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
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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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