Aromatic hydrocarbon degrading bacterium as well as screening method and application thereof
By screening and isolating the aromatic hydrocarbon degrading strain BO3-2, the problem of low degradation rate of various pollutants under low temperature conditions was solved, achieving efficient and low-disturbance groundwater remediation, which is suitable for pollution remediation in petrochemical enterprises.
Patent Information
- Application Number
- CN202410972760.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-19
- Publication Date
- 2026-01-20
AI Technical Summary
The aromatic hydrocarbon degrading strains screened in the existing technology have low degradation rates for various pollutants under low temperature conditions, which is difficult to meet the needs of groundwater pollution remediation in operating petrochemical enterprises, and there is also a risk of secondary pollution.
A strain of aromatic hydrocarbon degrading bacterium, BO3-2, belonging to the genus Sphingobium sp., was screened and isolated. Through specific culture media and gradient dilution methods, strains that efficiently degrade multiple pollutants were screened under low-temperature conditions and applied to groundwater remediation.
Under conditions of 15-28℃, pH 6.0-8.0, BTEXS concentration of 50mg/L-200mg/L and naphthalene concentration of 5mg/L-20mg/L, strain BO3-2 exhibits a degradation rate of over 99% for various aromatic hydrocarbon pollutants without the accumulation of toxic intermediate products, making it suitable for low-disturbance remediation.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of environmental microorganisms, in particular to a strain of aromatic hydrocarbon-degrading bacteria and a screening method and application thereof. BACKGROUND
[0002] In the process of rapid development of petrochemical industry, environmental pollution problems in the production petrochemical site are also faced, and the leakage in the production, transportation and processing links will cause serious pollution to the soil and groundwater in the site. Among them, BTEX and PAHs are often detected in the soil or groundwater of the production petrochemical site such as oil depot, oil refining and chemical enterprise and gas station, and the pollution concentration is high and accompanied by complex pollution.
[0003] Unlike the remediation of retired petrochemical sites, the remediation process of production petrochemical enterprises needs to meet the requirements of safe production and green and sustainable site remediation, and it is urgent to form a remediation scheme or technology with low disturbance and high safety. Among them, in-situ microbial enhanced remediation technology has the characteristics of simple operation, small secondary pollution and small disturbance to the site. However, in the existing research, only strains with degradation ability to single substrate are screened, and the degradation rate of pollutants under low temperature conditions is low. Therefore, screening functional strains that can efficiently degrade multiple pollutants under low temperature environment is the key to solving the groundwater pollution remediation of production petrochemical enterprises.
[0004] The applicant screens and isolates a strain of aromatic hydrocarbon-degrading bacteria from a contaminated site of a production petrochemical enterprise, and explores its degradation characteristics and degradation pathway of multiple pollutants under low temperature conditions. SUMMARY
[0005] The purpose of the present application is to meet the needs of microorganisms that can degrade multiple pollutants under low temperature conditions in actual groundwater pollution scenarios, and to provide a strain of aromatic hydrocarbon-degrading bacteria and a screening method and application thereof.
[0006] To solve the above technical problems, the technical solution adopted by the present application is as follows: a strain of aromatic hydrocarbon-degrading bacteria, which is Sphingobium sp., and the strain is named BO3-2. It has been preserved in China Center for Type Culture Collection on September 28, 2023, and the preservation number is CCTCC M 20231828B03-2 Sphingobium sp..
[0007] The aromatic hydrocarbon-degrading bacteria is gram-negative, and its colony morphology in Luria-Bertani culture medium is round, yellow in color, and opaque strain with smooth surface and slight protrusions. It is observed as an ellipse by scanning electron microscope, and the length is 0.75-1.0 μm.
[0008] The application further provides a screening method of the aromatic hydrocarbon-degrading bacteria, comprising the following steps:
[0009] (1) mixing a basic inorganic salt medium with a benzene series solution and a naphthalene solution to obtain a liquid culture medium;
[0010] (2) taking a soil sample from a soil contaminated by a petrochemical enterprise to mix with the liquid culture medium to obtain a first mixed liquid containing bacteria, and after culturing for 2-3 days, 10% of the culture solution is removed to another fresh liquid culture medium, and the process is repeated until an nth mixed liquid is obtained, wherein n=5-8;
[0011] (3) gradient dilution and coating separation culture of the nth mixed liquid, so as to obtain the aromatic hydrocarbon-degrading bacteria.
[0012] The solvent for the benzene series solution in step (1) is N,N-dimethylformamide, and the concentration is 100 mg / mL (B:T:E:X:S=1:1:1:1:1, w / w); the solvent for the naphthalene solution is N,N-dimethylformamide, and the concentration is 10 mg / mL.
[0013] The volume ratio of the basic inorganic salt medium, the benzene series solution and the naphthalene solution added in the liquid culture medium of step (1) is 10 6 :2:2.
[0014] In step (2), when the first mixed liquid is prepared, the ratio of the added amount of the soil sample to the liquid culture medium is 3-6 g:20 mL.
[0015] In step (2), a serum bottle is used as a culture container, and the culture process is carried out under a closed condition at a temperature of 20-25 °C.
[0016] The gradient dilution and coating separation culture process specifically comprises: taking 1 mL of the nth culture solution, diluting it with a phosphate buffer to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , and then transferring 100 μL of the liquid at each concentration gradient to a Luria-Bertani culture medium plate, coating and separating with a coating rod, picking a single colony on the LB plate with an inoculation ring, and streaking and separating on a Luria-Bertani culture medium plate, and the process is repeated twice until a single colony is obtained.
[0017] The application also provides application of the aromatic hydrocarbon-degrading bacteria in degrading benzene, toluene, ethylbenzene, dimethylbenzene, styrene and naphthalene.
[0018] The application has the following beneficial effects:
[0019] 1. The degradation bacteria can degrade 20mg / L of toluene, ethylbenzene, styrene, dimethylbenzene and 10mg / L of naphthalene by more than 99% within 48h under the culture condition of 15 DEG C, and the degradation rate of benzene is 67% after 72h.
[0020] 2. The degradation bacteria can degrade target pollutants under a mixed pollutant system with a wide temperature range (15-28 DEG C), a pH range (pH 6.0-8.0), a BTEXS concentration range (50mg / L-200mg / L) and a naphthalene concentration range (5mg / L-20mg / L), and no toxic intermediate metabolites are accumulated, and no secondary pollutants are generated.
[0021] 3. The degradation bacteria can stably degrade various aromatic hydrocarbon pollutants under low-temperature conditions, can be applied to various application scenarios such as groundwater remediation of petrochemical enterprises, have little negative impact on the environment, and have good development and utilization prospect. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 It is a colony morphology diagram of the aromatic hydrocarbon-degrading bacteria BO3-2.
[0023] Figure 2 It is a scanning electron microscope diagram of the aromatic hydrocarbon-degrading bacteria BO3-2.
[0024] Figure 3 It is a phylogenetic tree of the aromatic hydrocarbon-degrading bacteria BO3-2.
[0025] Figure 4 It is a degradation rate diagram of the aromatic hydrocarbon-degrading bacteria BO3-2 under different temperatures.
[0026] Figure 5 It is a degradation rate diagram of the aromatic hydrocarbon-degrading bacteria BO3-2 under different pHs.
[0027] Figure 6 It is a degradation rate diagram of the aromatic hydrocarbon-degrading bacteria BO3-2 under different substrate concentrations.
[0028] Figure 7 It is a degradation rate diagram of the aromatic hydrocarbon-degrading bacteria BO3-2 under different yeast powder concentrations.
[0029] Figure 8 Gas chromatogram of aromatic hydrocarbon degradation by aromatic hydrocarbon-degrading bacteria BO3-2. DETAILED DESCRIPTION
[0030] In order to better understand the present application, the content of the present application is further illustrated below in combination with examples, but the content of the present application is not limited to the following examples.
[0031] An aromatic hydrocarbon-degrading bacterium is Sphingobium sp., and the strain is named BO3-2, which has been preserved in China Center for Type Culture Collection on September 28, 2023, and the preservation number is CCTCC M 20231828B03-2 Sphingobium sp.. The degradation bacterium is gram-negative, and its colony morphology in Luria-Bertani culture medium is round, yellow in color, and is an opaque strain with smooth and slightly raised surface. It is observed as an ellipse by scanning electron microscope, and the length is 0.75-1.0 μm.
[0032] The screening method of the aromatic hydrocarbon-degrading bacterium comprises the following steps:
[0033] (1) Mixing the basic inorganic salt culture medium with benzene series solution and naphthalene solution to obtain a liquid culture medium.
[0034] The solvent used in the benzene series solution is N,N-dimethylformamide, and the concentration is 100 mg / mL (B:T:E:X:S = 1:1:1:1:1, w / w); the solvent used in the naphthalene solution is N,N-dimethylformamide, and the concentration is 10 mg / mL.
[0035] The volume ratio of the basic inorganic salt culture medium, the benzene series solution and the naphthalene solution in the liquid culture medium is 10 6 :2:2.
[0036] (2) Taking the soil sample from the contaminated soil of the petrochemical enterprise to mix in the liquid culture medium to obtain the first mixed liquid containing bacteria, and after 2-3 days of culture, 10% of the culture solution is removed to another fresh liquid culture medium, and the process is repeated until the nth mixed liquid is obtained, wherein n = 5-8.
[0037] The ratio of the addition amount of the soil sample to the liquid culture medium is 3-6 g:20 mL when the first mixed liquid is prepared.
[0038] The serum bottle is used as the culture container, and the culture process is carried out in a closed condition, and the temperature is 20-25℃.
[0039] (3) Gradient dilution and plating separation culture of the nth mixed solution, i.e. to obtain aromatic hydrocarbon degrading bacteria.
[0040] The gradient dilution and plating separation culture process is as follows: 1 mL of the nth culture solution is diluted with phosphate buffer to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 μL of liquid is taken at each concentration gradient and transferred to a Luria-Bertani culture medium plate, and then plated with a spreader. A single colony is picked on the LB plate with a loop and streaked on a Luria-Bertani culture medium plate. This process is repeated twice until a single colony is obtained.
[0041] The degradation bacteria can degrade target pollutants under a mixed pollutant system with a temperature range of 15-18℃, a pH range of 6.0-8.0, a BTEXS concentration range of 50mg / L-200mg / L, and a naphthalene concentration range of 5mg / L-20mg / L.
[0042] <Example 1>
[0043] An aromatic hydrocarbon degrading bacteria is Sphingobium sp. BO3-2, which is preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC M 20231828B03-2 Sphingobium sp. and a preservation time of September 28, 2023.
[0044] The aromatic hydrocarbon degrading bacteria is gram-negative, with regular colony morphology on Luria-Bertani culture medium plates, round shape, yellow color, and opaque strain, smooth surface and slight protrusion. Meanwhile, 16S rDNA sequencing is performed on the strain, and the obtained 16S rDNA sequence is subjected to BLAST comparison. The comparison result shows that the 16S rDNA nucleotide sequence of the strain BO3-2 has more than 99% homology with the nucleotide sequences of different strains of Sphingobium sp.
[0045] The single colony is inoculated on the LB solid medium, and the plate is inverted in a constant temperature incubator for 2 days. The colony morphology is regular, yellow, round, and opaque, as shown in Figure 1 . The strain is oval under a scanning electron microscope, as shown in Figure 2 .
[0046] <Example 2>
[0047] The contaminated soil from a certain in-production petrochemical enterprise in Wuhan was taken to the laboratory in a brown bottle, the soil was added to the culture medium to prepare a mixture, and strain screening work was carried out. The basic inorganic salt culture medium was mixed with the benzene series and naphthalene solution to obtain a liquid culture medium.
[0048] (1) A soil sample derived from contaminated soil of a petrochemical enterprise in production was mixed in a portion of liquid culture medium, wherein the basic inorganic salt component comprises: ammonium chloride 0.2 g / L, sodium chloride 7.95 g / L, magnesium chloride hexahydrate 0.77 g / L, magnesium sulfate heptahydrate 1.05 g / L, calcium chloride 0.07 g / L, potassium chloride 0.22 g / L, sodium bicarbonate 0.01 g / L, sodium bromide 0.02 g / L, dipotassium hydrogen phosphate 0.25 g / L and 1% trace elements.
[0049] The benzene series solution concentration was 100 mg / mL (B:T:E:X:S = 1:1:1:1:1, w / w).
[0050] The naphthalene solution concentration was 10 mg / mL; the ratio of the addition volumes of the basic inorganic salt culture medium, the benzene series solution and the naphthalene solution was 10 6 :2:2.
[0051] (2) A soil sample derived from contaminated soil of a petrochemical enterprise in production was mixed in a portion of liquid culture medium to obtain a first mixed liquid, and after 3 days of culture, 10% was transferred to another fresh liquid culture medium to obtain a second mixed liquid, and this process was repeated until the eighth mixed liquid was obtained. When the first mixed liquid was prepared, the addition ratio of the soil sample to the liquid culture medium was 4 g:20 mL. The screening conditions were in a closed and dark condition, and the temperature was 20°C.
[0052] (3) The obtained eighth mixed liquid was spread on LB solid culture medium plates, different morphological colonies were picked from the plates, and the degradation ability verification was carried out, and a strain capable of efficiently degrading benzene series and naphthalene was obtained, and 16S rRNA sequencing was carried out, and a strain Sphingobium sp. B03-2 was obtained.
[0053] The components of the LB solid culture medium are: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.
[0054] BO3-2 16S rDNA sequence:
[0055]
[0056] The Sphingobium sp. BO3-2 16S rRNA sequence is amplified by PCR using universal primers with strain DNA as a template, and a high similarity sequence is selected on GenBank, and a phylogenetic tree is constructed by MEGA X, see Figure 3 .
[0057] <Embodiment 3>
[0058] Strain degradation effect of benzene series and naphthalene under different conditions and growth
[0059] The BO3-2 strain is inoculated into Luria-Bertani medium containing 100 mg / L of BTEXS (B:T:E:X:S = 1:1:1:1:1, w / w) and grown overnight, centrifuged at 8000 rpm for 5 min, and the bacterial body is resuspended twice with phosphate buffer, and the initial OD600 is 0.2. The bacterial solution is cultured at 150 rpm in the dark, and the residual concentration of benzene series and naphthalene is determined by headspace gas chromatography.
[0060] 1. Different temperatures:
[0061] Degradation conditions: pH 7.0, 100 mg / L BTEXS, 10 mg / L naphthalene; temperature: 15℃, 20℃, 28℃;
[0062] The results show that: Figure 4 The strain BO3-2 has degradation effect on benzene series and naphthalene in the environment of 15℃-28℃, and has good degradation effect on benzene, toluene, ethylbenzene, p-xylene, m-xylene, o-xylene, styrene, and naphthalene in the environment of 20℃-28℃, and the degradation rate can reach 66.9% or more, and the degradation rate is 33 mg / (L·d) in low temperature environment (15℃).
[0063] 2. Different pH:
[0064] Degradation conditions: 100 mg / L BTEXS, 10 mg / L naphthalene; temperature: 20℃; pH: 6.0, 7.0, 8.0;
[0065] The results show that: Figure 5The figure shows the effect of different pH on the degradation rate of benzene series and naphthalene by aromatic hydrocarbon-degrading strain Sphingobium sp. B03-2. The strain B03-2 has degradation effect on benzene series and naphthalene in the environment with pH 6.0-8.0, wherein the degradation effect is the best at pH 7, and the degradation rate is 66.9%-100%, and the degradation rate of benzene in neutral and alkaline environment (pH 7.0-8.0) can reach 66.3%-66.9%.
[0066] 3. Different substrate concentrations
[0067] Degradation conditions: pH 7.0; temperature: 20℃; BTEXS and naphthalene concentrations: 50mg / L BTEXS and 5mg / L naphthalene, 100mg / L BTEXS and 10mg / L naphthalene, 150mg / L BTEXS and 15mg / L naphthalene, 200mg / L BTEXS and 20mg / L naphthalene
[0068] The results show that: Figure 6 The figure shows the effect of different substrate concentrations on the degradation rate of benzene series and naphthalene by aromatic hydrocarbon-degrading strain Sphingobium sp. B03-2. The benzene series and naphthalene can be degraded at the substrate concentrations of 50mg / L BTEXS and 5mg / L naphthalene and 100mg / L BTEXS and 10mg / L naphthalene, and the degradation rate is 48.7%-100%. When the strain is inoculated into the degradation system with the substrate of 150mg / L BTEXS and 15mg / L naphthalene, the strain B03-2 has good degradation effect on toluene, ethylbenzene, p-xylene, m-xylene, o-xylene, styrene and naphthalene, and the degradation rate is 69.2%-100%. When the strain is inoculated into the degradation system with the substrate of 200mg / L BTEXS and 20mg / L naphthalene, the strain B03-2 has good degradation effect on ethylbenzene, p-xylene, m-xylene, o-xylene, styrene and naphthalene, and the degradation rate is 77.4%-99.2%.
[0069] 4. Different yeast powder concentrations
[0070] Degradation conditions: pH 7.0; temperature: 20℃; benzene series 100mg / L, naphthalene 10mg / L; yeast powder concentration: 0mg / L, 10mg / L, 50mg / L
[0071] The results show that: Figure 7 The figure shows the effect of different substrate concentrations on the degradation rate of benzene series and naphthalene by aromatic hydrocarbon-degrading strain Sphingobium sp. B03-2. The addition of yeast powder can enhance the ability of B03-2 to degrade BTEXS and naphthalene, and the degradation rate of benzene series and naphthalene is 70.4%-100% in the environment with yeast powder concentration of 50mg / L.
[0072] <Embodiment 4>
[0073] Metabolite analysis of BTEXS and naphthalene degradation by strain BO3-2
[0074] 5 serum bottles of the degradation system at 48h and 72h were taken respectively, and after extraction with equal volume of ethyl acetate, the extract was combined, dried with anhydrous sodium sulfate, and then evaporated to dryness with a rotary evaporator, redissolved with acetonitrile, filtered with a 0.22μm organic solvent-resistant filter, and derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide as the derivatization reagent. Qualitative analysis was performed by gas chromatography-mass spectrometry, and the TIC gas chromatogram is shown in Figure 8 . The mass spectrometry data of the substances with changing abundance over time in the extracted gas chromatogram were extracted, and compared with the NIST standard database. It was found that a variety of aromatic hydrocarbon biological metabolites were produced, and their abundance changed over time, including phenol (11.48min), 2-ethylphenol (15.58min), phenylethanol (16.47min), 1-phenyl-1,2-ethanediol (22.09min), phenyl glycolic acid (23.07min), 4-carboxybenzoic acid (26.79min), 2,5-dihydroxybenzoic acid (27.72min), and other silane derivatives.
[0075] The specific embodiments of the present application are described above. It should be understood that the present application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which do not affect the essential content of the present application.
Claims
1. Aromatic hydrocarbon-degrading bacteria, which are Sphingobium sp., and the strain is named BO3-2, and has been preserved in the China Center for Type Culture Collection on September 28, 2023, and the preservation number is CCTCC M 20231828B03-2 Sphingobium sp.
2. The aromatic hydrocarbon-degrading bacterium of claim 1, wherein The gram staining is negative, the colony morphology in Luria-Bertani medium is round, the color is yellow, and it is an opaque strain with smooth and slightly raised surface, and the scanning electron microscope observation is oval, the length is 0.75-1.0 μm.
3. The screening method for aromatic hydrocarbon degrading bacteria as described in claim 1, characterized in that, It comprises the following steps: (1) mixing the base inorganic salt medium with benzene series solution and naphthalene solution to obtain a liquid medium; (2) taking a soil sample from a contaminated soil of a petrochemical production enterprise and placing it in the liquid medium to obtain a first mixed liquid containing bacteria, and after 2-3 days of culture, 10% of the culture solution is removed to another fresh liquid medium, and the process is repeated until the nth mixed liquid is obtained, wherein n = 5-8; (3) gradient dilution and plating separation culture of the nth mixed liquid, i.e. obtaining aromatic hydrocarbon-degrading bacteria.
4. The screening method for aromatic hydrocarbon degrading bacteria as described in claim 3, characterized in that, The solvent used in the benzene series solution in step (1) is N,N-dimethylformamide, and the concentration is 100 mg / mL (B:T:E:X:S = 1:1:1:1:1, w / w); the solvent used in the naphthalene solution is N,N-dimethylformamide, and the concentration is 10 mg / mL.
5. The screening method for aromatic hydrocarbon degrading bacteria as described in claim 3, characterized in that, The ratio of the added volumes of the basal inorganic salt medium, the benzene series solution, and the naphthalene solution in the liquid medium of step (1) is 10 6 :2:
2.
6. The screening method for aromatic hydrocarbon degrading bacteria as described in claim 3, characterized in that, In step (2), when preparing the first mixed liquid, the ratio of the amount of soil sample to the amount of liquid medium is 3-6 g:20 mL.
7. The screening method for aromatic hydrocarbon degrading bacteria as described in claim 3, characterized in that, In step (2), a serum bottle is used as a culture container, and the culture process is carried out in a closed condition, and the temperature is 20-25℃.
8. The screening method for aromatic hydrocarbon degrading bacteria as described in claim 3, characterized in that, Gradient dilution and coating separation culture process is as follows: taking 1 mL of the nth culture solution, gradually diluting with phosphate buffer solution to 10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -5 ,10 -6 , taking 100 μL of each concentration gradient liquid and transferring it to Luria-Bertani culture medium plate, coating separation with a coating step rod, picking single colonies on the LB plate with a loop and streaking on the Luria-Bertani culture medium plate, repeating twice, until single colonies are obtained. 9.The aromatic hydrocarbon-degrading bacteria of claim 1 are used for degrading benzene, toluene, ethylbenzene, xylene, styrene and naphthalene.
10. Use of the aromatic hydrocarbon-degrading bacteria according to claim 9 for degrading benzene, toluene, ethylbenzene, xylene, styrene and naphthalene, characterized in that, The degradation bacteria can degrade target pollutants under the following conditions: temperature range of 15-18℃, pH range of 6.0-8.0, BTEXS concentration range of 50 mg / L-200 mg / L, and naphthalene concentration range of 5 mg / L-20 mg / L in a mixed pollutant system.