CN113215015B - Efficient cyhalothrin degrading bacterium and application thereof - Google Patents
Efficient cyhalothrin degrading bacterium and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of pesticide pollution removal and bioremediation in the environment, and discloses a high-efficiency cyhalothrin degrading bacterium and application thereof. The efficient cyhalothrin degrading bacteria is preserved in China general microbiological culture Collection center (CGMCC) No.19810 on 5, 12 and 2020, the GenBank accession number of the strain 16SrDNA is MT320017, the strain is identified as ochrobactrum intraradicale, the bacteria are from soil polluted by the efficient cyhalothrin for a long time, and the strain is obtained by artificial enrichment, domestication, culture, separation and purification and is named as LB1. The bacterium is gram-negative bacterium, the thallus is short rod-shaped, the colony color is white, opaque, the surface is moist and smooth, the bulge is realized, the edge is neat, the picking is easy, and the most suitable growth condition is as follows: pH7.0-8.0, and temperature 25-30 deg.C.
Description
Technical Field
The invention belongs to the technical field of pesticide pollution removal and bioremediation in the environment, and particularly relates to screening of a high-efficiency cyhalothrin degrading bacterium and application of the high-efficiency cyhalothrin degrading bacterium in bioremediation treatment of polluted soil and water.
Background
Efficient cyhalothrin (Lambda-cyhalothrin), also known as cyhalothrin or cyhalothrin, is prepared by substituting methyl on the ethylene side chain of cyclopropane carboxylic acid of a photolabile chrysanthemic acid side chain with three fluorines and chlorines on the basis of the chemical structure of natural pyrethrin, so that the structure is more stable, the toxicity is stronger, the insecticidal spectrum is wider, and the pesticide effect is faster. Therefore, the insecticidal composition is widely applied to deinsectization of crops such as vegetables, cotton, tea, fruit trees and the like in China. The efficient cyhalothrin has good light stability under natural light irradiation, has strong adsorption affinity to plants and particles, and is easy to remain in large quantity in the environment. The residual and overproof phenomena of the lambda-cyhalothrin are common in China, the frequent excessive use of the lambda-cyhalothrin can increase the environmental risk of non-target organisms and can cause serious damage to a plurality of systems such as the movement system, the reproductive system, the respiratory system, the immune system and the like of human beings and other organisms.
The residual lambda-cyhalothrin in the environment can be removed by various ways such as chemical oxidation, adsorbent adsorption and biodegradation, and the removal by microbial degradation is considered to be the most economical and effective means. China is a large country with biological resources, the species, metabolism and ecological types of microorganisms are rich and various, and a large amount of bacteria, actinomycetes, fungi, algae and other microorganisms exist in the natural environment, so that sufficient resources are provided for screening strains. The efficient degradation strain is screened and separated from the long-term high-concentration pesticide-polluted soil, and then the efficient degradation strain and the indigenous microbial community synergistically act to transform and degrade the efficient cyhalothrin pesticide residue, so that the efficient degradation strain is an effective way for repairing the pesticide-polluted soil and water, and has wide application prospect due to the characteristics of high degradation efficiency, low cost, good ecological restoration, no secondary pollution and the like.
Disclosure of Invention
The invention aims to provide a high-efficiency cyhalothrin degrading bacterium and application thereof in an actual environment.
The efficient cyhalothrin degrading bacteria is preserved in China general microbiological culture Collection center (CGMCC) at 12.5.2020, with the preservation number of CGMCC No.19810 and the preservation address: no. 3 of Xilu No.1 of Beijing Korean Yang district, the GenBank accession number of the strain 16S rDNA is MT320017. The efficient cyhalothrin degrading bacteria is identified as Ochrobactrum intermedium, is obtained by artificial enrichment, domestication, culture, separation and purification of the efficient cyhalothrin degrading bacteria from soil polluted by the efficient cyhalothrin for a long time, and is named as LB1. The bacterium is gram-negative bacterium, the thallus is short rod-shaped, the colony color is white, opaque, the surface is moist and smooth, the bulge is realized, the edge is neat, the picking is easy, and the most suitable growth condition is as follows: pH7.0-8.0, and temperature 25-30 deg.C.
The separation method of the efficient cyhalothrin degrading bacteria comprises the following steps:
(1) And (3) screening strains with degradation effect on the lambda-cyhalothrin from the soil sprayed with the lambda-cyhalothrin for a long time by an enrichment domestication method. The specific method comprises the following steps: weighing 5g of soil sample, adding the soil sample into a culture bottle filled with 100mL of inorganic salt culture medium, and placing the culture bottle in a constant-temperature shaking incubator with the temperature of 30 ℃ and the rotating speed of 150r/min for culture. Wherein the inorganic salt culture medium (g/L): NH 4 NO 3 1.0g，NaCL 1.0g，KH 2 PO41.5g， K 2 HPO 4 0.5g，MgSO 4 0.1g，FeSO 4 0.025g and 1000mL of deionized water, and the pH is adjusted to 7.0.
(2) And (2) after the culture bottle is cultured for 5 days in the step (1), taking out and standing, taking 5mL of supernatant, transferring the supernatant into a culture bottle containing 100mL of an inorganic salt culture medium containing 50mg/L of efficient cyhalothrin, and placing the culture bottle under the constant temperature condition of 30 ℃ and 150r/min of rotation speed to perform oscillation enrichment culture by taking the efficient cyhalothrin as the only carbon source and energy source.
(3) And (3) repeating the process of the step (2), continuing the culture according to the method, and sequentially increasing the concentration of the lambda-cyhalothrin to 100mg/L, 150mg/L, 200mg/L, 250mg/L, 300mg/L and 400mg/L to finish the acclimatization process of the strain.
(4) Starting from the concentration of the lambda-cyhalothrin of 100mg/L, 0.5mL of culture solution obtained after domestication in each period is taken on a super clean bench, the culture solution is placed in a centrifuge tube with 4.5mL of sterile water to be shaken up, and the bacterial suspension is diluted into 10 -1 In this way, the dilution is performed in a gradient manner, and 10 is taken -4 -10 -7 And (3) coating the bacterial suspension under the gradient, wherein 0.2mL of the bacterial suspension is taken from each gradient, the bacterial suspension is coated on an LB plate culture medium which is separated and purified, each gradient is repeatedly coated for 3 times, and the coated plate is inversely placed in an incubator for culture. Selecting bacteria from bacterial colonies with different shapes, sizes and good growth, streaking and coating on a plate, separating and purifying the bacterial strains, transferring the obtained single bacterial colony to a test tube LB culture medium, culturing for 2-3d, and storing in a refrigerator at 4 ℃.
Respectively picking the thalli of the single bacterial strain preserved in the step (4) into 50mL beef extract peptone culture medium under the aseptic condition, carrying out oscillation activation culture under the constant temperature condition of 30 ℃ and 150r/min of rotation speed for 2d, then centrifuging the culture solution at 4000rmp for 10min, pouring out the supernatant, reserving the bottom thalli, adding the thalli into phosphoric acid buffer solution with the same volume as the culture solution, fully shaking up to prepare bacterial suspension (OD) 600 = 1.0), for standby. According to the inoculation amount of 10%, the bacterial suspensions are respectively added into 50mL of inorganic salt culture medium with the concentration of the high-efficiency cyhalothrin of 100mg/L, the medium is placed at 30 ℃ and cultured in a shaking table at a constant temperature of 150r/min, the degradation rate of each strain to the high-efficiency cyhalothrin is measured after 5d, and the strain with the highest degradation rate is screened out to serve as a target strain. The beef extract peptone medium comprises the following components (g/L): 3g of beef extract, 10g of peptone, 5g of sodium chloride and 1000mL of deionized water, and adjusting the pH value to 7.0-7.2. The phosphate buffer comprises the following components: KH (Perkin Elmer) 2 PO 4 8.5g、K 2 HPO 4 21.75g，Na 2 HPO 4 33.4g、NH 4 Cl 5g and deionized water 1000mL.
The invention relates to application of high-efficiency cyhalothrin degrading bacteria in degrading polluted water containing high-efficiency cyhalothrin. Preparation of bacterial suspension of the strain LB 1: selecting a small amount of LB1 thallus into beef extract peptone culture medium, placing into a constant temperature double-layer shaking incubator at 30 deg.C and 150r/min for culturing, centrifuging the culture solution at 4000rmp for 20min at low speed, pouring out supernatant, adding sterilized phosphoric acid buffer solution, shaking, centrifuging again, repeating the above steps for 3 times, and adjusting OD of the final bacterial suspension 600 =1.0, and the degradation rate of the efficient cyhalothrin in the water body after 5 days when the initial concentration of the efficient cyhalothrin is 100mg/L can reach 58.41%.
The inventionApplication of efficient cyhalothrin degrading bacteria in repairing contaminated soil containing efficient cyhalothrin. Preparing the strain into bacterial suspension OD 600 =1.0, and the degradation rate after 20 days for the initial concentration of the efficient cyhalothrin in the soil is 100mg/kg can reach 62.47%.
The invention relates to application of efficient cyhalothrin degrading bacteria in repairing soil polluted by organochlorine pesticides DDT and chlordane.
Detailed Description
Example 1
The invention relates to a separation method of high-efficiency cyhalothrin degrading bacteria, which screens bacterial strains with degrading effect on the high-efficiency cyhalothrin from soil sprayed with the high-efficiency cyhalothrin for a long time by an enrichment domestication method, and comprises the following steps:
(1) Weighing 5g of soil sample sprayed with the efficient cyhalothrin for a long time, adding the soil sample into a culture bottle filled with 100mL of inorganic salt culture medium, and placing the culture bottle in a constant-temperature oscillation incubator with the temperature of 30 ℃ and the rotating speed of 150r/min for culture. Wherein the inorganic salt culture medium (g/L): NH (NH) 4 NO 3 1.0g，NaCL 1.0g，KH 2 PO 4 1.5g，K 2 HPO 4 0.5g，MgSO 4 0.1g，FeSO 4 0.025g and 1000mL of deionized water, and the pH is adjusted to 7.0.
(2) And (2) after the culture bottle in the step (1) is cultured for 5 days, taking out and standing, taking 5mL of supernatant, transferring the supernatant into a culture bottle containing 100mL of 50mg/L efficient cyhalothrin inorganic salt culture medium, and placing the culture bottle under the constant temperature condition of 30 ℃ and 150r/min of rotating speed to perform oscillation enrichment culture by taking the efficient cyhalothrin as a unique carbon source and energy source.
(3) And (3) repeating the process in the step (2), continuing to culture according to the method, and sequentially increasing the concentration of the lambda-cyhalothrin to 100mg/L, 150mg/L, 200mg/L, 250mg/L, 300mg/L and 400mg/L to finish the acclimation process of the strain.
(4) Starting from the concentration of the lambda-cyhalothrin of 100mg/L, 0.5mL of culture solution obtained after domestication in each period is taken on a super clean bench, the culture solution is placed in a centrifuge tube with 4.5mL of sterile water to be shaken up, and the bacterial suspension is diluted into 10 -1 And the dilution is carried out by gradient by analogy, and 10 is taken -4 -10 -7 Gradient of gradientTaking 0.2mL of the bacterial suspension, coating each gradient on an LB plate culture medium which is separated and purified, repeating the coating for 3 times by each gradient, and inversely placing the coated plate in an incubator for culture. Selecting bacteria from bacterial colonies with different shapes, sizes and good growth, streaking and coating on a plate, separating and purifying the bacterial strains, transferring the obtained single bacterial colony to a test tube LB culture medium, culturing for 2-3d, and storing in a refrigerator at 4 ℃.
(5) Respectively picking the single bacterial strain preserved in the step (4) into 50mL beef extract peptone culture medium under the aseptic condition, carrying out oscillation activation culture under the constant temperature condition of 30 ℃ and 150r/min of rotation speed for 2d, taking the culture solution, centrifuging for 10min at 4000rmp, pouring out the supernatant, reserving the bottom bacterial strain, adding the bottom bacterial strain into phosphoric acid buffer solution with the same volume as the culture solution, and fully shaking up to prepare bacterial suspension (OD) 600 = 1.0), for standby. According to the inoculation amount of 10%, the bacterial suspensions are respectively added into 50mL of inorganic salt culture medium with the concentration of the high-efficiency cyhalothrin of 100mg/L, the medium is placed at 30 ℃ and cultured in a shaking table at a constant temperature of 150r/min, the degradation rate of each strain to the high-efficiency cyhalothrin is measured after 5d, and the strain with the highest degradation rate is screened out to serve as a target strain. The beef extract peptone medium comprises the following components (g/L): 3g of beef extract, 10g of peptone, 5g of sodium chloride and 1000mL of deionized water, and adjusting the pH value to 7.0-7.2. The phosphate buffer solution comprises the following components: KH (Perkin Elmer) 2 PO 4 8.5g、K 2 HPO 4 21.75g，Na 2 HPO 4 33.4g、NH 4 Cl 5g and deionized water 1000mL.
The efficient cyhalothrin degrading bacteria is preserved in China general microbiological culture Collection center (CGMCC) at 12.5.2020, with the preservation number of CGMCC No.19810, and the preservation address: no. 3 of Xilu No.1 of Beijing Korean Yang district, the GenBank accession number of the strain 16S rDNA is MT320017. The efficient cyhalothrin degrading bacteria are identified as Ochrobactrum intermedium, are gram-negative bacteria, are short rod-shaped, are white in colony color, are opaque, are wet and smooth in surface, are convex, are neat in edge and are easy to pick, and the most suitable growth conditions are as follows: pH7.0-8.0, temperature 25-30 deg.C
Example 2
The degradation performance of the ochrobactrum intermedius on the efficient cyhalothrin in the water body is as follows:
culturing Ochrobactrum intermedium in domesticated high efficiency degrading strain medium in beef extract peptone culture medium to prepare OD 600 Bacterial suspension = 1.0. Under the aseptic condition, inoculating 10% of bacterial suspension of a bacterial strain LB1 into a sterilized inorganic salt culture medium, then adding a standard solution of efficient cyhalothrin to ensure that the concentration of the efficient cyhalothrin in the final inorganic salt culture solution is 100mg/L, using the inorganic salt culture medium without inoculation as a blank experiment control for non-biological action degradation, setting 3 parallel samples, and carrying out all operations under the aseptic condition. And (3) placing the culture solution in a constant-temperature double-layer shaking incubator for culturing for 5d at the temperature of 30 ℃ at 150r/min, and sampling and determining the residual quantity of the lambda-cyhalothrin in the 1d, the 2d, the 3d, the 4d and the 5d respectively. The result shows that the Ochrobactrum intermedium (Ochrobactrum intermedium) has higher degradation performance on the lambda-cyhalothrin in the water environment, and the degradation rate of the lambda-cyhalothrin with the initial concentration of 100mg/L after 5 days can reach 58.41 percent.
Example 3
The degradation performance of the ochrobactrum intermedius on the efficient cyhalothrin in the soil is as follows:
the soil sample used in this example was taken from farmland soil not contaminated with lambda-cyhalothrin. The soil sample is crushed and sieved by a 100-mesh sieve, and is placed in a ventilated place to be dried for standby. Adding a standard solution of high-efficiency cyhalothrin into an inorganic salt culture medium to ensure that the concentration of the standard solution is 100mg/L, uniformly spraying the inorganic salt culture solution into a prepared soil sample to ensure that the concentration of the high-efficiency cyhalothrin in the soil is 100mg/kg, and dividing the soil sample into 6 parts. Taking 3 parts of the prepared soil sample, and respectively adding the prepared suspension of Ochrobactrum intermedium (OD) 600 = 1.0) so that the soil sample contains 100mL/kg degradation microbial inoculum, and the soil sample is fully and uniformly stirred, three repeated experiments are set, and three soil samples without bacteria but with the same volume of sterile water are used as blank controls for non-biological degradation. All the soil samples are placed in a ventilated place for curing for 20 days, a proper amount of sterile water is sprayed every 5 days to keep the soil samples moist, and after 20 days, the soil samples are taken for efficient chlorineAnd (4) measuring the residual quantity of the cyfluthrin. The result shows that the ochrobactrum intermedium has good degradation effect on the lambda-cyhalothrin in the soil, and when the bacterial dose is 100mL/kg, the degradation rate of the lambda-cyhalothrin with the initial concentration of 100mg/L in the soil can reach 62.47% after 20 d.
Example 4
The influence of each environmental factor on the effect of the ochrobactrum intraradicum on degrading the lambda-cyhalothrin is as follows:
selecting pH, temperature, inoculum size and an added carbon source as influencing factors, setting single-factor experiments with different gradients, and researching and analyzing the influence of different environmental factors on the performance of the ochrobactrum intermedium for degrading the lambda-cyhalothrin. The gradient of each factor was set to pH 5, 6, 7, 8 and 9, respectively; the temperature is 20 ℃, 25 ℃,30 ℃, 35 ℃ and 40 ℃; the inoculation amount is 6%, 8%, 10%, 12% and 14%; the carbon source is 3g/L soybean meal, 3g/L cyclodextrin and 3g/L glucose. The specific degradation procedure was the same as in example 2. The result shows that the optimum degradation condition of the ochrobactrum intermedium in the experimental range is pH7, the temperature is 25-30 ℃, the inoculation amount is 10%, and 3g/L glucose is added as a carbon source.
Example 5
The performance of the ochrobactrum intermedium for degrading organochlorine pesticides DDT and chlordane is as follows:
propagating the Ochrobactrum anthropi in the domesticated high-efficiency degradation strain medium in an activation culture medium to prepare a bacterial suspension (OD) 600 = 1.0). And adding 10mL of the bacterial suspension into a triangular flask filled with an inorganic salt mixed solution of 50mL LDDT and chlordane, culturing at the temperature of 30 ℃ and at the speed of 150r/min in a constant-temperature incubator, and inoculating no ochrobactrum ankanum in the other triangular flask filled with the inorganic salt mixed solution of DDT and chlordane as a control for non-biological degradation. Set up 3 replicates. After 30 days, sampling and analyzing the residual quantity of 2 organochlorine pesticides in the culture solution. The result shows that when the initial concentration of DDT and chlordane is 5mg/L, the degradation rates of DDT and chlordane reach 21.37 percent and 19.25 percent respectively after 30 days. This example illustrates that Ochrobactrum intermedium (Ochrobactrum intermedium) also has a good effect of degrading organochlorine pesticides.
Claims (4)
1. AThe strain of efficient cyhalothrin degrading bacteria is characterized in that the preservation number of the efficient cyhalothrin degrading bacteria is CGMCC No.19810, and the efficient cyhalothrin degrading bacteria is identified as ochrobactrum intracellulare (Xanthium falcatum) (A. Xanthium Chinense)Ochrobactrumintermedium)。
2. The use of the bacteria for degrading cyhalothrin according to claim 1, wherein the bacteria are used for degrading cyhalothrin contained in polluted water.
3. The use of a lambda-cyhalothrin-degrading bacterium according to claim 1, which is used for remedying a contaminated soil containing lambda-cyhalothrin.
4. The use of the lambda-cyhalothrin degrading bacteria of claim 1, which is characterized in that the application of the lambda-cyhalothrin degrading bacteria in restoring the soil polluted by the organic chlorine pesticide DDT and chlordane.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101096644A (en) * | 2006-06-26 | 2008-01-02 | 谢明 | Pseudomonas stutzeri JSD-008 and its degradation function for organophosphorus pesticide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101096644A (en) * | 2006-06-26 | 2008-01-02 | 谢明 | Pseudomonas stutzeri JSD-008 and its degradation function for organophosphorus pesticide |
Non-Patent Citations (4)
| Title |
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| Biodegradation of beta-cypermethrin and 3-phenoxybenzoic acid by a novel Ochrobactrum lupini DG-S-01;Shaohua Chen 等;《Journal of Hazardous Materials》;20110118;第433-440页 * |
| Brucella intermedia strain LB1 16S ribosomal RNA gene, partial sequence;Xu,H. 等;《GenBank Database》;20200415;Accession NO.MT320017 * |
| 氟氯氰菊酯降解菌的筛选与降解特性的研究;卫正 等;《生物技术通报》;20160929;第114-122页 * |
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