Method for dynamically monitoring microbial corrosion risk of pipeline

Abstract

The invention discloses a pipeline microbial corrosion risk dynamic monitoring method which comprises the following three steps: S1, continuously collecting a pipeline water sample for multiple times, and carrying out viable bacteria qPCR analysis to obtain water sample microbial activity mu in different periods; the time interval between two times of collection of the pipeline water sample is 7-30 days, and the number of times of continuous collection of the pipeline water sample is not less than 3; s2, collecting the pipe wall sediment sample for qPCR analysis while collecting the pipeline water sample each time, and obtaining the total bacterial count of the pipe wall sediment in different periods; and S3, analyzing the variation trend of the dynamic data of the viable bacteria in the water sample and the dynamic data of the total bacterial count of the pipe wall sediment along with time, and evaluating the microbial corrosion risk condition in the pipeline. According to the method disclosed by the invention, the microbial corrosion risk in the pipeline is evaluated by defining the microbial activity index mu in a water sample, combining with the total bacterial count of pipe wall sediments and analyzing the change trends of the two parameters in a long-term monitoring process, so that the crossing from microbial quantity detection to microbial corrosion risk evaluation is realized.

Description

Technical Field

[0001] This invention relates to the field of pipeline corrosion monitoring technology, and in particular to a method for dynamic monitoring of pipeline microbial corrosion risk. Background Technology

[0002] Microbial corrosion poses a serious threat to oil and gas production. In shale gas production, due to the extensive use of flowback fluids, microbial corrosion is a major factor affecting pipeline transportation. For on-site microbial testing, according to industry standard SY / T0532-2012 "Analysis Method for Bacteria in Oilfield Injection Water - Extinction Dilution Method," bacterial culture is typically performed. After a growth period (>=7 days), the bacterial count is determined by examining the color change of the culture medium and / or the formation of precipitates and the dilution order. However, the international standard NACE TM0212-2018 requires a culture time (MPN method) of 7-28 days. The lengthy testing time and delayed results are unacceptable for production and maintenance companies seeking effective bacterial control.

[0003] Biofilm is generally considered the direct cause of microbial corrosion on pipeline walls. Conventional water sample testing only indicates the number of planktonic bacteria, which is not directly related to the number of bacteria attached to the biofilm. Therefore, it is common to see cases where almost no corrosive bacteria are detected in the water sample, yet microbial corrosion and perforation have occurred; conversely, there are also cases where the number of bacteria in the water sample is high, but the degree of pipe wall corrosion is low. Therefore, only by detecting / monitoring the pipeline biofilm can the microbial corrosion status of the pipeline be directly and accurately determined. Currently available biofilm sensors are affected by the detection location, and their results may not be representative. Due to technical and sampling limitations, accurate detection / monitoring of pipeline biofilm in the field is currently difficult to achieve.

[0004] With the development of technologies such as molecular biology, qPCR quantitative analysis, based on the principle of DNA amplification, has been widely used in laboratory analysis of bacteria and other microorganisms. By extracting microbial DNA, qPCR analysis can be completed within two hours, offering an unparalleled speed advantage compared to traditional bacterial culture methods. However, conventional qPCR analysis cannot distinguish between dead and live cells, meaning it cannot evaluate sterilization efficiency, thus limiting its application in the detection and control of microbial corrosion in pipelines. Summary of the Invention To address the issue of poor accuracy in existing pipeline microbial corrosion monitoring methods, this invention provides a dynamic monitoring method for pipeline microbial corrosion risk. This method is applicable to bacteria such as sulfate-reducing bacteria (SRB) and iron bacteria (FB), which can be detected using qPCR.

[0005] The method for dynamic monitoring of pipeline microbial corrosion risk provided by this invention comprises the following steps: S1. Collect pipeline water samples multiple times for live bacteria qPCR analysis to obtain the activity μ of microorganisms in the water samples at different times.

[0006] The time interval between two pipe water samples is 7-30 days. The time interval between the two water samples can be the same or different, and the number of consecutive pipe water samples collected is no less than 3. Preferably, the time interval between two pipe water samples is 15 days, and the total duration of continuous sampling and testing exceeds 3 months.

[0007] The method for viable bacteria qPCR analysis of collected water samples is as follows: S11. Take two water samples of the same volume from the collected water samples and filter them separately using two 0.2μm polycarbonate filter membranes. Label the two filter membranes as filter membrane T and filter membrane uT, respectively. Transfer the two filtered filter membranes to different centrifuge tubes under sterile conditions and label them as centrifuge tube T and centrifuge tube uT, respectively. Add sterile 1xPBS phosphate buffer solution to the two centrifuge tubes. S12. Add the active agent to centrifuge tube T and mix well. The active agent is propidium azidoide or ethidium bromide monoazide. Then, perform qPCR analysis on the samples in both centrifuge tubes. The specific method is as follows: incubate both centrifuge tubes at 37°C for 60-90 minutes; then add neutralization buffer to each centrifuge tube, mix well, incubate at room temperature for 15 minutes, and then centrifuge. Perform routine nucleic acid purification on the sediment at the bottom of the centrifuge tubes. Finally, test using a quantitative polymerase chain reaction (qPCR) instrument. The bacterial content measured in centrifuge tube T is recorded as C. T The bacterial count measured in centrifuge tubes is recorded as C. uT .

[0008] S13. Calculate microbial activity μ: .

[0009] S2. At the same time as collecting water samples from the pipeline, samples of sediment from the pipe wall were collected for qPCR analysis to obtain the total number of bacteria in the sediment from the pipe wall at different times.

[0010] The pipe wall deposits can originate from pigging materials, corrosion-resistant pads, or biofilm probes. Preferably, the pipe wall deposits are pigging materials obtained through a pigging operation. The pigging operation is preferably a combination of a foam pig and a wire brush pig.

[0011] If the deposits on the tube wall are mud-like samples, use a sterile syringe to draw 2-5 mL of mud, expel the air introduced by the syringe tip, and then immediately inject it into an anaerobic bottle pre-filled with inert gas or an anaerobic tube containing transport culture medium. After purging with nitrogen, seal and package for later use.

[0012] If the deposits on the tube wall are solid samples, collect the solid samples using sterile tools, then quickly place them into a wide-mouth bottle or anaerobic bag pre-filled with inert gas, fill the container to reduce the air at the top, add a small amount of sterile anaerobic saline to keep it moist, purge with nitrogen, seal and package for later use.

[0013] The method for performing qPCR analysis is as follows: Under aseptic conditions, the sealed sample was taken out and mixed with sterile 1xPBS phosphate buffer solution. After shaking for 1 minute, it was sonicated for 30-60 seconds, then centrifuged. The supernatant was used for DNA purification and qPCR analysis to determine the total bacterial count C0.

[0014] S3. Analyze the dynamic data of live bacteria in water samples and the dynamic data of total bacteria count in pipe wall sediments over time to assess the risk of microbial corrosion in the pipeline.

[0015] Specifically, the analysis focuses on the changing trends of microbial activity μ in the water sample obtained in step S1 over time, and the changing trends of the total bacterial count in the pipe wall sediment obtained in step S2 over time. If the microbial activity μ in the water sample remains below the threshold and the total bacterial count in the pipe wall sediment shows a decreasing trend during the same period, it indicates that biofilm activity in the pipe is inhibited and the risk of microbial corrosion in the pipe is low. If the microbial activity μ in the water sample is above the threshold, or the total bacterial count in the pipe wall sediment shows an increasing trend, it indicates that biofilm activity in the pipe is high and the risk of microbial corrosion in the pipe is high, requiring an increase in the amount of bactericide used.

[0016] Compared with the prior art, the advantages of the present invention are: (1) This invention combines water sample monitoring in pipelines with analysis of pipe wall cleaning materials to assess the risk of microbial corrosion in pipelines, thus solving the problem of unreliable results obtained from a single data source.

[0017] (2) This invention redefines the “waste residue” (pipe cleaning material) generated during pipeline cleaning operations as a “standard sample” for evaluating the activity of biofilm on the pipe wall, and provides a complete method for anaerobic collection, preservation and transportation and pretreatment.

[0018] (3) The method of this invention can be used for microbial corrosion monitoring of oil and gas field gathering and transportation pipelines, such as for microbial corrosion risk monitoring of gathering and transportation pipelines in shale gas production. It is the first to systematically apply PMA / EMA-qPCR (viability-qPCR) technology to directly quantify metabolically active corrosive bacteria (such as SRB), making the monitoring results directly correlated with corrosion risk, and the corrosion risk monitoring results are highly accurate. It overcomes the problem of poor accuracy of existing oil and gas pipeline microbial corrosion detection methods.

[0019] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0020] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0021] Taking the monitoring of microbial corrosion risk in shale gas pipelines as an example, this paper illustrates the specific application of the pipeline microbial corrosion risk dynamic monitoring method of the present invention.

[0022] Step S1: Collect pipeline water samples at equal time intervals multiple times for live bacteria qPCR analysis to obtain the microbial activity μ of water samples at different times.

[0023] First, collect water samples from the field. Generally, a single measurement of water from an oil and gas field requires 50ml-200ml. Therefore, prepare at least twice the amount of field water samples for testing. The time interval between two pipeline water sample collections is 15 days, and the total continuous sampling and testing period exceeds 3 months.

[0024] Two equal volumes of water samples were taken from the collected water samples and filtered through two 0.2 μm polycarbonate membranes. The two membranes were labeled as membrane T and membrane uT, respectively. The filtered membranes were then transferred to two 2 ml centrifuge tubes under sterile conditions and labeled as centrifuge tube T and centrifuge tube uT, respectively. 700 μl of sterile 1xPBS phosphate buffer solution was added to each centrifuge tube.

[0025] Add propidium azide (PMA) to centrifuge tube T and mix thoroughly; control the PMA concentration to 10-50 μM. This surfactant is prepared using dimethyl sulfoxide (DMSO) as the stock solution. During the processing steps, the final concentration of DMSO is controlled between 0.1% and 1% (v / v). Its functions are: 1) to ensure the hydrophobic staining molecules are fully dissolved, forming a homogeneous reaction system; 2) to gently enhance cell membrane permeability, ensuring consistent staining efficiency of the staining agent on all target bacterial cells, thereby improving the accuracy and repeatability of live bacteria quantitative detection.

[0026] Then, incubate the two centrifuge tubes at 37°C for 60-90 minutes, gently shaking occasionally to mix. Next, add 70 μl of neutralization buffer to each centrifuge tube. The neutralization buffer is a solution containing dithiothreitol (DTT) and L-cysteine, with each DTT and L-cysteine ​​having a mass concentration of 1-5%. Mix thoroughly and incubate at room temperature for 15 minutes. Then, centrifuge at 10,000 rpm for 1 minute. Carefully aspirate the supernatant from the centrifuge tube, avoiding aspirating any precipitated bacterial sediment. Perform routine nucleic acid purification on the bacterial sediment at the bottom of the centrifuge tube, following standard procedures known in the field and not detailed here. Finally, perform quantitative polymerase chain reaction (qPCR) testing. The bacterial content measured in centrifuge tube T is recorded as C. T The bacterial count measured in centrifuge tubes is recorded as C. uT .

[0027] Quantitative polymerase chain reaction (qPCR) instruments amplify DNA templates using specific primers. By monitoring changes in fluorescence signals, an amplification curve is obtained, and the content of the original template can be calculated.

[0028] Finally, the microbial activity μ was calculated: .

[0029] A higher μ value indicates higher microbial community activity, while a lower μ value indicates lower microbial community activity.

[0030] S2. At the same time as collecting water samples from the pipeline, samples of sediment from the pipe wall were collected for qPCR analysis to obtain the total number of bacteria in the sediment from the pipe wall at different times.

[0031] The deposits on the pipe wall can come from cleaning materials, corrosion pads, or biofilm probes, etc. (1) Since cleaning materials reflect the overall biofilm activity of the pipe wall, this method prefers cleaning materials obtained through cleaning operations. The cleaning operation preferably adopts a combination process of 'foam cleaning softening + wire brush cleaning stripping' to ensure that the deposit coverage is ≥90%.

[0032] The collection of pigging materials is carried out using the following two methods: Method 1: If the sediment on the tube wall is a mud-like sample, use a sterile syringe to insert into the bottom of the tube and extract 2-5 mL of mud to expel any air that may have been introduced into the syringe. Then immediately inject the mud into a prepared anaerobic bottle pre-filled with inert gas or an anaerobic tube containing transport culture medium. After purging with a portable nitrogen cylinder (99.99%), seal the package for later use.

[0033] Method 2: If the deposits on the tube wall are solid samples, collect them using sterile tools. Multiple samples should be taken from different locations and mixed to form a representative sample, or the sampling points should be labeled separately. Then, quickly place the sample into a wide-mouthed bottle or anaerobic bag pre-filled with inert gas, filling the container as much as possible to reduce air at the top. A small amount of sterile anaerobic saline can be added simultaneously to keep the container moist. After purging with a portable nitrogen cylinder (99.99%), seal the package for later use.

[0034] Place the obtained sample bags or sample bottles in a 4°C refrigerator and deliver them to the laboratory for processing within 24 hours, with a maximum time not exceeding 48 hours.

[0035] In the laboratory, under aseptic conditions, the sealed sample was taken out and mixed with sterile 1xPBS phosphate buffer solution at a mass ratio of 1:10. The mass of the sample and the 1xPBS solution was recorded. After shaking at room temperature for 1 minute, the mixture was sonicated for 30 seconds, and then centrifuged at 500-1000g for 5-10 minutes. 700 μl of the supernatant was transferred to a new 2 ml centrifuge tube for DNA purification and qPCR analysis to determine the total bacterial count (C0).

[0036] S3. Analyze the dynamic data of live bacteria in water samples and the dynamic data of total bacteria count in pipe wall sediments over time to assess the risk of microbial corrosion in the pipeline.

[0037] If the microbial activity μ in the water sample is controlled at a low level (meeting the standard requirements) during long-term monitoring (more than 3 months), it indirectly indicates that biofilm activity has decreased.

[0038] If the total bacterial count (C0) in the pig feed continuously decreases during long-term monitoring (more than 3 months), it indicates a decrease in biofilm activity.

[0039] Through long-term monitoring (more than 3 months), the following evaluation criteria were established: If the microbial activity μ in the water sample remains below the threshold and the total bacterial count C0 in the pipe wall sediment shows a downward trend during the same period, it indicates that the biofilm activity in the pipeline is inhibited and the risk of microbial corrosion in the pipeline is low. If the microbial activity μ in the water sample is above the threshold, or the total bacterial count C0 in the pipe wall sediment shows an upward trend, it indicates that the biofilm activity in the pipeline is high and the risk of microbial corrosion in the pipeline is high, requiring an increase in the dosage of bactericide.

[0040] In summary, this invention, through a combination of surfactant treatment and qPCR detection technology, enables rapid detection (approximately 4 hours) of the content of corrosive bacteria SRB and FB in oil and gas fields, which can greatly improve the decision-making efficiency for bacterial control within pipelines.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for dynamic monitoring of microbiologically influenced corrosion risk in a pipeline, characterized in that, It comprises the following three steps: S1, continuously collect pipeline water samples for viable bacteria qPCR analysis to obtain water sample microbial activity μ at different time periods; the time interval between two water sample collections is 7-30 days, and the number of consecutive water sample collections is not less than 3 times; S2, collect a pipe wall deposit sample at the same time as each water sample collection for qPCR analysis to obtain the total number of bacteria in the pipe wall deposit at different time periods; S3, analyze the change trend of the water sample viable bacteria dynamic data and the change trend of the total number of bacteria in the pipe wall deposit with time to evaluate the pipeline microbial corrosion risk.

2. The method for dynamic monitoring of microbiologically influenced corrosion risk in a pipeline of claim 1, wherein, In step S1, the time interval between two water sample collections is the same or different.

3. The method for dynamic monitoring of microbiologically influenced corrosion risk in a pipeline of claim 2, wherein, In step S1, the water sample is analyzed by viable bacteria qPCR as follows: S11, take two water samples of the same volume from the collected water sample and filter them with two 0.2 μm polycarbonate membranes, respectively, and mark them as filter T and filter uT on the two membranes; transfer the filtered two membranes to different centrifuge tubes under sterile conditions, respectively, and mark them as centrifuge tube T and centrifuge tube uT, and add sterile 1x PBS phosphate buffer solution to the two centrifuge tubes; S12, adding active agent to centrifuge tube T, mixing well, the active agent is propidium iodide or ethidium bromide monoazide; then performing qPCR analysis on the samples in the two centrifuge tubes, the measured bacterial content of centrifuge tube T is denoted as C T , and the measured bacterial content of centrifuge tube uT is denoted as C uT ; S13, calculate the microbial activity μ: 。 4. The method for dynamic monitoring of microbiologically influenced corrosion risk in a pipeline of claim 3, wherein, In step S12, the qPCR analysis of the samples in the two centrifuge tubes is as follows: Two centrifuge tubes are incubated at 37°C for 60-90 minutes; then neutralization buffer is added to each of the two centrifuge tubes, mixed evenly, and incubated at room temperature for 15 minutes before centrifugal separation. The sediment at the bottom of the centrifuge tube is subjected to conventional nucleic acid purification treatment, and finally tested by quantitative polymerase chain reaction (qPCR). The bacterial content measured by centrifuge tube T is denoted as C T , and the bacterial content measured by centrifuge tube uT is denoted as C uT .

5. The method for dynamic monitoring of microbiologically influenced corrosion risk in a pipeline of claim 1, wherein, The pipe wall deposit is from a pig, a corrosion coupon, or a biofilm probe.

6. The method for dynamic monitoring of microbiologically influenced corrosion risk in a pipeline of claim 5, wherein, In step S2, if the pipe wall deposit is a slurry sample, use a sterile syringe to extract 2-5 mL of slurry, discharge the air brought in at the front end of the syringe, and then immediately inject it into an anaerobic bottle pre-filled with inert gas or an anaerobic tube containing transport medium, and then seal and package after purging with nitrogen; if the pipe wall deposit is a solid sample, use sterile tools to collect the solid sample, and then quickly place it in a wide-mouth bottle or anaerobic bag pre-filled with inert gas, fill the container to reduce the air at the top, and add a small amount of sterile anaerobic saline to keep it moist, and then seal and package after purging with nitrogen.

7. The method for dynamic monitoring of microbiologically influenced corrosion risk in a pipeline of claim 6, wherein, In step S2, the qPCR analysis is as follows: Under sterile conditions, mix the sealed and packaged sample with sterile 1x PBS phosphate buffer solution, shake for 1 minute, then ultrasonic for 30-60 seconds, then centrifuge, take the supernatant for DNA purification and qPCR analysis, and measure the total number of bacteria C0.

8. The method for dynamic monitoring of microbiologically influenced corrosion risk of a pipeline according to any one of claims 1 to 7, characterized in that, In step S3, analyze the change trend of the water sample microbial activity μ obtained in step S1 with time, and the change trend of the total number of bacteria in the pipe wall deposit obtained in step S2 with time; if the microbial activity μ in the water sample is continuously below the threshold value and the total number of bacteria in the pipe wall deposit at the same period shows a downward trend, it indicates that the activity of the biofilm in the pipeline is inhibited, and the pipeline microbial corrosion risk is low; if the microbial activity μ in the water sample is higher than the threshold value, or the total number of bacteria in the pipe wall deposit shows an upward trend, it indicates that the activity of the biofilm in the pipeline is high, and the pipeline microbial corrosion risk is high, and the amount of bactericide needs to be increased.

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