A strain of Paracoccus mutans capable of degrading benzo[a]pyrene and its application

Abstract

The present invention provides a strain of Paracoccus mutans capable of degrading benzo[a]pyrene. The provided Paracoccus mutans strain LYCW-1-6 has a deposit number of CGMCC No. 24841. The Paracoccus mutans provided by the present invention can be used to degrade benzo[a]pyrene. When the benzo[a]pyrene-degrading bacteria is inoculated at a ratio of 2% into a liquid culture medium containing 100 mg / L of benzo[a]pyrene, after 7 days of culture, the benzo[a]pyrene in the culture medium can be degraded by up to 70%. Furthermore, compared to other reported benzo[a]pyrene-degrading bacteria, the strain of the present invention is capable of treating soil or water contaminated with high concentrations of benzo[a]pyrene.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental remediation microorganism screening and application, and specifically relates to a strain of Paracoccus mutans capable of degrading benzopyrene and an application thereof. Background Art

[0002] Benzopyrene (B[a]pyrene) is a highly carcinogenic polycyclic aromatic hydrocarbon (PAH) found in automobile exhaust, petrochemical refinery exhaust, and flue gases from the combustion of various types of carbon black, coal, and petroleum. It is highly carcinogenic, mutagenic, and teratogenic, and can also produce phototoxicity. Its presence in soil is highly insidious, with a long incubation period, widespread impact, and difficulty in control.

[0003] Years of research have shown that microbial decomposition is the safest and most effective method for degrading benzo[a]pyrene. Reported bacteria that can degrade benzo[a]pyrene include strains such as Alcanivorax xenomutans, Microbacterium sp., Sphingomonas sp., Pseudomonas sp., Mycobacterium sp., and Acinetobacter sp.

[0004] However, due to the different polluted environments, screening new strains capable of degrading benzopyrene is still one of the research hotspots in the field of environmental governance. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain of Paracoccus mutans with the function of degrading benzopyrene and its application, thereby making up for the shortcomings of the existing technology.

[0006] The Paracoccus versutus LYCW-1-6 strain provided by the present invention was deposited in the General Microbiology Center of the China Culture Collection Administration on May 6, 2022. The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.24841.

[0007] The 16S gene sequence of the Paracoccus multocida is SEQ ID NO: 1;

[0008] The Paracoccus mutans provided by the present invention can be used for the degradation of polycyclic aromatic hydrocarbons;

[0009] The polycyclic aromatic hydrocarbon, as a specific example, is benzopyrene.

[0010] The present invention also provides a method for degrading benzo[a]pyrene, which uses the above-mentioned Paracoccus mutans as a degrading bacterium to degrade benzo[a]pyrene.

[0011] In another aspect, the present invention provides a preparation for degrading benzopyrene, wherein the preparation contains live bacteria of the above-mentioned Paracoccus mutans.

[0012] The Paracoccus mutans strain provided by the present invention can be used to degrade benzo(a)pyrene. When the benzo(a)pyrene-degrading bacteria is inoculated at a 2% concentration into a liquid culture medium containing 100 mg / L of benzo(a)pyrene, after seven days of culture, the benzo(a)pyrene in the culture medium can be degraded by up to 70%. Furthermore, compared to other reported benzo(a)pyrene-degrading bacteria, the strain of the present invention is more capable of treating soil or water contaminated with high concentrations of benzo(a)pyrene. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : A culture photograph of the Paracoccus mutans LYCW-1-6 strain screened by the present invention,

[0014] Figure 2 : Benzopyrene degradation efficiency curve of LYCW-1-6 strain from day 0 to day 7. DETAILED DESCRIPTION

[0015] The present invention is described in detail below with reference to the embodiments and accompanying drawings.

[0016] Example 1: Enrichment, screening and identification of strains

[0017] The samples for screening strains were soil samples from the vicinity of the Dagang Oilfield Refinery. 100 ml of normal saline was added to 10 g of soil sample, shaken for 3 hours, and allowed to stand for 10 minutes. After that, 5 ml was inoculated into benzopyrene inorganic salt culture medium for enrichment.

[0018] The formula for the benzopyrene inorganic salt medium is as follows: 3.58g of Na2HPO4, 2.32g of NaCl, 0.15g of KCl, 0.54g of NH4Cl, 0.1g of MgSO4.7H2O, 0.04g of CaCl2·2H2O, and 10ml of trace element solution. Dissolve and mix the above substances in pure water, dilute to 1L, and adjust the pH to 7.0. The trace element solution contains (per liter): 200mg of FeSO4·7H2O, 10mg of ZnSO4·7H2O, 3mg of MnCl2·4H2O, 20mg of CoCl2·6H2O, 1mg of CuCl2·2H2O, 2mg of NiCl2·6H2O, 500mg of Na2MoO4·2H2O, and 30mg of H3BO3. The final concentration of benzopyrene is 100 mg / L (100 mg is dissolved in 10 ml of acetone, and 1 ml of benzopyrene acetone solution is added to every 100 ml of culture medium)

[0019] Culture at 30°C in a shaker at 180 rpm, transferring every 7 days. After 20 generations of subculture, isolate the individual strains. For single-strain isolation, add 20% agar to the aforementioned benzopyrene inorganic salt medium to create a solid medium. After solidification, apply benzopyrene acetone solution to the surface. Apply 10 μl of 10 mg / ml benzopyrene acetone solution to each 90 mm solid medium. Allow to evaporate at room temperature for one day before use.

[0020] After diluting and smearing the benzopyrene-degrading bacterial colony, the plate was cultured at 30°C for 3-5 days, and the single colony on the plate was streaked and purified to obtain the pure culture of the bacterial strain LYCW-1-6 ( Figure 1 ).

[0021] The 16S gene of the strain was amplified and sequenced using bacterial 16S universal primers 27F and 1492R, and the 16S sequence obtained was as follows:

[0022]

[0023] Blast comparison identified it as Paracoccus versutus and named it Paracoccus versutus LYCW-1-6 strain. It was deposited in the General Microbiology Center of China Culture Collection Administration on May 6, 2022. The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.24841.

[0024] Example 2: Identification of Benzopyrene Degradation Ability of Benzopyrene-Degrading Bacteria

[0025] The strain LYCW-1-6 was cultured using the above-mentioned inorganic salt culture medium, wherein the final concentration of benzopyrene added was still 100 mg / L, and the inoculation amount was 2%. The residual benzopyrene in the culture system was detected on days 0-7 after inoculation to calculate the degradation rate of benzopyrene by the strain.

[0026] The detection method of benzopyrene is as follows: add 10mL of dichloromethane to the reaction system, shake and extract for 5 minutes, transfer to a separatory funnel, let it stand, extract 3 times, combine the extracts, and finally adjust the volume to 25mL. The concentration of benzopyrene was determined using a high performance liquid chromatograph (C18 reverse phase column). The detection method selected was the ultraviolet method, the detection wavelength was set to 295nm, the mobile phase was 90% acetonitrile + 10% water, and the flow rate was 1.0mL / min. The results of the degradation of benzopyrene by the LYCW-1-6 strain are shown in Table 1, and the degradation efficiency curve is shown in Figure 2 shown.

[0027] Table 1: Benzopyrene degradation efficiency of LYCW-1-6 strain on days 0-7

[0028] Time (days) Control concentration mg / L Treatment concentration mg / L Degradation efficiency% 0 138.32 126.22 8.75 1 134.81 75.91 43.69 2 146.73 62.55 57.37 3 144.14 60.17 58.25 4 129.34 44.86 65.32 5 131.67 42.54 67.69 6 124.94 40.08 67.92 7 108.14 34.42 68.17

[0029] Example 3: Identification of the ability of Paracoccus mutans LYCW-1-6 to utilize other polycyclic aromatic hydrocarbons

[0030] The carbon source in the above-mentioned inorganic salt culture medium was replaced by the following polycyclic aromatic hydrocarbons: pyrene, fluorene, carbazole, fluoranthene, anthracene, phenanthrene, acenaphthene, and naphthalene, each at a concentration of 100 mg / L. The culture conditions were the same as those in Example 1.

[0031] The specific method is as follows: Preparation of PAH preparation solution: Dissolve 500 mg of each of fluorene, carbazole, fluoranthene, acenaphthene, and naphthalene in 50 mL of acetone, store in a brown bottle, and protect from light at 4°C; dissolve 500 mg of pyrene in 50 mL of anhydrous ethanol, store in a brown bottle, and protect from light at 4°C; dissolve 250 mg of anthracene in 50 mL of acetone, store in a brown bottle, and protect from light at 4°C. Prepare the aforementioned inorganic salt culture medium, with each culture volume 30 mL. After sterilization, add 300 μL (600 μL for anthracene) of the PAH preparation solution to each culture volume in a clean hood, resulting in a final concentration of 100 mg / L. The PAH-supplemented culture medium was inoculated with Paracoccus mutans LYCW-1-6 at a 2% ratio and cultured in a shaker at 180 rpm and 30°C, protected from light. PAH utilization capacity was measured on day 7 of the culture.

[0032] After 7 days of culture, the utilization capacity of the Paracoccus mutans LYCW-1-6 strain for the above-mentioned polycyclic aromatic hydrocarbons is shown in Table 2.

[0033] Table 2: Identification of the ability of Paracoccus mutans LYCW-1-6 strain to utilize polycyclic aromatic hydrocarbons

[0034] Substrate type Utilization ability Fluoranthene ﹢ Fluorine ﹢ Naphthalene ﹢ Acenaphthene ﹢ carbazole ﹢ Philippines ﹢ anthracene ﹢ pyrene ﹢

[0035] Note: "+" means it can be used, "-" means it cannot be used

[0036] Example 4: Identification of the ability of Paracoccus mutans LYCW-1-6 to repair contaminated soil

[0037] 1. Laboratory Benzopyrene-Contaminated Soil Experiment Design: Benzopyrene degradation experiments were conducted using Paracoccus mutans LYCW-1-6 on a natural benzopyrene-contaminated soil located in Dongli District, Tianjin. The initial benzopyrene concentration was 4-5 mg / kg. The experimental design consisted of a control group (CK) without the addition of the benzopyrene-degrading strain LYCW-1-6, and an experimental group (D) supplemented with the benzopyrene-degrading strain LYCW-1-6, 5% bacterial inoculant, 3% wheat straw, and 2.4% activated carbon. Each experimental group was set up in triplicate.

[0038] 2. Laboratory Benzopyrene-Contaminated Soil Remediation: Each experiment was conducted on 500g of soil, placed in a 30°C incubator in the dark, and incubated. The soil surface was covered with gauze and sprayed with tap water daily to replenish lost moisture. Soil samples were collected every ten days to measure residual benzopyrene concentrations. An equal proportion of microbial inoculant was added every 30 days to maintain sufficient soil microbial activity. The soil remediation experiment was tentatively scheduled for 60 days. The results after 60 days of degradation are shown in Table 3 below.

[0039] 3. Selection and activation of microbial agents: Paracoccus mutans LYCW-1-6 was used as the microbial agent for this soil remediation.

[0040] Select screening medium: 3.58g Na2HPO4, 2.32g NaCl, 0.15g KCl, 0.54g NH4Cl, 0.1g MgSO4.7H20, 0.04g of CaCl2-2H2O, 10ml trace element solution; initial pH 7.0.

[0041] Glucose (3 g / L) was added to the culture medium to activate the microbial agent. When the bacterial count grew to 1×10 9 Inoculate into the soil.

[0042] 4. Detect the BaP degradation efficiency of the soil at 0d, 10d, 20d, 30d, 40d, 50d, and 60d, and use ultrasonic extraction to detect the BaP content in the soil. The specific method is as follows:

[0043] 4.1 Principle: BaP in soil was extracted by ultrasonic extraction with acetonitrile-tetrahydrofuran, and the content of BaP was quantitatively detected by high performance liquid chromatography-fluorescence detector.

[0044] 4.2 Detection process:

[0045] 4.2.1 Sample preparation: Air-dry the soil samples at room temperature in the dark and remove gravel, plant roots and other debris. Grind the air-dried samples, sieve them (60 mesh), and then quarter the samples.

[0046] 4.2.2 Sample extraction: Weigh 1 g of sample to the nearest 0.001 g, add 10 mL of acetonitrile-tetrahydrofuran mixed solution, mix well, extract by ultrasonication for 20 min, centrifuge for 3-5 min (5000 r / min), remove the supernatant, add 10 mL of acetonitrile-tetrahydrofuran mixed solution, extract for 30 min according to the above conditions, combine the two supernatants, make up to 25 mL with acetonitrile-tetrahydrofuran mixed solution, filter through a microporous filter membrane, and use the filtrate as the test solution.

[0047] 4.2.3 Determination of BaP sample: Inject 10 μL of the test solution into the liquid chromatograph and perform liquid chromatography determination according to the conditions for preparing the BaP standard. Calculate the BaP concentration in the test solution based on the standard curve.

[0048] 4.2.4 Calculation results: The mass concentration of BaP in the sample is calculated according to formula ①.

[0049] W=CV / m ①

[0050] Where:

[0051] W----BaP content in the sample, in milligrams per kilogram;

[0052] C-----BaP concentration obtained from the standard curve, in micrograms per milliliter;

[0053] V------The volume of the liquid to be tested, in milliliters

[0054] m------sample mass, in g.

[0055] Table 3: Benzopyrene contaminated soil remediation data

[0056] time CK group concentration mg / kg Group D concentration mg / kg 0d 3.54 3.57 10d 2.54 1.87 20d 1.44 1.76 30d 3.42 2.10 40d 2.51 1.34 50d 2.16 1.55 60d 2.00 1.36

[0057] These results demonstrate that the Paracoccus mutans strain provided by the present invention can be used to degrade benzo(a)pyrene. When the benzo(a)pyrene-degrading bacteria were inoculated at a 2% concentration into a liquid culture medium containing 100 mg / L of benzo(a)pyrene, up to 70% of the benzo(a)pyrene in the culture medium was degraded after seven days of culture. Furthermore, compared to other reported benzo(a)pyrene-degrading bacteria, the strain of the present invention is more capable of treating soil or water contaminated with high concentrations of benzo(a)pyrene.

Claims

1. A Paracoccus mutans, characterized in that The Paracoccus mutans ( Paracoccus versutus ) is deposited in China with a number of CGMCC NO. 24841.

2. Use of the Paracoccus mutans according to claim 1 in degrading benzo[a]pyrene.

3. A method for degrading benzopyrene, characterized in that: The method is to use the Paracoccus mutans described in claim 1 as a degrading bacterium to degrade benzopyrene.

4. A preparation for degrading benzopyrene, characterized in that: The preparation contains live bacteria of Paracoccus mutans according to claim 1.

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