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WO2018145329A1 - Procédé de prétraitement d'échantillon de sol permettant la détection de bactéries oxydant les hydrocarbures dans la prospection microbienne de pétrole et de gaz - Google Patents

Procédé de prétraitement d'échantillon de sol permettant la détection de bactéries oxydant les hydrocarbures dans la prospection microbienne de pétrole et de gaz Download PDF

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Publication number
WO2018145329A1
WO2018145329A1 PCT/CN2017/074109 CN2017074109W WO2018145329A1 WO 2018145329 A1 WO2018145329 A1 WO 2018145329A1 CN 2017074109 W CN2017074109 W CN 2017074109W WO 2018145329 A1 WO2018145329 A1 WO 2018145329A1
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soil
oxidizing bacteria
sample
oil
pretreatment method
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English (en)
Chinese (zh)
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郝纯
梅海
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Advanced Energy & Environmental Technologies Inc
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Advanced Energy & Environmental Technologies Inc
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    • 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
    • C12Q1/64—Geomicrobiological testing, e.g. for petroleum

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  • the invention belongs to the technical field of soil microbial detection, and particularly relates to a pretreatment method for soil samples for oil and gas microbial exploration of hydrocarbon oxidizing bacteria.
  • the sample preservation method for environmental soil microbial detection is mainly to freeze at a low temperature after on-site collection, or to keep it at 1-5 ° C, that is, after taking a certain amount of soil sample in the field to seal, use a refrigerator, liquid nitrogen, ice pack. Either dry ice or other methods are used to freeze or maintain 1-5 ° C, and the whole process of transportation also needs to be kept frozen or low temperature.
  • the use of preserved soil samples to detect microorganisms, the microbial information obtained is of great significance in the fields of agriculture, environmental protection and oil and gas exploration.
  • hydrocarbon oxidizing bacteria are a general term for a class of microorganisms capable of metabolizing hydrocarbons. Studies have shown that common hydrocarbon oxidizing bacteria are Streptomyces, Pseudomonas, Rhodococcus and Glycyrrhiza. Such microorganisms can be concentrated using very low light hydrocarbon gas as long as they have a continuous stream of light hydrocarbons, and are only enriched under the surface of the hydrocarbon-containing structure. The use of appropriate microbial techniques to detect microbial anomalies can accurately predict the presence of underlying reservoirs and the nature of their reservoirs.
  • the temperature is strict. Freezing or cryopreservation must ensure that the sample requires a strict low temperature process from acquisition to transportation, and it is often difficult to ensure low temperatures in field construction.
  • the present application provides a pretreatment method for soil samples for oil and gas microbial exploration of hydrocarbon oxidizing bacteria.
  • the pretreatment method provided by the present application is simple, effective, and low in cost, and can be used for oil and gas microbial exploration of hydrocarbon oxidizing bacteria detection. Especially convenient for long-distance transportation of soil samples.
  • the invention provides a pretreatment method for soil samples for oil and gas microbial exploration of hydrocarbon oxidizing bacteria, comprising the following steps:
  • step 2) The ventilated container sealed in step 1) is subjected to constant temperature blasting to dry the soil therein.
  • the temperature of the constant temperature blast is 25 to 50 °C.
  • the temperature of the constant temperature blast is 30 to 38 °C.
  • the constant temperature blasting time is from 1 to 72 hours.
  • the constant temperature blasting time is 3 to 24 hours.
  • the open venting container is a kraft paper bag.
  • the manner of the constant temperature blast is that the individual blasts are separately thermostated or the thermostat and the blast are used simultaneously.
  • the method further comprises:
  • step 2) The sample dried in step 2) is stored at room temperature.
  • Dry dehydration is a microbial preservation method, such as sand pipe preservation method.
  • the sand pipe preservation method is applicable to spore-producing actinomycetes, Bacillus, Aspergillus, Penicillium and a few yeasts, and the preservation time is about 2-10 years.
  • the application of the sand tube preservation method to vegetative cells is not effective. Because dry dehydration treatment will cause some microorganisms to die, and the preservation effect on vegetative cells is not good, it has not been used for soil microbiology research, especially in the study of hydrocarbon oxidizing bacteria in oil and gas microbial exploration. method.
  • the present invention places the soil collected in the field in an open air permeable container, and seals the opening of the container; then, the sealed ventilated container is subjected to constant temperature blasting to dry the soil therein; and the dried sample is at room temperature. save.
  • the test results show that the soil after pretreatment using the method of the present invention is subjected to the detection of a hydrocarbon oxidizing bacteria, and the results are similar to those of the conventional freezing method.
  • the soil after drying of the present invention was placed at room temperature for two weeks, and then tested again, and the results were similar to those of the freshly dried sample.
  • the above results illustrate that the method of the present invention as an alternative to conventional cryopreservation in oil and gas microorganisms In the study of hydrocarbon oxidizing bacteria in exploration, it has high applicability.
  • FIG. 2 is a comparison diagram of detection results of two soil pretreatment methods according to Embodiment 2 of the present invention.
  • Example 3 is a comparison diagram of detection results of samples 1 to 16 after different drying time treatments in Example 3 of the present invention.
  • Example 4 is a comparison diagram of detection results of samples 1 to 16 treated at different temperatures in Example 4 of the present invention.
  • the invention provides a pretreatment method for soil samples for oil and gas microbial exploration of hydrocarbon oxidizing bacteria, comprising the following steps:
  • step 2) The ventilated container sealed in step 1) is subjected to constant temperature blasting to dry the soil therein.
  • the object of the invention is to overcome the disadvantages of cryopreservation of soil samples, and provide a simple and new pretreatment method for long-distance transportation of soil samples, and has practical effects and effects in the study of hydrocarbon oxidizing bacteria for oil and gas microbial exploration.
  • the soil of the surface is collected in the field, put into a non-sealed bag such as a kraft paper bag, and sealed to prevent leakage.
  • a non-sealed bag such as a kraft paper bag
  • the environmental area for collecting the soil sample is generally a region or a work area for carrying out oil and gas microbial exploration work; the soil collected in the field contains a certain amount of hydrocarbon oxidizing bacteria.
  • the soil pretreatment method of the present application is also applicable to the pretreatment of water-containing soil such as farmland, forest, Gobi.
  • the area involved is a high-altitude Gobi area on the Vietnamese Plateau, which is a permafrost with a few vegetation on the surface.
  • oil and gas microbial exploration work is to be carried out in the area, and a survey line or a test net is needed to collect the soil on the surface.
  • the area involved is the eastern plain of the Songliao Basin, which belongs to the high latitude cold area, the surface is farmland, and rice is grown.
  • oil and gas microbial exploration work is to be carried out in the area, and a survey line or a test net is needed to collect the soil on the surface.
  • the method for collecting soil samples in the present application is not particularly limited, and the conventional collection method in the art can be used.
  • the soil sample collection interval may be 250 m and the collection depth is 20 cm.
  • 8 to 20 samples can be collected for pretreatment, and the mass of each sample is 20 g to 30 g.
  • the soil collected in the field can be directly pretreated, or it can be pre-treated after being frozen and stored in a sealed plastic container.
  • the venting container used in the present application is a vented and open, non-sealed container through which at least water vapor can pass, such as a non-sealed bag having an open kraft paper bag. Then, the embodiment of the present application seals the bag sealed with soil to prevent sample leakage.
  • the sealed soil-filled ventilated container is placed in a device having a blast and a constant temperature function, and is subjected to constant temperature blasting, and the soil treated therein is dried.
  • the invention mainly utilizes two measures of constant temperature and blasting to dehydrate and dry the soil.
  • dry dehydration treatment may cause partial Gram-negative bacteria and vegetative cells such as Pseudomonas in the soil sample oxidizing bacteria to die, but Gram-like bacteria such as Streptomyces in the soil sample oxidizing bacteria Positive bacteria and some spore-forming hydrocarbon oxidizing bacteria have little effect.
  • the applicant unexpectedly found that although the dehydration treatment of this application will cause some of the hydrocarbon oxidizing bacteria to die, the hydrocarbon oxidizing bacteria that have survived dormancy are detected and statistically studied, and then various geological studies are carried out according to the results, and the low temperature can be achieved and frozen. Sample processing was similar to the results.
  • the temperature of the constant temperature blast is 25 to 50 ° C, preferably 30 to 38 ° C.
  • the time of the constant temperature blast is 1 to 72 hours, such as 8 hours, 12 hours, 24 hours, 48 hours, etc.; the time of the constant temperature blast of the present invention is preferably 3 to 24 hours. More preferably, it is 8 hours - 12 hours.
  • the time of the constant temperature blast treatment of the present application mainly varies depending on the degree of moisture of the soil, and it is possible to treat from 1 hour in desert soil to 72 hours in soil after rain. In order to facilitate the operation in the field and to consider the treatment effect, it can be treated by constant temperature blasting overnight, that is, it is preferably treated for about 12 hours.
  • the device with the blast and constant temperature function described in the embodiment of the present application may be a blast oven.
  • the drying method of the constant temperature blast treatment may be referred to as drying.
  • the constant temperature blasting method may be a single blast, a single constant temperature, or a simultaneous use of a constant temperature and a blast. Among them, depending on the size of the blast oven, the blast volume can be 10-500 m 3 /h, and the number of cycles is 100-1000 times/h.
  • test results show that the soil after pretreatment using the method of the present invention is subjected to the detection of a hydrocarbon oxidizing bacteria, and the results are similar to those of the conventional freezing method.
  • Some embodiments of the present application further comprise: preserving the dried soil sample at room temperature.
  • the normal temperature may be referred to as room temperature, generally 20 to 30 ° C which is well known in the art; and the storage time may be within two weeks.
  • the soil after drying of the present invention was placed at room temperature for two weeks, and then tested again, and the results were similar to those of the freshly dried sample.
  • the above results indicate that the method of the present invention, as an alternative method for conventional cryopreservation, has high applicability to the study of hydrocarbon oxidizing bacteria in oil and gas microbial exploration.
  • the soil collected in the field is put into a non-sealed bag such as a kraft paper bag, and sealed; the bag with the soil is placed in a box with a blast and a constant temperature function, and the air temperature is constant.
  • the soil is dried after treatment; the sample after drying is stored at room temperature.
  • the method of the invention dehydrates and drys the soil, causes a part of the microorganism to die, and another part of the microorganism enters a dormant state, and stores and transports the sample at normal temperature, and the treated sample is still suitable for hydrocarbon oxidation in oil and gas microbial exploration. Bacterial detection.
  • the pretreatment method provided by the present application is simple, effective, and low in cost, and can be used for oil and gas microbial exploration, and is particularly convenient for long-distance transportation of soil samples.
  • the soil pretreatment method is suitable for pretreatment of water-containing soils such as farmland, forests, and Gobi.
  • the area involved is a high-altitude Gobi area on the Qinghai-Tibet Plateau, which is a permafrost with a few vegetation on the surface.
  • Conventional frozen soil pretreatment and transportation methods require preparation of temperature control equipment such as refrigerators and ice packs, which are time consuming and difficult to transport, and it is difficult to achieve rapid freezing of samples at high altitudes.
  • Each sampling point was collected according to conventional methods, placed in a sealed plastic bag, and immediately stored frozen, and all samples were quickly transported back to the laboratory.
  • the conditions and operation of the freezing are: after collecting the soil in the field, put the incubator with the frozen ice bag, and after the camp, use the freezer to freeze at -20 °C, transport the incubator with the frozen ice bag, and ensure that it is frozen when it arrives at the laboratory. Store at the -20 °C freezer after arriving at the laboratory.
  • the frozen soil samples were divided into two parts in the laboratory. One sample was transferred from the sealed plastic bag to the kraft paper bag, sealed, placed in a blast oven at 30 ° C, and dried for 8 hours. The wind parameter is 25 m 3 /h, the number of cycles is 400 times / h, and a dry sample (referred to as a dry sample) is obtained, followed by subsequent detection of the hydrocarbon oxidizing bacteria. Another frozen sample, called a wet sample, was taken and the soil samples were tested for subsequent hydrocarbon oxidizing bacteria without any treatment. The frozen sample used in this example was 25 g, and the dried sample was a sample obtained by drying 25 g of the frozen sample.
  • FIG. 1 is a comparison diagram of the detection results of the two soil pretreatment methods according to the first embodiment of the present invention. From the overall view of Figure 1, after the two soil pretreatment methods, the amount of hydrocarbon oxidizing bacteria cultured by the sample is equivalent.
  • the working area involved in 1 is the same as that in the first embodiment. According to the actual exploration situation, the selected survey lines are selected in the work area, and 8 samples are collected. The sample collection distance is 250m and the collection depth is 20cm.
  • FIG. 2 is a comparison diagram of detection results of two soil pretreatment methods according to Example 2 of the present invention.
  • the hydrocarbon oxidizing bacteria were detected immediately after the soil blast drying treatment, and the treatment was carried out for two weeks at room temperature before detection, and the amount of the hydrocarbon oxidizing bacteria cultured was determined to be equivalent. It can be seen that the soil sample after the constant temperature blast dehydration treatment of the present application can be stored for a long time at room temperature, and the applicability of the method described in the present application is high.
  • the area involved is the eastern plain of the Songliao Basin, which belongs to the high latitude cold area, the surface is farmland, and rice is planted. To carry out oil and gas microbial exploration work in the area, it is necessary to lay up a survey line or test net to collect the soil on the surface.
  • Conventional frozen soil pretreatment and transportation methods require the preparation of temperature control equipment such as refrigerators and ice packs, which are time consuming and difficult to transport.
  • the frozen soil samples were divided into five parts in the laboratory.
  • One sample was transferred from the sealed plastic bag to the kraft paper bag, sealed and placed in a 38 ° C blast oven for drying for 12 h;
  • the samples were transferred from a sealed plastic bag to a kraft paper bag, sealed and placed in a 38 ° C blast oven for 24 h;
  • a sample was transferred from a sealed plastic bag into a kraft paper bag.
  • After sealing it was placed in a blast oven at 38 ° C for drying for 48 h;
  • a sample was transferred from a sealed plastic bag to a kraft paper bag, sealed, and placed in a 38 ° C blast oven for drying.
  • After 72 h treatment subsequent detection of the hydrocarbon oxidizing bacteria was carried out.
  • Another frozen sample that is, a sample that is not dried, is subjected to subsequent detection of a hydrocarbon oxidizing bacteria without any treatment.
  • FIG. 3 is a comparison diagram of the detection results of samples 1 to 16 after different drying time treatments in Example 3 of the present invention.
  • the frozen soil samples were divided into five parts in the laboratory.
  • One sample was transferred from the sealed plastic bag to the kraft paper bag, sealed and placed in a blast oven at 20 ° C for drying for 72 hours.
  • the samples were transferred from the sealed plastic bag to the kraft paper bag, sealed and placed in a blast oven at 30 ° C for drying for 72 h; a sample was transferred from the sealed plastic bag to the kraft paper bag. After sealing, it was placed in a blast oven at 40 ° C for drying for 72 hours; a sample was transferred from a sealed plastic bag to a kraft paper bag, sealed and placed in a 50 ° C blast oven for drying.
  • 72 h treatment subsequent detection of the hydrocarbon oxidizing bacteria was carried out.
  • Another frozen sample that is, a sample that is not dried, is subjected to subsequent detection of a hydrocarbon oxidizing bacteria without any treatment.
  • FIG. 4 is a comparison diagram of the detection results of samples 1 to 16 after different temperatures in Example 4 of the present invention.
  • the soil after pretreatment using the method of the present invention was subjected to the detection of a hydrocarbon oxidizing bacteria, and the results were similar to those of the conventional freezing method.
  • the soil after drying of the present invention was placed at room temperature for two weeks, and then tested again, and the results were similar to those of the freshly dried sample.
  • the invention provides a simple and new pretreatment method for long-distance transportation of soil samples, overcomes the disadvantages of cryopreservation of soil samples, has practical effects and effects in research of hydrocarbon microbial hydrocarbon oxidizing bacteria, and has high applicability. .

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Abstract

La présente invention concerne un procédé de prétraitement d'échantillon de sol pour la détection de bactéries d'oxydation d'hydrocarbures dans la prospection microbienne de pétrole et de gaz comprenant les étapes suivantes : 1) placer le sol collecté sur le terrain dans un récipient perméable ouvert et sceller l'ouverture du récipient ; et 2) effectuer un soufflage à température constante sur le récipient perméable scellé à l'étape 1) pour sécher le sol contenu dans ce dernier. Les résultats de test ont montré que le sol prétraité selon le procédé a été utilisé pour la détection de bactéries oxydant les hydrocarbures, et les résultats obtenus étaient similaires à ceux du procédé de congélation classique. Après séchage, le sol a été placé à température ambiante pendant deux semaines puis testé à nouveau. Le résultat obtenu est similaire à celui de l'échantillon séché immédiatement. Les résultats ci-dessus indiquent que le procédé, en tant que procédé alternatif à la cryoconservation classique, présente une applicabilité élevée dans l'étude de bactéries d'oxydation d'hydrocarbures dans la prospection microbienne de pétrole et de gaz.
PCT/CN2017/074109 2017-02-10 2017-02-20 Procédé de prétraitement d'échantillon de sol permettant la détection de bactéries oxydant les hydrocarbures dans la prospection microbienne de pétrole et de gaz Ceased WO2018145329A1 (fr)

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CN201710074144.5A CN106701894B (zh) 2017-02-10 2017-02-10 一种用于油气微生物勘探烃氧化菌检测的土壤样品的预处理方法

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Cited By (2)

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CN114958964A (zh) * 2022-07-08 2022-08-30 英索油能源科技（北京）有限责任公司 一种基于微生物基因的油气勘探方法
CN115975788A (zh) * 2023-03-02 2023-04-18 英索油能源科技（北京）有限责任公司 一种油气勘探用的微生物基因检测装置

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CN109423459B (zh) * 2017-08-30 2022-08-19 中国石油化工股份有限公司 一种假单胞菌及其鉴定方法和应用
CN109423457B (zh) * 2017-08-30 2022-02-01 中国石油化工股份有限公司 一种纳士维尔链霉菌及其鉴定方法和应用
CN109423458B (zh) * 2017-08-30 2022-08-19 中国石油化工股份有限公司 一种红球菌及其鉴定方法和应用

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CN105699168A (zh) * 2016-03-04 2016-06-22 湖南农业大学 一种土壤灭菌方法在测定土壤微生物生物量中的应用

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