Method for separating nitrogen-fixing bacteria from petroleum pollutants

A technology for oil pollution and nitrogen-fixing bacteria, applied in microorganism-based methods, biochemical equipment and methods, separation of microorganisms, etc. performance, improved accuracy and separation efficiency

Inactive Publication Date: 2018-10-09
CHINA AGRI UNIV
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  • Application Information

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Problems solved by technology

However, there are many types of autogenous nitrogen-fixing bacteria, including aerobic, anaerobic, micro-aerobic, facultative anaerobic, and photosynthetic bacteria. Because nitrogenase is highly sensitive to oxygen, many nitrogen-fixing bacteria can only be produced under anaerobic or micro-aerobic conditions. Nitrogen fixation, easily missed in conventional separation methods
Therefore, th

Method used

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  • Method for separating nitrogen-fixing bacteria from petroleum pollutants
  • Method for separating nitrogen-fixing bacteria from petroleum pollutants
  • Method for separating nitrogen-fixing bacteria from petroleum pollutants

Examples

Experimental program
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Example Embodiment

[0035] Example 1 The growth of nitrogen-fixing bacteria added with different concentrations of glucose in a nitrogen-free medium

[0036] (1) Bacterial cell culture: The strain Klebsiella sp PJ35, which is determined to have the ability to fix nitrogen, was selected as the experimental material. After the strain was cultured to the logarithmic phase with LB medium, the initial density was adjusted to OD 600 =0.2;

[0037] (2) Detection of growth ability: Inoculate the cultivated nitrogen-fixing bacteria into nitrogen-free medium containing different glucose concentrations. The glucose concentrations are: 10g / L, 30g / L, 50g / L, 80g / L. Measure the OD of the bacteria every two hours 600 value. See the result figure 1 .

[0038] It shows that under the glucose concentration of 30g / L and 50g / L, the growth ability of nitrogen-fixing bacteria in nitrogen-free medium is the best. When the glucose concentration exceeds 80g / L, its growth will be inhibited, while the glucose concentration of 10g...

Example Embodiment

[0039] Example 2 Isolation of nitrogen-fixing bacteria in soil contaminated by the explosion of crude oil leakage from Sinopec’s Huangwei pipeline in Huangdao District, Qingdao

[0040] (1) Sample collection: collect the soil contaminated by the explosion of the Qingdao Huangwei oil pipeline to screen nitrogen-fixing bacteria

[0041] (2) Isolation of nitrogen-fixing bacteria: Weigh 2.5g of soil sample and add 10ml of sterile water to suspend, vortex shaker for 10 minutes to promote dispersion, stand still for 2-5 minutes, absorb the suspension and dilute to 10 -3 After melting the nitrogen-free solid medium, heat it at 55°C, add 1 ml of each dilution to 25ml of the nitrogen-free solid medium, mix well, immediately prepare a plate, and repeat 3 for each dilution. After culturing at 30°C for 2-4 days, there will be colonies growing on the surface or inside of the medium, that is, nitrogen-fixing bacteria in petroleum-contaminated soil are isolated and single colonies are picked to GM...

Example Embodiment

[0049] Example 3 Isolation of Nitrogen Fixing Bacteria in Xinmin High Pour Point Oil-Contaminated Soil

[0050] (1) Sample collection: collect petroleum contaminated soil from oil fields.

[0051] (2) Isolation of nitrogen-fixing bacteria: Weigh 2.5g of soil sample and add 10ml of sterile water to suspend, vortex shaker for 10 minutes to promote dispersion, stand still for 2-5 minutes, absorb the suspension and dilute to 10 -3 After melting the nitrogen-free solid medium, heat it at 55°C, add 1 ml of each dilution to 25ml of the nitrogen-free solid medium, mix well, immediately prepare a plate, and repeat 3 for each dilution. After culturing at 30°C for 2-4 days, there will be colonies growing on the surface or inside of the medium, that is, nitrogen-fixing bacteria in petroleum-contaminated soil are isolated and single colonies are picked to GMCY medium for purification and stored at -80°C.

[0052] (3) Analysis of the phylogenetic status of nitrogen-fixing bacteria: After purificat...

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Abstract

The invention provides a method for separating nitrogen-fixing bacteria from petroleum pollutants, and belongs to the technical field of microorganism separation culture. The method comprises the following steps: (1) collecting the petroleum pollutants, adding water for suspension, and carrying out gradient dilution on suspension liquid; (2) preparing a nitrogen-free solid medium, separately adding suspension liquids with different dilutions when the medium is in a melted state, blending and then standing for culturing, and if bacterial colonies grow on the surface or in the medium, separatingthe bacterial colonies to obtain the nitrogen-fixing bacteria in the petroleum pollutants. The problems that bacteria capable of fixing nitrogen under an aerobic condition only can be obtained by a conventional method, and bacteria capable of fixing nitrogen under an microaerobic or anaerobic condition are easily missing during screening can be solved, diversified demands of the nitrogen-fixing bacteria to oxygen are met, therefore, the accuracy and efficiency of separation of the nitrogen-fixing bacteria from the petroleum pollutants are improved, and the application prospect is wide.

Description

technical field [0001] The invention relates to the technical field of microorganism isolation and cultivation, in particular to a method for isolating nitrogen-fixing bacteria from oil-polluted soil or oil-polluted water. Background technique [0002] Biological nitrogen fixation refers to the process of nitrogen-fixing microorganisms converting nitrogen in the atmosphere into a nitrogen source that can be used by organisms. It can be divided into symbiotic nitrogen fixation, joint nitrogen fixation and autogenous nitrogen fixation. Symbiotic nitrogen fixation generally means that microorganisms live closely with other organisms and form a special symbiotic structure for nitrogen fixation; combined nitrogen fixation means that some autogenous nitrogen-fixing bacteria can colonize the rhizosphere or body of plants, and there is a mutually beneficial relationship with the host. And have a certain specificity; autogenous nitrogen-fixing bacteria usually live independently in t...

Claims

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Application Information

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IPC IPC(8): C12N1/02C12N1/20C12R1/065
CPCC12N1/02C12N1/20
Inventor 王磊陈琳张心昱郑国砥徐志凌
Owner CHINA AGRI UNIV
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