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Method for screening facultative anaerobic bacteria producing biosurfactant

A technology of facultative anaerobic bacteria and biological surface, applied in the field of screening facultative anaerobic bacteria producing biosurfactant, can solve the problems of complicated operation, impure bacterial species, mixed and other problems, achieve simple operation, convenient acquisition, improved The effect of work efficiency

Active Publication Date: 2020-08-18
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005]These screening methods either require molecular biology identification technology, and then need to be equipped with specialized technical equipment and professional technicians, or require strict anaerobic culture conditions, and the operation is complicated; At the same time, there may also be defects such as impure strains, strict anaerobic bacteria (or strict aerobic bacteria) mixed with facultative anaerobic bacteria, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Collect soil samples polluted by crude oil for a long time, add to enrichment medium (composition: NaCl5g, (NH 4 ) 2 SO 4 2.5g, KH 2 PO 4 10.0g, Na 2 HPO 4 4.0g, MgSO 4 ·7H 2 O 0.5g, distilled water 1000mL, crude oil 5g, medium pH 7.2), the shake flask was cultivated in a shaker at 150rpm and a temperature of 30°C for 5 days, and the crude oil in the shake flask obviously emulsified. Take 10 mL of the culture solution and add it to the sterilized enrichment medium for cultivation under the same conditions, so that the enrichment culture is continuously transferred for 3 times, and the enrichment is completed. Take 1mL of the third enrichment culture solution, and serially dilute to 10 -8 Spread on the LB solid medium plate, place the plate in a constant temperature 30°C incubator and cultivate it for 1 day, pick different colonies and connect it to the fermentation medium (composition: glucose 10.0g, (NH 4 ) 2 SO 4 2.5g, KH 2 PO 4 10.0g, Na 2 HPO 4 ...

Embodiment 2

[0031] The soil sample contaminated by the above-mentioned crude oil was added to the enrichment medium (composition: NaCl 5g, (NH 4 ) 2 SO 4 2.5g, KH 2 PO 4 10.0g, Na 2 HPO 4 4.0g, MgSO 4 ·7H 2 O 0.5g, distilled water 1000mL, crude oil 5g, medium pH 7.2), the shake flask was cultivated in a shaker at 200rpm and a temperature of 50°C for 7d, and the crude oil in the shake flask obviously emulsified. Take 10 mL of the culture solution and add it to the sterilized enrichment medium for cultivation under the same conditions, so that the enrichment culture is continuously transferred for 3 times, and the enrichment is completed. Take 1 mL of the third enrichment culture solution, spread it on the LB solid medium plate after dilution, put the plate upside down in a constant temperature 50°C incubator and cultivate it for 2 days, pick different colonies, and transfer them to the fermentation medium (composition : Glucose 10.0g, (NH 4 ) 2 SO 4 2.5g, KH 2 PO 4 10.0g, ...

Embodiment 3

[0033] Collect Huabei Oilfield oil well production fluid sample, add to enrichment medium (composition: NaCl5g, (NH 4 ) 2 SO 4 2.5g, KH 2 PO 4 10.0g, Na 2 HPO 4 4.0g, MgSO 4 ·7H 2 O 0.5g, distilled water 1000mL, crude oil 5g, culture medium pH 7.2), 150rpm, constant temperature 37 ℃ in shaker culture 5d, shake flask crude oil appears obvious emulsification phenomenon. Take 10 mL of the culture solution and add it to the sterilized enrichment medium, and cultivate under the same conditions, so that the enrichment culture is continuously transferred for 3 times, and the enrichment is completed. Take 1mL of the enriched culture solution, spread it on the LB solid medium plate after dilution, place the plate upside down in a constant temperature 37°C incubator and cultivate it for 1.5 days, pick different colonies, and transfer to the fermentation medium (composition: glucose 10.0 g, (NH 4 ) 2 SO 4 2.5g, KH 2 PO 4 10.0g, Na 2 HPO 4 4.0g, MgSO 4 ·7H 2 O 0.5g,...

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Abstract

The invention relates to a method for screening facultative anaerobic bacteria producing biosurfactant, which comprises the following steps: (1) collecting an oil-polluted environment sample, and storing in an environment of 4 DEG C; (2) taking a small amount of samples, adding the samples into an enrichment medium for culture, inoculating the obtained culture solution into a newly prepared enrichment medium for culture, and continuously transferring and culturing for three times; (3) taking the enrichment culture solution transferred for the third time, and coating the enrichment culture solution on an LB plate for culture to obtain a single colony; (4) inoculating the single colony into a fermentation medium for culture, measuring the surface tension of a culture solution, and selectinga strain with the surface tension of the culture solution lower than 30mN / m; and (5) inoculating the selected strain on a crude oil flat plate, and culturing to obtain the strain with an obvious oil discharge ring, namely the anaerobic strain for producing the biosurfactant. Compared with the prior art, the method has the advantage that facultative anaerobic product surfactant strains can be obtained through quick and efficient screening.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a method for screening facultative anaerobes producing biosurfactants. Background technique [0002] Surfactants are an important class of chemical raw materials, which have the functions of emulsification, dispersion, and washing, and have been widely used in industries such as petroleum, cosmetics, and cleaning products. In recent years, with the enhancement of people's environmental awareness, environmental pollution and human injury caused by chemical surfactants have attracted more and more attention, and the limitations of the large-scale use of chemical surfactants have also become increasingly prominent. People place high hopes on biosurfactants that are environmentally friendly and have little harm to the human body. [0003] Biosurfactants are surface-active metabolites produced by microorganisms under certain culture conditions, mainly including phospholipids, glycolipid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/02C12N1/26C12R1/10
CPCC12N1/02C12N1/26
Inventor 牟伯中解胜男刘金峰杨世忠周蕾
Owner EAST CHINA UNIV OF SCI & TECH
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