Benzene series degrading bacterium and application thereof

Abstract

The invention relates to the technical field of microorganisms and environmental protection, and discloses a benzene series degrading bacterium and application thereof.The benzene series degrading bacterium is stenotrophomonas sp. YB2, and the preservation number of the benzene series degrading bacterium is GDMCC No: 63283. The benzene series degrading bacterium provided by the invention is stenotrophomonas YB2, the strain can grow by using benzene series as a unique carbon source, and the tolerance concentration to the benzene series can reach more than 500mg / L; the strain not only can efficiently degrade benzene, but also has good degradation performance on toluene and xylene. Therefore, the benzene-series degrading bacterium provided by the invention can be well applied to benzene-series-containing sewage treatment and benzene-series-polluted soil remediation, and has important significance in relieving benzene-series environmental pollution.

Description

Technical Field

[0001] The present invention relates to the technical fields of microorganisms and environmental protection, and particularly relates to a benzene series compound-degrading bacterium and its application. Background Art

[0002] Benzene series compounds (BTEX) are characteristic pollutants frequently detected in polluted sites in the petrochemical industry. In recent years, oil leakage accidents have occurred in the underground facilities of petrochemical enterprises in production due to reasons such as poor maintenance, resulting in pollution of benzene series compounds in groundwater. The pollution of benzene series compounds in petrochemical enterprises in production and retired ones has become an important risk source affecting the soil and groundwater environment, posing a potential threat to human health. The World Health Organization has determined benzene as a human carcinogen.

[0003] At present, a series of remediation technologies have been formed for benzene series compound-polluted sites. However, due to the large number of sensitive targets and high safety requirements around petrochemical enterprises in production, it is difficult to carry out traditional thermal desorption technology. Bioremediation can supplement oxygen to the soil and groundwater through measures such as biopiling, injection of oxygen / air, hydrogen peroxide, etc., but these are all invasive measures with high costs. Therefore, taking appropriate artificial measures to strengthen the degradation process of indigenous microorganisms will be a more widely used bioremediation technology for benzene, toluene, and xylene-polluted soil. Many research results have proved that benzene is biodegradable, especially under aerobic conditions, and a variety of aerobic benzene-degrading bacteria have been identified. However, the existing strains used for remediating benzene series compound-polluted soil have problems of great difficulty in remediation or even inability to remediate sites with relatively high benzene series compound pollution concentrations (above 100 mg / L). Summary of the Invention

[0004] The purpose of the present invention is to overcome the problems in the prior art that the strains used for remediating benzene series compound-polluted soil have great difficulty in remediation or even inability to remediate sites with relatively high benzene series compound pollution concentrations, and to provide a benzene series compound-degrading bacterium and its application. The benzene series compound-degrading bacterium strain has tolerance in an environment with a relatively high benzene series compound concentration, and in addition to having a high degradation efficiency for benzene, it also shows good degradation performance for toluene and xylene.

[0005] To achieve the above purpose, on the one hand, the present invention provides a benzene series compound-degrading bacterium, which is Stenotrophomonas sp. YB2, and its preservation number is GDMCC No: 63283.

[0006] 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.

[0007] Preferably, the benzene series compound is at least one of benzene, toluene, and xylene.

[0008] Preferably, it is used in the treatment of benzene series-containing sewage or the remediation of benzene series-polluted soil.

[0009] The third aspect of the present invention provides an application of the benzene series-degrading bacterium as described above in the preparation of a product for degrading benzene series.

[0010] The fourth aspect of the present invention provides a bacterial agent, which contains the benzene series-degrading bacterium as described above.

[0011] Preferably, the bacterial agent further contains a carrier.

[0012] Preferably, the carrier is selected from one or more of sodium alginate, chitosan, polyacrylamide, polyvinyl alcohol, corn starch, starch, clay, talc, kaolin, zeolite, and biochar.

[0013] The fifth aspect of the present invention provides a method for preparing a bacterial agent, which includes: fermenting and culturing the benzene series-degrading bacterium described in claim 1.

[0014] Preferably, the method further includes: mixing the fermented liquid obtained by fermentation with a carrier.

[0015] Preferably, the conditions for fermentation include: temperature 25-35°C, rotation speed 100-300 rpm, gas-liquid ratio 1:(1-2), and dissolved oxygen ≥20%.

[0016] The sixth aspect of the present invention provides a bacterial agent prepared by the method as described above.

[0017] The seventh aspect of the present invention provides an application of the bacterial agent as described above in the degradation of benzene series.

[0018] The benzene series-degrading bacterium provided by the present invention is Stenotrophomonas YB2, which can use benzene series as the sole carbon source for growth, and the tolerance concentration to benzene series can reach more than 500 mg / L; this strain can not only efficiently degrade benzene, but also has good degradation performance for toluene and xylene. Therefore, the benzene series-degrading bacterium provided by the present invention can be well applied to the treatment of benzene series-containing sewage and the remediation of benzene series-polluted soil, which has important significance for alleviating benzene series environmental pollution.

[0019] Biological preservation

[0020] The benzene series-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: 63283, and the taxonomic name: Stenotrophomonas sp. YB2. The deposit address is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province, and the postal code is 510070. Brief description of the drawings

[0021] Figure 1 It is the scanning electron microscope image of Stenotrophomonas sp. YB2 provided by the present invention;

[0022] Figure 2 It is the colony morphology image of Stenotrophomonas sp. YB2 provided by the present invention;

[0023] Figure 3 It is the 16S rDNA phylogenetic tree image of Stenotrophomonas sp. YB2 provided by the present invention;

[0024] Figure 4 It is the test result image of the degradation rate of the bacterial agent a on benzene series compounds in Test Example 3. Detailed Embodiments

[0025] The following further elaborates on the detailed embodiments of the present invention with reference to the accompanying drawings. It should be understood that the detailed 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.

[0026] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and 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 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 herein.

[0027] The present invention provides a benzene series compound degrading bacterium, which is Stenotrophomonas sp. YB2, and its preservation number is GDMCC No: 63283. This strain was preserved in the Guangdong Provincial Microbial Culture Collection Center on the 5th floor of Building 59, 100th Yard, Xianlie Middle Road, Guangzhou City, Guangdong Province on March 20, 2023.

[0028] Stenotrophomonas sp. YB2 provided by the present invention not only has a high degradation efficiency for benzene, but also shows high degradation performance for toluene and xylene. It has good economy, good versatility, and is suitable for engineering applications.

[0029] In the present invention, the Stenotrophomonas sp. YB2 was isolated and screened from the activated sludge contaminated with benzene series compounds in a petrochemical plant in Qilu. Among them, Stenotrophomonas sp. YB2 can grow with benzene series compounds as the sole carbon source and has the ability to efficiently degrade benzene series compounds (such as benzene, toluene, and xylene).

[0030] In the present invention, the screening method of the benzene series compound degrading bacterium includes the following steps:

[0031] (1) Add the benzene series - contaminated activated sludge collected from a petrochemical plant in Qilu to sterile water. After shaking for 10 - 20 h, centrifuge and take the supernatant containing bacteria.

[0032] (2) Spread the supernatant containing bacteria on an LB solid medium and culture for 24 - 50 h to obtain colonies.

[0033] (3) Inoculate the colonies into an enrichment medium and shake - culture for 10 - 30 h to obtain a culture solution.

[0034] (4) Separate and purify - culture the culture solution until a purified colony with a single morphology is obtained, thereby obtaining benzene - series - degrading bacteria.

[0035] Among them, in step (3), the enrichment medium is an inorganic salt medium with benzene series as the sole carbon source. Among them, the benzene series is one or more of benzene, toluene, and xylene, preferably a mixture of benzene, toluene, and xylene.

[0036] Furthermore, the enrichment medium contains 150 - 250 mg / L benzene series.

[0037] In a preferred embodiment, in step (3), the formula of the enrichment medium includes: 200 mg / L benzene series (a mixture of benzene, toluene, and xylene), 0.5 g / L potassium dihydrogen phosphate (KH2PO4), 0.5 g / L dipotassium hydrogen phosphate (K2HPO4), 0.1 g / L calcium chloride (CaCl2), 0.2 g / L magnesium sulfate (MgSO4), 0.1 g / L sodium chloride (NaCl), and 2 g / L ammonium sulfate ((NH4)2SO4), pH 7.0.

[0038] More preferably, each 1 L of the enrichment medium further contains 1 mL of trace element solution. Among them, the formula of the trace element solution is: dissolved in every 100 mL of deionized water are 0.022 g of ZnSO4·4H2O, 0.039 g of Na2MoO4·2H2O, 0.186 g of MnCl2·4H2O, 0.005 g of Co(NO3)2·6H2O, and 0.008 g of CuSO4·5H2O.

[0039] In the screening method described in the present invention, a strain capable of efficiently degrading benzene series is finally screened, and its number is YB2.

[0040] In the present invention, the strain morphology of the strain YB2 is rod - shaped.

[0041] In the present invention, the colony characteristics of the strain YB2 are: the colony shape is circular, the color is milky white, the edge is neat, and it is not sticky.

[0042] Furthermore, the 16S rDNA gene sequencing result of the strain YB2 is as shown in SEQ ID NO: 1.

[0043] Based on the comprehensive physiological and biochemical characteristics and molecular biological characteristics, it is determined that the strain YB2 belongs to Stenotrophomonas sp., and it is named Stenotrophomonas YB2.

[0044] In a specific embodiment, in step (1), the activated sludge contaminated with benzene series compounds is collected from the sludge at a depth of 0 - 30 cm.

[0045] In a specific embodiment, in step (2), the formula of the LB solid medium is: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, and agar 10 g / L, with a pH of 7.0.

[0046] Specifically, step (4) includes: diluting the culture solution at three gradients of 10 -5 、10 -6 and 10 -7 , then taking 100 μL of the diluted bacterial solution and spreading it on a fresh LB solid medium, and culturing it in a constant temperature incubator. Repeat the above steps until single colonies grow on the plate; select colonies with faster growth, different colors and morphologies, and inoculate them on a new LB solid medium by the method of streaking on the plate. Repeat the above streaking and isolation process until a purified colony with a single morphology is formed.

[0047] The present invention also provides an application of the benzene series compound-degrading bacterium (i.e., Stenotrophomonas YB2) as described above in degrading benzene series compounds.

[0048] The present invention does not limit the specific types of the benzene series compounds. In a preferred embodiment, the benzene series compounds are at least one of benzene, toluene, and xylene.

[0049] In a preferred embodiment, it is an application of the benzene series compound-degrading bacterium (i.e., Stenotrophomonas YB2) in the treatment of sewage containing benzene series compounds or the remediation of soil contaminated with benzene series compounds.

[0050] When the required treatment time is relatively long, in order to make the treatment effect of Stenotrophomonas YB2 on sewage containing benzene series compounds or soil contaminated with benzene series compounds better, preferably, during the treatment process, elements such as C, N, and P are added to the material to be treated (sewage containing benzene series compounds or soil contaminated with benzene series compounds), so that the reproduction rate of Stenotrophomonas YB2 is fast and its activity is good.

[0051] Furthermore, the present invention also provides an application of the benzene series compound-degrading bacterium (i.e., Stenotrophomonas YB2) as described above in the preparation of products for degrading benzene series compounds.

[0052] The present invention also provides a bacterial agent, which contains the benzene series compound-degrading bacteria as described above.

[0053] The present invention does not limit the specific components in the bacterial agent, and it is sufficient to contain a bacterial suspension, a fermentation broth or a culture concentrate of the benzene series compound-degrading bacteria as described above. It can be understood that other strains that can be used in combination with the benzene series compound-degrading bacteria may also be contained in the bacterial agent to improve the degradation effect on benzene series compounds.

[0054] In a preferred embodiment, in addition to the above active ingredients, the benzene series compound-degrading bacterial agent also contains a carrier. The carrier can be a carrier commonly used in the field of bacterial agents and is biologically inert.

[0055] In the benzene series compound-degrading bacterial agent of the present invention, the carrier can be a solid carrier or a liquid carrier.

[0056] 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.

[0057] In a preferred embodiment, the carrier is a solid carrier, more preferably one or more of 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 commercially available products can be used, or it can be prepared by methods well-known to those skilled in the art.

[0058] 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, a granule, a wettable powder or a water dispersible granule.

[0059] The present invention also provides a method for preparing a bacterial agent, which includes: fermenting and culturing the benzene series compound-degrading bacteria as described above.

[0060] During specific implementation, the benzene series compound-degrading bacteria are first activated, then enlarged cultured, and then fermented and cultured.

[0061] In a specific embodiment, the method for preparing the bacterial agent includes the following steps:

[0062] (1) Strain activation: Spreading Stenotrophomonas sp. YB2 on an LB solid medium and culturing it at a constant temperature to activate the strain;

[0063] (2) Primary seed culture: Inoculate the activated colony YB2 into LB liquid medium and shake culture at 25 - 35°C and 100 - 300 rpm for 16 - 48 h to obtain the primary seed culture solution;

[0064] (3) Secondary seed culture: Inoculate the primary seed solution into liquid LB medium at an inoculation amount of 1 - 10% by volume ratio and shake culture at 25 - 35°C and 100 - 300 rpm for 16 - 48 h to obtain the secondary seed culture solution;

[0065] (4) Fermentation: Inoculate the secondary seed culture solution into the fermentation medium at an inoculation amount of 5 - 10% by volume ratio for fermentation culture. During the fermentation process, the conditions are controlled as follows: temperature 27 - 35°C, rotation speed 120 - 300 rpm, gas - liquid ratio (aeration ratio) 1:(1 - 2), and stop fermentation when the dissolved oxygen rises.

[0066] In a preferred embodiment, the formula of the LB liquid medium is: peptone 10 g / L, yeast extract 5 g / L, and NaCl 10 g / L, pH 7.0.

[0067] In a preferred embodiment, the formula of the LB solid medium is: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, and agar 10 g / L, pH 7.0.

[0068] In a preferred embodiment, in step (4), the fermentation medium contains glucose, corn steep liquor dry powder, soybean meal, ammonium sulfate, magnesium sulfate, sodium chloride, ferrous sulfate, calcium chloride, and polyether defoamer.

[0069] Further preferably, the formula of the fermentation medium is: glucose 1.2 - 1.8 wt%, corn steep liquor dry powder 0.85 - 1 wt%, soybean meal 3 - 4 wt%, ammonium sulfate 0.01 - 0.02 wt%, magnesium sulfate 0.03 - 0.05 wt%, sodium chloride 0.01 - 0.03 wt%, ferrous sulfate 0.002 - 0.005 wt%, calcium chloride 0.01 - 0.02 wt%, polyether defoamer 0.05 - 0.2 wt%, and the balance is water.

[0070] In a specific embodiment, the method for preparing the bacterial agent includes the following steps:

[0071] (1) Strain activation: Spread Stenotrophomonas YB2 on LB solid medium, place it in a constant - temperature and humidity - controlled incubator, and culture at 27°C for 24 h to activate the colonies;

[0072] (2) Preparation of primary seed liquid: The activated colony YB2 was inoculated into LB liquid medium and cultured with shaking at 27°C and 120 rpm for 24 h to obtain the primary seed liquid;

[0073] (3) Preparation of secondary seed liquid: The primary seed liquid was inoculated into 1 L of LB liquid medium at an inoculation amount of 5%, cultured with shaking at 27°C and 120 rpm, and aerated with an aeration rate of 0.5 L / min for 24 h to obtain the secondary seed liquid;

[0074] (4) Fermentation: The fermentation medium was added to the fermenter. After sterilization, the secondary seed liquid was inoculated at a volume ratio of 5%. During the fermentation process, the temperature was controlled at 27°C, the tank pressure was 0.05 MPa, the initial rotation speed was 200 rpm, the dissolved oxygen was ≥20%, and the gas-liquid ratio was 1:1; when the dissolved oxygen dropped to 20% and the pH rose to 8.8, the fermentation was completed to obtain the microbial agent for benzene series degradation.

[0075] In a preferred embodiment, the method further includes: mixing the fermented liquid with a carrier.

[0076] In the method for preparing the microbial agent of the present invention, the carrier is a solid carrier, more preferably one or more of sodium alginate, chitosan, polyacrylamide, polyvinyl alcohol, corn starch, starch, clay, talc, kaolin, zeolite, and biochar.

[0077] The present invention also provides a microbial agent prepared by the method as described above.

[0078] The present invention also provides an application of the microbial agent as described above in the degradation of benzene series.

[0079] In a preferred embodiment, the benzene series is one or more of benzene, toluene, and xylene.

[0080] In a preferred embodiment, it is the application of the microbial agent in the treatment of sewage containing benzene series or the remediation of soil polluted by benzene series. The microbial agent can efficiently degrade benzene series in sewage or soil, and can be directly used for the treatment of sewage containing benzene series or the remediation of soil polluted by benzene series, or can be used as one component in combination with other components (such as chemical agents, other strains that can synergistically act with the microbial agent, culture medium components, etc.).

[0081] When the treatment time is long, in order to make the treatment effect of the microbial agent on the sewage containing benzene series or the soil polluted by benzene series better, preferably, during the treatment process, elements such as C, N, and P are added to the material to be treated (sewage containing benzene series or soil polluted by benzene series) to make the reproduction rate and activity of Stenotrophomonas YB2 in the microbial agent better.

[0082] Further, when the bacterial agent is used for repairing benzene series - contaminated soil, it can be used alone or in combination with other remediation techniques (such as chemical oxidation / reduction techniques, vapor extraction techniques, bioventing techniques, etc.).

[0083] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0084] In the following examples, the medium formulations are as follows:

[0085] Trace element solution: Add 0.022 g of ZnSO4·4H2O, 0.039 g of Na2MoO4·2H2O, 0.186 g of MnCl2·4H2O, 0.005 g of Co(NO3)2·6H2O, and 0.008 g of CuSO4·5H2O to every 100 mL of deionized water;

[0086] Enrichment medium: 200 mg / L of benzene series (a mixture of benzene, toluene, and xylene, where the weight ratio of benzene:toluene:xylene = 1:1:1), 0.5 g / L of potassium dihydrogen phosphate (KH2PO4), 0.5 g / L of dipotassium hydrogen phosphate (K2HPO4), 0.1 g / L of calcium chloride (CaCl2), 0.2 g / L of magnesium sulfate (MgSO4), 0.1 g / L of sodium chloride (NaCl), 2 g / L of ammonium sulfate ((NH4)2SO4), and 0.1 vol% of trace element solution, pH 7.0;

[0087] LB liquid medium: 10 g / L of peptone, 5 g / L of yeast extract, and 10 g / L of NaCl, pH 7.0;

[0088] LB solid medium: Add 10 g / L of agar based on the LB liquid medium;

[0089] Fermentation medium: 1.5 wt% of glucose, 0.9 wt% of corn steep liquor powder, 3 wt% of soybean meal, 0.015 wt% of ammonium sulfate, 0.04 wt% of magnesium sulfate, 0.02 wt% of sodium chloride, 0.004 wt% of ferrous sulfate, 0.015 wt% of calcium chloride, 0.1 wt% of polyether defoamer, and the balance is water.

[0090] Example 1

[0091] This example is used to illustrate the screening of the Stenotrophomonas YB2 of the present invention.

[0092] (1) Collect the benzene - series - contaminated activated sludge from a petrochemical plant in Qilu. Take 15 g of the activated sludge and add it to a 250 - mL conical flask containing 100 mL of sterile water. Oscillate and culture at 27 °C and 120 rpm for 16 h, then centrifuge at 2000 rpm and take the supernatant containing bacteria;

[0093] (2) Spread the supernatant containing bacteria on LB solid medium and culture for 48 h to obtain colonies.

[0094] (3) Inoculate the colonies into enrichment medium and shake culture at 27 °C and 120 rpm for 24 h to obtain a culture solution.

[0095] (4) Dilute the culture solution in three gradients of 10 -5 、10 -6 and 10 -7 . Then take 100 μL of the diluted bacterial solution and spread it on fresh LB solid medium, and culture it in a constant temperature incubator. Repeat the above steps until single colonies grow on the plate. Select colonies with faster growth, different colors and morphologies, and inoculate them on a new LB solid medium by the method of streaking. Repeat the above streaking separation process until a purified colony with a single morphology is formed, thus obtaining the strain YB2 that can efficiently degrade benzene series compounds.

[0096] Example 2

[0097] This example is used to illustrate the identification of the oligotrophic bacterium YB2 of the present invention.

[0098] 1. Morphological identification

[0099] (1) Observe the strain YB2 obtained in Example 1 under a scanning electron microscope, and the results are as Figure 1 shown.

[0100] It can be seen from Figure 1 that the strain YB2 is all rod-shaped.

[0101] (2) Figure 2 is a schematic diagram of the colony morphology of the strain YB2 obtained in Example 1.

[0102] It can be seen from Figure 2 that the colony shape of the strain YB2 is round, the color is milky white, the edge is neat, the surface is smooth and not sticky.

[0103] 2. Molecular biology identification

[0104] Identification method: After the strain YB2 obtained in Example 1 is enlarged and cultured, it is sent to Shanghai Sangon Biotech Co., Ltd. for bacterial genome extraction, PCR amplification and sequencing. The primers used for PCR amplification are the bacterial universal primers 27F and 1492R.

[0105] Results: The 16S rDNA gene sequencing results of strain YB2 are shown in SEQ ID NO: 1. Its DNA sequence length is 1481 bp. The sequencing sequence was aligned in NCBI-Nucleotide BLAST and a phylogenetic tree was constructed ( Figure 3 ), and it was found that strain YB2 was highly homologous to Stenotrophomonas, and the consistency of the gene sequences with multiple species in the genus Stenotrophomonas was more than 98%;

[0106] Combined with physiological and biochemical characteristics and molecular biological characteristics, it was determined that strain YB2 belongs to the genus Stenotrophomonas, named Stenotrophomonas sp. YB2 (i.e., Stenotrophomonas YB2), and was deposited in the Guangdong Provincial Microbial Strain Preservation Center on March 20, 2023, with the deposit number GDMCC No: 63283.

[0107] Among them, the sequence of SEQ ID NO: 1 is as follows:

[0108] AGTTTGATTCCCGGCTCAGAGTGAACGCTGGCGGTAGGCCTAACA

[0109] CATGCAAGTCGAACGGCAGCACAGTAAGAGCTTGCTCTTACGGGTGGC

[0110] GAGTGGCGGACGGGTGAGGAATGCATCGGAATCTACTCTGTCGTGGGG

[0111] GATAACGTAGGGAAACTTACGCTAATACCGCATACGACCTACGGGTGA

[0112] AAGCAGGGGATCTTCGGACCTTGCGCGATTGAATGAGCCGATGCCCGA

[0113] TTAGCTAGTTGGCGGGGTAAGAGCCCACCAAGGCGACGATCGGTAGCT

[0114] GGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGA

[0115] CTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCC

[0116] TGATCCAGCCATACCGCGTGGGTGAAGAAGGCCTTCGGGTTGTAAAGC

[0117] CCTTTTGTTGGGAAAGAAAAGCATTCGGTTAATACCCGATTGTTCTGAC

[0118] GGTACCCAAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGT

[0119] AATACGAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTG

[0120] CGTAGGTGGTTGTTTAAGTCTGTCGTGAAAGCCCTGGGCTCAACCTGG

[0121] GAATTGCGATGGAAACTGGGCGACTAGAGTGTGGCAGAGGGTAGTGG

[0122] AATTCCTGGTGTAGCAGTGAAATGCGTAGAGATCAGGAGGAACATCCG

[0123] TGGCGAAGGCGACTGCCTGGGCCAACACTGACACTGAGGCACGAAAG

[0124] CGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAAC

[0125] GATGCGAACTGGATGTTGGGTGCAATTTGGCACGCAGTATCGAAGCTA

[0126] ACGCGTTAAGTTCGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTC

[0127] AAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAAT

[0128] TCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATGCACGGAACTT

[0129] TCCAGAGATGGATTGGTGCCTTCGGGAACCGTGACACAGGTGCTGCAT

[0130] GGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG

[0131] AGCGCAACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTA

[0132] AGGAGACCGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAA

[0133] GTCATCATGGCCCTTACGGCCAGGGCTACACACGTACTACAATGGTAGG

[0134] GACAGAGGGCTGCAAGCCGGCGACGGTGAGCCAATCCCAGAAACCCT

[0135] ATCTCAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAAT

[0136] CGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCC

[0137] TTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGCA

[0138] GGTAGCTTAACCTTCGGGAGGGCGCTTGCCACGGTGTGGCCGATGACT

[0139] GGGGTGAAGTCGTAA。

[0140] Example 3

[0141] This example is used to illustrate the preparation method of Stenotrophomonas sp. YB2 bacterial liquid.

[0142] (1) Spread the Stenotrophomonas sp. YB2 obtained in Example 1 on the LB solid medium, place it in a constant temperature and humidity incubator, and culture it at 27 °C for 24 h to activate the colonies;

[0143] (2) Inoculate the activated colony YB2 into LB liquid medium and shake culture at 27 °C and 120 rpm for 24 h to obtain the seed liquid YB2;

[0144] (3) Inoculate the seed liquid YB2 into 1 L of LB liquid medium at an inoculation amount of 5%, shake culture at 27 °C and 120 rpm, and perform aeration with an aeration rate of 0.5 L / min. Culture for 24 h to obtain the enriched bacterial liquid of Stenotrophomonas YB2. Adjust its concentration to make the OD 600 value = 1 to obtain the bacterial liquid of Stenotrophomonas YB2.

[0145] Example 4

[0146] This example is used to illustrate the bacterial agent and its preparation method of the present invention.

[0147] (1) Spread the Stenotrophomonas YB2 obtained in Example 1 on LB solid medium, place it in a constant temperature and humidity incubator, and culture at 27 °C for 24 h to activate the colonies. Inoculate the activated colony YB2 into LB liquid medium and shake culture at 27 °C and 120 rpm for 24 h to obtain the primary seed liquid YB2;

[0148] (2) Inoculate the primary seed liquid YB2 into 1 L of LB liquid medium at an inoculation amount of 5%, shake culture at 27 °C and 120 rpm, and perform aeration with an aeration rate of 0.5 L / min. Culture for 24 h to obtain the enriched bacterial liquid of Stenotrophomonas YB2. Adjust its concentration to make the OD 600 value = 1 to obtain the secondary seed liquid;

[0149] (3) Add fermentation medium to the fermenter. After sterilization, inoculate the secondary seed liquid at a volume ratio of 5%. Control the temperature at 27 °C, the tank pressure at 0.05 MPa, the initial rotation speed at 200 rpm, the dissolved oxygen ≥ 20%, and the gas-liquid ratio at 1:1 during the fermentation process; when the dissolved oxygen drops to 20% and the pH rises to 8.8, the fermentation is completed to obtain the fermentation broth;

[0150] (4) Dissolve 3 parts by weight of sodium alginate in distilled water, add 12 parts by weight of urea and 1 part by weight of dipotassium hydrogen phosphate, stir and mix for 30 min, then heat-treat and sterilize at 121 °C for 15 min, and cool to obtain the gel;

[0151] (5) Prepare a calcium chloride solution with 3 parts by weight of calcium chloride and perform heat-treatment sterilization, then add the above fermentation broth and the above gel to it. Among them, the volume ratio of the fermentation broth to the gel is 1:10, crosslink for 4 h, stand, filter, and dry at low temperature (40 °C) to obtain the bacterial agent a.

[0152] Example 5

[0153] This example is used to illustrate the bacterial agent and its preparation method described in the present invention.

[0154] (1) The Stenotrophomonas YB2 obtained in Example 1 was spread on an LB solid medium and placed in a constant temperature and humidity incubator for culturing at 27 °C for 24 h to activate the colonies. The activated colonies YB2 were inoculated into an LB liquid medium and cultured with shaking at 27 °C and 120 rpm for 24 h to obtain the primary seed liquid YB2.

[0155] (2) The primary seed liquid YB2 was inoculated into 1 L of LB liquid medium at an inoculation amount of 5%, cultured with shaking at 27 °C and 120 rpm, and aerated. The aeration rate was 0.5 L / min, and the culture was carried out for 24 h to obtain the enriched bacterial liquid of Stenotrophomonas YB2. Adjust its concentration to make the OD 600 value = 1 to obtain the secondary seed liquid.

[0156] (3) Fermentation medium was added to the fermenter. After sterilization, the secondary seed liquid was inoculated at a volume ratio of 5%. During the fermentation process, the temperature was controlled at 27 °C, the tank pressure was 0.05 MPa, the initial rotation speed was 200 rpm, the dissolved oxygen was ≥ 20%, and the gas-liquid ratio was 1:1; when the dissolved oxygen dropped to 20% and the pH rose to 8.8, the fermentation was completed to obtain the fermentation broth.

[0157] (4) Under the condition of limited oxygen (4%), corn cob was pyrolyzed into biochar at 500 °C, pulverized, sieved through an 80-mesh sieve to obtain biochar particles. The particle size of the biochar particles was < 0.08 mm, the pH value was 6.5 - 8.5, the specific surface area was 1200 m 2 / g, the total pore volume was 0.5 cm 3 / g, and the average pore diameter was 2 nm.

[0158] (5) The above biochar particles were autoclaved at 121 °C for 20 min. The above fermentation broth was inoculated on the biochar particles at an inoculation amount of 10% and cultured on a shaker at 35 °C and 150 rpm. After culturing for 20 h, the supernatant was poured out, and then the precipitate was washed with physiological saline. After repeating the washing 3 times, the bacterial agent b was obtained.

[0159] Example 6

[0160] This example is used to illustrate the bacterial agent and its preparation method described in the present invention.

[0161] (1) The Stenotrophomonas sp. YB2 obtained in Example 1 was spread on an LB solid medium and placed in a constant temperature and humidity incubator. It was cultured at 27 °C for 24 h to activate the colonies. The activated colonies YB2 were inoculated into an LB liquid medium and cultured with shaking at 27 °C and 120 rpm for 24 h to obtain the primary seed liquid YB2;

[0162] (2) The primary seed liquid YB2 was inoculated into 1 L of LB liquid medium at an inoculation amount of 5%. It was cultured with shaking at 27 °C and 120 rpm and aerated. The aeration rate was 0.5 L / min. After culturing for 24 h, an enriched bacterial liquid of Stenotrophomonas sp. YB2 was obtained. Its concentration was adjusted to make the OD value of the bacterial liquid 600 = 1 to obtain the secondary seed liquid;

[0163] (3) Fermentation medium was added to the fermenter. After sterilization, the secondary seed liquid was inoculated at a volume ratio of 5%. During the fermentation process, the temperature was controlled at 27 °C, the tank pressure was 0.05 MPa, the initial rotation speed was 200 rpm, the dissolved oxygen was ≥20%, and the gas-liquid ratio was 1:1; When the dissolved oxygen dropped to 20% and the pH rose to 8.8, the fermentation was completed to obtain the bacterial agent c.

[0164] Example 7

[0165] This example is used to illustrate the bacterial agent and its preparation method of the present invention.

[0166] (1) The Stenotrophomonas sp. YB2 obtained in Example 1 was spread on an LB solid medium and placed in a constant temperature and humidity incubator. It was cultured at 27 °C for 24 h to activate the colonies. The activated colonies YB2 were inoculated into an LB liquid medium and cultured with shaking at 27 °C and 120 rpm for 24 h to obtain the primary seed liquid YB2;

[0167] (2) The primary seed liquid YB2 was inoculated into 1 L of LB liquid medium at an inoculation amount of 5%. It was cultured with shaking at 27 °C and 120 rpm and aerated. The aeration rate was 0.5 L / min. After culturing for 24 h, an enriched bacterial liquid of Stenotrophomonas sp. YB2 was obtained. Its concentration was adjusted to make the OD 600 value = 1 to obtain the secondary seed liquid;

[0168] (3) Fermentation medium was added to the fermenter. After sterilization, the secondary seed liquid was inoculated at a volume ratio of 5%. During the fermentation process, the temperature was controlled at 27 °C, the tank pressure was 0.05 MPa, the initial rotation speed was 200 rpm, the dissolved oxygen was ≥20%, and the gas-liquid ratio was 1:1; When the dissolved oxygen dropped to 20% and the pH rose to 8.8, the fermentation was completed to obtain the fermentation broth;

[0169] (4) Inoculate the above fermentation broth onto zeolite at an inoculation amount of 10%, and culture it on a shaker at 35 °C and 150 rpm. After culturing for 20 h, pour off the supernatant, then wash the precipitate with physiological saline, and repeat the washing 3 times to obtain bacterial agent d.

[0170] Test Example 1

[0171] Inoculate the Stenotrophomonas YB2 bacterial solution obtained in Example 3 and the bacterial agent a obtained in Example 4 into an inorganic salt medium with a benzene concentration of 200 mg / L at an inoculation amount of 5% by volume, and treat it at 27 °C and 120 rpm for 14 h. Set 3 replicates for each treatment group, and use an inorganic salt medium containing benzene at a concentration of 200 mg / L without adding any bacterial solution or bacterial agent as the control group. Sample and analyze every 2 h, use headspace gas chromatography to determine the benzene content in the sample, and calculate the degradation rate of benzene by the bacterial solution and the bacterial agent. The results are shown in Table 1.

[0172] Table 1 Benzene degradation rate at different treatment times

[0173]

[0174] As can be seen from Table 1, after 14 h of treatment, the degradation rate of benzene by Stenotrophomonas YB2 is 94%, and the degradation rate of benzene by bacterial agent a is 98%. This shows that both the Stenotrophomonas YB2 bacterial solution and bacterial agent a can efficiently degrade benzene. Among them, the degradation rate of benzene by bacterial agent a is higher. This may be because when free bacteria are used alone, they are easily affected by the toxicity of benzene in the environment and have poor survival rate, so the benzene degradation rate is relatively low. However, the bacterial agent a prepared by the immobilization technology has a high microbial density, fast reaction speed, and can reduce the toxic effect of benzene, so its degradation rate is higher.

[0175] Test Example 2

[0176] Inoculate the Stenotrophomonas YB2 bacterial solution obtained in Example 3, the bacterial agent a obtained in Example 4, and the bacterial agent b obtained in Example 5 into sludge with toluene concentrations of 150, 200, and 250 mg / L respectively at an inoculation amount of 5% by volume, and treat it at 27 °C and 120 rpm for 8 h. Set 3 replicates for each treatment group, and calculate the degradation rate of toluene by the bacterial solution and the bacterial agent. The results are shown in Table 2.

[0177] Table 2

[0178]

[0179] As can be seen from Table 2, the bacterial solution and bacterial agent containing Stenotrophomonas YB2 can efficiently degrade toluene, indicating that the Stenotrophomonas YB2 provided by the present invention can efficiently degrade toluene.

[0180] Meanwhile, for the same inoculation amount, after 8 hours of treatment, the immobilized bacterial agents a and b showed higher degradation rates than the Stenotrophomonas YB2 bacterial solution. This may be because the immobilized bacterial agents have a relatively large biomass loading, and the microorganisms have strong environmental tolerance, high activity, and good stability. Therefore, the bacterial agents prepared by the sodium alginate embedding method and the biochar adsorption method can both improve the degradation efficiency of free strains.

[0181] Test Example 3

[0182] The bacterial agent a obtained in Example 4 and the inoculation amount of 5% by volume were inoculated into 200 mg / L of benzene series compounds (a mixture of benzene, toluene, and xylene, where the weight ratio of benzene:toluene:xylene = 1:1:1), and treated at 27°C and 120 rpm for 16 hours. Three replicates were set for each treatment group, and the degradation rates of the bacterial agent a for benzene, toluene, and xylene were calculated. The results are as Figure 4 shown.

[0183] It can be Figure 4 seen that the bacterial agent a has high degradation efficiency for benzene, toluene, and xylene, and can be applied to the remediation of water environments or soils contaminated with benzene series compounds.

[0184] 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 solutions 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 Stenotrophomonas sp. YB2, and its preservation number is GDMCC No: 63283.

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 The benzene series substances are at least one of benzene, toluene and xylene.

4. The application according to claim 2 or 3, characterized in that, It is an application in sewage treatment containing benzene series substances or remediation of benzene series substance-polluted soil.

5. Application of the benzene series degrading bacterium according to claim 1 in preparing a product for degrading benzene series substances.

6. A bacterial agent, characterized in that, The bacterial agent contains the benzene series degrading bacterium according to claim 1.

7. The microbial agent according to claim 6, characterized in that, The bacterial agent also contains a carrier.

8. The microbial agent according to claim 7, characterized in that, The carrier is selected from one or more of sodium alginate, chitosan, polyacrylamide, polyvinyl alcohol, corn flour, starch, clay, talc, kaolin, zeolite and biochar.

9. A method for preparing a bacterial agent, characterized in that, The method includes: fermentatively culturing the benzene series degrading bacterium according to claim 1.

10. The method according to claim 9, characterized in that, The method also includes: mixing the fermented liquid obtained by fermentation with the carrier.

11. The method according to claim 9, wherein The conditions for the fermentation include: temperature 25 - 35 °C, rotation speed 100 - 300 rpm, gas-liquid ratio 1:(1 - 2), dissolved oxygen ≥ 20%.

12. The bacterial agent prepared by the method according to any one of claims 9 - 11.

13. Application of the bacterial agent according to any one of claims 6 - 8 and 12 in degrading benzene series substances.

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