Benzene series degrading bacterium and application thereof
The HB2 strain of dye phage bacteria efficiently degrades benzene under low temperature and low dissolved oxygen conditions, solving the problem of low tolerance to benzene degradation bacteria in the prior art, achieving effective repair of benzene in groundwater and soil, and has low cost and environmental protection characteristics.
Patent Information
- Application Number
- CN202410110152.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-25
- Publication Date
- 2025-07-25
AI Technical Summary
The existing benzene degraded bacteria have low tolerance to benzene and are difficult to adapt to low temperature and low oxygen conditions in the underground environment, resulting in poor treatment of benzene contamination in groundwater and soil.
Using the HB2 strain of Pigmentiphaga sp., the strain has high tolerance to benzene, toluene, ethylbenzene and xylene, and can efficiently degrade benzene under low temperature and low dissolved oxygen conditions, and biorepair is performed by preparing bacteria agents injected into contaminated underground sites.
It realizes efficient benzene degradation under low temperature and low dissolved oxygen conditions, provides a low-cost, green and environmentally friendly underground pollution repair method, reduces site disturbances, and has good application prospects.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a benzene series compound-degrading bacterium and its application. Background Art
[0002] Benzene, toluene, ethylbenzene, and three isomers of xylene are collectively referred to as benzene series compounds, which are important volatile organic pollutants in the environment. Due to the continuous development of the chemical industry, more and more benzene series compound pollutants are discharged into the environment for various reasons, resulting in increasingly serious benzene series compound pollution of soil and water bodies. Benzene series compounds entering groundwater will cause serious environmental and human health impacts.
[0003] Compared with other components in petroleum, benzene series compounds have stronger water solubility, and they can cause benzene series compound pollution of surface water bodies and groundwater bodies along with surface runoff and infiltration of water bodies. Therefore, the treatment of soil and water bodies polluted by benzene series compounds is an urgent problem to be solved in the petrochemical industry. The treatment of water bodies polluted by benzene series compounds is divided into chemical, physical, and biological treatment methods. Among them, the physical method has low treatment efficiency and often requires the combined action of chemical and biological methods to achieve the treatment effect. The chemical method treats pollutants through chemical reactions, with a large amount of medicine input, high energy consumption, and secondary pollution.
[0004] There have been reports on benzene series compound-degrading bacteria, but the application of biological treatment methods for benzene series compounds is very few, and the treatment effect often fails to achieve the expected effect. Underground site pollution is mostly concentrated in the middle and lower layers of the soil vadose zone. The underground environment has characteristics such as low temperature, low dissolved oxygen, complex water-soil mixing system, and difficult degradation of benzene series compounds. The current benzene series compound-degrading bacteria have low tolerance to benzene series compounds and poor tolerance to low oxygen and low temperature conditions in the underground environment. They can only be applied to open water bodies such as aeration tanks and polluted rivers, and it is difficult to adapt to the treatment of high-concentration benzene series compounds in underground environments such as groundwater and underground soil. Summary of the Invention
[0005] The purpose of the present invention is to overcome the problems existing in the prior art, such as low tolerance of strains to benzene series compounds, low degradation performance, and low tolerance to low oxygen and low temperature conditions in the underground environment. The present invention provides a benzene series compound-degrading bacterium and its application. This benzene series compound-degrading bacterium can adapt to low temperature and low oxygen environments in the underground environment and is suitable for the remediation of benzene series compound pollution in groundwater environments.
[0006] To achieve the above purpose, on the one hand, the present invention provides a benzene series compound-degrading bacterium, which is Pigmentiphaga sp. HB2, and its preservation number is GDMCC NO: 63281.
[0007] On the second hand, the present invention provides an application of the benzene series compound-degrading bacterium as described above in degrading benzene series compounds.
[0008] Preferably, it is applied to the remediation of benzene series compound-contaminated groundwater and benzene series compound-contaminated subsurface soil.
[0009] Preferably, the benzene series compounds are one or more of benzene, toluene, ethylbenzene, m-xylene, o-xylene, and p-xylene.
[0010] Preferably, the applicable temperature of the benzene series compound-degrading bacteria is 10-37 °C.
[0011] The third aspect of the present invention provides an application of the benzene series compound-degrading bacteria as described above in the preparation of products for degrading benzene series compounds.
[0012] The fourth aspect of the present invention provides a bacterial agent, which contains the benzene series compound-degrading bacteria described in claim 1.
[0013] Preferably, the dosage form of the bacterial agent is a liquid agent, an emulsion, a suspension agent, a powder agent, or a granule agent.
[0014] The fifth aspect of the present invention provides a method for remediating a benzene series compound-contaminated underground site, which method comprises the following steps:
[0015] (1) Prepare a culture medium containing nitrogen and phosphorus elements;
[0016] (2) Add the benzene series compound-degrading bacteria to the culture medium for cultivation until the bacterial concentration reaches 5×10 9 ~10×10 9 cell / mL to obtain a bacterial liquid;
[0017] (3) Inject the bacterial liquid into the benzene series compound-contaminated underground site;
[0018] Among them, in step (2), the benzene series compound-degrading bacteria are the benzene series compound-degrading bacteria as described above.
[0019] Preferably, the benzene series compound-contaminated underground site is benzene series compound-contaminated groundwater and benzene series compound-contaminated subsurface soil.
[0020] Preferably, in the culture medium, the concentration of nitrogen element is 20-60 mmol / L.
[0021] Preferably, in the culture medium, the molar ratio of nitrogen element to phosphorus element is 5-15:1.
[0022] Preferably, in step (3), the bacterial liquid is injected into the benzene series compound-contaminated underground site at a pressure of 0.3-0.8 MPa.
[0023] The benzene-degrading bacterium provided by the present invention is Pigmentiphaga sp. HB2. This strain has a high tolerance to the toxicity of benzene series compounds (benzene, toluene, ethylbenzene, and xylene). Its tolerance concentration to benzene can reach 300 mg / L, to toluene can reach 500 mg / L, to ethylbenzene can reach 800 mg / L, and to xylene can reach 1200 mg / L. This strain can efficiently degrade benzene, toluene, ethylbenzene, and xylene, and can adapt to low temperature and low dissolved oxygen conditions in the underground environment, and can be directly used for in-situ bioremediation of groundwater environment polluted by benzene series compounds.
[0024] In addition, the benzene-degrading bacterium provided by the present invention can achieve the remediation of benzene series compound pollution in underground sites through low-cost, green and low-carbon, no secondary pollution, and small site disturbance, etc., and can be widely used, with good application prospects.
[0025] Biological deposit
[0026] The benzene-degrading bacterium provided by the present invention was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 20, 2023, with the deposit number GDMCC No: 63281. The taxonomic name is Pigmentiphaga sp. HB2. The deposit address is: 5th Floor, Building 59, 100th Yard, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code: 510070. Brief description of the drawings
[0027] Figure 1 is a schematic diagram of single-layer injection of the bacterial liquid injection method in the method provided by the present invention;
[0028] Figure 2 is a schematic diagram of multi-layer injection of the bacterial liquid injection method in the method provided by the present invention;
[0029] Figure 3 is a plate culture diagram of Pigmentiphaga sp. HB2 provided by the present invention;
[0030] Figure 4 is an electron microscope image of Pigmentiphaga sp. HB2 provided by the present invention;
[0031] Figure 5 is a phylogenetic tree of Pigmentiphaga sp. HB2 provided by the present invention;
[0032] Figure 6 is an effect diagram of the degradation of benzene at different concentrations by Pigmentiphaga sp. HB2 provided by the present invention;
[0033] Figure 7 is the growth situation of Pigmentiphaga sp. HB2 under different concentrations of benzene exposure provided by the present invention;
[0034] Figure 8It is the degradation effect diagram of the toluene-dyeing bacterium HB2 provided by the present invention on toluene at different concentrations;
[0035] Figure 9 It is the growth situation of the toluene-dyeing bacterium HB2 provided by the present invention under the exposure of toluene at different concentrations;
[0036] Figure 10 It is the degradation effect diagram of the toluene-dyeing bacterium HB2 provided by the present invention on ethylbenzene at different concentrations;
[0037] Figure 11 It is the growth situation of the toluene-dyeing bacterium HB2 provided by the present invention under the exposure of ethylbenzene at different concentrations;
[0038] Figure 12 It is the degradation effect diagram of the toluene-dyeing bacterium HB2 provided by the present invention on o-xylene at different concentrations;
[0039] Figure 13 It is the growth situation of the toluene-dyeing bacterium HB2 provided by the present invention under the exposure of o-xylene at different concentrations;
[0040] Figure 14 It is the degradation effect diagram of the toluene-dyeing bacterium HB2 provided by the present invention on m-xylene at different concentrations;
[0041] Figure 15 It is the growth situation of the toluene-dyeing bacterium HB2 provided by the present invention under the exposure of m-xylene at different concentrations;
[0042] Figure 16 It is the degradation effect diagram of the toluene-dyeing bacterium HB2 provided by the present invention on p-xylene at different concentrations;
[0043] Figure 17 It is the growth situation of the toluene-dyeing bacterium HB2 provided by the present invention under the exposure of p-xylene at different concentrations;
[0044] Figure 18 It is the degradation effect diagram of the toluene-dyeing bacterium HB2 provided by the present invention on the degradation performance of benzene and toluene under low temperature and low oxygen conditions;
[0045] Figure 19 It is the growth situation of the toluene-dyeing bacterium HB2 provided by the present invention under the exposure of benzene and toluene under low temperature and low oxygen conditions. Specific Embodiments
[0046] The following further details the specific embodiments of the present invention with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of illustration and explanation of the present invention, and are not intended to limit the present invention.
[0047] The endpoints and any values within the ranges disclosed in this document are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this document.
[0048] The present invention provides a benzene series compound-degrading bacterium, which is Pigmentiphaga sp. HB2, and its preservation number is GDMCC NO: 63281. This strain was preserved in the Guangdong Provincial Microbial Culture Collection Center on the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou City, Guangdong Province on March 20, 2023.
[0049] The Pigmentiphaga sp. HB2 described in the present invention can adapt to the low temperature (15 °C) and low dissolved oxygen (<2.5 mg / L) conditions in the underground environment and can be directly used for in-situ bioremediation of groundwater environments polluted by benzene series compounds.
[0050] The Pigmentiphaga sp. HB2 provided by the present invention was obtained from a groundwater sample polluted by benzene series compounds in the northwest after enrichment, domestication, separation, purification, and mutagenesis. By using this strain to biodegrade the groundwater site polluted by benzene series compounds, the effect of site purification and remediation can be achieved. Among them, the groundwater sample polluted by benzene series compounds is a soil-water mixture.
[0051] The Pigmentiphaga sp. HB2 has a high toxicity tolerance to benzene series compounds (benzene, toluene, ethylbenzene, and xylene). Specifically, the tolerance concentration to benzene can reach 300 mg / L, the tolerance concentration to toluene can reach 500 mg / L, the tolerance concentration to ethylbenzene can reach 800 mg / L, and the tolerance concentration to xylene can reach 1200 mg / L.
[0052] In addition, the Pigmentiphaga sp. HB2 has strong degradation performance for benzene series compounds, has good application prospects in the treatment of water bodies polluted by benzene series compounds, is non-toxic and harmless to humans, and has high safety.
[0053] In the present invention, the screening method of the Pigmentiphaga sp. HB2 includes the following steps:
[0054] (1) Mix an inorganic salt medium, a trace element mixture solution, and a benzene series compound solution to obtain a liquid medium with a benzene series compound content of 50-150 mg / L;
[0055] (2) Place the collected groundwater sample polluted by benzene series compounds in the liquid medium and incubate it at a constant temperature to obtain a culture solution;
[0056] (3) Mutagenize the culture medium, and adjust the mutagenesis intensity to make the lethality of microorganisms in the culture medium reach 65-95%, so as to obtain the mutagenized culture medium;
[0057] (4) Increase the concentration of benzene series compounds in the liquid medium in step (1) by 3-5 times, then add the remaining surviving bacteria in the mutagenized culture medium, and culture for 2-5 days under the conditions of low temperature (8-15°C) and low dissolved oxygen (<2.5 mg / L) to obtain the target culture medium;
[0058] (5) Separate and purify the target culture medium, and the obtained pure strain is the benzene series compound degrading bacterium.
[0059] In a specific embodiment, in step (1), the benzene series compound is one or more of benzene, toluene, ethylbenzene, m-xylene, o-xylene and p-xylene.
[0060] In a specific embodiment, in step (1), the inorganic salt medium contains (NH4)2SO4, KH2PO4, K2HPO4, CaCl2, MgSO4 and NaCl.
[0061] Preferably, in the liquid medium, the content of (NH4)2SO4 is 1-4 g / L, the content of KH2PO4 is 0.3-1 g / L, the content of K2HPO4 is 0.3-1 g / L, the content of CaCl2 is 0.03-0.5 g / L, the content of MgSO4 is 0.1-0.4 g / L, and the content of NaCl is 0.05-0.3 g / L.
[0062] In the present invention, in step (1), the trace element mixture contains Zn, Mn, Co, Fe, Mo and B.
[0063] In a preferred embodiment, in the liquid medium, the content of Zn is 0.3-0.8 mM, the content of Mn is 1-3 mM, the content of Co is 0.1-0.3 mM, the content of Fe is 10-30 mM, the content of Mo is 0.2-0.6 mM, and the content of B is 0.5-2 mM. Here, mM refers to mmol / L.
[0064] In the present invention, the benzene series compound solution contains benzene series compounds and a cosolvent, wherein the cosolvent is N,N-dimethylformamide. In a preferred embodiment, the concentration of the benzene series compound solution is 20-80 g / L, more preferably 40-60 g / L.
[0065] In a specific embodiment, step (2) includes: placing the benzene series compound-polluted groundwater sample in a liquid culture medium (in the liquid culture medium, the content of the benzene series compound-polluted groundwater sample is 50-100 g / L), culturing in a constant temperature shaker for 3-7 days to obtain a mixed solution, transferring 5-10% by volume of the mixed solution to a new liquid culture medium, and culturing again for 3-7 days; repeating this process 4-6 times to obtain a culture solution.
[0066] In the present invention, in step (3), the mutagenesis treatment technology can be ultraviolet mutagenesis, microwave mutagenesis, plasma mutagenesis, etc., and preferably plasma mutagenesis.
[0067] In a preferred embodiment, in step (3), the mutagenesis intensity of the mutagenesis treatment is adjusted to make the lethality of the microorganisms in the culture solution reach 75-85%.
[0068] In a preferred embodiment, step (4) includes: increasing the concentration of the benzene series compound in the liquid culture medium in step (1) by 3-5 times, then adding the remaining surviving bacteria in the mutagenized culture solution, and culturing under low temperature (15 °C) and low dissolved oxygen (2 mg / L) conditions for 2-5 days to obtain a target culture solution.
[0069] In a preferred embodiment, in step (5), the steps of separation and purification include: diluting the target culture solution with phosphate buffer to 10 -4 , 10 -6 , and 10 -8 , sucking 150 μL of liquid from each concentration gradient and coating it on a solid culture medium plate containing benzene series compounds, with at least 3 parallels for each gradient, culturing, and then picking appropriate single colonies with an inoculation loop and culturing them in the liquid culture medium in step (1). When the culture reaches the exponential growth phase, the bacteria are coated on the solid culture medium plate again. Repeat this process 2 -3 times, and the obtained pure bacteria are the benzene series compound-degrading bacteria. Among them, the solid culture medium is prepared by adding 10 g / L of agar to the liquid culture medium in step (1).
[0070] In the screening method described in the present invention, a strain capable of efficiently degrading benzene series compounds was finally screened and named HB2.
[0071] The strain HB2 is Gram-positive and the strain morphology is bacillus.
[0072] The strain HB2 has regular colony morphology on Luria -Bertani medium (LB medium), showing white, opaque and smooth morphology.
[0073] The strain HB2 was subjected to 16S rDNA sequencing, and the sequencing results are shown in SEQ ID NO: 1. The sequencing results were subjected to BLAST alignment, and the alignment results showed that the nucleotide sequence of the 16S rDNA of strain HB2 had a homology greater than 99% with the nucleotide sequences of different strains of the genus Pigmentiphaga sp.
[0074] Based on the comprehensive physiological and biochemical characteristics and molecular biological characteristics, it was determined that the screened strain HB2 capable of efficiently degrading benzene series compounds belongs to Pigmentiphaga sp., and it was named Pigmentiphaga sp. HB2.
[0075] The present invention also provides an application of the benzene series compound degrading bacterium as described above in degrading benzene series compounds.
[0076] Furthermore, it is an application of the benzene series compound degrading bacterium in the remediation of groundwater contaminated with benzene series compounds and soil contaminated with benzene series compounds.
[0077] In the application described in the present invention, the benzene series compounds are one or more of benzene, toluene, ethylbenzene, m-xylene, o-xylene, and p-xylene.
[0078] The benzene series compound degradation of the present invention has good temperature tolerance, and it also has good ability to degrade benzene series compounds at low temperatures. In the present invention, the applicable temperature of the Pigmentiphaga sp. HB2 is 10 - 37 °C. Specifically, the lowest temperature for Pigmentiphaga sp. HB2 to repair the site contaminated with benzene series compounds can be 10 °C, and the highest is 37 °C.
[0079] The present invention also provides an application of the benzene series compound degrading bacterium (i.e., Pigmentiphaga sp. HB2) as described above in the preparation of products containing benzene series compounds.
[0080] Since the benzene series compound degrading bacterium has high toxicity tolerance to benzene series compounds (benzene, toluene, ethylbenzene, and xylene) and can efficiently degrade benzene series compounds, therefore, the product also has high toxicity tolerance to benzene series compounds and can efficiently degrade benzene series compounds.
[0081] The present invention also provides a bacterial agent, which contains the benzene series compound degrading bacterium (i.e., Pigmentiphaga sp. HB2) as described above.
[0082] The present invention does not limit the specific components in the bacterial agent, as long as it contains a bacterial suspension, fermentation broth, or culture concentrate of the benzene series compound degrading bacterium as described above. Among them, the bacterial suspension, fermentation broth, and culture concentrate of the benzene series compound degrading bacterium are used as the active components of the bacterial agent. It can be understood that the bacterial agent can also contain other strains used in combination with the benzene series compound degrading bacterium to improve the degradation effect of benzene series compounds.
[0083] In a preferred embodiment, in addition to the above active ingredients, the benzene series compound degrading bacterial agent further contains a carrier. The carrier can be a carrier commonly used in the field of bacterial agents and biologically inert.
[0084] In the benzene series compound degrading bacterial agent of the present invention, the carrier can be a solid carrier or a liquid carrier.
[0085] In a specific embodiment, the solid carrier can be an organic material, a plant material, a mineral material or a composite material. The liquid carrier can be an organic solvent, a vegetable oil or a mineral oil, wherein the organic solvent can be decane and / or dodecane.
[0086] In a preferred embodiment, the carrier is a solid carrier, more preferably one or more selected from sodium alginate, chitosan, polyacrylamide, polyvinyl alcohol, corn starch, starch, clay, talc, kaolin, zeolite and biochar. The present invention has no special limitation on the source of the carrier, and commercial products can be used, or it can be prepared by itself according to the methods well known to those skilled in the art.
[0087] The present invention also does not limit the specific dosage form of the bacterial agent, which can be selected according to actual needs. Specifically, for example, it can be a liquid agent, an emulsion, a suspension, a powder or a granule.
[0088] The present invention does not limit the specific preparation method of the bacterial agent. In a specific embodiment, the preparation method of the bacterial agent includes: fermenting and culturing the benzene series compound degrading bacteria.
[0089] During specific implementation, the benzene series compound degrading bacteria are first activated, then expanded in culture, and then fermented and cultured.
[0090] In a preferred embodiment, the preparation method of the bacterial agent further includes: mixing the fermentation broth obtained by fermentation with the carrier.
[0091] The present invention also provides a method for repairing a benzene series compound contaminated underground site. In a specific embodiment, a bacterial solution is prepared on-site at the benzene series compound contaminated site and then used for repair. During specific implementation, methods such as on-site expansion culture of strains, on-site activation of concentrated bacterial solution, and on-site activation of immobilized bacterial agent can be used to prepare the bacterial solution.
[0092] In a preferred embodiment, the method for repairing a benzene series compound contaminated underground site includes the following steps:
[0093] (1) Prepare a culture medium containing nitrogen and phosphorus elements;
[0094] (2) Add the benzene series compound degrading bacteria to the culture medium for culture until the bacterial concentration reaches 5×10 9 ~10×10 9 cell / mL to obtain a bacterial solution;
[0095] (3) Inject the bacterial solution into the underground site contaminated with benzene series compounds;
[0096] Among them, in step (2), the benzene series compound-degrading bacterium is the benzene series compound-degrading bacterium as described above (i.e., dye-degrading bacterium HB2).
[0097] In a preferred embodiment, the underground site contaminated with benzene series compounds is groundwater contaminated with benzene series compounds and underground soil contaminated with benzene series compounds.
[0098] In a preferred embodiment, in step (1), in the culture medium, the concentration of nitrogen element is 20-60 mmol / L. Specifically, for example, it can be 20 mmol / L, 30 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L or 60 mmol / L.
[0099] In a preferred embodiment, in step (1), in the culture medium, the molar ratio of nitrogen element to phosphorus element is 5-15:1. Specifically, for example, it can be 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1 or 15:1.
[0100] In a specific embodiment, step (2) includes: adding the benzene series compound-degrading bacterium to the culture medium to make the bacterial concentration reach 5×10 8 cell / mL or more, culturing for 2-48 h until the bacterial concentration reaches 5×10 9 ~10×10 9 cell / mL to obtain the bacterial solution.
[0101] In a preferred embodiment, in step (2), the benzene series compound-degrading bacterium is added to the culture medium in the form of bacterial species, concentrated bacterial solution or immobilized bacterial agent.
[0102] In a preferred embodiment, in step (3), the bacterial solution is injected into the underground site contaminated with benzene series compounds at a pressure of 0.3-0.8 MPa, so that the bacterial solution diffuses circularly underground by pressure.
[0103] Please refer to Figure 1-2 , the connection line between multiple injection points is perpendicular to the direction of underground water flow. When the underground water flow is too fast (greater than 20 cm / day) or the concentration of benzene series compounds is too high, multiple rows of bacterial solutions are injected. According to the pollution depth range of the underground site, multi-layer injection can be carried out. The bacterial cells injected underground can adsorb on the soil particles in the groundwater and form a bacterial film, which can "intercept" the benzene series compound pollutants migrating downstream from the upstream through degradation to achieve the repair purpose.
[0104] In a preferred embodiment, in step (3), the radius of the injection range of the bacterial solution is 0.5 to 2 meters, and for every 1 m 2 the amount of the bacterial solution injected into each layer of the site is 50 to 200 L.
[0105] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.
[0106] Example 1
[0107] This example is used to illustrate the screening of the dye-degrading bacterium HB2 described in the present invention.
[0108] (1) Mix an inorganic salt medium (a mixture of (NH4)2SO4, KH2PO4, K2HPO4, CaCl2, MgSO4 and NaCl), trace elements (Zn, Mn, Co, Fe, Mo and B) and a benzene series solution to obtain a liquid medium.
[0109] Among them, the contents of each inorganic salt component in the liquid medium are: (NH4)2SO4 2 g / L, KH2PO4 0.5 g / L, K2HPO4 0.5 g / L, CaCl2 0.1 g / L, MgSO4 0.2 g / L, NaCl 0.1 g / L;
[0110] Among them, the contents of each trace element in the liquid medium are: ZnSO4·7H2O 0.2 g / L, MnSO4·H2O 0.20 g / L, CoCl2·6H2O 0.03 g / L, FeSO4·7H2O 3 g / L, Na2MoO4·2H2O 0.1 g / L, H3BO3 0.05 g / L;
[0111] Among them, the benzene series solution is obtained by dissolving a benzene series in a co-solvent (N,N-dimethylformamide), and the concentration of the benzene series solution is 50 g / L; in the liquid medium, the content of the benzene series is 100 mg / L, and the benzene series is composed of benzene and toluene mixed in a molar ratio of 1:1.
[0112] (2) Weigh the collected benzene series-contaminated groundwater sample and place it in the liquid medium. Among them, in the liquid medium, the content of the benzene series-contaminated groundwater sample is 50 g / L. After culturing in a constant temperature shaker for 3 days, a mixed solution is obtained. Transfer 5% by volume of the mixed solution to a new liquid medium and culture for another 3 days. Repeat this 4 times to obtain a culture solution.
[0113] (3) Use the culture solution for mutagenesis treatment. Adopt the plasma mutagenesis technology, with a power of 120 w, a gas flow rate of 10 SLM, and a mutagenesis time of 10 s, to achieve a lethality rate of 82%.
[0114] (4) Increase the concentration of benzene series compounds in the liquid medium in step (1) to 500 mg / L. Add the remaining surviving bacteria in the mutagenized culture solution into the liquid medium, and culture it in an anaerobic incubator at 15 °C and a dissolved oxygen condition of 2 mg / L for 3 days to obtain the target culture solution containing benzene series compound highly tolerant bacteria.
[0115] (5) Perform spread dilution streak plate isolation culture on the target culture solution. Specifically: Dilute the target culture solution with phosphate buffer to 10 -4 , 10 -6 , and 10 -8 . After sucking 150 μL of liquid for each concentration gradient, coat it on a solid medium plate containing 500 mg / L benzene series compounds, with 3 parallels for each gradient. After culturing for a certain period of time, pick single colonies with an inoculation loop and culture them in the liquid medium in step (4). After culturing for 36 h until the exponential growth phase, coat the bacteria on the solid medium plate (prepared by adding 10 g / L agar to the liquid medium in step (1)) again. After repeating 2 times, strain HB2 is obtained. Among them, the plate culture diagram of strain HB2 is as shown in Figure 3 .
[0116] Example 2
[0117] This example is used to illustrate the identification of the dye-consuming bacterium HB2 described in the present invention.
[0118] 1. Morphological identification
[0119] (1) Observe the strain HB2 obtained in Example 1 under a scanning electron microscope, and the result is as shown in Figure 4 .
[0120] It can be seen from Figure 4 that the strain morphology of strain HB2 is bacillus.
[0121] (2) After detection, the strain HB2 shows a positive Gram stain. The colony morphology of the strain HB2 on the LB medium is regular, showing a white, opaque and smooth morphology.
[0122] 2. Molecular biology identification
[0123] (1) Streak-culture the strain HB2 obtained in Example 1 on a purified solid medium. After cultivation, extract its whole genome using a DNA kit, and perform PCR amplification with primers 27-F and 1492-R. The PCR reaction system is 25 μL: 1 μL each of the upstream and downstream primers (10 μmol / L), 1 μL of DNA template (10 ng / μL), 12.5 μL of 2×Taq Master Mix, and make up to 25 μL with ultrapure water. The PCR reaction conditions are: 94°C for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 1 min 30 s, for 35 cycles; final extension at 72°C for 7 min.
[0124] (2) Entrust the PCR amplification product to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing result of the 16S rRNA gene of strain HB2 is shown in SEQ ID NO: 1. Compare the obtained gene sequence with GenBank for homology and construct a phylogenetic tree ( Figure 5 ).
[0125] (3) The obtained gene sequence belongs to the genus Pigmentiphaga sp. after comparison and analysis with the database. Name it Pigmentiphaga sp. HB2 (i.e., Pigmentiphaga HB2), and deposit this strain at the Guangdong Provincial Microbial Strain Collection Center on March 20, 2023, with the deposit number GDMCC No: 63281.
[0126] Among them, the sequence of SEQ ID NO: 1 is as follows:
[0127]
[0128] Example 3
[0129] This example is used to illustrate the determination of the benzene degradation performance of the dye - eating bacterium HB2 of the present invention.
[0130] The degradation effect and growth of the dye - eating bacterium HB2 on benzene at different concentrations were measured. The test method was as follows: The dye - eating bacterium HB2 obtained in Example 1 was enlarged in culture to obtain a sufficient amount of bacterial solution with a bacterial density of 10 9 cells / mL; a benzene solution with a concentration of 50 g / L was prepared according to the method of Example 1. Different volumes of the benzene solution with a concentration of 50 g / L were added to 500 - mL Erlenmeyer flasks, and then the bacterial solution was added to the Erlenmeyer flasks until the volume reached 200 mL. The Erlenmeyer flasks were sealed with rubber stoppers and cultured in a constant - temperature shaker at 30 °C and 130 rpm. The test results are as Figure 6 and Figure 7 shown.
[0131] From Figure 6 and Figure 7 it can be seen that the dye - eating bacterium HB2 can tolerate and degrade up to 300 mg / L of benzene.
[0132] Example 4
[0133] This example is used to illustrate the determination of the toluene degradation performance of the dye - eating bacterium HB2 of the present invention.
[0134] The degradation effect and growth of the dye - eating bacterium HB2 on toluene at different concentrations were measured. The test method was carried out according to the method described in Example 3. The difference was that different concentrations of benzene solutions were replaced with different concentrations of toluene solutions. The test results are as Figure 8 and Figure 9 shown.
[0135] From Figure 8 and Figure 9 it can be seen that the dye - eating bacterium HB2 can tolerate and degrade up to 500 mg / L of toluene.
[0136] Example 5
[0137] This example is used to illustrate the determination of the ethylbenzene degradation performance of the dye - eating bacterium HB2 of the present invention.
[0138] The degradation effect and growth of the dye - eating bacterium HB2 on ethylbenzene at different concentrations were measured. The test method was carried out according to the method described in Example 3. The difference was that different concentrations of benzene solutions were replaced with different concentrations of ethylbenzene solutions. The test results are as Figure 10 and Figure 11 shown.
[0139] From Figure 10 andFigure 11 It can be seen that the dye - degrading bacterium HB2 can tolerate and degrade up to 800 mg / L of ethylbenzene.
[0140] Example 6
[0141] This example is used to illustrate the determination of the o - xylene degradation performance of the dye - degrading bacterium HB2 described in the present invention.
[0142] The degradation effect and growth of the dye - degrading bacterium HB2 on different concentrations of o - xylene were measured. The test method was carried out according to the method described in Example 3. The difference is that different concentrations of benzene solution were replaced with different concentrations of o - xylene solution. The test results are as Figure 12 and Figure 13 shown.
[0143] From Figure 12 and Figure 13 it can be seen that the dye - degrading bacterium HB2 can tolerate and degrade up to 1200 mg / L of o - xylene.
[0144] Example 7
[0145] This example is used to illustrate the determination of the m - xylene degradation performance of the dye - degrading bacterium HB2 described in the present invention.
[0146] The degradation effect and growth of the dye - degrading bacterium HB2 on different concentrations of m - xylene were measured. The test method was carried out according to the method described in Example 3. The difference is that different concentrations of benzene solution were replaced with different concentrations of m - xylene solution. The test results are as Figure 14 and Figure 15 shown.
[0147] From Figure 14 and Figure 15 it can be seen that the dye - degrading bacterium HB2 can tolerate and degrade up to 1200 mg / L of m - xylene.
[0148] Example 8
[0149] This example is used to illustrate the determination of the p - xylene degradation performance of the dye - degrading bacterium HB2 described in the present invention.
[0150] The degradation effect and growth of the dye - degrading bacterium HB2 on different concentrations of p - xylene were measured. The test method was carried out according to the method described in Example 3. The difference is that different concentrations of benzene solution were replaced with different concentrations of p - xylene solution. The test results are as Figure 16 and Figure 17 shown.
[0151] From Figure 16 and Figure 17 it can be seen that the dye - degrading bacterium HB2 can tolerate and degrade up to 1200 mg / L of p - xylene.
[0152] Example 9
[0153] The degradation effect and growth of dye - degrading bacterium HB2 on benzene and toluene at different concentrations under low - temperature and low - oxygen conditions were measured. The test method was carried out according to the method described in Example 3. The difference was that different concentrations of benzene solution were replaced with different concentrations of benzene or toluene solution.
[0154] The low - temperature (15 °C) and low - oxygen (1.5 - 2 mg / L) conditions were provided by a low - oxygen anaerobic incubator. The test results are as Figure 18 and Figure 19 shown.
[0155] From Figure 18 and Figure 19 it can be seen that under low - temperature and low - oxygen conditions, the dye - degrading bacterium HB2 described in the present invention also has good degradation ability for benzene and toluene.
[0156] Application Example 1
[0157] The actual application effect of the dye - degrading bacterium HB2 obtained in Example 1 of the present invention in the remediation of a benzene - series - contaminated site in an oil and gas field in Inner Mongolia Autonomous Region was tested. The contaminated site was mainly contaminated with benzene and toluene, with average concentrations of 126 mg / L and 206 mg / L respectively.
[0158] 1. Test method:
[0159] (1) A on - site activated culture medium was prepared. 920 g of ammonium hydrogen phosphate compound fertilizer and 2100 g of ammonium sulfate granular fertilizer were added to each ton of water respectively, so that the concentration of nitrogen element in the culture medium reached 40 mmol / L, and the molar ratio of nitrogen element to phosphorus element was 10:1.
[0160] (2) 1 L of dye - degrading bacterium HB2 was added to 100 L of the culture medium and cultured for 8 h. Then, 100 L of the obtained culture solution was taken and added to 3000 L of the on - site activated culture medium. After culturing for another 8 h, the bacterial concentration reached 15×10 9 cell / mL, and a bacterial solution was obtained.
[0161] (3) The site to be repaired was 10 m wide, and the groundwater flowed from northwest to southeast with a flow rate of 12 cm / day. The low - pressure injection method was used to inject into the underground site at 0.4 Mpa. There were 5 injection points in each layer, arranged horizontally as shown in Figure 1-2 so that the bacterial solution diffused circularly underground by pressure. It was divided into 3 m, 5 m, and 7 m. Each injection point had 3 injection layers, and 200 L of the bacterial solution was injected into each injection layer. A total of 600 L of the bacterial solution was injected into each injection point, and a total of 3000 L of the bacterial solution was injected.
[0162] After the injection was completed, monitoring wells were established downstream of the site flow field to monitor the degradation situation.
[0163] 2. Test results:
[0164] 40 days after the injection of the bacterial solution, the remediation rates of benzene and toluene reached 82% and 93% respectively.
[0165] Application of Comparative Example 1
[0166] It was implemented according to the test method described in Application Example 1. The difference was that the dye-consuming bacterium HB2 was replaced with a commercial benzene-series compound-degrading bacterium.
[0167] The results showed that 40 days after the injection of the bacterial solution, the remediation rates of benzene and toluene were 22% and 37% respectively.
[0168] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.
Claims
1. A benzene-degrading bacterium, characterized in that, The benzene-series degrading bacterium is Pigmentiphaga sp. HB2, and its preservation number is GDMCC NO: 63281.
2. Application of the benzene-series degrading bacterium according to claim 1 in degrading benzene-series substances.
3. The application according to claim 2, wherein It is applied in the remediation of groundwater polluted by benzene-series substances and underground soil polluted by benzene-series substances.
4. The application according to claim 2 or 3, characterized in that, The benzene-series substances are one or more of benzene, toluene, ethylbenzene, m-xylene, o-xylene, and p-xylene.
5. The application according to claim 2 or 3, characterized in that, The applicable temperature of the benzene-series degrading bacterium is 10-37 °C.
6. Application of the benzene-series degrading bacterium according to claim 1 in preparing a product for degrading benzene-series substances.
7. A bacterial agent, characterized in that, The bacterial agent contains the benzene-series degrading bacterium according to claim 1.
8. The microbial agent according to claim 7, characterized in that, The dosage form of the bacterial agent is liquid, emulsion, suspension, powder or granule.
9. A method for remediating benzene series contaminated underground sites, characterized in that, This method includes the following steps: (1) Prepare a culture medium containing nitrogen and phosphorus elements; (2) Add benzene-degrading bacteria to the culture medium and cultivate until the bacterial concentration reaches 5×10 9 ~10×10 9 cell / mL to obtain a bacterial solution; (3) Inject the bacterial liquid into the underground site polluted by benzene-series substances; Among them, in step (2), the benzene-series degrading bacterium is the benzene-series degrading bacterium according to claim 1.
10. The method according to claim 9, characterized in that, The underground site polluted by benzene-series substances is groundwater polluted by benzene-series substances and underground soil polluted by benzene-series substances.
11. The method according to claim 9, wherein In the culture medium, the concentration of nitrogen element is 20-60 mmol / L.
12. The method according to claim 9 or 11, characterized in that, In the culture medium, the molar ratio of nitrogen element to phosphorus element is 5-15:
1.
13. The method according to claim 9, wherein In step (3), inject the bacterial liquid into the underground site polluted by benzene-series substances at a pressure of 0.3-0.8 MPa.