Method for enriching and separating dominant nitrogen fixing bacteria from petroleum-contaminated soil

A technology of oil pollution and nitrogen-fixing bacteria, applied in the direction of microorganism-based methods, isolated microorganisms, biochemical equipment and methods, etc., can solve the problems of high energy consumption and diversity, reduction of microbial diversity, restrictions, etc., and achieve good application prospects, The effect of promoting nitrogenase activity

Active Publication Date: 2018-10-26
CHINA AGRI UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the presence of various components in petroleum affects and inhibits the growth of most microorganisms including nitrogen-fixing

Method used

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  • Method for enriching and separating dominant nitrogen fixing bacteria from petroleum-contaminated soil
  • Method for enriching and separating dominant nitrogen fixing bacteria from petroleum-contaminated soil
  • Method for enriching and separating dominant nitrogen fixing bacteria from petroleum-contaminated soil

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Nitrogenase activity analysis of nitrogen-fixing bacteria under different oxygen concentration conditions of embodiment 1

[0041] (1) Activation of strains: GMCY medium for aerobic nitrogen-fixing bacteria Azotobacter sp.PJ12, facultative anaerobic nitrogen-fixing bacteria Klebsiella sp.PJ3-5, microaerobic Azospirillum sp.FW2 and facultative anaerobic Pseudoxanthomonassp.FW8 cultured in LB Activation culture at 30°C;

[0042] (2) Pick a single colony of the activated strain and inoculate it into 5ml of liquid medium, and culture it at 30°C and 200rpm (culture for 2 days for FW12, and 12 hours for H5, FW2 and FW8);

[0043] (3) Insert the cultured bacterial solution into 100ml of fresh medium according to the corresponding amount (H5, FW2 and FW8 are inserted into 1% amount, and FW12 is inserted into 4% inoculum amount), and the culture time is as above;

[0044] (4) Collect fresh bacterial liquid and wash once with sterile deionized water;

[0045] (5) Suspend the ba...

Embodiment 2

[0049] Example 2 Isolation of dominant nitrogen-fixing bacteria in Xinmin high pour point oil polluted soil

[0050] (1) Sample collection: oil-contaminated soil was collected from oil fields.

[0051] (2) Enrichment of dominant nitrogen-fixing bacteria: Weigh 5 g of oil-contaminated soil, transfer it to a Hungate anaerobic tube, and then add filter-sterilized glucose concentrate to make the final concentration 0.01 g / g of soil sample, 30 ℃ induction for 2-4 days.

[0052] (3) Infuse 10% volume of acetylene gas (i.e. 1.4ml), take 100μl of gas in 24 hours and measure the ethylene content in the gas by gas spectrometer (SQ-204 type gas chromatograph, FID detector, 2m packed column, built-in GDX502). The purpose of this step is to screen under the condition of carbon and nitrogen imbalance, the high activity of soil nitrogenase means the enrichment of nitrogen-fixing bacteria. This high activity can be the enrichment of strains with high nitrogenase activity or the increase of t...

Embodiment 3

[0065] Example 3 Isolation of dominant nitrogen-fixing bacteria in oil-contaminated soil of Panjin Oilfield

[0066] (1) Sample collection: oil-contaminated soil was collected from oil fields.

[0067] (2) Enrichment of dominant nitrogen-fixing bacteria: Weigh 5 g of oil-contaminated soil, transfer it to a Hungate anaerobic tube, and then add filter-sterilized glucose concentrate to make the final concentration 0.01 g / g of soil sample, 30 ℃ induction for 2-4 days.

[0068] (3) Infuse 10% volume of acetylene gas (i.e. 1.4ml), take 100μl of gas in 24 hours and measure the ethylene content in the gas by gas spectrometer (SQ-204 type gas chromatograph, FID detector, 2m packed column, built-in GDX502).

[0069] (4) Isolation of nitrogen-fixing bacteria: Add 15mL of sterile water to the sample with high soil nitrogenase activity to make soil leachate, vortex for 10 minutes to promote dispersion, stand still for 2-5 minutes, draw the suspension, and perform gradient dilution (10 0...

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Abstract

The invention provides a method for enriching and separating dominant nitrogen fixing bacteria from petroleum-contaminated soil, and belongs to the technical field of microbial isolation and culture.The method comprises the following steps: (1) collecting the petroleum-contaminated soil; adding glucose for induction; measuring the nitrogenase activity of a sample by an acetylene reduction process; selecting the sample with high nitrogen fixing activity or large quantity of nitrogen fixing bacteria; preparing a suspension, and performing gradient dilution; (2) preparing a nitrogen-free solid medium; adding suspensions different in dilution in a molten state of the medium; mixing uniformly and transferring into an anaerobic tube to prepare a thin-layer medium; culturing with 0-2% oxygen concentration, wherein if a colony grows on the surface or the inside of the medium, the dominant nitrogen fixing bacteria enriched in the petroleum-contaminated soil are separated and obtained. According to the method disclosed by the invention, with the imbalance of carbon nitrogen ratio and by adding a quick-acting carbon source, indigenous nitrogen fixing bacteria are activated and enriched; meanwhile, by adopting the nitrogen-free medium and culturing with 0-2% oxygen concentration, the nitrogenase expression is promoted so as to obtain dominant nitrogen-fixing bacteria in the petroleum-contaminated soil; thus, the method has a wide application prospect.

Description

technical field [0001] The invention relates to the technical field of microorganism isolation and cultivation, in particular to a method for enriching and separating dominant nitrogen-fixing bacteria from oil-contaminated soil. Background technique [0002] Biological nitrogen fixation refers to the process of nitrogen-fixing microorganisms converting nitrogen in the atmosphere into nitrogen sources that can be used by organisms. It can be divided into symbiotic nitrogen fixation, joint nitrogen fixation and autogenous nitrogen fixation. Autotrophic nitrogen-fixing bacteria are usually able to fix molecular nitrogen and reduce it to ammonia by living independently in the natural environment or culture medium. [0003] Nitrogen-fixing bacteria are widely distributed in nature, and the analysis of oil-contaminated soil microorganisms shows that the number of nitrogen-fixing bacteria is positively correlated with a certain concentration of oil pollution in the soil. With the ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/02C12R1/065
CPCC12N1/02C12N1/20
Inventor 王磊李唯陈琳张心昱郑国砥张雅茜
Owner CHINA AGRI UNIV
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