CN112779194A

CN112779194A - Gordoniella alkalophaga and application thereof in degrading insecticide imidacloprid

Info

Publication number: CN112779194A
Application number: CN202110280182.2A
Authority: CN
Inventors: 葛峰; 程茜; 戴亦军
Original assignee: Nanjing National Environmental Research Institute Co ltd; Nanjing Normal University
Current assignee: Nanjing National Environmental Research Institute Co ltd; Nanjing Normal University
Priority date: 2021-03-16
Filing date: 2021-03-16
Publication date: 2021-05-11
Anticipated expiration: 2041-03-16
Also published as: CN112779194B

Abstract

The invention discloses Gordonia alkalophaga and application thereof in degrading imidacloprid serving as a nicotine insecticide, wherein the Gordonia alkanivorans strain is Gordonia alkanivorans C403 which is preserved in the China general microbiological culture Collection center, and the preservation numbers are as follows: CGMCC No. 21704. Compared with the prior pesticide degradation technology, the Gordonia alkalophaga CGMCC No.21704 of the new strain can effectively degrade the nicotine pesticide imidacloprid in resting cells, can be used as a pesticide degradation microbial inoculum, and has good application prospect.

Description

Gordoniella alkalophaga and application thereof in degrading insecticide imidacloprid

Technical Field

The invention belongs to the technical field of microbial biology, and particularly relates to gordonia alkalophaga and application thereof in degrading neonicotinoid insecticides.

Background

Imidacloprid is a nitro methylene systemic insecticide, belonging to chloronicotinyl insecticides, also known as neonicotinyl insecticides. The insecticidal composition is mainly suitable for various crops such as rice, wheat, vegetables, fruit trees and the like, and is mainly used for preventing and controlling various sucking mouthparts pests such as aphids, leafhoppers, thrips, trialeurodes vaporariorum and the like. Environmental homing data shows that imidacloprid has a long half-life in soil and that only a small fraction of imidacloprid is absorbed by the crops after application and the vast majority will enter the soil or penetrate into the surface water. Studies have shown that imidacloprid residues have a great toxicity to non-targeted insects, aquatic organisms, birds, mammals and other organisms, and can cause death of the organisms through calcium ion imbalance, mitochondrial dysfunction and DNA damage. The imidacloprid residue in the ecosystem enters a food chain along with food and water, and has great influence on the life health of human beings. The presence of imidacloprid in drinking water was found by the Klarich et al survey and demonstrated their general persistence during conventional water treatment. Thus, removal of imidacloprid residues from ecosystems has attracted public attention on a global scale, and it is of great importance to find a simple method for removing imidacloprid from surface and groundwater and removing it from contaminated aqueous wastewater.

There are many researches on the degradation of imidacloprid at home and abroad, mainly including three methods: the physical method comprises the following steps: ultrasonic techniques, washing, ionizing radiation, etc.; a chemical method: oxidative decomposition, adsorption, hydrolysis, light engineering and the like: ③ the biological method: microbial degrading enzyme, engineering bacteria and the like. Among them, microbial metabolism is the most effective, least harmful, and environmentally friendly removal method, and can be applied to bioremediation of contaminated soil. It has been reported that various strains have been demonstrated to transduce imidacloprid, such as Stenotrophomonas maltophilia CGMCC 1.1788, Pseudomonas putida microbial Z-4, Salmonella heterotrimenter species CGMCC 16346, Pseudoxanthomonas indica CGMCC 6648, Pseudomonas sp.1G, Klebsiella pneumoniae BCH1, Leifsoniasp, PC-21, Aspergillus terreus strain YESM, and the like. However, no report and literature on degrading the neonicotinoid insecticide imidacloprid by Gordonia alkalophaga is found.

Disclosure of Invention

The invention aims to provide Gordoniaalkali and application thereof in degrading nicotine pesticide imidacloprid.

The invention provides Gordonia, which is classified and identified as Gordonia alkanivorans (Gordonia alkannivorans) with the strain name of C403, and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of: CGMCC No. 21704.

The culture method of the Gordonia alkalophaga comprises the following steps: inoculating the Gordonia alkalophaga CGMCC No.21704 on an LB agar culture medium, and culturing in an incubator at the temperature of 30 +/-1 ℃; or inoculating into LB liquid culture medium, and shake culturing at 30 + -1 deg.C in shaking table.

The invention also provides application of the Gordonia alkanivorans (Gordonia alkannivorans) CGMCC No.21704 in degrading neonicotinoid insecticides.

The neonicotinoid insecticide is imidacloprid.

The preferred application method is as follows: inoculating the Gordonia alkalophaga CGMCC No.21704 in a nutrient culture solution, performing shake culture, and then performing centrifugal collection and washing on thalli; and suspending the thallus in phosphate buffer solution containing imidacloprid or surface water, performing shaking culture, and degrading the imidacloprid by Gordonia alkalophaga CGMCC No. 21704.

The nutrient culture solution is preferably LB culture medium.

Compared with the prior art, the new strain Gordonia alkalophaga CGMCC No.21704 has the advantages that resting cells can effectively degrade the nicotine pesticide imidacloprid, can be used as a pesticide degradation microbial inoculum, and has good application prospect.

Drawings

FIG. 1 shows the colony morphology of Gordonia alkalophaga CGMCC No.21704 of the present invention.

FIG. 2 shows the microscopic morphology of Gordonia alkalophaga CGMCC No.21704 of the present invention.

FIG. 3 is HPLC chart of degradation of imidacloprid by resting cells of Gordonia alkalophaga CGMCC No.21704 of the present invention.

FIG. 4 is an HPLC chart of the imidacloprid degradation process by Gordonia alkalophaga CGMCC No.21704 of the invention with different co-metabolism matrixes added in the resting cells. A: no sugar or organic acid salt was added, B: substrate control without inoculated cells, C: adding glucose, D: adding sodium succinate and E: adding sodium citrate, and F, adding sodium pyruvate.

FIG. 5 is an HPLC chart of degradation of imidacloprid in surface water by Gordonia alkalophaga CGMCC No.21704 resting cells at different times; wherein A: control day 0, B: 2 days, C: 4 days, D: and 6 days.

Detailed description of the invention

The Gordonia alkalophaga CGMCC No.21704 and its use of the present invention are explained in detail with reference to the following examples.

Example 1: separation, purification, screening and identification of biodegradable imidacloprid microorganisms

1. Strain isolation

Collecting sample from effluent pool of pesticide production in Jiangsu, adding 2mL of effluent sample into 18mL of sterile water, shaking and incubating for 2h in shaking table at 30 deg.C and 220rpm, standing for 20min, collecting 100 μ l of supernatant, and diluting to 10 μ l^-4、10^-5And 10^-6And spread on MSM solid medium containing 50mg/L imidacloprid. The composition of MSM medium is (g/L): KH (Perkin Elmer)₂PO₄1.36，Na₂HPO₄ 2.13，MgSO₄·7H₂O0.5 and 10mL of metal ion solution, pH 7.0. The composition of the metal ionic liquid is (g/L): CaCl₂·2H₂O 0.40,H₃BO₃ 0.30,CuSO₄·5H₂O 0.04，KI0.10,FeSO₄·7H₂O 0.20，MnSO₄·7H₂O 0.40,NaMoO₄·2H₂O0.20 and 10.0mL/L concentrated HCl. The plate is placed in an incubator at 30 ℃ for culture, the single colony growing out is picked up and transferred to an LB plate to draw a short line, and the plate is cultured at 30 ℃ until the colony grows out again. The formula of the LB culture medium is (g/L): yeast powder 5, peptone 10, sodium chloride 10, water 1000mL, pH 7.2. From the above samples, 18 microorganisms having different colony morphologies were isolated.

2. Determination of imidacloprid degradation ability of screened strains

The cells were scraped from the above plate grown for 48 hours with a sterile spatula and placed in a 50mL sterile centrifuge tube, 2mL phosphate buffer (pH7.5) containing 100mg/L imidacloprid was added, the cells were washed by shaking, and then centrifuged at 8000rpm for 10min, the washed cells were resuspended in 2mL of the above buffer, and the cells were sealed with a gas-permeable sealing film and incubated in a shaker at 30 ℃ and 220 rpm. After 4 days of incubation, the samples were centrifuged at 12000rpm for 5min to remove the biomass and the supernatant was collected. 1/5 volumes of acetonitrile were added, and the mixed solution was filtered through a filter (0.22 μm). The above filtrate was used for HPLC analysis. And analyzing and detecting the imidacloprid content change by using an Agilent 1200 type HPLC instrument. HPLC conditions: the mobile phase was 25% acetonitrile and 75% deionized water (containing 0.01% acetic acid) at a flow rate of 1 ml/min; the HPLC column is Agilent HC-C18 reversed phase column (4.6 × 250mm, 5 μm), and the column temperature is 30 deg.C; the detection wavelength was 269 nm. The imidacloprid content change in the zero time sample and the 4d sample was compared.

3. Identification of Gordonia alkalophaga CGMCC No.21704 strain

The HPLC analysis results (fig. 3) show that the sample inoculated with the C403 strain had a significant reduction in the substrate peak area compared to the control group not inoculated with the microorganism, indicating that the C403 strain had a significant imidacloprid degradation capacity. The samples inoculated with the other 17 microorganisms respectively had no reduction in the area of the substrate peak in the HPLC analysis, indicating that these strains had no imidacloprid degradation ability. Identifying the strain of the C403 strain by observing the microscopic morphology of the strain by an optical microscope; secondly, extracting genome amplification 16S rRNA gene, and performing blastn analysis on the amplified gene sequence. Strain C403 in generalThe colony morphology of the strain on LB plate is: orange, dry texture, small and round colony, smooth and neat edge, and non-transparent colony. The morphology of the C403 single colony is shown in FIG. 1, and the shape of the optical microscope of the crystal violet single stain is shown in FIG. 2. The method for extracting the genome amplification 16SrRNA gene of the strain comprises the following steps: the Takara MiniBEST bacterial Genomic DNA Extraction Kit Ver 3.0 genome Extraction Kit was used and the genome was extracted using Gram-negative Positive lysis. The primers are 16S rRNA gene amplification universal primers K1 and K2. The K1 primer sequence is: 5'-AACTGAAGAGTTTGATCC-3' (SEQ ID No: 1), the K2 primer sequence is: 5'-TAGGTTACCTTGTTGTTACGACTT-3' (SEQ ID No: 2). The primers were synthesized by Biotechnology engineering (Shanghai) Co., Ltd. Reaction system: 10 × LA buffer 2.0 μ L, MgCl₂2.0 μ L, dNTP (2.5mmol/L)2.0 μ L, K1(50mmol/L)0.4 μ L, K2(50mmol/L)0.4 μ L deionized water 11.0 μ L LA Taq enzyme (2U/. mu.L) 0.2 μ L, DNA template 2.0 μ L for a total reaction volume of 20 μ L. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 50s, annealing at 58 ℃ for 40s, extension at 72 ℃ for 2min for 30 cycles, and extension at 72 ℃ for 10 min. The obtained sequence was sequenced by Biotechnology engineering (Shanghai) Ltd, and the obtained partial 16S rRNA gene sequence (SEQ ID No: 3) was aligned in the Genbank database of the national center for Biotechnology information. The alignment results showed that the C403 strain was Gordonia alkanivorans.

The C403 strain is preserved in China general microbiological culture Collection center (CGMCC) at 22.1.2021, the preservation unit addresses are as follows: the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, is classified and named Gordonia alkanivorans with the preservation number of CGMCC No. 21704.

Example 2: imidacloprid degradation by resting cells of Gordonia alkalophaga CGMCC No.21704

The Gordonia alkalophaga CGMCC No.21704 preserved at-80 ℃ is streaked on an LB solid culture medium, and is cultured in an incubator at 30 ℃ for 4 days until a single colony grows out. Selecting single colony with toothpick, inoculating into 100mL conical flask containing 30mL LB liquid culture medium, shake culturing at 30 deg.C and 220rpm for 60 hr to obtain seed solution. Then, the cells were inoculated in an amount of 1% into a 500mL Erlenmeyer flask containing 100mL of LB liquid medium, and cultured at 30 ℃ for 36 hours with shaking at 220 rpm. After the completion of the culture of the cells, the cell concentration of the resting transformant was adjusted to OD₆₀₀Is 5. The cells were centrifuged at 8000rpm for 8min at 4 ℃ to collect the cells. Subsequently, the mixture was washed twice with a phosphate buffer (pH7.5) and centrifuged again. It was resuspended in a volume of 5mL in phosphate buffer containing 200mg/L imidacloprid. Then, the cells were incubated at 30 ℃ on a shaker at 220rpm, and periodically sampled. Meanwhile, a substrate control group without inoculation is set. The sample was centrifuged at 12000rpm for 5min, and the supernatant was diluted 1-fold with acetonitrile and passed through a 0.22 μm microporous membrane for HPLC analysis. As shown in FIG. 4A, the metabolite was present in the group inoculated with Gordonia alkalophaga CGMCC NO.21704, and the imidacloprid content was significantly reduced compared to the substrate control (FIG. 4B). HPLC quantitative analysis shows that the imidacloprid content is reduced by 442.4 mu mol/L, and the degradation rate is 53.5%.

Example 3: glucose-promoted degradation of imidacloprid by resting cells of Gordonia alkalophaga CGMCC No.21704

Essentially the same as example 2, except that 1% glucose was added to the transformation broth as a co-metabolizing substrate during transformation. The result shows that after 4d of transformation, as shown in figure 4, the imidacloprid content in the experimental group (containing co-metabolism substrate glucose, figure 4C) inoculated with the Gordonia alkalophaga CGMCC NO.21704 is reduced by 650.81 mu mol/L, and the degradation rate is 78.6%; the imidacloprid content of the control group without glucose (fig. 4A) was the same as the degradation rate and example 2.

Example 4: sodium succinate for promoting gobang-feeding bacteria CGMCC No.21704 resting cells to degrade imidacloprid

Essentially the same as example 3, except that the co-metabolic substrate was changed to sodium succinate. Results As shown in FIG. 4, the imidacloprid content in the experimental group inoculated with Gordonia alkalophaga No. CGMCC No.21704 (containing the co-metabolism matrix sodium succinate, FIG. 4D) was reduced by 576.6. mu. mol/L, and the degradation rate was 70.8%; the imidacloprid content and degradation rate of the control group (fig. 4A) which did not contain sodium succinate were the same as example 2.

Example 5: sodium citrate promotes Gordonia alkalophaga CGMCC No.21704 resting cells to degrade imidacloprid

Essentially the same as example 3, except that the co-metabolic substrate was changed to sodium citrate. The results are shown in FIG. 4, which shows that the imidacloprid content in the experimental group inoculated with Gordonia alkalophaga CGMCC No.21704 (containing the co-metabolism substrate sodium citrate, FIG. 4E) is reduced by 642.4 mu mol/L, and the degradation rate is 78.2%. The imidacloprid content and degradation rate of the control group (fig. 4A) which did not contain sodium succinate were the same as example 2.

Example 6: sodium pyruvate promotes Gordonia alkalophaga CGMCC No.21704 resting cells to degrade imidacloprid

Essentially the same as example 3, except that the co-metabolic substrate was changed to sodium pyruvate. The results are shown in figure 4, and the imidacloprid content in the experimental group (containing co-metabolism substrate sodium pyruvate, figure 4F) inoculated with Gordonia alkalophaga CGMCC No.21704 is reduced by 577.1 mu mol/L, and the degradation rate is 72.8%; the imidacloprid content and degradation rate of the control group (fig. 4A) which did not contain sodium succinate were the same as example 2.

Example 7: degradation of imidacloprid in surface water by resting cells of Gordonia alkalophaga CGMCC No.21704

Collecting surface water sample in the lake of China, Yanjing university, Xianlin school, filtering with 0.22 μm filter membrane, adding imidacloprid into the filtered surface water, and setting the final concentration at 25 mg/L. Gordonia alkalophaga CGMCC No.21704 strain was cultured by the method described in example 1, and the absorbance (OD) of the strain was measured₆₀₀) After the cells were adjusted to 1, collected and washed 2 times with a phosphate buffer (pH7.5), the cells were resuspended in the above surface water containing 25mg/L imidacloprid and dispensed into 100mL flasks per 30 mL. Surface water without inoculated bacteria was used as a control. Placing the triangular flask in a shaking table with the rotation speed of 220rpm at the temperature of 30 ℃ for culture, sampling after 2 days, 4 days and 6 days of culture respectively, and carrying out HPLC analysis after the samples are processed. The results show (FIG. 5), after 2 days, the imidacloprid concentration in the water was reduced from 109. mu. mol/L to 76.85. mu. mol/L, and the degradation rate of the imidacloprid was 29.5%; after 4 days of degradation, the concentration of the imidacloprid is reduced to 65.9 mu mol/L from 109 mu mol/L, and the degradation rate of the imidacloprid is 39.5 percent; 6 days after the conversion, the imidacloprid concentration is at the beginning 109. mu. mol-L is reduced to 56.2 mu mol/L, and the degradation rate of imidacloprid is 48.4 percent.

By adopting the method of the embodiment, the detection result shows that the Gordonia alkalophaga CGMCC No.21704 of the invention has the degradation effect on the neonicotinoid insecticide imidacloprid, can be used as a pesticide degradation microbial inoculum and has good application prospect.

The invention is not limited to the embodiments described above only.

SEQUENCE LISTING

<110> Nanjing national environmental research institute Co., Ltd

NANJING NORMAL University

<120> Gordonia alkalophaga and application thereof in degrading insecticide imidacloprid

<130>

<160> 3

<170> PatentIn version 3.3

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<212> DNA

<213> Artificial sequence

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aactgaagag tttgatcc 18

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<212> DNA

<213> Artificial sequence

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taggttacct tgttgttacg actt 24

<210> 3

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<212> DNA

<213> Gordonia alkaninvorans

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agtggcggcg tgcttacaca tgcagtcgaa cggaaaggcc cagcttgctg ggtactcgag 60

tggcgaacgg gtgagtaaca cgtgggtgat ctgccctgaa ctttgggata agcctgggaa 120

actgggtcta ataccggata tgaccttgga gtgcatgctc tggggtggaa agcttttgcg 180

gttcaggatg ggcccgcggc ctatcagctt gttggtgggg taatggccta ccaaggcgac 240

gacgggtagc cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact 300

cctacgggag gcagcagtgg ggaatattgc acaatgggcg caagcctgat gcagcgacgc 360

cgcgtgaggg atgacggcct tcgggttgta aacctctttc accagggacg aagcgcaagt 420

gacggtacct ggagaagaag caccggccaa ctacgtgcca gcagccgcgg taatacgtag 480

ggtgcgagcg ttgtccggaa ttactgggcg taaagagctc gtaggcggtt tgtcgcgtcg 540

tctgtgaaat tctgcaactc aattgtaggc gtgcaggcga tacgggcaga cttgagtact 600

acaggggaga ctggaattcc tggtgtagcg gtgaaatgcg cagatatcag gaggaacacc 660

ggtggcgaag gcgggtctct gggtagtaac tgacgctgag gagcgaaagc gtgggtagcg 720

aacaggatta gataccctgg tagtccacgc cgtaaacggt gggtactagg tgtggggctc 780

atttcacgag ttccgtgccg tagctaacgc attaagtacc ccgcctgggg agtacggccg 840

caaggctaaa actcaaagga attgacgggg gcccgcacaa gcggcggagc atgtggatta 900

attcgatgca acgcgaagaa ccttacctgg gtttgacata caccagacgc atgtagagat 960

acatgttccc ttgtggttgg tgtacaggtg gtgcatggct gtcgtcagct cgtgtcgtga 1020

gatgttgggt taagtcccgc aacgagcgca acccttgtcc tgtattgcca gcgggttatg 1080

ccggggactt gcaggagact gccggggtca actcggagga aggtggggat gacgtcaagt 1140

catcatgccc cttatgtcca gggcttcaca catgctacaa tggctggtac agagggctgc 1200

gataccgtga ggtggagcga atcccttaaa gccagtctca gttcggattg gggtctgcaa 1260

ctcgacccca tgaagtcgga gtcgctagta atcgcagatc agcaacgctg cggtgaatac 1320

gttcccgggc cttgtacaca ccgcccgtca cgtcatgaaa gtcggtaaca cccgaagccg 1380

gtggcctaac cccttgtggg agggagctgt cgaaggggat tcgt 1424

Claims

1. Gordoniaalkanivorans (Gordonia alkannivorans) CGMCC No. 21704.

2. A method for culturing Gordonia alkalophaga according to claim 1, wherein said Gordonia alkanivorans (Gordonia alkannivorans) CGMCC No.21704 is inoculated on LB agar medium and cultured in an incubator at 30. + -. 1 ℃; or inoculating into LB liquid culture medium, and shake culturing in shaking table at 30 + -1 deg.C.

3. The use of Gordonia alkanivorans (Gordonia alkannivorans) CGMCC No.21704 in degrading neonicotinoid insecticide imidacloprid as claimed in claim 1.

4. The application of claim 3, wherein the application method comprises: inoculating the strain of Gordonia alkanivorans (Gordonia alkannivorans) CGMCC No.21704 in a liquid nutrient medium, and collecting the strain after shaking culture; suspending the thallus in phosphate buffer solution or surface water containing imidacloprid, shaking and mixing, and degrading imidacloprid with dead cells of Gordonia alkanivorans (Gordonia alkannivorans) CGMCC No. 21704.

5. Use according to claim 4, wherein the liquid nutrient medium is LB liquid medium.