BTEX degrading bacterium in petrochemical enterprise polluted site in coastal saline-alkali area as well as screening method and application of BTEX degrading bacterium

Abstract

The invention relates to the technical field of microbial remediation of environmental pollution, in particular to a BTEX degrading bacterium in a contaminated site of petrochemical enterprises in a coastal saline-alkali area as well as a screening method and application of the BTEX degrading bacterium, the BTEX degrading bacterium is Pseudomonas sp. LO-A1 and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the BTEX degrading bacterium is CCTCC No: M20231888. The pseudomonas sp. LO-A1 has the advantages that the degradation efficiency of the pseudomonas sp. LO-A1 on the benzene series is high, and the good degradation effect on the benzene series under the condition of high salinity and alkalinity is also realized. Benzene series in water are degraded through the strain, the purpose of removing pollutants can be achieved, intermediate products are thoroughly degraded, the operation cost is low, negative influences on the environment are small, and the strain has good development and utilization prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial remediation of environmental pollution, and particularly relates to a BTEX-degrading bacterium for a polluted site of a petrochemical enterprise in production in a coastal saline-alkali area, a screening method thereof, and an application thereof. Background Art

[0002] Benzene series compounds (BTEX) include benzene, toluene, ethylbenzene, and xylene (o-xylene, m-xylene, p-xylene), and are a class of aromatic compounds containing a benzene ring structure. Benzene series compounds are abundantly present in petroleum and its refined products and are widely used in industries such as paint, synthetic rubber, synthetic resin, dye, plastic, synthetic fiber, synthetic drug, pesticide, and pharmaceutical organic chemical industry. Due to their characteristics of being easily soluble, volatile, and highly toxic, benzene series compounds can quickly penetrate into soil and groundwater and then migrate and diffuse, and remain in the groundwater environment for a long time, causing serious impacts on the ecological environment and human health. For the above reasons, it is urgent to eliminate this pollutant from the environment. However, conventional physical or chemical methods for removing benzene series compounds will cause secondary pollution problems and are costly. In contrast, the biological method uses the metabolism of microorganisms to catalytically degrade benzene series compounds and has the advantages of simple equipment, low investment, easy operation, and less secondary pollution, providing a feasible way for the green and efficient degradation of benzene series compounds.

[0003] At present, multiple strains of benzene series compound-degrading bacteria have been isolated. For example: CN114292775A discloses Pseudomonas stutzeri YJY21-01 with the ability to degrade benzene series compounds and its application. This bacterium can efficiently degrade toluene at 34°C, but the degradation efficiency decreases by about 20% at pH 8, and when the temperature drops to 25°C, its degradation rate is only half of that under the optimal conditions. CN112251378A conducts a systematic study on the efficient degradation of benzene by Corynebacterium AL-5. Strain AL-5 can completely degrade 100 mg / L of benzene and 80% of toluene within 10 h at 28°C, but does not have the ability to degrade xylene. CN101624576A discloses Mycobacterium cosmeticum byf-4 with the ability to degrade benzene series compounds and its application. This bacterium can degrade benzene, toluene, ethylbenzene, and o-xylene at 30°C and pH 7.2-7.4, but under low temperature (20°C) or alkaline (pH = 8) conditions, its degradation rate of BETX also decreases by more than 20% compared with the optimal conditions.

[0004] It can be seen that the degradation effect of existing degradation bacteria on benzene series compounds is restricted by environmental factors such as temperature, pH, and salinity. However, some petrochemical enterprises are located in coastal areas, where the groundwater and soil often show high alkalinity, and the groundwater temperature is usually in the range of 10°C to 20°C, which greatly limits the utilization efficiency and purification ability of ordinary microorganisms for BTEX. Therefore, it is crucial to develop highly efficient BTEX-degrading functional microorganisms that are tolerant to salt and alkali. Summary of the Invention

[0005] The purpose of the present invention is to provide a BTEX-degrading bacterium for the polluted site of a petrochemical enterprise in production in a coastal saline-alkali area, as well as its screening method and application. By screening and acclimating at low temperature, it can adapt to the actual groundwater and soil environment, so as to overcome the problems of difficult survival and low degradation efficiency of microorganisms in the actual site.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The present invention provides Pseudomonas sp. LO-A1 that can efficiently degrade benzene series compounds. The Pseudomonas sp. LO-A1 was deposited at the China Center for Type Culture Collection on October 13, 2023. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC No: M20231888.

[0008] Pseudomonas sp. LO-A1 is Gram-negative stained, without a capsule and is a pseudomonad in terms of strain morphology, and has regular colony morphology on Luria-Bertani medium, being white, raised, opaque, and smooth.

[0009] The 16S rRNA gene sequence of Pseudomonas sp. LO-A1 provided by the present invention is:

[0010]

[0011]

[0012]

[0013] The screening method of Pseudomonas sp. LO-A1 provided by the present invention includes the following steps:

[0014] (1) Mix the basal inorganic salt medium and the trace element mixture to obtain the SSDM medium;

[0015] (2) Filter and pump the groundwater samples from the polluted areas of the petrochemical plants to enrich the microorganisms on the filter membrane. Place the filter membrane in a 30 mL SSDM culture medium, and then inject a certain amount of BTEX solution with a syringe to obtain a first mixed solution containing bacteria. After culturing, transfer 10% of the first mixed solution to another 30 mL SSDM culture medium, and then inject a certain amount of BTEX solution with a syringe to obtain a second mixed solution. Repeat this process several times until the nth mixed solution is obtained, where n = 6 - 8. The BTEX solution is a mixed solution of benzene, toluene, ethylbenzene, and xylene with a volume ratio of 1:1:1:1.

[0016] (3) Perform streak plate isolation culture on the obtained nth mixed solution to obtain Pseudomonas sp. LO - A1.

[0017] Preferably, in step (1), the inorganic salt components in the basic inorganic salt culture medium include at least: 0.2 g / L NH4Cl, 7.95 g / L NaCl, 0.77 g / L MgCl2·6H2O, 1.05 g / L MgSO4·7H2O, 0.076 g / L CaCl2, 0.22 g / L KCl, 0.01 g / L NaHCO3, 0.026 g / L NaBr, 0.25 g / L K2HPO4.

[0018] Preferably, in step (1), the solute components in the trace element mixture include at least: 0.15 g / L ZnSO4·7H2O, 0.26 g / L MnSO4·H2O, 0.03 g / L CoCl2·6H2O, 4.5 g / L FeSO4·7H2O, 0.02 g / L NiCl2·6H2O, 0.01 g / L CuCl2, 0.1 g / L Na2MoO4·2H2O, 0.06 g / L H3BO3.

[0019] Preferably, in step (1), the solvent used in the BTEX solution is N,N - dimethylformamide, and the total concentration of benzene, toluene, ethylbenzene, and xylene is 100 g / L.

[0020] And / or, the volume ratio of the inorganic salt culture medium and the trace element solution in each SSDM culture medium to the BTEX solution injected each time with a syringe is 10 3 :1:1.

[0021] Preferably, in step (2), when preparing the first mixed solution, the groundwater sample used for filtration is 400 - 500 mL (the filter membrane used for filtration is 0.22 μm).

[0022] Preferably, in step (2), the cultivation is carried out under airtight conditions at a temperature of 20 °C, and the time for each cultivation is 5 - 7 days.

[0023] Preferably, in step (3), the separation and cultivation process is as follows: Take 0.1 mL of the nth mixed solution and add it to 0.9 mL of sterilized liquid inorganic salt medium and mix well to obtain 10 -1 , and successively dilute it step by step in a gradient manner to 10 -2 , 10 -3 , 10 -4 , 10 -5 . Then spread the diluted solution on a Luria - Bertani medium plate containing BTEX (benzene, toluene, ethylbenzene, xylene with a volume ratio of 1:1:1:1, and in xylene, the volume ratio of o - xylene:m - xylene:p - xylene = 1:1:1). There are 3 parallels for each gradient. Then use an inoculation loop to pick single colonies and streak - separate them on the Luria - Bertani medium plate. The streak - separation is repeated 2 - 3 times. Inoculate the obtained single colonies into the degradation system to verify the degradation ability. If it is observed that the bacteria can grow using BTEX as the sole carbon source and the BTEX concentration decreases, it is Pseudomonas sp. LO - A1.

[0024] The present invention also provides the application of the described Pseudomonas sp. LO - A1 in degrading benzene series compounds.

[0025] Furthermore, the present invention provides a bacterial agent containing the described Pseudomonas sp. LO - A1.

[0026] In the present invention, the bacterial agent containing Pseudomonas sp. LO - A1 can be a bacterial agent prepared by using conventional technical means and adding excipients allowed in the field of bacterial agent preparation.

[0027] Preferably, the bacterial agent is a liquid bacterial agent or a solid bacterial agent.

[0028] Specifically, the liquid bacterial agent is a bacterial suspension containing the described Pseudomonas sp. LO - A1 or a processed bacterial suspension; the solid bacterial agent is prepared by mixing a bacterial suspension containing the described Pseudomonas sp. LO - A1 or a processed bacterial suspension with an adsorbent.

[0029] The beneficial effects of the present invention are as follows: Through scientific domestication and screening, a strain of BTEX highly efficient degrading bacterium, Pseudomonas sp. LO-A1 (GenBank accession number: OR608112), is obtained. This strain has a high degradation efficiency for BTEX and also has a good degradation effect on benzene series compounds under different saline-alkali conditions.

[0030] By using Pseudomonas sp. LO-A1 provided by the present invention to degrade benzene series compounds in water, the purpose of removing pollutants can be achieved, and the intermediate products are completely degraded. This operation has a low cost and little negative impact on the environment, and has good prospects for development and utilization. Description of the Drawings

[0031] Figure 1 It is a picture of the medium becoming turbid over time during the degradation of BTEX by the strain in Example 2.

[0032] Figure 2 It is a colony morphology diagram of Pseudomonas sp. LO-A1.

[0033] Figure 3 It is a phylogenetic tree of Pseudomonas sp. LO-A1.

[0034] Figure 4 It is a growth curve and degradation curve diagram of Pseudomonas sp. LO-A1 degrading benzene series compounds.

[0035] Figure 5 It is a schematic diagram of the degradation rate of benzene series compounds by Pseudomonas sp. LO-A1 at different temperatures.

[0036] Figure 6 It is a schematic diagram of the degradation rate of benzene series compounds by Pseudomonas sp. LO-A1 at different pH values.

[0037] Figure 7 It is a schematic diagram of the degradation rate of benzene series compounds by Pseudomonas sp. LO-A1 at different salinities.

[0038] Figures 5-7 In each column chart, from left to right are benzene, toluene, ethylbenzene, m,p-xylene, o-xylene, and DO600 nm . Detailed Embodiments

[0039] The preferred embodiments of the present invention will be described in detail below in conjunction with embodiments. It should be understood that the following embodiments are given only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0041] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial sources.

[0042] Example 1 Obtaining of Strains

[0043] Contaminated groundwater was collected from a petrochemical plant in Tianjin and placed in a brown bottle and transported back to the laboratory. After enrichment by a filter membrane, it was added to a culture medium to obtain a mixture. A highly efficient BTEX-degrading bacterium was obtained through screening and domestication. The degradation of benzene series compounds by this strain was verified, and it was found that the strain could efficiently degrade BTEX and had stable passaging characteristics.

[0044] This strain was Gram-negative, with regular morphology, white, convex, opaque, and smooth.

[0045] 16S rDNA sequencing was performed on it, and the obtained 16S rDNA sequence was subjected to BLAST alignment. The alignment results showed that the nucleotide sequence of the 16S rDNA of strain LO-A1 had more than 99% homology with the nucleotide sequences of different strains of the genus Pseudomonas.

[0046] The specific process of obtaining the strain is as follows:

[0047] (1) Mix the basal inorganic salt medium and the trace element mixture to obtain a liquid medium;

[0048] (2) Take a groundwater sample from a contaminated area of a petrochemical plant in Tianjin, filter it, enrich the microorganisms on the filter membrane, place it in a 30 mL liquid medium, and then inject a certain amount of BTEX solution with a syringe to mix, obtaining a first mixed solution containing bacteria. After culturing, 10% of the first mixed solution was taken and transferred to another 30 mL liquid medium, and then a certain amount of benzene series compound solution was injected with a syringe to obtain a second mixed solution. This was repeated several times until the nth mixed solution was obtained, where n = 6;

[0049] (3) Perform streak plate isolation culture on the obtained 6th mixed solution to obtain the BTEX-degrading bacterium.

[0050] In step (1), the inorganic salt components in the basic inorganic salt medium include: 0.2 g / L NH4Cl, 7.95 g / L NaCl, 0.77 g / L MgCl2·6H2O, 1.05 g / L MgSO4·7H2O, 0.076 g / L CaCl2, 0.22 g / L KCl, 0.01 g / L NaHCO3, 0.026 g / L NaBr, 0.25 g / L K2HPO4.

[0051] In step (1), the solute components in the trace element mixture include: 0.15 g / L ZnSO4·7H2O, 0.26 g / L MnSO4·H2O, 0.03 g / L CoCl2·6H2O, 4.5 g / L FeSO4·7H2O, 0.02 g / L NiCl2·6H2O, 0.01 g / L CuCl2, 0.1 g / L Na2MoO4·2H2O, 0.06 g / L H3BO3.

[0052] In step (1), the solutes of the BTEX solution (100 g / L) are benzene, toluene, ethylbenzene, and xylene with a volume ratio of 1:1:1:1, and the solvent is N,N-dimethylformamide.

[0053] In step (1), the volume ratio of the inorganic salt medium and the trace element solution in each liquid medium to the BTEX solution injected each time through a syringe is 10 3 :1:1.

[0054] In step (2), when preparing the first mixture, the filtration volume of the groundwater sample is 400 - 500 mL.

[0055] In step (2), the acclimation conditions can be 150 r / min. The culture process is carried out under airtight conditions at a temperature of 20°C, and the time for each culture is about 5 - 7 days.

[0056] In step (3), the specific process of dilution coating and separation culture is as follows: Take 0.1 mL of the nth transferred degrading bacterial liquid and add it to 0.9 mL of sterilized liquid inorganic salt medium (0.9% NaCl solution) and mix well to obtain 10 -1 , and sequentially perform gradient dilution step by step to 10 -2 , 10 -3 , 10 -4 , 10 -5, and spread it on the Luria-Bertani medium plate containing BTEX. There are 3 parallels for each gradient. Then, pick single colonies with an inoculation loop and isolate them on the Luria-Bertani medium plate in a line. Repeat this 2-3 times. Inoculate the obtained single bacteria into the degradation system to verify the degradation ability. If it is observed that the bacteria can grow using BTEX as the sole carbon source and the BTEX concentration decreases, it is a BTEX-degrading bacterium. The colony morphology of LO-A1 is as Figure 2 shown, with regular morphology, being white, convex, opaque, and smooth. Figure 3 shows the phylogenetic relationship between strain LO-A1 and other bacteria in the genus Pseudomonas. It is in the same branch of the phylogenetic tree as Pseudomonas vancouverensis BApL12, indicating a relatively close phylogenetic relationship.

[0057] Example 2: Study on the Degradation of BTEX in Wastewater by Strains

[0058] Place 20 mL of an inorganic salt medium containing 100 mg / L BTEX (benzene, toluene, ethylbenzene, xylene in a volume ratio of 1:1:1:1, and in xylene, o-xylene:m-xylene:p-xylene = 1:1:1) in a 150 mL serum bottle. The inoculation procedure is the same as in Example 1. To prevent the volatilization of benzene series compounds, seal it with a lid. Incubate it with shaking at 20 °C, 150 rpm, in the dark. Take samples at regular intervals and measure the residual concentration of BTEX.

[0059] Figure 1 is a picture of the medium becoming turbid over time during the degradation of BTEX by the strain in Example 2.

[0060] Figure 4 shows that under the condition of a temperature of 20 °C, Pseudomonas sp. LO-A1 can degrade 80% of the benzene series compounds with an initial concentration of 100 mg / L within 7 days, indicating that it can adapt to the actual groundwater temperature and maintain the degradation activity of BTEX.

[0061] Example 3: Study on the Degradation Characteristics (Different Temperatures, pH Values, and Salinities) of Strains

[0062] After LO-A1 is expanded and cultured in Luria-Bertani medium, centrifuge it at 8000 r / min for 5 min, and resuspend it with 0.9% normal saline to wash away carbon sources such as yeast powder. Repeat this 2 times with the total volume remaining unchanged. Measure the absorbance value at a wavelength of 600 nm and add it to a 150 mL serum bottle. The serum bottle contains 20 mL of an inorganic salt medium containing BTEX. The initial OD600 value in the degradation system is 0.1. To prevent the volatilization of benzene series compounds, seal it with a lid. Incubate it with shaking at 150 rpm, in the dark. Change the pH value, salinity, and temperature respectively. Take samples after 96 h and measure the residual concentration of BTEX.

[0063] 1. Different temperatures

[0064] Degradation conditions: pH 7.0, 100 mg / L BTEX; salinity 1%; temperatures: 15°C, 20°C, 28°C;

[0065] The results show that: Figure 5 This is a schematic diagram of the degradation rate of BTEX by the aromatic hydrocarbon-degrading strain Pseudomonas sp. LO-A1 at different temperatures provided in the present invention. Strain LO-A1 has good degradation effects on toluene, m / p-xylene at 20°C, and the degradation rate can reach over 85.3%; it has good degradation effects on toluene, ethylbenzene at 28°C, and the degradation rate can reach over 78.5%, and it also has certain degradation effects on benzene, m / p-xylene.

[0066] 2. Different pH values:

[0067] Degradation conditions: 100 mg / L BTEXS; salinity 1%; temperature: 20°C; pH values: 7.0, 8.0, 9.0;

[0068] The results show that: Figure 6 This is a schematic diagram of the degradation rate of BTEX by the aromatic hydrocarbon-degrading strain Pseudomonas sp. LO-A1 at different pH values provided in the present invention. Strain LO-A1 has degradation effects on BTEX in the pH 7.0 - 9.0 environment. Among them, the degradation effect is the best in the alkaline environment (pH 8), and the degradation rates of benzene, toluene, and ethylbenzene are 90.2%, 99.7%, and 99.5% respectively. And in the neutral environment (pH 7.0), the degradation rates of toluene, m / p-xylene reach over 85.3%.

[0069] 3. Different salinities:

[0070] Degradation conditions: pH 7.0; temperature: 20°C; 100 mg / L BTEX; salinities 1%, 3%, 5%;

[0071] The results show that: Figure 7 This is a schematic diagram of the degradation rate of BTEX by the aromatic hydrocarbon-degrading strain Pseudomonas sp. LO-A1 at different salinities provided in the present invention. Strain LO-A1 has degradation effects on BTEX in the salinity 1%, 3%, 5% environments. Among them, the degradation effect on BTEX is the best under the salinity 3% - 5% conditions, and the degradation rates of benzene, toluene, and ethylbenzene reach over 81.9%. It also has removal effects on m / p-xylene and o-xylene. Under the salinity 1% condition, the removal rate of toluene reaches 85.3%.

[0072] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A strain of Pseudomonas sp. LO-A1, characterized in that, It is preserved in the China Center for Type Culture Collection, with the preservation number: CCTCC NO: M20231888, and the preservation date is October 13, 2023.

2. The Pseudomonas sp. LO-A1 according to claim 1, characterized in that, Its Gram stain is negative. The strain morphology has no capsule and is Pseudomonas, and the colony morphology on Luria-Bertani medium is regular, white, convex, opaque and smooth.

3. A screening method for the Pseudomonas sp. LO-A1 according to claim 1 or 2, characterized in that, It includes the following steps: (1) Mix the basal inorganic salt medium and the trace element mixture to obtain the SSDM medium. (2) Take the groundwater sample from the polluted area of the petrochemical plant and filter it by suction to enrich the microorganisms on the filter membrane. Place the filter membrane in a 30 mL portion of the SSDM medium, and then inject a certain amount of BTEX solution with a syringe to obtain the first mixed solution containing bacteria. After cultivation, transfer 10% of the first mixed solution to another 30 mL portion of the SSDM medium, and then inject a certain amount of BTEX solution with a syringe to obtain the second mixed solution. Repeat this several times until the nth mixed solution is obtained, where n = 6 - 8. The BTEX solution is a mixed solution of benzene, toluene, ethylbenzene, and xylene with a volume ratio of 1:1:1:

1. (3) Perform streak plate isolation culture on the obtained nth mixed solution to obtain Pseudomonas sp. LO-A1.

4. A screening method for a Pseudomonas sp. LO-A1 according to claim 3, characterized in that, In step (1), the inorganic salt components in the basal inorganic salt medium at least include: 0.2 g / L NH4Cl, 7.95 g / L NaCl, 0.77 g / L MgCl2·6H2O, 1.05 g / L MgSO4·7H2O, 0.076 g / L CaCl2, 0.22 g / L KCl, 0.01 g / L NaHCO3, 0.026 g / L NaBr, 0.25 g / L K2HPO4.

5. A screening method for a Pseudomonas sp. LO-A1 according to claim 3 or 4, characterized in that, In step (1), the solute components in the trace element mixture at least include: 0.15 g / L ZnSO4·7H2O, 0.26 g / L MnSO4·H2O, 0.03 g / L CoCl2·6H2O, 4.5 g / L FeSO4·7H2O, 0.02 g / L NiCl2·6H2O, 0.01 g / L CuCl2, 0.1 g / L Na2MoO4·2H2O, 0.06 g / L H3BO3.

6. A screening method for a Pseudomonas sp. LO-A1 according to any one of claims 3-5, characterized in that, In step (1), the solvent used for the BTEX solution is N,N-dimethylformamide, and the total concentration of benzene, toluene, ethylbenzene, and xylene is 100 g / L. And / or, the volume ratio of the inorganic salt medium and the trace element solution in each SSDM medium to the BTEX solution injected each time through the syringe is 10 3 :1:

1.

7. A screening method for a Pseudomonas sp. LO-A1 according to any one of claims 3-6, characterized in that, In step (2), when preparing the first mixed solution, the suction filtration volume of the groundwater sample is 400 - 500 mL.

8. A screening method for a Pseudomonas sp. LO-A1 according to any one of claims 3-7, characterized in that, In step (2), the cultivation process is carried out under closed conditions, the temperature is 20 °C, and the cultivation time for each time is 5 - 7 d.

9. A screening method for a Pseudomonas sp. LO-A1 according to any one of claims 3-8, characterized in that, In step (3), the separation and culture process is specifically as follows: Take 0.1 mL of the nth mixed solution, add it to 0.9 mL of sterilized liquid inorganic salt medium and mix well, that is, 10 -1 , and sequentially perform gradient dilution step by step to 10 -2 , 10 -3 , 10 -4 , 10 -5 . Then spread the diluted mixed solution on the Luria-Bertani medium plate containing BTEX, with 3 replicates for each gradient. Next, use an inoculation loop to pick single colonies and streak them on the Luria-Bertani medium plate for separation. The streak separation is repeated 2 - 3 times. Inoculate the obtained single bacteria into the degradation system to verify the degradation ability. If it is observed that the bacteria can grow using BTEX as the sole carbon source and the BTEX concentration decreases, it is Pseudomonas sp. LO-A1.

10. An application of the Pseudomonas sp. LO-A1 according to claim 1 or 2 in degrading benzene series compounds.

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