One strain of gene recombinant Rhodocoddus erythropolis and its use for removing harmful substance-sulphur and nitrogen in crude oil

A technology of Rhodococcus erythropolis and crude oil, applied in the direction of bacteria, etc., can solve the problems of limiting the application of biological denitrification and loss of combustion value

Active Publication Date: 2006-09-20
上海道多生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This pathway completely metabolizes carbazole, and the application of this strain will lose the combustion value in comparison, so it also limits the application of biological denitrification

Method used

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  • One strain of gene recombinant Rhodocoddus erythropolis and its use for removing harmful substance-sulphur and nitrogen in crude oil
  • One strain of gene recombinant Rhodocoddus erythropolis and its use for removing harmful substance-sulphur and nitrogen in crude oil
  • One strain of gene recombinant Rhodocoddus erythropolis and its use for removing harmful substance-sulphur and nitrogen in crude oil

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Construction of the strain of Rhodococcus erythropolis (Rhodococcus erythropolis) XPDN CCTCC No. M205141.

[0047]Using the polymerase chain reaction (PCR) method, primers P1: 5'-gCCgACTAgTAAggAgATggACgTggCg-3' and primers P2: 5'-gACgAgTACTgCAgCgCCgTCATACgTTgC-3' were obtained from the carbazole-degrading strain Pseudomonas sp. XLDN4- The carbazole degrading gene carABC was obtained in the genome of 9, and the enzyme system encoded by this gene fragment was responsible for degrading carbazole to generate anthranilic acid. The gene fragment was double-digested with SpeI and ScaI and ligated to the plasmid vector pRESQ treated with SpeI and SnaBI double-digested, wherein both the ScaI and SnaBI restriction sites are blunt ends and can be ligated under the action of ligase Together, the recombinant plasmid pCarABC was constructed using the above method, which can express the carABC gene in Rhodococcus erythropolis. The recombinant plasmid pCarABC was introduced...

Embodiment 2

[0056] Example 2: Extraction of the carbazole degradation gene contained in the above-mentioned Rhodococcus erythropolis (Rhodococcus erythropolis) XPDN CCTCC No. M205141.

[0057] Use LB medium to cultivate 5ml of bacterial culture to saturation, take 1.5ml of culture, and centrifuge at 5000 rpm for 5 minutes; add 567μl of TE buffer to the precipitate, and the formula of TE buffer is as follows: 10mmol / L trihydroxy Methylaminomethane (Tris), 1mmol / L ethylenediaminetetraacetic acid (EDTA), adjust the pH to 8.0 with hydrochloric acid, resuspend it by repeated blowing with a pipette, add 30 μl of 10% dodecyl Sodium sulfonate (SDS) and 3μl 20mg / mL proteinase K, mix well, and incubate at 37°C for 2 hours; add 100μl, 5mol / L NaCl, mix well, then add 80μl CTAB / NaCl solution, the CTAB / NaCl solution formula is as follows: the cetyltriethylammonium bromide (CTAB) that mass volume ratio is 10% is dissolved in the NaCl of 0.7mol / L, mixes, and incubates 10 minutes at 65 ℃; chloroform / iso...

Embodiment 3

[0145] Example 3: A method for simultaneously degrading carbazole and dibenzothiophene in a water system by using Rhodococcus erythropolis cells, and the treatment temperature is 30°C.

[0146] The present invention utilizes Rhodococcus erythropolis to simultaneously degrade the application of DBT and carbazole in the water system, and the sequence of steps involved in the method is as follows:

[0147] (1) Strain selection: Rhodococcus erythropolis (Rhodococcus erythropolis) XPDN CCTCC No.M205141;

[0148] (2) Strain culture: Rhodococcus erythropolis (Rhodococcus erythropolis) XPDN CCTCC No.M205141 was inoculated in 100 mL of DMSO liquid inorganic salt basic medium containing 0.008% mass-volume ratio, and shaken for 48 hours at 30°C;

[0149] (3) Collect cells: take the culture medium obtained in step (2) and centrifuge at 4,500 rpm for 10 minutes, collect the precipitated cells, resuspend the cells with pH 7.0 phosphate buffer, and then centrifuge under the same conditions, ...

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Abstract

This invention discloses Rhodococcus erythropolis XPDN strain, which is preserved in China Center for Type Culture Collection (Wuhan University, Wuhan, China) with a serial number: M205141. This invention also discloses the application of Rhodococcus erythropolis in desulfurization and denitrification for crude oil, which comprises the steps of: culturing Rhodococcus erythropolis, collecting Rhodococcus erythropolis, preparing resting cells, treating crude oil samples and inspecting. This invention has such advantages as easy operation and high desulfurization and denitrification capacity, and can degrade dibenzo thiophene and carbazole into 2-hydroxybiphenyl and ortho-aminobenzoic acid, thus have important applications in contamination treatment.

Description

technical field [0001] The present invention relates to a gene recombinant bacterium and its application in the removal of crude oil harmful substances-sulfur and nitrogen, in particular, to a genetically modified Rhodococcus erythrophylla and its application in the removal of crude oil harmful substances- Sulfur and nitrogen applications. Background technique [0002] Although non-hydrocarbon compounds in petroleum do not occupy a dominant position in quantity, their composition and content determine the quality of petroleum. The organic sulfur and organic nitrogen contained in fossil fuel coal and petroleum are important sources of pollution to the environment. When these compounds are burned, they emit a large amount of toxic gases such as sulfur oxides and nitrogen oxides, and the resulting large-scale acid rain seriously pollutes the air and water sources. , destroy the ecological balance, and endanger human existence. Nitrogen-containing compounds in fossil fuels are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12S1/02
Inventor 许平于波马翠卿李福利冯进辉周文娟陶飞王颖蔡晓凤
Owner 上海道多生物科技有限公司
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