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CN-121991804-A - Separation and screening method for crude oil degradation functional bacteria

CN121991804ACN 121991804 ACN121991804 ACN 121991804ACN-121991804-A

Abstract

The invention is applicable to the technical field of crude oil treatment, and provides a separation and screening method of crude oil degradation functional bacteria; the method comprises the steps of S1, collecting an environment sample polluted by crude oil for a long time, S2, inoculating the environment sample into a gradient carbon source inorganic salt enrichment culture medium for enrichment culture to obtain an enrichment culture solution, S3, carrying out gradient dilution on the enrichment culture solution, then coating the enrichment culture solution on a selective solid culture medium containing crude oil, culturing to obtain single bacterial colonies, S4, picking the single bacterial colonies, inoculating the single bacterial colonies into a liquid culture medium containing crude oil, and measuring the degradation rate of the crude oil, and S5, screening out bacterial strains with the degradation rate of the crude oil higher than a preset threshold value, namely the crude oil degradation functional bacteria. According to the invention, crude oil is taken as the only carbon source, so that the mixed bacteria which can only utilize the easily degradable carbon source are eliminated from the source, the mixed bacteria are enriched to the rare bacterial strain truly provided with the crude oil-enzyme system, the abundance of the target bacteria can be improved by 2-3 orders of magnitude after first passage, and the subsequent purification period is shortened by more than 50%.

Inventors

  • QIU TAO
  • XIA JIAQI
  • LI QING
  • FU CHUNHUI

Assignees

  • 宝利恒(天津)生物科技有限公司

Dates

Publication Date
20260508
Application Date
20251226

Claims (10)

  1. 1. The separation and screening method for the crude oil degradation functional bacteria is characterized by comprising the following steps of: s1, collecting an environmental sample polluted by crude oil for a long time; S2, inoculating the environmental sample into a gradient carbon source inorganic salt enrichment culture medium for enrichment culture to obtain an enrichment culture solution; S3, carrying out gradient dilution on the enrichment culture solution, then coating the enrichment culture solution on a selective solid culture medium containing crude oil, and culturing to obtain single colonies; s4, picking single bacterial colony, inoculating the bacterial colony into a liquid culture medium containing crude oil, and measuring the degradation rate of the crude oil; s5, screening out strains with the crude oil degradation rate higher than a preset threshold value, namely the functional bacteria for degrading the crude oil.
  2. 2. The method for separating and screening functional bacteria for degrading crude oil according to claim 1, wherein the environmental sample is crude oil polluted soil or oily wastewater, and the surface layer sample with the collection depth of 5-20cm is obtained.
  3. 3. The method for separating and screening the crude oil degradation functional bacteria according to claim 1, wherein the inorganic salt enrichment culture medium takes crude oil as a sole carbon source, the concentration of the crude oil increases gradually according to the gradient from 5g/L to 10g/L to 15g/L, and meanwhile, 0.05-0.1g/L of crude oil degradation inducer is added, and the crude oil is continuously enriched for 3-4 times under the corresponding habitat simulation condition, and is cultivated for 4-6 days each time, so that enriched bacterial liquid is obtained.
  4. 4. The method for separating and screening functional bacteria for degrading crude oil according to claim 3, wherein the composition of the inorganic salt enrichment medium is ：Na 2 HPO 4 0.6-0.8g/L、KH 2 PO 4 0.2-0.3g/L、NaN0 3 3.0-4.0g/L、CaCl 2 0.01-0.02g/L、FeSO 4 0.01-0.015g/L、MgSO 4 0.3-0.5g/L,pH to 7.0-8.0.
  5. 5. The method for separating and screening functional bacteria for degrading crude oil according to claim 4, wherein the enrichment culture conditions are a temperature of 25-35 ℃ and a rotation speed of 150-200rpm, and the culture time is 3-7 days.
  6. 6. The method for separating and screening the crude oil degradation functional bacteria according to claim 1 is characterized in that the preparation method of the selective solid culture medium comprises the steps of adding 1.5-2.0% of agar into an inorganic salt enrichment culture medium, sterilizing and solidifying, diluting crude oil to a preset concentration by using normal hexane, uniformly coating the crude oil on the surface of the culture medium, and completely volatilizing the normal hexane to obtain the crude oil degradation functional bacteria.
  7. 7. The method for separating and screening functional bacteria for degrading crude oil according to claim 1, wherein the step S5 of screening out strains with a crude oil degradation rate higher than a preset threshold, namely the functional bacteria for degrading crude oil, comprises the following steps: and screening out strains with the degradation rate of the crude oil higher than 80%, namely the functional bacteria for degrading the crude oil.
  8. 8. The method for separating and screening the functional bacteria for degrading crude oil according to claim 1, further comprising the step of carrying out 16S rRNA gene sequencing identification on the screened functional bacteria and determining the species information of the functional bacteria.
  9. 9. The method for separating and screening the functional bacteria for degrading crude oil according to claim 8, further comprising the step of verifying degradation performance of the screened functional bacteria, and comprising the following steps: Measuring the degradation rate of crude oil under the conditions of different pH values of 5-9, temperature of 20-40 ℃ and salinity of 1-10%; The degradability of the modified cellulose to different crude oils is measured.
  10. 10. The method for separating and screening the functional bacteria for degrading the crude oil according to claim 9, further comprising the steps of constructing a composite bacterial system of the screened functional bacteria, mixing 2-3 strains with the highest degradation rate according to the volume ratio of 1:1-1:3, and measuring the synergistic degradation effect of the 2-3 strains.

Description

Separation and screening method for crude oil degradation functional bacteria Technical Field The invention relates to the technical field of crude oil treatment, in particular to a separation and screening method of crude oil degradation functional bacteria. Background With the increasing severity of petroleum pollution problems, bioremediation technology has received attention for its environmental friendliness and economy. In the prior art, the conventional enrichment medium often contains yeast powder, peptone and other easily available carbon sources, so that fast food type mixed bacteria are excessively proliferated, high-efficiency strains capable of really cracking long-chain alkane and aromatic hydrocarbon are inhibited or submerged, the abundance of target bacteria is low, the subsequent purification period is long, and the success rate is low. In order to solve the technical problem, a separation and screening method for crude oil degradation functional bacteria is provided. Disclosure of Invention The invention aims to provide a separation and screening method of crude oil degradation functional bacteria, which aims to solve the problems in the background technology. In order to achieve the above purpose, the present invention provides the following technical solutions: a separation and screening method of crude oil degradation functional bacteria comprises the following steps: s1, collecting an environmental sample polluted by crude oil for a long time; S2, inoculating the environmental sample into a gradient carbon source inorganic salt enrichment culture medium for enrichment culture to obtain an enrichment culture solution; S3, carrying out gradient dilution on the enrichment culture solution, then coating the enrichment culture solution on a selective solid culture medium containing crude oil, and culturing to obtain single colonies; s4, picking single bacterial colony, inoculating the bacterial colony into a liquid culture medium containing crude oil, and measuring the degradation rate of the crude oil; s5, screening out strains with the crude oil degradation rate higher than a preset threshold value, namely the functional bacteria for degrading the crude oil. As a still further scheme of the invention, the environment sample is crude oil polluted soil or oily wastewater, and the surface layer sample with the depth of 5-20cm is collected. As a still further scheme of the invention, the inorganic salt enrichment culture medium takes crude oil as a unique carbon source, the concentration of the crude oil is gradually increased according to the gradient from 5g/L to 10g/L to 15g/L, and simultaneously, 0.05 to 0.1g/L of crude oil degradation inducer is added, and the inorganic salt enrichment culture medium is continuously enriched for 3 to 4 times under the corresponding habitat simulation condition, and is cultivated for 4 to 6 days each time to obtain enriched bacterial liquid. As a still further aspect of the invention, the composition of the inorganic salt enrichment medium is ：Na2HPO40.6-0.8g/L、KH2PO40.2-0.3g/L、NaN033.0-4.0g/L、CaCl20.01-0.02g/L、FeSO40.01-0.015g/L、MgSO40.3-0.5g/L,pH adjusted to 7.0-8.0. As a still further scheme of the invention, the enrichment culture conditions are that the temperature is 25-35 ℃, the rotating speed is 150-200rpm, and the culture time is 3-7 days. The preparation method of the selective solid culture medium comprises the steps of adding 1.5-2.0% of agar into an inorganic salt enrichment culture medium, sterilizing and solidifying, diluting crude oil to a preset concentration by using normal hexane, uniformly coating the crude oil on the surface of the culture medium, and completely volatilizing the normal hexane to obtain the selective solid culture medium. The invention further provides a further proposal that S5, strains with the crude oil degradation rate higher than a preset threshold are screened out, namely the functional bacteria for crude oil degradation, which comprises the following steps: and screening out strains with the degradation rate of the crude oil higher than 80%, namely the functional bacteria for degrading the crude oil. As a still further scheme of the invention, the separation and screening method of the crude oil degradation functional bacteria further comprises the step of carrying out 16S rRNA gene sequencing identification on the screened functional bacteria and determining the species information of the functional bacteria. As a still further scheme of the invention, the separation and screening method of the crude oil degradation functional bacteria further comprises the step of carrying out degradation performance verification on the screened functional bacteria, and comprises the following steps: Measuring the degradation rate of crude oil under the conditions of different pH values of 5-9, temperature of 20-40 ℃ and salinity of 1-10%; The degradability of the modified cellulose to different crude oils is measured. As a s