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A Bordetella petrii NS and applications of an immobilized enzyme thereof in the soil

A technology of immobilizing enzymes and degrading bacteria, applied in the restoration of polluted soil, bacteria, and microorganism-based methods, etc., can solve the problems of remediation of endosulfan, polluted soil, etc. Effect

Inactive Publication Date: 2013-04-24
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no way to remediate endosulfan-contaminated soil except to ban the production and use of endosulfan

Method used

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  • A Bordetella petrii NS and applications of an immobilized enzyme thereof in the soil
  • A Bordetella petrii NS and applications of an immobilized enzyme thereof in the soil
  • A Bordetella petrii NS and applications of an immobilized enzyme thereof in the soil

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Screening and Identification of Endosulfan Degrading Bacteria NS

[0038] Medium:

[0039] Inorganic salt basal medium: 5.8g K 2 HPO 4 , 4.5g KH 2 PO 4 , 2.0g (NH 4 ) 2 SO 4 , 0.16g MgSO 4 , 0.02g CaCl 2 , 0.002g Na 2 MoO 4 , 0.001g FeSO 4 , 0.001g MnCl 2 , 1L of deionized water, adjusted to pH 7.0, and sterilized at 121°C for 30min.

[0040] Medium containing a small amount of carbon source: Add 5.0 g of peptone to the above inorganic salt basic medium, adjust to pH 7.0, and sterilize at 121°C for 30 minutes.

[0041] Separation and purification medium: add endosulfan to the medium containing a small amount of carbon source, so that the concentration of endosulfan in the medium is 100 μg·mL -1, 15.0 g of agar, adjusted to pH 7.0, and sterilized at 121 °C for 30 min.

[0042] LB medium: 10.0g peptone, 5.0g yeast extract, 10.0g NaCl dissolved in 1L deionized water, adjust the pH to 7.0, and sterilize at 121°C for 30min.

[0043] 1) Strain enric...

Embodiment 2

[0051] Example 2 Analysis of endosulfan-degrading bacteria NS16S rDNA sequence

[0052] 1. Extraction of total DNA of endosulfan-degrading bacteria NS

[0053] In the experiment, the MI BIO PowerSoil DNA Isolation Kit was used to extract the total DNA in the soil. The main operation steps were slightly improved on the basis of the original instructions. The specific process is as follows:

[0054] 1) Bacterial culture: Inoculate endosulfan-degrading bacteria NS on LB medium, shake and culture at 30°C for 18 hours;

[0055] 2) Cell collection: Weigh and record the weight of the empty 10ml centrifuge tube. Take 5mL culture solution in a 10mL centrifuge tube, 8000r min -1 Centrifuge for 8 minutes, discard the supernatant, and collect the bacteria. Weigh and record the weight of the 10ml centrifuge tube containing the bacteria again, and the difference between the two weighing values ​​is the weight of the bacteria. Dilute and mix with sterile water: bacteria (mass ratio) 3:1 ...

Embodiment 3

[0083] Embodiment 3: the degradation characteristics of degrading bacteria NS

[0084] Extraction of endosulfan from the separation and purification medium: Inoculate the endosulfan-degrading bacteria NS into the separation and purification medium and cultivate for 5 days, add 5mL of n-hexane, fully oscillate and extract on the vortex mixer, let it stand for stratification, and take the organic phase.

[0085] Determination of endosulfan: gas chromatographic analysis: Shimadzu GC-14C gas chromatograph, capillary column (OV-1701) 0.53mm×30m, FID detector. Injection port temperature 260°C, column temperature 240°C, detector 260°C, N 2 Flow rate 25mL·min -1 , the injection volume is 2 μL.

[0086] The formula for calculating the degradation rate of endosulfan by bacterial suspension:

[0087] R = ρ ck - ρ ρ ck ...

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Abstract

The present invention provides a Bordetella petrii NS and applications of an immobilized enzyme thereof in the soil, wherein the accession number is CCTCC No: M 2012327. The Bordetella petrii NS has the ability of degrading endosulfan in a slightly alkaline environment, and is capable of degrading residual endosulfan in water, soil and other objects safely, efficiently and rapidly, thereby reducing damage to the environment caused by the endosulfan harm, and repairing endosulfan contaminated soil and protecting the environment. The immobilized enzyme prepared by using the strain of the invention is simple in preparation process, low in cost, high in efficiency, and free of secondary pollution, thus having important practical values and good application prospects.

Description

technical field [0001] The invention relates to the application of an endosulfan-degrading bacterium NS and its immobilized enzyme in soil, and belongs to the technical field of biodegradation treatment. Background technique [0002] The chemical name of Endosulfan is 1,2,3,4,7,7-hexachlorobicyclo(2,2,1)hept-2-ene-5,6-bishydroxymethyl sulfite, molecular formula for C 9 h 6 Cl 6 o 3 S, with a molecular weight of 406.91, endosulfan technical is solid at normal temperature and pressure, usually a mixture of two isomers (α-endosulfan and β-endosulfan, the ratio of the two is 7:3), the chemical structure is as follows: [0003] [0004] Pure endosulfan is white crystal, and the mixture is brown crystal. It has the smell of sulfur dioxide; the relative density of endosulfan is 1.745g / cm 3 , the saturated vapor pressure at 25°C is 1.33×10 3 Pa, melting point is 70-100°C; insoluble in water, soluble in organic solvents such as xylene, chloroform, acetone, etc.; unstable i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20B09C1/10C12R1/01
Inventor 朱鲁生孔玲芬王军王金花谢慧王凤花魏坤苏坤昌
Owner SHANDONG AGRICULTURAL UNIVERSITY
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