Application of Ochrobactrum sp. bacterial (H1) in quinoline degradation

A technology of quinoline and strains, applied in the direction of bacteria, microbe-based methods, microbes, etc.

Active Publication Date: 2013-12-11
CHINA UNIV OF MINING & TECH (BEIJING)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The applicant screened a strain that can efficiently degrade quinoline from the sludge of the secondary sedimentation tank of Shougang coking wastewater, which was identified as Ochrobactrum sp. by 16S rDNA. There have been no related reports on degradation conditions, degradation rate, and degradation kinetics.

Method used

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  • Application of Ochrobactrum sp. bacterial (H1) in quinoline degradation
  • Application of Ochrobactrum sp. bacterial (H1) in quinoline degradation
  • Application of Ochrobactrum sp. bacterial (H1) in quinoline degradation

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Experimental program
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Effect test

Embodiment 1

[0018] The screening of embodiment 1 bacterial strain

[0019] 1. Source and enrichment of strains

[0020] Take 5 mL of sludge from the secondary settling tank of the wastewater treatment system of Shougang Coking Plant, add 50 mL of beef extract liquid medium, and add 50 mg / L quinoline to it. Put it in a constant temperature shaker at 30°C, 150r / min, and culture for 3 days to obtain enriched culture solution.

[0021] Enrichment medium components: beef extract 3g, peptone 10g, NaCl 5g, distilled water 1000mL, pH 7.0.

[0022] Screening of strains

[0023] Put 5 mL of the enriched culture solution into a 10 mL centrifuge tube and centrifuge at 10000 r / min at 23 °C for 5 min in a high-speed refrigerated centrifuge. Take the precipitate and add it to normal saline to mix well, then centrifuge and repeat twice to remove residual medium and secondary metabolites. The precipitated bacterial cells obtained by centrifugation were inserted into 50 mL of the only carbon source med...

Embodiment 2

[0027] The identification of embodiment 2 bacterial strains

[0028] Using the Solarbio Bacteria Genomic DNA Extraction Kit, the genomic DNA of the strain was extracted and used as a template to perform PCR amplification using the 16SrDNA gene universal primers. Primer design for amplifying 16SrDNA:

[0029] F-primerF27: 5'-AGA GTT TGA TCC TGG CTC AG-3'

[0030] R-primer R1492: 5'-GGC TAC CTT GTT ACG ACT T-3'

[0031] Using the total DNA of the bacteria as a template, the reaction system is 50 μL:

[0032]

[0033] The PCR reaction conditions were specifically set as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 55°C for 30 s, extension at 72°C for 90 s, 40 cycles, final extension at 72°C for 5 min, and storage at 4°C. Take 2 μL of the PCR reaction product for 1% agarose gel electrophoresis experiment.

[0034] The obtained 16SrDNA sequence amplified non-purified DNA product was sequenced by Shanghai Sangon Bioengineering Tech...

Embodiment 3

[0035] The optimal condition screening of embodiment 3 bacterial strain H1 degradation

[0036] The conditions that affect the growth of bacteria mainly include temperature, pH value, shaker speed, bacterial inoculum amount, etc. Design an L9 (3^4) orthogonal experiment according to above-mentioned four conditions, every group of experiments is set three parallel, test the degradation rate of 250mg / L quinoline in 24 hours. List the obtained test results (see Table 1). Wherein, Kn (n=1,2,3) represents the sum of the degradation rates of each factor at the same level; kn (n=1,2,3) represents the average number of corresponding K values ​​(kn=Kn / 3); R It is the extreme difference sought for the value of k. The higher the degradation rate of the test index, the better. It can be seen directly from Table 4.1 that the test result of No. 4 test combination condition A3B1C2D3 is the largest, and it is the best effect among the nine tests. In order to find the best conditions, calc...

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Abstract

The present invention relates to an application of bacterial in quinoline degradation, wherein the bacterial is Ochrobactrum sp.. According to the present invention, the optimum degradation conditions are screened, wherein the optimum bacterial H1 growth temperature through utilizing quinoline is 30 DEG C, the initial growth pH value is 5.0, and a shaking table rotation speed is 150 r/min; the bacterial H1 meets a first order kinetic model when the initial quinoline concentration is within 250 mg/L, wherein a first order reaction rate is proportional to a quinoline concentration, the reaction that the bacterial grows through utilizing quinoline belongs to the first order reaction within the concentration range, the specific growth rate is increased along with concentration increase, and bacterial growth is not limited by the concentration within the concentration range; after the bacterial H1 is immobilized by using sodium alginate, a 400 mg/L quinoline degradation rate can be 87.5% within 24h; and the crude enzyme solution in the bacterial cell is extracted, wherein the crude enzyme solution can induce catechol 1,2-dioxygenase (C12O), nitrogen heterocyclic ring is firstly oxidized into quinolone during quinoline metabolism, and then the nitrogen heterocyclic ring is subjected to chain opening to form benzaldehyde, xylene, 2-pentanone, hydroxyphenyl propionic acid and other intermediate products.

Description

technical field [0001] The invention relates to the application of a strain Ochrobactrum sp. in degrading quinoline Background technique [0002] Quinoline is a colorless, hygroscopic liquid with a pungent odor. Soluble in ethanol, ether, benzene, carbon disulfide, slightly soluble in water. Judging from its chemical structure, it belongs to the organic heterocyclic aromatic hydrocarbons, which are formed during the processing of coal, wood and petroleum. Quinoline in coking wastewater is mostly produced in the process of detarring and washing benzene. Quinoline pollution is mainly transmitted by air, water bodies, underground coal mining, etc., and the gas emitted by cigarettes also contains quinoline components. Some researchers have found that the liver is the target organ of quinoline, which has a certain impact on the occurrence of liver cancer. [0003] Although quinoline is toxic, some microorganisms can use it as the only carbon and nitrogen source. Moreover, en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A62D3/02C12R1/01A62D101/26
Inventor 于彩虹胡琳娜黄莹刘畅罗京于广飞王建兵张春辉何绪文
Owner CHINA UNIV OF MINING & TECH (BEIJING)
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