Composite accelerant for accelerating degradation of polycyclic aromatic hydrocarbon by microorganisms as well as preparation method and application of composite accelerant

Abstract

The invention relates to the technical field of water pollution remediation, and discloses a composite accelerant for accelerating microorganisms to degrade polycyclic aromatic hydrocarbons as well as a preparation method and application of the composite accelerant. The composite accelerant contains low-molecular-weight organic acid and ferric salt at the same time. The method for degrading the polycyclic aromatic hydrocarbon by the microorganisms comprises the step of culturing the microbial agent in an inorganic culture medium containing the polycyclic aromatic hydrocarbon and the composite accelerant. The iron and organic acid composite accelerant microbial method provided by the invention is simple in process, environment-friendly and efficient, and by utilizing the composite accelerant, the extracellular and intracellular enzyme generation of microorganisms can be improved, and the microbial degradation process of polycyclic aromatic hydrocarbon is enhanced. According to the method for promoting phenanthrene degradation through microbial stimulation, more efficient degradation is achieved, and the polycyclic aromatic hydrocarbon degradation process can be completed in a short time.

Description

Technical Field

[0001] This invention relates to the technical field of water pollution remediation, and in particular to a composite promoter for accelerating the degradation of polycyclic aromatic hydrocarbons by microorganisms, its preparation method, and its application. Background Technology

[0002] With the acceleration of modern urbanization and the large-scale development and use of energy sources such as petroleum and coal, pollution from polycyclic aromatic hydrocarbons (PAHs) has become increasingly serious. PAHs are volatile hydrocarbons produced by the incomplete combustion of coal, petroleum, wood, tobacco, and organic polymers. There are more than 500 types of PAHs. Besides some natural sources, PAHs in soil also come from the incomplete combustion of fossil fuels such as coal and petroleum, as well as atmospheric deposition and sewage irrigation. PAHs are adsorbed by atmospheric particulate matter and enter aquatic and soil ecosystems through rainwater deposition, scouring, and transport. PAHs are difficult to degrade through natural environments such as light and oxygen, easily enter ecosystems through the food chain, and have a cumulative effect, seriously endangering human health and ecological security. In particular, PAHs such as phenanthrene, acenaphthene, fluorene, fluoranthene, and pyrene have been proven to have strong carcinogenic potential. For example, phenanthrene, with its three benzene ring structure, low solubility, and strong chemical stability, is listed as a key environmental pollutant for monitoring.

[0003] Currently, there are three main categories of methods for remediating polycyclic aromatic hydrocarbon (PAH) contaminated water bodies: physical methods, chemical methods, and biological methods. Among them, bioremediation has a very broad development potential and application prospect due to its advantages such as low treatment cost, no secondary pollution, on-site treatment, high public acceptance, and good efficiency. Microbial remediation includes biostimulation and bioenhancement. However, most current microbial degradation technologies are based on single or combined microbial agents, and rarely use accelerators to directly improve the degradation efficiency of PAHs in the short term. The ability of microorganisms to rapidly enter a highly efficient degradation state of PAHs in the short term has a unique advantage in dealing with more complex and sudden pollution emergency remediation. Therefore, existing technologies for microbial degradation of PAHs suffer from problems such as slow start-up, insufficient secretion of biological enzymes, and long degradation cycles, necessitating a technical method that can accelerate PAH degradation in a short period of time.

[0004] The microbial degradation promoter disclosed in CN114621907A can achieve efficient degradation of polycyclic aromatic hydrocarbons through various organic acids such as citric acid, oxalic acid, and humic acid. After adding the promoter, the degradation efficiency increased from 54.75% to 69.12% within 1 day, but the efficiency improvement within 1 day was relatively low compared with that without adding the promoter.

[0005] The microbial agent disclosed in CN116987601B can achieve a polycyclic aromatic hydrocarbon degradation rate of over 85% within 7 days, but it fails to accelerate microbial degradation in a shorter time, resulting in a long degradation time.

[0006] Based on this, the present invention provides a method for preparing a composite promoter for accelerating the degradation of polycyclic aromatic hydrocarbons by microorganisms and a method for microbial degradation of polycyclic aromatic hydrocarbons. Summary of the Invention

[0007] To address the technical problem that the degradation effect of simple bacterial strains on polycyclic aromatic hydrocarbons is weak in existing technologies, this invention provides a composite promoter for accelerating the degradation of polycyclic aromatic hydrocarbons by microorganisms, its preparation method, and its application.

[0008] The first objective of this invention is to provide a method for preparing a composite promoter that accelerates the microbial degradation of polycyclic aromatic hydrocarbons, comprising the following steps: A solution of an iron salt and a solution of a low molecular weight organic acid are provided. Iron salt solution and low molecular weight organic acid solution are mixed and stirred to fully chelate iron ions with organic acids to form a composite accelerator.

[0009] In some embodiments of the present invention, the iron source is selected from ferric salts and / or ferrous salts, and further, the iron source is selected from ferric sulfate; the concentration of the iron salt solution is 1000-5000 μM.

[0010] In some embodiments of the present invention, the concentration of the low molecular weight organic acid is 1000-5000 μM; the low molecular weight organic acid is a phenolic organic acid and / or an aliphatic carboxylic acid; further, the low molecular weight organic acid is selected from one or more of caffeic acid, ferulic acid, citric acid and oxalic acid.

[0011] In some embodiments of the present invention, the chelation molar ratio of iron ions to low molecular weight organic acids is (1:5)-(1:10).

[0012] In some embodiments of the present invention, the mixing conditions are: temperature of 15-35°C, stirring speed of 100-300 rpm, and time of 0.5-2 h.

[0013] The second objective of this invention is to provide a composite promoter for accelerating the degradation of polycyclic aromatic hydrocarbons by microorganisms, which is prepared by the aforementioned preparation method.

[0014] A third objective of this invention is to provide the application of the aforementioned composite promoter in accelerating the microbial degradation of polycyclic aromatic hydrocarbons, wherein the method of application includes the following steps: (1) Prepare and sterilize an inorganic salt culture medium containing 0.8-1.2 g / L ammonium salt, 1.8-2.2 g / L potassium salt, 0.1-0.3 g / L magnesium salt and 0.01-0.03 g / L calcium salt; add polycyclic aromatic hydrocarbons to the sterilized culture medium at a concentration of 50-250 mg / L.

[0015] (2) Inoculate the Bacillus seed culture into a sterile inorganic salt culture medium and add the compound promoter to the inorganic culture medium.

[0016] In some embodiments of the present invention, in step (1), the ammonium salt is ammonium chloride, the potassium salt is potassium dihydrogen phosphate, the magnesium salt is magnesium sulfate heptahydrate, and the calcium salt is calcium chloride dihydrate.

[0017] In some embodiments of the present invention, in step (1), the concentration of the polycyclic aromatic hydrocarbon is 50-250 mg / L, and the polycyclic aromatic hydrocarbon includes one or more of phenanthrene, anthracene, pyrene or benzo[a]pyrene.

[0018] In some embodiments of the present invention, in step (2), the inoculation amount of the Bacillus seed liquid is 1-5%.

[0019] In some embodiments of the present invention, in step (2), the molar concentration of iron salt in the inorganic salt culture medium in the composite promoter is 50-300 μM, the molar concentration of low molecular weight organic acid in the inorganic salt culture medium is 250-3000 μM, and the molar ratio of iron salt to low molecular weight organic acid is 1:5-10.

[0020] The technical solution of the present invention has the following advantages over the prior art: Unlike other existing biostimulation-driven microbial degradation technologies, this invention employs a composite promoter with a pre-chelation step involving ferric sulfate, phenolic organic acids, and aliphatic carboxylic acids to achieve bio-promoted degradation of polycyclic aromatic hydrocarbons (PAHs). By forming soluble ligands with ferric ions through phenolic organic acids or aliphatic carboxylic acids, the bioavailability of iron as a coenzyme factor is improved, enhancing the synthetic function of intracellular and extracellular enzymes in microorganisms. This increases the yield of extracellular non-specific degradation enzymes within 0.5 days, accelerates the ring-opening pretreatment efficiency of PAHs, and thus improves the degradation effect, achieving a degradation efficiency of over 82% within 2 days.

[0021] This study solves the problem of slow degradation rate of polycyclic aromatic hydrocarbons by single strains and provides an economical, environmentally friendly solution with short degradation time. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 These are the degradation curves of phenanthrene in each example and comparative example over 6 days; Figure 2 This is the concentration change curve of intracellular and extracellular laccase during degradation in Example 1; Figure 3 This is the concentration change curve of intracellular and extracellular laccase during degradation in Example 4; Figure 4 This is the concentration change curve of intracellular and extracellular laccase during degradation in Comparative Example 1; Figure 5 The curves showing the concentration changes of intracellular and extracellular laccase during degradation in Comparative Example 2 are shown. Figure 6 The relative abundance of gene transcription of polycyclic aromatic hydrocarbon degrading enzymes in Examples 1 and 2. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0024] Example 1 This embodiment provides a method for preparing an accelerator that accelerates the degradation of polycyclic aromatic hydrocarbons by microorganisms, as detailed below: (1) Dissolve ferric sulfate in deionized water to prepare a ferric sulfate mother liquor with a concentration of 2000 μM; at the same time, dissolve caffeic acid in a small amount of ethanol and then dissolve it in deionized water to form a 2000 μM caffeic acid mother liquor.

[0025] (2) The 100 μM ferric sulfate diluted mother liquor and the 500 μM caffeic acid diluted mother liquor were contacted and mixed at 30 °C and 120 rpm for 1 h to make the ratio of iron ions to caffeic acid fully chelated to 1:5, thus obtaining the composite promoter.

[0026] (3) Prepare 200 mL of inorganic salt culture medium containing 1.2 g / L ammonium chloride, 2.2 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.03 g / L calcium chloride dihydrate. Adjust the pH to 7.5. After sterilizing the inorganic salt culture medium at 121°C for 20 minutes, add phenanthrene to the culture medium at a concentration of 250 mg / L.

[0027] (4) The number of viable bacteria is 3×10 8 Bacillus seed culture (strain purchased from the China Agricultural Microbial Culture Collection Center, strain number ACCC02754) at a concentration of 2% (v / v) was inoculated into sterile inorganic salt medium. Finally, 10 mL of a compound accelerator was added to the inorganic salt medium. The inorganic medium was then incubated at 30°C and shaken at 150 rpm for 6 days for degradation experiments. The degradation results are shown in Table 1.

[0028] Example 2 This embodiment provides a method for preparing an accelerator that accelerates the degradation of polycyclic aromatic hydrocarbons by microorganisms, as detailed below: (1) Dissolve ferric sulfate in deionized water to prepare a ferric sulfate mother liquor with a concentration of 2000 μM. At the same time, dissolve caffeic acid in a small amount of ethanol and then dissolve it in deionized water to form a 2000 μM caffeic acid mother liquor.

[0029] (2) The 200 μM ferric sulfate diluted mother liquor and the 600 μM caffeic acid diluted mother liquor were contacted and mixed at 35°C and 150 rpm for 1.5 h to make the ratio of iron ions to caffeic acid fully chelated to 1:3, thus obtaining a composite accelerator.

[0030] (3) Prepare 200 mL of inorganic salt culture medium containing 1.2 g / L ammonium chloride, 2.2 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.03 g / L calcium chloride dihydrate. After sterilizing the inorganic salt culture medium at 121°C for 20 minutes, add phenanthrene to the culture medium at a concentration of 250 mg / L.

[0031] (4) The number of viable bacteria is 3×10 8 Bacillus seed culture at a concentration of 2 CFU / mL was inoculated into sterile inorganic salt medium. Finally, a compound stimulant was added to the inorganic salt medium. The inorganic medium was then subjected to a degradation experiment at 30°C and 150 rpm for 6 days with shaking. The degradation results are shown in Table 1.

[0032] Example 3 This embodiment provides a method for preparing an accelerator that accelerates the degradation of polycyclic aromatic hydrocarbons by microorganisms, as detailed below: (1) Dissolve ferric sulfate in deionized water to prepare a ferric sulfate mother liquor with a concentration of 2000 μM. At the same time, dissolve caffeic acid in a small amount of ethanol and then dissolve it in deionized water to form a 1000-5000 μM caffeic acid mother liquor. (2) The 200 μM ferric sulfate diluted mother liquor and the 1000 μM caffeic acid diluted mother liquor were contacted and mixed at 35°C and 200 rpm for 2 h to fully chelate the iron ions and organic acids to a ratio of 1:5 to form a composite accelerator.

[0033] (3) Prepare 200 mL of inorganic salt culture medium containing 1.2 g / L ammonium chloride, 2.2 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.03 g / L calcium chloride dihydrate. After sterilizing the inorganic salt culture medium at 121°C for 20 minutes, add phenanthrene to the culture medium at a concentration of 250 mg / L.

[0034] (4) The number of viable bacteria is 3×10 8Bacillus seed culture at CFU / mL was inoculated into sterile inorganic salt medium at an inoculum volume of 2%. Finally, a compound stimulant was added to the inorganic medium. The inorganic medium was then subjected to a degradation experiment at 30°C and 150 rpm for 6 days with shaking. The degradation results are shown in Table 1.

[0035] Example 4 This embodiment provides a method for preparing an accelerator that accelerates the degradation of polycyclic aromatic hydrocarbons by microorganisms, as detailed below: (1) Dissolve ferric sulfate in deionized water to prepare a ferric sulfate mother liquor with a concentration of 2000 μM. At the same time, dissolve caffeic acid in a small amount of ethanol and then dissolve it in deionized water to form a 1000-5000 μM oxalic acid mother liquor. (2) Mix 100 μM ferric sulfate diluted mother liquor and 1000 μM oxalic acid diluted mother liquor at 30 °C and 120 rpm for 1 h to fully chelate iron ions with organic acids in a ratio of 1:10 to form a composite accelerator.

[0036] (3) Prepare 200 mL of inorganic salt culture medium containing 1.2 g / L ammonium chloride, 2.2 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.03 g / L calcium chloride dihydrate. After sterilizing the inorganic salt culture medium at 121°C for 20 minutes, add phenanthrene to the culture medium at a concentration of 250 mg / L.

[0037] (4) The number of viable bacteria is 3×10 8 Bacillus seed culture at CFU / mL was inoculated into sterile inorganic salt medium at an inoculum volume of 2%. Finally, a compound stimulant was added to the inorganic medium. The inorganic medium was then subjected to a degradation experiment at 30°C and 150 rpm for 6 days with shaking. The degradation results are shown in Table 1.

[0038] Example 5 This embodiment provides a method for preparing an accelerator that accelerates the degradation of polycyclic aromatic hydrocarbons by microorganisms, as detailed below: (1) Dissolve ferrous sulfate in deionized water to prepare a ferrous sulfate mother liquor with a concentration of 2000 μM. At the same time, dissolve caffeic acid in a small amount of ethanol and then dissolve it in deionized water to form a 1000-5000 μM oxalic acid mother liquor. (2) Mix 100 μM ferrous sulfate diluted mother liquor and 500 μM caffeic acid diluted mother liquor at 30 °C and 120 rpm for 1 h to fully chelate iron ions with organic acids in a ratio of 1:5 to form a composite accelerator.

[0039] (3) Prepare 200 mL of inorganic salt culture medium containing 1.2 g / L ammonium chloride, 2.2 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.03 g / L calcium chloride dihydrate. After sterilizing the inorganic salt culture medium at 121°C for 20 minutes, add phenanthrene to the culture medium at a concentration of 250 mg / L.

[0040] (4) The number of viable bacteria is 3×10 8 Bacillus seed culture at CFU / mL was inoculated into sterile inorganic salt medium at an inoculum volume of 2%. Finally, a compound stimulant was added to the inorganic medium. The inorganic medium was then subjected to a degradation experiment at 30°C and 150 rpm for 6 days with shaking. The degradation results are shown in Table 1.

[0041] Comparative Example 1 A method for preparing an accelerator to accelerate the degradation of polycyclic aromatic hydrocarbons by microorganisms is basically the same as that in Example 1, except that an equal amount of ferric sulfate is used in the accelerator, but no organic acid is added.

[0042] Comparative Example 2 A method for preparing an accelerator to accelerate the degradation of polycyclic aromatic hydrocarbons by microorganisms is basically the same as that in Example 1, except that an equal amount of caffeic acid is used in the accelerator, but ferric sulfate is not added.

[0043] Comparative Example 3 A method for preparing an accelerator to accelerate the degradation of polycyclic aromatic hydrocarbons by microorganisms is basically the same as that in Example 1, except that an equal amount of oxalic acid is used in the accelerator, but ferric sulfate is not added.

[0044] Comparative Example 4 A method for preparing an accelerator to accelerate the degradation of polycyclic aromatic hydrocarbons by microorganisms is basically the same as that in Example 1, except that no organic acid or ferric sulfate is added to the accelerator.

[0045] Table 1

[0046] As shown in Table 1, the remediation method of the present invention achieved better remediation results. In Example 1, the degradation rate of phenanthrene reached 82% on the second day after using ferric salt and caffeic acid. In Example 4, the degradation rate of phenanthrene reached 72% on the second day after using ferric salt and oxalic acid. In addition, Comparative Examples 2 and 3 used only caffeic acid and oxalic acid, respectively, with degradation rates of 50% and 52%; Comparative Example 1 used only iron, with a degradation rate of 48%. However, the degradation rate of Comparative Example 4, without the addition of iron and organic acids, was lower than that of the present invention.

[0047] Performance testing Relative abundance of gene transcription of polycyclic aromatic hydrocarbon degrading enzymes in Examples 1, 2 and the comparative examples.

[0048] Experimental procedures: including extracellular laccase assay and prokaryotic transcriptomics assay. Results of the extracellular laccase assay are shown below. Figures 2-5 The results of the prokaryotic transcriptomics test are shown in [link to test]. Figure 6 .

[0049] Extracellular laccase in Bacillus is an important non-specific enzyme of polycyclic aromatic hydrocarbons (PAHs). The assay procedure for extracellular laccase is as follows: Prepare 0.1M sodium acetate buffer (pH=4.5) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) substrate solution (ABTS solution). Centrifuge the culture medium in inorganic medium at 10000 rpm for 10 minutes, resuspend twice in inorganic salt medium, and separate the supernatant extracellular laccase and the crude enzyme solution containing the broken precipitate. Add 2.4 mL of sodium acetate buffer solution, 0.5 mL of ABTS solution, and the crude enzyme solution sequentially to a quartz cuvette, mix well, start the timer, and monitor the absorbance change at 420 nm using a spectrophotometer. The experiment is conducted at 30℃, and the linear increase in absorbance over 3-5 minutes is recorded. The change in absorbance per minute (ΔA) is expressed as the percentage change. 420 The calculation was based on the enzyme activity (U / L), with an equivalent amount of enzyme solution that had been boiled and inactivated as a blank control. Enzyme activity (U / L) is defined as the amount of enzyme required to oxidize 1 μmol ABTS per minute under the above reaction conditions, calculated using the formula U / L = (ΔA / min). 420 ×V total ×10 6 ) / ɛ×d×V sample Calculations are performed, where α is the molar extinction coefficient of ABTS (36,000 M). -1 ·cm -1 ), d is the optical path of the cuvette (1 cm).

[0050] Prokaryotic transcriptomics is a method used to analyze gene transcription levels in microbial samples. The procedure is as follows: Take an appropriate amount of bacterial cells centrifuged at 10,000 rpm for 10 min, grind them thoroughly with liquid nitrogen, and transfer them to a 1.5 ml centrifuge tube containing 1 ml of Trizol total RNA extraction reagent, pre-chilled. Shake to ensure complete lysis and incubate at room temperature for 5 min. Centrifuge at 13,000 rpm for 5 min at 4°C, then transfer the supernatant to another centrifuge tube. Add 200 μl of pre-chilled chloroform at a ratio of 0.2 ml chloroform / 1 ml Trizol, vortex to mix, and incubate at room temperature for 5 min. Centrifuge at 13,000 rpm for 15 min at 4°C, then transfer the upper aqueous phase (avoiding the pipette tip from touching the intermediate protein layer) to another centrifuge tube (400 μl). Add an equal volume of pre-chilled isopropanol and incubate at room temperature for 10 min. Centrifuge at 13,000 rpm for 10 min at 4°C, discard the supernatant, and add 1 ml of pre-chilled 75% ethanol to resuspend the precipitate. Centrifuge at 12000 rpm for 5 min at 4℃, discard the supernatant, centrifuge the tube for a few more seconds, remove any residual liquid hanging on the tube wall, aspirate the residual liquid with a 10 μL pipette tip, and air dry at room temperature for 3-5 min. Dissolve in 20-50 μL of sterile 0.1% DEPC water. Further construct sequencing libraries using the rRNA strand-specific method, which can simultaneously sequence mRNA, lncRNA, and circRNA. Use the Illumina® Stranded TotalRNAPrep kit for strand-specific library construction. After extracting total RNA, removing rRNA, reversing to synthesize cDNA, ligating the adapter, digesting the cDNA double strand with UNG enzyme, library enrichment, and sequencing on the NovaSeqXPlus platform, use the FASTP tool to remove adapter sequences from reads. Remove low-quality (quality value less than 20) bases from the 3' end of the sequences, remove reads containing more than 10% N, and discard sequences with adapter removal and those shorter than 20 bp after quality trimming. The quality-controlled raw data was aligned with a reference genome to obtain reads for subsequent transcriptome assembly, expression level calculation, etc. Simultaneously, the quality of the transcriptome sequencing alignment results was assessed, primarily including sequencing saturation, gene coverage, read distribution in different regions of the reference genome, and read distribution across different chromosomes. After obtaining the ReadCounts for each gene, differential gene expression analysis was performed on multiple samples (≥2) to identify differentially expressed genes and further investigate their functions. The software used for differential expression analysis was DESeq2, and the default screening criteria for significantly differentially expressed genes were: FDR < 0.05 and |log2FC| ≥ 1.

[0051] like Figure 2 As shown, caffeic acid and ferric sulfate increased the degradation of extracellular enzymes within the first 3 days. Compared to Figure 3-5 As shown, oxalic acid reacts with ferric sulfate or ferric sulfate to induce a more than 1.5-fold upregulation of extracellular laccase activity within 3 days prior to degradation. Example 1 exhibits a higher extracellular laccase production, enabling the completion of more extracellular pretreatment processes for phenanthrene. Figure 6 As shown, the transcription level of the enzyme-producing gene CYP102, including laccase, in Example 1 is higher than that in Example 4. The results indicate that the degradation efficiency of phenanthrene by Bacillus in Example 1 is superior to that in Comparative Examples 1, 2, 3, and 4.

[0052] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a composite accelerator that accelerates the degradation of polycyclic aromatic hydrocarbons by microorganisms, characterized in that, Includes the following steps: A solution of an iron salt and a solution of a low molecular weight organic acid are provided. The iron salt solution and the low molecular weight organic acid solution are mixed and stirred to allow the iron ions to fully chelate with the low molecular weight organic acid, forming a composite accelerator.

2. The method for preparing a composite promoter for accelerating the microbial degradation of polycyclic aromatic hydrocarbons according to claim 1, characterized in that, The iron source is selected from ferric salts and / or ferrous salts; the concentration of the iron salt solution is 1000-5000 μM.

3. The method for preparing a composite promoter for accelerating the microbial degradation of polycyclic aromatic hydrocarbons according to claim 1, characterized in that, The concentration of the low molecular weight organic acid is 1000-5000 μM; the low molecular weight organic acid includes phenolic organic acids and / or aliphatic carboxylic acids.

4. A method for preparing a composite accelerator for accelerating the microbial degradation of polycyclic aromatic hydrocarbons according to claim 1, characterized in that, The chelation molar ratio of iron ions to low molecular weight organic acids is (1:5)-(1:10).

5. The method for preparing a composite promoter for accelerating the microbial degradation of polycyclic aromatic hydrocarbons according to claim 1, characterized in that, Mixing conditions: temperature 15-35℃, stirring speed 100-300rpm, time 0.5-2h.

6. A composite accelerator for accelerating the microbial degradation of polycyclic aromatic hydrocarbons, characterized in that, Prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the composite promoter according to claim 6 in accelerating the microbial degradation of polycyclic aromatic hydrocarbons, characterized in that, The application method includes the following steps: (1) Prepare and sterilize an inorganic salt culture medium, wherein the inorganic salt culture medium contains ammonium salt, potassium salt, magnesium salt, calcium salt and polycyclic aromatic hydrocarbons; (2) Inoculate the Bacillus seed culture into a sterile inorganic salt culture medium and add the compound promoter to the inorganic culture medium.

8. The application according to claim 7, characterized in that, In step (1), the concentration of the polycyclic aromatic hydrocarbon is 50-250 mg / L, and the polycyclic aromatic hydrocarbon includes one or more of phenanthrene, anthracene, pyrene or benzo[a]pyrene; The inorganic salt culture medium has a pH of 6.5-8.

5.

9. The application according to claim 7, characterized in that, In step (2), the inoculation amount of the Bacillus seed solution is 1-5%; the viable count of the Bacillus seed solution is (2.5-3.5)×10⁻⁶. 8 cfu / mL.

10. The application according to claim 7, characterized in that, In step (2), the molar concentration of iron salt in the inorganic salt medium in the composite promoter is 50-300 μM, the molar concentration of low molecular weight organic acid in the inorganic salt medium is 250-3000 μM, and the molar ratio of iron salt to low molecular weight organic acid is 1:5-10.

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