Arthrobacter pseudospermum capable of efficiently decomposing polycyclic aromatic hydrocarbons and its application

Abstract

The present invention relates to a strain of Pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons (PAHs) and its application. The Pseudoarthrobacter is named L1SW and has a deposit number of CGMCC25541. The Pseudoarthrobacter L1SW can utilize the PAH phenanthrene as its sole carbon source, can grow normally in a culture medium containing 5g / L phenanthrene, and can tolerate up to 65mg / L of zinc ions. The strain can completely decompose phenanthrene with an initial concentration of 500mg / L within 60 hours, and within 3 days, the decomposition rates of 3g / L, 4g / L, and 5g / L phenanthrene are 95%, 77%, and 57%, respectively. The Pseudoarthrobacter L1SW of the present invention can efficiently degrade phenanthrene, has good environmental adaptability, and can be used for removing PAH pollutants from soil and for soil remediation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a strain of Pseudomonas aeruginosa that can efficiently decompose polycyclic aromatic hydrocarbons and an application thereof. Background Art

[0002] PAHs are typical petrochemical pollutants. They can be produced by both natural activities (forest fires, volcanic eruptions) and human activities (oil spills, exhaust emissions, etc.), with human activities being the primary driver of their generation and pollution. PAHs are widely present in natural environments, including water, soil, and air, and exhibit potent teratogenicity, triatomine, and bioaccumulative effects, posing a direct threat to organisms and human health. Phenanthrene, a typical condensed-ring PAH, is widely distributed and poses a high level of environmental pollution, making it a common indicator for monitoring PAH pollution.

[0003] At present, microbial degradation is the main and effective method for remediating PAH pollution. It has developed rapidly in recent years and has broad application prospects. Compared with physical and chemical methods, microbial remediation technology has low cost, simple operation, and low secondary pollution due to the abundant microbial resources and easy cultivation. Among them, the core of microbial degradation of PAH compounds is to screen and obtain PAH-degrading strains with good environmental adaptability and high degradation efficiency, which is also the basis for realizing microbial remediation of PAH pollution in actual environments. In addition, since actual PAH-contaminated environments are often mixed with heavy metal components, this requires that the screened strains can not only efficiently degrade PAHs, but also effectively tolerate high heavy metal environments. Therefore, screening and identifying microbial strains that can effectively degrade PAHs under heavy metal conditions will help promote effective bioremediation of PAH-contaminated environments.

[0004] Chinese patent CN104946568A discloses a heavy metal-resistant polycyclic aromatic hydrocarbon (PAH)-degrading bacterium, a composition, and its uses. The bacterium is Mycobacterium gilvum, with a deposit number of CGMCC No. 10941. The bacterium can be used to remove or degrade PAHs, especially those in soil and water environments. It can also be used for bioremediation of PAH-contaminated soil and water environments, especially soil or water contaminated with both heavy metals and PAHs. The bacterium can also synergize with surfactants to enhance the ability to remove or degrade PAHs. However, the degradation efficiency of this strain is not very good. When the PAH content is 50 mg / L, the strain's degradation rate of phenanthrene on the fourth day is only 92.1%.

[0005] Chinese patent CN102943052A discloses a strain of Klebsiella pneumoniae (Klebsiella pneumoniae etzyx1) that degrades polycyclic aromatic hydrocarbons (PAHs) and its use in the remediation of compositely contaminated soil. The strain, Klebsiella pneumoniae Tzyx1, has the China Center for Type Culture Collection accession number CCTCCM2012239 and is capable of degrading PAHs. However, the degradation efficiency of this strain is not very good. The patent states that after 15 days of KL+M treatment, the PAH degradation rates in soil were 86.6% for naphthalene and 91.0% for phenanthrene.

[0006] Chinese patent CN113583899A discloses a polycyclic aromatic hydrocarbon-degrading strain of the genus Sphingobacterium, JT-M9-H, and its patent application. The strain, designated Sphingobacterium sp. JT-M9-H and deposited with CCTCC NO: M2021248, was submitted to the China Center for Type Culture Collection, Wuhan University, Wuhan, China, for deposit on March 18, 2021. However, the patent only describes the treatment effects of low concentrations of phenanthrene (initial concentrations of 80 and 20 mg / L, respectively), and does not address the effects of high concentrations.

[0007] Therefore, the degradation efficiency and substrate tolerance of the bacteria for decomposing polycyclic aromatic hydrocarbons disclosed in the prior art need to be improved. Summary of the Invention

[0008] The present invention aims to provide a strain of Pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons and its application. The Pseudoarthrobacter can stably and efficiently decompose polycyclic aromatic hydrocarbons under heavy metal conditions and has application potential in repairing polycyclic aromatic hydrocarbon-contaminated environments.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] The present invention first provides a strain of Pseudomonas aeruginosa that can efficiently decompose polycyclic aromatic hydrocarbons, named L1SW, which was deposited in the General Microbiology Center of the China Culture Collection Administration on August 17, 2022, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 25541.

[0011] The present invention provides a pseudoarthrobacter L1SW strain derived from contaminated soil at the Lanzhou Petrochemical Company's oil refining site, obtained through artificial enrichment and screening. L1SW colonies are round, raised, milky white, opaque, Gram-positive, grow singly or in pairs, are aerobic, and grow well at 30°C. Identification and sequence analysis of the strain's 16S rDNA indicate that the strain is a pseudoarthrobacter ( Pseudarthrobactersp. ).

[0012] The present invention also provides the 16S rDNA of the pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons, and the sequence thereof is shown in SEQ ID NO.1.

[0013] The present invention further provides a method for obtaining the pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons, comprising the following steps:

[0014] A. Collect contaminated soil from oil refining sites as a microbial source;

[0015] B. Prepare 100 mL of inorganic salt medium containing 500 mg / L phenanthrene using PAHs as the sole carbon source, adjust the pH to 7.4 with hydrochloric acid, and sterilize at 121°C for 20 min.

[0016] C. When the culture medium temperature drops to about 30°C, inoculate under a clean bench at a soil inoculum rate of 3%;

[0017] D. Carry out enrichment culture in a constant temperature shaker incubator at 30°C and 180 rpm;

[0018] E. After repeated cultivation three times, repeatedly streak the culture on a solid culture medium plate until a single strain is isolated. Then, the strain is inoculated into the above-mentioned inorganic culture medium for acclimation. The above operation is repeated several times to obtain a purified pseudoarthrobacter, which is named L1SW.

[0019] In the above-mentioned method for obtaining Arthrobacter pseudospermum, preferably, the composition and concentration of the inorganic salt culture medium are as follows: K2HPO4·3H2O, 13.75 g / L; KH2PO4, 4.5 g / L; (NH4)2SO4, 2.0 g / L; MgSO4·7H2O, 0.16 g / L; FeSO4·7H2O, 0.005 g / L; CaCl 2· 2H2O, 0.011 g / L; MnCl2·4H2O, 0.002 g / L.

[0020] The present invention further provides an application of the Pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons. The Pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons is used to decompose polycyclic aromatic hydrocarbons.

[0021] Furthermore, the Arthrobacter pseudotyped, which can efficiently decompose polycyclic aromatic hydrocarbons, is used for the decomposition and removal of polycyclic aromatic hydrocarbons in a heavy metal environment.

[0022] In one embodiment of the present invention, the heavy metal environment is a soil environment or a water environment.

[0023] In one embodiment of the present invention, the heavy metal ions are one or more of Cu, Cd, Pb, Zn, and As. Specifically, the heavy metal conditions may be an environment containing 50-100 mg / L zinc ions, for example, 65 mg / L zinc ions.

[0024] In one embodiment of the present invention, the polycyclic aromatic hydrocarbon is one or more of naphthalene, fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Preferably, the polycyclic aromatic hydrocarbon is phenanthrene.

[0025] The pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons described in the present invention uses polycyclic aromatic hydrocarbons (preferably phenanthrene) as the sole carbon source.

[0026] The present invention further provides a bacterial agent, wherein the bacterial agent contains the pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons.

[0027] In one embodiment of the present invention, the bacterial agent further contains a surfactant.

[0028] The present invention also provides a method for removing polycyclic aromatic hydrocarbons in a heavy metal environment, and adopts the pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons to remove polycyclic aromatic hydrocarbons in the heavy metal environment.

[0029] The Pseudoarthrobacter strain L1SW of the present invention can grow normally in a culture medium containing 5 g / L of polycyclic aromatic hydrocarbons (PAHs) using PAHs as the sole carbon source and can tolerate up to 65 mg / L of zinc ions. The strain can completely decompose phenanthrene at an initial concentration of 500 mg / L within 60 hours, and within three days, the decomposition rates for 3 g / L, 4 g / L, and 5 g / L of phenanthrene reach 95%, 77%, and 57%, respectively.

[0030] According to the present invention, Pseudomonas aeruginosa Pseudarthrobactersp. The L1SW strain is highly efficient in degrading polycyclic aromatic hydrocarbons (PAHs) in the presence of heavy metals and is therefore suitable for the remediation and remediation of complex polluted environments containing these pollutants. Compared to previously discovered PAH-degrading strains, the strain described in this paper exhibits higher degradation efficiency, substrate tolerance, and environmental adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Arthrobacter pseudorhizobacter Pseudarthrobactersp. Flat plate line drawing of L1SW;

[0032] Figure 2 Arthrobacter pseudorhizobacter Pseudarthrobactersp. Growth curve of strain L1SW when low concentration (50 mg / L; 250 mg / L; 500 mg / L) of phenanthrene was used as the sole carbon source;

[0033] Figure 3 Arthrobacter pseudorhizobacterPseudarthrobactersp. Growth curve of strain L1SW when high concentration (3 g / L; 4 g / L; 5 g / L) of phenanthrene was used as the sole carbon source;

[0034] Figure 4 Arthrobacter pseudorhizobacter Pseudarthrobactersp. Degradation curves of L1SW for low concentrations (50 mg / L; 250 mg / L; 500 mg / L) of phenanthrene;

[0035] Figure 5 Arthrobacter pseudorhizobacter Pseudarthrobactersp. Degradation curves of L1SW for high concentrations (3 g / L; 4 g / L; 5 g / L) of phenanthrene;

[0036] Figure 6 Arthrobacter pseudorhizobacter Pseudarthrobactersp. Degradation of 250 mg / L phenanthrene by L1SW at different zinc ion concentrations (0.65 mg / L; 6.5 mg / L; 32.5 mg / L; 65 mg / L);

[0037] Figure 7 Arthrobacter pseudorhizobacter Pseudarthrobactersp. Growth of L1SW at different zinc ion concentrations (0.65 mg / L; 6.5 mg / L; 32.5 mg / L; 65 mg / L) with 250 mg / L phenanthrene as the sole carbon source. DETAILED DESCRIPTION

[0038] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Example 1

[0040] 1. Pseudoarthrobacter Pseudarthrobactersp. Acquisition and preservation of L1SW

[0041] Contaminated soil from the Lanzhou Petrochemical Company's oil refining site was collected as a microbial source and transported to the laboratory in refrigerated condition. An inorganic salt medium containing 500 mg / L of polycyclic aromatic hydrocarbons was prepared and inoculated into a 250-mL Erlenmeyer flask containing 100 mL of the inorganic salt medium at a 3% soil inoculum. The composition of the inorganic salt medium was as follows: K₂HPO₄·3H₂O, 13.75 g / L; KH₂PO₄, 4.5 g / L; (NH₄)₂SO₄, 2.0 g / L; MgSO₄·7H₂O, 0.16 g / L; FeSO₄·7H₂O, 0.005 g / L; and CaCl₃. 2·2H2O, 0.011 g / L; MnCl2·4H2O, 0.002 g / L. Adjust the pH to 7.4 with hydrochloric acid and sterilize in an autoclave at 121°C for 20 min. When the culture medium temperature drops to about 30°C, inoculate the sludge under the clean bench. Then enrich and culture in a constant temperature shaker incubator at 30°C and 180 r / min for 7 days. After repeating the culture three times, repeatedly streak the culture on a solid culture medium plate (agar 20.0 g / L) until a single strain is isolated. Afterwards, the strain is inoculated into the above-mentioned inorganic culture medium for acclimation. The above operation is repeated many times to obtain a purified L1SW strain that can degrade polycyclic aromatic hydrocarbons. The plate streak diagram is shown as follows: Figure 1 As shown. L1SW colonies are round, raised, milky white, and opaque; Gram staining is positive, single or in pairs, aerobic, and grows well at 30°C. The identification and sequence analysis of the 16S rDNA of the strain showed that it was a pseudoarthrobacter ( Pseudarthrobactersp. The Pseudomonas aeruginosa L1SW strain was deposited on August 17, 2022 at the General Microbiology Center of the China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number CGMCC No. 25541.

[0042] 2. Pseudoarthrobacter Pseudarthrobactersp. Identification of L1SW

[0043] The isolated L1SW strain was subjected to 16S rDNA amplification, sequencing, and database comparison. Genomic DNA from the L1SW strain was extracted using a genome extraction kit (TIANGEN, Beijing). The 16S rDNA fragment was then amplified and sequenced using a 16S rDNA fragment amplification and detection kit (Takara, Dalian). The 16S rDNA sequence of strain L1SW is shown in SEQ ID NO. 1. Comparison with the NCBI database (http: / / www.ncbi.nlm.nih.gov / ) indicated that the L1SW strain was a pseudoarthrobacter. Pseudarthrobactersp. .

[0044] SEQ ID NO.1

[0045] ttacacatgc aagtcgaacg atgatcccag cttgctgggg gattagtggc gaacgggtga 60

[0046] gtaacacgtg agtaacctgc ccttaactct gggataagcc tgggaaactg ggtctaatac 120

[0047] cggatatgac tcctcatcgc atggtgggg gtggaaagct ttttgtggtt ttggatggac 180

[0048] tcgcggccta tcagcttgtt ggtgaggtaa tggcttacca aggcgacgac gggtagccgg 240

[0049] cctgagaggg tgaccggcca cactgggact gagacacggc ccagactcct acgggaggca 300

[0050] gcagtgggga atattgcaca atgggcgcaa gcctgatgca gcgacgccgc gtgagggatg 360

[0051] acggccttcg ggttgttaac ctctttcagt agggaagaag cgtaagtgac ggtaacctgca 420

[0052] gaaagcgc cggctacta cgtgccagca gccgcggtaa tacgtagggc gcaagcgtta 480

[0053] tccggaatta ttgggcgtaa agagctcgta ggcggtttgt cgcgtctgcc gtgaaagtcc 540

[0054] ggggctcaac tccggatctg cggtgggtac gggcagacta gagtgatgta ggggagactg 600

[0055] gaattcctgg tgtagcggtg aaatgcgcag atatcaggag gaacaccgat ggcgaaggca 660

[0056] 720

[0057] taccctggta gtccatgccg taaacgttgg gcactaggtg tgggggacat tccacgtttt 780

[0058] tccgcgccgt agctaacgca ttaagtgccc cgcctgggga gtacggccgc aaggctaaaa 840

[0059] ctcaaaggaa ttgacggggg cccgcacaag cggcggagca tgcggattaa ttcgatgcaa 900

[0060] cgcgaagaac cttaccaagg cttgacatga accggtaata cctggagaca ggtgccccgc 960

[0061] ttgcggtcgg tttacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt1020

[0062] taagtcccgc aacgagcgca accctcgttc tatgttgcca gcacgtgatg gtggggactc1080

[0063] ataggagact gccggggtca actcggagga aggtggggac gacgtcaaat catcatgccc1140

[0064] cttatgtctt gggcttcacg catgctacaa tggccggtac aaagggttgc gatactgtga1200

[0065] ggtggagcta atcccaaaaa gccggtctca gttcggattg gggtctgcaa ctcgacccca1260

[0066] tgaagtcgga gtcgctagta atcgcagatc agcaacgctg cggtgaatac gttcccgggc1320

[0067] cttgtacaca ccgcccgtca agtcacgaaa gttggtaaca cccgaagccg gtggcctaac1380

[0068] cccttgtggg agggagctgt cgaaggtgg 1409

[0069] 3. Pseudoarthrobacter Pseudarthrobactersp. Degradation efficiency of polycyclic aromatic hydrocarbons by strain L1SW

[0070] Will Pseudarthrobactersp. The L1SW strain was streaked onto solid culture plates and incubated at 30°C for 3 days. Single colonies were selected and inoculated into liquid culture medium containing 50-5000 mg / L of polycyclic aromatic hydrocarbons (phenanthrene) as the sole carbon source. Cultures were performed in a constant-temperature shaking incubator at 30°C and 180 rpm for 3-7 days. Two 3 mL replicate samples were collected at different time points. The absorbance of one sample at 600 nm was measured using a UV-2100 spectrophotometer (Unic) to indicate microbial growth. The results are shown in the table below. Figure 2 and Figure 3 As shown. An equal volume of acetonitrile was added to another sample and mixed, ultrasonicated for 5 minutes, filtered with a 0.22 μm filter membrane, 100 μl was taken for filtration, and the filtrate was injected into the inner tube of the injection bottle. The residual amount of polycyclic aromatic hydrocarbons phenanthrene was determined by ultra-high performance liquid chromatography (UPLC, Agilent 1290) using a ZORBAX Eclipse Plus C18 reverse phase column (5 μm, 4.6×150 mm). The residual concentration of polycyclic aromatic hydrocarbons phenanthrene at different initial concentrations is shown in the figure. Figure 4 and Figure 5 As shown. The strain L1SW can grow normally in a culture medium containing 5 g / L of the polycyclic aromatic hydrocarbon phenanthrene as the sole carbon source. The strain can completely decompose phenanthrene with an initial concentration of 500 mg / L within 60 hours, and the decomposition rates of 3 g / L, 4 g / L, and 5 g / L phenanthrene reach 95%, 77%, and 57% respectively within 3 days. The strain can still grow in a culture medium containing 65 mg / L zinc ions with phenanthrene as the sole carbon source, and completely degrade 250 mg / L phenanthrene within 96 hours. The results are shown as follows. Figure 6 and Figure 7 shown.

[0071] Based on the description of the examples of this application, it can be seen that Pseudomonas aeruginosa Pseudarthrobactersp.The L1SW strain is highly efficient at degrading polycyclic aromatic hydrocarbons (PAHs) in the presence of heavy metals and can be used to treat and remediate complex polluted environments containing these pollutants. Compared to previously discovered PAH-degrading strains, the strain possesses higher degradation efficiency, substrate tolerance, and improved environmental adaptability.

[0072] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A strain of Pseudomonas aeruginosa that can efficiently decompose polycyclic aromatic hydrocarbons ( Pseudarthrobacter sp. ) L1SW, characterized in that The deposit number is CGMCC No.25541.

2. The use of the pseudoarthrobacterium that can efficiently decompose polycyclic aromatic hydrocarbons as claimed in claim 1, characterized in that: The pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons is used for decomposing and removing polycyclic aromatic hydrocarbons in a heavy metal environment, wherein the polycyclic aromatic hydrocarbons are phenanthrene and the heavy metal is Zn.

3. The use according to claim 2, characterized in that The heavy metal environment is a soil environment or a water environment.

4. A bacterial agent, characterized in that The bacterial agent contains the pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons as claimed in claim 1.

5. The microbial agent according to claim 4, characterized in that The bacterial agent also contains a surfactant.

6. A method for removing polycyclic aromatic hydrocarbons in a heavy metal environment, characterized in that: The polycyclic aromatic hydrocarbons in a heavy metal environment are removed by using the pseudoarthrobacter that can efficiently decompose polycyclic aromatic hydrocarbons as claimed in claim 1, wherein the heavy metal is Zn and the polycyclic aromatic hydrocarbons are phenanthrene.

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