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Low temperature-resistant petroleum-degrading bacillus sp. strain, culture method and application thereof

A technology of petroleum hydrocarbons and degradation rate, applied in the field of biological treatment of environmental pollutants, to achieve the effects of good activity and degradation performance, priority bioavailability, and high-efficiency degradation ability

Inactive Publication Date: 2015-06-17
BEIJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide soil microorganisms capable of efficiently degrading petroleum in view of the problems existing in using microorganisms to remediate petroleum hydrocarbon-contaminated soils under the above-mentioned existing low temperature conditions.

Method used

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  • Low temperature-resistant petroleum-degrading bacillus sp. strain, culture method and application thereof
  • Low temperature-resistant petroleum-degrading bacillus sp. strain, culture method and application thereof
  • Low temperature-resistant petroleum-degrading bacillus sp. strain, culture method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0066] Isolation and domestication of embodiment 1 Bacillus sp.DG24

[0067] The polluted soil near the oil wells of Dagang Oilfield in Tianjin was collected on site. Crude oil was used as the only carbon source. The mother liquid of inorganic salt liquid culture medium was diluted with sterile water to constant volume, and crude oil was added.

[0068] Inorganic salt (10×) liquid medium mother solution (ultrapure aqueous solution): 4g Na 2 HPO 4 , 1.5g KH 2 PO 4 , 1gNH 4 Cl, 0.5g MgSO 4 ·7H 2 O, 0.015g CaCl 2 , 1g NaNO 3 , 0.01g FeSO 4 ·7H 2 O, dilute to 1000ml with ultrapure water;

[0069] Inorganic salt liquid medium: Inorganic salt liquid medium mother solution (10×) 100ml, add 1ml trace elements (add 0.5mg CuSO to 100ml pure water 4 ·5H 2 O, 1.0 mg H 3 BO 3 ,1.0mg MnSO 4 ·5H 2 O, 7.0mg ZnSO 4 Prepared), dilute to 1000ml with ultrapure water, adjust pH=7.0 with 0.2M sodium hydroxide or 0.1M hydrochloric acid;

[0070] Crude oil inorganic salt-containing ...

Embodiment 2

[0077] Preservation and enrichment culture method of embodiment 2 Bacillus sp.DG24

[0078] Test example 2-1 Preservation method of crude oil degrading bacteria Bacillus sp.DG24

[0079] Components of crude oil inorganic salt liquid medium: 0.4g Na per 1000ml 2 HPO 4, 0.15g KH 2 PO 4 , 0.1gNH 4 Cl, 0.05g MgSO 4 ·7H 2 O, 0.0015g CaCl 2 , 0.1g NaNO 3 , 0.001g FeSO 4 ·7H 2 O, trace elements 1ml (add 0.5mg CuSO to 100ml pure water 4 ·5H 2 O, 1.0 mg H 3 BO 3 ,1.0mg MnSO 4 ·5H 2 O, 7.0mg ZnSO 4 prepared), dilute to 1000ml with ultrapure water, and use 2mol·L -1 HCl or 2mol·L -1 NaOH adjusts the pH of the inorganic salt liquid medium to 7.0, and 1 g of sterilized crude oil.

[0080] Emulsified crude oil liquid medium: use a sterilized inoculation loop to pick, purify, and repeatedly domesticate 5 times the Bacillus sp.DG24 colony cultured in the crude oil inorganic salt solid medium by streaking, insert it into the crude oil inorganic salt liquid medium, and put it...

Embodiment 3B

[0085] Embodiment 3Bacillus sp.DG24 is to the optimum environmental condition of crude oil degradation

[0086] By setting different salinity, different pH and different temperature, determine the optimum environmental conditions for Bacillus sp.DG24 to degrade crude oil.

[0087] Test Example 3-1 Effect of salinity on degradation of crude oil by Bacillus sp.DG24

[0088] LB liquid medium: yeast extract powder 5g, peptone 10g, NaCl 5g, distilled water to 1000ml, pH7.0~7.2.

[0089] Crude oil stock solution (20000mg·L -1 ): Add 2g of crude oil into a 150ml ground-neck Erlenmeyer flask, then quantitatively add 100ml of chromatographic grade dichloromethane, seal the bottle with a parafilm, shake it slowly and place it in a 4°C refrigerator for later use.

[0090] NaCl standard stock solution (30% NaCl): add 30g NaCl to a 250ml Erlenmeyer flask filled with 100ml sterile water, stir well with a glass rod and set aside.

[0091] Inorganic salt liquid medium composition: 0.4g Na ...

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Abstract

The invention relates to a low temperature-resistant petroleum-degrading bacillus sp. strain and a culture method and application thereof. The culture method for the low temperature-resistant petroleum-degrading bacillus sp. strain comprises the following steps: dissolving a petroleum-contaminated soil sample with normal saline, successively carrying out shake oscillation and standing for layering, inoculating a bacterium suspension into a crude oil inorganic salt liquid medium, taking a proper amount of lower-layer fermentation liquid in the liquid medium after shake cultivation, inoculating the lower-layer fermentation liquid to a fresh inorganic salt liquid medium for continued culture and repeating previous operation 5 times; subjecting obtained bacterium liquid to gradient dilution, coating the diluted bacterium liquid on crude oil inorganic salt solid mediums for culture, observing the numbers of bacterium colonies of the mediums and choosing a medium with a moderate number of bacterium colonies for streak culture; and repeatedly carrying out domestication and streak culture until a plurality of pure bacterium strains are obtained. The low temperature-resistant petroleum-degrading bacillus sp. strain provided by the invention preferentially degrades medium-long-chain saturated alkane in components of crude oil and is applicable to degradation of petroleum hydrocarbon and bioremediation of soil or water bodies contaminated by petroleum, especially to a low temperature environment.

Description

technical field [0001] The invention belongs to the technical field of biological treatment of environmental pollutants, and in particular relates to a bacterial strain for degrading petroleum hydrocarbons in a low-temperature environment, a cultivation method thereof, and an application thereof in restoration of soil environmental pollution. Background technique [0002] Petroleum is a viscous, flammable, yellow to black mixture of gaseous, liquid and solid hydrocarbons that naturally exist below the surface of the earth and contain various hydrocarbons (normal alkanes, branched alkanes, aromatics , alicyclic hydrocarbons) and a small amount of other organic compounds (sulfides, nitrides, naphthenic acids, etc.) As one of the most important primary energy sources in the world, it is also the raw material of many chemical industrial products such as fertilizers, pesticides, plastics, etc. Petroleum has a very important strategic position and huge consumption. All countries i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A62D3/02C02F3/34B09C1/10C12R1/085A62D101/20
Inventor 王红旗花菲赵一村
Owner BEIJING NORMAL UNIVERSITY
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