Petroleum hydrocarbon-degrading bacteria and application thereof

Abstract

This invention relates to a petroleum hydrocarbon-degrading bacterium and its application, belonging to the field of microbial technology. To address the technical problems in existing petroleum-contaminated soil bioremediation, such as the scarcity of highly efficient degrading strains, low degradation efficiency, and insufficient capacity to handle complex hydrocarbons, this invention provides a petroleum hydrocarbon-degrading bacterium, *Acinetobacter pylori* AFRC34, with accession number CCTCC No:2026007. This strain can use petroleum hydrocarbon organic matter in diesel fuel as the sole carbon source and grows on inorganic salt culture media. It maintains a high petroleum hydrocarbon degradation rate even at a low temperature of 15℃. In an inorganic salt culture medium containing 10,000 mg / L high-concentration diesel fuel at 37℃, the degradation rate reaches 92.46%. The strain and inoculant provided by this invention can efficiently degrade petroleum hydrocarbon pollutants, and are particularly suitable for in-situ bioremediation in cold regions and heavily contaminated sites.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a petroleum hydrocarbon degrading bacterium and its application. Background Technology

[0002] In modern industry, petroleum plays a vital role in production and is therefore often overexploited. However, during extraction, processing, and transportation, petroleum is inevitably released into the environment, causing pollution that has become a widespread environmental concern worldwide. As a complex natural organic compound, petroleum is primarily composed of hydrocarbons (including alkanes, alkenes, and aromatics), which are also its main source of toxicity. When these toxic substances enter the soil, they not only affect and harm the soil's permeability, the structure of the soil's microbial community, and the growth of plants, but also seep into the ground with precipitation, polluting groundwater and ultimately entering the human or animal body through the food chain.

[0003] Faced with widespread oil pollution, physical methods (such as dilution, adsorption, and volatilization), chemical methods (such as chemical inactivation and chemical oxidants), and biotechnology are currently widely used for the remediation of oil-contaminated soils. Among these, biotechnology is more efficient and environmentally friendly than physical and chemical methods. This technology utilizes the normal metabolic activities of microorganisms to degrade toxic and harmful pollutants into harmless inorganic substances such as CO, CH4, and H2O, without causing secondary pollution to the environment. It is a safe, economical, non-toxic, and pollution-free remediation method.

[0004] In recent years, research on the use of microorganisms to degrade petroleum hydrocarbon pollutants has been increasing, but existing technologies still have significant shortcomings: on the one hand, the discovery of highly efficient degrading strains is still insufficient, and resources of strains that can adapt to complex polluted environments, have a broad degradation spectrum, and high activity remain scarce; on the other hand, existing degradation technologies still have significant deficiencies in degradation efficiency, degradation cycle, and the ability to treat high concentrations or mixed hydrocarbon pollutants, making it difficult to meet the needs of rapid and thorough remediation of actual contaminated sites. Therefore, developing new, highly efficient petroleum hydrocarbon degrading strains and improving their degradation performance has important research significance and application value. Summary of the Invention

[0005] This invention addresses the technical problems in existing bioremediation of petroleum-contaminated soils, such as the scarcity of highly efficient degrading strains, low degradation efficiency, and insufficient capacity to handle complex hydrocarbons, by providing a petroleum hydrocarbon degrading bacterium and its application.

[0006] One objective of this invention is to provide a petroleum hydrocarbon-degrading bacterium, wherein the petroleum hydrocarbon-degrading bacterium is Acinetobacter pylori (… Acinetobacter pittii AFRC34, accession number CCTCC No:2026007, accession date January 4, 2026.

[0007] In a preferred embodiment of the present invention, the fermentation method of the petroleum hydrocarbon degrading bacterium Acinetobacter p. ferrogenes AFRC34 includes the following steps: S1: Acinetobacter pylori AFRC34 was inoculated into LB medium under aseptic conditions and cultured at 37℃ and 180 r / nim for 12 h to obtain primary seed culture medium; S2: The primary seed culture obtained in S1 was inoculated into fresh LB medium and cultured at 37℃ and 180 r / min for 12-24 h to obtain the secondary seed culture. S3: The secondary seed culture obtained in S1 was inoculated into a sterilized fermentation medium and cultured at 37℃, 180 r / min, and aeration for 6-8 h. The OD was then measured. 600 Fermentation is stopped when the value is ≥1.2, and the fermentation broth of petroleum hydrocarbon degrading bacteria is obtained.

[0008] In a preferred embodiment of the present invention, the amount of the primary seed culture medium inoculated into the new LB medium in S1 is 1%.

[0009] In a preferred embodiment of the present invention, the amount of the secondary seed culture medium inoculated into the fermentation medium in S2 is 1%.

[0010] In a preferred embodiment of the present invention, the LB culture medium in S1 and S2 consists of: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10.0 g / L NaCl.

[0011] In a preferred embodiment of the present invention, the ventilation condition in S3 is: the ratio of the volume of liquid introduced during fermentation to the volume of sterile air is 1:1.5.

[0012] In a preferred embodiment of the present invention, the fermentation medium in S3 is: 1 / 4 concentration LB medium.

[0013] A second objective of this invention is to provide a microbial agent containing the aforementioned petroleum hydrocarbon degrading bacteria, specifically Acinetobacter pylori AFRC34.

[0014] The third objective of this invention is to provide the application of the above-mentioned petroleum hydrocarbon degrading bacteria or the above-mentioned microbial agents in the degradation of petroleum hydrocarbons. The application refers to the efficient degradation of petroleum hydrocarbons by Acinetobacter pylori AFRC34 under conditions of 15℃-37℃.

[0015] The fourth objective of this invention is to provide the application of the above-mentioned petroleum hydrocarbon degrading bacteria or the above-mentioned microbial agents in the purification of petroleum-polluted wastewater or soil.

[0016] Compared with the prior art, the beneficial effects of the present invention are: the present invention provides a petroleum hydrocarbon degrading bacterium, namely Acinetobacter pylori AFRC34, with accession number CCTCC No:2026007 and accession date of January 4, 2026.

[0017] Compared with the prior art, the Acinetobacter pylori AFRC34 provided by the present invention has the following advantages: 1. Unique carbon source utilization capability: The Acinetobacter pylori provided by this invention can grow normally on inorganic salt culture medium using petroleum hydrocarbon organic matter in diesel as the sole carbon source, indicating that it has a highly efficient petroleum hydrocarbon metabolic pathway, avoiding the cost and secondary pollution risks brought about by additional nutrient addition.

[0018] 2. Outstanding low-temperature degradation performance: This strain can still maintain a high degradation rate (80.16%) of petroleum hydrocarbon pollutants at a low temperature of 15℃, breaking through the technical bottleneck that most existing degradation strains are limited to mesophilic conditions (25-37℃), and is suitable for in-situ bioremediation of petroleum-contaminated soil in cold regions or winter.

[0019] 3. Excellent tolerance to high concentrations and degradation efficiency: In an inorganic salt culture medium at 37°C with a diesel concentration as high as 10,000 mg / L, this strain achieved a degradation rate of 92.46% for petroleum hydrocarbons, indicating that it not only tolerates high concentrations of petroleum pollution stress but also has the potential to treat heavily polluted sites.

[0020] 4. Environmental friendliness and rapid adaptability: This strain grows rapidly and is highly adaptable to the environment. It does not damage the original soil ecology during the remediation process, and its metabolites are harmless inorganic substances with no secondary pollution, thus truly achieving green and safe bioremediation.

[0021] In summary, the Acinetobacter pylori AFRC34 and its biological agent provided by this invention have shown significant improvements in low-temperature adaptability, high-concentration pollutant degradation efficiency, and carbon source utilization specificity.

[0022] [Biological Preservation Information]: The petroleum hydrocarbon degrading bacteria described are *Acinetobacter pylori* AFRC34, with accession number CCTCC No: 2026007, and classified as... Acinetobacter pittii It is deposited at the China Center for Type Culture Collection on January 4, 2026. Attached Figure Description

[0023] Figure 1 This is a streak line purification diagram of a single colony from the initial screening. Figure 2 This is a graph showing the screening results of petroleum hydrocarbon degrading strains; the horizontal axis 1-6 represent the strain numbers. Figure 3A is a streak pattern of single colonies purified through secondary screening; A is a streak pattern on an LB plate, and B is a streak pattern on a diesel-inorganic salt plate. Figure 4 Morphological observation of Acinetobacter pylori AFRC34; Figure 5 Electron micrograph of Acinetobacter pylori AFRC34; Figure 6 The image shows the electrophoresis results of Acinetobacter pylori AFRC34. Figure 7 The genome circos diagram of Acinetobacter pylori AFRC34; Figure 8 A bar chart showing the KEGG Pathway classification of Acinetobacter pylori AFRC34. Detailed Implementation

[0024] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0026] In the following examples, the petroleum hydrocarbon degrading bacteria described in this invention are all referred to as Acinetobacter piriformis AFRC34.

[0027] The reagents used in the following examples: 1. Inorganic salt culture medium: K2HPO4 1.6 g, KH2PO4 0.4 g, (NH4)2SO4 1.5 g, MgSO4 0.5 g, trace element solution 10 mL. After the reagents are fully dissolved, add water to make up to 1000 mL and adjust the pH to 7.0-7.2.

[0028] 2. Trace element solution: 3 g CaCl2, 0.5 g FeSO4·7H2O. After the reagents are fully dissolved, add water to make up to 1000 mL and store at 4℃.

[0029] 3. LB medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L.

[0030] 4. Beef extract peptone medium: 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl. After the reagents are fully dissolved, add water to make up to 1000 mL and adjust the pH to 7.0-7.2.

[0031] 5. Diesel degradation medium: 1 L of inorganic salt medium, a certain amount of 0# diesel (calculate the amount to be added based on the density of diesel, which is 0.82-0.86 g / mL; for example, to prepare 50 mL of inorganic salt medium with a concentration of 10000 mg / L, add 600 μL of diesel).

[0032] Example 1: Screening, isolation and purification of Acinetobacter pylori AFRC34 S1: Take 10 g of oily sludge sample collected from Lingwu City, sieve to remove stones, branches and other impurities, place it in sterilized inorganic salt medium, add glass beads to disperse the sludge sample, and culture in a shaker at 37℃ and 180 r / min; after enrichment for 96 h, take the enriched bacterial solution from the conical flask, inoculate it into a new inorganic salt medium at a ratio of 5% for further enrichment, and increase the diesel concentration to 1500 mg / L to acclimate the microorganisms in the culture system; repeat the above process for a total of 4 generations, with diesel concentrations of 1500, 2000, 2500 and 3000 mg / L respectively; S2: Take 1 mL of the bacterial culture enriched and domesticated for 4 generations from S1, and dilute it with sterile water in a 10-fold serial dilution ratio. Take 10 mL of each of these dilutions. -4 -10 -6 Three gradient bacterial solutions of 200 μL were spread on beef extract peptone medium using a spreader. The morphological characteristics of the colonies were observed. Single colonies were picked and streaked for purification. After purification three times, the test strain was obtained. S3. Initial Screening: Take an inorganic salt solid culture medium plate, add 2-3 drops of diesel oil, and spread it evenly with a spreader to obtain an inorganic salt solid culture medium containing diesel oil. Use an inoculation loop to pick up the purified test strains from S2 and inoculate them at a ratio of 5% into the inorganic salt solid culture medium containing diesel oil. Incubate upside down in a 37℃ incubator and observe the microbial growth regularly. Select the best-growing strains for low-temperature preservation. Six strains were obtained from the initial screening (the results of the initial screening single colony streak purification are shown in the figure). Figure 1 (as shown) S4. Secondary Screening: Strains 1-6 obtained in S3 were inoculated at a ratio of 5% into diesel degradation medium and cultured in a constant temperature shaker at 37℃ and 180 r / min for 7 days. The remaining diesel in the medium was then tested. Figure 2As shown, the pure strain numbered "2" with the best growth status and highest degradation efficiency was selected from the 6 strains for preservation and named AFRC34 (the results of the single-colony streak purification after secondary screening are shown in the figure). Figure 3 (As shown).

[0033] Example 2: Identification of Acinetobacter pylori AFRC34 1. Morphological observation The AFRC34 strain obtained in Example 1 was morphologically observed. The AFRC34 strain was inoculated on LB solid medium plates, and the colony morphology characteristics were observed.

[0034] like Figure 4 As shown, the colonies of strain AFRC34 are round, white, raised, smooth and moist, and about 2-4 mm thick.

[0035] like Figure 5 As shown, under an electron microscope, the AFRC34 strain individuals are rod-shaped, without flagella, with a cell diameter of approximately 0.6-0.8 μm and a length of approximately 1-1.5 μm.

[0036] 2. Molecular biological identification Genomic DNA was extracted from the AFRC34 strain obtained in Example 1, and the 16S rDNA of the AFRC34 strain was rapidly amplified using polymerase chain reaction (PCR) technology. During the amplification process, the universal bacterial primers 27F (nucleotide sequence SEQ ID NO.2: AGAGTTTGATCMTGGCTCAG) and 1492R (nucleotide sequence SEQ ID NO.3: TACGGYTACCTTGTTACGACTT) were used.

[0037] The PCR reaction system consisted of: 2 µL upstream primer, 2 µL upstream primer, 4 µL template DNA (bacterial culture), 17 µL ddH2O, and 25 µL 2×Taq PCR MasterMix. The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ high-temperature denaturation for 30 s, 55℃ low-temperature annealing for 15 s, 72℃ medium-temperature extension for 30 s, for 35 cycles; 72℃ medium-temperature extension for 5 min. The amplified products were stored at 4℃.

[0038] After PCR amplification, agarose gel electrophoresis was used for determination. If the size of the fragment is about 1.5kb, it indicates that the PCR amplification was successful and the gel can be cut and recovered.

[0039] The gel electrophoresis results of the amplified products are as follows: Figure 6 As shown, the amplification product was sequenced, and the nucleotide sequence obtained from the sequencing results is shown in SEQ ID NO.1.

[0040] Using the NCBI Blast program, the sequence file with the nucleotide sequence shown in SEQ ID NO.1 was compared with the data in the NCBI 16S database. The results showed that strain AFRC34 had the highest sequence homology with species of the Acinetobacter genus; therefore, it was identified as *Acinetobacter pirelli*. Acinetobacter pittii ).

[0041] Example 3: Application of Acinetobacter pylori AFRC34 in the degradation of petroleum hydrocarbons 1. Low temperature tolerance test S1: Under aseptic conditions, Acinetobacter pylori AFRC34 was inoculated at a ratio of 5% into a 100 mL Erlenmeyer flask containing 50 mL of inorganic salt medium, and the strain was activated by shaking in a shaker at 37℃ and 180 r / min for 12 h.

[0042] S2: The activated Acinetobacter pylori AFRC34 from S1 was cultured to the logarithmic growth phase to obtain the seed culture of Acinetobacter pylori AFRC34. This seed culture was placed in a 50 mL centrifuge tube and centrifuged at 5000 r / min for 10 min. The supernatant was discarded, and the centrifuged bacterial cells were washed. The lower precipitate was dispersed and mixed thoroughly with PBS buffer, and then centrifuged again at 5000 r / min for 10 min. The supernatant was discarded. This washing process was repeated three times. The bacterial culture was diluted with ultrapure water, and the OD... 600 Adjust to 1.0 and store at 4℃ for later use to obtain Acinetobacter pylori AFRC34 bacterial suspension.

[0043] S3: The Acinetobacter pylori AFRC34 bacterial suspension obtained in S2 was inoculated into 50 mL of sterile diesel degradation medium at an inoculation rate of 5%. The diesel concentration was controlled at 10000 mg / L and a blank control was set up. The medium was cultured for 7 days at 15, 20, 25, 30 and 37 ℃, pH 7.0 and 180 r / min respectively. After 7 days of culture, the remaining diesel content in the conical flask was measured and its degradation rate was calculated.

[0044] The results are shown in Table 1. At 37℃, the degradation rate of petroleum hydrocarbons by Acinetobacter pylori AFRC34 reached 92.46%; at 15℃, the degradation rate was 80.16%, close to the degradation rate at 30℃. Therefore, the Acinetobacter pylori AFRC34 provided by this invention has a high degradation effect on petroleum hydrocarbons.

[0045] Table 1

[0046] 2. Discovery of highly efficient degradation genes The genome of Acinetobacter pylori strain AFRC34 was sequenced using the platform of Shanghai Sangon Biotech Co., Ltd.

[0047] The raw data from second-generation sequencing were statistically analyzed and quality assessed using Fastp, and quality cuts were performed to obtain relatively accurate and effective data. The raw data from third-generation sequencing were also quality cut to obtain high-quality data. PacBio / Nanopore single-molecule sequencing data were assembled using Canu / NextDenovo / Unicycler. Second-generation sequencing data were introduced, and GapFiller was used to fill gaps in the assembled contigs. Pilon was used for sequence correction to correct editing errors and small fragment insertions / deletions during the assembly process. NCBI-PGAP / Prokka software was used to predict gene elements, including CDS, tRNA, and rRNA.

[0048] The gene protein sequence was compared with multiple databases, including CDD, KOG, COG, NR, NT, PFAM, Swissprot, and TrEMBL, using NCBI Blast+ to obtain its functional annotation information. GO functional annotation information was obtained based on the gene's annotation results with Swissprot and TrEMBL, and KEGG annotation information was obtained using KAAS.

[0049] like Figure 7 As shown, the genome circos diagram of Acinetobacter pylori AFRC34 demonstrates its complete genome information: the total length of the genome is approximately 3.81 Mb (3812095 bp), the average GC content is 38.75%, and the sequencing coverage reaches 100%, proving that the genome assembly is complete and there is no obvious contamination or structural abnormality. Figure 6 The distribution of functional elements in the genome is clearly presented: the coding region (CDS) accounts for the largest proportion, and it also contains complete rRNA, tRNA and non-coding RNA. Through COG functional annotation, it can be clearly seen that the genome covers all core life function categories, including replication, recombination and repair, energy metabolism, transcription and translation, material transport, signal transduction and defense mechanisms. Among them, the number of genes in the categories of replication, recombination and repair, general function prediction, unknown function, amino acid metabolism and transcription is the largest, which not only reflects the species' complete life activity capacity, but also reflects its active metabolism and strong environmental adaptability.

[0050] like Figure 8As shown in the KEGG Pathway classification bar chart, this chart counts the number of genes annotated to each functional pathway in the genome. Genes are divided into five primary categories: cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems. Metabolic pathway genes are overwhelmingly dominant, with amino acid metabolism and general pathway genes having the highest number (≥200). The abundance of genes in core metabolic pathways such as carbohydrates, lipids, and energy is also significantly higher than in other categories, directly demonstrating that this species has highly active metabolic functions and possesses complete and vigorous basal metabolism and energy conversion capabilities. The abundance of genes in membrane transport and signal transduction pathways within environmental information processing indicates strong environmental perception, material transport, and stress adaptation capabilities. Genetic information processing pathway genes are complete, ensuring stable genome replication and gene expression. Organismal system pathway genes account for a very low proportion, consistent with the genomic characteristics of prokaryotic microorganisms. Overall, this chart demonstrates that the genome is functionally complete, covering all core life activities such as growth and reproduction, environmental adaptation, genetic transmission, and metabolism, while also suggesting its potential to synthesize secondary metabolites.

[0051] As shown in Table 2, the genome of Acinetobacter pylori AFRC34 contains one alkB1_2 gene (alkane 1-monooxygenase), which is a gene encoding alkane monooxygenase and a functional gene for alkane degradation.

[0052] Table 2

[0053] 3. Preparation of fermentation broth for Acinetobacter pylori AFRC34 S1: Acinetobacter pylori AFRC34 was inoculated into LB medium at a rate of 1% using aseptic techniques and cultured at 37°C and 180 r / min for 12 h to obtain primary seed culture. S2: The primary seed culture obtained in S1 was inoculated into fresh LB medium and cultured at 37℃ and 180 r / min for 12 h to obtain the secondary seed culture. S3: The secondary seed culture obtained in S1 was inoculated into a sterilized fermentation medium (1 / 4 concentration LB medium) and cultured at 37℃, 180 r / min, and an aeration ratio of 1:1.5 (the ratio of liquid volume to sterile air during fermentation was 1:1.5) for 6 h. OD was then measured. 600 Fermentation was stopped when the value was ≥1.2, and the fermentation broth of Acinetobacter pylori AFRC34 was obtained.

[0054] 4. Degradation rate experiment of contaminated soil Add 5% of the above-obtained Acinetobacter pylori AFRC34 fermentation broth to the straw. Add urea and potassium dihydrogen phosphate evenly to the straw mixture at a nitrogen-to-phosphorus ratio of 5:1 and stir well. Then mix the straw mixture with 20 tons of petroleum hydrocarbon-contaminated soil, adjust the moisture content to 50-70%, and spread the soil evenly (1.5 m high and 3 m long and wide). After mixing evenly, pile the soil for petroleum hydrocarbon degradation fermentation. During the fermentation process, maintain the soil moisture content at 50% and turn the soil over to maintain air circulation.

[0055] Sampling was conducted on petroleum hydrocarbon-contaminated soil at 5, 10, 15, 20, 25, and 30 days of fermentation. The sampling principle was to collect samples from different directions and depths of the soil pile at multiple locations, ensuring thorough mixing before testing. The concentration of C in the petroleum hydrocarbon-contaminated soil was analyzed according to the national standard gas chromatography method HJ1021-2019. 10 -C 40 Measurement.

[0056] The results are shown in Table 3. In treatment groups 1-2, which were treated with Acinetobacter pylori AFRC34, the content of petroleum hydrocarbons in the contaminated soil showed a certain trend of decrease. This indicates that Acinetobacter pylori AFRC34 provided by the present invention has the ability to degrade petroleum hydrocarbons in heavily contaminated soil.

[0057] Table 3

[0058] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A petroleum hydrocarbon degrading bacterium, characterized in that, The petroleum hydrocarbon-degrading bacteria is Acinetobacter pittii (ATCC 7703) Acinetobacter pittii ) AFRC34, the preservation number is CCTCC No:2026007, and the preservation date is January 4, 2026.

2. The petroleum hydrocarbon degrading bacteria according to claim 1, characterized in that, The fermentation method for the petroleum hydrocarbon degrading bacterium Acinetobacter AFRC34 includes the following steps: S1: Acinetobacter pylori AFRC34 was inoculated into LB medium under aseptic conditions and cultured at 37℃ and 180 r / nim for 12 h to obtain primary seed culture medium; S2: The primary seed culture obtained in S1 was inoculated into fresh LB medium and cultured at 37℃ and 180 r / min for 12-24 h to obtain the secondary seed culture. S3: The secondary seed culture solution obtained in S1 was inoculated into the fermentation medium after high temperature sterilization, and cultured at 37°C, 180 r / min, and under aeration conditions for 6-8 h, and the OD value was measured 600 When the OD value≥1.2, the fermentation was stopped, and the petroleum hydrocarbon degrading bacteria fermentation solution was obtained.

3. The petroleum hydrocarbon degrading bacteria according to claim 2, characterized in that, The inoculation amount of the primary seed culture medium described in S1 into the new LB medium is 1%.

4. The petroleum hydrocarbon degrading bacteria according to claim 2, characterized in that, The inoculation amount of the secondary seed culture medium described in S2 into the fermentation medium is 1%.

5. The petroleum hydrocarbon degrading bacteria according to claim 2, characterized in that, The LB medium composition described in S1 and S2 is: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10.0 g / L NaCl.

6. The petroleum hydrocarbon degrading bacteria according to claim 2, characterized in that, The ventilation conditions described in S3 are: the ratio of the volume of liquid introduced during fermentation to the volume of sterile air is 1:1.

5.

7. The petroleum hydrocarbon degrading bacteria according to claim 2, characterized in that, The fermentation medium described in S3 is: 1 / 4 concentration of LB medium.

8. A microbial inoculant, characterized in that, The microbial agent contains the petroleum hydrocarbon degrading bacteria according to any one of claims 1 to 7, wherein the petroleum hydrocarbon degrading bacteria is Acinetobacter pylori AFRC34.

9. The application of the petroleum hydrocarbon degrading bacteria according to any one of claims 1 to 7 or the microbial agent according to claim 8 in the degradation of petroleum hydrocarbons, characterized in that, The application refers to the efficient degradation of petroleum hydrocarbons by Acinetobacter pylori AFRC34 at 15℃-37℃.

10. The application of the petroleum hydrocarbon degrading bacteria according to any one of claims 1 to 7 or the microbial agent according to claim 8 in the purification of petroleum-polluted wastewater or soil.

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