CN113897314A - Method for degrading tris (2-chloropropyl) phosphate by using amycolatopsis and application thereof - Google Patents
Method for degrading tris (2-chloropropyl) phosphate by using amycolatopsis and application thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
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- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/22—Organic substances containing halogen
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen
Abstract
The invention discloses a method for degrading tris (2-chloropropyl) phosphate by adopting Amycolatopsis sp.FT-1 and application thereof, belonging to the technical field of biological treatment of environmental organic pollutants. The method comprises the following specific steps: the degradation effect of Amycolatopsis (Amycolatopsis sp.FT-1) on tris (2-chloropropyl) phosphate was analyzed by inoculating Amycolatopsis sp.FT-1) into a degradation medium containing tris (2-chloropropyl) phosphate, performing shake degradation at a constant temperature for 5 days, and then measuring the residual concentration of tris (2-chloropropyl) phosphate in the medium by gas chromatography-mass spectrometry (GC-MS). The method has strong environmental adaptability and low cost, the degradation rate of the tris (2-chloropropyl) phosphate with the initial concentration of 5mg/L can reach 85.84 percent after the tris (2-chloropropyl) phosphate is degraded for 5 days, the actual application value is high, and a reference is provided for solving the problem of controlling the pollution of the tris (2-chloropropyl) phosphate in the environment.
Description
Technical Field
The invention belongs to the technical field of biological treatment of environmental organic pollutants, and particularly relates to a method for degrading tris (2-chloropropyl) phosphate by using Amycolatopsis sp.FT-1 and application thereof.
Background
In recent years, with the gradual exit from the flame retardant market due to the defects of large smoke generation amount, strong corrosivity, easy release of toxic gas and the like of the traditional brominated flame retardant, the novel organic phosphorus flame retardant is widely applied to the production of various commercial products such as textiles, electronic products, plastics, furniture, coatings and the like as an ideal substitute of the brominated flame retardant. The phosphoric acid tri (2-chloropropyl) ester is the most common organic phosphorus flame retardant at present, is an additive chemical auxiliary agent, is usually added into various products in physical mixing modes such as doping, adsorption and the like, and is not organically combined with the products in a chemical bonding mode, so that the phosphoric acid tri (2-chloropropyl) ester is easily separated from the products through ways such as volatilization, abrasion, corrosion, leakage and the like, is released into the natural environment, and causes environmental pollution along with the migration of the atmosphere and water. At present, the existence of the tris (2-chloropropyl) phosphate can be detected in various environmental media and organisms such as air dust, water, soil, river sediment, sediment and the like. The residual phosphoric acid tri (2-chloropropyl) ester in the organism can generate toxic effects on multiple aspects of endocrine interference, reproductive toxicity, neurotoxicity, immunotoxicity and the like on the organism. As a new class of organic pollutants, the ecological risks and health safety effects of such organic phosphorus flame retardants in the environment have attracted extensive attention of environmental researchers.
At present, the degradation and removal of the organophosphorus flame retardant are mainly carried out by a physical-chemical method and a microbial degradation method. The physical chemical method cannot be the best choice for pollution control because of the problems of high operation cost, easy secondary pollution and the like. The microbial degradation method has the advantages of environmental friendliness and low cost, so that the method becomes one of the safest, economic and effective organic pollution remediation methods, but the microbial degradation of the tris (2-chloropropyl) phosphate is limited due to the lack of effective degradation strains, so that the screening and application of the tris (2-chloropropyl) phosphate high-efficiency degradation bacteria have certain research significance. From the current patent application situation, no related patent of tris (2-chloropropyl) phosphate degrading bacteria exists.
Disclosure of Invention
The invention aims to provide a method for degrading tris (2-chloropropyl) phosphate by using Amycolatopsis sp.FT-1 and application thereof, and provides a technical method for pollution treatment and bioremediation of tris (2-chloropropyl) phosphate by rapidly and efficiently degrading the tris (2-chloropropyl) phosphate by using Amycolatopsis sp.FT-1.
The purpose of the invention is realized by the following technical scheme.
A method for degrading tris (2-chloropropyl) phosphate by using Amycolatopsis sp.FT-1 and application thereof comprise the following steps:
(1) amycolatopsis sp.FT-1 was inoculated into sterilized enrichment medium and cultured with shaking on a shaker.
(2) Collecting strains, and inoculating the strains in a sterilized tris (2-chloropropyl) phosphate degradation culture medium for shake culture.
(3) The degradation effect of Amycolatopsis sp.FT-1 on tris (2-chloropropyl) phosphate is analyzed by measuring tris (2-chloropropyl) phosphate in the degradation medium by a gas chromatography-mass spectrometer.
Preferably, the enrichment medium in step (1) consists of: 20g/L glucose, 3g/L KH2PO4，1.5g/L MgSO40.008g/L thiamine hydrochloride and 1000mL of potato extract, and the pH value is 6.0.
Preferably, the potato extract of step (1) is prepared by the following method: taking 200g of peeled potatoes, cutting into small pieces, adding 500mL of ultrapure water, boiling for 30min, crushing the potato pieces, filtering by adopting double-layer gauze to remove potato residues, and supplementing the obtained filtrate to 1000 mL.
The culture conditions in the step (1) are as follows: the temperature is 20-40 ℃, the rotating speed is 150-; preferred culture conditions are: the temperature is 30 ℃, the rotation speed is 160rpm, and the culture time is 96 h.
The tris (2-chloropropyl) phosphate degradation medium in the step (2) comprises the following components: 2g/L (NH)4)2SO4，1.5g/L Na2HPO4·12H2O，1.5g/L K2HPO4，0.2g/L MgSO4·7H2O，0.01g/L CaCl23-10g/L glucose, 5-20mg/L tris (2-chloropropyl) phosphate, 0.1mL/L trace element solution and 1000mL of ultrapure water, wherein the pH value is 4.0-9.0. Wherein the microelement solution comprises the following components: 50g/L FeSO4·7H2O，2.2g/L ZnSO4·7H2O，0.3g/L CuSO4·5H2O,14.3g/L MnSO4·H2O, 1000mL of ultrapure water. A preferred glucose concentration is 5g/L and a preferred pH is 7.0.
The culture conditions in the step (2) are as follows: the inoculation amount is 2-4g/L, the temperature is 20-40 ℃, the rotation speed is 150-170rpm, and the culture time is 3-5 d. Preferred culture conditions are: the inoculation amount is 3g/L, the temperature is 30 ℃, the rotation speed is 160rpm, and the culture time is 5 d.
The Amycolatopsis sp.FT-1 is used for screening the activated sludge of a Hunter sewage treatment plant in Guangzhou city, the acclimation of the strain adopts a method for increasing the pollutants in a gradient manner, tris (2-chloropropyl) phosphate is added into an inorganic salt culture medium, an acclimation culture medium with tris (2-chloropropyl) phosphate concentration gradient of 5, 10, 20, 30, 50, 70 and 100mg/L is prepared, and the acclimation method for increasing the pollutant concentration in a gradient manner is used for acclimation. The bacteria are amycolatopsis, the external shape is observed, the bacteria are dry and wrinkled, and the surface is a white round bacterial colony with convex edge, concave middle and irregular and opaque.
The Amycolatopsis sp.FT-1 is preserved in Guangdong province microorganism culture collection center, and the preservation number is GDMCC No: 61664, date of deposit: 12/5/2021, storage address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.
When the degradation culture is carried out for 5 days under the condition, the degradation rate of the tris (2-chloropropyl) phosphate with the initial concentration of 5mg/L reaches 85.84 percent.
Compared with the prior art, the invention has the following advantages and technical effects:
1. the invention provides a method for degrading tris (2-chloropropyl) phosphate by using Amycolatopsis sp.FT-1 and application thereof, and provides a technical method for pollution treatment and bioremediation of tris (2-chloropropyl) phosphate by the rapid and efficient degradation of tris (2-chloropropyl) phosphate by the Amycolatopsis.
2. The amycolatopsis has strong environmental adaptability, good degradation effect on the tris (2-chloropropyl) phosphate, and low cost for degrading the tris (2-chloropropyl) phosphate by using the amycolatopsis.
Drawings
FIG. 1 is a standard graph of tris (2-chloropropyl) phosphate peak area;
FIG. 2 is a graph showing the degradation profile of Amycolatopsis sp.FT-1 to tris (2-chloropropyl) phosphate at various times.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Example 1
1. Screening of degrading strains
(1) Activated sludge of a Hunter sewage treatment plant in Guangzhou city is collected, 100mL of the activated sludge is added into 250mL of an inorganic salt culture medium containing 5mg/L of tris (2-chloropropyl) phosphate, and the mixture is cultured in a constant temperature shaking table at 30 ℃ and 160 rpm. The inorganic salt culture medium mainly comprises the following components: 2g/L (NH)4)2SO4，1.5g/L Na2HPO4·12H2O,1.5g/L K2HPO4，0.2g/L MgSO4·7H2O，0.01g/L CaCl20.1mL/L of a trace element solution and 1000mL of ultrapure water, and has a pH of7.0. Wherein the microelement solution comprises the following components: 50g/L FeSO4·7H2O，2.2g/L ZnSO4·7H2O，0.3g/L CuSO4·5H2O，14.3g/L MnSO4·H2O, 1000mL of ultrapure water.
(2) Transferring for 1 time every 10 days, increasing the concentration of the tris (2-chloropropyl) phosphate (increasing according to the concentration gradient of 5, 10, 20, 30, 50, 70 and 100 mg/L) while transferring every time until the concentration of the tris (2-chloropropyl) phosphate reaches 100mg/L, separating and purifying the strains by adopting a dilution gradient method and a plate coating method, selecting bacterial colonies with fast growth and different forms, inoculating the pure single strains to an inorganic salt solid culture medium containing 0.5mg of the tris (2-chloropropyl) phosphate after ensuring that the pure single strains are pure, storing the strains in a refrigerator at 4 ℃, and carrying out a subsequent degradation experiment. The inorganic salt solid culture medium comprises the following components: 2g/L (NH)4)2SO4，1.5g/L Na2HPO4·12H2O， 1.5g/L K2HPO4，0.2g/L MgSO4·7H2O，0.01g/L CaCl2Agar 18g/L, trace element solution 0.1mL/L, and ultrapure water 1000mL, and the pH was 7.0. Wherein the microelement solution comprises the following components: 50g/L FeSO4·7H2O，2.2g/L ZnSO4·7H2O，0.3 g/L CuSO4·5H2O，14.3g/L MnSO4·H2O, 1000mL of ultrapure water.
2. Identification of degrading strains
(1) Morphological characteristics of thallus and bacterial colony
The bacteria are amycolatopsis, the external shape is observed, the bacteria are dry and wrinkled, and the surface is a white round bacterial colony with convex edge, concave middle and irregular and opaque.
(2) ITS rDNA sequence
The strain was identified as Amycolatopsis sp by 99.38% homology to the ITS sequence of Amycolatopsis as determined by ITS sequence determination and analytical comparison, and the ITS sequence is shown in Table 1 and designated Amycolatopsis sp.FT-1, where the ITS sequence (1458 bp in total) was submitted to gene bank under the accession number MZ 040131. The strain can be obtained from Guangdong province microorganism strain collection center, and the preservation number is GDMCC No: 61664.
example 2 degradation analysis of tris (2-chloropropyl) phosphate by Amycolatopsis
Amycolatopsis sp.FT-1 was inoculated into sterilized enrichment medium and cultured at 30 ℃ and 160rpm with shaking at constant temperature for 96 hours. The enrichment medium comprises the following main components: 20g/L glucose, 3g/L KH2PO4，1.5g/L MgSO40.008g/L thiamine hydrochloride and 1000mL of potato extract, and the pH value is 6.0.
The potato extract is prepared by the following method: taking 200g of peeled potatoes, cutting into small pieces, adding 500mL of ultrapure water, boiling for 30min, crushing the potato pieces, filtering by adopting double-layer gauze to remove potato residues, and supplementing the obtained filtrate to 1000 mL.
Centrifuging the enriched and cultured bacterial liquid at 3000rpm for 1min, collecting thalli, repeatedly washing the thalli for 3 times by using a degradation culture medium, then adding the degradation culture medium to prepare bacterial suspension with the concentration of 100g/L, adding 600 mu L of the bacterial suspension into 19.4mL of sterilized degradation culture medium (namely, the inoculation amount is 3g/L) to carry out degradation experiments, wherein the culture conditions are 30 ℃, 160rpm, and after shaking at constant temperature for 5d, determining the sample by GC-MS (gas chromatography-Mass spectrometer), and analyzing the degradation effect of the thalli on tris (2-chloropropyl) phosphate. The degradation medium consists of: 2g/L (NH)4)2SO4，1.5g/L Na2HPO4·12H2O，1.5g/L K2HPO4，0.2g/L MgSO4·7H2O，0.01g/L CaCl25g/L glucose, 5mg/L tris (2-chloropropyl) phosphate, 0.1mL/L trace element solution, 1000mL of ultrapure water, and pH 7.0. Wherein the microelement solution comprises the following components: 50g/L FeSO4·7H2O，2.2g/L ZnSO4·7H2O，0.3g/L CuSO4·5H2O，14.3 g/L MnSO4·H2O, 1000mL of ultrapure water.
The concentration of tris (2-chloropropyl) phosphate was determined by GC-MS under the following analytical conditions:
(1) gas chromatography conditions
A chromatographic column: an SH-Rxi-5Sil MS (30m multiplied by 0.25mm multiplied by 0.25 mu m) capillary column, ultra-high purity helium as carrier gas, a constant flow mode, a column flow of 1.0mL/min, no shunt sample injection, a sample injection amount of 1uL and a sample injection port temperature of 280 ℃; the temperature raising procedure is that the initial temperature is 50 ℃ (keeping for 1min), the temperature is raised to 200 ℃ (keeping for 1min) at the speed of 15 ℃/min, and the temperature is raised to 220 ℃ at the speed of 4 ℃/min and kept for 1 min.
(2) Mass spectrum conditions: the ion source temperature is 230 ℃, and the injector temperature is 280 ℃; the ion source is an EI source, and the solvent delay time is 3 min.
Preparing standard solutions with the concentrations of the tris (2-chloropropyl) phosphate of 0.1, 0.3, 0.5, 1.0, 3.0 and 5.0mg/L respectively, and drawing a standard curve of the tris (2-chloropropyl) phosphate-peak area, wherein the standard curve is shown in figure 1. After 5 days of degradation, the final residual concentration of tris (2-chloropropyl) phosphate in the degradation medium of the control group was 4.980 mg/L and the final residual concentration of triphenyl phosphate in the degradation medium of the experimental group was 0.705mg/L, as determined by comparing the control group with the experimental group (the method provided in example 2), the degradation efficiency of the experimental group was 85.84%, and the results are shown in FIG. 2.
Example 3
Example 3 as example 2 except that the glucose concentration in the degradation medium was 3g/L and 10g/L, respectively, during the degradation, the degradation rates of tris (2-chloropropyl) phosphate by Amycolatopsis sp.FT-1 were 28.30% and 88.76%, respectively.
Example 4
Example 4 same as example 2 except that the initial concentrations of tris (2-chloropropyl) phosphate in the degradation medium were 10mg/L and 20mg/L, respectively, during degradation, and the degradation rates of tris (2-chloropropyl) phosphate by Amycolatopsis sp.FT-1 were 85.22% and 84.05%, respectively, for the final Amycolatopsis.
Example 5
Example 5 as in example 2, except that during the degradation process, the pH of the degradation medium was 4, 5, 6, 8 and 9, respectively, and the degradation rate of the final Amycolatopsis sp.FT-1 on tris (2-chloropropyl) phosphate was 67.71%, respectively,
65.85%, 77.06%, 54.22% and 52.04%.
Example 6
Example 6 is the same as example 2 except that the inoculum size was 2g/L and 4g/L respectively during degradation, and the final degradation rate of the Amycolatopsis sp.FT-1 on tris (2-chloropropyl) phosphate was 69.37% and 88.72%, respectively.
Example 7
Example 7 as in example 2, except that the temperature during the enrichment and degradation was 20 ℃ and the rotation speed was 150rpm, the degradation rate of tris (2-chloropropyl) phosphate by Amycolatopsis sp.ft-1 was 53.62%.
Example 8
Example 8 as in example 2, except that the temperature was 40 ℃ and the rotation speed was 170rpm during the enrichment and degradation, the degradation rate of tris (2-chloropropyl) phosphate by Amycolatopsis sp.ft-1 was 77.93%.
Example 9
Example 9 was the same as example 2 except that the enrichment culture was carried out for 84h and 108h, respectively, and the degradation rates of tris (2-chloropropyl) phosphate by Amycolatopsis sp.ft-1 were 80.45% and 73.29%, respectively, in the final stage of the enrichment culture.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Guilin university of science
<120> method for degrading tris (2-chloropropyl) phosphate by using amycolatopsis and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1458
<212> DNA
<213> Amycolatopsis halotolerans
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tggaaagttc cggcggtgca ggatgaaccc gcggcctatc agcttgttgg tggggtaatg 240
gcctaccaag gcgacgacgg gtagccggcc tgagagggtg accggccaca ctgggactga 300
gacacggccc agactcctac gggaggcagc agtggggaat attgcacaat gggcgcaagc 360
ctgatgcagc gacgccgcgt gagggatgac ggccttcggg ttgtaaacct ctttcgccag 420
ggacgaagcg caagtgacgg tacctggata agaagcaccg gctaactacg tgccagcagc 480
cgcggtaata cgtagggtgc gagcgttgtc cggaattatt gggcgtaaag agctcgtagg 540
cggtttgtcg cgtcggccgt gaaatctcca cgcttaacgt ggagcgtgcg gtcgatacgg 600
gcagacttga gttcggcagg ggagactgga attcctggtg tagcggtgaa atgcgcagat 660
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aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgctgtaa acgttgggcg 780
ctaggtgtgg gcgacatcca cgttgtccgt gccgtagcta acgcattaag cgccccgcct 840
ggggagtacg gccgcaaggc taaaactcaa aggaattgac gggggcccgc acaagcggcg 900
gagcatgtgg attaattcga tgcaacgcga agaaccttac ctgggcttga catgcgccag 960
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Claims (6)
1. A method for degrading tris (2-chloropropyl) phosphate by using Amycolatopsis sp.FT-1 and application thereof are characterized by comprising the following steps:
(1) inoculating Amycolatopsis sp.FT-1 into sterilized enrichment medium, and shake culturing in shaker;
(2) collecting strains, and then inoculating the strains into a sterilized tris (2-chloropropyl) phosphate degradation culture medium for shaking culture;
(3) the degradation effect of Amycolatopsis sp.FT-1 on tris (2-chloropropyl) phosphate is analyzed by measuring tris (2-chloropropyl) phosphate in the degradation medium by a gas chromatography-mass spectrometer.
2. The method for degrading tris (2-chloropropyl) phosphate by using Amycolatopsis sp.ft-1 and the use thereof according to claim 1, wherein the method comprises the following steps: the enrichment medium in the step (1) comprises the following components: 20g/L glucose, 3g/L KH2PO4，1.5g/L MgSO40.008g/L thiamine hydrochloride and 1000mL of potato extract, and the pH value is 6.0.
3. The method for degrading tris (2-chloropropyl) phosphate by using Amycolatopsis sp.ft-1 and the use thereof according to claim 1, wherein the method comprises the following steps: the culture conditions in the step (1) are as follows: the temperature is 20-40 ℃, the rotation speed is 150-.
4. The method for degrading tris (2-chloropropyl) phosphate by using Amycolatopsis sp.ft-1 and the use thereof according to claim 1, wherein the method comprises the following steps: the tris (2-chloropropyl) phosphate degradation medium in the step (2) comprises the following components: 2g/L (NH)4)2SO4，1.5g/L Na2HPO4·12H2O，1.5g/L K2HPO4，0.2g/L MgSO4·7H2O，0.01g/L CaCl23-10g/L glucose, 5-20mg/L tris (2-chloropropyl) phosphate, 0.1mL/L trace element solution, 1000mL of ultrapure water, and the pH value is 4.0-9.0, wherein the trace element solution comprises: 50g/L FeSO4·7H2O，2.2g/L ZnSO4·7H2O，0.3g/L CuSO4·5H2O，14.3g/L MnSO4·H2O, 1000mL of ultrapure water.
5. The method for degrading tris (2-chloropropyl) phosphate by using Amycolatopsis sp.ft-1 and the use thereof according to claim 1, wherein the method comprises the following steps: the culture conditions in the step (2) are as follows: the inoculation amount is 2-4g/L, the temperature is 20-40 ℃, the rotation speed is 150-170rpm, and the culture time is 3-5 d.
6. Use of the method for degrading tris (2-chloropropyl) phosphate by using Amycolatopsis sp.ft-1 according to any one of claims 1 to 5 in bioremediation of organophosphorus flame retardant contamination.
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| WO2024087263A1 (en) * | 2022-10-26 | 2024-05-02 | 广东工业大学 | Zavarzinia compransoris capable of degrading organophosphorus flame retardant and use of zavarzinia compransoris |
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