Microorganism real-time visual monitoring method and device based on fluorescence labeling

Abstract

The invention belongs to the technical field of microbial remediation, and particularly relates to a real-time visual monitoring method and device for microorganisms based on fluorescence labeling, and the method comprises the following steps: labeling petroleum hydrocarbon degrading bacteria with fluorescent protein to obtain microorganisms containing fluorescent plasmids; microorganisms containing fluorescent plasmids are cultured to a stable phase and then added into a saturated porous medium, a portable excitation light source is combined with emitted light filtering, the migration process of the marked microorganisms in the soil medium can be directly observed with naked eyes, and a common camera or a mobile phone can be adopted for shooting. Real-time in-situ visualization of migration of microorganisms in a soil medium is achieved, and the method has great significance in soil pollution remediation and treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial remediation, and in particular relates to a real-time visual monitoring method and device for microorganisms based on fluorescent labeling. Background Art

[0002] With the increasing attention paid to risk control of operating enterprises and groundwater remediation, the number of applications of technologies such as microbial remediation, in situ biological / chemical reduction, and monitoring of natural attenuation will increase. In particular, microbial in situ remediation technology has high application prospects due to its low investment cost, small impact on the surrounding environment, and no secondary pollution. During the in situ remediation process, whether active microorganisms can migrate to the contaminated site is a limiting factor that determines the remediation effect. At present, there is a lack of in-depth understanding of the migration laws of microorganisms in porous media, which leads to poor in-situ remediation effects at actual contaminated sites. This is also a bottleneck limiting the large-scale application of microbial in situ remediation. Therefore, exploring the migration behavior of microorganisms in underground environments is of great significance for soil pollution remediation and control.

[0003] At present, the research objects of migration and transformation laws are mostly pollutants, and there are few studies on the migration laws of microorganisms themselves. The migration mechanism of microorganisms is very complex, not only affected by the external environment, but also by their own characteristics. However, there is currently a lack of effective research methods. The existing research methods usually take out and process the samples and observe them under a microscope. For example, the patent with application number 201610292624.4 discloses a foodborne microorganism visualization analysis method based on nano-fluorescence microscopic hyperspectral imaging technology, using nano-fluorescent probes to specifically mark foodborne microorganisms. The patent with application number 202011326237.0 discloses an experimental device and method for visualization research of microbial diffusion and migration, and the transparent cylinder is removed and observed under a microscope. The patent with application number 202211493017.6 discloses a detection method and a visualization fluorescence detection device for pathogenic microorganisms, and the marking of pathogenic microorganisms is achieved by designing target-specific crRNA. The above methods all have the problems of complex operation, disturbance to in situ samples and poor timeliness. Summary of the invention

[0004] In order to solve or partially solve the above problems, the present invention discloses a method for real-time visual monitoring of microorganisms based on fluorescent labeling, comprising the following steps:

[0005] Fluorescent proteins are used to label petroleum hydrocarbon-degrading bacteria to obtain fluorescently labeled microorganisms;

[0006] The microorganisms containing the fluorescent marker are cultured to a stable period and then added to a saturated porous medium to observe and mark the migration process of the microorganisms in the saturated porous medium.

[0007] Furthermore, the fluorescent protein is used to label petroleum hydrocarbon degrading bacteria as follows:

[0008] The pGFP4412 plasmid was transformed into Bacillus subtilis to make Bacillus subtilis carry green fluorescence, and the pRGC plasmid was transformed into Pseudomonas aeruginosa and Burkholderia cepacia to make Pseudomonas aeruginosa and Burkholderia cepacia carry red fluorescence.

[0009] Furthermore, the saturated porous medium is constructed according to the following steps:

[0010] Build a sand trough experimental device;

[0011] Filling the sand trough experimental device with fine sand as a filling medium;

[0012] After each 1 to 5 cm of fine sand is filled, compaction is performed and the total mass of the filling medium is recorded, wherein the total height of the fine sand is 20 to 80 cm;

[0013] After filling is completed, the sand tank experimental device is saturated with water from bottom to top to form a saturated porous medium for the migration of degradation bacteria.

[0014] Furthermore, the particle size of the fine sand is 0.075-0.125 mm, the porosity is 40%, and the permeability coefficient is 2.0-5.0*10-5 m / s.

[0015] Furthermore, the petroleum hydrocarbon degrading bacteria are obtained according to the following steps:

[0016] Obtain crude oil contaminated soil, remove impurities, grind and sieve, take 1g of soil sample and place it in 10mL of sterile water, shake it on a shaker at 150rpm for 2h and then let it stand;

[0017] The supernatant was taken and 10% was inoculated into a liquid culture medium with crude oil as the sole carbon source, and cultured at 30°C and 150 rpm for 5 days;

[0018] The bacterial liquid was spread on crude oil plates, single colonies were screened and the strains were identified.

[0019] Furthermore, using fluorescent protein to label petroleum hydrocarbon degrading bacteria to obtain fluorescently labeled microorganisms includes the following steps:

[0020] After activating a single colony on an LB plate, competent cells were cultured;

[0021] Add fluorescent plasmids into the competent cells and culture to obtain microorganisms containing fluorescent plasmids; wherein the fluorescent plasmids are pGFP4412 and pRGC.

[0022] Further, after activating a single colony on an LB plate, culturing to obtain competent cells comprises the following steps:

[0023] Activate petroleum hydrocarbons onto LB plates and culture at 37°C overnight;

[0024] Pick a single colony and inoculate it into 5 mL of LB medium, shake at 200 rpm at 37°C overnight;

[0025] Inoculate 50 μL of overnight bacterial solution into a test tube containing 5 mL of LB and culture at 37°C, 200 rpm for about 2 h until the OD600 of the bacterial solution reaches 0.4-0.6;

[0026] Place the culture solution on ice for 10 min to cool it to 0°C; centrifuge at 7000 g for 10 min at 4°C, discard the supernatant, and recover the cells;

[0027] Add 1 mL of pre-cooled 150 mM MgCl2 to resuspend the precipitate, place on ice for 30 min, centrifuge at 7000 g for 5 min at 4 °C, discard the supernatant, and recover the cells;

[0028] Resuspend the precipitate with 800 μL pre-cooled 150 mM MgCl2 and dispense into pre-cooled 1.5 mL EP tubes, 100 μL per tube.

[0029] Furthermore, adding a fluorescent plasmid to the competent cells and culturing to obtain a microorganism containing the fluorescent plasmid comprises the following steps:

[0030] Add the target fluorescent plasmid to 100 μL competent cells, mix well and place on ice for 1 hour;

[0031] After heat shock in a 42°C water bath for 3 min, transfer to ice and cool for 5 min;

[0032] Add 1 mL of LB liquid medium and culture at 37°C with shaking at 200 rpm for 45 min to rejuvenate;

[0033] The cells were spread on LB plates containing antibiotics and cultured at 37°C overnight;

[0034] Pick a single colony and inoculate it into 5 mL of LB medium containing antibiotics, shake at 200 rpm at 37°C overnight;

[0035] The plasmid was extracted using a plasmid extraction kit, and gel electrophoresis was used to verify that the plasmid was transformed into the strain.

[0036] Furthermore, the observation and marking of the migration process of microorganisms in saturated porous media is specifically as follows:

[0037] Use a portable excitation light source to illuminate the sand tank, and use emission light filtering to observe and record the location of the fluorescence;

[0038] Observe and mark the migration process of microorganisms in saturated porous media according to the location of the fluorescence;

[0039] The observation method includes naked eye observation or instrument photography.

[0040] On the other hand, the present invention proposes a sand tank experimental device for completing the real-time visual monitoring method of microorganisms based on optical marking as described in the claims, comprising a base and a tank body above the base, wherein a filling medium is arranged in the tank body;

[0041] The tank body is made of transparent material, and the filling medium is fine sand with a particle size of 0.075-0.125 mm, a porosity of 40%, and a permeability coefficient of 2.0-5.0*10-5 m / s.

[0042] Beneficial effects of the present invention:

[0043] The present invention uses fluorescent protein to label petroleum hydrocarbon degrading bacteria to obtain microorganisms containing fluorescent plasmids; the microorganisms containing fluorescent plasmids are then cultured to a stable period and then added to a saturated porous medium. Through a portable excitation light source combined with emission light filtering, the migration process of the labeled microorganisms in the soil medium can be directly observed with the naked eye, and photographed with an ordinary camera or mobile phone to achieve real-time in-situ visualization of the migration of microorganisms in the soil medium. The method proposed by the present invention is simple to operate and has great significance for soil pollution remediation and control.

[0044] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0046] Figure 1 A flow chart of a method for real-time visual monitoring of microorganisms based on fluorescent labeling in an embodiment of the present invention is shown;

[0047] Figure 2 A diagram showing the growth of petroleum hydrocarbon degrading bacteria on a crude oil plate in an embodiment of the present invention is shown;

[0048] Figure 3 The diagram shows the migration of fluorescently labeled microorganisms in a saturated porous medium in a sand tank according to an embodiment of the present invention. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] The present invention uses a fluorescent labeling method to achieve real-time visual monitoring of microorganisms and to achieve in-situ real-time monitoring of the migration behavior of microorganisms in porous media. Figure 1 As shown, it mainly includes the following steps:

[0051] Fluorescent proteins are used to label petroleum hydrocarbon-degrading bacteria to obtain fluorescently labeled microorganisms;

[0052] The microorganisms containing the fluorescent plasmid are cultured to a stable period and then added into a saturated porous medium to observe and mark the migration process of the microorganisms in the saturated porous medium.

[0053] In the actual monitoring process, it is also necessary to build a sand trough experimental device, which includes a base and a trough body. The material is transparent acrylic plate or high borosilicate glass. Uniform fine sand is selected as the filling medium (particle size 0.075-0.125mm, porosity 40%, permeability coefficient 2.0-5.0*10-5m / s), and compacted every 5 cm. The total mass of the filling medium is recorded, and the total height of the filling medium is 20-80 cm. After filling, the sand trough is saturated with water from bottom to top to form a saturated porous medium for the migration of degradation bacteria.

[0054] The method of using fluorescent protein to label petroleum hydrocarbon degrading bacteria comprises the following steps: petroleum hydrocarbon degrading bacteria are Bacillus subtilis and Pseudomonas aeruginosa, both of which are screened from the soil of a certain oil field. The pGFP4412 plasmid is transformed into Bacillus subtilis, so that Bacillus subtilis carries green fluorescence. The pRGC plasmid is transformed into Pseudomonas aeruginosa and Burkholderia cepacia, so that Pseudomonas aeruginosa and Burkholderia cepacia carry red fluorescence.

[0055] The microorganisms with fluorescent plasmids are cultured in LB medium until the stable period. The cultured microorganisms containing fluorescent markers are added to the saturated porous medium. In a dark environment, a portable excitation light source is used to illuminate the sand tank, and the emission light filter is used to observe and record the location of the fluorescence with the naked eye. The migration process of microorganisms in the saturated porous medium can be directly observed and marked with the naked eye, and can also be photographed with an ordinary camera or mobile phone to achieve real-time in-situ visualization of the migration of microorganisms in the saturated porous medium. This system can be used not only for the migration law of a single microorganism, but also for the migration law of two or more microorganisms.

[0056] In order to more clearly illustrate the fluorescent protein labeling of petroleum hydrocarbon degrading bacteria and the observation process in the present invention, the acquisition of petroleum hydrocarbon degrading bacteria, the transformation of petroleum hydrocarbon degrading bacteria and the migration process of fluorescently labeled microorganisms in the saturated porous medium of the sand tank are described in detail in combination with the embodiments below.

[0057] Example 1: Acquisition of petroleum hydrocarbon degrading bacteria

[0058] Obtain crude oil contaminated soil from an oil field, remove impurities, grind and pass through a 2mm soil sieve, take 1g of soil sample and place in 10mL of sterile water, shake on a shaker at 150rpm for 2h, and then let stand for 30mi;

[0059] The supernatant after standing was taken and 10% was inoculated into a liquid culture medium with crude oil as the sole carbon source, and cultured at 30°C and 150 rpm for 5 days;

[0060] The cultured bacterial solution was applied to a crude oil plate for growth. In this embodiment, the growth of petroleum hydrocarbon degrading bacteria on the crude oil plate was as follows: Figure 2 As shown, single colonies were screened for bacterial species identification.

[0061] Example 2: Transformation of petroleum hydrocarbon degrading bacteria

[0062] The petroleum hydrocarbon-degrading bacteria were activated onto LB plates and cultured at 37°C overnight;

[0063] The single colony identified in Example 1 was picked and inoculated into 5 mL of LB medium, and shaken at 200 rpm at 37°C overnight;

[0064] Inoculate 50 μL of overnight bacterial solution into a test tube containing 5 mL of LB and culture at 37°C, 200 rpm for about 2 h until the OD600 of the bacterial solution reaches 0.4-0.6;

[0065] Place the mixture on ice for 10 min to cool the bacterial solution to 0°C. Centrifuge at 7000 g for 10 min at 4°C, discard the supernatant, and recover the cells.

[0066] Add 1 mL of pre-cooled 150 mM MgCl2 to gently resuspend the pellet and place on ice for 30 min. Centrifuge at 7000 g for 5 min at 4°C, discard the supernatant, and recover the cells;

[0067] Gently resuspend the pellet with 800 μL pre-cooled 150 mM MgCl2 and dispense into pre-cooled 1.5 mL EP tubes, 100 μL per tube;

[0068] Add target DNA (pGFP4412 or pRGC) to 100 μL competent cells, mix gently, and place on ice for 1 h;

[0069] After heat shock in a 42°C water bath for 3 min, the cells were quickly transferred to ice and cooled for 5 min.

[0070] Add 1 mL of LB liquid culture medium and culture at 37°C with shaking at 200 rpm for 45 min to rejuvenate.

[0071] The cells were spread on LB plates containing antibiotics and cultured at 37°C overnight.

[0072] A single colony was picked and inoculated into 5 mL of LB medium containing antibiotics and shaken at 200 rpm at 37°C overnight.

[0073] The plasmid was extracted using a plasmid extraction kit, and gel electrophoresis was used to verify that the plasmid was transformed into the strain.

[0074] Example 3: Migration of fluorescently labeled microorganisms in saturated porous media in a sand tank

[0075] Build a sand trough experimental device, including a base and a trough body. The trough body is made of transparent acrylic plate or high borosilicate glass;

[0076] Choose uniform fine sand as the filling medium (particle size 0.075-0.125mm, porosity 40%, permeability 2.0-5.0*10-5m / s), compact it every 5 cm, and the filling medium height is 50 cm;

[0077] After filling, the sand trough is saturated with water from bottom to top to form a saturated porous medium for the migration of degradation bacteria, and the soil moisture content at this time is measured;

[0078] The microorganisms containing the fluorescent markers are cultured in LB medium until the stable period, and the cultured microorganisms containing the fluorescent markers are added to the saturated porous medium of the sand tank;

[0079] After a period of time, in a dark environment, a portable excitation light source is used to illuminate the sand tank, and the emission light filter is used to observe and record the location of the fluorescence with the naked eye, so that the migration process of microorganisms in the saturated porous medium can be directly observed and marked with the naked eye. The migration diagram of the fluorescent-labeled microorganisms in the saturated porous medium of the sand tank in this embodiment is as follows: Figure 3 As shown, the migration of microorganisms can be clearly seen.

[0080] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A real-time visual monitoring method for microorganisms based on fluorescent labeling, characterized in that: The following steps are involved: Fluorescent proteins are used to label petroleum hydrocarbon-degrading bacteria to obtain fluorescently labeled microorganisms; The microorganisms containing the fluorescent marker are cultured to a stable period and then added to a saturated porous medium to observe and mark the migration process of the microorganisms in the saturated porous medium.

2. The method for real-time visual monitoring of microorganisms based on fluorescent labeling according to claim 1, characterized in that: The specific method of using fluorescent protein to label petroleum hydrocarbon degrading bacteria is as follows: The pGFP4412 plasmid was transformed into Bacillus subtilis to make Bacillus subtilis carry green fluorescence, and the pRGC plasmid was transformed into Pseudomonas aeruginosa and Burkholderia cepacia to make Pseudomonas aeruginosa and Burkholderia cepacia carry red fluorescence.

3. The method for real-time visual monitoring of microorganisms based on fluorescent labeling according to claim 1 or 2, characterized in that: The saturated porous medium is constructed according to the following steps: Build a sand trough experimental device; Filling the sand trough experimental device with fine sand as a filling medium; After each 1 to 5 cm of fine sand is filled, compaction is performed and the total mass of the filling medium is recorded, wherein the total height of the fine sand is 20 to 80 cm; After filling is completed, the sand tank experimental device is saturated with water from bottom to top to form a saturated porous medium for the migration of degradation bacteria.

4. The method for real-time visual monitoring of microorganisms based on fluorescent labeling according to claim 3, characterized in that: The particle size of the fine sand is 0.075-0.125 mm, the porosity is 40%, and the permeability coefficient is 2.0-5.0*10-5 m / s.

5. The method for real-time visual monitoring of microorganisms based on fluorescent labeling according to claim 1, characterized in that: The petroleum hydrocarbon degrading bacteria are obtained according to the following steps: Obtain crude oil contaminated soil, remove impurities, grind and sieve, take 1g of soil sample and place it in 10mL of sterile water, shake it on a shaker at 150rpm for 2h and then let it stand; The supernatant was taken and 10% was inoculated into a liquid culture medium with crude oil as the sole carbon source, and cultured at 30°C and 150 rpm for 5 days; The bacterial liquid was spread on crude oil plates, single colonies were screened and the strains were identified.

6. The method for real-time visual monitoring of microorganisms based on fluorescent labeling according to claim 1, characterized in that: Using fluorescent protein to label petroleum hydrocarbon degrading bacteria, obtaining microorganisms containing fluorescent labels includes the following steps: After activating a single colony on an LB plate, competent cells were cultured; Add fluorescent plasmids into the competent cells and culture to obtain microorganisms containing fluorescent plasmids; wherein the fluorescent plasmids are pGFP4412 and pRGC.

7. The method for real-time visual monitoring of microorganisms based on fluorescent labeling according to claim 6, characterized in that: After activating a single colony on an LB plate, culturing competent cells includes the following steps: The petroleum hydrocarbon-degrading bacteria were activated onto LB plates and cultured at 37°C overnight; Pick a single colony and inoculate it into 5 mL of LB medium, shake at 200 rpm at 37°C overnight; Inoculate 50 μL of overnight bacterial solution into a test tube containing 5 mL of LB and culture at 37°C, 200 rpm for about 2 h until the OD600 of the bacterial solution reaches 0.4-0.6; Place the culture solution on ice for 10 min to cool it to 0°C; centrifuge at 7000 g for 10 min at 4°C, discard the supernatant, and recover the cells; Add 1 mL of pre-cooled 150 mM MgCl2 to resuspend the precipitate, place on ice for 30 min, centrifuge at 7000 g for 5 min at 4 °C, discard the supernatant, and recover the cells; Resuspend the precipitate with 800 μL pre-cooled 150 mM MgCl2 and dispense into pre-cooled 1.5 mL EP tubes, 100 μL per tube.

8. The method for real-time visual monitoring of microorganisms based on fluorescent labeling according to claim 7, characterized in that: Adding the fluorescent plasmid to the competent cells and culturing to obtain a microorganism containing the fluorescent plasmid comprises the following steps: Add the target fluorescent plasmid to 100 μL competent cells, mix well and place on ice for 1 hour; After heat shock in a 42°C water bath for 3 min, transfer to ice and cool for 5 min; Add 1 mL of LB liquid medium and culture at 37°C with shaking at 200 rpm for 45 min to rejuvenate; The cells were spread on LB plates containing antibiotics and cultured at 37°C overnight; Pick a single colony and inoculate it into 5 mL of LB medium containing antibiotics, shake at 200 rpm at 37°C overnight; The plasmid was extracted using a plasmid extraction kit, and gel electrophoresis was used to verify that the plasmid was transformed into the strain.

9. The method for real-time visual monitoring of microorganisms based on fluorescent labeling according to claim 1, characterized in that: The specific process of observing and marking the migration of microorganisms in saturated porous media is as follows: Use a portable excitation light source to illuminate the sand tank, and use emission light filtering to observe and record the location of the fluorescence; Observe and mark the migration process of microorganisms in saturated porous media according to the location of the fluorescence; The observation method includes naked eye observation or instrument photography.

10. A sand trough experimental device, used to implement the real-time visual monitoring method of microorganisms based on optical markers according to any one of claims 1 to 9, characterized in that: It comprises a base and a tank body above the base, wherein a filling medium is arranged in the tank body; The tank body is made of transparent material, and the filling medium is fine sand with a particle size of 0.075-0.125 mm, a porosity of 40%, and a permeability coefficient of 2.0-5.0*10-5 m / s.

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