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Pseudomonas nitroreducens and application thereof

A technology of pseudomonas and nitro, applied in the field of environmental microorganisms, can solve the problems of high quinoline concentration, increase the difficulty of biotechnology treatment, and the degradation rate of less than 20%, so as to improve the ecological environment, enrich microbial resources, and ensure safety effect

Inactive Publication Date: 2011-05-11
NORTH CHINA ELECTRIC POWER UNIV (BAODING)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A small number of Gram-positive bacteria have a strong ability to degrade quinoline. For example, Lactobacillus sp. Q5 can degrade 72.6% of quinoline with an initial concentration of 500 mg / L within 72 hours. A strain of Bacillus sp. )Q5 reaches 60% to 800mg / L quinoline degradation rate, and limit concentration is 1000mg / L (Fang Miaomiao etc., Petroleum Sinica, 2007,23 (5): 111-116), but degradation rate is less than 20%
In the actual pollution site, the concentration of quinoline is either high or coexists with other pollution sources, which increases the difficulty of biotechnology treatment

Method used

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  • Pseudomonas nitroreducens and application thereof
  • Pseudomonas nitroreducens and application thereof
  • Pseudomonas nitroreducens and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Isolation and purification of Pseudomonas nitroreducens CQ1

[0044] Inoculate 10mL of oxidation pond wastewater from the wastewater treatment system of Beijing Yanshan Petrochemical Company into 100mL enrichment medium shake flasks containing 200mg / L quinoline, and incubate at 30°C and 160rpm on a shaking table for 5 days. The culture liquid in the shake flask was used as the strain, and transferred to the fresh enriched culture liquid containing the same concentration of quinoline, the inoculum size was 5 mL, and the culture was acclimatized again for 5 days, and the transfer was done 3 times. Inoculate 5 mL of the last cultured bacterial liquid into 100 mL of inorganic salt liquid medium containing quinoline 500 mg / L, culture under the same conditions, transfer once every 5 days, and transfer 3 times. Dilute the last culture solution with the enriched culture solution to 10× and 100× bacteria solutions, and spread them on the 500mg / L quinoline inorganic...

Embodiment 2

[0045] Example 2: 16S rDNA Identification of Nitroreductive Pseudomonas (Pseudomonas nitroreducens) CQ1

[0046] Using CTAB method to extract the genomic DNA of CQ1 bacteria as a template, using the conservative primers 27F and 1495R (Lane DJ, Wiley, Chichester, 1991, 115-175) to amplify the full length of bacterial 16S rDNA, amplify about 1.5kb of 16S rDNA of CQ1 strain .

[0047] 27F: 5'-GAG AGT TTG ATC CTG GCT CAG-3' (SEQ ID No.2)

[0048] 1495R: 5'-CTA CGG CTA CCT TGT TAC GA-3' (SEQ ID No.3)

[0049] The PCR reaction components are:

[0050] 10×PCR buffer 5μl

[0051] dNTP (2.5mM) 5μl

[0052] Primer 27F (10μM) 2μl

[0053] Primer 1495R (10μM) 2μl

[0054] gDNA (0.1μg) 1μl

[0055] Pfu Taq (5U / μL) 0.5μl

[0056] wxya 2 O 34.5 μl

[0057] Total volume 50μl

[0058] The PCR amplification reaction conditions were: denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 53°C for 40 s, extension at 72°C for 1 min, and a cycle number of 35; full ex...

Embodiment 3

[0061] Example 3: Detection of the degradation effect of quinoline by Pseudomonas nitroreductor CQ1

[0062] Take the purified strain, inoculate it in LB medium, and cultivate it to OD 600 =0.6, inoculate in 100ml, pH7.5 inorganic salt liquid culture medium by 1: 500 (V: V) inoculum size, wherein quinoline content is respectively 200mg / L, 300mg / L, 400mg / L, 500mg / L, 600mg / L, 700mg / L and 800mg / L. Cultivate on a shaker at 30°C and 170rpm, take out 3ml every 6h to measure OD 600 value. Then the bacterial solution was centrifuged at 12000 rpm for 10 min, and the supernatant was taken for high-pressure liquid chromatography (HPLC). Each treatment was repeated 3 times.

[0063] Detection method:

[0064] 1), determination of quinoline concentration: the chromatographic column adopts Agilent ZORBAX SB-C18 chromatographic column (250 × 4.6mm, 5 μ m); VWD UV-Vis Detector; mobile phase is V methanol: V water = 4: 1, and the flow rate is 1mL / min; the detection wavelength is 275nm, ...

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Abstract

The invention discloses Pseudomonas nitroreducens and application thereof, belonging to the field of environmental microorganism. The Pseudomonas nitroreducens CQ1 of the invention is derived from the sewage of oxidation ponds in the sewage treatment system of Beijing Yanshan Petrochemical Co., Ltd., and obtained by enriching, separating and purifying. The Pseudomonas nitroreducens CQ1 is cultured and collected in the China General Microbiological Culture Collection Center (hereinafter referred to as CGMCC) with the collection number being CGMCC No.3159. The invention further discloses the application of Pseudomonas nitroreducens in the biodegradation of quinoline or derivatives thereof. Quinoline or derivatives thereof with the concentration reaching 700mg / L can be degraded by Pseudomonas nitroreducens of the invention, and the degradability can reach 100% after treating for 72h. Moreover, the Pseudomonas nitroreducens CQ1 of the invention is sensitive to kanamycin sulfate, tetracycline and gentamicin and does not carry plasmids.

Description

technical field [0001] The invention belongs to the field of environmental microorganisms, and in particular relates to a strain of pseudomonas nitroreductor and its application in biodegrading quinoline or its derivatives. Background technique [0002] Quinoline (also known as azanaphthalene) is a typical representative of nitrogen-containing heterocyclic compounds, and it is also the basic structural unit of a large number of complex nitrogen-containing heterocyclic compounds. Quinoline and its derivatives are widely found in natural products such as coal tar, mineral oil, creosote, fossil fuels and plant alkaloids, and are also important refractory organic compounds in various industrial wastewaters such as coking wastewater, rubber wastewater, and pharmaceutical wastewater polluting ingredients. Because of their high water solubility, these compounds have become common pollutants in soil and groundwater, especially near solid waste landfills, steel mills and fossil fuel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A62D3/02C02F3/34C02F101/38A62D101/26C12R1/38
Inventor 张薇
Owner NORTH CHINA ELECTRIC POWER UNIV (BAODING)
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