Bacillus for degrading 1, 2-bis (2, 4, 6-tribromophenoxy) ethane and screening and application thereof
By screening and applying the Bacillus sp. FX-1 degradation system, the problem of difficult degradation of the new bromine flame retardant 1,2-bis(2,4,6-tribromophenoxy)ethane was solved, and efficient, low-cost and environmentally friendly pollution control effect was achieved.
Patent Information
- Application Number
- CN202411873597.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-04-15
AI Technical Summary
The prior art is difficult to effectively degrade the new bromine flame retardant 1,2-bis(2,4,6-tribromophenoxy)ethane, and the traditional degradation methods have high costs and secondary pollution risks.
By screening and identifying Bacillus sp. FX-1, a degraded strain, from the soil and applying it to the degradation system, efficient degradation of 1,2-bis(2,4,6-tribromophenoxy)ethane is achieved.
This method can achieve a 73.2% degradation rate of 1,2-bis(2,4,6-tribromophenoxy)ethane at a lower cost, avoid secondary pollution, and provide an environmentally friendly pollution control solution.
Smart Images
Figure CN119818895A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological treatment of environmental organic pollutants, and particularly relates to a method for degrading 1,2-bis(2,4,6-tribromophenoxy)ethane by using Bacillus sp. FX-1. Background Art
[0002] Brominated flame retardants are the most widely used flame retardants in the world and are added to products such as electronic and electrical equipment, building materials and plastic products. Traditional brominated flame retardants represented by polybrominated diphenyl ethers have been gradually eliminated by the United States, the European Union, China and other countries due to their difficulty in degradation, biotoxicity and bioaccumulation, and new brominated flame retardants that replace traditional brominated flame retardants are being used in large quantities. At present, new brominated flame retardants have been detected in the atmosphere, water bodies, sediments, soil and organisms. As a new type of organic pollutant, the ecological risks and health and safety effects of new brominated flame retardants in the environment have attracted widespread attention from environmental researchers. Among them, 1,2-bis(2,4,6-tribromophenoxy)ethane is a new type of brominated flame retardant with a high detection rate and is relatively common.
[0003] At present, the degradation methods of new brominated flame retardants mainly include photodegradation and chemical degradation. These degradation methods cannot be the best choice for pollution control of new brominated flame retardants due to their high operating costs and the possibility of causing secondary pollution. Microbial degradation has the advantages of being environmentally friendly and low-cost, making it one of the safest, most economical and effective methods for remediating organic pollutants. However, the microbial degradation of 1,2-bis(2,4,6-tribromophenoxy)ethane is limited due to the lack of effective degradation strains. Therefore, the screening and application of 1,2-bis(2,4,6-tribromophenoxy)ethane degradation bacteria has certain research significance. From the current patent application situation, there are no related patents for 1,2-bis(2,4,6-tribromophenoxy)ethane degradation strains. Summary of the invention
[0004] The primary purpose of the present invention is to provide a Bacillus sp. FX-1 capable of degrading 1,2-bis(2,4,6-tribromophenoxy)ethane and a screening method and application thereof. The method is to screen 1,2-bis(2,4,6-tribromophenoxy)ethane degrading bacteria from soil and add them to a degradation system, thereby providing an effective technical method for 1,2-bis(2,4,6-tribromophenoxy)ethane pollution control.
[0005] The purpose of the present invention is achieved through the following technical solutions:
[0006] The method for degrading 1,2-bis(2,4,6-tribromophenoxy)ethane by Bacillus sp. FX-1 comprises the following steps:
[0007] (1) Inoculate Bacillus sp. FX-1 into a sterilized enrichment medium and culture under constant temperature shaking;
[0008] (2) Collecting the bacterial strains, adding them to the sterilized 1,2-bis(2,4,6-tribromophenoxy)ethane degradation medium, and culturing them under constant temperature shaking;
[0009] (3) The residual concentration of 1,2-bis(2,4,6-tribromophenoxy)ethane in the degradation medium was determined using gas chromatography-tandem mass spectrometry, and the degradation effect of Bacillus sp. FX-1 on 1,2-bis(2,4,6-tribromophenoxy)ethane was analyzed.
[0010] Preferably, the enrichment medium in step (1) is composed of: 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast powder, pH 7.0.
[0011] The culture conditions in step (1) are: temperature of 20-40°C, constant temperature shaking culture time of 20-30 h; the preferred culture conditions are: temperature of 30°C, culture time of 24 h.
[0012] The composition of the 1,2-bis(2,4,6-tribromophenoxy)ethane degradation culture medium in step (2) is: 2 g / L (NH4)2SO4, 1.5 g / L Na2HPO4·12H2O, 1.5 g / L K2HPO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2, 0.005 g / L FeSO4·7H2O, 1 mg / L 1,2-bis(2,4,6-tribromophenoxy)ethane, 0.1 mL / L trace element solution, 1000 mL ultrapure water, pH 7.0; wherein the trace element solution is composed of: 2.2 g / L ZnSO4·7H2O, 0.3 g / LCuSO4·5H2O, 14.3 g / L MnSO4·H2O, 1000 mL ultrapure water. The culture conditions in step (2) are: the dosage OD 600 =0.85-3.0, temperature is 20-40°C, reaction time is 1-5 days. The preferred degradation conditions are: dosage is OD 600 =2.5, temperature is 30°C, and reaction time is 5 days.
[0013] The Bacillus sp. FX-1 was screened from mixed soil in a contaminated area near a chemical plant in Foshan. 1,2-bis(2,4,6-tribromophenoxy)ethane was added to the inorganic salt medium, and the acclimation medium with a concentration gradient of 1,2-bis(2,4,6-tribromophenoxy)ethane was prepared at 5, 10, 20, 30, 50, 70, 100, 150 and 200 mg / L, and the acclimation was carried out by increasing the concentration gradient of the pollutant. The external morphology of the bacterium is white, the surface and edges are smooth, and the viscous state is preserved in the Guangdong Provincial Microbiological Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, preservation time: June 2024, and the preservation number is GDMCC No.65585.
[0014] Under this condition, the degradation rate of 1,2-bis(2,4,6-tribromophenoxy)ethane with an initial concentration of 1 mg / L reached 73.2% after 5 days of degradation.
[0015] Compared with the prior art, the present invention has the following advantages and effects:
[0016] 1. The present invention provides a method for degrading 1,2-bis(2,4,6-tribromophenoxy)ethane by using Bacillus sp. FX-1. The rapid and efficient degradation of 1,2-bis(2,4,6-tribromophenoxy)ethane by Bacillus sp. FX-1 can provide a technical method for the pollution control and bioremediation of 1,2-bis(2,4,6-tribromophenoxy)ethane.
[0017] 2. The Bacillus sp. FX-1 in the present invention has strong adaptability to the environment and has a good degradation effect on 1,2-bis(2,4,6-tribromophenoxy)ethane. The cost of using this Bacillus sp. FX-1 to degrade 1,2-bis(2,4,6-tribromophenoxy)ethane is low. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 It is a standard curve of 1,2-bis(2,4,6-tribromophenoxy)ethane concentration-peak area;
[0019] Figure 2 The degradation of 1,2-bis(2,4,6-tribromophenoxy)ethane by Bacillus sp. FX-1 at different time points. DETAILED DESCRIPTION
[0020] The present invention is further described in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto.
[0021] Example 1
[0022] 1. Screening of degradation strains
[0023] (1) Soil was collected from a contaminated area near a chemical plant in Foshan City. After mixing evenly, 5 g of soil was added to 100 mL of an inorganic salt culture medium containing 5 mg / L 1,2-bis(2,4,6-tribromophenoxy)ethane and cultured in a constant temperature shaker at 30°C and 160 rpm. The main components of the inorganic salt culture medium are: 2 g / L (NH4)2SO4, 1.5 g / L Na2HPO4·12H2O, 1.5g / L K2HPO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2, 0.005 g / L FeSO4·7H2O, 0.1 mL / L trace element solution, 1000 mL ultrapure water, pH 7.0; the trace element solution consists of: 2.2 g / L ZnSO4·7H2O, 0.3g / L CuSO4·5H2O, 14.3 g / L MnSO4·H2O, 1000 mL ultrapure water.
[0024] (2) The culture medium was transferred once every 7 days. Each time the concentration of 1,2-bis(2,4,6-tribromophenoxy)ethane was increased (according to the concentration gradient of 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 and 200 mg / L) until the concentration of 1,2-bis(2,4,6-tribromophenoxy)ethane reached 200 mg / L. The strains in the acclimation solution were then separated and purified by the dilution gradient method and the plate coating method. After ensuring that it was a pure single strain, it was inoculated onto an inorganic salt solid culture medium containing 0.5 mg 1,2-bis(2,4,6-tribromophenoxy)ethane on the surface and cultured in a constant temperature incubator at 30°C for 5 days. After the colonies were grown, the degradation ability of each single strain on 1,2-bis(2,4,6-tribromophenoxy)ethane was tested. The strain numbered FX-1 was tested to have a better degradation effect on 1,2-bis(2,4,6-tribromophenoxy)ethane. The components of the inorganic salt solid culture medium were: 2 g / L (NH4)2SO4, 1.5 g / L Na2HPO4·12H2O, 1.5 g / L K2HPO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2, 0.005 g / L FeSO4·7H2O, 0.1 mL / L trace element solution, 1000 mL ultrapure water, pH 7.0; the trace element solution was composed of: 2.2 g / L ZnSO4·7H2O, 0.3 g / L CuSO4·5H2O, 14.3 g / LMnSO4·H2O, 16 g / L agar, 1000 mL ultrapure water.
[0025] 2. Identification of degradation strains
[0026] (1) Morphological characteristics
[0027] The fungus is white in appearance, with a smooth and sticky surface and edges.
[0028] (2) 16S rRNA sequence
[0029] The bacteria were sent to Guangdong Microbiological Culture Collection Center for 16S rRNA sequencing to identify the strain species. The strain was identified as Bacillus sp., and the 16S rRNA sequence is shown in Sequence Table 1. The strain was named Bacillus sp. FX-1, of which the 16S rRNA sequence (1393 bp in total) has been submitted to Gene Bank with the accession number PQ764545. The bacteria can be obtained from Guangdong Microbiological Culture Collection Center with the accession number GDMCC No.65585.
[0030] Embodiment 2:
[0031] Degradation Analysis of 1,2-Bis(2,4,6-tribromophenoxy)ethane by Bacillus sp. FX-1
[0032] Bacillus sp. FX-1 was inoculated into the sterilized enrichment medium and cultured at 30°C, 160 rpm, with constant temperature shaking for 24 h. The main components of the enrichment medium were: 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast powder, and pH 7.0.
[0033] The enriched culture solution was centrifuged at 4000 rpm for 1 min, the bacteria were collected and washed three times with sterile saline, and then sterilized degradation medium was added for degradation experiment. The dosage was OD 600=2.5, the culture conditions were 30°C, 160 rpm, and after constant temperature oscillation reaction for 5 days, the bacteria were crushed and extracted. The obtained samples were measured by gas chromatography-tandem mass spectrometry to analyze the degradation effect of the bacteria on 1,2-bis(2,4,6-tribromophenoxy)ethane. The composition of the degradation medium was: 2 g / L (NH4)2SO4, 1.5 g / L Na2HPO4·12H2O, 1.5 g / L K2HPO4, 0.2 g / L MgSO4·7H2O, 0.01 g / LCaCl2, 0.005 g / L FeSO4·7H2O, 1 mg / L 1,2-bis(2,4,6-tribromophenoxy)ethane, 0.1 mL / L trace element solution, 1000 mL ultrapure water, pH 7.0; the trace element solution consisted of: 2.2 g / L ZnSO4·7H2O, 0.3 g / LCuSO4·5H2O, 14.3 g / L MnSO4·H2O, 1000 mL ultrapure water.
[0034] The concentration of 1,2-bis(2,4,6-tribromophenoxy)ethane was determined by GC-MS / MS, and the analysis conditions were as follows:
[0035] (1) Gas chromatography conditions:
[0036] Chromatographic column: HP-5MS (15m × 250 μm × 0.25 μm);
[0037] Inlet temperature: 280°C;
[0038] Heating program: initial temperature 80 °C, hold for 1 min, increase to 300 °C at a rate of 22 °C / min, hold at 300 °C for 24 min;
[0039] Carrier gas: high-purity helium, purity greater than 99.999%;
[0040] Constant flow control: 1 mL / min;
[0041] Mode: splitless injection;
[0042] Injection volume: 2 μL;
[0043] (2) Mass spectrometry conditions:
[0044] Ion source: EI source;
[0045] The transmission line temperature is 280°C;
[0046] Ion source temperature 230°C;
[0047] Solvent delay time: 4 min;
[0048] Scan mass range: 50-600 m / z;
[0049] Characteristic ion: 358.7 m / z.
[0050] Prepare standard solutions of 1,2-bis(2,4,6-tribromophenoxy)ethane with concentrations of 0.1, 0.3, 0.5, 0.7, and 1.0 mg / L, and draw a standard curve of 1,2-bis(2,4,6-tribromophenoxy)ethane-peak area. The standard curve is as follows: Figure 1 As shown. The sample without Bacillus sp. FX-1 was used as the control group (except for the addition of bacteria, the other operations and parameters of the control group were the same as those of the experimental group). After 5 days of degradation, the final residual concentration of 1,2-bis(2,4,6-tribromophenoxy)ethane in the degradation medium of the control group was measured to be 0.97 mg / L, while the final residual concentration of 1,2-bis(2,4,6-tribromophenoxy)ethane in the degradation medium of the experimental group was 0.26 mg / L. By comparison and calculation, it can be obtained that the degradation efficiency of the experimental group is 73.2%. The results are shown in Figure 2 shown.
[0051] Example 3
[0052] Example 3 is the same as Example 2, except that the concentration of 1,2-bis(2,4,6-tribromophenoxy)ethane in the degradation medium is 1.5 mg / L, and the degradation rate of 1,2-bis(2,4,6-tribromophenoxy)ethane by Bacillus sp. FX-1 is 62.1%.
[0053] Example 4
[0054] Example 4 is the same as Example 2, except that the degradation reaction time is 3 days. Finally, the degradation rate of 1,2-bis(2,4,6-tribromophenoxy)ethane by Bacillus sp. FX-1 is 43.7%.
[0055] Example 5
[0056] Example 5 is the same as Example 2, except that the pH of the degradation medium is 4.0, 6.0 and 9.0, respectively. Finally, the degradation rates of 1,2-bis(2,4,6-tribromophenoxy)ethane by Bacillus sp. FX-1 are 45.2%, 60.8% and 54.5%, respectively.
[0057] Example 6
[0058] Example 6 is the same as Example 2, except that the dosage during degradation is OD 600= 0.85, 1.5 and 3.0, and finally the degradation rates of 1,2-bis(2,4,6-tribromophenoxy)ethane by Bacillus sp. FX-1 were 20.3%, 45.8% and 72.6%, respectively.
[0059] Example 7
[0060] Example 7 is the same as Example 2, except that the degradation temperatures are 20°C and 40°C, respectively. Finally, the degradation rates of 1,2-bis(2,4,6-tribromophenoxy)ethane by Bacillus sp. FX-1 are 40.6% and 62.5%, respectively.
[0061] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A method for degrading 1,2-bis(2,4,6-tribromophenoxy)ethane using Bacillus sp. FX-1, characterized in that: The following steps are involved: (1) Inoculate Bacillus sp. FX-1 into a sterilized enrichment medium and culture under constant temperature shaking; (2) Collecting the bacterial strains, adding them to the sterilized 1,2-bis(2,4,6-tribromophenoxy)ethane degradation medium, and culturing them under constant temperature shaking; (3) The residual concentration of 1,2-bis(2,4,6-tribromophenoxy)ethane in the degradation medium was determined using a gas chromatography-tandem mass spectrometer, and the degradation effect of Bacillus sp. FX-1 on 1,2-bis(2,4,6-tribromophenoxy)ethane was analyzed; The Bacillus sp. FX-1 is deposited in Guangdong Microbiological Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, preservation time: December 2024, preservation number is GDMCC No. 65585.
2. The method for degrading 1,2-bis(2,4,6-tribromophenoxy)ethane using Bacillus sp. FX-1 according to claim 1, characterized in that: The enrichment medium described in step (1) is composed of: 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast powder, and a pH of 7.
0.
3. The method for degrading 1,2-bis(2,4,6-tribromophenoxy)ethane using Bacillus sp. FX-1 according to claim 1, characterized in that: The culture conditions in step (1) are: temperature of 20-40°C and culture time of 20-30 h.
4. The method for degrading 1,2-bis(2,4,6-tribromophenoxy)ethane using Bacillus sp. FX-1 according to claim 1, characterized in that: The composition of the 1,2-bis(2,4,6-tribromophenoxy)ethane degradation culture medium in step (2) is: 2 g / L (NH4)2SO4, 1.5 g / L Na2HPO4·12H2O, 1.5 g / L K2HPO4, 0.2 g / LMgSO4·7H2O, 0.01 g / L CaCl2, 0.005 g / L FeSO4·7H2O, 1 mg / L 1,2-bis(2,4,6-tribromophenoxy)ethane, 0.1 mL / L trace element solution, 1000 mL ultrapure water, pH 4.0-9.0; wherein the trace element solution is composed of: 2.2 g / L ZnSO4·7H2O, 0.3 g / L CuSO4·5H2O, 14.3 g / L MnSO4·H2O, 1000 mL ultrapure water.
5. The method for degrading 1,2-bis(2,4,6-tribromophenoxy)ethane using Bacillus sp. FX-1 according to claim 1, characterized in that: The degradation reaction conditions in step (2) are: dosage OD 600 =0.85-3.0, temperature is 20-40℃, reaction time is 1-5 days.
6. Application of the method for degrading 1,2-bis(2,4,6-tribromophenoxy)ethane by using Bacillus sp. FX-1 as claimed in any one of claims 1 to 5 in the bioremediation of water bodies polluted by new brominated flame retardants; The novel brominated flame retardant is 1,2-bis(2,4,6-tribromophenoxy)ethane.