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VBNC biphenyl degrading bacterium isolation and screening method and application

A technology for separating, screening, and degrading bacteria, applied in the field of microbial degradation of pollutants, can solve the problems of difficult separation of bacteria, low degradation efficiency, and lack of efficient degradation bacteria, and achieve the effects of low cost, easy access to raw materials, and easy operation

Inactive Publication Date: 2015-04-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a screening method for VBNC biphenyl-degrading bacteria and its application in view of the problems of difficult strain separation, low degradation efficiency and lack of efficient degrading bacteria in the microbial remediation technology of the existing PCBs polluted environment

Method used

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  • VBNC biphenyl degrading bacterium isolation and screening method and application
  • VBNC biphenyl degrading bacterium isolation and screening method and application
  • VBNC biphenyl degrading bacterium isolation and screening method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the preparation of accelerator SRpf

[0038] 1. Source of strains: Micrococcus Luteus IAM14879 can be purchased from the Micrococcus Luteus Culture Collection Center of RIKEN.

[0039] 2. LMM medium: 4.0g / L NH 4 Cl, 1.4g / L KH 2 PO 4 , 0.005g / L biotin, 0.02g / L L-methionine, 0.04g / L vitamin B1, 1.0g / L inosine, 0.03g / L MgSO 4 , 8.75g / L L-lithium lactate, 1.5ml / L mineral salt solution (0.375g / L CuSO 4 ·5H 2 O, 0.785g / L MnCl 2 4H 2 O, 0.18g / L FeSO 4 ·7H 2 O, 0.029g / L Na 2 MoO 4 2H 2 O, 0.089g / L ZnSO 4 ·7H 2 O), the pH is 7.5.

[0040] 3. Pre-fermentation: M.luteus was inoculated on LMM plates from inclined test tubes, and cultured at 30°C for 3 days. Pick 3 rings of M.luteus lawn into a 250mL Erlenmeyer flask filled with 50mL LMM liquid medium, culture at 30°C, 160r / min, for 36h.

[0041] 4. Fermentation culture: take the seed culture liquid and transfer it into a 50 mL Erlenmeyer flask containing 15 mL of LMM culture liquid according to the ino...

Embodiment 2

[0043] Example 2: Enrichment screening and isolation and purification of VBNC biphenyl degrading bacteria

[0044] 1. Source of sediment: The sediment used to isolate VBNC biphenyl-degrading bacteria was taken from a PCB-contaminated area in Luqiao District, Taizhou City, Zhejiang Province.

[0045] 2. Screening culture medium: there are two kinds of screening culture medium, namely the treatment group and the control group, and the promoter SRpf is added to the inorganic salt medium at a volume fraction of 10% to obtain the treatment group culture medium. The culture medium of the control group was the inorganic salt medium without the promoter SRpf. Wherein the composition of the inorganic salt medium is: 1-2g / L of KH 2 PO 4 , K of 2.5-3.5g / L 2 HPO 4 ·3H 2 O, 0.2g / L MgSO 4 , 0.02g / L FeSO 4 ·7H 2 O, 1g / L NaCl, 2-4g / L (NH 4 ) 2 SO 4 , 0.01g / L CaCl 2 , 2mL / L trace salt solution (4mg / L MoO 3 , 28mg / L of ZnSO 4 ·5H 2 O, 0.02mg / L CuSO 4 ·5H 2 O, 4 mg / L of H 3 BO ...

Embodiment 3

[0048] Example 3: Among the VBNC biphenyl-degrading strains, the screening, molecular identification and naming preservation of strain TG9

[0049] 1. Screening of strain TG9: The 11 strains unique to the treatment group were compared with the existing strains for 16S rRNA gene sequence, phenotype, and chemical characteristics, and a new strain TG9 different from the existing strains was screened out.

[0050] 2. Strain TG916S rRNA gene sequence: (1) Use the DNA extraction kit (UNIQ-10 Column Bacterial Genomic DNA Extraction Kit) to extract bacterial DNA and amplify the 16S rDNA of the strain. The 50 μL PCR reaction system includes: EasyTaq SuperMix: 25 μL, 1 μL each of primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1541R (5'-AAGGAGGTGATCCAGCCGCA-3'), template DNA 2 μL, deionized water: 21 μL. The PCR reaction conditions were: pre-denaturation at 95°C for 4 min, denaturation at 95°C for 3 min, annealing at 53°C for 30 s, extension at 72°C for 1 min, and 30 cycles, final extension...

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Abstract

The invention discloses a VBNC biphenyl degrading bacterium isolation and screening method and applications. The method includes the steps that an accelerant SRpf is added into a processing group for enrichment culturing; the accelerant SRpf is not added into a control group; and a special bacterial strain in the processing group is isolated and purified to screen a VBNC state bacterium. A bacterial strain TG9 isolated and screened with the method is characterized in that 16S rRNA is a gene sequence shown in SEQ ID No.1; the bacterial strain TG9 can grow in the wide temperature, pH and salinity range, and can grow with biphenyl as a unique carbon source; meanwhile, the biphenyl tolerance concentration is as high as 3000 mg / L; and low chloro polychlorinated biphenyl and low chloro benzoic acid can also be degraded. In this way, the VBNC state bacterium isolation and screening method can be used for screening efficient biphenyl degrading bacterium groups and can also be used for screening other organic pollutant degrading bacterium groups; and a safe, low-cost and efficient method is provided for exploring a new culture resource and a new environment function bacterium.

Description

technical field [0001] The invention belongs to the field of microbial degradation of pollutants, and in particular relates to a separation and screening method and application of VBNC biphenyl-degrading bacteria. Background technique [0002] There are a large number of microorganisms in the natural environment, and the use of pure culture technology can only isolate and identify 0.01%-10% of the microorganisms in nature, and more than 90% of the microorganisms are living but non-culturable ("viable but non-culturable) ", referred to as VBNC) state and not recognized. VBNC bacteria are the vast majority of microbial resources that have not been developed and utilized in nature. Although the diversity and flora structure information can be obtained based on non-cultivation methods, it is impossible to isolate and purely cultivate the relevant ecological functions and genotypes of the strains. Or missing. The research on the culturability of bacteria in VBNC state will prov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/38C12N1/36C12N1/20A62D3/02C12R1/01A62D101/20A62D101/22
CPCA62D3/02A62D2101/20A62D2101/22C12N1/36C12N1/38C12N1/205C12R2001/01
Inventor 苏晓梅沈超峰刘殷东胡金星秦智慧
Owner ZHEJIANG UNIV
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