Salt-tolerant Rhodococcus sp. JH for generating biological emulsifier and degrading alkane and application thereof in bioremediation of petroleum polluted saline-alkali soil

A biological emulsifier and oil pollution technology, applied in the field of microbial biotechnology and environmental biology, can solve the problems of oil pollution and achieve the effect of improving the degradation rate

Inactive Publication Date: 2012-02-01
BINZHOU UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0010] One of the purposes of the present invention is to provide a kind of salt-tolerant Rhodococcus (Rhodococcus sp.) JH and its biological treatment technology for petroleum-contaminated soils for the increasingly serious problem of oil pollution of soil

Method used

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  • Salt-tolerant Rhodococcus sp. JH for generating biological emulsifier and degrading alkane and application thereof in bioremediation of petroleum polluted saline-alkali soil
  • Salt-tolerant Rhodococcus sp. JH for generating biological emulsifier and degrading alkane and application thereof in bioremediation of petroleum polluted saline-alkali soil
  • Salt-tolerant Rhodococcus sp. JH for generating biological emulsifier and degrading alkane and application thereof in bioremediation of petroleum polluted saline-alkali soil

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Isolation and screening of Rhodococcus sp.JH bacterial strain provided by the invention

[0049] Get 1g of the oil-contaminated saline-alkaline soil sample, and inoculate it into 100ml of inorganic salt liquid wax medium aseptically (the medium composition: Na 2 HPO 4 1.5g; KH 2 PO 4 3.48g; MgSO 4 0.7g; (NH 4 ) 2 SO 4 4g; yeast powder 0.01g; liquid paraffin 2% (v / w), distilled water 1000ml; pH natural; sterilized at 121°C for 30min), cultured on a shaker at 37°C for 5 days. The enriched culture solution was thoroughly mixed, 100 μl was taken and spread on a 1.5% LB solid plate, cultured in a constant temperature incubator at 37°C for 2-3 days, and the grown colonies were observed. Single colonies were picked and checked for purity using a microscope. Each grown colony was inoculated into LB medium and cultivated to OD 630 It is about 0.8, and it is inserted into 100 ml of inorganic salt liquid wax medium as a seed solution at a ratio of 10% (v / v), and cultured ...

Embodiment 2

[0053] PCR Amplification and Sequence Determination of 16S rRNA Gene of Rhodococcus sp.JH Strain

[0054] Rhodococcus sp.JH CGMCC No.3388 was inoculated in LB medium, cultured on a shaker at 37°C (130rpm) for 16 hours, collected by centrifugation, resuspended, added lysozyme and SDS to break the wall, and extracted genomic DNA by the phenol-chloroform method. And using the forward primer 27F (5'-GAGAGTTTGATCCTGGCTCAG-3') and the reverse primer 1541R (5'-AAGGAGGTGATCCAGCC-3'), use this pair of primers to perform PCR amplification on its 16S rRNA gene, and send the amplification primers to Shanghai Workers were sequenced. The PCR conditions are: 94°C, 10min; 94°C, 45s, 55°C, 45s, 72°C, 90s, 30 cycles; 72°C, 10min, 4°C storage. The length of the 16S rRNA gene sequence is 1479bp, and the accession number in GenBank is HM134277, which has a sequence similarity of 97% with the 16S rRNA (accession number: NR 026185) of Rhodococcus ruber strain: DSM43338. The sequence of its 16S rRN...

Embodiment 3

[0056] The Rhodococcus sp.JH bacterial strain provided by the present invention degrades alkanes and crude oil from Binnan Oil Production Plant of Shengli Oilfield. Activate the bacterial strain on an LB plate, take a single colony and insert it into 5ml LB liquid medium for overnight culture, and inoculate with 1% inoculum Into 100ml LB liquid culture medium, 37 ℃ of shaking cultures 5h to logarithmic growth phase, inoculate with 100ml inorganic salt culture medium that contains different alkanes or crude oil as carbon source with 10% inoculum size (medium composition is the same as embodiment 1 ) in an Erlenmeyer flask, cultured at 37°C for 3 days, analyzed by gas chromatography, the degradation rate of the strain to alkanes with different carbon numbers, measured the degradation rate of crude oil with infrared oil measuring instrument and carried out gas chromatography of residual crude oil with high pressure gas chromatography analysis, the result is as figure 1 and figu...

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Abstract

The invention belongs to the field of microbial biotechnologies and environmental biotechnologies, in particular relates to salt-tolerant Rhodococcus sp. JH, a biological emulsifier generated by fermenting the Rhodococcus sp. JH and application of the Rhodococcus sp. JH in the bioremediation of petroleum polluted saline-alkali soil. The salt-tolerant Rhodococcus sp. JH is a Rhodococcus sp. JH strain, is preserved in the China general microbiological culture collection (CGMCC) on November fourth, 2009 and has CGMCC No.3388. Biological emulsifier-contained fermenting liquor which is produced by fermenting and culturing the CGMCC No.3388 bacterium provided by the invention in an inorganic salt culture medium using hydrocarbon as a unique carbon source can be directly applied to the petroleum polluted saline-alkali soil to improve the degrading rate of microorganisms on petroleum hydrocarbon and the bioremediation of the petroleum polluted saline-alkali soil.

Description

【Technical field】 [0001] The invention belongs to the field of microbial biotechnology and environmental biotechnology, in particular, it relates to a strain of Rhodococcus sp. Application in repair. 【Background technique】 [0002] With the increasing demand for petroleum products, crude oil mining, processing and transportation activities are becoming more and more frequent. These processes make a large amount of petroleum and its processed products enter the soil, breaking the original ecological balance of nature, and bringing harm to animals, plants and human beings. At present, this has become a worldwide environmental problem. Now the world's total oil production is nearly 3×10 per year 10 tons, and my country's current annual oil production has already exceeded 1×10 8 tons, and the annual newly polluted soil reaches 1×10 5 tons. The Yellow River Delta region is rich in oil and gas resources and is the second largest oil base in my country. The oil wells here are...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20B09C1/10C12R1/01
Inventor 王君范延辉姚志刚
Owner BINZHOU UNIV
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