CN112375718B - Pymetrozine degrading bacterium IURM F17 and application thereof - Google Patents
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Abstract
The invention discloses a pymetrozine degrading bacterium IURM F17 and application thereof, belonging to the technical field of biology. Experiments prove that the pymetrozine-degrading bacterium IURM F17 can survive in an environment with pymetrozine as a unique carbon source, has high pymetrozine-degrading activity, can be applied to the degradation of pymetrozine pesticides in polluted environments, and has good application value in the aspects of soil pollution treatment and environment restoration.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a pymetrozine degrading bacterium IURM F17 and application thereof.
Background
China is a traditional big agricultural country, agriculture in China has gone through the history of more than five thousand years, and with the increasing number of people, the demand for grains is increasing day by day, so that new agricultural technologies such as chemical fertilizers and pesticides are urgently needed to put into production to achieve the purpose of grain quality increase. The use of chemical pesticide can greatly reduce the loss caused by diseases and insect pests and improve the quality of agricultural products. However, with the long-term use of these pesticides, their disadvantages become increasingly prominent: the pesticide is continuously accumulated in the environment, so that soil, water and atmospheric environment are seriously polluted, the diversity of surrounding biological populations is reduced, the balance of an ecological system is damaged, the health of people and the export of agricultural products are influenced, along with the continuous improvement of the living level and the environmental protection consciousness of people, the harm of the pesticide is deeply known and understood by people, and therefore, the elimination of pesticide residue pollution becomes a social science problem to be urgently solved by scientific research workers.
At present, pesticide degradation methods are numerous at home and abroad, and among different degradation methods, a microbial degradation method is one of the most important, most clean and efficient methods. Microorganisms are often the final degradation way of pollutants due to the characteristics of multiple types, wide distribution, rapid propagation, easy variation, strong biochemical adaptability and the like. The microbial remediation is a biological measure for realizing environmental purification and ecological effect recovery by utilizing the metabolism, the absorption, the transformation, the removal or the degradation of chemical pesticide components of specific microorganisms, and has the characteristics of high efficiency, economy, no secondary pollution and the like. A large number of researches prove that various microorganisms existing in natural environment play an important role in the aspect of pesticide degradation, and researchers have found a plurality of microorganisms capable of degrading pesticides through technologies such as enrichment culture, separation screening and the like. However, the pymetrozine degrading strains are rarely reported, further research is needed, and the method has great practical significance for eliminating pollution of waste pymetrozine in the environment and purifying the environment.
Disclosure of Invention
The invention aims to provide pymetrozine degrading bacteria and application thereof in the aspects of pymetrozine degradation in soil and water and the like.
In order to solve the technical problems, the invention provides the following technical scheme: pymetrozine degrading bacterium IURM F17; wherein the pymetrozine-degrading bacteria IURM F17 has been preserved in China general microbiological culture Collection center (CGMCC) at 12.10.2020, the preservation unit address is No. 3 of Xilu No. 1 of Beijing province facing Yang, the preservation number is CGMCC No.20854, and the classification is named as Pseudomonas stutzeri (Pseudomonas stutzeri) ((R))Pseudomonas stutzeri）。
The pymetrozine-degrading bacterium IURM F17 has a repairing effect on an environment polluted by pymetrozine, and particularly has a repairing effect on a soil environment polluted by pymetrozine and a water body polluted by pymetrozine.
The pymetrozine degrading bacteria IURM F17 have a degrading effect on waste pymetrozine in soil and pymetrozine contained in a water body, and can be directly applied to the field of purification treatment of farmlands.
The screening method of the pymetrozine-degrading bacterium IURM F17 comprises the following steps:
1) Weighing soil from sludge polluted by pymetrozine, adding pymetrozine-degrading bacteria IURM F17 to screen liquid culture medium for enrichment, and culturing in a constant temperature shaking table at 35-40 ℃ and 150-200r/min for 4-10 days;
2) Absorbing the culture medium obtained in the step 1, adding a new pymetrozine degrading bacterium IURM F17 screening liquid culture medium, and carrying out passage for 3-6 times;
3) Streaking and separating the bacterial liquid obtained in the step 2 on an LB (Langmuir-Blodgett) plate to obtain pymetrozine degrading bacteria IURM F17;
wherein the pymetrozine degrading bacterium IURM F17 screening culture medium comprises the following components: each 1L of deionized water contains 100mg/L of pymetrozine pesticide, 0.66 g of ammonium chloride and dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5 g of potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.5 g magnesium sulfate (MgSO) 4 ) 0.1 g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5 g, 0.35 g of sodium chloride, and pH 7.0-7.2;
the LB solid medium comprises the following components: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 15g of agar powder, and the volume is made up to 1L by deionized water.
The preferable screening method of the pymetrozine-degrading bacterium IURM F17 comprises the following steps:
1) Weighing 5g of soil from sludge polluted by pymetrozine, adding 100mL of pymetrozine degrading bacteria IURM F17 screening liquid culture medium for enrichment, and culturing in a constant temperature shaking table at 37 ℃ and 180r/min for 5-7 days;
2) 3 mL of the culture medium obtained in the step 1 is sucked and added with a new pymetrozine degrading bacterium IURM F17 screening liquid culture medium for passage, and the passage is carried out for 4-5 times;
3) Streaking and separating the bacterial liquid obtained in the step 2 on an LB (Langmuir-Blodgett) plate to obtain pymetrozine degrading bacteria IURM F17;
the dry powder of the pymetrozine-degrading bacteria IURM F17 is prepared by drying the pymetrozine-degrading bacteria IURM F17 subjected to amplification culture by a conventional method.
The pymetrozine degrading bacterium IURM F17 is obtained by screening according to the following method:
1. soil separation strain is weighed from sludge polluted by pymetrozine in Changzhou Wucheng area of Jiangsu
Weighing 5g of sludge, adding 95 mL of pymetrozine degrading bacteria IURM F17 screening liquid culture medium for enrichment, and culturing for 5-7 days in a constant temperature shaking table at 37 ℃ and 180 r/min; then 3 mL of culture medium is sucked and added with a new pymetrozine degrading bacterium IURM F17 screening liquid culture medium for passage, the passage is carried out for 4-5 times, streaking separation is carried out on an LB (Langmuir-Blodgett) plate, and a batch of strains which can survive in the culture medium taking pymetrozine as a unique carbon source are finally obtained through hundreds of times of separation test screening;
the pymetrozine degrading bacteria IURM F17 screening culture medium comprises the following components: every 1L of deionized water contains 100mg/L of pymetrozine pesticide, 0.66 g of ammonium chloride and dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5 g, monopotassium phosphate (KH) 2 PO 4 ) 0.5 g magnesium sulfate (MgSO) 4 ) 0.1 g of dipotassium hydrogenphosphate (K) 2 HPO 4 ) 1.5 g, 0.35 g of sodium chloride, and pH 7.0-7.2;
the LB solid medium comprises the following components: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 15g of agar powder, and the volume is made up to 1L by deionized water.
2. Analyzing the degradation efficiency of different strains obtained by separation on pymetrozine in the culture medium, and screening to obtain the strain with the best pymetrozine degradation effect.
The specific determination method is as follows:
1. preparation of the Medium
Basic salt culture medium: NH 4 Cl 0.66 g， K 2 HPO 4 1.5 g， KH 2 PO 4 0.5 g， MgSO 4 0.1 g， K 2 HPO 4 1.5 g, naCl 0.35 g, pH 7.0-7.2; sterilizing at 121 deg.C for 20 min.
LB liquid medium: 5g of yeast powder, 10g of sodium chloride, 10g of peptone and 1000 mL of deionized water, wherein the pH value is 7.0, and the sterilization is carried out at 121 ℃ for 20 min.
2. Method for measuring pymetrozine degradation efficiency
2.1 Drawing of pymetrozine standard curve
Taking 0.05 g of pymetrozine standard substance, dissolving the standard substance by using chromatographic acetonitrile, and carrying out constant volume to a 50 mL volumetric flask to prepare 1000 mg/L mother liquor. Diluting the mother liquor with chromatographic pure acetonitrile, preparing 10, 20, 40, 50, 60, 80 and 100mg/L series standard solutions step by step, and measuring peak area by using a high performance liquid chromatograph under the condition of 298 nm wavelength to obtain a concentration-peak area standard curve.
2.2 Method for measuring pymetrozine degradation rate
The culture solution was taken, and eluted by high performance liquid chromatography at 298 nm with a mobile phase of a mixture containing methanol/water/formic acid (100, 900, v/v/v) at a flow rate of 1.0 mL/min. The injection volume was 20. Mu.L, the column temperature was kept at 40 ℃ and the peak area was determined. And (4) calculating the pymetrozine concentration by using a linear regression equation established by a pymetrozine standard curve, and further calculating to obtain the pymetrozine degradation rate. Three replicates were set up for all experiments.
2.3 obtaining the optimal strain through the degradation rate of the pymetrozine by pymetrozine degrading bacteria IURM F17
Culturing pymetrozine-degrading IURM F17 strain in LB liquid culture medium, centrifuging, collecting thallus, resuspending with sterile water, and diluting to OD 600 =1, inoculating the selected strain into 1-100.0 mL of a basic liquid inorganic salt culture medium containing 100mg/L pymetrozine, placing the culture medium in a constant temperature shaking table for culture at 37 ℃ at 180r/min, and sampling every 6 hours to determine the degradation rate.
The screened pymetrozine-degrading bacterium IURM F17BYT7 has an obvious effect on the degradation of pymetrozine, the strain grows well, and the degradation rate of the pymetrozine for 30 hours is 84.67%.
2.4 degradation of waste pymetrozine in wastewater by pymetrozine-degrading bacterium IURM F17
Inoculating 1% of pymetrozine-degrading bacteria IURM F17 into 100mL of sterilized waste water containing pymetrozine, determining the content of pymetrozine in the waste water to be 58 mg/L by a high performance liquid chromatograph, culturing for 30h under the conditions of 37 ℃ and 180r/min, and determining the degradation rate of pymetrozine as follows: 77.67 percent.
2.5 degradation of waste pymetrozine in soil by pymetrozine-degrading bacterium IURM F17BYT7
Inoculating 1% of pymetrozine-degrading bacteria IURM F17BYT7 into 100mL of sterilized soil liquid, determining by a high performance liquid chromatograph that the content of pymetrozine in the soil is 54 mg/Kg, culturing for 30h under the constant temperature condition of 37 ℃, and determining that the pymetrozine degradation rate is 73.27%.
Identification of strains
Obtaining the genome DNA of pymetrozine degrading bacteria IURM F17by adopting an oscillation crushing method, amplifying the 16S rRNA gene of the strain by a PCR method, and sending the amplified gene to the Chairi biological company of Shanghai for sequencing analysis; the upstream primer sequences used to amplify the 16S rRNA gene were: 5 'AGAGAGTTTGATCCTGGCTCAG 3' and the sequence of the downstream primer is 5 'GGTTACCTTGTTACGACTT 3'.
The PCR reaction conditions were: pre-denaturation at 98 ℃ for 5min, denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 15 s, extension at 72 ℃ for 40 s, setting 30 cycles for reaction, and keeping the temperature at 10 ℃ for 5min. Using the BLAST function of NCBI for sequence alignment in the GeneBank database, the classification of the analyzed strains was: pseudomonas stutzeri (A)Pseudomonas stutzeri）
The gene sequence of the IURM F1716S rRNA of the pymetrozine degrading bacteria is shown in a sequence table.
The invention has the beneficial effects that:
1. the pymetrozine degrading bacteria IURM F17 which can survive in the environment with pymetrozine as the only carbon source is screened out, and degradation analysis shows that the bacterial strain can grow well in a solid culture medium and a liquid culture medium containing 100mg/L of pymetrozine, and has high pymetrozine degrading characteristics.
2. The pymetrozine-degrading bacterium IURM F17 has application prospects in the degradation of pymetrozine in soil, the degradation of pymetrozine in water body pollution, the degradation of waste pymetrozine in soil and the restoration of environmental pollution.
3. The application of the pymetrozine degrading bacterium IURM F17 is beneficial to avoiding secondary pollution caused by physical or chemical method degradation of pymetrozine.
Drawings
FIG. 1 is a morphological diagram of a single colony of the pymetrozine-degrading bacterium IURM F17.
FIG. 2 shows the rate of degradation of pymetrozine measured by inoculating 1% of an inoculum of IURM F17 into 100mL of sterile MSM.
FIG. 3 shows the pymetrozine degradation rate measured by inoculating 1% of pymetrozine-degrading bacteria IURM F17 into 100mL of pymetrozine-containing wastewater.
FIG. 4 shows the rate of degradation of pymetrozine measured by inoculating 1% of an inoculum of IURM F17 to 100 g of sterilized soil.
Detailed Description
Screening process of pymetrozine-degrading bacterium IURM F17
(I) main material
1. Culture medium
Pymetrozine screening liquid medium components: each 1L of deionized water contains 100mg/L of pymetrozine pesticide, 0.66 g of ammonium chloride and dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5 g of potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.5 g magnesium sulfate (MgSO) 4 ) 0.1 g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5 g, 0.35 g of sodium chloride, and 7.0-7.2 of pH;the corresponding solid culture medium is prepared by adding 15g of agar powder into 1L of liquid culture medium.
Composition of LB liquid Medium per 1L: 10g of peptone, 5g of yeast powder and 10g of NaCl, adjusting the pH of the prepared liquid culture medium to 7.0-7.2 by using HCl or NaOH, and then adding deionized water to make up to 1L. The corresponding LB solid medium is prepared by adding 15g of agar powder into 1L of liquid medium.
2. Instrumentation apparatus
The kit comprises a FlexCycler PCR amplification instrument, a Tanon-3500 gel imaging system, an electrophoresis apparatus of six instrument factories in Beijing, a sterilization pot, an electronic balance, a UV1900 ultraviolet visible spectrophotometer, a constant temperature incubator, an eppendorf high-speed refrigerated centrifuge, a shaking table and a Heire low-temperature refrigerator.
(II) screening process of pymetrozine degrading bacterium IURM F17
(1) Weighing an earth separation strain from sludge polluted by pymetrozine in Changzhou Wuji area of Jiangsu, weighing 5g of sludge, adding 100mL of pymetrozine degrading bacteria IURM F17 screening liquid culture medium for enrichment, and culturing for 5-7 days in a constant temperature shaking table at 37 ℃ and 180 r/min; then 3 mL of culture medium is sucked and added with a new pymetrozine degrading bacterium IURM F17 screening liquid culture medium for passage, the passage is carried out for 4-5 times, streaking separation is carried out on an LB (Langmuir-Blodgett) plate, and a batch of strains which can survive in the culture medium taking pymetrozine as a unique carbon source are obtained finally after hundreds of separation test screening;
the pymetrozine degrading bacteria IURM F17 screening culture medium comprises the following components: each 1L of deionized water contains 100mg/L of pymetrozine pesticide, 0.66 g of ammonium chloride and dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5 g, monopotassium phosphate (KH) 2 PO 4 ) 0.5 g magnesium sulfate (MgSO) 4 ) 0.1 g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 1.5 g, 0.35 g of sodium chloride, and pH 7.0-7.2;
the LB solid medium comprises the following components: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 15g of agar powder, and the volume is made up to 1L by deionized water.
(2) Streaking and separating the culture on an LB plate, and culturing in a constant-temperature incubator at 37 ℃ for 12-26 hours to obtain a morphological diagram of a single colony of pymetrozine degrading bacteria IURM F17 strain IURM F17 (shown in figure 1);
the following is a detailed description of the embodiments.
Example 1
Culturing pymetrozine-degrading bacteria IURM F17 in LB liquid culture medium, centrifuging to collect thallus, resuspending with sterile water, and diluting to OD 600 =1, inoculating the selected strain into 1-100 mL inorganic salt culture medium containing 100.0 mg/L pymetrozine, placing the strain in a constant temperature shaking table for culturing for 30h at 37 ℃ at 180r/min, wherein the pymetrozine degrading bacterium IURM F17 has obvious growth and higher pymetrozine degrading efficiency.
Example 2
The pymetrozine-degrading bacteria IURM F17 is inoculated into 100mL of sterile MSM according to the inoculation amount of 1%, pymetrozine is added to the final concentration of 100mg/L, the mixture is cultured for 30h under the conditions of 37 ℃ and 180r/min, and the pymetrozine degradation rate is determined to be 84.67% (see figure 2).
Example 3
Inoculating 1% of pymetrozine-degrading bacteria IURM F17 into 100mL of waste water containing pymetrozine, determining that the content of pymetrozine in the waste water is 58 mg/L, culturing for 30h at 37 ℃ and 180r/min, and determining that the pymetrozine degradation rate is as follows: 77.67% (see fig. 3).
Example 4
The pymetrozine-degrading bacteria IURM F17 are inoculated into 100 g of sterilized soil according to the inoculation amount of 1%, the content of pymetrozine in the soil is measured to be 54 mg/Kg, the soil is cultured for 30h under the constant temperature condition of 37 ℃, and the pymetrozine degradation rate is measured to be 73.27% (see figure 4).
Example 5
The preparation method of the dry powder of the pymetrozine-degrading bacterium IURM F17 comprises the steps of carrying out amplification culture on the pymetrozine-degrading bacterium IURM F17 and drying the obtained product by a conventional method.
Sequence listing
<110> university of Changzhou
Jiangsu bio Environmental Protection Technology Co.,Ltd.
HEIBEI CIXIN ENVIRONMENTAL PROTECTION AND TECHNOLOGY Co.,Ltd.
<120> pymetrozine degrading bacterium IURMF17 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1436
<212> DNA
<213> Pseudomonas stutzeri (Pseudomonas stutzeri)
<400> 1
ggctacagta ctactatgct agtcgagcgg atgagtggag cttgctggat gattcagcgg 60
cggacgggtg agtaatgcct aggaatctgc ctggtagtgg gggacaacgt ttcgaaagga 120
acgctaatac cgcatacgtc ctacgggaga aagtggggga tcttcggacc tcacgctatc 180
agatgagcct aggtcggatt agctagttgg cgaggtaaag gctcaccaag gcgacgatcc 240
gtaactggtc tgagaggatg atcagtcaca ctggaactga gacacggtcc agactcctac 300
gggaggcagc agtggggaat attggacaat gggcgaaagc ctgatccagc catgccgcgt 360
gtgtgaagaa ggtcttcgga ttgtaaagct ctttaagttg ggaggaaggg cagtaagtta 420
ataccttgct gtcttgacgt taccgacaga ataagcaccg gctaacttcg tgccagcagc 480
cgcggtaata cgaagggtgc aagcgttaat cggaattact gggcgtaaag cgcgcgtagg 540
tggttcgtta agttggatgt gaaagccccg ggctcaacct gggaactgca tccaaaactg 600
gcgagctaga gtatggcaga gggtggtgga atttcctgtg tagcggtgaa atgcgtagat 660
ataggaagga acaccagtgg cgaaggcgac cacctgggct aatactgaca ctgaggtgcg 720
aaagcgtggg gagcaaacag gattagatac cctggtatgc cacgccgtaa acgatgtcta 780
ctagccgttg ggatccttga gatcttagtg gcgcagctaa cgcattaagt cgaccgcctg 840
gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca caagcggtgg 900
agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggccttgac atgctgagaa 960
cctgccagag atggcggggt gccttcggga actcagacac aggtgctgca tggctgtcgt 1020
cagctcgtgt cgtgagatgt tgggttaagt cccgtaacga gcgcaaccct tgtccttagt 1080
taccagcacg ttatggtggg cactctaagg gactgccggt gacaaaccgg aggaaggtgg 1140
ggatgacgtc aagtcatcat ggcccttacg gcctgggcta cacacgtgct acaatggtcg 1200
gtacaaaggg ttgccaagcc gcgaggtgga gctaatccca taaaaccgct cgtagtccgg 1260
atcgcagtct gcaactcgac tgcgtgaagt cggaatcgct agtaatcgtg aatcagaatg 1320
tcacggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagtgggtt 1380
gctccagaag tagctagtct aaccttcggg gggcacagta caactgagat catcgc 1436
Claims (7)
1. A pymetrozine-degrading bacterium IURM F17 is characterized in that the pymetrozine-degrading bacterium IURM F17 is pseudomonas stutzeri (Pseudomonas stutzeri)Pseudomonas stutzeri) Wherein the pymetrozine-degrading bacterium IURM F17 is preserved in China general microbiological culture Collection center (CGMCC) at 10/12 of 2020, and the preservation number is CGMCC NO.20854.
2. The use of the bacterium IURM F17 for the degradation of pymetrozine as claimed in claim 1, wherein said bacterium IURM F17 has a repairing effect on the environment contaminated by pymetrozine.
3. The use of the IURM F17 as claimed in claim 2, wherein said IURM F17 has a repairing effect on the environment contaminated by the waste pymetrozine in the soil.
4. The use of the pymetrozine-degrading bacterium IURM F17 as claimed in claim 2, wherein said pymetrozine-degrading bacterium IURM F17 has a repairing effect on the water environment polluted by pymetrozine in a water body.
5. The use of the pymetrozine-degrading bacterium IURM F17 as claimed in claim 1, wherein said pymetrozine-degrading bacterium IURM F17 has a degrading effect on waste pymetrozine in soil.
6. The use of the pymetrozine-degrading bacterium IURM F17 as claimed in claim 1, wherein said pymetrozine-degrading bacterium IURM F17 has a degrading effect on pymetrozine contained in a water body.
7. A dry powder which is prepared by using the pymetrozine-degrading bacterium IURM F17 as claimed in claim 1 and performing scale-up culture and drying.
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| CN101096644A (en) * | 2006-06-26 | 2008-01-02 | 谢明 | Pseudomonas stutzeri JSD-008 and its degradation function for organophosphorus pesticide |
| CN110699287A (en) * | 2019-10-25 | 2020-01-17 | 黄河三角洲京博化工研究院有限公司 | Bacterial strain with function of degrading organic amine substances and application thereof |
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|---|---|---|---|---|
| CN101096644A (en) * | 2006-06-26 | 2008-01-02 | 谢明 | Pseudomonas stutzeri JSD-008 and its degradation function for organophosphorus pesticide |
| CN110699287A (en) * | 2019-10-25 | 2020-01-17 | 黄河三角洲京博化工研究院有限公司 | Bacterial strain with function of degrading organic amine substances and application thereof |
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| 特丁噻草隆降解菌的分离鉴定及降解特性;任建军等;《江苏农业学报》;20200430(第02期);第276-278页 * |
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