WO2018145329A1

WO2018145329A1 - Pretreatment method of soil sample for hydrocarbon oxidizing bacteria detection in microbial prospecting of oil and gas

Info

Publication number: WO2018145329A1
Application number: PCT/CN2017/074109
Authority: WO
Inventors: 郝纯; 梅海
Original assignee: 盎亿泰地质微生物技术（北京）有限公司
Priority date: 2017-02-10
Filing date: 2017-02-20
Publication date: 2018-08-16
Also published as: CN106701894B; CN106701894A

Abstract

A pretreatment method of soil sample for hydrocarbon oxidizing bacteria detection in microbial prospecting of oil and gas comprises the following steps: 1) placing soil collected in the field into an open permeable container and sealing the opening of the container; and 2) performing a constant temperature blast to the permeable container sealed in step 1) to dry the soil therein. The test results showed that the soil pretreated with the method was used for hydrocarbon oxidizing bacteria detection, and the results thereof were similar to those of the conventional freezing method. After drying, the soil was put at room temperature for two weeks and then tested again. The result thereof was similar to that of the immediately dried sample. The above results indicate that the method, as an alternative method to conventional cryopreservation, has a high applicability in the study of hydrocarbon oxidizing bacteria in microbial prospecting of oil and gas.

Description

Pretreatment method for soil samples for oil and gas microbial exploration of hydrocarbon oxidizing bacteria

This application claims priority to Chinese Patent Application filed on Dec. 10, 2017, the Chinese Patent Office, Application No. 201710074144.5, entitled "Pretreatment Method for Soil Samples for Oil and Gas Microbial Exploration of Hydrocarbon Oxidase Detection" The entire content of which is incorporated herein by reference.

Technical field

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.

Background technique

At present, 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.

Among them, the microbiological basis of oil and gas microbial exploration technology is the abnormally high adaptability and wide distribution of bacteria to different nutrient sources. Oil and gas microbial exploration is carried out by measuring the biochemical characteristics of hydrocarbon oxidizing bacteria and the characteristics of population distribution. 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.

However, in the study of hydrocarbon oxidizing bacteria in oil and gas microbial exploration, the soil samples were pretreated by the above-mentioned freezing or low temperature method, and the method has the following problems:

1) 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.

2) Transportation is difficult. The process of transporting frozen or low-temperature samples stored in the refrigerator, liquid nitrogen, ice pack or dry ice from the field to the laboratory is difficult, costly, and the risk of sample warming is high.

Summary of the invention

In view of this, 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:

1) placing the soil collected in the field in an open air permeable container to seal the opening of the container;

2) The ventilated container sealed in step 1) is subjected to constant temperature blasting to dry the soil therein.

Preferably, the temperature of the constant temperature blast is 25 to 50 °C.

Preferably, the temperature of the constant temperature blast is 30 to 38 °C.

Preferably, the constant temperature blasting time is from 1 to 72 hours.

Preferably, the constant temperature blasting time is 3 to 24 hours.

Preferably, the open venting container is a kraft paper bag.

Preferably, the manner of the constant temperature blast is that the individual blasts are separately thermostated or the thermostat and the blast are used simultaneously.

Preferably, the method further comprises:

3) 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. However, 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.

Compared with the prior art, 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.

DRAWINGS

1 is a comparison diagram of detection results of two soil pretreatment methods according to Embodiment 1 of the present invention;

2 is a comparison diagram of detection results of two soil pretreatment methods according to Embodiment 2 of the present invention;

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;

4 is a comparison diagram of detection results of samples 1 to 16 treated at different temperatures in Example 4 of the present invention.

detailed description

The technical solutions in the embodiments of the present invention are clearly and completely described below. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.

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.

According to the actual exploration situation, 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.

In the embodiment of the present application, 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. In addition, the soil pretreatment method of the present application is also applicable to the pretreatment of water-containing soil such as farmland, forest, Gobi.

In some embodiments of the present application, the area involved is a high-altitude Gobi area on the Tibetan Plateau, which is a permafrost with a few vegetation on the surface. In the embodiment of the present application, 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. Another in this application In some embodiments, 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. In the embodiment of the present application, 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. In an embodiment of the present application, the soil sample collection interval may be 250 m and the collection depth is 20 cm. In the embodiment of the present application, 8 to 20 samples can be collected for pretreatment, and the mass of each sample is 20 g to 30 g. In the present application, 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.

In the embodiment of the present application, 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. In the present invention, such 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.

In an embodiment of the present application, the temperature of the constant temperature blast is 25 to 50 ° C, preferably 30 to 38 ° C. In the embodiment of the present application, 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. At this time, 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.

In the examples of the present application, after the drying treatment in the air-blast oven, the subsequent detection of the hydrocarbon oxidizing bacteria is performed. For the detection method of the hydrocarbon oxidizing bacteria, refer to US Patent 3880142 (Hitzman, 1959) Hitzman, DO, 1959. Prospecting for petroleum deposits (detecting hydrocarbon consumption bacteria colonies by artificial hydrocarbon nutrient culturing). US Patent, 3, 880, 142, assigned to Phillips Petroleum Co. The result of the counting is directly recorded as the microbial value of the sample.

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.

Some embodiments of the present application further comprise: preserving the dried soil sample at room temperature. Wherein, 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.

In summary, in the embodiment of the present invention, 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.

Therefore, 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. In addition, the soil pretreatment method is suitable for pretreatment of water-containing soils such as farmland, forests, and Gobi.

In order to further understand the present application, a pretreatment method for a soil sample for oil and gas microbial exploration hydrocarbon oxidizing bacteria detection provided by the present invention will be specifically described below in conjunction with examples.

Example 1

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. To carry out oil and gas microbial exploration work in the area, it is necessary to lay a survey line or test net. Collect soil from 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.

The specific implementation steps of the soil sample pretreatment of the present invention are as follows:

1 According to the actual exploration situation, select the deployed survey lines in the work area and collect 20 samples. The sample collection distance is 250m and the collection depth is 20cm.

2 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.

3 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.

4 The results obtained by the above two methods are compared to a line graph. The results are shown in FIG. 1. 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.

Example 2

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.

2 Each sampling point was collected according to a conventional method, placed in a sealed plastic bag, and immediately frozen and stored in the same manner as in Example 1, and all samples were quickly transported back to the laboratory.

3 Transfer the sample from the sealed plastic bag to the kraft paper bag in the laboratory, seal it, place it in a blast oven at 30 °C, and dry it for 8 hours. The sample is then divided into two parts, one is called a dried sample, and the subsequent hydrocarbon oxidizing bacteria are immediately tested; the other is stored at room temperature, and the hydrocarbon oxidizing bacteria are detected two weeks later.

4 The results obtained by the above two methods are compared to a line graph, and the result is shown in FIG. 2 . 2 is a comparison diagram of detection results of two soil pretreatment methods according to Example 2 of the present invention.

From the overall view of Fig. 2, 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.

Example 3

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.

In order to compare the effects of different drying times on the results of hydrocarbon oxidizing bacteria counts, the following tests were carried out:

1 According to the actual exploration situation, select the known oil and gas fields in the work area, design 16 lines of samples through the oil and gas field, numbered 1-16, 5-10 samples are located above the oil field, the sample collection distance is 250m, the collection depth It is 20cm.

3 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.

4 The results of the five methods were compared to a line graph. The results are shown in FIG. 3. 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.

It can be seen from Fig. 3 that different drying times have little effect on the number of hydrocarbon oxidizing bacteria obtained at the last detection. Samples 5-10 exhibit high values, which are consistent with the reservoirs that are present beneath them.

Example 4

The study area involved is the same as in the third embodiment. In order to compare the effects of different drying temperatures on the results of hydrocarbon oxidizing bacteria counts, the following tests were carried out:

1 According to the actual exploration situation, select the known oil and gas fields in the work area, design 16 lines of samples through the oil and gas field, numbered 1-16, 5-10 samples are located above the oil field, the sample collection distance is 250m, the collection depth It is 20cm. The sample collected was the same as in Example 3.

3 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. 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.

4 The results of the above five methods were compared to a line graph. The results are shown in FIG. 4. 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.

It can be seen from Fig. 4 that the microbial value after treatment at 50 ° C is lower than other temperature treatments; except for drying at 50 ° C, different drying temperatures have little effect on the amount of hydrocarbon oxidizing bacteria obtained at the last detection. Samples 5-10 of all processing temperatures exhibited relatively high values, consistent with the occurrence of reservoirs below.

As is apparent from the above examples, 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. .

The above description is only a preferred embodiment of the present invention, and it should be noted that A person skilled in the art can make various modifications to these embodiments without departing from the principles of the present invention, and such modifications are also considered to be within the scope of the invention.

Claims

A pretreatment method for soil samples for oil and gas microbial exploration of hydrocarbon oxidizing bacteria, characterized in that it comprises the following steps:

1) placing the soil collected in the field in an open air permeable container to seal the opening of the container;

2) The ventilated container sealed in step 1) is subjected to constant temperature blasting to dry the soil therein.
The pretreatment method according to claim 1, wherein the temperature of the constant temperature blast is 25 to 50 °C.
The pretreatment method according to claim 2, wherein the temperature of the constant temperature blast is 30 to 38 °C.
The pretreatment method according to claim 1, wherein the constant temperature blasting time is from 1 to 72 hours.
The pretreatment method according to claim 4, wherein the constant temperature blasting time is 3 to 24 hours.
The pretreatment method according to claim 1, wherein the open gas permeable container is a kraft paper bag.
The pretreatment method according to claim 1, characterized in that 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 preprocessing method according to any one of claims 1 to 7, further comprising:

3) The sample dried in step 2) is stored at room temperature.