Application of Oligotrophomonas microacidophilus in the degradation of ethylbenzene

Abstract

The invention discloses an application of Stenotrophomonas acididaminiphila in degrading ethylbenzene and relates to the field of microorganisms. An embodiment of the invention provides a strain of Stenotrophomonas acididaminiphila JPEB-1, which can degrade ethylbenzene under inorganic salt culture conditions and can achieve biodegradation of high-concentration ethylbenzene in combination with Micrococcus luteus JPEB-2.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to the application of oligotrophic microacidophilus in degrading ethylbenzene. Background Art

[0002] Ethylbenzene is a kind of alkylbenzene, which is widely used in industrial production as a solvent and the main precursor of styrene. It is also a component of naphtha and asphalt, and a by-product of the production of high-octane gasoline. Therefore, the spillage and leakage of petroleum and various chemical products often cause soil, groundwater and atmosphere to be polluted by organic substances such as ethylbenzene, which endangers environmental safety and threatens human life and health. Therefore, the treatment of organic waste gas containing ethylbenzene and groundwater and soil contaminated by ethylbenzene has become an urgent task.

[0003] Conventional physical or chemical methods for removing ethylbenzene will cause secondary pollution problems and are costly. Therefore, biological treatment can be used as another effective and environmentally friendly means of treating wastewater and contaminated soil containing organic compounds such as ethylbenzene. At present, many strains of ethylbenzene-degrading bacteria have been isolated. For example: CN100489087C discloses Staphylococcus xtlosus Y1, which has the ability to degrade ethylbenzene, and its application. The bacteria removes 91.28% of ethylbenzene in a packed tower under the operating conditions of 30°C, 100mg / L inlet ethylbenzene concentration, and 80L / h gas flow. CN113717878A discloses a strain of ethylbenzene-degrading bacteria, Achromobacter sp.ED 2, which can completely degrade 50mg / L ethylbenzene within 92h. When the ethylbenzene concentration is higher than 200mg / L, the growth of the bacteria is significantly inhibited, and the ethylbenzene degradation rate is less than 10%. CN101514329A discloses Bacillus amyloliquefaciens byf-5 capable of degrading benzene compounds and its application. The bacterium can degrade benzene, toluene, ethylbenzene and o-xylene at 30°C and pH 8.0. When the concentration of ethylbenzene is higher than 125 mg / L, the bacterium is inhibited and basically cannot degrade ethylbenzene. It can be seen that the degradation effect of existing degradation bacteria on specific substances such as ethylbenzene is not particularly ideal.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide the application of oligotrophic microacidophilus in degrading ethylbenzene.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides a microacidophilic Stenotrophomonas, whose name is Stenotrophomonas acidaminiphila JPEB-1 and whose deposit number is GDMCC No:62575.

[0008] In a second aspect, an embodiment of the present invention provides the use of ethylbenzene-degrading bacteria and / or its metabolites in the preparation of a product for degrading ethylbenzene, wherein the ethylbenzene-degrading bacteria include the microacidophilic oligotrophic monocytogenes described in the above embodiment.

[0009] In a third aspect, an embodiment of the present invention provides a product for degrading ethylbenzene, which includes the ethylbenzene-degrading bacteria and / or its metabolites described in the above embodiments.

[0010] In a fourth aspect, embodiments of the present invention provide use of ethylbenzene-degrading bacteria and / or its metabolites in degrading ethylbenzene, and the ethylbenzene-degrading bacteria and / or its metabolites are as described in the above embodiments.

[0011] The present invention has the following beneficial effects:

[0012] The embodiment of the present invention provides a strain of Stenotrophomonasacidaminiphila JPEB-1, which can degrade ethylbenzene under inorganic salt culture conditions, and can achieve biodegradation of high-concentration ethylbenzene in combination with Micrococcus luteus JPEB-2. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0014] Figure 1 This is the phylogenetic tree of Stenotrophomonas acidaminiphila JPEB-1;

[0015] Figure 2 This is the phylogenetic tree of Micrococcus luteus JPEB-2;

[0016] Figure 3 is a schematic diagram of a biodegradation device;

[0017] Figure 4 is the ethylbenzene degradation curve of S.acidaminiphila JPEB-1;

[0018] Figure 5 This is the ethylbenzene degradation curve of the combined bacterial solution of S.acidaminiphila JPEB-1 and M.luteus JPEB-2;

[0019] Figure 6 This is the ethylbenzene degradation effect of the combined bacterial solution. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0021] First, an embodiment of the present invention provides a microacidophilic oligotrophic mononas, which has a round shape in LB medium, neat edges, light yellow, translucent, and a raised and sticky center of the colony. Its name is Stenotrophomonasacidaminiphila JPEB-1, and JPEB-1 has a 100% similarity with microacidophilic oligotrophic mononas Stenotrophomonasacidaminiphila JCM 13310 after sequencing. The taxonomic name is Stenotrophomonasacidaminiphila, which is preserved in the Guangdong Provincial Microbial Culture Collection Center, and the preservation address is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou; the preservation date is June 30, 2022, and the preservation number is GDMCC No: 62575.

[0022] The 16S rDNA sequence of S. acidaminiphila JPEB-1 is shown in SEQ ID No. 1, and the phylogenetic tree is shown in Figure 1 .

[0023]

[0024] On the other hand, an embodiment of the present invention further provides the use of ethylbenzene-degrading bacteria and / or its metabolites in the preparation of products for degrading ethylbenzene, wherein the ethylbenzene-degrading bacteria include the microacidophilic oligotrophic monocytogenes described in the above embodiment.

[0025] In some embodiments, the ethylbenzene-degrading bacteria further include the Micrococcus luteus described in the preceding embodiments.

[0026] In some embodiments, the Micrococcus luteus is named Micrococcus luteus JPEB-2. After sequencing, JPEB-2 has a similarity of 99.3% with Micrococcus luteus NCTC 2665, and is named Micrococcus luteus JPEB-2.

[0027] The 16S rDNA sequence of M.luteus JPEB-2 is shown in SEQ ID No. 2, and the phylogenetic tree is shown in Figure 2 .

[0028]

[0029] In other embodiments, the Micrococcus luteus can also be selected from the currently commercially available or preserved Micrococcus luteus, such as the Micrococcus luteus GDMCC 1.59 preserved in the Guangdong Provincial Microbiological Culture Collection Center.

[0030] Preferably, the degrading ethylbenzene includes degrading ethylbenzene with a concentration of 100 to 650 mg / L, specifically any one of 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, 300 mg / L, 350 mg / L, 400 mg / L, 450 mg / L, 500 mg / L, 550 mg / L, 600 mg / L and 650 mg / L.

[0031] On the other hand, an embodiment of the present invention further provides a product for degrading ethylbenzene, which includes the ethylbenzene-degrading bacteria and / or its metabolites as described in any of the aforementioned embodiments.

[0032] In some embodiments, the product may further include: a culture medium for culturing the ethylbenzene-degrading bacteria.

[0033] In some embodiments, the culture medium includes at least one of an inorganic salt culture medium and LB medium.

[0034] The main functions of the inorganic salt culture medium are as follows: when degrading ethylbenzene, the inorganic salt culture medium is used to resuspend the bacteria so as to contact with the sample containing ethylbenzene, so that the bacteria can use ethylbenzene as the only carbon source and avoid the growth of miscellaneous bacteria.

[0035] LB culture medium can be used to prepare a fermentation broth of bacteria, and the fermentation broth preparation method can include the following steps: using an inoculation loop to pick a ring of S. acidaminiphila JPEB-1 or M. luteus JPEB-2 from the slant culture medium and inoculate it into 100 ml of sterilized LB culture medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, 121°C, 15 min high pressure sterilization) for activation, the culture temperature is 35°C, the shaking speed is 150 r / min, and the culture time is 24 hours.

[0036] In some embodiments, the inorganic salt culture solution includes the following components and their final concentrations, measured by the effective concentrations of the components: NaNO3 45-55 g / L, K2HPO4 35-45 g / L, MgSO4 30-40 g / L, CaCl2 23-32 g / L, EDTANa2 0.1-3 g / L, Na2CO3 15-25 g / L, H3BO3 1-5 g / L, MnCl2·4H2O 0.1-3 g / L, ZnSO4 0.01-0.5 g / L, Na2MoO4·2H2O 0.1-2 g / L, CuSO4·5H2O 0.001-0.1 g / L and CoCl2·6H2O 0.001-0.1 g / L.

[0037] On the other hand, an embodiment of the present invention further provides the use of ethylbenzene-degrading bacteria and / or its metabolites in degrading ethylbenzene, wherein the ethylbenzene-degrading bacteria and / or its metabolites are as described in any of the above embodiments.

[0038] In some embodiments, degrading ethylbenzene comprises: contacting a sample containing ethylbenzene with ethylbenzene-degrading bacteria and / or its metabolites.

[0039] In some embodiments, when the ethylbenzene-degrading bacteria include Stenotrophomonas microacidophilus and Micrococcus luteus, the step of degrading ethylbenzene includes: contacting the ethylbenzene-containing sample with a mixed bacterial solution of a bacterial solution of Stenotrophomonas microacidophilus and a bacterial solution of Micrococcus luteus.

[0040] In some embodiments, the mixed bacterial solution is obtained by mixing the bacterial solutions of Microacidophilus Oligotrophomonas and Micrococcus luteus at the same bacterial solution concentration in a volume ratio of (5-9): (1-3). At this volume ratio, it has a good ethylbenzene degradation efficiency. The volume ratio can be any one of 5:1, 5:2, 5:3, 6:1, 6:2, 6:3, 7:1, 7:2, 7:3, 8:1, 8:2, 8:3, 9:1 and 9:2 or a range between any two of them. Preferably, the volume ratio is (7-9): (1-2), at which the ethylbenzene degradation efficiency can be maximized.

[0041] In some embodiments, the same bacterial concentration refers to OD 600 The bacterial solution is 0.1 to 0.5, which can be specifically OD 600 It is any one of 0.1, 0.2, 0.3, 0.4 and 0.5 or a range between any two of them, and is preferably 0.2.

[0042] In some embodiments, the bacterial liquid of Oligotrophomonas microacidophilus and the bacterial liquid of Micrococcus luteus are obtained by resuspending the bacterial cells in an inorganic salt culture medium.

[0043] In some embodiments, the inorganic salt culture solution is as described in any of the above embodiments. In some embodiments, the sample is selected from at least one of waste gas, soil and sewage.

[0044] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0045] Example 1

[0046] Isolation and cultivation of S.acidaminiphila JPEB-1 and Micrococcus luteus JPEB-2.

[0047] 1. Strain Isolation

[0048] Use a 500mL conical flask to prepare 90mL of screening culture medium, steam sterilize at 121℃ for 15min, cool to room temperature, add 0.2mL of ethylbenzene filtered through a 0.22μm filter membrane, and then add 10mL of aerobic activated sludge from petrochemical wastewater treatment. After sealing, culture in a shaking incubator at 35℃ and 150r / min for 48h. Transfer the cultured screening bacterial solution to fresh culture medium at a transfer volume of 10% each time, and increase the amount of ethylbenzene by 0.2mL each time. Transfer 5 times.

[0049] Wherein, the components of the screening culture medium (inorganic salt culture medium) are shown in Table 1.

[0050] Table 1 Inorganic salt culture medium composition

[0051]

[0052]

[0053] According to the preparation method of the screening medium, 1.8% agar was added to make a solid separation medium. 1 mL of the fifth transfer solution was added to 9 mL of sterile liquid inorganic salt medium and mixed. -1 , and then diluted step by step to 10 -2 , 10 -3 , 10 -4 , 10 -5 , and spread the dilution on the LB medium plate, with 3 parallels for each gradient. Then use an inoculation loop to pick up a single colony and separate it on the LB medium plate. Repeat the streak separation 2 to 3 times and inoculate the obtained single bacteria into the biodegradation device to verify the degradation ability. If it is observed that the bacteria can grow using ethylbenzene as the only carbon source and the ethylbenzene concentration is reduced, it is an ethylbenzene-degrading bacteria.

[0054] In this example, S. acidaminiphila JPEB-1 and Micrococcus luteus JPEB-2 were screened and obtained.

[0055] 2. Strain culture

[0056] Use an inoculation loop to pick up the colony and inoculate it into a conical flask for enrichment culture, and then culture it in a constant temperature shaker. The culture conditions are: temperature 35℃; rotation speed 150rpm, and culture time 24h. After centrifugation or filtration of the bacterial liquid, the bacterial body is obtained, which can be directly used for ethylbenzene degradation.

[0057] Example 2

[0058] S.acidaminiphila JPEB-1 degrades ethylbenzene at different concentrations:

[0059] ① Centrifuge the bacterial solution obtained by fermentation of S.acidaminiphila JPEB-1 at 7000r / min and 4℃, discard the supernatant, and wash it repeatedly with phosphate buffer for 3 times. Resuspend the washed bacteria with inorganic salt culture medium and adjust its OD 600 to 0.2.

[0060] ② Take two 500mL blue-cap bottles, add 200mL of the resuspended bacterial solution in step ① to one, and add 200mL of the sterilized screening culture medium to the other as a control.

[0061] ③ Connect the 500mL blue-cap bottle in step ② with the 1L blue-cap bottle, as shown in Figure 3 Ethylbenzene is added from the detection port, evaporates in the bottle and is pumped into the bacterial solution through an air pump.

[0062] ④ Add 1mL, 0.8mL, 0.6mL, 0.4mL and 0.2mL of ethylbenzene into the device through the detection port respectively, and exchange gas between the two blue-capped bottles through an air pump.

[0063] The whole set of equipment was placed in a constant temperature environment of 30℃, and the operating parameters of the device were: air pump flow rate 3L / h, gas-liquid ratio 7, and the maximum concentration of ethylbenzene was 612.5mg / L. Sampling analysis was performed after 48h, and the degradation effect of ethylbenzene is shown in Table 2.

[0064] Table 2. Degradation effect of S.acidaminiphila JPEB-1 on different concentrations of ethylbenzene

[0065]

[0066] Degradation of high concentration ethylbenzene:

[0067] Referring to steps ①, ② and ③ of Example 2, after the device was connected, 1 mL of ethylbenzene was added to the device through the detection port. The initial ethylbenzene concentration was 612.5 mg / L. After sealing, the device was placed in a constant temperature environment at 30°C. Samples were taken and analyzed at 12, 24, 36, 48, 60 and 72 hours. The degradation effect of ethylbenzene was as follows: Figure 4 shown.

[0068] Example 3

[0069] JPEB-1 and JPEB-2 jointly degraded ethylbenzene.

[0070] (a) A batch of S.acidaminiphila JPEB-1 and M.luteus JPEB-2 bacterial cultures were enriched using the method described in Example 1, and the cultures were repeatedly washed three times with phosphate buffer, and the washed cultures were resuspended with an inorganic salt culture medium, and the OD 600 to 0.2.

[0071] (b) Take two 500 mL blue-capped bottles, add 200 mL of the resuspended bacterial solution in step (a) to one of them, wherein the volume ratios of the S.acidaminiphila JPEB-1 and M.luteus JPEB-2 bacterial resuspensions are adjusted to 100:0, 95:5, 90:10, and 80:20, respectively, and add 200 mL of the sterilized screening culture medium to the other one as a control.

[0072] (c) Connect the 500 mL blue-cap bottle in step (b) to the 1 L blue-cap bottle, and the connection method is as described in Example 1.

[0073] (d) Add 1 mL of ethylbenzene dropwise into the device through the detection port.

[0074] (e) Take samples every 6 hours to analyze the ethylbenzene concentration.

[0075] Ethylbenzene degradation effect of combined bacterial solution Figure 5 In order to better illustrate the advantages of the combination of S.acidaminiphila JPEB-1 and M.luteus JPEB-2 in degrading ethylbenzene, it was compared with several ethylbenzene-degrading bacteria. As shown in Table 3, this combination can degrade ethylbenzene with a higher load and a faster degradation rate, which has certain advantages.

[0076] Table 3 Comparison of degradation characteristics of several ethylbenzene-degrading bacteria

[0077]

[0078] Note: Micromonospora aurantiaca ZHY1-5 is derived from the Chinese patent with application number "CN201410261781.X" and the invention name is a micromonospora strain and its application; Pseudomonasviridilivida FB1 is derived from the Chinese patent with application number " CN201811060093.1 ", a Chinese patent with the invention name of "Benzene-degrading bacteria FB1 and its screening method and application in degrading benzene-degrading substances".

[0079] Example 4

[0080] S.acidaminiphila JPEB-1 and M.luteus GDMCC 1.59 jointly degrade ethylbenzene.

[0081] (a) A batch of S.acidaminiphila JPEB-1 and M.luteus GDMCC1.59 bacterial cultures were enriched using the method in Example 1, and washed three times with phosphate buffer, and the washed bacterial cultures were resuspended with an inorganic salt culture medium to adjust the OD 600 to 0.2.

[0082] (b) Take two 500 mL blue-cap bottles, add 200 mL of the resuspended bacterial solution in step (a) to one of them, wherein the volume ratio of the S.acidaminiphila JPEB-1 and M.luteus GDMCC 1.59 bacterial resuspensions is adjusted to 90:10, respectively, and add 200 mL of the sterilized screening culture medium to the other one as a control.

[0083] (c) Connect the 500 mL blue-cap bottle in step (b) to the 1 L blue-cap bottle, and the connection method is as described in Example 1.

[0084] (d) Add 1 mL of ethylbenzene dropwise into the device through the detection port.

[0085] (e) Take samples every 6 hours to analyze the ethylbenzene concentration.

[0086] Ethylbenzene degradation effect of combined bacterial solution Figure 6 shown.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A microacidophilic oligotrophic monocytogenes, characterized in that Its name is Stenotrophomonas acidaminiphila JPEB-1, the deposit number is GDMCC No:62575.

2. Use of ethylbenzene-degrading bacteria in the preparation of products for degrading ethylbenzene, characterized in that: The ethylbenzene-degrading bacteria include the microacidophilic oligotrophic monocytogenes described in claim 1.

3. The use according to claim 2, characterized in that: The ethylbenzene degrading bacteria also include Micrococcus luteus.

4. The use according to claim 2, characterized in that: The degrading ethylbenzene includes degrading ethylbenzene with a concentration of 100-650 mg / L.

5. A product for degrading ethylbenzene, characterized in that: It comprises the ethylbenzene-degrading bacteria as described in claim 2 or 3.

6. The product for degrading ethylbenzene according to claim 5, characterized in that: The product also includes a culture medium for culturing the ethylbenzene-degrading bacteria.

7. The use of ethylbenzene-degrading bacteria in degrading ethylbenzene, characterized in that: The ethylbenzene-degrading bacteria is as described in claim 2 or 3.

8. The use according to claim 7, characterized in that: When the ethylbenzene-degrading bacteria include oligotrophic mononas and Micrococcus luteus, the ethylbenzene-degrading method includes: contacting the ethylbenzene-containing sample with a mixed bacterial solution prepared by mixing a bacterial solution of oligotrophic mononas and a bacterial solution of Micrococcus luteus with the same bacterial solution concentration in a volume ratio of (5-9):(1-3).

9. The use according to claim 8, characterized in that: The same bacterial solution concentration refers to OD 600 The concentration of the bacterial solution is 0.1~0.

5.

10. The use according to claim 8, characterized in that: The sample is selected from at least one of exhaust gas, soil and sewage.

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