High-temperature-resistant gene engineering bacterium for producing extracellular water-insoluble polysaccharide and application thereof

A technology of water-insoluble polysaccharides and genetically engineered bacteria, which is applied in the application field of selectively plugging high-permeability layers of high-temperature oil reservoirs, high-temperature-resistant extracellular water-insoluble polysaccharide-producing genetically engineered bacteria, and improving oil recovery. Problems such as the limitation of the scope of application in the mine field and the different performance of wild strains, etc., to achieve the effect of improving oil recovery

Inactive Publication Date: 2011-07-20
CHINA UNIV OF PETROLEUM (BEIJING)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The wild strains screened from nature have different properties, and it is difficult to have both excellent oil displacement performance and good environmental adaptability at the same time, and the field application range is often greatly limited.

Method used

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  • High-temperature-resistant gene engineering bacterium for producing extracellular water-insoluble polysaccharide and application thereof
  • High-temperature-resistant gene engineering bacterium for producing extracellular water-insoluble polysaccharide and application thereof
  • High-temperature-resistant gene engineering bacterium for producing extracellular water-insoluble polysaccharide and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the construction of genetically engineered bacteria

[0040] Step 1: Screening of Parental Strains

[0041] The screening method of the recipient strain JD is as follows: according to the conventional strain screening method, the water sample collected from Jilin Oilfield is diluted 10000 times, spread on the agar plate containing 4% (v / v) molasses, and cultivated at 30°C for 24h , the isolated single colonies of various microorganisms were placed in 50mL 4% molasses liquid medium, and anaerobically cultured at 30°C for 1 day to judge the production of insoluble polymers. After repeated isolation and purification, a strain was obtained JD bacteria producing water-insoluble polymers. The strain can utilize monosaccharides to synthesize extracellular water-insoluble polysaccharides at a temperature lower than 37°C.

[0042] The screening method of the donor bacteria GW is as follows: According to the conventional strain screening method, the produced water...

Embodiment 2

[0049] Embodiment 2, JD bacteria and genetically engineered bacteria extracellular water-insoluble polysaccharide hydrolyzate comparison

[0050] Inoculate the LB medium containing 1% (M / V) glucose with JD bacteria and genetically engineered bacteria GJ and cultivate until the end of producing extracellular water-insoluble polysaccharides, centrifuge the fermentation broth at a speed of 3000rpm for 20min, remove the supernatant, and take the centrifugal precipitate Add 2mL of 4% NaOH solution to the substance, pipette and suspend it, remove bacterial protein in a boiling water bath at 100°C for 30 minutes, then centrifuge to collect the precipitate, add an appropriate amount of neutral detergent and heat it in a water bath at 100°C for 1 hour to remove miscellaneous sugars and lipids in the precipitated substance Impurities such as species were washed repeatedly with deionized water, and after drying, cellulase was added to one third of the weight of extracellular water-insolub...

Embodiment 3

[0052] Embodiment 3, the impact of salt concentration on the extracellular water-insoluble polysaccharide produced by genetically engineered bacteria GJ

[0053] The medium is: molasses 4% (V / V), (NH 4 ) 2 HPO 4 0.1% (W / V), NaCl concentration gradients are 15000mg / L, 20000mg / L, 25000mg / L, 30000mg / L respectively, inoculated with activated JD and GJ, the inoculum size is 4%, the cultivation temperature of genetically engineered bacteria GJ The culture temperature of JD was 50°C, and the culture temperature of JD was 37°C, and cultured statically for 5 days.

[0054] After the cultivation, extracellular water-insoluble polysaccharides were obtained by filtering with filter paper, and weighed after drying. The effect of salt concentration on the extracellular water-insoluble polysaccharide produced by genetically engineered bacteria GJ is shown in image 3 , the genetically engineered strain GJ can still produce polysaccharides at a high salt concentration of 30,000mg / L, and ...

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Abstract

The invention provides a high-temperature-resistant gene engineering bacterium for producing an extracellular water-insoluble polysaccharide and application thereof, which relates to the application of a biological engineering technology to microbial enhanced oil recovery. The gene engineering bacterium is a high-temperature-resistant gene engineering bacterium for producing an extracellular water-insoluble polysaccharide and has the collection number of CGMCC No.3909; a recipient strain of the gene engineering bacterium is a JD bacterium (Enterobacter sp.), which is not resistant to high temperature and can produce the extracellular water-insoluble polysaccharide at the temperature of 37 DEG C; and a donor bacterium of the gene engineering bacterium is a GW (Geobacillus kaustophilus) bacterium of a thermophile bacterium. Genome DNA (Deoxyribonucleic Acid) of the GW bacterium is transferred into the JD bacterium by adopting an electric shock transformation method, so that a gene engineering bacterium which can produce the extracellular water-insoluble polysaccharide at the high temperature of over 50 DEG C is obtained. The gene engineering bacterium can be applied to profile control of higher temperature oil reservoirs. The aim of increasing the crude oil recovery ratio can be fulfilled by selectively plugging a high-permeability zone. The invention also provides an effective method for expanding the application range of a bacterial strain applied to microbial enhanced oil recovery.

Description

technical field [0001] The present invention relates to the application of genetic engineering technology in microbial oil recovery breeding, in particular to a high-temperature-resistant extracellular water-insoluble polysaccharide-producing genetically engineered bacterium constructed by an electric shock transformation method, and the genetically engineered bacterium is highly effective in selectively plugging high-temperature oil reservoirs. Permeable layer and its application in enhanced oil recovery. Background technique [0002] Today, with the increasing demand for various energy sources, the world is paying unprecedented attention to oil extraction, and finding effective and cheap new technologies for tertiary oil recovery is the goal that countries have been exploring. Years of experience have proved that the most effective tertiary oil recovery method must be selected in order to ensure the maximum recovery of crude oil under the condition of limited capital const...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/87C12P19/04C12S1/00E21B43/22C12R1/19
Inventor 张忠智孙珊珊罗一菁乔玮岳湘安张立娟
Owner CHINA UNIV OF PETROLEUM (BEIJING)
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