High-efficiency extraction of bacterial polycyclic aromatic hydrocarbon metabolites and identification of degradation pathways

A polycyclic aromatic hydrocarbon and pathway technology, applied in the field of biochemical analysis, can solve the problems of long time, no consideration of metabolites, complicated operation, etc., and achieve the effects of complete peak shape, simple and efficient extraction method, and good resolution.

Active Publication Date: 2018-11-16
杭州质谱检测技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above two methods do not consider the metabolites in the bacteria, and part of the extraction method needs to be extracted step by step according to the acidity and alkalinity. The operation is cumbersome and time-consuming, which greatly reduces the extraction efficiency of metabolites.

Method used

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  • High-efficiency extraction of bacterial polycyclic aromatic hydrocarbon metabolites and identification of degradation pathways
  • High-efficiency extraction of bacterial polycyclic aromatic hydrocarbon metabolites and identification of degradation pathways
  • High-efficiency extraction of bacterial polycyclic aromatic hydrocarbon metabolites and identification of degradation pathways

Examples

Experimental program
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Effect test

Embodiment 1

[0111] Embodiment 1, extract the degradation product of Mycobacterium sp.WY6 (CCTCC NO: M2017373) with pyrene as the only carbon source, and carry out the following steps successively:

[0112] 1) Draw 200 μL of 25% (V / V) glycerol to preserve the strains in 20 mL of LB medium, activate and culture at 28°C and 130 rpm for 12 hours; draw 200 μL of activated bacteria liquid into a new 20 mL of LB medium, Cultivate at 130 rpm for 14-16 hours to obtain the bacterial liquid.

[0113] 2) Centrifuge the bacterial liquid at 4°C and 5000 rpm for 10 minutes to obtain degrading bacteria; vortex the obtained bacterial cells with sterile water, resuspend the bacterial cells in sterile water; centrifuge again, repeat this step twice, and finally Bacteria resuspended in sterile water to obtain bacterial suspension;

[0114] 3), adjust bacterial suspension in ultraviolet spectrophotometer OD 600nm =1.0 (at this time, the cell concentration is about 5×10 8 CFU mL -1 );

[0115] 4), draw 1m...

Embodiment 2

[0134] Example 2, extract the degradation product of Massilia sp.WG5 (CCTCC AB 2015362) with phenanthrene as the only carbon source, and perform the following steps in sequence:

[0135] 1) Take 200 μL of 25% glycerol to preserve the strains in 20 mL of LB medium, and culture at 28°C and 130 rpm for 12 hours; draw 200 μL of activated bacteria liquid and transfer it to a new 20 mL of LB medium, and culture at 28°C and 130 rpm for 14 to 16h to obtain the bacterial liquid.

[0136] 2) Centrifuge the bacterial solution at 4°C and 5000 rpm for 10 minutes to obtain degrading bacteria; vortex the obtained bacterial cells with sterile water, resuspend the bacterial cells in sterile water; centrifuge again, repeat this step twice, and finally Body weight suspended in sterile water to obtain bacterial suspension;

[0137] 3) Regulate the bacteria suspension in the OD of the ultraviolet spectrophotometer 600nm =1.0 (at this time, the cell concentration is about 5×10 8 CFU mL -1 );

...

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Abstract

The invention discloses a method for high-efficiency extraction of polycyclic aromatic hydrocarbon metabolites in bacteria and determination of degradation approaches. The method comprises the following steps: culturing degrading bacteria with phenanthrene / pyrene as a sole carbon source; extracting degradation products; and carrying out HPLC detection, determining the peak appearance time of substances and lower detection limit by using a standard sample, carrying out conversion via marked lines so as to obtain the contents of the degradation products, and calculating the proportion of pollutant degradation approaches. The method provided by the invention can be used for assessing the possibility that a bacterial strain has novel degradation approaches and provides a theoretical basis forfurther exploration of degradation approaches and treatment of polycyclic aromatic hydrocarbon pollution with microbes.

Description

technical field [0001] The invention belongs to the field of biochemical analysis, in particular to a method for simply and efficiently extracting degradation products of polycyclic aromatic hydrocarbons in a culture medium and a method for determining the proportion of degradation pathways of polycyclic aromatic hydrocarbons. Background technique [0002] Polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbons, PAHs) refer to compounds in which two or more benzene rings are connected in the form of condensed rings. They are persistent organic pollutants (Persistent Organic Pollutants, POPs) regulated by international conventions, mainly derived from fossil fuels. , gas and coal tar production, wood processing, oil spills, and waste incineration, etc., are carcinogenic, teratogenic, and mutagenic, and can exist in the atmosphere, water, and soil for a long time, bringing harm to human health and the ecological environment. . Therefore, how to control PAHs in the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 汪海珍杨黎楼骏孙姗姗陈远志徐建明
Owner 杭州质谱检测技术有限公司
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