Oil and gas exploration method based on microbial gene

Abstract

An oil and gas exploration method based on a microbial gene is provided, where samples are collected from shallow surface layers above a known oil well, a gas well, and a dry well in an exploration area, DNA is extracted and subjected to high-throughput sequencing (HTS), and a pattern map of a microbial community composition in the exploration area is established according to sequencing results; and characteristic microorganisms in surface soil above an oil/gas well in the exploration area are screened out according to the pattern map, then primers are designed according to attribute characters of the characteristic microorganisms, and samples throughout the exploration area are subjected to fluorescence quantitative polymerase chain reaction (PCR) to detect a number of the characteristic microorganisms.

Claims

1. An oil and gas exploration method based on a microbial gene, comprising the following steps: S1) determining a sampling site in an exploration area; S2) determining a sampling site in a forward-modeling area, wherein the forward-modeling area is established with a known oil containing well, and/or with a known gas containing well in the exploration area; or wherein the forward-modeling area is established with a known well within an area that is adjacent to the exploration area; wherein the sampling site of the forward-modeling area is above the known oil and/or gas containing well in the exploration area, or wherein the sampling site of the forward-modeling area is above the known well within an area adjacent to the exploration area; S3) designing a scheme for sampling the forward-modeling area, wherein the sampling scheme involves collecting and preserving a soil sample from one or more sampling sites above each well in the forward modeling area; wherein the soil samples include microorganisms and each collected soil sample is preserved with a preservation agent comprising of the following components: a Tris-HCl solution pH 8.0 comprising a 0.5% (w/v) cetyltrimethylammonium bromide (CTAB), a 2% (v/v) ethanol, a 1% (v/v) ?-mercaptoethanol, 0.75 mM ascorbic acid, 0.25 mM ascorbyl palmitate, 10 mM ethylenediaminetetraacetic acid disodium salt, 0.5 mM NaCl, and 1 mg/L DNase inhibitor; S4) extracting DNA from the preserved soil samples of the one or more sampling sites in the forward modeling area; S5) subjecting the extracted DNA from each soil sample from the forward-modeling area to a high-throughput sequencing (HTS), and reading operational taxonomic unit (OTU) values of species at each sampling site; and S6) identifying gas-indicating effective bacteria and/or oil-indicating effective bacteria at each sampling site within the exploration area; calculating oil indexes and/or gas indexes according to the OTU values of the soil sample at each sampling site within the exploration area, and/or the OTU values of the gas-indicating effective bacteria in a known well that is a dry well and in a known well that is an oil well; and/or the OTU values of the oil-indicating effective bacteria in a known well that is a dry well and in a known well that is an oil well; comparing the values of the oil indexes and/or the gas indexes to determine effective oil indexes and/or effective gas indexes at each sampling site within the exploration area; and establishing an isopleth of oil and gas-indicating microorganism values at each sampling site across the exploration area using the values of the effective oil indexes and/or effective gas indexes at each sampling site.

2. The method according to claim 1, wherein the known well is a dry well, a water well, a display well, or an industrial well.

3. The method according to claim 1, wherein the HIS is a HIS of a 16S rDNA gene or a hydrocarbon oxidation-associated gene, and the hydrocarbon oxidation-associated gene comprises one or more selected from the group consisting of a pmoA gene, an mmoX gene, a bmoX gene, an alk gene, and a P450 gene.

4. The method according to claim 1, wherein the effective oil indexes are selected for the first 5 species with a maximum oil/gas value greater than 2 and wherein the effective gas indexes are selected for the first 5 species with a maximum gas/oil value greater than 2.

5. The method according to claim 1, wherein the soil sample is collected at a sampling depth that is from 20 cm to 100 cm, and wherein the soil sample is collected at a sampling amount of 50 g to 100 g.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows an isopleth of oil and gas-indicating microbial values of a specified area obtained according to a patented method of the patent (granted patent publication No.: CN 107267623 B); and

(2) FIG. 2 shows an isopleth of oil and gas-indicating microbial values of the same area obtained according to an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(3) The embodiments of the present disclosure are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present disclosure from the contents disclosed in this specification. The present disclosure can also be implemented or applied through other different specific implementations. Based on different viewpoints and applications, various modifications or alterations can be made to various details of this specification without departing from the spirit of the present disclosure.

Example 1

(4) This example provided an oil and gas exploration method based on a microbial gene for oil and gas exploration in a specified exploration area, including the following steps:

(5) Early sample collection was the same as the sample collection for oil and gas microbial exploration.

(6) Specific steps were as follows:

(7) 1. Based on a geological background of an exploration area and preliminary research findings, a technical service scheme was customized in view of the problems and difficulties in the current exploration.

(8) A. The scheme design is very flexible, and the design should consider an expected size and shape of an exploration target, whether sampling is allowed along or between key seismic survey lines, construction feasibility, and complexity of a surface environment.

(9) B. According to different exploration targets, a spacing between sampling sites varies greatly, and can range from 50 m to 100 m to 500 m to 1,000 m.

(10) C. A forward model must be established with a known well in an exploration area or an area adjacent to the exploration area as a target, and the known well preferably includes a dry well, a water well, a display well, and an industrial well.

(11) 2. According to the design scheme, a surveyor calculated coordinates of each site.

(12) 3. A sampling depth was determined according to surface characteristics and human activities of the exploration area. Studies of the present disclosure showed that the sampling depth was 20 cm to 60 cm.

(13) 4. A sampling amount was determined according to indexes to be detected. Studies of the present disclosure showed that the sampling amount was 50 g to 100 g.

(14) 5. A preservation manner of a sample was determined according to indexes to be detected. Studies of the present disclosure showed that a sample was preserved at ?20? C.

(15) 6. DNA was extracted from each sample.

(16) 7. Samples from a forward-modeling area (a known oil/gas well) were subjected to HTS of a 16S rDNA gene or a hydrocarbon oxidation-associated gene. The hydrocarbon oxidation-associated gene included one or more selected from the group consisting of the identified pmoA gene, mmoX gene, bmoX gene, alk gene, and P450 gene.

(17) OTU values of species were read. Data were shown in Table 1.

(18) TABLE-US-00001 TABLE 1 HTS data Dry Oil Gas No. Species well well well 1 s_Acidobacteria_bacterium_WX27 11.00 20.00 21.00 2 s_agricultural_soil_bacterium_SC-I-11 1.30 2.60 0.88 3 s_agricultural_soil_bacterium_SC-I-39 1.00 7.00 14.60 4 s_Bacillus_aryabhattai 1.67 2.20 0.00 5 s_bacterium_Ellin517 4.17 6.20 2.56 6 s_bacterium_Ellin6537 2.33 2.60 6.78 7 s_bacterium_Ellin6543 9.00 39.60 42.00 8 s_bacterium_endosymbiont_of_Onthophagus_Taurus 1.83 0.00 15.00 9 s_bacterium_enrichment_culture_clone_auto10_4W 1.00 8.00 5.11 10 s_bacterium_enrichment_culture_clone_auto112_4W 1.00 1.20 2.89 11 s_bacterium_enrichment_culture_clone_auto195_4W 4.17 2.00 4.22 12 s_bacterium_enrichment_culture_clone_auto84_4W 7.00 5.20 7.56 13 s_bacterium_enrichment_culture_clone_B302011 4.83 3.60 16.33 14 s_Candidatus_Yanofskybacteria_bacterium_GW2011_GWA2_41_22 3.00 3.00 4.00 15 s_Catellatospora_sp._KC-EP-S6 2.17 0.20 39.44 16 s_Cylindrotheca_closterium 1.50 13.80 2.44 17 s_delta_proteobacterium_WX81 2.17 1.80 7.78 18 s_Gemmata_sp._F11-1 5.00 9.20 13.00 19 s_Gemmatirosa_kalamazoonesis 0.33 0.00 2.11 20 s_groundwater_metagenome 1.00 5.40 2.89 21 s_marine_metagenome 1.67 4.00 23.56 22 s_Mesorhizobium_ciceri 2.83 4.20 1.33 23 s_Methylobacterium_radiotolerans 0.33 1.60 0.67 24 s_miscellaneous_Crenarchaeota_group_archaeon_SMTZ-80 4.67 39.40 2.11 25 s_Oryza_longistaminata 0.33 3.00 8.00 26 s_Parcubacteria_group_bacterium_GW2011_GWA2_40_14 3.17 9.60 1.89 27 s_Planctomycetaceae_bacterium_LX124 1.83 1.20 2.11 28 s_Planctomycetaceae_bacterium_SCGC_AG-212-F19 2.33 2.40 0.67 29 s_planctomycete_A-2 4.17 13.00 2.33 30 s_planctomycete_WY69 6.17 6.40 17.44 31 s_Planctomycetia_bacterium_WSF3-27 13.83 16.60 72.67 32 s_Pythium_insidiosum 0.00 0.60 0.78 33 s_Rhodococcus_erythropolis 1.00 1.20 1.11 34 s_soil_bacterium_WF55 0.33 2.00 1.00 35 s_soil_bacterium_WWH121 1.50 1.20 5.00 36 s_Sphingomonas_paucimobilis 1.17 3.00 5.00 37 s_Spirochaeta_sp._MWH-HuW8 1.50 1.40 1.44 38 s_Streptomyces_cinnamonensis 5.00 5.20 3.44 39 s_Turneriella_parva 2.00 3.00 4.56 40 s_wastewater_metagenome 1.00 7.60 9.00 41 s_Weissella_confusa 0.83 1.20 12.78 Note: The OTU value indicates an average OTU value of samples collected above a well.

(19) 8. An oil and gas-indicating microbial fingerprint of the exploration area was established, and gas-indicating effective bacteria and/or oil-indicating effective bacteria in the exploration area were identified.

(20) Oil indexes and/or gas indexes were calculated according to OTU values.

(21) (1) Oil index=OTU value in an oil well/OTU value in a dry well, and gas index=OTU value in a gas well/OTU value in a dry well. The first 10 with a maximum value were selected from each of the oil indexes and the gas indexes. Species corresponding to each of the oil indexes and the gas indexes were screened out. Results were shown in Tables 2 and 3.

(22) TABLE-US-00002 TABLE 2 Oil indexes Dry Oil Gas Oil Gas No. Species well well well index index Gas/oil 7 s_bacterium_Ellin6543 9.00 39.60 42.00 4.40 4.67 0.94 23 s_Methylobacterium_radiotolerans 0.33 1.60 0.67 4.80 2.00 2.40* 20 s_groundwater_metagenome 1.00 5.40 2.89 5.40 2.89 1.87 34 s_soil_bacterium_WF55 0.33 2.00 1.00 6.00 3.00 2.00 3 s_agricultural_soil_bacterium_SC-I-39 1.00 7.00 14.60 7.00 14.60 0.48 40 s_wastewater_metagenome 1.00 7.60 9.00 7.60 9.00 0.84 9 s_bacterium_enrichment_culture_clone_auto10_4W 1.00 8.00 5.11 8.00 5.11 1.57 24 s_miscellaneous_Crenarchaeota_group_archaeon_SMTZ-80 4.67 39.40 2.11 8.44 0.45 18.66* 25 s_Oryza_longistaminata 0.33 3.00 8.00 9.00 24.00 0.38 16 s_Cylindrotheca_closterium 1.50 13.80 2.44 9.20 1.63 5.65* *effective index

(23) TABLE-US-00003 TABLE 3 Gas indexes Dry Oil Gas Oil Gas No. Species well well well index index Gas/oil 9 s_bacterium_enrichment_culture_clone_auto10_4W 1.00 8.00 5.11 8.00 5.11 0.64 31 s_Planctomycetia_bacterium_WSF3-27 13.83 16.60 72.67 1.20 5.25 4.38* 19 s_Gemmatirosa_kalamazoonesis 0.33 0.00 2.11 0.00 6.33 8 s_bacterium_endosymbiont_of_Onthophagus_Taurus 1.83 0.00 15.00 0.00 8.18 40 s_wastewater_metagenome 1.00 7.60 9.00 7.60 9.00 1.18 21 s_marine_metagenome 1.67 4.00 23.56 2.40 14.13 5.89* 3 s_agricultural_soil_bacterium_SC-I-39 1.00 7.00 14.60 7.00 14.60 2.09 41 s_Weissella_confusa 0.83 1.20 12.78 1.44 15.33 10.65* 15 s_Catellatospora_sp._KC-EP-S6 2.17 0.20 39.44 0.09 18.21 197.22* 25 s_Oryza_longistaminata 0.33 3.00 8.00 9.00 24.00 2.67* *effective index

(24) (2) Then values of the oil indexes and gas indexes were compared. Among the oil indexes, the first 5 species with a maximum oil/gas value greater than 2 were determined as effective oil indexes; and among the gas indexes, the first 5 species with a maximum gas/oil value greater than 2 were determined as effective gas indexes.

(25) In this embodiment, the determined oil indexes were s_Methylobacterium_radiotolerans(O-1), s_miscellaneous_Crenarchaeota_group_archaeon_SMTZ-80(O-2), and s_Cylindrotheca_closterium(O-3); and

(26) the determined effective gas indexes were s_Catellatospora_sp._KC-EP-S6(G-1), s_Weissella_confusa(G-2), s_marine_metagenome(G-3), s_Oryza_longistaminata(G-4), and s_Planctomycetia_bacterium_WSF3-27(G-5).

(27) In actual oil and gas exploration, there is relatively low probability that both an oil well and a gas well exist in a same work area. If there is only an oil well or a gas well, there is no need to consider the results of oil/gas when oil indexes and gas indexes are screened.

(28) In this embodiment, some invalid data were processed at an early stage of data processing, such that the later data processing was convenient and the later data processing results were intuitive.

(29) The elimination of inefficient bacteria (with a relatively small proportion in each of the dry well, oil well, and gas well) during PCR detection could significantly reduce a reading when there was no oil and gas, namely, a background noise reading, which improved a signal-noise ratio (SNR) and improved the accuracy of exploration prediction.

(30) 9. Corresponding primers were designed according to the effective indexes identified based on the oil and gas-indicating microbial fingerprint, and fluorescence quantitative PCR was conducted to obtain an oil-indicating microbial value and/or a gas-indicating microbial value.

(31) 10. According to microbial results and geophysical and petroleum geological data, an oil and gas area was comprehensively determined through a multi-disciplinary and multi-field technology.

(32) FIG. 2 shows an isopleth of microbial values of the exploration area obtained according to the method in Example 1 of the present disclosure. FIG. 1 shows an isopleth of the same area obtained according to a method of the patent (granted patent publication No.: CN 107267623 B). It can be seen from FIG. 1 that although the isopleth has a high coincidence rate with a known well, it is not so obvious in the background area and abnormal area, and there are still many high points outside a trap, which may form a pseudo-abnormal band with other undefined abnormal points. It can be seen from FIG. 2 that the isopleth not only has a high coincidence rate with a known well, but also can have an excellent coincidence rate with a trap line and finely characterize an oil area.

Example 2 Research on a Preservation Effect of a Sample Preservation Agent

(33) Formula 1 of the preservation agent:

(34) CTAB: 0.5% (w/v); ethanol: 2% (v/v); ?-mercaptoethanol: 1% (v/v); ascorbic acid: 0.9 mM; ascorbyl palmitate: 0.1 mM; ethylenediaminetetraacetic acid disodium salt: 10 mM; NaCl: 0.5 mM; DNase inhibitor: 1 mg/L; and pH 8.0 Tris-HCl: the balance.

(35) Formula 2 of the preservation agent:

(36) CTAB: 2% (w/v); ethanol: 4.5% (v/v); ?-mercaptoethanol: 3% (v/v); ascorbic acid: 0.9 mM; ascorbyl palmitate: 0.1 mM; ethylenediaminetetraacetic acid disodium salt: 5 mM; NaCl: 0.2 mM; DNase inhibitor: 2 mg/L; and pH 8.0 Tris-HCl: the balance.

(37) Formula 3 of the preservation agent:

(38) CTAB: 1% (w/v); ethanol: 3% (v/v); ?-mercaptoethanol: 2% (v/v); ascorbic acid: 0.9 mM; ascorbyl palmitate: 0.1 mM; ethylenediaminetetraacetic acid disodium salt: 8 mM; NaCl: 0.3 mM; DNase inhibitor: 2 mg/L; and pH 8.0 Tris-HCl: the balance.

(39) Formula 4 of the preservation agent:

(40) CTAB: 0.5% (w/v); ethanol: 2% (v/v); ?-mercaptoethanol: 1% (v/v); ascorbic acid: 0.5 mM; ascorbyl palmitate: 0.5 mM; ethylenediaminetetraacetic acid disodium salt: 10 mM; NaCl: 0.5 mM; DNase inhibitor: 1 mg/L; and pH 8.0 Tris-HCl: the balance.

(41) Formula 5 of the preservation agent:

(42) CTAB: 0.5% (w/v); ethanol: 2% (v/v); ?-mercaptoethanol: 1% (v/v); ascorbic acid: 0.75 mM; ascorbyl palmitate: 0.25 mM; ethylenediaminetetraacetic acid disodium salt: 10 mM; NaCl: 0.5 mM; DNase inhibitor: 1 mg/L; and pH 8.0 Tris-HCl: the balance.

(43) Preparation method of the preservation agent:

(44) According to the formula, the ethanol and ?-mercaptoethanol were added to the Tris-HCl, a resulting mixture was thoroughly stirred, then the CTAB, ascorbyl palmitate, ascorbic acid, ethylenediaminetetraacetic acid disodium salt, NaCl, and DNase inhibitor were added, and a resulting mixture was stirred for dissolution.

(45) The preparation of the preservation agent was conducted in a sterile environment, and the preservation agent was filtered through a 0.25 ?m filter membrane to allow sterilization for later use.

(46) The preservation agent of the above formula was added to a fresh sample obtained in Example 1 according to a volume ratio of 1:1, then a resulting mixture was preserved at 25? C. for 72 h, and total DNA was extracted according to a conventional method, which was an experimental group. Total DNA was extracted from a sample not preserved with the preservation agent of formula 1 (processing time: less than 4 h) and used as a control, which was recorded as 100%. Total DNA extraction rates obtained were shown in the table below:

(47) TABLE-US-00004 Total DNA extraction rate/% Preservation agent of formula 1 (preservation at 25? C. 90.2b for 72 h) Preservation agent of formula 2 (preservation at 25? C. 91.3b for 72 h) Preservation agent of formula 3 (preservation at 25? C. 92.7b for 72 h) Preservation agent of formula 4 (preservation at 25? C. 92.3b for 72 h) Preservation agent of formula 5 (preservation at 25? C. 96.4a for 72 h) Preservation agent of formula 1 (no preservation) 100a

(48) The results show that a total DNA extraction rate of a sample preserved with the preservation agent of formulas 1 to 5 for 3 d at room temperature is greater than 90%, which can meet the requirements of the agent; and the preservation agent of formula 5 has the optimal performance.

(49) The preferred specific implementations and examples of the present disclosure are described in detail above, but the present disclosure is not limited to the above implementations and examples. Within the knowledge of those skilled in the art, various variations can also be made without departing from the concept of the present disclosure.