CN111826288A - Method for degrading tricresyl phosphate by using Phanerochaete chrysosporium and application of method - Google Patents
Method for degrading tricresyl phosphate by using Phanerochaete chrysosporium and application of method Download PDFInfo
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Abstract
The invention discloses a method for degrading tricresyl phosphate by using Phanerochaete chrysosporium and application thereof. The method comprises the following steps: (1) culturing Phanerochaete chrysosporium to obtain spore liquid of the Phanerochaete chrysosporium; (2) inoculating spore liquid of phanerochaete chrysosporium into a liquid culture medium, culturing to logarithmic phase, adding tricresyl phosphate into the liquid culture medium, and continuing culturing to degrade the tricresyl phosphate. The phanerochaete chrysosporium has strong environmental adaptability and good degradation effect on tricresyl phosphate, and provides technical support for environmental pollution treatment and bioremediation through rapid and efficient degradation of the tricresyl phosphate.
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 tricresyl phosphate by using Phanerochaete chrysosporium and application thereof.
Background
In recent years, with the gradual exit from the market of the traditional brominated Flame Retardants with the defects of large smoke generation amount, strong corrosivity, easy toxic gas release and the like, Organic Phosphorus Flame Retardants (OPFRs) are produced and used in large quantities as ideal substitutes of the brominated Flame Retardants. Tricresyl phosphate (TCPs) is a commonly used organic phosphorus flame retardant, and is mainly added into various products in physical mixing modes such as doping, adsorption and the like, so that the Tricresyl phosphate is easily separated from the products by volatilization, product 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 tricresyl phosphate can be detected in various environmental media such as air dust, water, river sediment, sediment and the like. Tricresyl phosphate also accumulates to varying degrees in animals, and even humans, simultaneously through environmental exposure and transmission through the food chain. The tricresyl phosphate remained in organisms can generate toxic effects on multiple aspects of endocrine interference, reproductive toxicity, neurotoxicity, immunotoxicity and the like. Therefore, the research on the degradation and removal technology of tricresyl phosphate is a very urgent task, both from the health of the ecosystem and from the safety of human beings.
At present, tricresyl phosphate is removed mainly by adopting a physical chemical method and a biological method, wherein the physical chemical method cannot be a preferred technology for pollution treatment due to the problems of higher operation cost, easiness in causing secondary pollution and the like. The microbial method for degrading/converting organic pollutants has environmental friendliness and low cost, so that the method becomes the safest, economic and effective way for restoring organic pollution of water bodies. The microbial degradation of tricresyl phosphate is often low in efficiency in the practical application process, and the microbial degradation of tricresyl phosphate is limited due to the lack of effective degrading strains, so that the screening and the application of the efficient tricresyl phosphate degrading bacteria have certain research significance.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for degrading tricresyl phosphate by using Phanerochaete chrysosporium.
Another object of the present invention is to provide an application of the method for degrading tricresyl phosphate by using Phanerochaete chrysosporium.
The purpose of the invention is realized by the following technical scheme: a method for degrading tricresyl phosphate by using Phanerochaete chrysosporium comprises the following steps:
(1) culturing Phanerochaete chrysosporium to obtain spore liquid of the Phanerochaete chrysosporium;
(2) inoculating spore liquid of phanerochaete chrysosporium into a liquid culture medium, culturing to logarithmic phase, adding tricresyl phosphate into the liquid culture medium, and continuing culturing to degrade the tricresyl phosphate.
The spore liquid of the phanerochaete chrysosporium in the step (1) is preferably obtained by the following method:
(I) inoculating phanerochaete chrysosporium to a PDA (PDA) plate culture medium for culturing to obtain a phanerochaete chrysosporium spore layer;
(II) scraping off a spore layer of the phanerochaete chrysosporium from a plate culture medium, adding sterile normal saline, and filtering by using gauze to obtain a spore liquid of the phanerochaete chrysosporium.
The PDA plate culture medium in the step (I) comprises the following components: glucose 20g, KH2PO43g，MgSO4·7H2O1.5g, thiamine hydrochloride 0.001g, agar 20g, potato extract 1000mL, pH 6.0.
The potato extracting solution is prepared by the following method: removing peel of potato 200g, cutting into small pieces, adding 500mL of water, boiling for 30min, mincing potato pieces, filtering to remove potato residue pieces, and adding the obtained filtrate to 1000 mL.
The filtration is preferably carried out by adopting double-layer gauze.
The culture conditions in step (I) are as follows: standing and culturing for 4-5 days at 35-37 ℃; preferably: and (3) standing and culturing for 4-5 days at 35 ℃.
The gauze in step (II) is preferably a double-layer sterile gauze.
The Phanerochaete chrysosporium in the step (1) is Phanerochaete chrysosporium (Phanerochaete cchrysosporium) BKM-F-1767; preferably Phanerochaete chrysosporium (Phanerochaete chrysosporium) BKM-F-1767, available from the Guangdong province collection of microbial cultures under the strain accession number GIM 3.383.
The spore liquid of Phanerochaete chrysosporium in the step (1) is OD600Is 0.80.9 of spore liquid of phanerochaete chrysosporium; preferably OD600Is 0.85 (2.0X 10)6CFU/mL) of spore liquid of Phanerochaete chrysosporium.
The inoculation amount of spore liquid of the phanerochaete chrysosporium in the step (2) is 3-5% by volume percent; preferably 4% by volume.
The liquid culture medium in the step (2) comprises the following components: 5g of glucose, 0.2g of ammonium tartrate and KH2PO42g，MgSO4·7H2O 0.5g，CaCl20.1g，MnSO4·H20.035g of O, 0.07g of NaCl, 0.001g of thiamine hydrochloride and CoCl20.007g，CuSO4·5H2O 0.007g，ZnSO4·7H2O 0.007g，AlK(SO4)2·12H2O 0.007g，H3BO30.007g，NaMoO4·2H20.0007g of O, 0.105g of nitrilotriacetic acid, 1000mL of ultrapure water and the pH value of 4-6.
The culture conditions of the step (2) from the culture to the logarithmic growth phase are as follows: carrying out constant temperature shaking culture at 30 ℃ and 160r/min for 3-4 days; preferably: shaking and culturing at 30 deg.C and 160r/min constant temperature for 3 days.
The dosage of the tricresyl phosphate in the step (2) is calculated according to the addition of the tricresyl phosphate in the reaction system with the final concentration of 5-10 mg/L.
The conditions for continuing the culture in the step (2) are preferably: shaking and culturing at constant temperature of 160r/min for 5-6 days at 30 ℃.
The method for degrading tricresyl phosphate by using Phanerochaete chrysosporium is applied to degrading tricresyl phosphate.
The tricresyl phosphate is tricresyl phosphate contained in a water body.
Compared with the prior art, the invention has the following advantages and effects:
1. the invention provides a method for degrading tricresyl phosphate by using Phanerochaete chrysosporium and application thereof, and provides a technical method for pollution treatment and bioremediation of the tricresyl phosphate by quickly and efficiently degrading the tricresyl phosphate by the Phanerochaete chrysosporium.
2. The phanerochaete chrysosporium has strong environmental adaptability, has good degradation effect on tricresyl phosphate, and has low cost for degrading pollutants by using the phanerochaete chrysosporium.
Drawings
FIG. 1 is a graph showing the standard curve of the concentration of tricresyl phosphate versus the peak area.
FIG. 2 is a graph showing the degradation of tricresyl phosphate after inoculation with Phanerochaete chrysosporium.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents used in the present invention are commercially available.
The Phanerochaete chrysosporium referred to in the invention is Phanerochaete chrysosporium (Phanerochaete cchrysosporium) BKM-F-1767 purchased from Guangdong province microorganism culture collection center, and the culture collection number is GIM 3.383.
Example 1 degradation assay of Phanerochaete chrysosporium for tricresyl phosphate
(1) Transferring Phanerochaete chrysosporium strains stored on a 4 ℃ inclined plane onto a PDA (personal digital assistant) plate culture medium by a scribing method, placing the plate into a constant-temperature incubator at 35 ℃ for standing culture for 4-5 days until a large amount of white powdery spore layers grow on the surface of the plate, flushing the plate with sterile normal saline, gently scraping off the spores on the surface of the plate by using a sterile inoculating loop, and filtering the mixture by using double-layer sterile gauze to obtain Phanerochaete chrysosporium spore liquid. Measuring absorbance of spore solution at 600nm with spectrophotometer, diluting with sterile physiological saline, and adjusting absorbance to OD600At 0.85, the concentration of the spore liquid was about 2.0 × 106CFU/mL; wherein:
the PDA plate culture medium comprises the following components: glucose 20g, KH2PO43g，MgSO4·7H2O1.5g, thiamine hydrochloride 0.001g, agar 20g, potato extract 1000mL, pH 6.0.
The preparation method of the potato extracting solution comprises the following steps: taking 200g of peeled potatoes, cutting into small pieces, adding 500mL of water, boiling for 30min, crushing the potato pieces, filtering out potato residue pieces by using double-layer gauze, and supplementing the filtrate to 1000 mL.
(2) Adding 0.8mL of the phanerochaete chrysosporium spore liquid into 19.2mL of a liquid culture medium, culturing for 3d under the constant-temperature oscillation culture condition of 30 ℃ and 160r/min, starting the phanerochaete chrysosporium to enter a logarithmic growth phase and forming mycelium pellets with uniform sizes in a system, adding 5mg/L of tricresyl phosphate into the culture system, culturing for 6d at 30 ℃ and 160rpm, and analyzing the degradation effect of the phanerochaete chrysosporium on the tricresyl phosphate by measuring a sample through GC-MS; wherein:
the liquid culture medium comprises the following components: 5g of glucose, 0.2g of ammonium tartrate and KH2PO42g，MgSO4·7H2O0.5g，CaCl20.1g，MnSO4·H20.035g of O, 0.07g of NaCl, 0.001g of thiamine hydrochloride and CoCl20.007g，CuSO4·5H2O 0.007g，ZnSO4·7H2O 0.007g，AlK(SO4)2·12H2O0.007g，H3BO30.007g，NaMoO4·2H2O0.0007 g, nitrilotriacetic acid 0.105g, ultra pure water 1000mL, pH 5.0.
(3) The tricresyl phosphate concentration was determined by GC-MS under the following analytical conditions:
chromatographic conditions: an SH-Rxi-5SilMS capillary column (30m multiplied by 0.25mm, 0.25um) with high-purity helium as carrier gas and a constant flow mode, wherein the column flow is 1mL/min, the split-flow sample injection is not carried out, the sample injection amount is 1uL, and the sample injection port temperature is 280 ℃; the temperature program starts at 100 deg.C (1 min hold), increases at 15 deg.C/min to 250 deg.C (1 min hold), increases at 2 deg.C/min to 210 deg.C (1 min hold), increases at 25 deg.C/min to 250 deg.C (1 min hold), and increases at 2 deg.C/min to 280 deg.C (1 min hold).
Mass spectrum conditions: the ion source temperature is 230 ℃, and the transmission line temperature is 280 ℃; EI source (70eV), ion detection Scan (SRM) is selected.
(4) Preparing standard solutions with tricresyl phosphate concentrations of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2mg/L, and drawing a tricresyl phosphate concentration-peak area standard curve, wherein the standard curve is shown in figure 1. The liquid culture medium which is not inoculated with phanerochaete chrysosporium is taken as a control group, the concentration of the tricresyl phosphate in the control group after 6 days of culture is 4.891mg/L, the concentration of the tricresyl phosphate in the experimental group (inoculated with the phanerochaete chrysosporium) is 2.289mg/L according to the test, the degradation efficiency of the experimental group is 53.2 percent according to the comparison between the control group and the experimental group, and the results are shown in FIG. 2 (measured once a day and repeated three times).
Example 2
The process is the same as example 1 except that: in the degradation process in the step (2), the concentration of tricresyl phosphate in the liquid culture medium is 10mg/L, the pH of the liquid culture medium is 6, and the culture conditions are as follows: the temperature is 30 ℃, the rotating speed of a shaking table is 160rpm, the culture time is 6d, and finally the degradation rate of the phanerochaete chrysosporium to the tricresyl phosphate is 42.6%.
Example 3
The process is the same as example 1 except that: in the degradation process in the step (2), the concentration of tricresyl phosphate in the liquid culture medium is 10mg/L, the pH of the liquid culture medium is 4, and the culture conditions are as follows: the temperature is 30 ℃, the rotating speed of a shaking table is 160rpm, the culture time is 6d, and finally the degradation rate of the phanerochaete chrysosporium to the tricresyl phosphate is 40.8%.
Example 4
The process is the same as example 1 except that: in the step (2), 0.6mL of the phanerochaete chrysosporium spore solution is added into 19.4mL of sterilized liquid nutrient medium, and finally, the degradation rate of the phanerochaete chrysosporium to triphenyl phosphate is 40.2%.
Example 5
The process is the same as example 1 except that: in the step (2), 1.0mL of the phanerochaete chrysosporium spore solution is added into 19.0mL of sterilized liquid nutrient medium, and finally, the degradation rate of the phanerochaete chrysosporium to triphenyl phosphate is 56.4%.
Example 6
The process is the same as example 1 except that: and (2) adding the phanerochaete chrysosporium spore liquid into a sterilized liquid nutrient medium, and culturing for 4d under the constant-temperature shaking culture condition of 30 ℃ and 160r/min, wherein the degradation rate of the phanerochaete chrysosporium to triphenyl phosphate is 50.3 percent finally.
Example 7
The process is the same as example 1 except that: and (3) in the degradation process of the step (2), adding triphenyl phosphate with the final concentration of 5mg/L into the culture system, culturing for 6d at 30 ℃ and 160rpm, and finally, ensuring that the degradation rate of the phanerochaete chrysosporium to triphenyl phosphate is 58.5%.
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.
Claims (10)
1. A method for degrading tricresyl phosphate by using Phanerochaete chrysosporium is characterized by comprising the following steps:
(1) culturing Phanerochaete chrysosporium to obtain spore liquid of the Phanerochaete chrysosporium;
(2) inoculating spore liquid of phanerochaete chrysosporium into a liquid culture medium, culturing to logarithmic phase, adding tricresyl phosphate into the liquid culture medium, and continuing culturing to degrade the tricresyl phosphate.
2. The method for degrading tricresyl phosphate using Phanerochaete chrysosporium according to claim 1, wherein:
the Phanerochaete chrysosporium in the step (1) is Phanerochaete chrysosporium (Phanerochaete cchrysosporium) BKM-F-1767.
3. The method for degrading tricresyl phosphate using Phanerochaete chrysosporium according to claim 1, wherein:
the spore liquid of Phanerochaete chrysosporium in the step (1) is OD6000.8-0.9 of spore liquid of phanerochaete chrysosporium;
the inoculation amount of the spore liquid of the phanerochaete chrysosporium in the step (2) is 3-5% by volume percent.
4. The method for degrading tricresyl phosphate using Phanerochaete chrysosporium according to claim 3, wherein:
the spore liquid of Phanerochaete chrysosporium in the step (1) is OD6000.85 of spore liquid of Phanerochaete chrysosporium;
the inoculation amount of the spore liquid of the phanerochaete chrysosporium in the step (2) is 4 percent by volume.
5. The method for degrading tricresyl phosphate using Phanerochaete chrysosporium according to claim 1, wherein:
the dosage of the tricresyl phosphate in the step (2) is calculated according to the addition of the tricresyl phosphate in the reaction system with the final concentration of 5-10 mg/L.
6. The method for degrading tricresyl phosphate using Phanerochaete chrysosporium according to claim 1, wherein:
the liquid culture medium in the step (2) comprises the following components: 5g of glucose, 0.2g of ammonium tartrate and KH2PO42g，MgSO4·7H2O 0.5g，CaCl20.1g，MnSO4·H20.035g of O, 0.07g of NaCl, 0.001g of thiamine hydrochloride and CoCl20.007g，CuSO4·5H2O 0.007g，ZnSO4·7H2O 0.007g，AlK(SO4)2·12H2O 0.007g，H3BO30.007g，NaMoO4·2H20.0007g of O, 0.105g of nitrilotriacetic acid, 1000mL of ultrapure water and the pH value of 4-6.
7. The method for degrading tricresyl phosphate using Phanerochaete chrysosporium according to claim 1, wherein:
the culture conditions of the step (2) from the culture to the logarithmic growth phase are as follows: carrying out constant temperature shaking culture at 30 ℃ and 160r/min for 3-4 days;
the conditions for continuing the culture in the step (2) are as follows: shaking and culturing at constant temperature of 160r/min for 5-6 days at 30 ℃.
8. The method for degrading tricresyl phosphate using Phanerochaete chrysosporium according to claim 1, wherein:
the spore liquid of the phanerochaete chrysosporium in the step (1) is obtained by the following method:
(I) inoculating phanerochaete chrysosporium to a PDA (PDA) plate culture medium for culturing to obtain a phanerochaete chrysosporium spore layer;
(II) scraping off a spore layer of the phanerochaete chrysosporium from a plate culture medium, adding sterile normal saline, and filtering by using gauze to obtain a spore liquid of the phanerochaete chrysosporium.
9. The method for degrading tricresyl phosphate using phanerochaete chrysosporium according to claim 8, wherein:
the PDA plate culture medium in the step (I) comprises the following components: glucose 20g, KH2PO43g，MgSO4·7H2O1.5g, thiamine hydrochloride 0.001g, agar 20g, potato extract 1000mL, pH 6.0;
the culture conditions in step (I) are as follows: and (3) standing and culturing for 4-5 days at 35-37 ℃.
10. Use of the method for degrading tricresyl phosphate according to any one of claims 1 to 9 by using Phanerochaete chrysosporium for degrading tricresyl phosphate.
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