JP7267588B2 - Detection method and piping maintenance method - Google Patents

Description

The present invention relates to a detection method and a pipe maintenance method.

In recent years, when mining fossil fuels (e.g. petroleum, natural gas, shale oil, shale gas, etc.), crushing of bedrock (hydraulic fracturing) using high-pressure water has been carried out. etc., microbial corrosion is observed.

Techniques for detecting biofilm formation, which is one of the signs of the occurrence of such microbial corrosion, are known.
In Patent Document 1, "a metal electrode arranged in a flow path, a reference electrode arranged in the flow path, and a non-metallic material are formed, and the metal electrode is upstream in the flow direction in the flow path. and a cover portion covering the side surface.

JP 2014-181963 A

The method of Patent Document 1 is an indirect method of placing a metal material for monitoring in the flow path and detecting the occurrence of biofilm on the metal material, and directly detects microbial corrosion of the metal pipe itself or its signs. It was nothing to detect.

SUMMARY OF THE INVENTION In view of the above problems, an object of the present invention is to provide a detection method for directly detecting microbial corrosion of a metal pipe through which a fluid is circulating, or a sign thereof. Another object of the present invention is to provide a pipe maintenance method.

As a result of intensive studies aimed at achieving the above object, the inventors of the present invention have found that the above object can be achieved with the following configuration.

[1] A detection method for detecting microbial corrosion of metal piping through which a fluid is circulating, wherein at least one selected from the group consisting of proteins, nucleic acids, and complexes thereof in the fluid A detection method comprising the steps of measuring a change per unit time in the content of a target molecule and comparing the change with a predetermined standard.
[2] The detection method according to [1], wherein the target molecule is a product derived from a bacteriophage.
[3] The detection method according to [1] or [2], wherein the content is the relative amount of labeled target molecules.
[4] The detection method according to any one of [1] to [3], wherein the measurement is fluorescence intensity measurement.
[5] The step of performing the detection method according to any one of [1] to [4], and adding an agent for suppressing the activity of microorganisms to the fluid when it is determined to be abnormal as a result of the comparison. A pipe maintenance method, comprising:

ADVANTAGE OF THE INVENTION According to this invention, the detection method which directly detects microbial corrosion of metal piping itself in which the fluid distribute|circulates, or its sign can be provided. The present invention can also provide a pipe maintenance method.

FIG. 4 is a schematic cross-sectional view of a metal pipe through which fluid flows; 1. It is explanatory drawing which shows the result of having measured the content of the product in the fluid in the P1 point in metal piping in sectional drawing of FIG. It is another example of a schematic cross-sectional view of a metal pipe through which a fluid flows. 2 is an explanatory diagram showing the result of measuring the content of target molecules in the fluid at point P2 in the metal pipe in the cross-sectional view of FIG. 1. FIG. It is an explanatory view for explaining a detection method concerning other embodiments of the present invention. FIG. 5 is a flow diagram for explaining a detection method according to another embodiment of the present invention;

The present invention will be described in detail below.
Although the description of the constituent elements described below may be made based on representative embodiments of the present invention, the present invention is not limited to such embodiments.
In this specification, a numerical range represented by "-" means a range including the numerical values before and after "-" as lower and upper limits.

[Detection method]
A detection method according to an embodiment of the present invention (hereinafter also referred to as "this detection method") is a detection method for detecting microbial corrosion of a metal pipe through which a fluid is flowing, comprising protein in the fluid, and measuring a change per unit time in the content of at least one target molecule selected from the group consisting of nucleic acids; and comparing the change with a predetermined standard. .

In addition, in this specification, a metal pipe means a pipe in which metal is used as a material at least in part. Piping means a set of members used for circulating fluid, and includes pipes, joints, gaskets, and the like.
Further, detecting microbial corrosion means detecting that corrosion has occurred due to the activity of microorganisms and/or that corrosion may occur due to the activity of microorganisms. It means to detect the occurrence of locally proliferating cell populations within. In addition, in general, the above cell populations are often microbial groups that have adhered to and proliferated on metal pipes, and moreover, the microbial groups often form biofilms.

One of the causes of corrosion in metal pipes used in hydraulic fracturing and the like is the activity of microorganisms. Such corrosion is commonly referred to as Microbiologically Influenced Corrosion. Microbial corrosion is caused by the combined contribution of microorganisms present in the fluid (e.g., sulfate-reducing bacteria, sulfur-reducing bacteria, sulfur-oxidizing bacteria, iron-oxidizing bacteria, iron-reducing bacteria, acid-producing bacteria, etc.). It is believed that

The above microorganisms may be contained in the fluid flowing through the inside of the metal pipe, but these microorganisms adhere to the metal pipe more than when they are present in the fluid in a floating state, Furthermore, the present inventors have found that when immobilized, particularly when biofilms are formed, metal pipes greatly contribute to microbial corrosion.
In general, biofilms have a lower oxygen content than fluids and are in a special environment. presumed to contribute.

In other words, the formation of locally proliferating cell populations (particularly biofilm-forming microorganisms) in metal pipes is considered to be one of the signs that microbial corrosion is occurring or that corrosion is occurring. Early detection of this is considered important for maintenance management of metal pipes.
However, it has been considered difficult to directly and accurately detect the generation of the cell population in a metal pipe having a vast surface area compared to the surface area occupied by the cell population.
In addition, when the cell population is a group of microorganisms that formed a biofilm, the group of microorganisms maintains homeostasis inside the biofilm, and microbial cells in a floating state are not necessarily released outside the biofilm. . Therefore, it has been difficult to detect changes resulting from biofilm formation by measuring the amount (number) of microorganisms in a fluid.

Therefore, conventionally, a method of indirectly examining the state of metal piping has been used. For example, in the method described in Patent Document 1, a sensor is placed in a metal pipe, a fluid retention portion is formed in the sensor, and biofilm generation in the retention portion is detected. It is intended to predict biofilm development. These methods cannot be said to directly detect local biofilm development.

When the generation of biofilms in metal pipes is predicted by the above method, measures such as adding a disinfectant to the fluid flowing through the metal pipes are often taken to suppress microbial corrosion. However, according to the above measurement method, it does not necessarily reflect the actual situation of the piping, and there were cases where the disinfectant was put in even though there was no actual biofilm generation. . This was both environmentally and economically disadvantageous.

On the other hand, even if biofilms are actually expected to occur, it is not possible to determine in which part of the metal pipes the biofilms are likely to occur. In some cases, the whole must be treated equally, and in this case also there were problems in terms of cost and environment.
In addition, even if an attempt is made to identify the position where biofilms are generated, it has been difficult to directly sample and analyze biofilms and the like in underground and/or buried metal pipes. Furthermore, in the state where the biofilm is formed, the internal microbial community maintains homeostasis, so the microorganisms themselves are less likely to be released into the fluid outside the biofilm, and the biofilm originates from the fluid. Microorganisms (or changes thereof) have been difficult to detect.

On the other hand, the present inventors found that the cell population generated in the metal pipe, typically the microorganism group that adheres to the metal pipe and is immobilized to form a biofilm, is a substance inside and outside the biofilm. Focusing on the fact that transport is performed, among others, a certain amount of proteins and at least one target molecule selected from the group consisting of nucleic acids are released from the group of microorganisms forming the biofilm. I paid attention.

Although the mechanism by which the target molecule is released is not necessarily clear, the present inventors speculate that products derived from bacteriophage contribute to this.

A bacteriophage is a virus that infects a microorganism (typically a bacterium) and in the process of infection many progeny bacteriophage are produced via the infecting microorganism.
Normally, a certain amount of microorganisms present in metal pipes through which fluid is circulated may also be infected with bacteriophages, and when fluid containing microorganisms is circulated in metal pipes, , bacteriophages are also present.
However, when fluid is flowing through a metal pipe, the content of bacteriophage-derived products contained in the fluid at a given location in the metal pipe often does not change significantly over time.

FIG. 1 shows a schematic cross-sectional view of a metal pipe through which fluid flows. In FIG. 1, a fluid 11 flows through a metal pipe 10 in the "Flow" direction in the drawing, and the fluid 11 contains microorganisms 12 . FIG. 2 is an explanatory diagram showing the result of measuring the content of the product in the fluid at point P1 in the metal pipe 10. As shown in FIG. In FIG. 2, the horizontal axis indicates time, and the vertical axis indicates the content of the target molecule in the fluid (relative amount, fluorescence intensity described later). 13 is plotted in the figure.

As described above, when there is no locally proliferated cell population in the pipe 10, the target molecule content does not change significantly over time. In this case, the detected product is derived from the microorganism 12 present in the fluid 11, but in an environment in which the fluid 11 is constantly flowing, the content of the target molecule at point P1 is Big changes are unlikely.

FIG. 3 shows another example of a schematic cross-sectional view of a metal pipe through which fluid flows. In FIG. 3, a fluid 11 flows in the metal pipe 10 in the "Flow" direction in the figure, the fluid 11 contains microorganisms 12, and some of the microorganisms adhere to the metal pipe 10, Immobilized to form a locally proliferated cell population 14 .
FIG. 4 is an explanatory diagram showing the result of measuring the target molecule content in the fluid at the point P2 in the metal pipe 10. As shown in FIG. In FIG. 4, the horizontal axis represents time, and the vertical axis represents the content of target molecules in the fluid (relative amount, fluorescence intensity described later). 15 is plotted in the figure.

As described above, when the locally proliferated cell population 14 is present in the pipe 10, the fluid 11 contains the locally proliferated cell population 14 in addition to the target molecule derived from the microorganism 12. The amount of target molecules released outside the biofilm is added, and the content of target molecules changes over time.

This change changes during the cell population formation process such as the growth of the microorganism group and the formation of the biofilm. For example, in the area of ar1 in FIG. It can be seen that a biofilm is formed in the pipe 10 in the region of ar2, and internal homeostasis is maintained.

Thus, changes in the content of target molecules in the fluid over time are thought to reflect movement of the target molecules inside and outside the biofilm. Therefore, by measuring the time-dependent change in the content of the target molecule in the fluid, it is possible to detect that the microorganisms are attached and immobilized in the metal pipe, and that the biofilm is formed. In addition, when the target molecule is a product of a bacteriophage, since there are many target molecules generated from one microbial cell, the signal from one microbial cell is substantially amplified, resulting in excellent sensitivity. can get.

As already explained, biofilm formation is a sign of microbial corrosion, and according to this detection method, microbial corrosion can be detected by examining the state of metal pipes in which fluid is flowing.
Below, each process of this detection method is explained in full detail.

[Measurement process]
The measuring step is a detection method for detecting microbial corrosion of a metal pipe through which a fluid is circulating, wherein at least one selected from the group consisting of proteins, nucleic acids, and complexes thereof in the fluid. This is the step of measuring the change in the target molecule content per unit time.

The change in the target molecule per unit time means the change in the content of the target molecule in the fluid when the measurement is continuously or intermittently performed at a predetermined position in the metal pipe for a predetermined period of time.

(target molecule)
The target molecule is at least one selected from the group consisting of proteins, nucleic acids, and complexes thereof, and is typically preferably a bacteriophage-derived product. As used herein, a product derived from a bacteriophage means the bacteriophage itself, an intermediate substance produced during the production of the bacteriophage in an infected cell, and a substance derived from a ruptured cell infected with a bacteriophage ( protein, nucleic acid, etc.).
In addition, the number of target molecules to be quantified in this detection method may be one or two or more.

When the target molecule is the bacteriophage itself, the bacteriophage is not particularly limited. One type of bacteriophage may be used, or two or more types may be used. As used herein, bacteriophages are capable of invading living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms and replicating themselves. includes any other term referring to viruses that use these to.

Here, "microscopic level" means one millimeter or less in its largest dimension. Bacteriophages are viruses that have evolved in nature to use bacteria as a means of replicating themselves. Phages do this by attaching themselves to a bacterium, injecting its DNA (or RNA) into the bacterium, and inducing the bacterium to replicate the phage hundreds or even thousands of times. This is called phage amplification.

Methods for detecting the content of target molecules are not particularly limited, but examples include matrix-assisted laser desorption/ionization/mass spectrometry (MALDI/TOF), ion mobility spectroscopy, optical spectroscopy, and antigen-antibody A known method such as a reaction method can be used.

Among others, the target molecule content is preferably the relative amount of the labeled target molecule, since it can be measured more easily. Since this detection method is characterized by detecting abnormal changes in the fluid that is constantly flowing through metal pipes, measuring the relative amount is sufficient to obtain the desired results. It is superior in that it is simpler in that it does not need to measure the absolute amount.

A labeled target molecule typically means a target molecule that has been dyed so that it can be quantitatively detected by spectroscopy.
A method for labeling the target molecule is not particularly limited, and a known dye may be used. Examples of known dyes that can be used include 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), Hoechst 33342, Propidium Iodide, and Coomassie Brilliant Blue.
Although the method for measuring the content of the labeled target molecule is not particularly limited, measurement of fluorescence intensity is preferred because it is simpler. In this case, the excitation and fluorescence wavelengths may be appropriately changed according to the dye used.

(fluid)
Although the fluid is not particularly limited, examples thereof include seawater and river water.

(Microorganism)
Although the microorganism is not particularly limited, it is preferably a microorganism that causes corrosion of pipes. Microorganisms that cause corrosion of piping are not particularly limited, but
For example, sulfur-reducing bacteria such as Desulfovibrio and Desulfuromonas; sulfur-oxidizing bacteria such as Thiobacillus; iron (or manganese)-oxidizing bacteria such as Galionella, Leptothrix, and Mariprofundus; iron-reducing bacteria such as the genera, Shewanella and Geothermobacter; acidogenic bacteria and fungi such as Clostridium, Fusarium, Penicillium and Hormoconis; In this paragraph, ";" is used as a symbol indicating the hierarchy of classification, and the same applies to the following in this specification.

[Comparison process]
The comparison step is a step of comparing the change with a predetermined criterion. The criteria for comparison are not particularly limited and can be determined as appropriate. For example, when comparing with the immediately preceding measured value, it is possible to determine an abnormality based on the fact that the measured value rises three times in a row as a reference.

[Pipe maintenance method]
The pipe maintenance method according to the embodiment of the present invention includes the steps of performing the already described detection method, and the steps of adding a chemical agent to the fluid to suppress the activity of microorganisms when it is determined to be abnormal as a result of the comparison. and a pipe maintenance method.

The agent to be added is not particularly limited as long as it can suppress the activity of microorganisms, and known bactericides and the like can be used.

[Another embodiment of the detection method]
A detection method according to another embodiment of the present invention is a detection method for detecting the occurrence of microbial corrosion in a metal pipe in which a fluid is circulating, and the location of the occurrence, wherein in the metal pipe, an upstream A first step of selecting a measurement point on the side and a measurement point on the downstream side, and at least one selected from the group consisting of proteins, nucleic acids, and complexes thereof in the fluid at each measurement point a second step of measuring a change in the content of the target molecule per unit time; side measurement point as a new downstream measurement point, select a new upstream measurement point from the upstream side of the upstream measurement point, and determine the amount of change at the new upstream measurement point is less than said amount of change at said new downstream measurement point, repeating said steps A and B until said step C is repeated.

According to this detection method, the occurrence of microbial corrosion and its location can be detected. FIG. 5 shows an explanatory diagram for explaining this method.
FIG. 5 shows a metal pipe through which fluid flows in the "Flow" direction. Here, A to D respectively indicate measurement points (places).

The four graphs in FIG. 5 show the fluorescence intensity (total) derived from the target molecule content versus the observation time at each point A to D of the metal pipe, in other words, the change in fluorescence intensity over time.
These measurement methods and the like have already been described in the description of the detection method according to the first embodiment, and description thereof will be omitted.

Graph B in FIG. 5 shows that the fluorescence intensity (denoted as “Intensity” in the figure) increases from time t1. The difference between the baseline and maximum value at this time is "IL-1". Similarly, in C and D, the fluorescence intensity increased at time t1 or later (the amounts of increase are shown as "IL-2" and "IL-3", respectively).

On the other hand, it can be seen that at point A, the fluorescence intensity is substantially constant. As already explained, a certain amount of microorganisms, bacteriophages, etc. are present in the fluid flowing through metal pipes. On the other hand, as at point B, the increase in the content of the target molecule over time means that local and abnormally proliferating cell populations (typically Specifically, it suggests the existence of a group of microorganisms adhering and proliferating on the inner wall of the pipe.

FIG. 6 is a diagram showing the flow of the method. In this method, first, an upstream measurement point and a downstream measurement point are selected along the flow direction of the fluid in the pipe. For example, assuming that point B in FIG. 5 is selected as the upstream side and point C is selected as the downstream side, the following steps will be described.

Next, the change in the target molecule content per unit time is measured at each measuring point. In FIG. 5, IL-1 and IL-2 correspond to the above.
Next, the amount of change is compared between the upstream measurement point and the downstream measurement point. At this time, the amount of change at the B point is greater than at the C point.

Next, point B is set as a new downstream measurement point, point A, which is upstream of point B, is selected as a new upstream measurement point, and again, the difference between the upstream measurement point and the downstream measurement point is between, compare the amount of change above. At this time, the amount of change at point A is smaller than at point B.

By doing so, microbial corrosion occurs in the area between the finally measured upstream measurement point (point A in the above case) and the downstream measurement point (point B in the above case). It is possible to detect that there is a possibility that

According to this method, it is possible to detect, with high accuracy, the occurrence and location of microbial corrosion in metal pipes for high-pressure water used, for example, in hydraulic fracturing.

10: Metal piping 11: Fluid 12: Microorganisms 13, 15: Measurement data 14: Microorganism group

Claims (4)

A detection method for detecting microbial corrosion of metal piping through which a fluid flows, comprising:
measuring changes per unit time in the content of at least one target molecule selected from the group consisting of proteins, nucleic acids, and complexes thereof in the fluid;
comparing the change to a predetermined criterion ;
a step of determining that a biofilm containing microorganisms that cause microbial corrosion is formed in the metal pipe when it is determined to be abnormal as a result of the comparison ;
The detection method, wherein the target molecule is a substance present in the fluid flowing through the metal pipe and a product derived from a bacteriophage that infects the microorganism. The detection method according to claim 1 , wherein the content is the relative amount of the labeled target molecule. The detection method according to claim 1 or 2 , wherein the measurement is fluorescence intensity measurement. A step of performing the detection method according to any one of claims 1 to 3 ;
and adding a chemical agent to the fluid for suppressing the activity of microorganisms when the fluid is determined to be abnormal as a result of the comparison.

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