A kind of petroleum degrading bacterium capable of degrading heavy crude oil and its separation method and application

A petroleum-degrading bacteria and petroleum-degrading technology, applied in the field of petroleum-degrading bacteria and its separation, can solve the problems of less attention to degradation of crude oil, limited utilization of petroleum hydrocarbon substrates, and difficult degradation, etc., achieving low application cost and high degradation efficiency , The effect of low process requirements

Active Publication Date: 2022-03-08
SOUTHEAST UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The existing screening of petroleum degrading microorganisms mainly focuses on refined oils such as gasoline and diesel, and less attention is paid to crude oils with more complex components and more difficult degradation
However, previous studies have shown that a single degrading bacteria has limited ability to utilize petroleum hydrocarbon substrates. In nature, no single strain with metabolic ability can degrade all components in crude oil, and crude oil contains colloids. Heavy components with complex structures such as asphaltene and asphaltenes are not easily biodegradable

Method used

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  • A kind of petroleum degrading bacterium capable of degrading heavy crude oil and its separation method and application
  • A kind of petroleum degrading bacterium capable of degrading heavy crude oil and its separation method and application
  • A kind of petroleum degrading bacterium capable of degrading heavy crude oil and its separation method and application

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Experimental program
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Effect test

Embodiment 1

[0040] Domestication, isolation and purification methods of petroleum degrading bacteria:

[0041]1) Put 5g of petroleum-contaminated soil samples into a 50mL centrifuge tube, add 25mL of ultrapure water, and shake on a vortex shaker for 5-10min to fully disperse the soil and let it settle. Among them, the oil-contaminated soil samples came from an oil-contaminated site.

[0042] 2) Take 10 mL of the supernatant and add it to 100 mL of petroleum liquid medium, and culture it on a shaker at 30° C. and 150 r / min for 7 days.

[0043] 3) After 7 days, inoculate at 5%, and re-transfer into fresh petroleum liquid medium. The culture conditions are the same as above, and the enrichment culture is continuously transferred for 3 times.

[0044] 4) Separation is carried out by the dilution coating plate method, and the culture solution is diluted to 10 -3 or 10 -4 Afterwards, 100 μL of the diluted bacterial solution was applied to fresh solid isolation medium.

[0045] 5) Cultivate ...

Embodiment 2

[0049] Molecular biological identification of strains

[0050] 1) Streak culture the preserved strain in LB solid medium, extract the whole genome with a DNA kit after culture, and perform PCR amplification with primers 27-F and 1492-R. The PCR reaction system was 25 μL: 1 μL of upstream and downstream primers (10 μmol / L), 1 μL of DNA template (10 ng / μL), 12.5 μL of 2×Taq Master Mix, and made up to 25 μL with ultrapure water. The PCR reaction conditions were: 94°C for 5 min; 94°C for 1 min, 52°C for 1 min, 72°C for 2 min, 30 cycles; 72°C for 10 min.

[0051] 2) The PCR amplification product was entrusted to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing, and the obtained gene sequence was compared with GenBank to determine its taxonomic status.

[0052] The 16S rRNA gene sequencing result of the strain is shown in SEQ ID NO: 1, the result shows that the gene sequence length is about 1348 (sequence), and the sequence comparison results show that it is similar to the...

Embodiment 3

[0056] Method for Determination of Petroleum Degradation Rate

[0057] 1) Add 0, 0.5, 1, 2, 3, 4mL standard oil solution to 5 volumetric flasks respectively, and dilute to the mark line with petroleum ether.

[0058] Pour the prepared standard solution into a quartz cuvette, use petroleum ether as a reference, and use a UV spectrophotometer to measure the absorbance at a wavelength of 226nm. Take the absorbance as the ordinate, and the concentration of crude oil as the abscissa, and draw after blank correction standard curve line.

[0059] 2) Inoculate the preserved strains back into 100mL petroleum liquid medium, and culture them on a shaking table at 30°C and 150r / min for 7 days (set up a sterile control group at the same time), extract undegraded petroleum in the medium with petroleum ether, and use ultraviolet spectroscopy to Measure the absorbance with a photometer at a wavelength of 226nm, and calculate the remaining oil concentration with reference to the standard curv...

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Abstract

The invention belongs to the technical field of microorganisms and environmental protection, and in particular relates to a petroleum-degrading bacterium capable of degrading heavy crude oil and its separation method and application. The petroleum-degrading bacterium belongs to the genus Ochrobactrum sp. It was preserved in the General Microorganism Center (CGMCC) of the China Microbiological Culture Collection Management Committee (CGMCC) on May 7, 2010. The strain preservation number is CGMCCNo.15739; Crude oil is the only carbon source for growth, and degrades and removes petroleum pollutants, without artificially adding energy, carbon sources, heat sources, etc., with low process requirements, low application costs, and no secondary pollution; heavy crude oil can be degraded within 7 days The degradation rate can reach more than 20%, the degradation efficiency is high, and the petroleum pollutants in the environment can be rapidly degraded, used for the bioremediation of petroleum-contaminated soil or water bodies, and can also be used to prepare composite bacterial agents with other strains for use in Efficient and thorough removal of petroleum pollutants in the environment.

Description

technical field [0001] The invention belongs to the technical field of microorganisms and environmental protection, and in particular relates to a petroleum-degrading bacterium capable of degrading heavy crude oil, a separation method and application thereof. Background technique [0002] Petroleum is one of the most important energy sources in modern society. Leakage accidents in the process of oil exploration, exploitation, storage, transportation and use will cause serious environmental hazards. Petroleum entering the soil will destroy the structure and properties of the soil, reduce soil activity, and destroy the soil ecosystem; some volatile organic compounds in petroleum hydrocarbons will affect human health, and some components have carcinogenic, mutagenic, and teratogenic effects. Traditional physical and chemical methods to solve the problem of oil pollution are not only expensive, but also easy to cause secondary pollution. attention. [0003] Microbial degradat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N1/02C02F3/34B09C1/10C12R1/01
CPCC12N1/02C12N1/20C02F3/34B09C1/10C12R2001/01C12N1/205
Inventor 余冉张晓东祝冲之祝欣石佳奇王磊陈樯段丹阳侯登峰贺梦凡张静董浩宇
Owner SOUTHEAST UNIV
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