Dimethoate degrading strain as well as preparation method and application thereof

Abstract

The invention discloses a dimethoate degrading strain as well as a preparation method and application thereof, and belongs to the technical field of biology. An individual of the dimethoate degrading strain is rod-shaped, a bacterial colony is a circle with irregular edges, and the middle position of the bacterial colony protrudes upwards; the bacterial colony has a smooth surface and is in a white mucus state; the degrading strain is named as Enterobacter sp. F13, and is preserved in the Guangdong Microbial Culture Collection Center, called GDMCC for short, the preservation date is May 14, 2023, and the preservation registration number is GDMCCN0: 63464. The in-situ strain capable of efficiently degrading the dimethoate is directionally screened from the sediment of the artificial river channel buffer zone, so that a biological basis is provided for production and application, and strain resources are provided for bioremediation of dimethoate polluted areas and optimization and blending of strains in wastewater biochemical treatment ponds.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a dimethoate-degrading strain, a preparation method and application thereof. Background Art

[0002] Dimethoate (chemical name: O, O-dimethyl-S-(N-methylcarbamoylmethyl) dithiophosphate, English name: Dimethoate, molecular weight: 229.12) is a widely used, highly effective, moderately toxic organophosphorus pesticide. Dimethoate has excellent killing effects on pests in cash crops such as vegetables, fruits, tea, and mulberry. However, due to the long half-life of dimethoate, its natural degradation rate is slow, and humans have abused pesticides for a long time, leading to water and soil pollution. When people eat contaminated food, these moderately toxic substances gradually accumulate in the human body, seriously endangering human health.

[0003] Microbial remediation of water environment pollution caused by pesticide residues is an important research direction. Microbial remediation is the preferred method in the remediation of water environment polluted by the application of dimethoate. In view of the impact of dimethoate pesticides on humans, scientists are committed to screening out microorganisms that can efficiently degrade dimethoate in the in situ environment, thereby achieving a key breakthrough in the treatment of dimethoate pollution. At present, research on screening dimethoate-degrading bacteria from vegetables and cultivated soil is relatively common, but these degrading bacteria cannot adapt well to the water environment. Therefore, the present invention directionally screens out in situ strains that can efficiently degrade dimethoate from the sediments of the artificial river buffer zone, aiming to provide a biological basis for production and application, and provide strain resources for the bioremediation of dimethoate-contaminated areas and the optimization and deployment of strains in wastewater biochemical treatment pools. Summary of the invention

[0004] The purpose of the present invention is to provide a dimethoate-degrading strain, a preparation method and an application thereof.

[0005] The present invention adopts the following technical scheme: a dimethoate-degrading strain, wherein the individual dimethoate-degrading strain is rod-shaped, the colony is circular with irregular edges, and the middle position is convex upward; the colony has a smooth surface and is white mucus-like;

[0006] The degradation strain was named Enterobacter sp. F13 and was deposited in Guangdong Provincial Microbiological Culture Collection Center, referred to as GDMCC. The deposit date was May 14, 2023, and the deposit registration number was GDMCCN0: 63464.

[0007] In a further embodiment, the in situ strain of the dimethoate-degrading strain is extracted from sediments in a river buffer zone.

[0008] The method for preparing the above-mentioned dimethoate-degrading strain comprises the following steps:

[0009] Step 1: Sampling sediment samples in the river buffer zone;

[0010] Step 2: dilute the sediment sample to obtain a bacterial suspension, further dilute the bacterial suspension and spread it on a complete solid culture medium, invert it and place it in a biochemical incubator for culture at 36-38° C. until a single colony is isolated;

[0011] Step 3: Pick a single colony and place it in an inorganic salt medium containing 10 mg / L dimethoate, and culture it in a shaker at 37°C and 180 rpm for 3-7 days; inoculate the bacterial solution grown in the 10 mg / L dimethoate inorganic salt medium into the 20 mg / L dimethoate inorganic salt medium, and culture it continuously. Stop acclimation when the final pesticide concentration reaches 100 mg / L;

[0012] Step 4: Store the strains that can survive on the culture medium containing 100 mg / L dimethoate in an inorganic salt culture medium containing 20% ​​glycerol and place it in a -80°C refrigerator.

[0013] In a further embodiment, the complete medium is glucose, yeast powder, ammonium sulfate, magnesium sulfate heptahydrate, manganese sulfate monohydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and 1 ml / L trace element solution, wherein the pH value of the medium is adjusted to 6.8;

[0014] The complete fixed medium is prepared by adding 1.5% agar to the complete medium.

[0015] According to the application of the dimethoate-degrading strain in bioremediation of polluted areas and biochemical treatment of wastewater as described above.

[0016] Beneficial effects of the present invention: The present invention selectively screens out in situ strains capable of efficiently degrading dimethoate from the sediments of the artificial river buffer zone, aiming to provide a biological basis for production and application, and to provide strain resources for the bioremediation of dimethoate-contaminated areas and the optimization and deployment of strains in wastewater biochemical treatment pools. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the morphological diagram of the dimethoate-degrading strain.

[0018] Figure 2 This is a Gram stain identification diagram.

[0019] Figure 3 This is a graph of PCR electrophoresis results.

[0020] Figure 4 This is a phylogenetic tree constructed using mega software.

[0021] Figure 5 This is a graph showing the growth curve of the strain.

[0022] Figure 6 This is a degradation comparison chart. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0024] Example 1

[0025] This embodiment discloses a malt-degrading strain, the individual of the malt-degrading strain is rod-shaped, the colony is a circle with irregular edges, and the middle position is convex upward; the colony is smooth on the surface and white mucus. The degradation strain is named Enterobacter sp. F13, which is preserved in the Guangdong Provincial Microbiological Culture Collection Center, referred to as GDMCC, with a preservation date of May 14, 2023, and a preservation registration number: GDMCCN0: 63464. The preservation address is Guangdong Provincial Institute of Scientific Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0026] It should be noted that the in situ strain of the dimethoate-degrading strain is extracted from the sediment of the river buffer zone.

[0027] Example 2

[0028] This embodiment discloses a method for preparing the dimethoate-degrading strain described in Example 1, comprising the following steps:

[0029] Step 1: Take samples from the river buffer zone to obtain sediment samples. For example, based on field investigations, 12 stations were selected in the buffer zone along the Yangtze River to Jichao. Mud samples were collected three times using a Peterson grab sampler and loaded into 15mL PE tubes. The sediment samples were refrigerated and transported back to the laboratory and stored at -20°C.

[0030] Step 2: dilute the sediment sample to obtain a bacterial suspension, further dilute the bacterial suspension and spread it on a complete solid culture medium, invert it and place it in a biochemical incubator at 36-38°C for incubation until a single colony is isolated. Specifically, use an electronic balance to weigh 1.0g of each sample from each site, add it to 99mL of saline containing glass beads with a diameter of 1mm, and shake and culture it at 37°C and 180rpm for 30min. At this time, the dilution multiple is recorded as 100 times; draw 1mL of the above bacterial suspension and add it to a test tube containing 9mL of saline, dilute it to 10-3 times, and dilute it to 10-7 times in this way; draw 100μL of bacterial suspensions with dilution multiples of 10-5 and 10-7 respectively, spread it on a complete solid culture medium, invert it and place it in a biochemical incubator at 37°C for incubation for 24h; try to pick colonies of different sizes, shapes, colors, etc. for streaking culture until a single colony is isolated.

[0031] Step 3: Pick a single colony and place it in an inorganic salt medium containing 10 mg / L dimethoate, and culture it in a shaker at 37°C and 180 rpm for 3-7 days; inoculate the bacterial solution grown in the 10 mg / L dimethoate inorganic salt medium into the 20 mg / L dimethoate inorganic salt medium, and culture it continuously. Stop acclimation when the final pesticide concentration reaches 100 mg / L.

[0032] Step 4: Store the strain that can survive on the inorganic salt medium containing 100 mg / L dimethoate in a complete medium containing 20% ​​glycerol and place it in a -80°C refrigerator.

[0033] Among them, the complete culture medium is 20.0g / L glucose, 5.0g / L yeast powder, 5.0g / L ammonium sulfate, 0.5g / L magnesium sulfate heptahydrate, 0.05g / L manganese sulfate monohydrate, 1.5g / L potassium dihydrogen phosphate, 3.4g / L dipotassium hydrogen phosphate, 1ml / L trace element solution (2g / L ferrous sulfate heptahydrate, 0.1g / L zinc sulfate heptahydrate, 0.3g / L boric acid, 0.2g / L cobalt chloride hexahydrate, 0.01g / L cupric chloride dihydrate, 0.02g / L nickel chloride hexahydrate, 0.03g / L sodium molybdate), wherein the pH value of the culture medium is adjusted to 6.8, sterilized at 115°C for 30min, and 1.5% agar is added to the solid on the above basis.

[0034] The inorganic salt culture medium is: ammonium sulfate 0.4g / L, ammonium nitrate 1.2g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 1.5g / L, magnesium sulfate 0.5g / L, ammonium chloride 1g / L, sodium chloride 0.5g / L, pH 7.0, sterilized at 121℃ for 30min, and 1.5% agar is added to the solid on the above basis.

[0035] Based on the above process, this example also performed Gram staining identification: after primary staining with ammonium oxalate crystal violet staining solution, mordanting with Lugol's iodine solution, decolorization with 95% ethanol, and re-staining with safranin staining solution, it showed red, and it was found that F13 was a Gram-negative bacterium. Figure 2 As shown. The steps are as follows: 1. Drip water in the center of the slide, then use a pipette to take a small amount of bacterial solution and mix it evenly with the water droplets on the slide, and then apply it into a thin bacterial film; 2. Fix the smear and dry it in the air. Hold one end of the slide with the bacterial film facing up, and pass the slide over a low fire 3 times. After cooling, add dye; 3. Place the slide on the slide rack and add ammonium oxalate crystal violet staining solution (the amount added should cover the bacterial film). Pour off the staining solution and rinse carefully with tap water; 4. Add Lugol's iodine solution, stain for 1-2 minutes, and wash with water; 5. Add 95% ethanol, shake the slide slightly to pour off the ethanol, repeat this 2-3 times, and immediately wash with water to stop decolorization; 6. Re-staining: Add safranin staining solution, stain for 2-3 minutes, and wash with water. Finally, gently dry with absorbent paper; 7. Microscopic examination.

[0036] In this example, DNA of the dimethoate-degrading strain was extracted by taking 10 mL of bacterial solution cultured to the logarithmic growth phase and using a DNA extraction kit (TaKaRa Mini BEST Bacteria Genomic DNA Extraction Kit Ver. 3.0) to extract DNA.

[0037] The DNA samples were reacted with universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TTGGYTACCTTGTTACGACT-3') at 95° for 5 min, 94° for 30 s, 57° for 30 s, and 72° for 90 s and 10 min, respectively; all the above reactions were carried out for 30 cycles.

[0038] PCR electrophoresis: Use 1.5% agarose gel, observe in 1xTAE, 150V, 100mA, 20min electrophoresis environment, the results are as follows Figure 3 , showing that the main band is clear and free of contamination. Bidirectional sequencing: The amplified product was sent to Chuzhou General Biological Company for first-generation bidirectional sequencing. The sequencing results are as follows:

[0039]

[0040]

[0041] The sequencing results were spliced ​​using contingexpress, and the 16srRNA sequences predicted by the genes were compared with the NCBI database using Blast. The parameter identify>95 was set, and then the 16srRNA sequences with higher identify were selected (if insufficient, all were selected). After multiple sequence alignment using muscle software, the phylogenetic tree was constructed using mega software. The results are shown in the figure. Figure 4 shown.

[0042] Determine the growth curve of the strain: Use a spectrophotometer to measure the concentration of the bacterial solution at OD600 nanometers over time. The results are as follows: Figure 5 As shown, the strain grew slowly from 0 to 3 hours and quickly entered the growth phase after 3 hours, indicating that the strain had a short incubation period and could quickly adapt to the growth environment.

[0043] In other embodiments, the method for extracting residual dimethoate is as follows: take 3 ml of the sample and filter it through a 0.22 μm glass fiber filter membrane, activate the sample with 12 mL of dichloromethane on a CleanertFlorisil solid phase extraction column, use a large-volume solid phase extraction device to pass the 3 ml sample through the CleanertFlorisil solid phase extraction column at a rate of 1 mL / min, then rinse the CleanertFlorisil solid phase extraction column with 10 ml of ultrapure water, and then elute with 15 ml of dichloromethane:n-hexane (1:1), blow the collected eluate to dryness with nitrogen, add 1 mL of n-hexane to dissolve it again, pass it through a 0.45 μm organic filter membrane and store it in a brown 1.5 mL injection bottle for machine detection.

[0044] The following method was used to detect residual dimethoate: GC-MS method was used, using the SH-Rxi-5SilMS chromatographic column (specifications: 30 mm × 0.25 μm × 0.25 mm) produced by Shimadzu, Japan, the injection port temperature was set to 250.0 °C, and non-split injection was performed. High-purity helium was used as the carrier gas, and the stable flow rate was set at 1.00 mL / min. The chromatographic temperature program was as follows: the initial temperature was 40.0 °C, maintained for 1 minute, then increased to 290.0 °C at a rate of 10.00 °C per minute and maintained for 1 minute, and then increased to 300 °C at the same heating rate and maintained for 1 minute. The mass spectrometry conditions were configured as follows: electron ionization source (EI), selected ion monitoring mode (SIM), ion source temperature was set to 230 °C, and the interface temperature was set to 250 °C.

[0045] The quantitative analysis was performed using the external standard method. A standard curve was established by preparing a mixed standard solution with a concentration range of 10.00 to 200.00 ng·L-1. The correlation coefficient r 2 All of them were greater than 0.998, ensuring a good linear relationship. The sample spike recovery test was carried out, and the recovery rate was between 80.1% and 91.2%. In each sample batch, a sample spike and blank spike experiment were carried out at the same time, in which the sample spike recovery rate was 82.2% to 93.3%, while the blank spike recovery rate fluctuated between 90.1% and 106.3%. The instrument detection limit was calculated based on 3 times the signal-to-noise ratio (S / N), and the detection limit of each target substance was 0.03 to 0.3ng·L-1.

[0046] Select the in-situ water samples from the sampling site and conduct experiments in groups. Sterilize the in-situ water and add F13 group. Figure 6 As shown, within the first 24 h of the reaction, the amount of dimethoate in the system decreased rapidly, and gradually reached an equilibrium state during the 24-60 h of the reaction. The degradation rate was 88.15% after 60 h of the reaction, indicating that F13 can promote the degradation of dimethoate in the in-situ water sample.

Claims

1. A dimethoate-degrading strain, characterized in that: The individual dimethoate-degrading strain is rod-shaped, and the colony is circular with irregular edges and bulges upward in the middle; the colony has a smooth surface and is white and mucous; The degradation strain was named Enterobacter sp. F13 and was deposited in Guangdong Provincial Microbiological Culture Collection Center, referred to as GDMCC. The deposit date was May 14, 2023, and the deposit registration number was GDMCCN0: 63464.

2. A dimethoate-degrading strain according to claim 1, characterized in that: The in-situ strain of the dimethoate-degrading strain is extracted from sediments in a river buffer zone.

3. A method for preparing the dimethoate-degrading strain as claimed in any one of claims 1 or 2, characterized in that: The following steps are involved: Step 1: Sampling sediment samples in the river buffer zone; Step 2: dilute the sediment sample to obtain a bacterial suspension, further dilute the bacterial suspension and spread it on a complete solid culture medium, invert it and place it in a biochemical incubator for culture at 36-38° C. until a single colony is isolated; Step 3: Pick a single colony and place it in an inorganic salt medium containing 10 mg / L dimethoate, and culture it in a shaker at 37°C and 180 rpm for 3-7 days; inoculate the bacterial solution grown in the 10 mg / L dimethoate inorganic salt medium into the 20 mg / L dimethoate inorganic salt medium, and culture it continuously. Stop acclimation when the final pesticide concentration reaches 100 mg / L; Step 4: Store the strain that can survive on the inorganic salt medium containing 100 mg / L dimethoate in an inorganic salt medium containing 20% ​​glycerol and place it in a -80°C refrigerator.

4. The method for preparing the dimethoate-degrading strain according to claim 3, characterized in that: The complete culture medium comprises glucose, yeast powder, ammonium sulfate, magnesium sulfate heptahydrate, manganese sulfate monohydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and 1 ml / L trace element solution, wherein the pH value of the culture medium is adjusted to 6.8; The complete solid medium is prepared by adding 1.5% agar to the complete medium. The inorganic salt culture medium is: ammonium sulfate, ammonium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ammonium chloride, sodium chloride, wherein the pH value of the culture medium is adjusted to 7.0; The inorganic salt solid culture medium is supplemented with 1.5% agar on the basis of the inorganic salt culture medium.

5. Use of the dimethoate-degrading strain according to claim 1 in bioremediation of polluted areas and biochemical treatment of wastewater.

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