of the fifth aspect, the carbon fixation and pollution removal bacterial population is loaded on a biochar carrier, and the biochar carrier is obtained by pyrolysis of plant material.

[0014] Compared with the prior art, the present application has the following beneficial effects.

[0015] (1) The present application provides a new Acinetobacter BJ03, and the bacterial agent prepared by using the Acinetobacter BJ03 has the advantages of simple culture, low compounding difficulty and easy operation. The bacterial agent can not only repair polycyclic aromatic hydrocarbon contaminated farmland soil and reduce the threat to the safety of agricultural products, but also fix CO2 and increase the SOC content in farmland soil.

[0016] (2) The use of the immobilized bacterial agent prepared by using the Acinetobacter BJ03 to repair PAHs contaminated farmland soil can provide technical support and theoretical support for carbon fixation and pollution removal of farmland soil. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a diagram showing the percentage of C atoms fixed by the carbon fixation and pollution removal immobilized bacterial agent prepared by using different immobilization times in the experimental example of the present application; 3 C / 12 C atom percentage diagram;

[0018] Figure 2 is a diagram showing the amount of assimilated CO2 in SOC by the carbon fixation and pollution removal immobilized bacterial agent prepared by using different immobilization times in the experimental example of the present application;

[0019] Figure 3 is a diagram showing the degradation rates of LMW PAHs, MMW PAHs, HMW PAHs and Total PAHs by the carbon fixation and pollution removal immobilized bacterial agent prepared by using different immobilization times in the experimental example of the present application;

[0020] Figure 4 is a diagram showing the percentage of C atoms fixed by the carbon fixation and pollution removal immobilized bacterial agent prepared by using different bacterial material ratios in the experimental example of the present application; 3 C / 12 C atom percentage diagram;

[0021] Figure 5 is a schematic diagram of the amount of assimilated CO2 in SOC of the carbon fixation and pollution removal immobilized bacterial agent prepared by using different ratios of bacteria and material in the experimental examples of the present application;

[0022] Figure 6 is a schematic diagram of the degradation rates of LMW PAHs, MMW PAHs, HMW PAHs and Total PAHs by the carbon fixation and pollution removal immobilized bacterial agent prepared by using different ratios of bacteria and material in the experimental examples of the present application. DETAILED DESCRIPTION

[0023] Example 1: The present example describes an Acinetobacter sp. BJ03, which was deposited with the China General Microbiological Culture Collection Center on May 26, 2025, and has a preservation number of CGMCC No. 34668 and a preservation address of No. 3, Yuanmingyuan West Road, Beijing, China Academy of Microbiology Institute.

[0024] It should be noted that the above-mentioned Acinetobacter sp. BJ03 is a new strain. The following gives the screening process of the above-mentioned Acinetobacter sp. BJ03.

[0025] Step 1, by enrichment and domestication, a high-efficiency PAEs degrading strain BJ03 was separated and purified from PAEs contaminated soil;

[0026] Step 2, the 16S rDNA gene sequence of the PAEs degrading strain was analyzed for homology comparison in the NCBI database, and the PAEs degrading strain BJ03 was identified as Acinetobacter sp.;

[0027] Step 3, the Acinetobacter sp. BJ03 was inoculated into LB liquid medium for activation, and the morphological characteristics of the bacterial colony were observed. The obtained Acinetobacter sp. BJ03 single colony was smooth, raised, grayish white, opaque and viscous, and the bacterial body was spherical and rod-shaped without flagella.

[0028] Example 2: The present example describes the application of the above-mentioned Acinetobacter sp. BJ03 in degrading polycyclic aromatic hydrocarbons.

[0029] Specifically, the above-mentioned Acinetobacter sp. BJ03 is used to prepare a bacterial agent for degrading polycyclic aromatic hydrocarbons, and the bacterial agent is used to degrade polycyclic aromatic hydrocarbons. The bacterial agent is obtained by loading the above-mentioned Acinetobacter sp. BJ03 on a biochar carrier, and the biochar carrier is obtained by pyrolysis of plant materials. The above-mentioned polycyclic aromatic hydrocarbons include naphthalene (NAP), acenaphthene (ANA), dihydroacenaphthene (ANY), fluorene (FLU), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLT), pyrene (PYR), benzo(a)anthracene (BaA), (CHR), benzo(a)pyrene (BaP).

[0030] Taking the degradation of polycyclic aromatic hydrocarbons in the organic contaminated soil by the above-mentioned polycyclic aromatic hydrocarbon-degrading bacterial agent as an example, the above-mentioned application comprises: putting the polycyclic aromatic hydrocarbon-degrading bacterial agent into the organic contaminated soil to degrade the polycyclic aromatic hydrocarbons in the organic contaminated soil.

[0031] It can be understood that the input amount of the carbon fixation and pollution removal immobilized bacterial agent can be 3% of the mass of the organic contaminated soil, or 4% or 5% of the mass of the organic contaminated soil, and the input amount of the carbon fixation and pollution removal immobilized bacterial agent is not limited in the embodiments of the present application.

[0032] In an implementation manner, the preparation process of the polycyclic aromatic hydrocarbon-degrading bacterial agent comprises S101-S103.

[0033] S101, preparing a liquid bacterial agent for degrading polycyclic aromatic hydrocarbons;

[0034] Optionally, the S101 comprises S1011-S1012.

[0035] S1011, activating and culturing Acinetobacter BJ03 to obtain activated Acinetobacter BJ03;

[0036] In the embodiments, the LB medium is used to activate and culture the Acinetobacter BJ03.

[0037] The process of the activation and culture will be described in detail below.

[0038] The seed liquid of the Acinetobacter BJ03 is added into the LB medium at a volume transfer amount of 5%, and is cultured to the logarithmic growth phase (temperature 30°C, oscillation culture at a speed of 150 rpm for 12 h). After centrifugal treatment, the supernatant is removed, and the Acinetobacter BJ03 precipitate is obtained. The inorganic salt medium is added into the Acinetobacter BJ03 precipitate for resuspension treatment. The steps of the centrifugal treatment and the resuspension treatment are repeated for 2-3 times. After the last resuspension, the activated Acinetobacter BJ03 is obtained.

[0039] The formula of the LB medium (1.0 L) comprises 5.0 g of yeast extract, 10.0 g of peptone and 10.0 g of NaCl. Since the LB medium is a commonly used medium in the technical field, the embodiments of the present application will not be described here.

[0040] The formula of the inorganic salt medium is shown in Table 1 below.

[0041] Table 1 Formula of the inorganic salt medium

[0042]

[0043]

[0044] The mod Trace Elements formulation in Table 1 above is shown in Table 2 below;

[0045] Table 2 mod Trace Elements formulation table

[0046] Ingredient Concentration H3BO3 0.5 mM MnCl2.4H2O 0.5 mM CoCl2-6H2O 0.8 mM [NiCl2·6H2O] 0.1 mM [CuCl2·2H2O] 0.01 mM ZnSO4·7H2O 0.5 mM Na2MoO4·2H2O 0.15 mM HCL (37%) 100 mM

[0047] S1012, the activated Acinetobacter BJ03 is added into the inorganic salt culture medium, and the OD600 of the inorganic salt culture medium is adjusted to 0.7 to obtain a liquid inoculum for degrading polycyclic aromatic hydrocarbons;

[0048] S102, a biochar carrier is prepared;

[0049] In this embodiment, the biochar carrier is obtained by pyrolyzing the wheat straw at 800°C. Since pyrolysis is a commonly used technical means in the technical field, the specific process of pyrolysis is not described in detail in this embodiment.

[0050] Alternatively, the wheat straw can be replaced by corn straw or rice straw, and the specific type of the pyrolysis material is not limited in this embodiment.

[0051] S103, the liquid inoculum for degrading polycyclic aromatic hydrocarbons is solidified with the biochar carrier to obtain an inoculum for degrading polycyclic aromatic hydrocarbons;

[0052] In this embodiment, the liquid inoculum for degrading polycyclic aromatic hydrocarbons is added to the biochar carrier at a ratio of 20 mL:1.5 g of the inoculum, and is solidified in a shaking incubator at 30°C and a rotation speed of 150 r / min for 0.5-4 days, and then is centrifuged using a disc centrifuge to obtain a carbon-fixing and pollution-removing immobilized inoculum.

[0053] Alternatively, the solidification temperature can be 25°C, the rotation speed can be 140 r / min, and the immobilization time for solidification can be 0.5 days or 3 days, and the above parameters are not limited in this embodiment. Since disc centrifugation is a commonly used technical means in the technical field, the rotation speed and time during disc centrifugation are not limited in this embodiment.

[0054] Embodiment 3: This embodiment describes a carbon-fixing and pollution-removing microbial population, which comprises the Acinetobacter BJ03 in embodiment 1. Specifically, the carbon-fixing and pollution-removing microbial population comprises polycyclic aromatic hydrocarbon-degrading bacteria and carbon-fixing bacteria; the polycyclic aromatic hydrocarbon-degrading bacteria comprise Pseudomonas putida CICC23685, the Acinetobacter BJ03, Kocuria BJ05, and Paenibacillus PHE-2; and the carbon-fixing bacteria are Nitrosospira viennensis EN76.

[0055] It should be noted that the other bacteria in the carbon fixation and odor removal bacterial population described above are existing bacteria, the accession number of the Pseudomonas putida. CICC23685 described above is CICC No. 23685; the accession number of the Kocuria sp. BJ05 described above is CGMCC No. 33674; the accession number of the Paenibacillus sp. PHE-2 described above is CGMCC No. 1.12900; and the accession number of the Nitrososphaera viennensis. EN76 described above is DSM 26422, wherein DSM refers to the German Microbial and Cell Culture Collection.

[0056] Example 4: This embodiment describes the application of the carbon fixation and odor removal bacterial population in the carbon fixation and odor removal of the organic contaminated soil in the above-mentioned example 3.

[0057] In the specific process of the application, the carbon fixation and odor removal bacterial population is first loaded on a biochar carrier to obtain a carbon fixation and odor removal immobilized bacterial agent, and then the carbon fixation and odor removal of the organic contaminated soil is realized by the carbon fixation and odor removal immobilized bacterial agent. The biochar carrier is obtained by pyrolysis of plant material. The plant material can be wheat straw, corn straw or rice straw.

[0058] In one implementation, the application includes introducing the carbon fixation and odor removal immobilized bacterial agent into the organic contaminated soil to degrade polycyclic aromatic hydrocarbons in the organic contaminated soil and to fix carbon in the organic contaminated soil. The polycyclic aromatic hydrocarbons include naphthalene (NAP), acenaphthene (ANA), acenaphthylene (ANY), fluorene (FLU), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLT), pyrene (PYR), benzo(a)anthracene (BaA), chrysene (CHR), and benzo(a)pyrene (BaP). (CHR), benzo(a)pyrene (BaP).

[0059] It can be understood that the amount of the carbon fixation and odor removal immobilized bacterial agent introduced can be 3% of the mass of the organic contaminated soil, or 4% or 5% of the mass of the organic contaminated soil. The amount of the carbon fixation and odor removal immobilized bacterial agent introduced is not limited in the embodiments of the present application.

[0060] The preparation method of the carbon fixation and odor removal immobilized bacterial agent for the organic contaminated soil is given below, including S201-S203;

[0061] S201, preparing a liquid bacterial agent of the carbon fixation and odor removal bacterial population;

[0062] Optionally, S201 includes S2011-S2013;

[0063] S2011, respectively, each of the polycyclic aromatic hydrocarbon degrading bacteria is activated and cultured, and activated Pseudomonas putida CICC23685, activated Acinetobacter BJ03, activated Kocuria BJ05 and activated Paenibacillus PHE-2 are obtained;

[0064] In this embodiment, LB medium is used for activating and culturing the above-mentioned Pseudomonas putida CICC23685, Acinetobacter BJ03, Kocuria BJ05 and Paenibacillus PHE-2, respectively;

[0065] The process of the above-mentioned activation and culture is described below by taking Pseudomonas putida CICC23685 as an example;

[0066] The seed liquid of Pseudomonas putida CICC23685 is added into the LB medium at a volume transfer amount of 5%, and is cultured to the logarithmic growth phase (temperature 30°C, oscillation culture at a speed of 150 rpm for 12 h). After centrifugal treatment, the supernatant is removed, and Pseudomonas putida CICC23685 precipitate is obtained. The inorganic salt medium is added into the Pseudomonas putida CICC23685 precipitate for resuspension treatment. The above-mentioned centrifugal treatment and resuspension treatment are repeated for 2-3 times. After the last resuspension, activated Pseudomonas putida CICC23685 is obtained.

[0067] The seed liquid of Pseudomonas putida CICC23685 is purchased from the above-mentioned preservation center of Pseudomonas putida CICC23685;

[0068] It should be noted that the activation and culture processes of the above-mentioned Acinetobacter BJ03, Kocuria BJ05 and Paenibacillus PHE-2 are the same as the activation and culture process of the above-mentioned Pseudomonas putida CICC23685. Therefore, the activation and culture processes of the above-mentioned Acinetobacter BJ03, Kocuria BJ05 and Paenibacillus PHE-2 are not described herein;

[0069] S2012, the carbon fixation bacteria are activated and cultured, and activated Nitrosospira vienensis EN76 is obtained;

[0070] In this embodiment, the Nitrosospira vienensis EN76 is continuously cultured in a constant-temperature incubator in the dark, and activated Nitrosospira vienensis EN76 is obtained. It should be understood that the activation and culture process of the above-mentioned Nitrosospira vienensis EN76 is the same as the activation and culture process of other strains in the above step, and the embodiment of the present application does not repeat the description herein;

[0071] S2013、mixing the activated Pseudomonas putida CICC23685, the activated Acinetobacter BJ03, the activated Kocuria BJ05, the activated Paenibacillus PHE-2 and the activated Nitrosomonas winogradskyi EN76, and adding the inorganic salt culture medium, and adjusting the OD600 of the inorganic salt culture medium to 0.7 to obtain the liquid inoculum of the carbon fixation and pollution removal bacterial community;

[0072] In this embodiment, the activated Pseudomonas putida CICC23685, the activated Acinetobacter BJ03, the activated Kocuria BJ05, the activated Paenibacillus PHE-2 and the activated Nitrosomonas winogradskyi EN76 are mixed in a ratio of 1:1:1:1:1 by cell number;

[0073] Alternatively, the above ratio can also be 1:1.5:1.5:1.5:1.5 or 1.5:1.5:1.5:1.5:1;

[0074] S202、preparing a biochar carrier;

[0075] In this embodiment, the biochar carrier is obtained after pyrolysis of wheat straw at 800°C. Since pyrolysis is a commonly used technical means in the technical field, the specific process of pyrolysis is not described in this embodiment;

[0076] Alternatively, the above wheat straw can be replaced by corn straw or rice straw;

[0077] S203、solidifying the liquid inoculum of the carbon fixation and pollution removal bacterial community with the biochar carrier to obtain a carbon fixation and pollution removal immobilized inoculum;

[0078] In this embodiment, the liquid inoculum of the carbon fixation and pollution removal bacterial community is added to the biochar carrier in a ratio of 20mL:1.5g, and is solidified in a shaking incubator at 30°C and a rotation speed of 150r / min for 0.5-4d, and then centrifuged using a disc centrifuge to obtain the carbon fixation and pollution removal immobilized inoculum;

[0079] Alternatively, the above ratio can also be 25mL:1.5g or 30mL:1.5g;

[0080] The above solidification temperature can also be 25°C, the rotation speed can also be 140r / min, and the immobilization time of the solidification treatment can also be 0.5d or 3d, and the above parameters are not limited in this embodiment. Since disc centrifugation is a commonly used technical means in the technical field, the rotation speed and time during disc centrifugation are not limited in this embodiment.

[0081] Experimental Example: This experimental example is used to optimize the immobilization time of the carbon sequestration and pollution removal immobilized bacterial agent in the preparation method of the carbon sequestration and pollution removal immobilized bacterial agent in the above embodiment 4 and the bacteria material ratio of the carbon sequestration and pollution removal bacterial population liquid bacterial agent and the biochar carrier.

[0082] The optimization process of the above two parameters is given below.

[0083] Collect the soil sample of a certain farmland soil, spread the soil sample to remove weeds, soil animals, stones and other impurities, dry in a cool place for more than 2 weeks, grind through a 20 mesh sieve, and obtain the experimental soil.

[0084] Select 11 polycyclic aromatic hydrocarbons, namely naphthalene, acenaphthene, dihydroacenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a) anthracene, and benzo(a) pyrene. Then, under the action of ultrasonic assistance, 250 mL of acetone (HPLC) is used to dissolve the above 11 polycyclic aromatic hydrocarbons to obtain a polycyclic aromatic hydrocarbon solution with a concentration of 3 ppm for each of the above 11 polycyclic aromatic hydrocarbons.

[0085] Set up 10 experimental groups, and the experimental device of each experimental group is a 100 mL serum bottle. The experimental device contains 5 g of experimental soil. Add sterile water to the 5 g of experimental soil in the experimental device to reach a water content of 60% in the experimental soil. Then add 100 μL of the above polycyclic aromatic hydrocarbon solution to the experimental soil. Each experimental treatment is set according to the following Table 3, and each treatment is set in triplicate.

[0086] Table 3 Experimental treatment parameter table of experimental groups

[0087]

[0088] After setting up the above 10 experimental groups, the experimental device in each experimental group is 13 The initial CO2 content is 5%, and after 90% of the CO2 is consumed, first flush the top of the experimental device with pressurized synthetic air (20% O2, 80% N2) for 1 min to maintain aerobic conditions, then add 13 CO2. Then place the experimental device in a 30°C incubator, and update the gas in the experimental device every 5 days. On the 20th day, collect the experimental soil from the above 10 experimental groups for freeze-drying. The freeze-dried experimental soil (hereinafter referred to as freeze-dried sample) is stored in a -20°C environment for subsequent analysis.

[0089] (1) Optimization of immobilization time of carbon sequestration and pollution removal immobilized bacterial agent.

[0090] Comparative experimental group 3-experimental group 7, the freeze-dried sample is measured by isotope ratio mass spectrometer and elemental analyzer to determine the δ 13 C value and SOC of labeled and unlabeled soil, which is used for subsequent 13 C / 12Analysis of the percentage of C atoms, the amount of CO2 assimilated in SOC, and carbon fixation efficiency.

[0091] Depend on Figure 1 and Figure 2 It can be seen that, for experimental groups 3-7, the carbon fixation effect of the carbon-fixing and pollution-eliminating immobilized bacterial agent is better when the immobilization time is 0.5d, 2d, and 3d. 13 C / 12 The percentage of carbon atoms reached 1.27, 1.30, and 1.27, respectively, and the amount of assimilated CO2 in the SOC after 2 and 3 days of fixation was 55.49 mg·kg⁻¹. -1 52.40 mg·Kg -1 The values ​​were significantly higher than those in experimental groups 1 and 2.

[0092] Compared with experimental groups 3-7, we will now use the immobilized bacterial agent prepared by the method of this invention to treat the soil and verify the ability of the immobilized bacterial agent to degrade polycyclic aromatic hydrocarbons in the soil.

[0093] After grinding the freeze-dried sample evenly, 2g of the freeze-dried sample was added to 10mL of a hexane and dichloromethane (volume ratio 1:1) solution, vortexed, and extracted by ultrasonication for 30min. The mixture was then centrifuged at 2000rpm for 10min, and the supernatant was collected. This process was repeated three times. The extract was purified by passing it through a column containing 2g of anhydrous sodium sulfate and 2g of silica gel. Finally, elution was performed with a mixture of 10mL of hexane and dichloromethane. The resulting mixture was dried using a rotary evaporator and then reconstituted with 2mL of methanol. The phenanthrene concentration was detected by HPLC, and the PAHs degradation rate for each experimental group was calculated. The formula for calculating the PAHs degradation rate is as follows.

[0094] PAHs degradation rate (%) = (1 - final PAHs content in the experimental soil / initial PAHs content in the experimental soil)

[0095] ×100%

[0096] Depend on Figure 3It can be seen that at 20 days, the carbon-fixing and pollution-removing immobilized bacterial agent with an immobilization time of 3 days (corresponding to experimental group 6) has a better degradation effect on PAHs. The total PAH removal rate of this carbon-fixing and pollution-removing immobilized bacterial agent is 82.18%, indicating that it has a good PAH degradation ability. The carbon-fixing and pollution-removing immobilized bacterial agent with an immobilization time of 3 days also has a better removal effect on LMW PAHs (low-ring PAHs), MMW PAHs (medium-ring PAHs), and HMW PAHs (high-ring PAHs), with removal rates of 87.39%, 80.27%, and 72.85%, respectively. Among them, LMW PAHs (low-ring PAHs) are 2-3 ring PAHs (NAP, ANA, ANY, FLU, PHE, ANT); MMW PAHs (medium-ring PAHs) are 4 ring PAHs (FLT, PYR, BaA, CHR); and HMW PAHs (high-ring PAHs) are 5-6 ring PAHs (BaP).

[0097] Therefore, considering the SOC content, the amount of CO2 assimilated in SOC, and the PAH removal rate at different immobilization times ( Figure 1-3 The overall effect of carbon fixation and pollution removal by immobilized microbial agents is optimal when the immobilization time is 3 days. At this immobilization time, the microbial agents will not fail to achieve saturation adsorption, or the outer biofilm of the biochar carrier will detach, resulting in a small number of microorganisms immobilized on the biochar carrier.

[0098] (2) Optimization of the ratio of liquid bacterial agent to biochar carrier for carbon-fixing and pollution-eliminating bacteria.

[0099] Compared with experimental groups 8-10, the δ¹² values ​​of labeled and unlabeled soils were determined by isotope ratio mass spectrometry and elemental analysis of freeze-dried samples. 13 C-value and SOC, used for subsequent... 13 C / 12 Analysis of the percentage of C atoms, the amount of CO2 assimilated in SOC, and carbon fixation efficiency.

[0100] Depend on Figure 4-5 It can be seen that at 20 days, the carbon fixation effect of the immobilized microbial agent with a microbial-to-substrate ratio of 20 mL:1.5 g was better (corresponding to experimental group 9). 13 C / 12 The percentage of carbon atoms reached 1.28, and the amount of CO2 assimilated in the SOC was 68.68 mg·kg⁻¹. -1 It was significantly higher than that of experimental group 1 and experimental group 2, and its molecular super-amplification was 47.03% higher than that of experimental group 2.

[0101] Compared with experimental groups 8-10, we will now use the carbon-fixing and pollution-reducing immobilized bacterial agent prepared by the method of this invention to treat the soil and verify the degradation ability of the carbon-fixing and pollution-reducing immobilized bacterial agent on polycyclic aromatic hydrocarbons in the soil.

[0102] After grinding the freeze-dried sample evenly, 2g of the freeze-dried sample was added to 10mL of hexane and dichloromethane (volume ratio 1:1), vortexed, and extracted by ultrasonication for 30min. After centrifugation at 2000rpm for 10min, the supernatant was collected. This process was repeated 3 times. The extract was purified by passing it through a column of 2g anhydrous sodium sulfate and 2g silica gel. Finally, 10mL of a mixture of hexane and dichloromethane was added for elution. The obtained mixture was dried by rotary evaporator and then diluted to volume with 2mL of methanol. The concentration of phenanthrene was detected by HPLC, and the degradation rate of PAHs in each experimental group was calculated.

[0103] Depend on Figure 6 It was found that at 20 days, both the carbon-fixing and pollution-eliminating immobilized bacterial agents with a bacterial-to-material ratio of 20 mL:1.5 g (corresponding to experimental group 9) and 30 mL:1.5 g (corresponding to experimental group 10) exhibited good PAH removal rates, with a removal rate of over 80% for all total PAHs. Among them, the carbon-fixing and pollution-eliminating immobilized bacterial agent with a bacterial-to-material ratio of 30 mL:1.5 g showed the highest removal efficiency for LMW PAHs, MMW PAHs, and HMW PAHs, at 87.29%, 85.82%, and 67.12%, respectively.

[0104] Considering the SOC content, CO2 assimilation in SOC, and PAH removal rate after adding different carbon-fixing and pollution-eliminating immobilized bacterial agents, the overall carbon-fixing and pollution-eliminating effect of the immobilized bacterial agents is optimal at a bacterial-to-material ratio of 20 mL: 1.5 g. At this ratio, the microbial density of the immobilized bacterial agent is neither too low nor too high, preventing the underutilization of some pollutants or carbon sources, or potentially leading to resource competition and limiting the carbon-fixing and pollution-eliminating efficiency.

Claims

1. Acinetobacter BJ03, characterized in that, Acinetobacter BJ03 was deposited on May 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34668.

2. The application of Acinetobacter BJ03 as described in claim 1 in the degradation of polycyclic aromatic hydrocarbons.

3. A microbial agent for degrading polycyclic aromatic hydrocarbons, characterized in that, It includes Acinetobacter BJ03 as described in claim 1.

4. The microbial agent for degrading polycyclic aromatic hydrocarbons as described in claim 3, characterized in that, The Acinetobacter BJ03 was loaded onto a biochar carrier, which was obtained by pyrolysis of plant material.

5. A carbon-fixing and pollution-eliminating bacterial community, characterized in that, It includes Acinetobacter BJ03 as described in claim 1.

6. The carbon-fixing and pollution-eliminating bacterial community as described in claim 5, characterized in that, It includes polycyclic aromatic hydrocarbon (PAH) degrading bacteria and carbon-fixing bacteria; the PAH degrading bacteria include *Pseudomonas putida* CICC23685, *Acinetobacter bacillus* BJ03, *Cochlearella* BJ05, and *Bacillus spp.* PHE-2; the carbon-fixing bacteria is *Nitrostrophomonas viennarifolia* EN76.

7. The application of the carbon-fixing and pollution-reducing bacterial community as described in claim 6 in carbon fixation and pollution reduction of organically polluted soil.

8. The application as described in claim 7, characterized in that, The carbon-fixing and decontamination bacteria were loaded onto a biochar carrier, which was obtained by pyrolysis of plant materials.

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