Method for high-throughput breeding of petroleum degradation mutant strains

A petroleum degradation and high-throughput technology, which is applied in the field of petroleum hydrocarbon degradation strains and high-throughput breeding of petroleum degradation mutant strains, can solve the problem that the degradation ability of petroleum hydrocarbons is not significantly improved, and achieves the avoidance of mutagenic saturation effect and mutagenic Fast, gentle results

Inactive Publication Date: 2019-01-11
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
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  • Application Information

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Problems solved by technology

This method has strong adaptability and good stability, but the improvement of the petroleum hydrocarbon degradation ability of the strain is not significant

Method used

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  • Method for high-throughput breeding of petroleum degradation mutant strains

Examples

Experimental program
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Effect test

Embodiment example 1

[0030] Comparison of the difference of petroleum hydrocarbon degradation rate between Gordonella strains without mutation and single chemical mutation (Licl)

[0031] The Gordon's strain was inoculated into TSB liquid medium, cultured with shaking at 30°C and 220r for 48h, centrifuged at 10000×g for 2min to collect the bacterial cells, washed twice with sterile normal saline, and made into bacterial suspension. Hemocytometer counts and adjusts the bacterial concentration to 0.7×10 7 / mL for use, inoculate a 1% petroleum-containing inorganic salt liquid medium with a 10% inoculum amount at 30°C and 200r shaking for 10 days to determine the degradation rate of petroleum hydrocarbons by gravimetric method.

[0032] Preparation of TSB medium containing lithium chloride: adding lithium chloride aqueous solution to the TSB solid medium according to a mass fraction of 0%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.5%.

[0033] Take 20 μL of the bacterial suspension and inject them into TSB solid...

Embodiment example 2

[0044] Comparison of the difference of petroleum hydrocarbon degradation rate between Gordonella strains without mutation and single atmospheric pressure plasma mutation

[0045] The Gordon's strain was inoculated into TSB liquid medium, cultured at 30°C and 200r shaking for 48h, centrifuged at 10000×g for 2min to collect the bacterial cells, washed twice with sterile normal saline, and made into bacterial suspension. Hemocytometer counts and adjusts the bacterial concentration to 0.7×10 7 Use about 10% of the inoculum amount to inoculate the liquid medium containing 1% petroleum, shaking culture at 30°C and 200r for 10 days, and measure the degradation rate of petroleum hydrocarbon by gravimetric method.

[0046] Take 10μL of bacterial suspension and inject it on the special iron plate for ARTP mutagenesis system, dry it with aseptic air to form a bacterial membrane, use helium as the working gas, set the gas flow to 10L / min, the working distance of 3mm, and the working power of 12...

Embodiment example 3

[0057] Comparison of the difference of petroleum hydrocarbon degradation rate between Gordonella strains without mutation and Licl-ARTP compound mutation

[0058] The Gordon's strain was inoculated into TSB liquid medium, cultured at 30°C and 200r shaking for 48h, centrifuged at 10000×g for 2min to collect the bacterial cells, washed twice with sterile normal saline, and made into bacterial suspension. Hemocytometer counts and adjusts the bacterial concentration to 0.7×10 7 Use about 10% of the inoculum amount to inoculate the liquid medium containing 1% petroleum, shaking culture at 30°C and 200r for 10 days, and measure the degradation rate of petroleum hydrocarbon by gravimetric method.

[0059] A TSB solid medium with a lithium chloride concentration with a strain lethality of about 80% was prepared.

[0060] Take 10μL of bacterial suspension and inject it on the special iron plate for ARTP mutagenesis system, dry it with aseptic air to form a bacterial membrane, use helium as th...

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Abstract

The invention discloses a method for high-throughput breeding of petroleum degradation mutant strains, which adopts Licl-ARTP combined mutagenesis of Gordon's strain to improve the ability of petroleum hydrocarbon degradation and comprises the steps: preparing bacterial suspension, preparing TSB solid culture medium containing different concentrations of lithium chloride, carrying out single chemical mutagenesis (Licl) and atmospheric pressure plasma mutagenesis (ARTP) on the strain, calculating lethality rate and determining degradation rate of petroleum hydrocarbons of the single mutagenic strain. (4) Licl-ARTP compound mutagenesis. (5) 96-well plate fermentation instead of shake flask fermentation is used to screen mutant strains by ultraviolet spectrophotometry with high throughput. The strain is Gordon's bacillus. The positive mutation rate of the mutant strain is high and the degradation ability of petroleum hydrocarbons is obviously improved. It is beneficial to industrial application and has great significance in economic, environmental and social benefits.

Description

Technical field [0001] The invention belongs to the technical field of mutagenesis and breeding of microbial strains, and relates to a method for breeding petroleum-degrading mutant strains with high throughput. According to this method, petroleum-hydrocarbon-degrading strains with high petroleum-hydrocarbon degradation rate can be selected. Background technique [0002] Petroleum is a complex compound containing a variety of hydrocarbons (normal alkanes, linear alkanes, aromatic hydrocarbons, alicyclic hydrocarbons) and a small amount of other organic compounds (sulfides, nitrides, naphthenic acids). Petroleum contains a variety of harmful substances. Among them, benzene series BTEX (benzene, toluene, ethylbenzene and xylene) and polycyclic aromatic hydrocarbons PAHs (phenanthrene, anthracene, pyrene, etc.) have significant "three causes" (carcinogenic, mutagenic, etc.) , Teratogenic) effect, it is imperative to take effective measures to control oil displacement. The use of Go...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/01C12R1/01
CPCC12N1/20C12N15/01
Inventor 钟成刘正田亚东贾士儒谢燕燕
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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