CN111004736B - Bacillus megaterium and application thereof in degrading pyrethroid insecticides - Google Patents
Bacillus megaterium and application thereof in degrading pyrethroid insecticides Download PDFInfo
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- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/04—Pesticides, e.g. insecticides, herbicides, fungicides or nematocides
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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
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- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
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- A62D2101/26—Organic substances containing nitrogen or phosphorus
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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
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- A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen
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- C—CHEMISTRY; METALLURGY
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/306—Pesticides
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/36—Organic compounds containing halogen
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
Abstract
The invention discloses bacillus megaterium and application thereof in degrading pyrethroid insecticides. The degrading strain is Bacillus megatherium (Bacillus megatherium) strain HLJ7, which is preserved in Guangdong province microorganism strain preservation center in 2019, 9, 4 days, and the preservation number is GDMCC No. 60760. The invention discloses the degradation effect of bacillus megaterium on pyrethroid insecticides such as allethrin for the first time, and the high-efficiency and rapid degradation strain HLJ7 is obtained by screening. Based on the invention, the bacillus megaterium has obvious biodegradation effect on pyrethroid insecticides such as allethrin, has high degradation efficiency and stable degradation performance, can tolerate higher-concentration pesticides, and can be used for bioremediation of polluted environments such as water and soil polluted by pyrethroid insecticides such as allethrin. The invention provides a new development approach for breaking the bottleneck of controlling pesticide residue pollution, enriches the germplasm resource library of pesticide degrading bacteria, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of pesticide pollution treatment. More particularly, relates to a Bacillus megalosporus strain HLJ7 for degrading pyrethroid pesticides such as allethrin and the like, and a microbial inoculum produced by the strain HLJ7 and application of the strain.
Background
The pyrethroid pesticide comprises allethrin and the like, the allethrin is a synthetic I-type pyrethroid formed by esterifying d-trans-first chrysanthemic acid and allyl ketone, and as the allethrin has the effects of killing, repelling and knocking down mosquitoes, most mosquito-repellent incense, aerosol, liquid spray and the like are taken as raw materials, and although some insects gradually generate drug resistance to the insecticide, the pyrethroid pesticide plays a vital role in controlling the spread of insect-borne diseases. With the increasing use range and dosage of allethrin, reports on the toxicological and environmental pollution effects of residues are increasing.
Allethrin inhibits the production of atpase, and allethrin residues are often detected in surface waters and foods. Allethrin is neurotoxic to rodents. Acute and chronic exposure to allethrin can damage tissues, including blood, nerves, fat, and other tissues. The allethrin remained in the air contacts corneal epithelial cells of human eyes to cause apoptosis through a mitochondrial pathway, and further causes negative influence on human eyesight. Numerous studies have shown that allethrin has a potential threat to the environment and even human health. Therefore, how to eliminate allethrin pesticide residues in the environment becomes a scientific research proposition which needs to be solved urgently by scientific researchers and has great economic and social significance.
The bioremediation technology is a new technology for degrading harmful pollutants in the environment into inorganic micromolecular compounds by using microorganisms or other organisms, has the advantages of high efficiency, safety, no residue, no secondary pollution and the like, and gradually becomes an optimal selection scheme for treating pesticide residue pollution.
However, the mineralization ability and the degradation performance of the pesticide by the microorganisms are unstable, so that the existing degrading bacteria resource library can not meet the actual requirement of the chemical pesticide residue pollution biodegradation. Particularly, no degradation preparation product specially aiming at allethrin exists at present.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art and provide a strain of pyrethroid pesticide degrading bacteria such as allethrin, namely Bacillus megalosporus (Bacillus megalosporus) HLJ 7. The strain can rapidly and efficiently degrade pyrethroid pesticides such as allethrin, has a degradation rate of more than 80% to allethrin of 50mg/L after being cultured for 11 days, and can be used for repairing the environments such as soil and water polluted by pesticide residues.
The invention aims to provide a Bacillus megatherium (Bacillus megalosporus) strain HLJ7 for efficiently degrading pyrethroid pesticides such as allethrin.
The invention also aims to provide application of the bacillus megaterium strain HLJ7 in degradation of pyrethroid pesticides such as allethrin.
The above purpose of the invention is realized by the following technical scheme:
the research of the invention finds that the Bacillus megaterium (Bacillus megalosporus) has good degradation effect on pyrethroid pesticides such as allethrin. And screening to obtain a high-efficiency degradation strain, namely a Bacillus megatherium (Bacillus megatherium) strain HLJ7, which is preserved in Guangdong province microorganism strain preservation center in 2019, 9, 4 days, wherein the preservation number is GDMCC No. 60760, and the preservation address is as follows: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.
The bacillus megaterium strain HLJ7 is derived from soil of suburban farmland of Harbin of Heilongjiang through artificial enrichment culture, separation and purification. The strain has efficient and rapid degradation efficiency on pyrethroid pesticides such as allethrin, the degradation rate of 50mg/L allethrin reaches more than 80% after the strain is cultured for 11 days, and the strain HLJ7 can be used as an excellent biodegradation bacterium to be applied to bioremediation of pyrethroid pesticide pollution such as allethrin.
Therefore, the following applications are also within the scope of the present invention:
application of Bacillus megatherium (Bacillus megatherium) in degrading pyrethroid pesticides such as allethrin or preparing degrading microbial inoculum of pyrethroid pesticides such as allethrin.
Application of Bacillus megatherium (Bacillus megatherium) in repairing natural environment of pyrethroid pesticides such as allethrin or preparing repairing microbial inoculum. The natural environment is water or soil.
Preferably, the bacillus megaterium is the bacillus megaterium strain HLJ7 described above.
Preferably, the pyrethroid pesticide is allethrin, permethrin, tetramethrin, permethrin or cypermethrin.
A microbial inoculum for efficiently degrading allethrin contains Bacillus megalosporus. Preferably, the bacillus megaterium is the bacillus megaterium strain HLJ7 described above.
The invention has the following beneficial effects:
the research of the invention finds that the bacillus megaterium has good degradation effect on pyrethroid pesticides such as allethrin, the high-efficiency and quick degradation strain HLJ7 is obtained by screening, the germplasm resource library of pesticide degradation bacteria is enriched, the degradation effect of the bacteria on pyrethroid pesticides such as allethrin is remarkable, the degradation rate of allethrin of 50mg/L reaches more than 80% after the bacteria are cultured for 11 days, and the strain HLJ7 has great development potential in the aspect of bioremediation of environment polluted by pyrethroid pesticides such as allethrin.
Drawings
FIG. 1 is a morphological characterization of strain HLJ 7.
FIG. 2 is a 16S rDNA phylogenetic tree of strain HLJ 7.
FIG. 3 is a standard curve of allethrin content versus peak area.
FIG. 4 is a high performance liquid chromatography peak of allethrin.
FIG. 5 shows the effect of an added mannitol carbon source on the growth of allethrin-degrading bacteria HLJ 7.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The media formulations described in the examples below are as follows:
inorganic salt culture medium: 2.0g (NH)4)2SO4；1.5g NaHPO4·12H2O；1.5g KH2PO4；0.2g MgSO4·7H2O；0.01g CaCl2·2H2O；0.001g FeSO4·7H2O; 1000mL of distilled water; pH 7.1.
Beef extract peptone solid medium: 10.0g peptone (typetone); 5.0g beef extract (yeast); 10.0g NaCl; 1000mL of distilled water; agar 1.5%.
Basic culture medium: 13.75g K2HPO4；4.50g KH2PO4；2g(NH4)2SO4(ii) a 2g of mannitol; 2g of glycerol; 0.2g MgSO4·7H2O；0.0132g CaCl2；0.009g FeSO4；0.003g MnCl2(ii) a 1000mL of distilled water.
All media were sterilized in an autoclave at 121 ℃ for 20 min.
Example 1 isolation, screening and identification of pesticide-degrading Strain HLJ7
1. Enrichment culture and separation screening of allethrin degrading bacteria:
collecting soil of suburban farmland of Harbin of Heilongjiang, weighing 5g of activated sludge sample, and adding into 50mL of the liquid basic culture medium containing allethrin (50 mg/L). After culturing for 7d at 30 ℃ and 200rpm, the mass concentration of the pesticide is increased from 50mg/L to 100mg/L, 200mg/L, 400mg/L and 800mg/L in turn according to the inoculation amount of 10% each time for continuous enrichment culture. The 4 transferred culture solution was then diluted in gradient and spread on LB solid plate containing 50mg/L allethrin, and cultured in 30 ℃ inverted for 2 d. After a single colony grows on the flat plate, selecting the single colony for a plurality of times of streaking and purifying, and separating to obtain a strain of bacteria with the number of HLJ 7.
2. Morphological identification of strain HLJ 7:
the strain HLJ7 is inoculated on a beef extract peptone medium plate and is subjected to inverted culture at 30 ℃ for 2d, and the colony morphology is observed. The bacterial colony cultured by beef extract peptone medium for 2d is in a faint yellow oval shape, large in bacterial colony, smooth in edge, soft in texture, non-sticky and easy to pick (see figure 1). The bacterium is a gram-positive bacterium, is aerobic and produces spores. The cells are rod-shaped, round-ended, single or in short-chain arrangement. Can liquefy gelatin, hydrolyzed starch, peptonized milk, and does not reduce nitric acid.
3. Molecular biological characterization of 16S rDNA of strain HLJ 7:
extracting genome DNA of the strain HLJ7, performing PCR amplification by using the extracted genome as a template and a 16S rDNA bacteria universal primer (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1429R: 5'-GGTTACCTTGTTACGACTT-3'), and entrusting a PCR product to Shanghai Yiwei Jie trading Limited company for sequencing. Comparing and analyzing the 16S rDNA sequence measured by the strain in a GenBank database by using BLAST, selecting related sequences with higher homology, constructing a phylogenetic tree by using Clustal 1.8.1 and MAGE 5.0 software, and analyzing the evolutionary relationship. As shown in FIG. 2, the 16S rDNA sequence of the strain HLJ7 obtained by separation and purification in the invention has the highest homology with Bacillus megaterium NBRC 15308 and the closest evolutionary distance.
Therefore, by combining morphological observation, physiological and biochemical characteristics and 16S rDNA sequence analysis, the strain HLJ7 is identified as Bacillus megatherium (Bacillus megalosporus) and is preserved in Guangdong province microorganism culture collection center in 2019, 9 and 4 days, wherein the preservation number is GDMCC No. 60760, and the preservation address is as follows: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.
Example 2 determination of the ability of Bacillus megaterium Strain HLJ7 to degrade allethrin
1. Experimental methods
(1) Preparing a seed solution:
the purified strain HLJ7 was inoculated into LB liquid medium containing 5mL overnight for activated culture to logarithmic phase, centrifuged at 4 ℃ and washed with physiological saline (0.9% NaCl) to obtain a cell, which was used as an inoculum.
(2) And (3) determining the degradation performance:
by 1.0X 107The inoculation amount of CFU/mL wet cells was inoculated into 50mL basal medium containing allethrin (50mg/L) and inorganic salt medium, respectively, and each set was repeated three times with no inoculation as a control. Shaking culturing at 30 deg.C and 200rpm for 11d, periodically sampling, and measuring OD with spectrophotometer600The values represent the growth of strain HLJ7, which was assayed for allethrin degradation by HPLC.
(3) Chromatographic conditions are as follows:
a model 2690 high performance liquid chromatograph (Water, USA) was used. The chromatographic column is C18Reversed phase column (Phenomenex,250nm × 4.60mm,5 μm), sample temperature of 28 deg.C, sample amount of 10 μ L, flow rate of 0.7mL/min, mobile phase of chromatographic acetonitrile: 80 parts of ultrapure water: 20, detection wavelength 254 nm.
The allethrin degradation rate was calculated according to the following formula:
wherein
In order to add the mass concentration of the residual pesticide in the bacterium group,
is the mass concentration of CK group pesticide residue, and the unit is mg/L.
(4) Quality control: and correcting the standard substance by adopting an external standard method to prepare a standard curve.
(5) And (3) determination of addition recovery rate, namely setting an addition recovery experiment, preparing a standard sample, adding allethrin into 20mL of sterile water to ensure that the mass concentration of the allethrin is respectively 5mg/L, 10mg/L, 25mg/L, 50mg/L and 100mg/L, adding 20mL of acetone into the sample, performing ultrasonic extraction for 30min, determining by HPLC (high performance liquid chromatography), and calculating the addition recovery rate.
2. The experimental results are as follows:
(1) standard curve of relationship between allethrin content and peak area: the content of allethrin has a good linear relation with the peak area. The standard curve equation of allethrin is: 2.6552X +88327 (R)20.9974). See fig. 3.
(2) Measurement of addition recovery rate the results of the test of addition recovery rate of allethrin in the standard sample are shown in table 1.
TABLE 1 recovery of allethrin addition from standard sample
The data analysis in the table shows that the addition recovery rate interval of the allethrin is 84.7-92.3%.
(3) Influence of additional carbon source on growth condition of allethrin degrading bacteria by OD of selected high-efficiency degrading strain HLJ7 in 1, 3, 5, 7 days under the condition of taking allethrin as sole carbon source and mannitol as additional carbon source in minimal medium600The values were measured to obtain an analysis chart as shown in FIG. 5. The data in the figure show that the added mannitol has a remarkable effect on allethrin degrading strain HLJ7, the added mannitol can increase the growth of degrading bacteria, the growth value of the strain is small and the increment is small in MSM taking allethrin as a unique carbon source, and the growth quantity of the degrading strain HLJ7 is obviously increased when the added mannitol is taken as a carbon source.
(4) The degradation effect of the allethrin degrading bacteria is as follows:
the results are shown in table 2, strain HLJ7 is able to rapidly degrade allethrin. In basal medium containing 50mg/L allethrin, strain HLJ7 grew rapidly without significant lag phase and entered the log phase of growth rapidly. After 5, 7, 9 and 11d of culture, the degradation rate of 50mg/L allethrin by the degrading strain HLJ7 reaches 48.6%, 58.3%, 69.4% and 82.3%, respectively, while the control (natural degradation rate) is only 8.6%.
The result shows that the strain HLJ7 can utilize allethrin as a growth substrate for growth and propagation, when the concentration of the allethrin is 50mg/L, the strain is cultured for 11 days, the degradation rate reaches 82.3%, and the strain has the capacity of efficiently and quickly degrading the allethrin.
TABLE 2 degradation of allethrin by the strain HLJ7
In addition, tests show that the bacillus megaterium strain HLJ7 has good degradation effect on other pyrethroid pesticides such as permethrin, tetramethrin, permethrin and cypermethrin.
Example 3 determination of the Effect of Bacillus megaterium Strain HLJ7 on degrading allethrin in soil
1. Soil sample for testing
The farmland topsoil (3-10 cm) is taken from the teaching farm test field of southern China agricultural university, belongs to red loam, and does not apply allethrin pesticide for more than 3 years.
Taking back the soil sample, naturally drying in the shade and ventilated place, grinding, sieving with a 2mm sieve, respectively dissolving a certain amount of allethrin in acetone, and soaking in diatomite to make the allethrin be completely adsorbed. And drying the soaked diatomite in a fume hood, and mixing the diatomite into the soil to ensure that the final concentration of allethrin in the soil is 50 mg/kg. 500g of soil sample is taken to be cultured in a constant temperature and humidity incubator at 30 ℃, and the culture is carried out according to the proportion of 1.0 multiplied by 107The inoculation amount of CFU/mL is inoculated into HLJ7 degradation bacterial suspension, and the water holding capacity of soil is kept at 40% by taking distilled water as a control. Continuously culturing for 15 days at 30 ℃ in the dark, periodically sampling, measuring the residual quantity of the allethrin by an HPLC method and calculating the degradation rate. The degradation rate was calculated as in example 2.
2. The measurement results are shown in table 3, and after the strain HLJ7 is cultured for 15 days, the degradation rate of allethrin in the soil can reach 77.1%.
TABLE 3 Effect of the Strain HLJ7 in degrading allethrin in soil
The results show that strain HLJ7 shows no non-degradation or degradation hysteresis effect after direct application into soil.
Claims (5)
1. A strain for efficiently degrading pyrethroid pesticides is a Bacillus megaterium (HLJ 7) strain, which is preserved in Guangdong province microorganism strain collection center in 2019, 9, 4 days, with the preservation number being GDMCC No. 60760 and the preservation address being as follows: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.
2. Use of Bacillus megaterium (Bacillus megalosporus) strain HLJ7 for degrading pyrethroid or preparing pyrethroid pesticide degrading bacteria according to claim 1, wherein the pyrethroid pesticide is allethrin, esfenpropathrin, tetramethrin, permethrin or cypermethrin.
3. The use of Bacillus megaterium (Bacillus megalosporus) strain HLJ7 for remediating the natural environment of pyrethroid pesticide residues or preparing a remediating microbial inoculum according to claim 1, wherein the pyrethroid pesticide is allethrin, esfenpropathrin, tetramethrin, permethrin or cypermethrin.
4. Use according to claim 3, wherein the natural environment is a body of water or soil.
5. An agent for efficiently degrading pyrethroid pesticide, which is characterized by comprising Bacillus megatherium (Bacillus megalosporus) strain HLJ7 as claimed in claim 1, wherein the pyrethroid pesticide is allethrin, esfenthrin, tetramethrin, permethrin or cypermethrin.
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| CN105408468A (en) * | 2013-03-15 | 2016-03-16 | 斯波根生物技术公司 | Plant growth-promoting bacteria and methods of use |
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