Pandoraea sp. For aerobically degrading pyrene as well as culture method and application of Pandoraea sp.

Abstract

The invention relates to a Pandoraea sp. For aerobically degrading pyrene, a culture method and application thereof, and belongs to the technical field of microbial biology. The bacterial strain is separated from rhizosphere soil of suaeda heteroptera in Liaoning estuary wetland, and is preserved in the China General Microbiological Culture Collection Center on April 24, 2024, and the preservation number is CGMCC NO.30434. The 16S rRNA gene sequence of the bacterial strain ZHL-3 is highly similar to that of Pandoraea, so that the classification name of the bacterial strain is Pandoraea sp.ZHL-3. The bacterial strain is named as Pandoraea sp.ZHL-3, and the bacterial strain is named as Pandoraea sp.ZHL-3. And the GenBank login number of the gene sequence of the rRNA of the strain 16S is OQ726226. The strain can grow by using various polycyclic aromatic hydrocarbon compounds as a unique carbon source, has relatively high pyrene degradation capacity, and also has good pH and salinity tolerance, so that the application prospect of the strain in the fields of sewage treatment, soil remediation and the like is shown.

Description

Technical Field

[0001] The invention belongs to the field of microbial biotechnology and relates to a strain of Pandora's bacterium capable of aerobically degrading pyrene, a cultivation method and application thereof. Technical Background

[0002] With the rapid development of the global economy, global demand for crude oil is increasing, and fossil fuels are increasingly accounting for a higher proportion of global energy use. However, the exploration, transportation, and refining of oil can lead to serious environmental pollution, resulting in the release of large quantities of polycyclic aromatic hydrocarbons (PAHs). Pyrene, a typical PAH, is difficult to degrade and highly toxic, posing a potential threat to both the environment and human health.

[0003] Bioremediation is a promising green technology for treating PAH-contaminated sites. Unlike physical and chemical processes, bioremediation methods are relatively low-cost, energy-efficient, and offer significant potential for improving environmental safety. Currently, numerous researchers at home and abroad have identified numerous strains capable of degrading pyrene, including bacteria such as Enterobacter, Bacillus, Mycobacterium, and Novosphingobium, as well as fungi such as Ganoderma lucidum, Penicillium, and Serratia. These strains can grow using pyrene as their sole carbon source. However, their pyrene-degrading abilities are limited, with only a few strains demonstrating significant pyrene degradation performance. Therefore, identifying new pyrene-degrading bacterial strains is crucial to advancing pyrene biodegradation research.

[0004] The genus Pandoraea has garnered widespread attention in recent years. Studies have shown that Pandoraea has broad applications in environmental remediation and pollutant degradation, including its ability to degrade organic pollutants such as lignin and polychlorinated biphenyls (PCBs). However, there are few reports on Pandoraea's ability to degrade pyrene. The Pandoraea sp. ZHL-3 employed in this study can grow using pyrene as its sole carbon and energy source and exhibits a strong pyrene-degrading ability. This bacterium has potential applications in the remediation of pyrene-containing wastewater, soil, and other contaminated sites. Summary of the Invention

[0005] The present invention aims to provide a Pandora bacteria strain that can aerobically degrade pyrene, a cultivation method, and its application. The strain can grow using a variety of polycyclic aromatic hydrocarbons as the sole carbon source, has a high ability to degrade pyrene, and has good pH and salinity tolerance, showing its application prospects in the remediation and treatment of contaminated sites such as pyrene-containing wastewater and soil.

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

[0007] A strain of Pandoraea that aerobically degrades pyrene has been deposited with the China General Microbiological Culture Collection under the registration number CGMCC No. 30434. The depositor's address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 24, 2024. The strain belongs to Pandoraea, and its 16S rRNA gene sequence shows high similarity to that of the genus Pandoraea. The strain has been designated Pandoraea sp. ZHL-3. The GenBank accession number for its 16S rRNA gene sequence is OQ726226.

[0008] The strain ZHL-3 16S rRNA gene sequence is as follows:

[0009] GCGGGGGCGGGCTGCTTAACATGCAGTCGAACGGCAGCACGGGTGCTTGCACCTG

[0010] GTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACGTACCTTGTAGTGGGGGATAGC

[0011] TCGGCGAAAGCCGGATTAATACCGCATACGCTCTGAGGAGGAAAGCGGGGGACCTTCG

[0012] GGCCTCGCGCTACAAGAGCGGCCGATGTCAGATTAGCTAGTTGGTGAGGTAAAAGCTCA

[0013] CCAAGGCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGAC

[0014] ACGGCCCAGACTCCTACGGGAGGCAGCAGTGKGGAATTTTGGACAATGGGCGAAAGCC

[0015] TGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCG

[0016] GAAAGAAATCCTCTGGGTTAATACCTCGGGGGGATGACGGTACCGGAAGAATAAGCACC

[0017] GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTA

[0018] CTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGACGGATGTGAAATCCCCGGGCTTAA

[0019] CCTGGGAACTGCATTCGTGACTGCAAGGCTAGAGTATGGCAGAGGGGGGTAGAATTCCA

[0020] CGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCT

[0021] GGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTG

[0022] GTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAACGTA

[0023] GCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGA

[0024] ATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAA

[0025] ACCTTACCTACCCTTGACATGTACGGAATCCTGCTGAGAGGTGGGAGTGCTCGAAAGAG

[0026] AACCGTAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAA

[0027] GTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGGCACTCCTARGGAG

[0028] ACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTAT

[0029] GGGTAGGGCTTCACACGTCATACAATGGTCGGTACAGAGGGCTGCCAAACCGCGAGGT

[0030] GGAGCTAACCCCAGAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTG

[0031] AAGCTGGAATCGCTAGTAATCGCGGATCAGCATGTCGCGGTGAATACGTTCCCGGGTCTT

[0032] GTACACACCGCCCGTCACACCATGGGAGTGGGTTTTGCCAGAAGTAGGTAGCCTAACCG

[0033] CAAGGAGGGCGCTACCACGGCAGAT

[0034] The strain ZHL-3 is isolated from the rhizosphere soil of Suaeda salsa in the Liaohe River estuary wetland in Panjin, Liaoning Province. The culture medium used for the isolation, purification and growth of the strain is a BH inorganic salt culture medium. The culture medium comprises 1.0 g / L KH2PO4, 1.0 g / L K2HPO4, 1.0 g / L (NH4)2SO4, and 0.2 g / L MgSO4. Each 100 mL of the BH inorganic salt culture medium contains 0.1 mL of a metal ion mother liquor. The metal ion mother liquor comprises: 10 g / L FeSO4·7H2O, 1.32 g / L CaCl2·2H2O, 3.56 g / L ZnSO4·7H2O, 0.30 g / L MnSO4·H2O, 0.06 g / L CuSO4, 0.12 g / L Na2MoO4·2H2O, and 0.02 g / L H3BO3. The strain is sterilized by passing through a 0.22 μm water filter membrane. 20 mg / L pyrene was added as required to the BH inorganic salt culture medium sterilized at 121°C and high temperature and high pressure for 20 min, and cultured at 30°C and 150 r / min under shaking conditions.

[0035] The strain ZHL-3 is applied to the degradation of pyrene. Within 8 days, the degradation rate of 20 mg / L pyrene by the strain ZHL-3 is kept above 60% in the range of pH 6.0-8.0 and salinity 0-20‰.

[0036] The present invention provides the following benefits: the strain ZHL-3 was isolated from the rhizosphere soil of Suaeda salsa in the Liaohe Estuary Wetland in Panjin, Liaoning Province. The strain ZHL-3 exhibits a slightly arcuate bacilli shape with obvious wrinkles and depressions on the bacterial surface. 16S rRNA gene sequence comparison reveals high similarity with Pandoraea, and the strain is therefore classified and named Pandoraea sp. ZHL-3. The GenBank accession number of the strain's 16S rRNA gene sequence is OQ726226. The strain can grow using a variety of polycyclic aromatic hydrocarbons as sole carbon sources, including phenanthrene, naphthalene, anthracene, salicylic acid, benzene, and toluene. It has a high ability to degrade pyrene. Within 8 days, the strain maintains a degradation rate of 20 mg / L pyrene above 60% within a pH range of 6.0-8.0 and a salinity range of 0-20‰. The strain has good pH and salinity tolerance, demonstrating its application prospects and potential in the treatment of pyrene-containing wastewater, soil, and other contaminated sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is an electron microscope image of Pandora strain ZHL-3.

[0038] Figure 2 This is the growth amount and degradation rate of Pandora's worm strain ZHL-3 under different pyrene concentrations.

[0039] Figure 3 The growth rate and pyrene degradation rate of Pandora strain ZHL-3 at different pH levels.

[0040] Figure 4 The growth rate and pyrene degradation rate of Pandora strain ZHL-3 under different salinities. DETAILED DESCRIPTION

[0041] The strain ZHL-3 was isolated by the inventor from the rhizosphere soil of Suaeda salsa in the Liaohe Estuary Wetland in Panjin, Liaoning Province in 2023. The strain was cultured with pyrene as the only carbon source, and a pure culture was obtained through repeated plate coating, separation and purification.

[0042] Culture medium: BH inorganic salt medium: KH2PO4 1.0g / L, K2HPO4 1.0g / L, (NH4)2SO4 1.0g / L, MgSO4 0.2g / L, each 100mL BH inorganic salt medium contains 0.1mL metal ion stock solution; the metal ion stock solution composition is: 10g / LFeSO4·7H2O, 1.32g / L CaCl2·2H2O, 3.56g / L ZnSO4·7H2O, 0.30g / L MnSO4·H2O, 0.06g / L CuSO4, 0.12g / L Na2MoO4·2H2O, 0.02g / L H3BO3; sterilize through a 0.22μm water filter membrane. LB solid medium: 10.0 g / L peptone, 5.0 g / L yeast extract powder, 10.0 g / L NaCl, 2% (W / V) agar, pH adjusted to 7.0-7.2 with NaOH / HCl.

[0043] Example 1: Obtaining Pandora bacteria ZHL-3

[0044] Rhizosphere soil samples from the Suaeda salsa plant from the Liaohe Estuary Wetland in Panjin, Liaoning Province were repeatedly diluted and plated onto LB solid medium plates and incubated at 30°C. Single colonies were selected and incubated in BH inorganic salt medium containing 20 mg / L pyrene at 30°C and 150 rpm to obtain a bacterial suspension. The cell morphology of strain ZHL-3 is slightly arcuate, with obvious wrinkles and depressions on the surface of the cell. The cell size is approximately (0.8-2) μm × (0.3-0.6) μm. Figure 1 .

[0045] Example 2: 16S rRNA molecular identification of strains

[0046] Colonies grown on LB solid medium were sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rRNA sequencing. Blast comparison of the sequencing results in Genbank revealed that the 16S rRNA sequence of ZHL-3 shared 99% similarity with that of Pandoraea , confirming that strain ZHL-3 belonged to the genus Pandoraea .

[0047] Example 3: Determination of the broad spectrum of substrates of Pandora bacteria ZHL-3

[0048] Using the bacterial solution from Example 1, BH inorganic salt medium with a 5% inoculum, the growth of strain ZHL-3 was investigated using various organic compounds as sole carbon sources, including phenanthrene, anthracene, naphthalene, benzene, toluene, benzoic acid, xylene, and p-xylene. The concentration of each compound was 50 mg / L. Table 1 shows the growth of Pandora's genus ZHL-3 using different compounds as sole carbon sources.

[0049] Table 1 Investigation on the broad spectrum of substrates of Pandora bacteria ZHL-3

[0050] pollutants result pollutants result Philippines + p-Xylene + anthracene + m-Xylene + Naphthalene + n-decane + benzene + n-Dodecane + Toluene + n-Hexadecane + benzoic acid + Catechol + o-Xylene + Salicylic acid +

[0051] Example 4: Effects of different pyrene concentrations on the growth and aerobic degradation of pyrene by Pandora bacteria ZHL-3

[0052] The bacterial solution in Example 1 was transferred to 100 mL of LB liquid medium at a 5% inoculum volume, cultured at 30°C, 150 rpm for 24 h, and centrifuged at 4°C, 8000 rpm for 10 min. The supernatant was discarded, and the cells were washed 2-3 times with BH inorganic salt medium and resuspended with BH inorganic salt medium. The OD was adjusted. 600 The initial bacterial solution was obtained by adding 100 mL of BH inorganic salt medium, 5% inoculum, pyrene concentrations of 10 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 80 mg / L, and 100 mg / L, pH 7.0, and shaking culture at 30 ° C and 150 r / min. After 8 days, the growth of the strain was detected (measured by OD 600 absorbance) and the residual concentration of pyrene. Figure 2 These are experimental results showing the growth and degradation rate of Pandora's bacteria ZHL-3 at different pyrene concentrations. The results show that strain ZHL-3 can degrade pyrene at various concentrations in a culture medium containing pyrene as the sole carbon source. It can degrade pyrene at concentrations ranging from 10 to 100 mg / L, with a degradation rate exceeding 85% for concentrations between 10 and 20 mg / L.

[0053] Example 5: Effects of different pH values ​​on the growth of Pandora bacteria ZHL-3 and aerobic degradation of pyrene

[0054] Prepare OD as in Example 4 600 The initial bacterial liquid was 1.0. 100 mL of BH inorganic salt medium, 5% inoculum, 20 mg / L pyrene concentration, pH values ​​of 5.0, 6.0, 7.0, 8.0, and 9.0 were used. Culture was performed at 30°C and 150 r / min, and the growth of the strain was detected after 8 days (measured by OD 600 absorbance) and the residual concentration of pyrene. Figure 3 These are experimental results showing the growth and pyrene degradation rates of Pandora's bacteria ZHL-3 at different pH levels. The results indicate that different pH levels affect the growth and aerobic pyrene degradation capabilities of strain ZHL-3. The strain grows well within a pH range of 6.0-8.0, achieving a pyrene degradation rate exceeding 60%. It also maintains good growth and high pyrene degradation rates at pH levels of 5.0 and 9.0, demonstrating excellent pH adaptability.

[0055] Example 6: Effects of different salinities on the growth of Pandora bacteria ZHL-3 and aerobic degradation of pyrene

[0056] Prepare OD as in Example 4 600 The initial bacterial liquid was 1.0. 100 mL of BH inorganic salt medium was used, with a 5% inoculum, a pyrene concentration of 20 mg / L, and a pH of 7.0. The salinity of the BH inorganic salt medium was adjusted to 0, 5‰, 10‰, 15‰, and 20‰ using NaCl. The culture was shaken at 30°C and 150 r / min. The growth of the strain was detected after 8 days (measured by OD 600 absorbance) and the residual concentration of pyrene. Figure 4 These are experimental results showing the growth and pyrene degradation rates of Pandora's bacteria ZHL-3 at different salinities. The results show that different salinities affect the growth and aerobic pyrene degradation ability of strain ZHL-3. The strain grows well in salinities ranging from 0 to 20‰, achieving a pyrene degradation rate exceeding 75% at low salinities of 0-10‰ and exceeding 60% at moderate salinities of 15‰ to 20‰, demonstrating its excellent salinity adaptability.

Claims

1. A strain of Pandora phylla that degrades pyrene, Its characteristics are: The registration number of the strain is CGMCCNO.30434, and is deposited in the China Common Microbial Sperm Collection Management Center. The deposit date is April 24, 2024, and the GenBank accession number of its 16SrRNA gene sequence is OQ726226.

2. A strain of Pandora pyrene-degrading strain according to claim 1, It is characterized in that The sequence of its strain ZHL-3 16S rRNA gene is as follows: GCGGGGCGGCTGCTTAACATGCAGTCGAACGGCAGCACGGGTGCTTGCACCTGGTGGCGAACGGGTGAGTAATACATCGGAACGTACCTTGTAGTGGGGATAGCTCGGCGAAAGCCGGATTAATACCGCATACGCTCTGAGGAAAGCGGGGGACCTTCGGCCTCG CGCTACAAGAGCGGCCGATGTCAGATTAGCTAGTTGGTGAGGTAAAAGCTCACCAAGGCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACACACACACACACTCCTACGGGAGGCAGCAGTGKGGAATTTGGACAATGGGCGAAAGCCTGATCCAA TGCCGCGTGTGTGAAGAAGGCCTTCGGTTGTAAAGCACTTTTGTCCGGAAAGAAATCCTCTGGGTTAATACCTCGGGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGTAATACGTAGGGTGCAAGCGTTAATCGGAATCGGAATTACTGGGCGTAAAGC GTGCGCAGGCGGTTTGTAAGACGGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTCGTGACTGCAAGGCTAGAGTATGGCAGAGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACG CTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTTAAACGATGTCAACTAGTTGGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCA CAAGCGGTGGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTACGGAATCCTGCTGAGAGGTGGGAGTGCTCGAAAGAGAACCGTAACACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCTTGTCCTTAGTTGCTACGCAAGAGGCACTCCTARGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGATGACGTCAAGTCCTCAATGGCCCTTATGGGTAGGCTTCACACGTCATACAATGGTCGGTACAGAGGGCTGCCAACCGCGAGGTGGAGCTAACCCCAGAAAACCG CGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCGTCACACCATGGGAGTGGGTTTTGCCAGAAGTAGGTAGCCTAACCGCAAGGAGGGCGCTACCACGGCAGAT .

3. The culture method of aerobic Pandora fungus degrading pyrene as claimed in claim 1, Its characteristics are: The strain is isolated from the rhizosphere soil of the winged pineapple wetland in Liaohekou, Panjin, Liaoning. The culture medium used for separation, purification and growth is BH inorganic salt medium, and the content of the medium is KH 2 PO 4 1.0g / L, K 2 HPO 4 1.0g / L, (NH4) 2 SO 4 1.0g / L, MgSO 4 0.2g / L, 0.1mL of metal ion mother liquor is contained in every 100mL of BH inorganic salt medium; the metal ion mother liquor is composed of: 10g / L FeSO 4 ·7H 2 O, 1.32g / L CaCl 2 ·2H 2 O, 3.56g / L ZnSO 4 ·7H 2 O, 0.30g / L MnSO 4 ·H 2 O, 0.06g / L CuSO 4 , 0.12g / L Na 2 MoO 4 ·2H 2 O, 0.02g / L H 3 BO 3 ; Sterilize the water-based filter membrane with a temperature of 0.22μm. 20 mg / L pyrene was added as required to the BH inorganic salt culture medium at high temperature autoclave 121°C and cultured under shaking and 150r / min.

4. The application of a strain of Pandora pyrene-degrading strain according to claim 1, Its characteristics are: The strain is used for the degradation of pyrene.

5. The application of claim 4, Its characteristics are: The degradation rate of strain ZHL-3 for 20 mg / L pyrene was maintained above 60% within the range of pH 6.0-8.0 and salinity of 0-20‰ within 8 days.

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