CN112557386A - Identification method of microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acid - Google Patents

Identification method of microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acid Download PDF

Info

Publication number
CN112557386A
CN112557386A CN202011462881.0A CN202011462881A CN112557386A CN 112557386 A CN112557386 A CN 112557386A CN 202011462881 A CN202011462881 A CN 202011462881A CN 112557386 A CN112557386 A CN 112557386A
Authority
CN
China
Prior art keywords
solution
petroleum
chain fatty
catalytic reaction
long
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202011462881.0A
Other languages
Chinese (zh)
Other versions
CN112557386B (en
Inventor
王恒伟
谢春杨迪
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Ocean University ZJOU
Original Assignee
Zhejiang Ocean University ZJOU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang Ocean University ZJOU filed Critical Zhejiang Ocean University ZJOU
Priority to CN202011462881.0A priority Critical patent/CN112557386B/en
Publication of CN112557386A publication Critical patent/CN112557386A/en
Application granted granted Critical
Publication of CN112557386B publication Critical patent/CN112557386B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/775—Indicator and selective membrane

Landscapes

  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plasma & Fusion (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The invention discloses an identification method of microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids, which comprises the following steps: putting microorganisms into initial catalytic reaction liquid containing petroleum and triton, oscillating for a period of time to obtain final catalytic reaction liquid, absorbing part of the final catalytic reaction liquid into another container, adding a copper amine solution and a long-chain fatty acid extracting agent solution in proportion, oscillating and mixing to obtain a mixed solution, centrifuging to enable the mixed solution to be layered and clarified, absorbing the uppermost layer solution into another container, adding a color developing agent to enable the mixed solution to be red, wherein the thicker the red color shows that the more concentrated the fatty acid substances of the final catalytic reaction liquid are, and the method can be used for identifying the microorganisms for catalyzing alkane molecules in the petroleum to produce the long-chain fatty acid. The identification method can quickly screen out the microorganisms capable of catalyzing the long carbon chain of the petroleum to generate carboxyl, is simple and visual, has low cost, and can simultaneously process a plurality of samples.

Description

Identification method of microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acid
Technical Field
The invention belongs to the field of biochemical engineering, and particularly relates to an identification method of a microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids.
Background
In the screening of petroleum degrading bacteria, the traditional work mainly takes the growth of bacteria in a culture medium containing petroleum as a unique carbon source as an index. For example: the pH tolerance range of the alkali-resistant sea spirillum is 4-11, the alkali-resistant sea spirillum is suitable for living in the sea oil spill environment, and can utilize and degrade petroleum in sea water to realize the microbial degradation of the sea oil spill. For another example, the method for screening petroleum-degrading bacteria uses an enrichment medium, a screening medium and an acclimatization medium which use petroleum as a sole carbon source, and specifically comprises adding petroleum and polysorbate-80 into the culture medium. The method is characterized in that the bacterial strain with petroleum degradation capability obtained by high-throughput primary screening is subjected to cell disruption to prepare a crude enzyme solution, and the crude enzyme solution is prepared by mixing the following raw materials in a volume ratio of 1: (90-110) adding the mixture into a petroleum degradation rate detection system for reaction, and taking the petroleum degradation rate as a screening index to obtain the screened strain, namely the high-efficiency petroleum degradation strain. The following steps are repeated: a method for quickly separating and screening pure petroleum hydrocarbon degrading strains for producing a surfactant from oil-contaminated soil. Firstly, collecting oil contaminated soil from an oil field, adding the oil contaminated soil into a hydrocarbon degradation culture medium which takes crude oil as a unique carbon source and has gradually increased content according to the amount of 5%, and carrying out enrichment culture on a constant-temperature shaking table. Diluting the enriched bacterial liquid to a certain degree, then coating the diluted bacterial liquid on a blood plate for culture, culturing the diluted bacterial liquid at 30 ℃ for 1-3 days, taking bacterial colonies with clear and large transparent ring edges on the blood plate, after culturing the bacterial liquid on a liquid meat peptone culture medium, dipping the bacterial liquid with a blunt end by using a sterilization toothpick for dibbling the bacterial liquid on an oil plate, culturing the bacterial colonies at 30 ℃ for 3 days, taking the bacterial colonies with obvious and large oil-eating spots for inoculation on a meat peptone inclined plane for storage and later use, culturing the bacterial colonies by using the meat peptone culture medium, successively inoculating a hydrocarbon degradation culture medium and a surfactant production culture medium, culturing the bacterial liquid at 30 ℃ for 7 days and 3 days, measuring the hydrocarbon degradation capacity and the surfactant production capacity, and obtaining the surfactant production petroleum hydrocarbon degradation bacterial strain. Therefore, the technical scheme focuses on screening out the microorganisms capable of degrading petroleum.
There is also a method of screening for a microorganism capable of catalyzing petroleum. The method mainly comprises the following steps: mixing an organic nitrogen source, buffer salt, a surfactant and solid gel in water to obtain an aqueous solution, wherein the initial pH of the aqueous solution is 6.0-8.0; sterilizing the aqueous solution at high temperature, and mixing the aqueous solution with petroleum hydrocarbon to obtain a liquid culture medium; mixing the liquid culture medium with a microorganism stock solution containing petroleum catalytic bacteria, and cooling to obtain a solid culture medium; culturing the solid culture medium until macroscopic bacterial colonies appear, and adding pH indicator solutions such as methyl orange, methyl yellow, methyl red and the like. The color change range of the pH indicator is 3.0-5.0, and if the solid culture medium around the bacterial colony changes color, the bacterial colony is a petroleum catalytic bacterium capable of metabolizing petroleum and generating acidic substances. At low concentrations, carboxylic acid content is not sensitive nor quantitative.
In the standard GB/T18609-2011, potassium hydroxide is used for titrating the content of fatty acid in petroleum. The operation characteristics are as follows: the sample was dissolved in a solvent consisting of toluene, isopropanol and a small amount of distilled water and titrated with a standard solution of potassium hydroxide isopropanol on a potentiometric titrator using a glass electrode and an Ag/AgCl reference electrode. And (3) drawing by taking the potential reading as a vertical coordinate and the volume of the standard solution consumed by titration, taking a jump point of a curve as a titration end point, and calculating the acid value of the petroleum. And when no obvious jump point exists on the obtained curve, taking the corresponding potential value reading on the potentiometer as the titration end point. The fatty acid extraction solution used for titration needs at least a glass bubble without a potential probe, which needs at least 3 to 5mL, so that the titration volume of the solution is difficult to operate by adopting a potassium hydroxide titration method, and the titration of trace fatty acid is difficult to realize.
Microorganisms exist in nature and can catalyze long-chain hydrocarbon molecules in petroleum to produce fatty acids with terminal carboxylic acid groups. In production practice, the function of such microorganisms is identified by quantifying the total carboxyl number of the fatty acids without the need for expensive liquid or gas phase means. However, the current method is lack of accurate, convenient and intuitive technical method, which can extract trace amount of long-chain fatty acid in water phase and petroleum mixed liquor and carry out color development and quantification of total fatty acid, so that the catalytic capability of the microorganism cannot be simply and conveniently identified.
Therefore, it is necessary to provide a further solution to the above problems.
Disclosure of Invention
The invention aims to provide a method for identifying microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids, and solves the problems.
The technical scheme of the invention is as follows:
a method for identifying a microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids comprises the following steps:
(1) mixing petroleum, triton and water to obtain initial catalytic reaction liquid;
(2) putting microbial thalli to be identified into the initial catalytic reaction liquid, and oscillating to obtain a final catalytic reaction liquid;
(3) dissolving triethanolamine and copper nitrate into water to obtain a copper amine solution;
(4) mixing chloroform and petroleum ether to obtain a long-chain fatty acid extractant;
(5) weighing 1,5-diphenyl carbonyl dihydrazide and symmetric diphenyl azocarbonyl hydrazide, and dissolving in methanol to obtain a color developing agent;
(6) absorbing the final catalytic reaction liquid into a container, adding the copper amine solution and the long-chain fatty acid extracting agent, vibrating, mixing and standing to obtain a final mixed liquid;
(7) centrifuging the final mixed solution to obtain liquid layers, namely an upper layer solution and a lower layer solution;
(8) and sucking the upper solution in the liquid layer into another container, adding the color developing agent to obtain a color developing solution, and judging the concentration of the long-chain fatty acid in the final catalytic reaction liquid according to the red shade of the color developing solution.
Further, in the step (1), the triton is selected from any one or more of X-100, X-114 or X-405, and the concentration of the triton in the initial catalytic reaction liquid is 0.1-10 g/L.
Further, in the step (3), the concentration of the triethanolamine in the copper amine solution is 90-180%
g/L, and the pH value of the copper amine solution is 7.7-8.5.
Further, the volume ratio of the chloroform to the petroleum ether in the step (4) is 1: 2-1: 20.
Further, in the step (7), the upper solution is a long-chain fatty acid extracting agent containing chloroform and petroleum ether.
The invention provides a method for identifying microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids, which has the following advantages:
(1) the sensitivity is high, and the content of fatty acid with the concentration as low as 0.025mM can be detected at the lowest. The invention utilizes a color development determination method, and the basic principle is that free long-chain fatty acid and alkaline copper salt are combined into a copper soap compound, copper ions in the copper soap compound are further complexed with a color developing agent in a proper organic solvent to form a red product, and the concentration of the red product has good linearity at a light absorption value of 0.1 to 1.7. When 1mL of the sample of the catalytic reaction solution was used, a concentration of 0.025mM was detected as a minimum, which corresponds to a total amount of 25nmol of long-chain fatty acids. Therefore, the method can easily realize trace quantification of the fatty acid;
(2) the amount of sample required is small, as low as 50. mu.L. Because the light absorption value of the color developing solution has good linearity from 0.1 to 1.7, when the content of fatty acid in a catalytic reaction solution sample exceeds 0.5mM, the light absorption value of the final color developing solution can be up to more than 0.20 by using 50 mu L of the catalytic reaction solution sample by adopting the technology of the invention, thereby being capable of quantifying extremely tiny samples;
(3) tens of samples containing long-chain fatty acids can be detected simultaneously, and the detection operation time is short. By adopting the technical method, only mu L-level catalytic reaction liquid is needed, the cuprammonium solution and the long-chain fatty acid extracting agent are added, the mixture is vibrated and mixed and centrifuged, the mixed solution can be layered and clarified, the mL-level uppermost layer solution is absorbed into another container, the color developing agent is added to be red, and finally the spectrophotometry is carried out for determination. As can be seen from the above steps, one person can simultaneously operate the detection of ten samples, and the quantitative time of the long-chain fatty acid can be shortened to be within 30 minutes;
(4) the invention can also judge the activity of the petroleum catalytic bacteria qualitatively by naked eyes by displaying the red shade. Specifically, the catalytic reaction liquid is developed by the method provided by the invention, and the shorter the time for color change is, the darker the red color is, which indicates that the capability of the microorganism for catalyzing petroleum to produce the long-chain fatty acid with the carboxylic acid group is stronger. The technology makes it possible to observe the ability of microbe to catalyze petroleum alkane to produce fatty acid by naked eyes, and avoids the adoption of expensive, time-consuming and labor-consuming means such as liquid phase or gas chromatography for screening and identification.
Drawings
FIG. 1 is a graph showing the peak shape scan of a developing solution in examples 1 to 8 according to the method for identifying a microorganism capable of catalyzing the production of a long-chain fatty acid from an alkane molecule in petroleum according to the present invention;
FIG. 2 is a schematic diagram showing the color stability of the microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids according to the method for identifying microorganisms capable of catalyzing alkane molecules in petroleum in examples 1 to 8;
FIG. 3 is a standard curve diagram of the method for identifying a microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids in example 6.
Detailed Description
A method for identifying a microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids comprises the following steps:
the method comprises the following steps: preparing an aqueous solution containing 0.1-10 g/L of triton and 0-5.0 g/L of organic nitrogen source at the lowest, sterilizing the aqueous solution containing the triton and the organic nitrogen source at high temperature, cooling to room temperature, and then adding 0.5-80 g/L of petroleum to obtain an initial catalytic reaction solution, wherein the petroleum is petroleum containing C5-C24 long-chain alkane and extracts thereof, including crude oil, diesel oil, gasoline, kerosene and the like, the organic nitrogen source is used for supporting the growth of microorganisms, one or more of peptone, yeast powder, yeast extract powder and yeast extract can be adopted, and the organic nitrogen source can also be derived from components carried by a microorganism sample;
step two: putting microbial thalli to be identified into the initial catalytic reaction liquid, and oscillating for a certain time to carry out dispersion and catalytic reaction to obtain final catalytic reaction liquid, wherein the microbial thalli to be identified can be from a fermentation liquid or a microbial colony on a solid culture medium or a solid or liquid sample containing microorganisms;
step three: dissolving triethanolamine and copper nitrate in water to obtain copper amine solution, wherein the copper nitrate is copper nitrate trihydrate and has a molecular formulaIs Cu (NO)3)2·3H2O, the final concentration of the copper ammonium salt in the water solution is 20-45 g/L, the concentration of triethanolamine is 90-180 g/L, and the mixture ratio of the triethanolamine and the copper nitrate is such that the pH value of the copper ammonium salt solution is 7.7-8.5;
in some embodiments, the concentration of copper nitrate in this step may be 30g/L and the concentration of triethanolamine may be 112g/L, when the pH of the copper amine solution is 8.1 + -0.1.
Step four: mixing chloroform and petroleum ether at a volume ratio of 1: 2-1: 20 to obtain a long-chain fatty acid extractant, wherein the higher the ratio of the petroleum ether is, the lower the density of the fatty acid extractant is, and the higher the density of the fatty acid extractant is, the longer the fatty acid extractant is, the higher the density of the fatty acid extractant is, the higher the fatty;
step five: weighing 1 part by weight of 1, 5-diphenylcarbonyldihydrazide and 19 parts by weight of symmetric diphenylazocarbonyl hydrazide, and dissolving in 4000 parts by volume of methanol to obtain a color developing agent;
in this step, 1, 5-diphenylcarbodihydrazide, also known as 1, 5-diphenylcarbazide, is known by the british name 1,5-Diphenylcabohydrazide, symmetric diphenylazocarbonyl hydrazide, also known as symmetric Diphenylcarbazone, is known by the british name Diphenylcarbazone.
Step six: absorbing a part of final catalytic reaction liquid into a container, adding a copper amine solution and a long-chain fatty acid extracting agent, inverting the container from top to bottom, shaking and mixing for 100-300 times, and standing for 1-30 minutes to obtain a final mixed liquid;
step seven: centrifuging the final mixed solution to change the turbid liquid into clear liquid and visually detect liquid layering, wherein the liquid layering can be performed in a 5mL plastic centrifuge tube, the rotating speed of a desk centrifuge is set to be more than 4000rpm, and the centrifuging time is at least 20 sec;
step eight: and (3) sucking a part of uppermost layer solution in the liquid layering into another container, and adding a color developing agent to obtain a color developing solution, wherein the thicker the red color of the color developing solution is, the thicker the long-chain fatty acid substances in the final catalytic reaction solution is.
In the step, 2.0mL of the uppermost layer solution can be absorbed to be mixed with 1.0mL of the color developing agent to develop red, so that the light absorption value after color development can be conveniently read by using a common cuvette with an optical path of 1 cm.
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanying the present invention are further described below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.
Reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic may be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
The embodiment shows a method for identifying microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids according to the following steps:
1) obtaining a catalytic reaction solution: adding 0.1g of triton X-114 into 1L of tap water to prepare an aqueous solution containing 0.1g/L of triton X-114, subpackaging and shaking the flask by 100 mL/bottle, sterilizing the flask by steam at 121 ℃ for 20min, adding 62 mu L/bottle of diesel oil before use to enable the final concentration to reach 0.5g/L to obtain an initial catalytic reaction solution, then adding 1mL of a microbial liquid sample to be identified, oscillating the sample in a shaking table at room temperature, and carrying out catalytic reaction for 5 hours to obtain a final catalytic reaction solution.
2) Preparing a copper amine solution: 40g of Cu (NO) are weighed out3)2·3H2Dissolving O in 1L of deionized water to obtain 40g/L of copper nitrate aqueous solution, weighing 180g of triethanolamine, metering the volume to 1L of deionized water to obtain 180g/L of triethanolamine aqueous solution, mixing the two aqueous solutions in equal volume, and obtaining the copper amine solution, namely the copper nitrate solution containing 20g/L and the triethanolamine solution containing 90g/L, wherein the final pH value is 8.2 +/-0.1.
3) Preparing a long-chain fatty acid extracting agent: mixing 100mL of chloroform and 200mL of petroleum ether, wherein the volume ratio of the chloroform to the petroleum ether is 1: 2.
4) Preparing a color developing agent: 0.10g of 1, 5-diphenylcarbonyldihydrazide and 1.9g of symmetric diphenylazocarbonyl hydrazide were weighed and dissolved in 400mL of methanol to obtain a color developer.
5) Absorbing 1mL of final catalytic reaction liquid into a 15mL clean centrifuge tube with a plug, adding 1mL of cuprammonium solution and 6mL of long-chain fatty acid extracting agent, reversing the mixture from top to bottom for about 200 times to obtain a mixed solution, standing for 5 minutes, centrifuging at 5000rpm for 20min to enable the mixed solution to be layered and clarified, absorbing 2mL of the uppermost layer solution into another glass test tube, adding 1mL of color developing agent, mixing and standing for 1 minute, measuring the light absorption value after color development at the wavelength of 530nm, and selecting the microorganism corresponding to the final catalytic reaction liquid with higher light absorption value.
Example 2
The embodiment shows a method for identifying microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids according to the following steps:
1) obtaining a catalytic reaction solution: adding 2g of triton X-114 into 1L of tap water to prepare an aqueous solution containing 2g/L of triton X-114, subpackaging and shaking 100 mL/bottle, sterilizing by steam at 121 ℃ for 20min, adding 0.6 mL/bottle of kerosene before use to enable the final concentration of the initial catalytic reaction liquid to reach 5g/L, then adding 5mL of a microbial liquid sample to be identified, shaking in a shaking table at room temperature, and carrying out catalytic reaction for 12 hours to obtain the final catalytic reaction liquid.
2) Preparing a copper amine solution: 40g of Cu (NO) are weighed out3)2·3H2Dissolving O in 1L of deionized water to obtain 40g/L of copper nitrate aqueous solution, weighing 224g of triethanolamine, metering the volume to 1L of deionized water to obtain 224g/L of triethanolamine aqueous solution, mixing the two aqueous solutions in equal volume, and obtaining the copper amine solution, namely the copper nitrate solution containing 20g/L and 110g/L of triethanolamine, wherein the final pH value is 8.4 +/-0.1.
3) Preparing a long-chain fatty acid extracting agent: mixing 500mL of chloroform and 2000mL of petroleum ether, wherein the volume ratio of the chloroform to the petroleum ether is 1: 4.
4) Preparing a color developing agent: 0.10g of 1, 5-diphenylcarbonyldihydrazide and 1.9g of symmetric diphenylazocarbonyl hydrazide were weighed and dissolved in 400mL of methanol to obtain a color developer.
5) Sucking 0.5mL of final catalytic reaction liquid into a 5mL clean centrifuge tube with a plug, adding 0.5mL of cuprammonium solution and 3mL of long-chain fatty acid extracting agent, reversing the mixture up and down for about 300 times to obtain a mixed solution, standing for 15 minutes, centrifuging at 8000rpm for 0.5min to allow the mixed solution to be layered and clarified, sucking 2mL of uppermost layer solution into another glass test tube, adding 1mL of color developing agent, mixing and standing for 5 minutes, measuring the light absorption value after color development at 530nm, and selecting the microorganism corresponding to the final catalytic reaction liquid with higher light absorption value.
Example 3
The embodiment shows a method for identifying microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids according to the following steps:
1) obtaining a catalytic reaction solution: adding 10g of triton X-100 into 1L of tap water to prepare an aqueous solution containing 10g/L of triton X-100, subpackaging and shaking 100 mL/bottle, sterilizing with steam at 121 ℃ for 20min, adding 8.8 mL/bottle of diesel oil before use to enable the final concentration of the initial catalytic reaction liquid to reach 70g/L, then adding 5mL of a microbial liquid sample to be identified, shaking in a shaking table at room temperature, and carrying out catalytic reaction for 48 hours to obtain the final catalytic reaction liquid.
2) Preparing a copper amine solution: 9g of Cu (NO) was weighed3)2·3H2O, using deionized water to fix the volume to 100mL to obtain 90g/L copper nitrate aqueous solution, weighing 22.4g of triethanolamine, using deionized water to fix the volume to 100mL to obtain 224g/L triethanolamine aqueous solution, mixing the two aqueous solutions in equal volume, wherein the obtained copper amine solution contains 45g/L copper nitrate and 110g/L triethanolamine, and the final pH value is 7.7 +/-0.1.
3) Preparing a long-chain fatty acid extracting agent: mixing 50mL of chloroform and 500mL of petroleum ether, wherein the volume ratio of the chloroform to the petroleum ether is 1: 10.
4) Preparing a color developing agent: 0.05g of 1, 5-diphenylcarbonyldihydrazide and 0.95g of symmetric diphenylazocarbonyl hydrazide were weighed and dissolved in 200mL of methanol to obtain a color developer.
5) Absorbing 1mL of final catalytic reaction liquid into a 15mL clean centrifuge tube with a plug, adding 1mL of cuprammonium solution and 4mL of long-chain fatty acid extracting agent, reversing the mixture from top to bottom for about 100 times to obtain a mixed solution, standing for 10 minutes, centrifuging at 8000rpm for 2min to stratify and clarify the mixed solution, absorbing 2mL of the uppermost layer solution into another glass test tube, adding 1mL of color developing agent, mixing and standing for 10 minutes, measuring the light absorption value after color development at 530nm wavelength, and selecting the microorganism corresponding to the final catalytic reaction liquid with higher light absorption value.
Example 4
The embodiment shows a method for identifying microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids according to the following steps:
1) obtaining a catalytic reaction solution: adding 5g of triton X-100, 5g of triton X-405 and 0.2g of yeast extract powder into 1L of tap water to prepare an aqueous solution containing 5g/L of triton X-100, 5g/L of triton X-405 and 0.2g/L of yeast extract powder, subpackaging and shaking 100 mL/bottle, carrying out steam sterilization at 115 ℃ for 30min, adding 5 mL/bottle of gasoline before use to enable the final concentration of an initial catalytic reaction solution to reach 38g/L, then adding 5mL of a microbial liquid sample to be identified, shaking the mixture in a shaking table at room temperature, and carrying out catalytic reaction for 24 hours to obtain a final catalytic reaction solution.
2) Preparing a copper amine solution: 60g of Cu (NO) are weighed out3)2·3H2Dissolving O in 1L of deionized water to obtain 60g/L of copper nitrate aqueous solution, weighing 224g of triethanolamine, metering the volume to 1L of deionized water to obtain 224g/L of triethanolamine aqueous solution, mixing the two aqueous solutions in equal volume, and obtaining the copper amine solution, namely the copper nitrate solution containing 30g/L and 110g/L of triethanolamine, wherein the final pH value is 8.1 +/-0.1.
3) Preparing a long-chain fatty acid extracting agent: mixing 25mL of chloroform and 500mL of petroleum ether, wherein the volume ratio of the chloroform to the petroleum ether is 1: 20.
4) Preparing a color developing agent: 0.05g of 1, 5-diphenylcarbonyldihydrazide and 0.95g of symmetric diphenylazocarbonyl hydrazide were weighed and dissolved in 200mL of methanol to obtain a color developer.
5) Absorbing 1mL of final catalytic reaction liquid into a 15mL clean centrifuge tube with a plug, adding 1mL of cuprammonium solution and 6mL of long-chain fatty acid extracting agent, reversing the mixture from top to bottom for about 100 times to obtain a mixed solution, standing for 20 minutes, centrifuging at 5000rpm for 20 minutes to layer and clarify the mixed solution, absorbing 2mL of the uppermost layer solution into another glass test tube, adding 1mL of color developing agent, mixing and standing for 2 minutes, measuring the light absorption value after color development at 530nm wavelength, and selecting the microorganism corresponding to the final catalytic reaction liquid with higher light absorption value.
Example 5
The embodiment shows a method for identifying microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids according to the following steps:
1) obtaining a catalytic reaction solution: adding 1g of triton X-405 and 5g of yeast extract into 1L of tap water to prepare an aqueous solution containing 1g/L of triton X-405 and 5g/L of yeast extract, subpackaging and shaking the aqueous solution with 100 mL/bottle, sterilizing the aqueous solution with steam at 121 ℃ for 30min, adding 7 mL/bottle of crude oil before use to enable the final concentration of the initial catalytic reaction solution to reach 55g/L, adding 10mL of microbial liquid sample to be identified, shaking the mixture in a shaking table at room temperature, and carrying out catalytic reaction for 96 hours to obtain the final catalytic reaction solution.
2) Preparing a copper amine solution: weigh 5.0g of Cu (NO)3)2·3H2Dissolving O in 100mL of deionized water to obtain 50g/L of copper nitrate aqueous solution, weighing 22.4g of triethanolamine, diluting to 100mL of deionized water to obtain 224g/L of triethanolamine aqueous solution, mixing the two aqueous solutions in equal volume, and obtaining the copper amine solution, namely the copper nitrate solution containing 25g/L and the triethanolamine solution containing 112g/L, wherein the final pH value is 8.2 +/-0.1.
3) Preparing a long-chain fatty acid extracting agent: mixing 100mL of chloroform and 500mL of petroleum ether, wherein the volume ratio of the chloroform to the petroleum ether is 1: 5.
4) Preparing a color developing agent: 0.05g of 1, 5-diphenylcarbonyldihydrazide and 0.95g of symmetric diphenylazocarbonyl hydrazide were weighed and dissolved in 200mL of methanol to obtain a color developer.
5) Absorbing 1mL of final catalytic reaction liquid into a 15mL clean centrifuge tube with a plug, adding 2mL of cuprammonium solution and 5mL of long-chain fatty acid extracting agent, reversing the upper part and the lower part for about 300 times to obtain a mixed solution, standing for 5 minutes, centrifuging at 5000rpm for 2min to enable the mixed solution to be layered and clarified, absorbing 2mL of the uppermost layer solution into another glass test tube, adding 1mL of color developing agent, mixing and standing for 30 minutes, measuring the light absorption value after color development at 530nm wavelength, and selecting the microorganism corresponding to the final catalytic reaction liquid with higher light absorption value.
Example 6
The embodiment shows a method for identifying microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids according to the following steps:
1) obtaining a catalytic reaction solution: adding 5g of triton X-100 and 1g of yeast extract powder into 1L of tap water to prepare an aqueous solution containing 5g/L of triton X-100 and 1g/L of yeast extract powder, subpackaging the aqueous solution into shake flasks in 100 mL/bottle, performing steam sterilization at 121 ℃ for 20min, adding 5 mL/bottle of kerosene to shake uniformly before use to enable the final concentration of the kerosene in the initial catalytic reaction solution to reach 40g/L, subpackaging the kerosene into 50mL centrifuge tubes subjected to steam sterilization in 5 mL/tube before catalytic reaction, sealing the centrifuge tubes with eight layers of gauze, adding a microbial sample to be identified into the initial catalytic reaction solution, shaking the centrifuge tubes at 30 ℃ to perform catalytic reaction for 72 hours, and obtaining the final catalytic reaction solution.
2) Preparing a copper amine solution: 6.5g of Cu (NO) are weighed out3)2·3H2Dissolving O in 100mL of deionized water to obtain 65g/L of copper nitrate aqueous solution, weighing 22.4g of triethanolamine, diluting with deionized water to 100mL to obtain 224g/L of triethanolamine aqueous solution, mixing the two aqueous solutions in equal volume, and obtaining the copper amine solution, namely the copper nitrate solution containing 32.5g/L and the triethanolamine solution containing 112g/L, wherein the final pH value is 8.1 +/-0.1.
3) Preparing a long-chain fatty acid extracting agent: mixing 100mL of chloroform and 200mL of petroleum ether, wherein the volume ratio of the chloroform to the petroleum ether is 1: 2.
4) Preparing a color developing agent: 0.05g of 1, 5-diphenylcarbonyldihydrazide and 0.95g of symmetric diphenylazocarbonyl hydrazide were weighed and dissolved in 200mL of methanol to obtain a color developer.
5) Sucking 0.50mL of final catalytic reaction liquid into a 5mL clean centrifuge tube with a plug, adding 0.5mL of copper amine solution and 3.0mL of long-chain fatty acid extractant, reversing the upper part and the lower part for about 300 times to obtain a mixed solution, standing for 15 minutes, centrifuging at 5000rpm for 30sec to enable the mixed solution to be layered and clarified, sucking 2mL of uppermost layer solution into another glass test tube, adding 1mL of color developing agent, mixing and standing for 2 minutes, measuring the light absorption value after color development at 530nm, and selecting the microorganism corresponding to the final catalytic reaction liquid with higher light absorption value. Referring to FIG. 3, FIG. 3 is a graph showing the standard curve of the method for identifying microorganisms capable of catalyzing the production of long chain fatty acids from paraffinic molecules in petroleum according to the present invention in example 6. As shown in FIG. 3, the calibration curve was prepared by dissolving lauric acid in kerosene at different concentrations and then adding the solution to an aqueous solution to develop color according to the procedure, and it was found that the linearity of the 530nm measurement value was good within 1 to 20 minutes of color development when the procedure of example 6 was employed, and it was possible to accurately quantify lauric acid, which is a long-chain fatty acid.
Example 7
The embodiment shows a method for identifying microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids according to the following steps:
1) obtaining a catalytic reaction solution: adding 1g of triton X-405 and 0.2g of peptone into 1L of tap water to prepare an aqueous solution containing 1g/L of triton X-405 and 0.2g/L of peptone, subpackaging the aqueous solution into a shake flask in 100 mL/bottle, performing steam sterilization at 121 ℃ for 20min, adding 5 mL/bottle of gasoline before use, shaking the bottle uniformly to enable the final concentration of the gasoline in the initial catalytic reaction solution to reach 38g/L, subpackaging the bottle into a 50mL centrifugal tube subjected to steam sterilization in 5 mL/tube, sealing the centrifugal tube with eight layers of gauze, adding sample solid particles containing the microorganisms to be screened, shaking the bottle at 30 ℃ to perform catalytic reaction for about 120 hours, and obtaining a final catalytic reaction solution.
2) Preparing a copper amine solution: weigh 8.2g of Cu (NO)3)2·3H2O is metered to 100mL by deionized water to obtain 82g/L of copper nitrate aqueous solution, 22.4g of triethanolamine is weighed, the metered to 100mL by deionized water to obtain 224g/L of triethanolamine aqueous solution, the two aqueous solutions are mixed in equal volume, the obtained copper amine solution contains 41g/L of copper nitrate and 112g/L of triethanolamine, and the final pH value is 7.9 +/-0.1.
3) Preparing a long-chain fatty acid extracting agent: mixing 100mL of chloroform and 500mL of petroleum ether, wherein the volume ratio of the chloroform to the petroleum ether is 1: 5.
4) Preparing a color developing agent: 0.05g of 1, 5-diphenylcarbonyldihydrazide and 0.95g of symmetric diphenylazocarbonyl hydrazide were weighed and dissolved in 200mL of methanol to obtain a color developer.
5) Sucking 1mL of final catalytic reaction solution into a 15mL clean centrifuge tube with a plug, adding 1mL of cuprammonium solution and 6mL of long-chain fatty acid extractant, reversing the mixture about 100 times from top to bottom to obtain a mixed solution, standing for 5 minutes, centrifuging at 5000rpm for 2min to layer and clarify the mixed solution, sucking 1mL of uppermost layer solution into another glass test tube, adding 1mL of color developing agent, mixing and standing for 1 minute, observing the color development result of each catalytic reaction solution, and selecting a microorganism sample corresponding to the final catalytic reaction solution with deep color development.
Example 8
The embodiment shows a method for identifying microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids according to the following steps:
1) obtaining a catalytic reaction solution: adding 10g of triton X-114 into 1L of seawater to prepare an aqueous solution containing 10g/L of triton X-114, subpackaging 100 mL/bottle into a shake flask, adding 10 mL/bottle of crude oil, shaking uniformly to enable the final concentration of the crude oil in the initial catalytic reaction solution to reach 80g/L, then adding 10 mL/bottle of numbered microbial liquid samples to be screened, sealing the bottle by eight layers of gauze, shaking in a shaking table at 30 ℃, and carrying out catalytic reaction for about 96 hours to obtain the final catalytic reaction solution.
2) Preparing a copper amine solution: 9.0g of Cu (NO) was weighed3)2·3H2And O, diluting with deionized water to 100mL to obtain 90g/L copper nitrate aqueous solution, weighing 37.5g of triethanolamine, diluting with deionized water to 100mL to obtain 375g/L triethanolamine aqueous solution, mixing the two aqueous solutions in equal volumes, and obtaining the copper amine solution, namely the copper amine solution contains 45g/L copper nitrate and 187.5g/L triethanolamine, wherein the final pH value is 8.2 +/-0.1.
3) Preparing a long-chain fatty acid extracting agent: mixing 100mL of chloroform and 600mL of petroleum ether, wherein the volume ratio of the chloroform to the petroleum ether is 1: 6.
4) Preparing a color developing agent: 0.1g of 1, 5-diphenylcarbonyldihydrazide and 1.9g of symmetric diphenylazocarbonyl hydrazide were weighed and dissolved in 400mL of methanol to obtain a color developer.
5) Sucking 50 mu L of final catalytic reaction liquid into a 15mL clean centrifuge tube with a plug, adding 200 mu L of cuprammonium solution and 5mL of long-chain fatty acid extracting agent, reversing the mixture up and down for about 100 times to obtain a mixed solution, standing for 5 minutes, centrifuging at 8000rpm for 2min to allow the mixed solution to be layered and clarified, sucking 2mL of the uppermost layer solution into another glass test tube, adding 2mL of color developing agent, mixing and standing for 1 minute, observing with naked eyes, and selecting the microorganism corresponding to the final catalytic reaction liquid with deeper red color.
Referring to fig. 1 to 2, fig. 1 is a graph showing the peak shape scanning of a developing solution in examples 1 to 8 according to a method for identifying a microorganism capable of catalyzing the production of a long-chain fatty acid from an alkane molecule in petroleum according to the present invention; FIG. 2 is a schematic diagram showing the color stability of the microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids in examples 1 to 8 according to the identification method of the microorganism. As shown in figure 1, the microorganisms capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids have maximum absorption at about 530nm, so that the 530nm can be used as a spectrophotometric method for quantitatively detecting the wavelength. As shown in FIG. 2, the measured value at 530nm is stable within 1 to 20 minutes of development, and the range of the fading is less than 5%, and therefore, it is suggested that spectrophotometric quantitative measurement can be performed within 1 to 20 minutes of development.
The identification method of the microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids, provided by the invention, has the following specific advantages:
(1) according to the method, the final concentration of triethanolamine in the copper amine solution is 90-180 g/L, and the copper amine solution only contains two components of copper nitrate and triethanolamine, so that the pH value of the copper amine solution can be stabilized at 7.7-8.5, and precipitates cannot be formed in the copper amine solution, so that the extraction effect of high-concentration long-chain fatty acid is stable as that of low-concentration long-chain fatty acid, and the identification method has good linearity in quantitative detection of the long-chain fatty acid.
In the prior art, the copper amine solution has a component containing acetic acid, while the copper amine solution in the method of the present invention does not contain acetic acid component. The procedure of example 6 was followed using a certain concentration of lauric acid as the long-chain fatty acid, and the final absorbance was affected by the obtained acetic acid as shown in Table 1:
Figure BDA0002833181100000121
Figure BDA0002833181100000131
TABLE 1
As shown in table 1, the presence of acetic acid significantly reduced the chromogenic absorbance. For example, 2% acetic acid content reduces the chromogenic absorbance by about 66%.
The copper amine solution in the process of the invention does not contain a high concentration of sodium chloride component. The composition of the copper amine solution contains sodium chloride with the concentration of up to 300-380g/L, which is used for increasing the density of the copper amine solution.
(2) The long-chain fatty acid extractant adopted in the invention can be positioned on the upper layer of the aqueous phase solution, and can also fully extract the chelate of the long-chain fatty acid and the copper ions, thereby being convenient for subsequent sampling of the extractant and effectively avoiding the influence of the aqueous phase copper ions.
Chloroform, a widely used organic solvent, is insoluble in water and has a density of about 1.5g/mL, compared to water, so that it sinks under water when extracting long-chain fatty acids from aqueous solutions. While petroleum ether has a density of about 0.74g/mL and floats on top of the aqueous solution. Chloroform can be used for efficiently extracting the chelate of long-chain fatty acid and copper ions, but the extraction rate of pure petroleum ether is very low, so that the color development light absorption value is about 5 percent of that of chloroform. Therefore, the volume ratio of chloroform to petroleum ether is optimized, the density of the long-chain fatty acid extracting agent obtained by mixing the chloroform and the petroleum ether is less than 0.95g/mL, floating is realized, and the chelate of copper ions and the long-chain fatty acid in the catalytic reaction liquid can be extracted more completely.
Chloroform and petroleum ether are used as extracting agents in the determination, the proportion of the extracting agents has a certain range, the optimization result provided by the invention can be used for conveniently and rapidly separating the upper layer solution and the lower layer solution by adopting simple centrifugal operation, and the detection result is more accurate. If the extractant is in the lower layer of the aqueous solution, the pipette must pass through the aqueous solution containing copper ions, which contaminate the solution and render the coloration result too high.
(3) In embodiments, the invention uses Triton X-100, X-114 or X-405 as an emulsifier for the petroleum component in the initial catalytic reaction solution, which allows for the dispersion of petroleum, thus facilitating the sufficient contact of the petroleum component with microorganisms, and which results in low background interfering absorbance values for chromogenic detection without unduly affecting the chromogenic light value of the catalytic solution. According to the detection data, even at a high concentration of 10g/L, when Triton X-100, X-114 or X-405 is used as an emulsifier, the increase of the chromogenic absorbance of the initial catalytic reaction solution is less than 0.08. If Tween-80 is used as an emulsifier, the increment of a color development light absorption value measured at 530nm of the initial catalytic reaction liquid is 0.2-0.80 at the concentration of 1-10 g/L, which causes excessive background interference on the color development of the final catalytic reaction liquid.
In conclusion, the identification method of the microorganism capable of catalyzing the alkane molecules in the petroleum to produce the long-chain fatty acid can extract and develop trace long-chain fatty acid in the water body and petroleum mixed liquor, and can quantify the total amount of the long-chain fatty acid by a spectrophotometry method, so that the method can be used for identifying the capability of the microorganism in catalyzing the alkane molecules in the petroleum to produce the long-chain fatty acid, has high sensitivity, simple operation and low cost, and can simultaneously process a plurality of samples.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims (5)

1. A method for identifying a microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acids, which is characterized by comprising the following steps:
(1) mixing petroleum, triton and water to obtain initial catalytic reaction liquid;
(2) putting microbial thalli to be identified into the initial catalytic reaction liquid, and oscillating to obtain a final catalytic reaction liquid;
(3) dissolving triethanolamine and copper nitrate into water to obtain a copper amine solution;
(4) mixing chloroform and petroleum ether to obtain a long-chain fatty acid extractant;
(5) weighing 1,5-diphenyl carbonyl dihydrazide and symmetric diphenyl azocarbonyl hydrazide, and dissolving in methanol to obtain a color developing agent;
(6) absorbing the final catalytic reaction liquid into a container, adding the copper amine solution and the long-chain fatty acid extracting agent, vibrating, mixing and standing to obtain a final mixed liquid;
(7) centrifuging the final mixed solution to obtain liquid layers, namely an upper layer solution and a lower layer solution;
(8) and sucking the upper solution in the liquid layer into another container, adding the color developing agent to obtain a color developing solution, and judging the concentration of the long-chain fatty acid in the final catalytic reaction liquid according to the red shade of the color developing solution.
2. The method of claim 1, wherein the microorganism is capable of catalyzing the production of long chain fatty acids from paraffinic molecules in petroleum, and the method comprises: in the step (1), the triton is selected from any one or more of X-100, X-114 or X-405, and the concentration of the triton in the initial catalytic reaction liquid is 0.1-10 g/L.
3. The method of claim 1, wherein the microorganism is capable of catalyzing the production of long chain fatty acids from paraffinic molecules in petroleum, and the method comprises: in the step (3), the concentration of the triethanolamine in the copper amine solution is 90-180 g/L, and the pH value of the copper amine solution is 7.7-8.5.
4. The method of claim 1, wherein the microorganism is capable of catalyzing the production of long chain fatty acids from paraffinic molecules in petroleum, and the method comprises: the volume ratio of the chloroform to the petroleum ether in the step (4) is 1: 2-1: 20.
5. The method of claim 1, wherein the microorganism is capable of catalyzing the production of long chain fatty acids from paraffinic molecules in petroleum, and the method comprises: and (4) in the step (7), the upper-layer solution is a long-chain fatty acid extracting agent containing chloroform and petroleum ether.
CN202011462881.0A 2020-12-14 2020-12-14 Identification method of microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acid Active CN112557386B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011462881.0A CN112557386B (en) 2020-12-14 2020-12-14 Identification method of microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acid

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011462881.0A CN112557386B (en) 2020-12-14 2020-12-14 Identification method of microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acid

Publications (2)

Publication Number Publication Date
CN112557386A true CN112557386A (en) 2021-03-26
CN112557386B CN112557386B (en) 2022-05-31

Family

ID=75062810

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011462881.0A Active CN112557386B (en) 2020-12-14 2020-12-14 Identification method of microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acid

Country Status (1)

Country Link
CN (1) CN112557386B (en)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1107790A (en) * 1966-03-29 1968-03-27 Boehringer & Soehne Gmbh A process and diagnostic agents for the detection of bacteria
JPS5931451A (en) * 1982-08-16 1984-02-20 Mitsubishi Heavy Ind Ltd Measurement of concentration of chlorine ion in water solution
US20050287677A1 (en) * 2004-05-26 2005-12-29 Andreas Bosmann Dissolvent for volumetric analysis
CN104897590A (en) * 2015-06-03 2015-09-09 常州大学 Method for quickly detecting content of petroleum hydrocarbon and thallus in oily sewage treatment system
CN105403557A (en) * 2015-10-23 2016-03-16 中国石油化工股份有限公司 Screening method of composite microbial oil displacement agent for oil extraction
CN109781668A (en) * 2019-02-11 2019-05-21 权冉(银川)科技有限公司 Sensor material and for medicinal material fruit-vegetable quality data informationization detect
CN110616248A (en) * 2019-10-10 2019-12-27 浙江海洋大学 Method for screening petroleum catalytic bacteria
CN111394419A (en) * 2019-01-03 2020-07-10 中国石油天然气集团有限公司 Method for detecting petroleum hydrocarbon degrading ability of petroleum hydrocarbon degrading bacteria in micro-scale mode

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1107790A (en) * 1966-03-29 1968-03-27 Boehringer & Soehne Gmbh A process and diagnostic agents for the detection of bacteria
JPS5931451A (en) * 1982-08-16 1984-02-20 Mitsubishi Heavy Ind Ltd Measurement of concentration of chlorine ion in water solution
US20050287677A1 (en) * 2004-05-26 2005-12-29 Andreas Bosmann Dissolvent for volumetric analysis
CN104897590A (en) * 2015-06-03 2015-09-09 常州大学 Method for quickly detecting content of petroleum hydrocarbon and thallus in oily sewage treatment system
CN105403557A (en) * 2015-10-23 2016-03-16 中国石油化工股份有限公司 Screening method of composite microbial oil displacement agent for oil extraction
CN111394419A (en) * 2019-01-03 2020-07-10 中国石油天然气集团有限公司 Method for detecting petroleum hydrocarbon degrading ability of petroleum hydrocarbon degrading bacteria in micro-scale mode
CN109781668A (en) * 2019-02-11 2019-05-21 权冉(银川)科技有限公司 Sensor material and for medicinal material fruit-vegetable quality data informationization detect
CN110616248A (en) * 2019-10-10 2019-12-27 浙江海洋大学 Method for screening petroleum catalytic bacteria

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ABDULATIF A. MANSUR等: "Assessing the hydrocarbon degrading potential of indigenous bacteria isolated from crude oil tank bottom sludge and hydrocarbon-contaminated soil of Azzawiya oil refinery,Libya", 《ENVIRON SCI POLLUT RES》 *
姚瑶等: "胜利油田土壤中石油降解菌分离和鉴定及代谢产物的研究", 《南京农业大学学报》 *

Also Published As

Publication number Publication date
CN112557386B (en) 2022-05-31

Similar Documents

Publication Publication Date Title
GB1604249A (en) Selective measurement of somatic and microbial cells
CN106755287B (en) Method for detecting and counting relative content of microorganisms
Berman et al. Metabolically active bacteria in Lake Kinneret
AU2005217026A1 (en) Measuring contamination
Varadi et al. Studying the bienzyme reaction with amperometric detection for measuring maltose
CN112557386B (en) Identification method of microorganism capable of catalyzing alkane molecules in petroleum to produce long-chain fatty acid
Fraticelli et al. On-line gas dialyzer for automated enzymatic analysis with potentiometric ammonia detection
Singh et al. Evaluation of biomass
CN103525896A (en) Quantitative high-vitality yeast cell screening method based on TTC (2,3,5-triphenyltetrazolium chloride) staining method
US5093236A (en) Microbiological oil prospecting
CN101029332A (en) Bacterium total testing fluid and its testing bottle
Parkes et al. Characterization of microbial populations in polluted marine sediments
CN108441491B (en) Method for rapidly screening and synthesizing miglitol key intermediate mutant strain and strain
CN113957116B (en) Method for detecting L-serine based on living cells of escherichia coli containing cysteine desulphurase
WO1992018642A1 (en) A method for the direct determination of the toxicity of particulate solids
Taniai et al. Fluorometric determination of ethanol in liquor samples by flow-injection analysis using an immobilized enzyme-reactor column with packing prepared by coupling alcohol oxidase and peroxidase onto chitosan beads
CN101806742A (en) Analysis method for fast detecting degradation rate of organophosphorus degrading bacteria
CN113156016B (en) Evaluation method for heavy metal pollution degree of sediment and application thereof
CN117169205B (en) Detection method of hypoxanthine based on colorimetric biosensor
Chen et al. A novel biosensor for the rapid determination of biochemical oxygen demand
CN1553187A (en) Rapid testing method for ayfactin fungal antibiotic
WO2003046136A2 (en) Single tube screen
WO1990002816A1 (en) Microbiological oil prospecting
Goriushkina et al. Application of amperometric enzyme biosensors for wine and must analysis
Dennis et al. A simple, rapid method for the detection of subspecies of Zymomonas mobilis

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant