Method for quantifying microorganisms for producing lipopeptide surfactant in microbial flooding reservoir

A surfactant and microbial oil displacement technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problem of poor repeatability, inability to quantitatively determine the amount of surfactant-producing microorganisms, and low accuracy. and other problems, to achieve the effect of simple operation, high sensitivity and accuracy, and strong specificity

Active Publication Date: 2013-02-13
CHINA PETROLEUM & CHEM CORP +1
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Problems solved by technology

The above two documents respectively use the size of the diameter of the oil discharge circle and the size of the surface tension to characterize the amount of biosurfactant, and then qualitatively reflect the amount of microorganisms producing surfactant. The operation method is si...

Method used

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  • Method for quantifying microorganisms for producing lipopeptide surfactant in microbial flooding reservoir
  • Method for quantifying microorganisms for producing lipopeptide surfactant in microbial flooding reservoir
  • Method for quantifying microorganisms for producing lipopeptide surfactant in microbial flooding reservoir

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Embodiment 1

[0047] 1. Design and synthesize primers

[0048] Search for the lipopeptide synthase gene of lipopeptide surfactant-producing microorganisms in GeneBank, compare their homology, select the sequence of the conserved region of the gene, and design primer pairs according to the primer design principles. Bao Bioengineering (Dalian) Co., Ltd. Company Synthesis.

[0049] The sequences of the primer pairs are

[0050] srfAF 5'-CAAAAKCGCAKCATACCACKTTGAG-3'

[0051] srfAR 5'-TCA TAR AGC GGC AYA TAT TGA TGC-3'

[0052] 2. Optimization and establishment of fluorescent quantitative PCR reaction program and reaction system

[0053] The optimization of reaction conditions mainly includes Mg 2+ concentration, primer-probe concentration ratio, and annealing temperature. In the Real-TimePCR reaction program, the reaction system was fixed, different annealing temperatures were adjusted (55°C-65°C), and the same concentration of lipopeptide-producing microorganism Bacillus subtilis TH-2...

Embodiment 2

[0081] Example 2: The comparison between the quantitative method of lipopeptide surfactant-producing microorganisms in microbial flooding reservoirs and the traditional method of measuring oil drainage circle and surface tension to analyze oil reservoir lipopeptide-producing microorganisms

[0082] The fluids produced by 6 oil wells in a certain block of Shengli Oilfield were taken respectively, and after corresponding treatment, they were tested by oil drainage circle method, surface tension test and quantitative analysis of lipopeptide-producing microorganisms.

[0083]

[0084] Table 3 Quantitative detection results of oil drainage ring, surface tension and lipopeptide-producing microorganisms in different oil wells

[0085]

[0086] It can be seen from the results that the oil expulsion circle method and surface tension measurement can indeed reflect the presence or absence of lipopeptide biosurfactant-producing microorganisms in reservoir samples, and these data can b...

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Abstract

The invention relates to a method for quantifying indigenous microorganisms for producing lipopeptide surfactant in microbial flooding reservoir. Primers are designed according to lipopeptide synthetase genes of microorganisms for producing lipopeptide surfactant, and fluorescent quantitative PCR (polymerase chain reaction) is performed. In optimized reaction procedures and reaction system, the lipopeptide synthetase genes obtained from a standard lipopeptide-producing strain is connected with a carrier to construct a recombinant plasmid used as a standard sample for real-time quantitative PCR; the standard sample is subjected to 10-time gradient dilution and used as a template for fluorescent quantitative PCR, and a standard curve is drawn; a microbial flooding reservoir sample is subjected to simple treatment and used as a template for fluorescent quantitative PCR; and whether data of replicate samples are identical and whether initial templates having different numbers of copies have the same amplification efficiency are judged according to linear relationship R2 and amplification efficiency E. When being used for detecting microorganisms for producing lipopeptide surfactant in reservoir under optimized reaction conditions, the lipopeptide synthetase specific primers have higher stability and sensitivity, thereby being widely used in the field of microbial enhanced oil recovery.

Description

technical field [0001] The invention relates to a quantitative method for microorganisms producing lipopeptide surfactants in oil flooding reservoirs, belonging to the field of microbial oil recovery. Background technique [0002] The production of biosurfactants by microorganisms is one of the main mechanisms for microorganisms to enhance oil recovery. Microbial surfactants mainly include lipopeptides and glycolipids. Lipopeptide is one of the most effective biosurfactants reported so far. Due to its special structure, it can help microbial cells adhere to the surface of hydrocarbons to decompose hydrocarbons, reduce surface tension, and maintain Its activity is of extremely important value to the microbial enhanced oil recovery technology. At present, biosurfactants are produced by microorganisms to enhance oil recovery. There are two methods. One is called surface method, which is to inject biosurfactants produced by microbial surface fermentation into oil reservoirs; t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/64C12Q1/06G01N21/64
Inventor 张绍东郝滨宋永亭郭辽原高光军宋智勇刘涛李彩风曹功泽吴晓玲
Owner CHINA PETROLEUM & CHEM CORP
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