CN114703071B - Method for degrading imidacloprid by using white rot fungi - Google Patents
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Abstract
The invention belongs to the technical field of microbial degradation, and discloses a method for degrading imidacloprid by using white rot fungi, which comprises the following steps: (1) Inoculating white rot fungi YK-624 to potato dextrose agar medium for subculture expansion; (2) Cutting out mycelium slices from a culture medium of subculture, and inoculating the mycelium slices into a liquid culture medium for static culture; (3) After the white rot fungi YK-624 is cultured in the liquid culture medium for a set time, the solution containing imidacloprid is added and the culture is continued. The invention adopts white rot fungi YK-624 with degradation capability higher than that of mode fungi, can effectively degrade imidacloprid in a liquid culture medium, and has the degradation rate as high as 89.4 percent in a preferred Kirk liquid culture medium based on the advantages that white rot fungi are easy to obtain and have high growth and propagation speed.
Description
Technical Field
The invention relates to the technical field of microbial degradation, in particular to a method for degrading imidacloprid by using white rot fungi.
Background
Imidacloprid (IMI), chemical name 1- (6-chloropyridin-3-ylmethyl) -N-nitroimidazolen-2-ylamine, is a new class of nicotinic pesticides. New nicotine pesticides have been developed rapidly since the market, have become the most commonly used type of pesticides worldwide, are registered and used in more than 120 countries, account for one fourth of the world's pesticide usage, and play an important role in maintaining crop yield. Imidacloprid is the first product of neonicotinoid insecticides, and has become one of the most widely used neonicotinoid insecticides in 1991 due to its strong activity, strong resistance to rain, and ease of seed treatment. However, the chemical nature of the neonicotinoids is relatively stable and highly water soluble and can migrate into the aquatic system in a variety of ways. According to various studies, imidacloprid is increasingly being detected in surface runoff and aquatic systems in canada, the united states, spain, bangladesh and china, and several studies have shown that imidacloprid can cause serious damage to some non-target organisms. Research shows that imidacloprid has high toxicity to bees, can cause 'colony collapse syndrome', causes the missing and massive death of bees, further causes serious crop pollination crisis in related countries, and causes massive yield reduction of crops (such as soybeans, sunflowers and the like). Other studies have shown that imidacloprid also presents a direct or indirect hazard to birds, aquatic organisms and mammals, and that there is a serious threat to human health with the accumulation of biological chains. Imidacloprid affects liver, thyroid and body weight, and exposure to high dose imidacloprid environment can cause degenerative changes in vital organs of the body in addition to cardiovascular and blood systems. Therefore, it is necessary to carry out a repair work for environmental pollution caused by imidacloprid.
After the environmental problems caused by imidacloprid are focused on, various repair technologies including chemical oxidation, physical adsorption, microbial degradation, photocatalyst and other methods are also presented in the prior art. The effect of direct photolysis is not ideal because of the low quantum efficiency of most pesticides. Chemical oxidation methods, including advanced oxidation and ozone oxidation, produce more toxic secondary products and are not suitable for practical restoration applications of imidacloprid to the environment. In the aspect of microbial degradation, the strong degradation capability of the bacterium Stenotrophomonas maltophilia on the imidacloprid is reported for the first time by the university of Nanjing, and the bacterium can utilize the co-metabolism to improve the degradation capability of the imidacloprid, and although the imidacloprid is degraded by the bacterium without secondary pollution, the bacterium can only degrade high-concentration organic pollutants and can not fully mineralize the imidacloprid.
Disclosure of Invention
Aiming at the problems, the invention provides a method for degrading imidacloprid by using white rot fungi, which aims to solve the problem of high-efficiency degradation of imidacloprid with low concentration.
The aim of the invention is realized by adopting the following technical scheme:
a method for degrading imidacloprid by using white rot fungi, which comprises the following steps:
(1) Subculture
Inoculating white rot fungi YK-624 to potato dextrose agar medium for subculture expansion;
(2) Stationary culture
Cutting out mycelium slices from a subcultured potato glucose agar medium, and inoculating the mycelium slices into a liquid medium, wherein the liquid medium is a potato glucose liquid medium or a Kirk liquid medium;
the Kirk liquid culture medium comprises the following components: per liter, comprising 10g glucose, 0.221g ammonium tartrate, 1.64g anhydrous sodium acetate, 1g Tween 80 and 100ml kirk salt solution, wherein Tween 80 is added during shaking culture;
the Kirk salt solution comprises the following components: comprises 20g KH per liter 2 PO 4 ,5g MgSO 4 ·7H 2 O,1.3g CaCl 2 ·2H 2 O,0.01g of ammonium sulfate hydrochloride, 16.7ml Kirk microelement solution;
the Kirk microelement solution comprises the following components: comprises 9g of trisodium nitrilotriacetate and 3g of MgSO per liter 4 ·7H 2 O,4.2g MnSO 4 ·H 2 O,6g NaCl,0.6g FeSO 4 ·7H 2 O,1.1g CoSO 4 ·7H 2 O,1.1g ZnSO 4 ·7H 2 O,0.6g CaCl 2 ·2H 2 O,0.06g CuSO 4 ·5H 2 O,0.11gAlK(SO 4 ) 2 ·12H 2 O,0.06g H 3 BO 3 ,0.07gNa 2 MoO 4 ·2H 2 O;
(3) Degradation culture
And after the white rot fungi YK-624 is subjected to shaking culture in the liquid culture medium for a set time, adding a solution containing imidacloprid and continuing to culture.
Preferably, the potato dextrose agar medium comprises 20% potato, 2% glucose and 2% agar by mass fraction.
Preferably, the diameter of the mycelium pellet is 10mm, and the volume of the liquid culture medium is 10mL.
Preferably, the potato dextrose broth comprises 20% potato and 2% dextrose by mass fraction.
Preferably, the stationary culture is performed for 3 days.
Preferably, the time of the degradation culture is 20d.
The beneficial effects of the invention are as follows:
1. the method provided by the invention adopts white rot fungi YK-624 with degradation capability higher than that of mode fungi, can effectively degrade imidacloprid in a liquid culture medium, is based on the advantages of easy acquisition and higher growth and propagation speed of white rot fungi, can effectively degrade imidacloprid, has degradation rate up to 89.4% in a preferred Kirk liquid culture medium, and has obvious degradation efficiency and degradation rate due to other schemes in the prior art.
2. The white rot fungi provided by the invention can bear and degrade medium-high concentration organic pollutants and can degrade lower concentration imidacloprid and other organic pollutants by combining proper process conditions and adopting a unique extracellular detoxification mechanism, and can be completely mineralized; can be applied to the environmental fields of sewage treatment, atmospheric pollution treatment, soil pollution control and the like caused by low-concentration pollutants.
Detailed Description
The invention is further described below with reference to examples.
Example 1
The method for degrading imidacloprid by using white rot fungi provided by the embodiment comprises the following steps:
(1) Subculture
Inoculating white rot fungi YK-624 to potato dextrose agar medium for subculture expansion; the potato glucose agar culture medium comprises 20% of potatoes, 2% of glucose and 2% of agar according to mass fraction, and the balance of water;
(2) Stationary culture
2 pieces of mycelium pieces with the diameter of 10mm are cut out from a subcultured potato dextrose agar medium and inoculated into a 100mL conical flask filled with 10mL of liquid medium, wherein the liquid medium is Kirk liquid medium;
the Kirk liquid culture medium comprises the following components: per liter, comprising 10g glucose, 0.221g ammonium tartrate, 1.64g anhydrous sodium acetate, 1g Tween 80 and 100ml kirk salt solution, wherein Tween 80 is added during shaking culture;
the Kirk salt solution comprises the following components: comprises 20g KH per liter 2 PO 4 ,5g MgSO 4 ·7H 2 O,1.3g CaCl 2 ·2H 2 O,0.01g of ammonium sulfate hydrochloride, 16.7ml Kirk microelement solution;
the Kirk microelement solution comprises the following components: comprises 9g of trisodium nitrilotriacetate and 3g of MgSO per liter 4 ·7H 2 O,4.2g MnSO 4 ·H 2 O,6g NaCl,0.6g FeSO 4 ·7H 2 O,1.1g CoSO 4 ·7H 2 O,1.1g ZnSO 4 ·7H 2 O,0.6gCaCl 2 ·2H 2 O,0.06g CuSO 4 ·5H 2 O,0.11gAlK(SO 4 ) 2 ·12H 2 O,0.06g H 3 BO 3 ,0.07gNa 2 MoO 4 ·2H 2 O;
(3) Degradation culture
After the white rot fungi YK-624 is cultured in the liquid culture medium for 3 days in a shaking way, the imidacloprid solution with the initial concentration of 100 mu M is added and the culture is continued for 20 days.
The white rot fungi Phanerochaete sordidaYK-624 adopted by the invention are separated from the Japanese Jiujiu island decay wood, and the embodiment of the invention further adopts ingenious process steps, so that the degradation capability of the white rot fungi to a plurality of organic pollutants such as imidacloprid, aflatoxin and the like is proved to be higher than that of mode fungi, the toxicity of degradation products is greatly reduced, and the white rot fungi have wide application prospect in degrading the organic pollutants.
The embodiment of the invention adopts white rot fungi YK-624 with higher degradation capacity than that of mode fungi, can effectively degrade imidacloprid in a liquid culture medium, and has the degradation rate of 89.4 percent in a preferred Kirk liquid culture medium based on the advantages that white rot fungi are easy to obtain and have high growth and propagation speed.
Example 2
The method for degrading imidacloprid by using white rot fungi provided by the embodiment comprises the following steps:
(1) Subculture
Inoculating white rot fungi YK-624 to potato dextrose agar medium for subculture expansion; the potato glucose agar culture medium comprises 20% of potatoes, 2% of glucose and 2% of agar according to mass fraction, and the balance of water;
(2) Stationary culture
2 pieces of mycelium slices with the diameter of 10mm are cut out from a subcultured potato dextrose agar medium and inoculated into a 100mL conical flask filled with 10mL of liquid medium, wherein the liquid medium is potato dextrose liquid medium;
the potato glucose liquid culture medium comprises 20% of potatoes, 2% of glucose and the balance of water according to mass fraction;
(3) Degradation culture
After the white rot fungi YK-624 is cultured in the liquid culture medium for 3 days, the imidacloprid solution with the initial concentration of 100 mu M is added and the culture is continued for 20 days.
Experimental example
The degradation rates of imidacloprid by the methods described in example 1 and example 2 were measured as follows:
after the degradation culture is completed, 20mL of acetone and an internal standard substance are added into the conical flask, bacterial cells in the degradation system are smashed by using a homogenizer, the culture solution is separated, and the quantitative analysis is performed by using a high performance liquid chromatograph after rotary evaporation concentration.
The quantitative analysis result shows that the degradation rate of white rot fungi YK-624 on imidacloprid in potato dextrose liquid medium is 8.2%, and the degradation rate of imidacloprid in Kirk medium is 89.4%.
In other embodiments, other components, proportions or values are specifically selected within the ranges of components, proportions and process parameters described in the present invention, so that the technical effects described in the present invention can be achieved, and therefore, the technical effects are not listed one by one.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and although the present invention has been 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 to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (6)
1. The method for degrading imidacloprid by using white rot fungi is characterized by comprising the following steps:
(1) Subculture
White rot fungiPhanerochaete sordida Inoculating YK-624 to potato glucose agar culture medium for subculture;
(2) Stationary culture
Cutting out mycelium slices from a subcultured potato glucose agar medium, and inoculating the mycelium slices into a liquid medium, wherein the liquid medium is a potato glucose liquid medium or a Kirk liquid medium;
the Kirk liquid culture medium comprises the following components: per liter, comprising 10g glucose, 0.221g ammonium tartrate, 1.64g anhydrous sodium acetate, 1g Tween 80 and 100mL Kirk salt solution, wherein Tween 80 is added during shaking culture;
the Kirk salt solution comprises the following components: comprises 20g KH per liter 2 PO 4 ,5g MgSO 4 ·7H 2 O,1.3g CaCl 2 ·2H 2 O,0.01g thiamine hydrochloride, 16.7mL Kirk microelement solution;
the Kirk microelement solution comprises the following components: comprises 9g of trisodium nitrilotriacetate and 3g of MgSO per liter 4 ·7H 2 O,4.2g MnSO 4 ·H 2 O,6g NaCl,0.6g FeSO 4 ·7H 2 O,1.1g CoSO 4 ·7H 2 O,1.1g ZnSO 4 ·7H 2 O,0.6g CaCl 2 ·2H 2 O,0.06g CuSO 4 ·5H 2 O,0.11g AlK(SO 4 ) 2 ·12H 2 O,0.06g H 3 BO 3 ,0.07g Na 2 MoO 4 ·2H 2 O;
(3) Degradation culture
The white rot fungiPhanerochaete sordida After YK-624 was shake-cultured in the liquid medium for a set period of time, the imidacloprid-containing solution was added and the culture was continued.
2. The method for degrading imidacloprid by white rot fungi according to claim 1, wherein the potato dextrose agar medium comprises 20% of potato, 2% of glucose and 2% of agar by mass fraction.
3. The method for degrading imidacloprid by white rot fungi according to claim 1, wherein the diameter of the mycelium pellet is 10mm and the volume of the liquid medium is 10mL.
4. The method for degrading imidacloprid by white rot fungi according to claim 1, wherein the potato dextrose liquid medium comprises 20% of potato and 2% of dextrose by mass fraction.
5. The method for degrading imidacloprid by white rot fungi according to claim 1, wherein the stationary culture time is 3d.
6. The method for degrading imidacloprid by white rot fungi according to claim 1, wherein the degradation culture time is 20d.
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