CN107699523B

CN107699523B - Bacterium capable of degrading pesticides buprofezin and bifenthrin and microbial inoculum produced by bacterium

Info

Publication number: CN107699523B
Application number: CN201711069143.8A
Authority: CN
Inventors: 闫新; 陈雪婷; 纪俊宾; 洪青; 何健; 蒋建东
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2017-11-03
Filing date: 2017-11-03
Publication date: 2020-06-05
Anticipated expiration: 2037-11-03
Also published as: CN107699523A; CN111394274B; CN111394274A

Abstract

The invention belongs to the field of environmental pollution bioremediation, and discloses a bacterium capable of stably degrading pesticides buprofezin and bifenthrin and a microbial inoculum produced by the bacterium, wherein the bacterium is preserved in China center for type culture collection in 2017, 9 and 25 months, and the preservation number of the strain is CCTCC NO. M2017537. The strain is gram-positive strain D-6, and is identified as Rhodococcus sp. The degrading strain D-6 can be used for degrading buprofezin and bifenthrin pesticides, the residual quantity of buprofezin in soil can be reduced by more than 95% in a short time, the residual quantity of bifenthrin in soil can be reduced by more than 70% in a certain time, the degrading property of the degrading strain D-6 is more stable than that of a reported degrading strain Rhodococcus sp.YL-1, and the degrading strain D-6 can be used for removing residual pollution of buprofezin and bifenthrin in the environment.

Description

Bacterium capable of degrading pesticides buprofezin and bifenthrin and microbial inoculum produced by bacterium

Technical Field

The invention belongs to the field of biological high technology, and discloses a bacterium capable of stably degrading pesticides buprofezin and bifenthrin and a microbial inoculum produced by the bacterium.

Background

Chemical pesticides play an important role in the comprehensive treatment of diseases, insects and weeds of crops, greatly promote the development of modern agriculture, but also bring about serious environmental problems. Firstly, the quality of agricultural products is reduced and the export is limited due to the overproof pesticide residue; secondly, pesticide residues can be enriched through a food chain, so that the life health of human beings is threatened; in addition, pesticide residues harm non-target organisms, indirectly change the structure of an ecosystem and destroy the functions of the ecosystem.

Buprofezin is an efficient broad-spectrum triazine pesticide, and has obvious control effect on pests such as plant hoppers, leafhoppers, whiteflies and the like. The problem of over standard buprofezin residue is frequently reported since the past 90 s widely used. Buprofezin has a half-life of 50 to 70 days in aerobic fields and 36 to 104 days in flooded rice fields. The buprofezin residue in the environment has serious harm to aquatic organisms and some non-target beneficial insects, is easy to be absorbed by human bodies through oral cavities, skin surfaces and respiratory tracts, and poses potential threat to human health. Bifenthrin is a high-efficiency pyrethroid insecticide and is widely applied to agricultural production and urban environment. Bifenthrin is not only seriously harmful to aquatic organisms, but also has potential harm to human health. Bifenthrin was listed in 2009 by the World Health Organization (WHO) in the list of secondary (toxic) industries.

Because the buprofezin and the bifenthrin have good control effect on tea small green leaf mites in tea trees and the non-standard use of pesticides by farmers leads to the compound pollution of the two pesticides in the tea garden soil. Pesticide residues in the soil enter a water body through rainwater washing, so that the environment is polluted, and potential threat to human health is caused. The bioremediation technology based on microbial degradation has the characteristics of safety, effectiveness, low price, no secondary pollution and wide application prospect. The microorganism capable of simultaneously degrading buprofezin and bifenthrin has more advantages in application. The structures of the buprofezin and the bifenthrin are similar, only Rhodococcus sp.YL-1 can be simultaneously degraded, but the degradation genes are easy to lose, the degradation characters are unstable, large-scale industrial production is not facilitated, and the repair of the buprofezin and the bifenthrin residues in the environment by using the bacterium is restricted. Therefore, it is necessary to continuously screen strains which can degrade the two pesticides simultaneously and have stable degradation properties to realize large-scale industrial production of the microbial inoculum and realize restoration of the two pesticide residues in the environment.

Disclosure of Invention

The invention aims to solve the problems, develop and develop a pesticide residue degradation microbial inoculum with stable properties, and use the microbial inoculum can reduce the residual quantity of buprofezin by more than 95 percent and the residual quantity of bifenthrin by more than 70 percent.

The technical scheme comprises the following main contents:

the invention provides a degrading bacterium for eliminating buprofezin and bifenthrin, wherein a bacterial strain is gram-staining reaction positive bacterium D-6 (deposited in a typical culture collection center in 2017 at 9 and 25 months, and the strain collection number is CCTCC NO. M2017537), and is identified as Rhodococcus sp. The main biological characteristics are gram positive staining, no spore and short rod shape of cell. The colony morphology was light pink opaque, smooth edged, raised, thick in texture and wet. No movement and good oxygen. 50 mg.L in 48h under the condition of laboratory shake flask^-1The degradation rate of the buprofezin is up to more than 95 percent, and 50 mg.L in 72 hours^-1The degradation rate of the bifenthrin is more than 70 percent. The degradation character of the strain D-6 is more stable than that of a reported buprofezin and bifenthrin degradation strain Rhodococcus sp.YL-1, the loss rate of the degradation character of the strain YL-1 is 1% after the strain YL-1 is continuously passaged for 3 times in an LB culture medium, the loss rate of the degradation character of the strain is 15% after the strain YL-1 is continuously passaged for 10 times in the LB culture medium, and the strain D-6 does not have a bacterial colony with the lost character after the same number of the passages, so that the strain D-6 has more advantages in fermentation industrial production.

The process for producing the microbial inoculum by using the buprofezin and bifenthrin degradation strain D-6 comprises the following steps: slant seeding-shake flask seed liquid-seeding tank-product (packing bacterial agent is liquid bacterial agent or solid adsorption bacterial agent).

The detailed implementation steps of the invention are as follows:

1. inoculating test tube slant strains of the buprofezin and bifenthrin residue degrading strain D-6 into a shake flask containing an LB culture medium, and performing shake culture to a logarithmic phase growth phase;

2. inoculating the cultured strain into a 500L seeding tank according to the inoculation amount of 10%, and culturing to logarithmic phase. The culture medium formula of the seeding tank is as follows: glucose 0.8%, (NH)₄)₂

SO

₄1％，K₂HPO₄0.2％，MgSO₄0.05％，NaCl 0.01％，CaCO₃0.3 percent of yeast extract, 0.02 percent of yeast extract and 7.2 to 7.5 of pH value.

3. Inoculating the seed liquid into a production tank for culture in an inoculation amount of 10%, wherein the culture medium of the production tank is the same as that of the seeding tank.

In the production process of the seeding tank and the production tank, the ventilation quantity of sterile air is 1:0.6-1.2 in volume ratio, the stirring speed is 180-. After fermentation is finished, the number of thalli reaches 10 hundred million/mL, and after fermentation is finished, culture solution is directly taken out of a tank and is subpackaged into liquid formulations by using a plastic packaging barrel or a packaging bottle or into solid microbial inoculum formulations by using a packaging bag for peat adsorption.

Advantageous effects

The invention provides a strain D-6 capable of efficiently, quickly and stably degrading buprofezin residue. 50 mg.L within 48h^-1The degradation rate of the buprofezin is up to more than 95 percent, and 50 mg.L in 72 hours^-1The degradation rate of the bifenthrin is as high as more than 70 percent, and the degradation character of the bifenthrin is more stable than that of the reported bacterial strain Rhodococcus qinshengi YL-1, so the bifenthrin has wide application potential and value.

The microbial inoculum has the advantages of low production and use cost, convenient use and good removal effect, and is suitable for large-area popularization and use in national grain, oil and vegetable production and export bases or places with green trademark marks. The invention has important significance for protecting ecological environment, protecting the health of people and improving the added value of agricultural products. The invention successfully solves the problem that pesticide residue exceeds the standard in agricultural production, not only exerts the high-efficiency and quick action of chemical pesticide in the prevention and treatment of plant insect pests, but also can produce non-toxic and pollution-free green agricultural products.

Biological material preservation information

D-6, is classified and named as Rhodococcus sp.D-6, the preservation unit is China center for type culture Collection, the preservation number of the strain is CCTCC NO.M2017537, the preservation date is 2017, 9 and 25 days, and the preservation address is Wuhan university, Wuhan, China.

Drawings

FIG. 1 photograph showing colony morphology of Strain D-6

FIG. 2 growth degradation curve of strain D-6 against buprofezin

FIG. 3 HPLC-MS spectrum of product generated by degrading buprofezin (A) and bifenthrin (B) with strain D-6

FIG. 4 Effect of temperature on the degradation of buprofezin by Strain D-6

FIG. 5 Effect of pH on the degradation of buprofezin by Strain D-6

FIG. 6 Effect of initial concentration of Buprofezin on Buprofezin degradation by Strain D-6

Detailed Description

Example 1 isolation and characterization of the strains

The invention provides a strain capable of degrading buprofezin and bifenthrin with stable properties and high efficiency and a microbial inoculum D-6 produced by the strain, which is separated from soil polluted by buprofezin for a long time in Jiangsu province. The screening method of the strain comprises the following steps: 5g of soil sample was added to 100mL of a solution containing 50 mg. L^-1The flask of the basal salt medium of buprofezin (hereinafter abbreviated as BMM) was cultured in a shaker at 30 ℃ and 150rpm, and after 5 days, the medium was transferred to a flask containing fresh BMM in an inoculum size of 5%, and enrichment culture was continuously carried out three times. Diluting the enrichment solution of the last passage, coating the enrichment solution on a BMM solid culture medium, culturing for 5 days at 30 ℃, and then picking out a single colony forming a transparent ring to culture in an LB test tube filled with 4 mL. After the thalli grow to be thick, one part of thalli is used for storing, and the other part of thalli is used for detecting degradation activity so as to obtain a buprofezin degradation strain. The method for detecting the degradation of buprofezin comprises the steps of transferring the thalli into a shake flask filled with 20mL of BMM culture medium, culturing for 5 days in a shaking table at 30 ℃ and 150rpm, extracting with dichloromethane with the same volume, and detecting the degradation effect by an ultraviolet spectrophotometer. The bifenthrin degradation detection method comprises transferring the thallus into a container containing 20mL of bifenthrin with a concentration of 50 mg.L^-1After culturing in a shake flask of a bifenthrin basic salt culture medium for 3 days at 30 ℃ and 150rpm, observing the color change of the culture medium, extracting with ethyl acetate of equal volume, and detecting the degradation effect by UHPLC-MS.

The strain D-6 is identified as Rhodococcus sp, and deposited in China center for type culture Collection in 2017, 9, 25 months, with the preservation number of CCTCCNo. m 2017537. The morphological characteristics of the growth of the strain D-6 on an LB plate are light pink, opaque, smooth edge, uplifted, viscous and moist, and compared with the reported buprofezin and bifenthrin degradation strain YL-1, the colony of the strain D-6 is lighter in color, larger in shape and moist in texture (FIGS. 1A and 1B). The main biological characteristics are gram positive (fig. 1C), inability to produce spores, inability to move aerobic bacteria; the methyl red test, the starch hydrolase test, the oxidase test and the nitrate reductase test are all negative; the V.P. test, the urease test and the catalase test are all positive. The 16S rRNA gene sequence of the strain D-6 was analyzed by alignment in the database EzBilCloud, and the result showed that the strain D-6 had the closest relationship with the genus Rhodococcus, among which Rhodococcus guinshengii JCM 15477^TThe similarity of (c) is 100%. According to colony morphology, physiological and biochemical characteristics and evolutionary developmental analysis of 16S rRNA gene sequences, the strain D-6 is preliminarily identified as Rhodococcus sp.

Example 2 laboratory degradation experiments

2.1 seed liquid preparation

Picking single colony activated on the plate, inoculating in LB culture medium, placing in shaker at 30 deg.C and 150rpm, and culturing to OD₆₀₀1.0. Centrifuging to collect thallus (4 deg.C, 6000rpm for 5min), washing twice with basal salt culture medium, and resuspending thallus to OD₆₀₀And the concentration is approximately equal to 2.0, and the result is the seed liquid.

2.2 degradation of buprofezin by Strain D-6

The seed solution of the strain D-6 was inoculated into BMM at an inoculum size of 2% and incubated for 48h at 30 ℃ on a shaker at 150 rpm. Taking 3mL of culture solution every 8h, detecting by HPLC, calculating the concentration and degradation rate of the residual buprofezin in the culture medium, diluting the regularly sampled culture solution and coating the LB solid culture medium, calculating the growth amount of thalli, and drawing a growth degradation curve of the strain D-6 to the buprofezin (as shown in figure 2). The strain D-6 can grow by taking buprofezin as a unique carbon source, and 50 mg.L can be grown in 48h^-1The buprofezin is degraded by more than 95 percent. FIG. 3A is an HPLC-MS spectrum of the product generated by degrading buprofezin by the strain D-6.

2.3 Effect of temperature on the degradation of buprofezin by Strain D-6

The seed solution of the strain D-6 was inoculated into BMM at an inoculum size of 2%, and cultured in shaking tables set uniformly at 150rpm and at 20 ℃, 25 ℃, 30 ℃ and 37 ℃ respectively (co-culture for 48 hours). Taking 3mL of culture solution every 8h, detecting by HPLC, calculating the concentration and degradation of the residual buprofezin in the culture medium, and determining the influence of the temperature on the degradation of the buprofezin by the strain D-6. As shown in FIG. 4, the strain D-6 has the highest degradation rate on buprofezin at 30 ℃, the optimal range of degradation is 25-30 ℃, the degradation efficiency can be influenced at low temperature (20 ℃) or high temperature (37 ℃), and the degradation efficiency is obviously influenced at high temperature (37 ℃).

2.4 Effect of initial pH on the degradation of buprofezin by Strain D-6

The pH of MSM was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 with HCl and NaOH, respectively. Adding 50 mg-L of basic salt culture medium with different pH values^-1The buprofezin is prepared by respectively inoculating the seed liquid into the culture medium with the pH of 4.0-10.0 according to the inoculation amount of 2%, and culturing for 48 hours in a shaking table at the temperature of 30 ℃ and the speed of 150 rpm. Taking 3mL of culture solution every 8h, detecting by HPLC, calculating the concentration and degradation of the residual buprofezin in the culture medium, and determining the influence of pH on the degradation of the buprofezin by the strain D-6. As shown in FIG. 5, strain D-6 showed the highest efficiency of degrading buprofezin at pH 7.0; when the pH is 5.0 and 9.0, the degradation activity of the compound has certain influence; when the pH is 4.0, the degradation efficiency of the strain D-6 is remarkably reduced, which shows that the degradation efficiency of the strain D-6 on the buprofezin in an alkaline environment is better than that in an acidic environment.

2.5 Effect of initial concentration of Buprofezin on degradation of Buprofezin by Strain D-6

Respectively inoculating the seed liquid with the inoculation amount of 2% to the buprofezin with the final concentration of 30 mg.L^-1，50mg·L^-1，100mg·L^-1And 200 mg. L^-1The basic salt medium of (4) was cultured for 48 hours at 30 ℃ on a shaker at 150 rpm. Taking 3mL of culture solution every 8h, detecting by HPLC, calculating the concentration and degradation of the residual buprofezin in the culture medium, and determining the influence of the initial concentration of the buprofezin on the degradation of the buprofezin by the strain D-6. As shown in FIG. 6, when the initial concentration of buprofezin is less than 50 mg.L^-1In the process, the degradation efficiency of the strain D-6 to the buprofezin can reach more than 95 percent within 48 hours; when the initial concentration of buprofezin is 100 mg.L^-1When the strain D-6 is used, the degradation efficiency of the strain D-6 to the buprofezin is reduced to 50% within 48 hours; when the initial concentration of buprofezin is 200 mg.L^-1In 48 hours, the degradation efficiency of the buprofezin is only 30 percent, which shows that the buprofezin with high concentration has an inhibiting effect on the degradation activity of the strain D-6.

2.6 degradation of Strain D-6 vs

The seed solution of the strain D-6 was inoculated at an inoculum size of 2% to a solution containing 50 mg.L^-1The bifenthrin is cultured in a basal salt culture medium at 30 ℃ and 150rpm in a shaking table. After 72h of incubation, a clear color change was observed in the culture broth, and the treated group turned yellow compared to the control group. FIG. 3B is an HPLC-MS spectrum of bifenthrin degradation product of strain D-6. And calculating the degradation efficiency of the strain on the bifenthrin by detecting the concentration of the residual bifenthrin in the culture solution. The strain D-6 can degrade more than 70% of bifenthrin within 72 hours.

Example 3 degradation stability experiment

Since the reported degradation trait of Rhodococcus qinshengi YL-1(CCTCC AB 2017132) buprofezin is unstable, it is necessary to evaluate the stability of the degradation trait of the strain D-6. Selecting fresh activated single colony forming transparent circle on BMM plate of strain D-6 and strain YL-1, inoculating in test tube containing 4mL LB culture medium, placing in shaker at 30 deg.C and 150rpm, and culturing to OD₆₀₀1.0. The resulting suspension was transferred to another tube containing 4mL of LB medium at an inoculum size of 1%, and cultured under the same culture conditions to OD₆₀₀1.0, this is passage 1. The bacterial suspension of the 1 st passage was diluted by an appropriate factor (10)^-4～10^-6) And coating a BMM (BMM) plate, and counting the percentage of the colony number which can not form a transparent ring to the total colony number after the colony grows out. The 10 passages were performed sequentially, and the percentage of colonies that failed to form clear circles was counted for each passage. Statistical results show that after the strain YL-1 is continuously passaged for 3 times in LB, the strain can be observed to be incapable of forming on a BMM flat plate coated with the diluted strain liquidThe bacterial colony of the transparent ring accounts for 1% of all the bacterial colonies, the proportion is increased along with the increase of the number of passages, and after 10 times of continuous passages, the degradation character loss rate of the bacterial strain YL-1 can reach 15%; in contrast, after 3 serial passages of the strain D-6 in LB, no colonies which could not form a transparent circle were observed on the BMM plate coated with the diluted strain, and the passage of the strain D-6 was continued. After the 10 th passage, no colonies that could not form a transparent circle were observed on the BMM plates coated after the dilution of the bacterial solution. Whether the bacterial colonies of the two strains can form transparent rings on a BMM solid culture medium or not and whether the buprofezin can be degraded exist a one-to-one correspondence relationship, so that the buprofezin degradation character of the strain D-6 is more stable.

Example 4 soil degradation experiment

The soil sample to be tested is collected from the soil of the farmland below the feet of the Zijin mountain and sieved by a 2mm sieve for later use. The diatomite is soaked in methanol mother liquor of buprofezin or bifenthrin, so that the pesticide is completely absorbed by the diatomite. Placing the soaked diatomite in a fume hood to be volatilized to dryness, and then stirring the diatomite into prepared field soil to ensure that the concentration of buprofezin and bifenthrin in the soil sample is respectively 10 mg-kg^-1. Each soil sample was 500g in mass, maintained at 35% water content, and incubated at 30 ℃ in the dark. Inoculating seed liquid into soil sample, and mixing to make the concentration of strain D-6 in soil reach 10⁸Each cell per gram of soil, soil without inoculation was used as a control. After 20d of incubation, the residual amounts of buprofezin and bifenthrin in the soil samples were measured by HPLC. The determination result shows that the strain D-6 has the degradation efficiency of 95.6 percent on the buprofezin residual in the soil and 73.5 percent on the bifenthrin residual in the soil.

Example 5 preparation of microbial inoculum

The stock of the buprofezin and bifenthrin degrading strain D-6 is activated on a culture dish, the degradation performance is measured, and the stock is inoculated on the inclined plane of a test tube for standby. The test tube seed was inoculated into a 1000mL shake flask containing 200mL LB medium (LB medium formulation: peptone 10g, yeast powder 5g, sodium chloride 5g, water 1L, pH 7.4), and shake-cultured at constant temperature until logarithmic phase, to prepare for inoculation of the first-stage seed tank. 50L of first-level seeding tank, 40L of material feeding amount and culture medium formulaComprises the following steps: glucose 0.8%, (NH)₄)₂SO₄1％，K₂HPO₄0.2％，MgSO₄0.05％，NaCl 0.01％，CaCO₃0.3 percent, 0.02 percent of yeast extract and 7.2 to 7.5 of pH value. After the feeding is finished, high-pressure damp-heat sterilization is carried out at 121 ℃, after the temperature is cooled to 30 ℃, the cultured shake flask strain is inoculated into a 50L first-class seed tank according to the inoculation amount of 10 percent, the shake flask strain is cultured to the logarithmic phase, the stirring speed is 220 r/min, and the introduction amount of sterile air is 1: 0.6-1.2. Inoculating the seed liquid reaching logarithmic phase into a secondary seed tank according to the inoculation amount of 10%. 500L of secondary seed tank, 400L of material feeding amount, and the formula and culture conditions of the culture medium are consistent with those of the primary seed tank. Inoculating the seed liquid reaching logarithmic phase into a production tank according to the inoculation amount of 10% for culture, wherein the culture medium composition of the production tank is the same as that of a seed tank. The capacity of the production tank is 5 tons, and the feeding amount is 4.5 tons. And (3) carrying out high-pressure damp-heat sterilization at 121 ℃ under the pressure of 1.1kg/cm2 in the production tank after feeding, cooling to 30 ℃ after sterilization, and introducing sterile air to keep a sterile state for later use. The temperature of the production tank after inoculation is controlled at 30 ℃, the ventilation quantity of sterile air in the culture process of the production tank is 1:0.6-1.2, the stirring speed is 180-. The number of the thalli after fermentation is up to 10 hundred million/mL.

After fermentation, the culture solution is directly taken out of the tank and is subpackaged into liquid dosage forms by using a plastic packaging barrel or a packaging bottle or into solid microbial inoculum dosage forms by adopting a packaging bag for peat adsorption.

Claims

1. A degrading strain Rhodococcus (Rhodococcus sp.) D-6 of pesticide buprofezin and bifenthrin is characterized in that the strain is preserved in China center for type culture collection in 2017, 9 and 25 months, and the preservation number of the strain is CCTCC number M2017537.

2. Use of the degrading strain Rhodococcus (Rhodococcus sp.) D-6 of claim 1 for degrading pesticides buprofezin and bifenthrin.

3. Use of the degrading strain Rhodococcus (Rhodococcus sp.) D-6 of claim 1 for preparing a fungicide for degrading pesticides buprofezin and bifenthrin.