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Ralstoniasp. and application thereof in bioremediation of petroleum-contaminated saline-alkali soil

A technology of bacterial strains and microbial strains, applied in the restoration of contaminated soil, methods based on microorganisms, microorganisms, etc., can solve the problems of few studies on halophilic bacteria

Inactive Publication Date: 2011-01-05
BINZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on halophilic microorganisms in artificial environments, especially halophilic bacteria.

Method used

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  • Ralstoniasp. and application thereof in bioremediation of petroleum-contaminated saline-alkali soil
  • Ralstoniasp. and application thereof in bioremediation of petroleum-contaminated saline-alkali soil
  • Ralstoniasp. and application thereof in bioremediation of petroleum-contaminated saline-alkali soil

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Isolation and screening of Ralstonia sp. XB strain provided by the invention.

[0030] Take 1g of oil-contaminated saline-alkali soil sample from the Yellow River Delta, and inoculate it into 100ml of inorganic salt liquid wax medium aseptically. 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 under natural conditions; 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 30° 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 100ml of inorganic salt liquid wax medium as a seed solution at a ratio of 10% (v / v), and cultured with reciprocating shaking on a shaker a...

Embodiment 2

[0033] PCR Amplification and Sequence Determination of 16SrDNA Gene of Ralstonia sp. XB Strain

[0034] CGMCC No.3389 was inoculated in LB medium, cultured on a shaker at 30°C (130rpm) for 16 hours, collected by centrifugation, resuspended, added lysozyme and SDS to break the wall, extracted genomic DNA by phenol-chloroform method, and used positive phase primers 27F (5'-GAG AGTTTGATCCTGGCTCAG-3') and reverse primer 1541R (5'-AAG GAG GTG ATC CAG CC-3'), use this pair of primers for PCR amplification of its 16S rDNA gene, and send the amplification primers to Beijing Sanbo company for sequencing. 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 rDNA gene sequence is 1488bp, and the sequence similarity with Ralstonia pickettii strain ATCC 27511 (Accession No. AY741342) is 99%. The sequence of its 16S rDNA is shown in the sequence listing.

Embodiment 3

[0036] Degradation of polycyclic aromatic hydrocarbons (PHAs) by Ralstonia sp. XB strain at different NaCl concentrations

[0037] Get the fresh slant strain of this bacterial strain, inoculate an inoculation loop in the Erlenmeyer flask that 100ml containing the inorganic salt culture medium (substrate composition is the same as embodiment 1) of naphthalene, anthracene, phenanthrene and pyrene each 50mg / L is housed, cultivate 0-5% NaCl was added to the base, cultured on a shaker at 30°C (130rpm) for 7 days, and the degradation rate of polycyclic aromatic hydrocarbons (PHAs) was determined by gas chromatography (GC method). The results are as follows figure 1 As shown, the degradation rate of strain XB to naphthalene can reach 92.1%, and in the presence of 5% NaCl, the degradation rate is still 14.2%; the degradation rate of anthracene can reach 57.8%, and in the presence of 5% NaCl, the degradation rate decreases. The degradation rate of phenanthrene can reach 87.4%. With the...

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Abstract

The invention discloses a Ralstoniasp. and application thereof in bioremediation of petroleum-contaminated saline-alkali soil. The Ralstoniasp. is collected by China General Microbiological Culture Collection Center (CGMCC), the preservation data is 4 November, 2009, and the preservation number is CGMCCNo.3389. The Ralstoniasp. is grown on an LB solid culture medium to form a white bacterial colony which is round and has regular edge and dry surface and is non-transparent; and the bacterial colony is in a round bar shap, dose not generate spores, is Gram-negative and has the highest growth temperature of 50 DEG C and the NaCl tolerance of 0-5%. The invention also discloses a nucleotide sequence of the Ralstoniasp. The Ralstoniasp. of the invention can degrade pollutants, such as petroleum hydrocarbon, polyaromatic hydrocarbon and the like in soil under the high salt environment; and the crude oil degradation rate can be above 60.0%. Thus, the invention can be applied in the biological control technology of soil pollution brought by polyaromatic hydrocarbon and petroleum.

Description

technical field [0001] The invention belongs to the field of microorganisms and their application, and in particular relates to a Ralstonia sp. and its application in bioremediation of oil-contaminated saline-alkali soil. Background technique [0002] The production and use of oil and its products will inevitably bring about soil pollution problems. For example, about 700 kt of ground crude oil produced by my country's oil companies every year, after recycling, 70 kt still enters the soil environment. Petroleum and petroleum products generally contain toxic and harmful substances such as polycyclic aromatic hydrocarbons (PAHs), benzene, toluene, ethylbenzene, xylene and phenols. Most of these toxic and harmful substances are considered to have "three causes" (carcinogenic, teratogenic, mutagenic) harmful substances, how to control oil pollution, especially the restoration of environmental pollution caused by polycyclic aromatic hydrocarbons, has become a common concern of sc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20B09C1/10A62D3/02C12R1/01A62D101/20
Inventor 姚志刚王君范延辉
Owner BINZHOU UNIV
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