CN109022295B - Method for degrading nitenpyram by using white rot fungi - Google Patents
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Abstract
The invention relates to a method for degrading nitenpyram by using white rot fungi, belonging to the technical field of microorganisms. The method for degrading nitenpyram by using white rot fungi comprises the following steps: (1) inoculating white rot fungus YK-624 to PDA culture medium (potato glucose agar culture medium), and storing in refrigerator at 4 deg.C; (2) after the hyphae grow full, cutting out the hyphae sheets of the white rot fungi with the diameter of 10mm, inoculating the hyphae sheets into a Kirk liquid culture medium, and standing and culturing for 3-5 days at the temperature of 24-30 ℃; (3) adding 0.05-0.15 mM nitenpyram mother liquor, and continuing to perform static culture at 24-30 ℃ for 3-5 days. The method for degrading nitenpyram by using white-rot fungi has the advantages of high degradation rate, safety and no secondary pollution risk by using the advantages of small volume, large specific surface area, strong reproductive capacity, strong adaptability and the like of the white-rot fungi.
Description
Technical Field
The invention relates to a method for degrading nitenpyram by using white rot fungi, belonging to the technical field of microorganisms.
Background
Nitenpyram (NIT), chemical name of which is (E) -N- (6-chloro-3-picolyl) -N-ethyl-N' -methyl-2-nitroethylenediamine, is one of neonicotinoid insecticides. Neonicotinoid insecticides have been developed in the 1990's as a replacement for organophosphorus pesticides, and have been widely used in recent years around the world as highly efficient and highly selective novel insecticides. Neonicotinoids kill insects by blocking their normal conduction in the central nervous system by agonizing the nicotinic acetylcholine receptors (nachrs) of the postsynaptic membrane. In mammals, however, neonicotinoid insecticides have poor ability to penetrate the blood brain barrier and have weak nAChR actions with the central and peripheral nervous systems, thus having low toxicity to mammals. Due to the unique action mechanism, the neonicotinoid insecticide becomes one of varieties with the fastest development, the most successful sale and the best insecticidal effect in the insecticide market. Since the introduction of imidacloprid into the market in 1991, neonicotinoid insecticides have rapidly grown to become the first insecticide in the world, and the usage amount thereof has increased every year. However, there is now increasing evidence that the use of neonicotinoid insecticides causes colony collapse syndrome, leading to loss of bees and massive mortality. Recently, it has been discovered that such insecticides are toxic to aquatic animals as well as mammals, including humans. The european food safety agency also reported that the two neonicotinoid insecticides, acetamiprid and imidacloprid, may have an effect on the developing human nervous system. If the fertilizer is used in large quantities in the future, the difficult degradability of the fertilizer pollutes soil and rivers, and the fertilizer may become 'second snivel'. With the constant disclosure of various experimental data, governments are also constantly issuing regulations or measures for neonicotinoid insecticides: the european union announced in 2013 that the use of three main neonicotinoid insecticides on certain crops is temporarily prohibited; in 2017, February of the European Union submits a directive to forbid neonicotinoid insecticides; canada also plans to stop using the neonicotinoid insecticide imidacloprid; france will disable neonicotinoid insecticides from 9 months in 2018. The problem of residual contamination of neonicotinoid insecticides and the study of toxicity mechanisms have attracted extensive attention worldwide.
Few degrading reports of nitenpyram are reported at home and abroad, and Li good reviews that nitenpyram solution is treated by low-temperature plasma generated by dielectric barrier discharge research on different dielectric barrier discharge powers and external factors such as Fe2+N-butanol, inorganic salt Na2CO3And H2O2The influence of plasma on the degradation of nitenpyram by the plasma (research on the degradation of nitenpyram pesticide wastewater by low-temperature plasma, high-voltage electric technology, 2011 10 th); the Nenjianfeng degrades nitenpyram in water by preparing a gas diffusion electrode, and the preparation of a catalyst, the preparation of the gas diffusion electrode and the degradation process of nitenpyram pesticide wastewater are optimized by adopting a star point design-effect surface curve method (the preparation of the gas diffusion electrode and the degradation mechanism of the nitenpyram pesticide wastewater, a Master academic paper, Shandong university 2011). The method has the advantages of high preparation cost of the used materials, complex operation management and easy secondary pollution.
The white rot fungi play an important role in the natural ecosystem, are precious biological resources, and are tools and research mode objects for environmental governance. The white rot fungi can degrade a large number of pollutants with different structures without the need of pre-conditioning specific pollutants before degrading pollutants difficult to degrade, has low requirement on nutritional conditions, and can save cost. The white rot fungus Phanerochaete sordida YK-624 has higher capability to a plurality of refractory pollutants such as aflatoxin and the like than that of a model strain Phanerochaete chrysosporium. The white-rot fungi has wide prospect in treating organic pollutants, and makes full use of the practical significance of the white-rot fungi on environmental remediation, thereby providing a theoretical basis for developing a bioremediation technology for degrading neonicotinoid insecticides by microorganisms.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for degrading nitenpyram by using white-rot fungi, and the developed method for degrading nitenpyram by using white-rot fungi has the advantages of high degradation rate, safety and no secondary pollution risk by using the advantages of small volume, large specific surface area, strong reproductive capacity, strong adaptability and the like of the white-rot fungi.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for degrading nitenpyram by using white rot fungi comprises the following steps:
(1) inoculating white rot fungi YK-624 to PDA culture medium, and storing in refrigerator at 4 deg.C;
(2) after the hyphae grow full, cutting out the hyphae sheets of the white rot fungi with the diameter of 10mm, inoculating the hyphae sheets into a Kirk liquid culture medium, and standing and culturing for 3-5 days at the temperature of 24-30 ℃;
(3) adding 0.05-0.15 mM nitenpyram mother liquor, and continuing to perform static culture at 24-30 ℃ for 3-5 days.
As a preferred embodiment of the method for degrading nitenpyram by using white rot fungi, the Kirk liquid medium comprises the following components: 10g of glucose, 0.221g of ammonium tartrate, 1.64g of anhydrous sodium acetate, 100mL of salt solution and water are added to reach the constant volume of 1L.
As a preferred embodiment of the method for degrading nitenpyram by using white rot fungi, the salt solution comprises the following components: 20g KH2PO4、5g MgSO4·7H2O、1.3g CaCl2·2H2O, 0.01g ammonium sulfate hydrochloride and 16.7mL of trace element solution, and adding water to a constant volume of 1L.
As a preferred embodiment of the method for degrading nitenpyram by using white rot fungi, the trace element solution comprises the following components: 9g Nitrilotriacetate, 3g MgSO4·7H2O、4.2g MnSO4·H2O、6g NaCl、0.6g FeSO4·7H2O、1.1g CoSO4·7H2O、1.1g ZnSO4·7H2O、0.6g CaCl2·2H2O、0.06g CuSO4·5H2O、0.11g AlK(SO4)2·12H2O、0.06g H3BO3、0.07g Na2MoO4·2H2And O, adding water to a constant volume of 1L.
As a preferred embodiment of the method for degrading nitenpyram by using white rot fungi, in the step (1), the PDA culture medium comprises the following components in percentage by mass: 20% of potatoes, 2% of glucose, 2% of agar and the balance of water.
As a preferred embodiment of the method for degrading nitenpyram by using white rot fungi, in the step (2), the Kirk liquid medium is 10 mL.
As a preferable embodiment of the method for degrading nitenpyram by using white rot fungi, in the step (2), the culture temperature is 30 ℃ and the culture time is 5 days.
As a preferable embodiment of the method for degrading nitenpyram by using white rot fungi, in the step (3), the culture temperature is 30 ℃ and the culture time is 5 days.
As a preferable embodiment of the method for degrading nitenpyram by using white rot fungi, in the step (3), the concentration of the nitenpyram mother liquor is 0.1 mM.
Compared with the prior art, the invention has the beneficial effects that: the method for degrading nitenpyram by using white-rot fungi has the advantages of high degradation rate, safety and no secondary pollution risk by using the advantages of small volume, large specific surface area, strong reproductive capacity, strong adaptability and the like of the white-rot fungi.
Drawings
FIG. 1 is a structural formula diagram of a metabolite for degrading nitenpyram by using white rot fungi.
FIG. 2 is an ESI-TOF-MS mass spectrum of a metabolite of nitenpyram degraded by white-rot fungi.
FIG. 3 shows that the white rot fungus is used for degrading metabolic products of nitenpyram13C-NMR spectrum.
FIG. 4 shows that the white rot fungus is used for degrading metabolic products of nitenpyram1H-NMR spectrum.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1
One embodiment of the method for degrading nitenpyram by using white rot fungi comprises the following steps:
(1) inoculating white rot fungi YK-624 to PDA culture medium, and storing in refrigerator at 4 deg.C;
(2) after the hypha grows over, cutting white rot fungi hypha slices with the diameter of 10mm, inoculating the white rot fungi hypha slices into 10mL Kirk liquid culture medium, and standing and culturing for 5 days at the temperature of 30 ℃;
(3) 0.1mM nitenpyram mother liquor is added, and standing culture is continued for 5 days at 30 ℃.
Wherein, the components of the Kirk liquid culture medium are as follows: 10g of glucose, 0.221g of ammonium tartrate, 1.64g of anhydrous sodium acetate, 100mL of salt solution and water are added to reach a constant volume of 1L; the salt solution comprises the following components: 20g KH2PO4、5g MgSO4·7H2O、1.3g CaCl2·2H2O, 0.01g ammonium sulfate hydrochloride and 16.7mL of trace element solution, and adding water to a constant volume of 1L; the trace element solution comprises the following components: 9g Nitrilotriacetate, 3g MgSO4·7H2O、4.2g MnSO4·H2O、6g NaCl、0.6g FeSO4·7H2O、1.1g CoSO4·7H2O、1.1g ZnSO4·7H2O、0.6g CaCl2·2H2O、0.06g CuSO4·5H2O、0.11g AlK(SO4)2·12H2O、0.06g H3BO3、0.07g Na2MoO4·2H2And O, adding water to a constant volume of 1L. The PDA culture medium comprises the following components in percentage by mass: 20% of potatoes, 2% of glucose, 2% of agar and the balance of water.
Example 2
One embodiment of the method for degrading nitenpyram by using white rot fungi comprises the following steps:
(1) inoculating white rot fungi YK-624 to PDA culture medium, and storing in refrigerator at 4 deg.C;
(2) after the hypha grows over, cutting white rot fungi hypha slices with the diameter of 10mm, inoculating the white rot fungi hypha slices into 10mL Kirk liquid culture medium, and standing and culturing for 3 days at 28 ℃;
(3) 0.05mM nitenpyram mother liquor is added, and standing culture is continued for 3 days at 28 ℃.
Wherein, the components of the Kirk liquid culture medium are as follows: 10g glucose, 0.221g of ammonium tartrate, 1.64g of anhydrous sodium acetate, 100mL of salt solution and water are added to the mixture until the volume is 1L; the salt solution comprises the following components: 20g KH2PO4、5g MgSO4·7H2O、1.3g CaCl2·2H2O, 0.01g ammonium sulfate hydrochloride and 16.7mL of trace element solution, and adding water to a constant volume of 1L; the trace element solution comprises the following components: 9g Nitrilotriacetate, 3g MgSO4·7H2O、4.2g MnSO4·H2O、6g NaCl、0.6g FeSO4·7H2O、1.1g CoSO4·7H2O、1.1g ZnSO4·7H2O、0.6g CaCl2·2H2O、0.06g CuSO4·5H2O、0.11g AlK(SO4)2·12H2O、0.06g H3BO3、0.07g Na2MoO4·2H2And O, adding water to a constant volume of 1L. The PDA culture medium comprises the following components in percentage by mass: 20% of potatoes, 2% of glucose, 2% of agar and the balance of water.
Example 3
One embodiment of the method for degrading nitenpyram by using white rot fungi comprises the following steps:
(1) inoculating white rot fungi YK-624 to PDA culture medium, and storing in refrigerator at 4 deg.C;
(2) after the hypha grows over, cutting white rot fungi hypha slices with the diameter of 10mm, inoculating the white rot fungi hypha slices into 10mL Kirk liquid culture medium, and standing and culturing for 5 days at 24 ℃;
(3) 0.15mM nitenpyram mother liquor is added, and the standing culture is continued for 5 days at 24 ℃.
Wherein, the components of the Kirk liquid culture medium are as follows: 10g of glucose, 0.221g of ammonium tartrate, 1.64g of anhydrous sodium acetate, 100mL of salt solution and water are added to reach a constant volume of 1L; the salt solution comprises the following components: 20gKH2PO4、5g MgSO4·7H2O、1.3g CaCl2·2H2O, 0.01g ammonium sulfate hydrochloride and 16.7mL of trace element solution, and adding water to a constant volume of 1L; the trace element solution comprises the following components: 9g Nitrilotriacetate, 3g MgSO4·7H2O、4.2g MnSO4·H2O、6g NaCl、0.6g FeSO4·7H2O、1.1g CoSO4·7H2O、1.1g ZnSO4·7H2O、0.6g CaCl2·2H2O、0.06g CuSO4·5H2O、0.11g AlK(SO4)2·12H2O、0.06g H3BO3、0.07g Na2MoO4·2H2And O, adding water to a constant volume of 1L. The PDA culture medium comprises the following components in percentage by mass: 20% of potatoes, 2% of glucose, 2% of agar and the balance of water.
Comparative example 1
A method for degrading nitenpyram by using white rot fungi comprises the following steps:
(1) inoculating white rot fungi YK-624 to PDA culture medium, and storing in refrigerator at 4 deg.C;
(2) after the hypha grows over, cutting white rot fungi hypha slices with the diameter of 10mm, inoculating into 10mL of PDB liquid culture medium (potato glucose liquid culture medium), and standing and culturing at 30 ℃ for 5 days;
(3) 0.1mM nitenpyram mother liquor is added, and standing culture is continued for 5 days at 30 ℃.
Wherein, the PDB liquid culture medium comprises the following components: 20% of potatoes, 2% of glucose and the balance of water. The PDA culture medium comprises the following components in percentage by mass: 20% of potatoes, 2% of glucose, 2% of agar and the balance of water.
Effect example 1
Degradation rate test of nitenpyram in examples 1-3 and comparative example 1 of the invention
After the degradation experiments of examples 1-3 and comparative example 1 were completed, the internal standard was added to the culture solutions of examples 1-3 and comparative example 1, and 20mL of acetone was added to break up the cells with a hand-held tissue homogenizer. The resulting homogenate was filtered, concentrated by rotary evaporation, and analyzed by high performance liquid chromatography (HPLC, column: Inertsil ODS-35 μm 4.6X250 mm). The nitenpyram is quantitatively analyzed by an internal standard method, and the result shows that the nitenpyram is completely degraded in the examples 1-3, while the degradation rate of the nitenpyram in the comparative example 1 is only 20%. Therefore, the method for degrading nitenpyram has the advantages of high degradation rate, safety and no secondary pollution risk.
Effect example 2
Example 1-3 identification of metabolites degrading nitenpyram
To identify the metabolites of nitenpyram, 148 μ M of nitenpyram was added to 5L of liquid medium and cultured under degradation experimental conditions. After 20 days of culture, the mixture is concentrated to 250mL by rotary evaporation, the obtained supernatant is extracted 3 times by using the same volume of ethyl acetate, and the extracted solution is distilled in a rotary evaporator under reduced pressure. The filtrate was subjected to gradient elution by silica gel column chromatography, followed by metabolites by thin layer chromatography, and analyzed for purity by HPLC (column: Inertsil C30S-Select 5 μm 4.6X250 mm). The product obtained by separation was further purified by preparative HPLC (column: Inertsil C30S-Select 5 μm 20X250mm) to obtain a high purity metabolite. Electrospray mass spectrometry and magnetic resonance spectroscopy were used to identify the metabolite (E) -N- ((6-chloropyridin-3-yl) methyl) -N-ethyl-N' -hydroxyyacetimidamide (CPMHA), which has the formula shown in FIG. 1, ESI-TOF-MS mass spectrometry as shown in FIG. 2,13the C-NMR spectrum is shown in figure 3,1the H-NMR spectrum is shown in FIG. 4.
Therefore, the method for degrading the nitenpyram by using the white rot fungi can effectively degrade the nitenpyram, and the degradation product is safe and has no secondary pollution risk.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (6)
1. A method for degrading nitenpyram by using white rot fungi is characterized by comprising the following steps:
(1) inoculating white rot fungi YK-624 to PDA culture medium, and storing at 4 deg.C in refrigerator;
(2) after the hyphae grow full, cutting out the hyphae sheets of the white rot fungi with the diameter of 10mm, inoculating the hyphae sheets into a Kirk liquid culture medium, and standing and culturing for 3-5 days at 24-30 ℃;
(3) adding 0.05-0.15 mM nitenpyram mother liquor, and continuing standing and culturing for 3-5 days at 24-30 ℃;
the Kirk liquid medium comprises the following components: 10g of glucose, 0.221g of ammonium tartrate, 1.64g of anhydrous sodium acetate and 100mL of salt solution, and adding water to a constant volume of 1L;
the salt solution comprises the following components: 20g KH2PO4、5 g MgSO4・7H2O、1.3 g CaCl2・2H2O, 0.01g ammonium sulfate hydrochloride and 16.7mL of trace element solution, and adding water to a constant volume of 1L;
the trace element solution comprises the following components: 9g Nitrilotriacetate, 3g MgSO4・7H2O、4.2 g MnSO4・H2O、6 g NaCl、0.6 gFeSO4・7H2O、1.1 g CoSO4・7H2O、1.1 g ZnSO4・7H2O、0.6 g CaCl2・2H2O、0.06 g CuSO4・5H2O、0.11 g AlK(SO4)2・12H2O、0.06 g H3BO3、0.07 g Na2MoO4・2H2And O, adding water to a constant volume of 1L.
2. The method for degrading nitenpyram by using white rot fungi according to claim 1, wherein in the step (1), the PDA culture medium comprises the following components in percentage by mass: 20% of potatoes, 2% of glucose, 2% of agar and the balance of water.
3. The method for degrading nitenpyram by using white rot fungi according to claim 1, wherein in the step (2), the Kirk liquid medium is 10 mL.
4. The method for degrading nitenpyram by using white rot fungi according to claim 1, wherein in the step (2), the culture temperature is 30 ℃ and the culture time is 5 days.
5. The method for degrading nitenpyram by using white rot fungi according to claim 1, wherein in the step (3), the culture temperature is 30 ℃ and the culture time is 5 days.
6. The method for degrading nitenpyram by using white rot fungi according to claim 1, wherein in the step (3), the concentration of the nitenpyram mother liquor is 0.1 mM.
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| CN114703071B (en) * | 2022-05-06 | 2023-06-16 | 广州大学 | Method for degrading imidacloprid by using white rot fungi |
| CN114699705B (en) * | 2022-05-06 | 2022-12-16 | 广州大学 | Method for degrading imidaclothiz by adopting white rot fungi |
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| Biotransformation of acetamiprid by the white-rot fungus Phanerochaete sordida YK-624;Jianqiao Wang et al.;《Appl Microbiol Biotechnol》;20110629;第93卷(第2期);摘要，第831页右栏第1段至第835页左栏第1段 * |
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