Natural hydrogen reservoir exploration method

Abstract

The invention provides a natural hydrogen reservoir exploration method which comprises the following steps: S1) collecting soil and / or sediments of a sampling point above a target to be researched, and analyzing hydrogen metabolism activity of microorganisms in the collected soil and / or sediments; and S2) comparing the microbial hydrogen metabolic activity of the natural hydrogen target area with the microbial hydrogen metabolic activity of the background area, and if the microbial hydrogen metabolic activity of the natural hydrogen target area is higher than the microbial hydrogen metabolic activity of the background area, determining that the natural hydrogen target area is an area containing natural hydrogen. Compared with the prior art, the method has the advantages that whether the underground structure target contains the natural hydrogen or not can be determined only by collecting the soil sample of the ground surface of the to-be-detected structure target and analyzing the metabolic activity of the microorganism hydrogen, and compared with an existing method for natural hydrogen reservoir exploration by measuring the soil hydrogen, the method has the advantages that the detection accuracy is high; the underground hydrogen micro-leakage can be identified more reliably and effectively, and higher prediction accuracy is obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of oil field exploration and development, and in particular relates to an exploration method for natural hydrogen reservoirs. Background Art

[0002] As the world transitions from fossil fuels to more sustainable low-carbon energy, hydrogen energy is becoming an important part of the future energy structure. Hydrogen energy development is gradually becoming an important direction of the energy technology revolution and an important way to achieve energy decarbonization. At present, the use of hydrogen energy is mainly based on artificial hydrogen production, but no matter what hydrogen production method is used, there are still shortcomings at this stage, such as high costs. Natural hydrogen is hydrogen generated in underground geological processes. Compared with artificial hydrogen production, it is a truly zero-carbon, renewable primary energy source with lower costs. It can fill the gap in energy demand and help clean energy substitution. According to incomplete statistics, more than 20 scientific research institutions and more than 40 companies around the world are involved in the exploration and development of natural hydrogen. The emerging energy industry of natural hydrogen is entering a rapid growth stage, and is showing a rapid development trend in hydrogen generation mechanism and reservoir formation mechanism research, exploration and discovery, and experimental development.

[0003] At present, the exploration methods of natural hydrogen include surface satellite image recognition, geochemical gas analysis and soil gas detection, geophysical methods such as gravity, magnetism, electric seismology, and downhole logging. But in general, the exploration technology of natural hydrogen reservoirs is not mature. The current natural hydrogen discovery is mainly based on the display of the surface "fairy circle" phenomenon, the hydrogen anomaly display of existing drilling and logging data, and soil gas measurement. However, further resource potential assessment requires the comprehensive application of multiple geophysical and geochemical exploration methods and drilling. Hydrogen has a very small molecular weight. After geological accumulation, it is very easy to escape to the near surface through leakage. When the amount of hydrogen leakage is large, it will affect the development of vegetation on the surface, forming depressions of varying sizes with little or no vegetation (also known as "fairy circles"), but when the hydrogen leakage flux is low, "fairy circles" cannot be observed. At this time, the intensity of hydrogen leakage is generally studied by conducting geochemical detection of the hydrogen content in the soil. However, hydrogen has undergone metabolism by hydrogen-oxidizing microorganisms near the surface, and its content has changed. Therefore, the reliability of hydrogen soil gas detection technology based on geochemical detection technology is sometimes not very high. Directly detecting and studying the activity of microbial hydrogen metabolism in the soil can avoid the problem of low reliability of geochemical hydrogen detection results. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for exploring natural hydrogen reservoirs, which utilizes microorganisms and has higher reliability and accuracy.

[0005] The present invention provides a method for exploring natural hydrogen reservoirs, comprising the following steps:

[0006] S1) collecting soil and / or sediment from sampling points above the target to be studied, and analyzing the hydrogen metabolism activity of microorganisms in the collected soil and / or sediment;

[0007] The sampling point includes a natural hydrogen target area and a background area surrounding the natural hydrogen target area; the area of ​​the background area is at least 0.5 times the area of ​​the natural hydrogen target area;

[0008] Alternatively, the sampling point includes a natural hydrogen target area and a background area located above the natural hydrogen target area in a cross-sectional direction; the cross-sectional length of the background area is at least 0.5 times the cross-sectional length of the natural hydrogen target area;

[0009] S2) comparing the hydrogen metabolic activity of microorganisms in the natural hydrogen target area with that in the background area. If the hydrogen metabolic activity of microorganisms in the natural hydrogen target area is higher than that in the background area, then the natural hydrogen target area is an area containing natural hydrogen.

[0010] Preferably, the microbial hydrogen metabolic activity includes the sum of the content of hydrogen oxidizing bacteria and the content of hydrogen oxidase genes after data processing, or the sum of the relative number of hydrogen oxidizing bacteria and the relative number of hydrogen oxidase genes after data processing.

[0011] Preferably, the data processing is as follows: sorting the contents of hydrogen oxidizing bacteria in the natural hydrogen target area and the background area or the relative number of hydrogen oxidizing bacteria in order of high and low, and assigning values ​​of 1, 2, 3 in order from low to high until the sample with the highest data, and when multiple data are the same, assigning the same value to obtain the content assignment of hydrogen oxidizing bacteria;

[0012] The contents of hydrogen oxidase genes in the natural hydrogen target area and the background area or the relative numbers of hydrogen oxidase genes are sorted in order, and the values ​​are assigned from low to high as 1, 2, 3 until the sample with the highest data. When multiple data are the same, the same value is assigned to obtain the content assignment of hydrogen oxidase genes;

[0013] The microbial hydrogen metabolism activity is the sum of the content assignment of hydrogen oxidizing bacteria and the content assignment of hydrogen oxidase genes.

[0014] Preferably, the content of the hydrogen oxidizing bacteria is measured by a solid culture detection method or a liquid culture detection method using hydrogen as an energy source;

[0015] The content of the hydrogen oxidase gene is detected by one or more methods selected from the group consisting of fluorescent quantitative PCR, digital PCR, sequencing and gene chip.

[0016] Preferably, the solid culture medium used in the solid culture detection method includes 1.5-2.5 g NH4Cl, 1-1.5 g K2HPO4, 0.1-0.2 g KH2PO4, 0.1-1 g KCl, 0.1-1 g MgSO4, 0.01-0.03 g FeSO4·7H2O, 10-20 g agar and 1000 mL water;

[0017] And / or, the solid culture detection method is performed under the condition that the volume concentration of hydrogen is 8% to 15%.

[0018] Preferably, the primer pair for fluorescent quantitative PCR includes a forward primer and a reverse primer;

[0019] The forward primer is: 5′-TCACCWCAACAAATWTTTGG-3′;

[0020] The reverse primer is: 5′-GCWGCTTCCATWACTCCACC-3′.

[0021] Preferably, the fluorescent quantitative PCR 20 μL reaction system includes: 7 μL of double distilled water, 10 μL of dye method fluorescent quantitative PCR mix, 2 μL of 10 μmol / L forward primer, 2 μL of 10 μmol / L reverse primer, and 2 μL of DNA template; the DNA template is DNA extracted from collected soil and / or sediment.

[0022] Preferably, the reaction procedure of the fluorescent quantitative PCR is: 95°C for 5 min; 95°C for 30 s, 52.7°C for annealing for 30 s, and 72°C for extension for 30 s, while collecting the fluorescent signal, for a total of 35 to 40 cycles.

[0023] Preferably, the steps of using fluorescent quantitative PCR to detect the content of hydrogen oxidase gene are specifically as follows:

[0024] A1) Extracting DNA from collected soil and / or sediment;

[0025] A2) amplifying the extracted DNA by fluorescent PCR to obtain fluorescent signals and amplification products;

[0026] A3) connecting the amplified product to the pUCm-T plasmid vector to prepare standard plasmids of different concentrations;

[0027] A4) performing PCR amplification on standard plasmids of different concentrations to establish a quantitative PCR standard curve;

[0028] A5) The content of hydrogen oxidase gene is obtained according to the fluorescence signal and the quantitative PCR standard curve.

[0029] The present invention also provides a natural hydrogen reservoir exploration kit, comprising a solid culture medium and a primer pair for fluorescent quantitative PCR;

[0030] The solid culture medium comprises 1.5-2.5 g NH4Cl, 1-1.5 g K2HPO4, 0.1-0.2 g KH2PO4, 0.1-1 g KCl, 0.1-1 g MgSO4, 0.01-0.05 g yeast extract, 0.01-0.03 g FeSO4·7H2O, 10-20 g agar and 1000 mL water;

[0031] The primer pair of the fluorescent quantitative PCR includes a forward primer and a reverse primer;

[0032] The forward primer is: 5′-TCACCWCAACAAATWTTTGG-3′;

[0033] The reverse primer is: 5′-GCWGCTTCCATWACTCCACC-3′.

[0034] The present invention provides a method for exploring natural hydrogen reservoirs, comprising the following steps: S1) collecting soil and / or sediments at sampling points above a target to be studied, and analyzing the hydrogen metabolic activity of microorganisms in the collected soil and / or sediments; the sampling points include a natural hydrogen target area and a background area surrounding the natural hydrogen target area; the area of ​​the background area is at least 0.5 times the area of ​​the natural hydrogen target area; or, the sampling points include a natural hydrogen target area and a background area located above the natural hydrogen target area in a cross-sectional direction; the cross-sectional length of the background area is at least 0.5 times the cross-sectional length of the natural hydrogen target area; S2) comparing the hydrogen metabolic activity of microorganisms in the natural hydrogen target area with that in the background area; if the hydrogen metabolic activity of microorganisms in the natural hydrogen target area is higher than that in the background area, the natural hydrogen target area is a region containing natural hydrogen. Compared with the existing technology, the present invention only needs to collect soil samples from the surface of the structural target to be detected for microbial hydrogen metabolic activity analysis to determine whether the underground structural target contains natural hydrogen. Compared with the existing method of exploring natural hydrogen reservoirs by measuring soil hydrogen, it can more reliably and effectively identify underground hydrogen micro-seepage and obtain higher prediction accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A line graph of the values ​​of hydrogen oxidizing bacteria in the samples according to the embodiment of the present invention;

[0036] Figure 2 A line graph showing the assignment of hydrogen oxidase genes to samples in an embodiment of the present invention;

[0037] Figure 3 It is a line graph of the hydrogen metabolism activity of the sample microorganisms in the embodiment of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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 making creative work are within the scope of protection of the present invention.

[0039] The present invention provides a method for exploring natural hydrogen reservoirs, comprising the following steps: S1) collecting soil and / or sediments at sampling points above a target to be studied, and analyzing the hydrogen metabolic activity of microorganisms in the collected soil and / or sediments; the sampling points include a natural hydrogen target area and a background area surrounding the natural hydrogen target area; the area of ​​the background area is at least 0.5 times the area of ​​the natural hydrogen target area; or, the sampling points include a natural hydrogen target area and a background area located above the natural hydrogen target area in a cross-sectional direction; the cross-sectional length of the background area is at least 0.5 times the cross-sectional length of the natural hydrogen target area; S2) comparing the hydrogen metabolic activity of microorganisms in the natural hydrogen target area with that in the background area; if the hydrogen metabolic activity of microorganisms in the natural hydrogen target area is higher than that in the background area, the natural hydrogen target area is a region containing natural hydrogen.

[0040] The present invention has no particular limitation on the sources of all raw materials, and any raw materials available on the market can be used.

[0041] Collect soil and / or sediment at sampling points above the target to be studied; the sampling points include a natural hydrogen target area and a background area surrounding the natural hydrogen target area; in this case, the natural hydrogen target area is located in the background area, and the two are located in the same plane; the area of ​​the background area is at least 0.5 times the area of ​​the natural hydrogen target area; or, the sampling points include the natural hydrogen target area and the background area located above the natural hydrogen target area in the cross-sectional direction; the cross-sectional length of the background area is at least 0.5 times the cross-sectional length of the natural hydrogen target area; in the present invention, the selection of sampling points and the selection of sampling point depths are well known to those skilled in the art. That is to say, those skilled in the art can determine the sampling points and sampling depths according to the medium uniformity and soil moisture of the target area. Further, the depth of the sampling points is preferably 10 to 30 cm, more preferably 15 to 25 cm, and even more preferably 20 cm. The more sampling points there are, the better the effect is. However, because of economic considerations, the number of sampling points can be 10 to 50. In the embodiment provided by the present invention, 17 sampling points are specifically taken as an example for illustration. The smaller the distance between adjacent sampling points, the higher the accuracy of the detection result. However, economic considerations must be taken at the same time. Specifically, the distance can be 100 to 1000 m. In the embodiment provided by the present invention, 500 m of distance between adjacent sampling points is specifically taken as an example for illustration.

[0042] Analyze the collected soil and / or sediment for microbial hydrogen metabolism activity; in the present invention, the microbial hydrogen metabolism activity preferably includes the content of hydrogen oxidizing bacteria and the content of hydrogen oxidase genes, and more preferably includes the sum of the content of hydrogen oxidizing bacteria and the content of hydrogen oxidase genes after data processing, or the sum of the relative number of hydrogen oxidizing bacteria and the relative number of hydrogen oxidase genes after data processing; hydrogen oxidase can be divided into three superfamilies according to the different metal compositions contained in the active center, namely [FeFe] hydrogen oxidase, [NiFe] hydrogen oxidase, and [Fe] hydrogen oxidase. Among them, [FeFe] hydrogen oxidase is generally considered to be a hydrogen-producing hydrogen oxidase, [NiFe] hydrogen oxidase is generally considered to be a hydrogen-consuming hydrogen oxidase, and [NiFe] hydrogen oxidase can be divided into two major categories according to group: hydrogen oxidation (H2 oxidation) and hydrogen fermentation (H2 generation). Specifically in the present invention, the detection object is limited to the genes of enzymes that oxidatively metabolize hydrogen. More specifically, the content of the hydrogen oxidizing bacteria is calculated by the colony count; the content of the hydrogen oxidase gene is calculated by its copy number; since the colony count and the copy number cannot be added directly, the present invention performs data processing to add them up and finally forms the "microbial hydrogen metabolic activity".

[0043] In the present invention, the content of the hydrogen oxidizing bacteria is preferably measured by a solid culture detection method or a liquid culture detection method using hydrogen as an energy source, and more preferably by a solid culture detection method using hydrogen as an energy source; the solid culture medium used in the solid culture detection method preferably includes 1.5-2.5g NH4Cl, 1-1.5g K2HPO4, 0.1-0.2g KH2PO4, 0.1-1g KCl, 0.1-1g MgSO4, 0.01-0.03g FeSO4·7H2O, 10-20g agar and 1000mL water, and more preferably includes 1.8-2.2g NH4Cl, 1.1-1.4g K2HPO4, 0.12-0.18g KH2PO4, 0.3-0.8g KCl, 0.3-0.8g MgSO4, 0.01-0.02g FeSO4·7H2O, 12-18 g agar and 1000 mL of water, more preferably 1.8-2.2 g NH4Cl, 1.1-1.3 g K2HPO4, 0.12-0.16 g KH2PO4, 0.4-0.6 g KCl, 0.4-0.6 g MgSO4, 0.01-0.02 g FeSO4·7H2O, 14-16 g agar and 1000 mL of water, most preferably 2.0 g NH4Cl, 1.2 g K2HPO4, 0.14 g KH2PO4, 0.5 g KCl, 0.5 g MgSO4, 0.01 g FeSO4·7H2O, 15 g agar and 1000 mL of water.

[0044] According to the present invention, the condition of the solid culture detection method is that the volume concentration of hydrogen is preferably 8% to 15%, more preferably 10% to 12%, and even more preferably 10%.

[0045] In a specific embodiment provided by the present invention, the content of hydrogen oxidizing bacteria is obtained according to the following steps: drying the collected soil and / or sediment to obtain the moisture content of the soil and / or sediment; mixing the collected soil and / or sediment with water, diluting it in a gradient manner, applying the dilution on the surface of a solid culture medium, placing it in a closed incubator filled with hydrogen for culturing after standing, counting, and obtaining the content of hydrogen oxidizing bacteria according to the dilution and moisture content; the drying temperature is preferably 100°C to 110°C, more preferably 105°C; the drying time is preferably 5 to 10h, more preferably 8h; the mass volume ratio of the collected soil and / or sediment to water is preferably 10g:90mL; the water is preferably sterile water; the gradient dilution is preferably carried out by a 10-fold gradient dilution method; specifically, the standing is to stand until the inoculation coating liquid is absorbed by the solid culture medium. In the present invention, the standing time can be 10 to 60 minutes, and can also be 20 to 40 minutes; the concentration of hydrogen in the closed incubator filled with hydrogen is preferably 8% to 15%, more preferably 10% to 12%, and more preferably 10%; the culture temperature is preferably 25°C to 35°C, more preferably 28°C to 32°C, and more preferably 30°C; the culture time is preferably 5 to 10 days, more preferably 6 to 8 days, and more preferably 7 days.

[0046] In the present invention, the content of the hydrogen oxidase gene is preferably detected by one or more methods including fluorescent quantitative PCR, digital PCR, sequencing and gene chip; wherein the primer pair of the fluorescent quantitative PCR includes a forward primer and a reverse primer; the forward primer is: 5′-TCACCWCAACAAATWTTTGG-3′ (SEQ ID NO: 1); the reverse primer is: 5′-GCWGCTTCCATWACTCCACC-3′ (SEQ ID NO: 2); the fluorescent quantitative PCR 20 μL reaction system preferably includes: 7 μL of double distilled water, dye method fluorescent quantitative PCR mix 10μL, 2μL of 10μmol / L forward primer, 2μL of 10μmol / L reverse primer, and 2μL of DNA template; the DNA template is DNA extracted from the collected soil and / or sediment; the DNA extracted from the collected soil and / or sediment can be extracted using a DNA extraction kit; the reaction procedure of the fluorescent quantitative PCR is preferably: 95℃5min; 95℃30s, 52.7℃ annealing 30s, 72℃ extension 30s, while collecting fluorescent signals, for a total of 35 to 40 cycles; after the reaction procedure is completed, it is preferred to detect the melting curve of the reaction product, and the final reactant can be stored at 4℃.

[0047] In a specific embodiment provided by the present invention, the step of using fluorescent quantitative PCR to detect the content of hydrogen oxidase gene is specifically as follows: A1) extracting DNA from the collected soil and / or sediment; A2) amplifying the extracted DNA by fluorescent PCR to obtain fluorescent signals and amplification products; A3) connecting the amplification products to the pUCm-T plasmid vector to prepare standard plasmids of different concentrations; A4) amplifying the standard plasmids of different concentrations by PCR to establish a quantitative PCR standard curve (i.e., a curve showing the relationship between fluorescence intensity and copy number); A5) obtaining the content of hydrogen oxidase gene according to the fluorescent signal and the quantitative PCR standard curve. More specifically, in step A5), the content of hydrogen oxidase gene is obtained according to the fluorescent signal and the quantitative PCR standard curve, combined with the moisture content of the soil and / or sediment.

[0048] In another specific embodiment provided by the present invention, the data processing is as follows: sorting the content of hydrogen oxidizing bacteria in the natural hydrogen target area and the background area (obtained by solid culture detection method, in units of CFU) or the relative number of hydrogen oxidizing bacteria (generally referring to liquid culture detection, the most common one is the maximum probable number method, i.e., MPN method. Liquid culture detection cannot obtain a specific absolute number, and the final result is generally a relative number) according to high and low, and assigning values ​​of 1, 2, 3 in order from low to high until the sample with the highest data. When multiple data are the same, they are assigned the same value to obtain the content assignment of hydrogen oxidizing bacteria; sorting the content of hydrogen oxidase genes in the natural hydrogen target area and the background area (the absolute content can be obtained by fluorescent quantitative PCR, digital PCR and quantitative sequencing technology) or the relative number of hydrogen oxidase genes (using sequencing or gene chip methods, the result generally obtained is a relative number) according to high and low, and assigning values ​​of 1, 2, 3 in order from low to high until the sample with the highest data. When multiple data are the same, they are assigned the same value to obtain the content assignment of hydrogen oxidase genes.

[0049] In a specific embodiment provided by the present invention, the microbial hydrogen metabolic activity is the sum of the content assignment of hydrogen oxidizing bacteria and the content assignment of hydrogen oxidase genes; specifically, the sum of the number or relative number of hydrogen oxidizing bacteria and hydrogen oxidase genes after data processing refers to the sum of the assignment of the number of hydrogen oxidizing bacteria and the assignment of the number of hydrogen oxidase genes of the same sample after the number or relative number of hydrogen oxidizing bacteria and hydrogen oxidase genes are assigned through data processing, that is, the microbial hydrogen metabolic activity.

[0050] The hydrogen metabolic activity of microorganisms in the natural hydrogen target area is compared with that in the background area. If the hydrogen metabolic activity of microorganisms in the natural hydrogen target area is higher than that in the background area, then the natural hydrogen target area is an area containing natural hydrogen.

[0051] The natural hydrogen reservoir exploration method provided by the present invention has the following advantages:

[0052] 1) Compared with geochemical natural hydrogen reservoir exploration methods such as soil hydrogen detection, since the hydrogen oxidizing bacteria detected by the present invention itself have a metabolic effect on the hydrogen in the soil, it can greatly change the concentration of hydrogen in the soil, thereby weakening the correlation between the hydrogen concentration in the soil and the underlying hydrogen reservoir. The detection object of the present invention is the hydrogen oxidizing bacteria that cause changes in the soil hydrogen concentration. If there is no continuous underground hydrogen supply, the number of such hydrogen oxidizing bacteria will decrease. Therefore, the results obtained by the present invention are more closely related to the underground hydrogen reservoir, and the reliability of the prediction is also better.

[0053] 2) The present invention uses a microbial hydrogen metabolism activity index to represent the degree of hydrogen metabolism by microorganisms in the soil. This index includes cultivable hydrogen oxidizing bacteria and hydrogen oxidase genes. This provides more effective information than using a single hydrogen oxidizing bacteria and hydrogen oxidase gene index, and the results are more reliable.

[0054] 3) Since there are many different methods for detecting hydrogen oxidizing bacteria and hydrogen oxidase genes, some methods obtain absolute quantitative results, some obtain relative quantitative results, and sometimes the absolute values ​​of the detection results of hydrogen oxidizing bacteria and hydrogen oxidase genes differ by more than an order of magnitude, it is necessary to study how to perform summation and other processing on the detection data of hydrogen oxidizing bacteria and hydrogen oxidase genes, and finally obtain the microbial hydrogen metabolism activity index data. The present invention provides a simple, convenient, and effective assignment processing method.

[0055] The present invention also provides a natural hydrogen reservoir exploration kit, comprising a solid culture medium and a primer pair for fluorescence quantitative PCR; the solid culture medium comprises 1.5-2.5g NH4Cl, 1-1.5g K2HPO4, 0.1-0.2g KH2PO4, 0.1-1g KCl, 0.1-1g MgSO4, 0.01-0.03g FeSO4·7H2O, 10-20g agar and 1000mL water; the primer pair for fluorescence quantitative PCR comprises a forward primer and a reverse primer; the forward primer is: 5′-TCACCWCAACAAATWTTTGG-3′; the reverse primer is: 5′-GCWGCTTCCATWACTCCACC-3′; wherein W=A / T.

[0056] More specifically, the solid culture medium more preferably comprises 1.8-2.2 g NH4Cl, 1.1-1.4 g K2HPO4, 0.12-0.18 g KH2PO4, 0.3-0.8 g KCl, 0.3-0.8 g MgSO4, 0.01-0.03 g yeast extract, 0.01-0.02 g FeSO4·7H2O, 12-18 g agar and 1000 mL of water, and more preferably comprises 1.8-2.2 g NH4Cl, 1.1-1.3 g K2HPO4, 0.12-0.16 g KH2PO4, 0.4-0.6 g KCl, 0.4-0.6 g MgSO4, 0.01-0.02 g FeSO4·7H2O, 14-16 g agar and 1000 mL of water, and most preferably comprises 2.0 g NH4Cl, 1.2 g K2HPO4, 0.14g KH2PO4, 0.5g KCl, 0.5g MgSO4, 0.01g FeSO4·7H2O, 15g agar and 1000mL water.

[0057] More specifically, the exploration kit also includes a DNA extraction kit; the DNA extraction kit can specifically be a soil DNA extraction kit.

[0058] In order to further illustrate the present invention, a natural hydrogen reservoir exploration method provided by the present invention is described in detail below in conjunction with embodiments.

[0059] The reagents used in the following examples are all commercially available.

[0060] Example 1

[0061] Detection of Hydrogen-Oxidizing Bacteria in Near-Surface Soil of a Natural Hydrogen-Containing Area

[0062] 1.1 Select a profile containing known hydrogen storage areas and background areas for sampling. The sampling depth is 20 cm and the sampling interval is 500 m. A total of 17 soil samples were collected and the total profile length is 8 km.

[0063] 1.2 The formula of the culture medium used for hydrogen hydride detection culture is as follows: 2.0g NH4Cl, 1.2g K2HPO4, 0.14g KH2PO4, 0.5g KCl, 0.5g MgSO4, 0.01g FeSO4·7H2O, 15g agar and 1000mL water, adjust the pH to 7.2-7.3; sterilize at 121℃ for 20min. When the culture medium cools to about 50℃, shake it and pour it into a 90mm sterile plate. After solidification, it is the hydrogen hydride detection culture medium.

[0064] 1.3 Weigh about 10g of soil and record the exact mass. Dry at 105℃ for 8 hours and place in a desiccator. After cooling, weigh the soil. Calculate the moisture content of the soil based on the difference in mass before and after drying.

[0065] 1.4 Weigh 10g of soil and dissolve it in 90mL of sterile water, shake and mix, then use the 10-fold gradient dilution method to make a gradient dilution solution, take 100μL of the dilution solution with the appropriate dilution gradient and add it to the above-mentioned hydrogen peroxide detection medium, use a sterile coating stick to spread it evenly, and inoculate three culture dishes for each sample.

[0066] 1.5 After the coating and inoculation is completed, the culture dish is placed horizontally for 30 minutes to absorb the inoculation solution, and then it is inverted in a closed incubator filled with hydrogen, the hydrogen concentration is maintained at about 10%, and the temperature is kept at 30°C for 7 days. After the incubation is completed, the colonies grown on the culture dish are counted.

[0067] 1.6 Take the average of the colony counts of three culture dishes of the same sample as the average colony count of the culture dishes at the dilution, and calculate the number of hydrogen oxidizing bacteria per gram of dry soil in the sample according to the dilution and the moisture content of the soil.

[0068] Example 2

[0069] Detection of Hydrogen Oxidase Genes in Near-Surface Soil of a Natural Hydrogen-Containing Area

[0070] 2.1 The samples used in this embodiment are the same as those used in Example 1, that is, a profile known to contain a hydrogen storage area and a background area is selected for sampling. The sampling depth is 20 cm, the sampling interval is 500 m, and a total of 17 soil samples are collected, with a total profile length of 8 km.

[0071] 2.2 Extract DNA from soil samples according to the operating procedures of the soil DNA extraction kit (FastDNA Spin Kit for Soil, MP Company, Cat. No. 116560).

[0072] 2.3 Fluorescence quantitative PCR (dye method) was used to detect and count the hydrogen oxidase gene hydA in soil samples. The detection primers were BHf (5′-TCACCWCAACAAATWTT TGG-3′); BHr (5′-GCWGCTTCCATWACTCCACC-3′).

[0073] 2.4 The fluorescence quantitative PCR detection system is 20 μL, which includes dye method fluorescence quantitative PCR pre-made MIX: 10 μL; primer BHf (10 μmol / L): 0.5 μL; primer BHr (10 μmol / L): 0.5 μL; sterile double distilled water: 7 uL; DNA template: 2 uL. The reaction procedure of fluorescence quantitative PCR is: reaction step one, 95℃5min; reaction step two, 95℃30s, 52.7℃ annealing 30s, 72℃ extension 30s, while collecting fluorescence signals, a total of 40 cycles. After the completion of 40 cycles, the melting curve of the reaction product is detected, and the final reaction product can be stored at 4℃.

[0074] 2.5 The hydrogen oxidase gene fragment obtained by the above primer amplification was connected to the pUCm-T plasmid vector to prepare a standard plasmid. The standard plasmid was used to establish a quantitative PCR standard curve, and the sample test results obtained in step 2.4 were converted into the number of copies of the hydrogen oxidase gene per gram of dry soil in combination with the soil moisture content data.

[0075] Example 3

[0076] Calculation and technical verification of hydrogen metabolic activity of soil microorganisms near the surface in a natural hydrogen-bearing area

[0077] 3.1 After obtaining the quantitative data of hydrogen oxidizing bacteria and hydrogen oxidase genes in the near-surface soil of the hydrogen storage area and background area profiles in Example 1 and Example 2, it is necessary to calculate the hydrogen metabolism activity of soil microorganisms.

[0078] 3.2 Sort all the measured hydrogen oxidizing bacteria numbers or relative numbers by high and low, and assign values ​​of 1, 2, 3, and the highest value to the sample in descending order of the sample measurement data. When multiple samples have the same measured values, they are assigned the same value. The number of hydrogen oxidizing bacteria in all samples of this embodiment and their assigned values ​​are shown in Table 1, and the line graph of their assigned values ​​is shown in Figure 1 In the figure, T01-72 to T01-80 are the areas where underground hydrogen is found after drilling, and the rest are background areas.

[0079] 3.3 Sort the number or relative number of all measured hydrogen oxidase genes by high and low, and assign values ​​of 1, 2, 3 in descending order of sample measurement data until the sample with the highest data. When multiple samples have the same measurement value, they are assigned the same value. The number of hydrogen oxidase genes of all samples in this embodiment and their assigned values ​​are shown in Table 2, and the line graph of their assigned values ​​is shown in Figure 2 In the figure, T01-72 to T01-80 are the areas where underground hydrogen is found after drilling, and the rest are background areas.

[0080] 3.4 Add the values ​​assigned to the number of hydrogen oxidizing bacteria and the number of hydrogen oxidase genes in the same sample to obtain the hydrogen metabolism activity of the microorganisms, and make a line graph comparing the hydrogen metabolism activity of the microorganisms in the near-surface soil of a section containing natural hydrogen in a region. The hydrogen metabolism activity of the microorganisms of all samples in this example is shown in Table 2, and the line graph of their values ​​is shown in Figure 2 In the figure, T01-72 to T01-80 are the areas where underground hydrogen is found after drilling, and the rest are background areas.

[0081] Depend on Figure 1 It can be seen that the number of hydrogen-oxidizing bacteria in the sample at point T01-72 presents a low value, which is inconsistent with its location above the hydrogen storage area. Figure 2 It shows that the number of hydrogen oxidase gene assignments at this point sample presents a high value feature. Finally, the microbial hydrogen metabolism activity index that combines the number of hydrogen oxidizing bacteria assignments and the number of hydrogen oxidase gene assignments presents a high value feature. Figure 3 This shows that the comprehensive index of microbial hydrogen metabolism activity provides more effective information than the single hydrogen oxidizing bacteria index, and the results are more reliable. Figure 2 It can be seen that the number of hydrogen oxidase genes in samples at the three points T01-76, T01-79 and T01-80 showed low values, which was inconsistent with their location above the hydrogen storage area. Figure 1 It shows that the number of hydrogen oxidizing bacteria in the sample at this point presents a high value feature. Finally, the microbial hydrogen metabolism activity index, which combines the number of hydrogen oxidizing bacteria and the number of hydrogen oxidase genes, presents a high value feature. Figure 3 This also shows that the comprehensive index of microbial hydrogen metabolic activity can obtain more effective information than using a single hydrogen oxidase gene index, and the results are more reliable.

[0082] Table 1 The number and value of hydrogen oxidizing bacteria in the near-surface soil of a natural hydrogen-containing area

[0083]

[0084] Table 2 Number and value of hydrogen oxidase genes in near-surface soil of a region containing natural hydrogen

[0085]

[0086]

[0087] Table 3 Hydrogen metabolism activity of soil microorganisms near the surface in a natural hydrogen-containing area

[0088]

[0089] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for exploring natural hydrogen reservoirs, characterized in that: The following steps are involved: S1) collecting soil and / or sediment from sampling points above the target to be studied, and analyzing the hydrogen metabolism activity of microorganisms in the collected soil and / or sediment; The sampling point includes a natural hydrogen target area and a background area surrounding the natural hydrogen target area; the area of ​​the background area is at least 0.5 times the area of ​​the natural hydrogen target area; Alternatively, the sampling point includes a natural hydrogen target area and a background area located above the natural hydrogen target area in a cross-sectional direction; the cross-sectional length of the background area is at least 0.5 times the cross-sectional length of the natural hydrogen target area; S2) comparing the hydrogen metabolic activity of microorganisms in the natural hydrogen target area with that in the background area. If the hydrogen metabolic activity of microorganisms in the natural hydrogen target area is higher than that in the background area, then the natural hydrogen target area is an area containing natural hydrogen.

2. The exploration method according to claim 1, characterized in that: The microbial hydrogen metabolism activity includes the sum of the content of hydrogen oxidizing bacteria and the content of hydrogen oxidase genes after data processing, or the sum of the relative number of hydrogen oxidizing bacteria and the relative number of hydrogen oxidase genes after data processing.

3. The exploration method according to claim 2, characterized in that: The data processing is as follows: sorting the contents of hydrogen oxidizing bacteria in the natural hydrogen target area and the background area or the relative number of hydrogen oxidizing bacteria in order of high and low, and assigning values ​​of 1, 2, 3 in order from low to high until the sample with the highest data, and when multiple data are the same, assigning the same value to obtain the content assignment of hydrogen oxidizing bacteria; The contents of hydrogen oxidase genes in the natural hydrogen target area and the background area or the relative numbers of hydrogen oxidase genes are sorted in order, and the values ​​are assigned from low to high as 1, 2, 3 until the sample with the highest data. When multiple data are the same, the same value is assigned to obtain the content assignment of hydrogen oxidase genes; The microbial hydrogen metabolism activity is the sum of the content assignment of hydrogen oxidizing bacteria and the content assignment of hydrogen oxidase genes.

4. The exploration method according to claim 2, characterized in that: The content of the hydrogen oxidizing bacteria is measured by a solid culture detection method or a liquid culture detection method using hydrogen as an energy source; The content of the hydrogen oxidase gene is detected by one or more methods selected from the group consisting of fluorescent quantitative PCR, digital PCR, sequencing and gene chip.

5. The exploration method according to claim 4, characterized in that: The solid culture medium used in the solid culture detection method includes 1.5-2.5 g NH4Cl, 1-1.5 g K2HPO4, 0.1-0.2 g KH2PO4, 0.1-1 g KCl, 0.1-1 g MgSO4, 0.01-0.03 g FeSO4·7H2O, 10-20 g agar and 1000 mL water; And / or, the solid culture detection method is performed under the condition that the volume concentration of hydrogen is 8% to 15%.

6. The exploration method according to claim 2, characterized in that: The primer pair of the fluorescent quantitative PCR includes a forward primer and a reverse primer; The forward primer is: 5′-TCACCWCAACAAATWTTTGG-3′; The reverse primer is: 5′-GCWGCTTCCATWACTCCACC-3′.

7. The exploration method according to claim 6, characterized in that: The fluorescent quantitative PCR 20 μL reaction system includes: 7 μL of double distilled water, 10 μL of dye method fluorescent quantitative PCR mix, 2 μL of 10 μmol / L forward primer, 2 μL of 10 μmol / L reverse primer, and 2 μL of DNA template; the DNA template is DNA extracted from collected soil and / or sediment.

8. The exploration method according to claim 7, characterized in that: The reaction procedure of the fluorescent quantitative PCR was as follows: 95° C. for 5 min; 95° C. for 30 s, 52.7° C. for annealing for 30 s, and 72° C. for extension for 30 s, while collecting the fluorescent signal, for a total of 35 to 40 cycles.

9. The exploration method according to claim 8, characterized in that: The specific steps of using fluorescent quantitative PCR to detect the content of hydrogen oxidase gene are: A1) Extracting DNA from collected soil and / or sediment; A2) amplifying the extracted DNA by fluorescent PCR to obtain fluorescent signals and amplification products; A3) connecting the amplified product to the pUCm-T plasmid vector to prepare standard plasmids of different concentrations; A4) performing PCR amplification on standard plasmids of different concentrations to establish a quantitative PCR standard curve; A5) The content of hydrogen oxidase gene is obtained according to the fluorescence signal and the quantitative PCR standard curve.

10. A kit for exploring natural hydrogen reservoirs, characterized in that: Including solid culture medium and primer pair for fluorescence quantitative PCR; The solid culture medium comprises 1.5-2.5 g NH4Cl, 1-1.5 g K2HPO4, 0.1-0.2 g KH2PO4, 0.1-1 g KCl, 0.1-1 g MgSO4, 0.01-0.05 g yeast extract, 0.01-0.03 g FeSO4·7H2O, 10-20 g agar and 1000 mL water; The primer pair of the fluorescent quantitative PCR includes a forward primer and a reverse primer; The forward primer is: 5′-TCACCWCAACAAATWTTTGG-3′; The reverse primer is: 5′-GCWGCTTCCATWACTCCACC-3′.

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