An atrazine-degrading bacterium and its application
A technology of atrazine and degrading bacteria, applied in the field of environmental microorganisms
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Embodiment 1
[0016] Example 1: Isolation of Atrazine Degrading Bacteria Y6X-2
[0017] Add 5% of atrazine-contaminated soil samples from the fine-species test field in Jizhou District, Tianjin into 100ml of sterilized 200mg / L atrazine-containing liquid enrichment medium, and shake culture at 30°C and 150rpm. After culturing for 7 days, take out 5ml of the culture medium and transfer it into 100mL of fresh enriched medium containing 200mg / L atrazine, and shake culture at 30°C and 150rpm. After culturing again for 7 days, take 5ml out of the culture medium and transfer it into 100mL fresh enriched medium containing 200mg / L atrazine, and shake culture at 30°C and 150rpm. After continuing to cultivate for 7 days, take the culture solution and dilute it step by step, spread it on the solid enrichment medium plate supplemented with 200 mg / L atrazine, and after culturing at 30°C for a period of time, isolate the colonies that can produce transparent circles for purification. The strain Y6X-2 was...
Embodiment 2
[0020] Example 2 Identification of Atrazine Degrading Bacteria Y6X-2
[0021] Inoculate activated Y6X-2, CGMCC No.15919 in liquid culture medium (peptone 0.5%, sodium chloride 0.5%, yeast powder 0.25%, the rest is water, add 1% filtered sterilized methanol), 30°C, 150rpm shaking culture for 24h. Take 1ml of fresh culture bacteria solution and centrifuge at 4°C and 8000rpm for 5min, collect the bacteria in a 2ml centrifuge tube, and extract DNA using a DNA extraction kit. After electrophoresis detection, the universal primer 8F / 1492R was used for PCR amplification. After electrophoresis detection of PCR products, such as figure 1 The electrophoresis band in lane 4 was used to determine the 16S rDNA gene sequence. The specific sequence is shown in SEQ ID NO: 1, which belongs to Methylopila bacteria after comparison. Morphological characteristics of Y6X-2: short rod shape, no spores, and small white colonies with a diameter of 1-2mm formed on the solid plate. Physiological an...
Embodiment 3
[0024] Example 3: Degradation of Atrazine by Y6X-2, CGMCC No.15919
[0025] The activated Y6X-2, CGMCC No.15919 was inoculated in liquid culture medium (sucrose 130g / L, peptone 2g / L, Na 2 HPO 4 1.4g / L, KH 2 PO 4 0.3g / L, the rest is water, pH 7), shake culture at 30°C and 150rpm for 24h. Fresh bacterial liquid was inoculated at 2% (V / V) into a liquid enrichment medium containing 200 mg / L atrazine, and cultured with shaking at 30° C. and 150 rpm for 7 days. Then extract with an equal volume of dichloromethane for 3 times, discard the aqueous phase, combine the organic phases, dehydrate with anhydrous sodium sulfate, and concentrate to dryness with a rotary evaporator under reduced pressure, use chromatographic acetonitrile to 10 mL, and pass through a 0.22 μm organic filter membrane .
[0026] The residual amount of atrazine was detected using the HPLC method in the literature "HPLC analysis of nicotinic-atrazine oil suspension concentrate". First, the external standard ...
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