Trinitrotoluene degradation strain as well as screening method and application thereof

A technology of trinitrotoluene and a screening method, applied in the field of trinitrotoluene degrading strains and screening thereof, can solve the problems of limited public reports and the like, and achieve the effect of rapid and efficient degradation and excellent microbial germplasm resources

Active Publication Date: 2021-08-06
INST OF GEOGRAPHICAL SCI & NATURAL RESOURCE RES CAS +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the sensitivity of trinitrotoluene, there are few domestic research teams dedicated

Method used

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  • Trinitrotoluene degradation strain as well as screening method and application thereof
  • Trinitrotoluene degradation strain as well as screening method and application thereof
  • Trinitrotoluene degradation strain as well as screening method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1 3

[0054] Example 1 Isolation and screening of trinitrotoluene-degrading bacterial strain BJ2 Pantoea wallsii

[0055] The separation and screening method of the trinitrotoluene-degrading bacterial strain BJ2 Pantoea wallsii of the present embodiment specifically comprises the following steps:

[0056] 1) Collect trinitrotoluene-contaminated soil at a trinitrotoluene-contaminated site in northern China, weigh 5g of the soil and add it to 50mL of M9 medium, and shake it at 30°C and 120rpm for 48 hours to obtain a suspension ;

[0057] 2) Transfer the obtained suspension to a modified M9 medium with trinitrotoluene as the only nitrogen source at a ratio of 10% (volume ratio), and culture at a constant temperature at 30°C and 120 rpm for 48 hours to obtain a bacterial suspension ;

[0058] 3) Carry out subculture three times according to the culture conditions described in step 2), take the culture solution of the last subculture cycle (i.e. the subculture cycle) and apply it to t...

Embodiment 2

[0061] Identification of embodiment 2 purified strains

[0062] In Example 2 of the present invention, trinitrotoluene-degrading bacterial strain BJ2 is obtained from trinitrotoluene-contaminated soil according to the screening method described in Example 1 (the bacterial strain in Example 2 is selected from the colony finally obtained in Example 1 to carry out Subsequent operations), the identification steps of the purified strain of trinitrotoluene-degrading bacterial strain BJ2 are as follows:

[0063] The purified strain BJ2 obtained in Example 1 was subjected to 16S rRNA amplification, using 16S rRNA universal primer (F27 (SEQ ID NO.1): 5'-AGAGTTTGATCMTGGCTCAG-3', R1492 (SEQ ID NO.2): 5 '-TACGYTACCTTGTTACGACT-3') for PCR amplification, the reaction program was: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 30s, 54°C annealing for 30s, 72°C extension for 90s, 24 cycles. The reaction system is: 10×Ex Taq buffer 5.0μL, 2.5mM dNTPMix 4.0μL, 10p Primer1 2.0μL, 10...

Embodiment 3 3

[0068] Example 3 Degradation effect of trinitrotoluene by trinitrotoluene degrading strain BJ2

[0069] Pick a single colony of the trinitrotoluene-degrading strain BJ2 screened in Example 1 and inoculate it in 50 mL of LB medium, enrich and culture it at 30°C and 120 rpm for 18 hours, and centrifuge the obtained culture solution at 4°C and 8000g for 10 minutes , collect the bacteria, wash three times with sterile phosphate buffer, resuspend, adjust OD 600 The value is 0.3, and the bacterial suspension is prepared. Gained bacterium suspension is inoculated into the improved M9 substratum with trinitrotoluene as the only nitrogen source with 10% dosing ratio (volume ratio), and trinitrotoluene concentration is 100mg / L in the nutrient solution; Control group (CK In the group), an equal amount of phosphate buffer was used instead of the bacterial suspension. The above-mentioned culture solution was shaken and cultured at a constant temperature at 30°C and 120 rpm for 72 hours, ...

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Abstract

The invention belongs to the technical field of microbial remediation, and particularly relates to a trinitrotoluene degradation strain as well as a screening method and application thereof. The invention provides a microbial strain capable of efficiently and rapidly degrading trinitrotoluene, and the microbial strain is applied to degradation of trinitrotoluene. The microbial strain is obtained by screening soil in a trinitrotoluene polluted site, and was preserved in China General Microbiological Culture Collection Center on March 23th, 2021, with the preservation number of CGMCC NO.22050. The strain provides a novel microbial germplasm resource for treating water and soil which are polluted by trinitrotoluene.

Description

technical field [0001] The invention belongs to the technical field of microbial remediation, and in particular relates to a trinitrotoluene-degrading bacterial strain and a screening method and application thereof. Background technique [0002] Trinitrotoluene (TNT) is a molecular structure containing energetic groups C-NO 2 The nitrobenzene compound, which can release a large amount of energy in a very short time when excited, is one of the typical energetic compounds. Trinitrotoluene has been used to manufacture various ammunition and explosives since its invention, and is widely used in various military activities and mining activities. The toxicity of trinitrotoluene to organisms is mainly concentrated in the blood, liver, spleen and other immune systems, which can oxidize or complex hemoglobin, cause hemoglobinemia, and cause headache, nausea, vomiting and other symptoms, leading to abnormal liver function and anemia , Seriously can cause cancer. In addition, studie...

Claims

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Application Information

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IPC IPC(8): C12N1/20B09C1/10C12R1/01
CPCC12N1/20B09C1/10
Inventor 廖晓勇罗俊鹏李尤朱勇兵刘晓东
Owner INST OF GEOGRAPHICAL SCI & NATURAL RESOURCE RES CAS
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