An in-situ monitoring device and method for microbial stress corrosion environment

Abstract

This invention discloses an in-situ monitoring and detection device and method for microbial stress corrosion environments, belonging to the technical field of stress corrosion experimental devices. The device includes a main frame, a servo-driven dual ball screw loading mechanism, a sterilizable experimental vessel with a bellows seal, an electrochemical detection component, and a laser confocal microscopy observation component. The device of this invention should be able to adjust and maintain key environmental parameters such as temperature, atmosphere, dissolved oxygen, pH, nutrients, and corrosive media in a controllable experimental environment to meet the directional culture needs of different types of microorganisms, including aerobic, anaerobic, microaerophilic, and facultative anaerobic microorganisms. Simultaneously, it should be able to apply different forms of mechanical action to the test sample, such as constant load, constant strain, constant displacement, or slow strain rate tension, to simulate the stress state of the material during actual service.

Description

Technical Field

[0001] This invention relates to the field of stress corrosion testing equipment, specifically to an in-situ monitoring and detection device and method for microbial stress corrosion environments. Background Technology

[0002] Microbiologically influenced corrosion (MIC) is a corrosion phenomenon caused by the life activities of microorganisms, and it is prevalent in various industrial environments such as marine environments, oil and gas pipeline transportation, water supply and drainage systems, and chemical facilities. Microbiological corrosion is estimated to account for approximately 20% of global corrosion-related losses among different types of corrosion. Microbiological corrosion not only accelerates the corrosion rate of metallic materials and shortens their service life, but it can also lead to serious production safety accidents. Although microbiological corrosion has attracted widespread attention in many industrial sectors, its mechanisms remain not fully understood due to limitations in traditional observation methods, necessitating in-depth research to reveal its occurrence mechanisms and influencing factors.

[0003] Stress corrosion cracking (SCC) is a phenomenon that causes brittle fracture in materials under the combined effects of sustained stress and a corrosive environment. Almost all metallic materials can undergo stress corrosion under specific conditions, including common structural materials such as carbon steel and aluminum alloys. Stress corrosion not only affects structural integrity but can also trigger sudden fractures without significant deformation, threatening production safety. In-situ testing is crucial for accurately assessing and understanding the mechanisms of stress corrosion. In-situ monitoring allows for real-time observation of material changes under corrosive conditions and provides reliable data support for research on corrosion mechanisms.

[0004] However, there is still a significant gap in experimental equipment for coupling microbial corrosion and stress corrosion. Most existing stress corrosion studies have failed to effectively consider the potential impact of microorganisms, resulting in a relative lack of research on the coupling effect of microorganisms and stress corrosion. Patent CN220854441U discloses an in-situ measurement device for stress corrosion environments, patent CN112284898A provides a stress corrosion simulation and in-situ observation device, and patent CN215866139U discloses a PID-controlled variable-rate tensile stress corrosion testing device for buried pipeline steel. Although these patents provide useful references for in-situ observation and testing of stress corrosion, their device designs have the following main problems: (1) they cannot strictly control the microbial growth environment, resulting in the microbial activity not accurately reflecting its role in the corrosion process; (2) the in-situ detection function is relatively simplified, lacking detailed monitoring of the corrosion process; (3) they only support stress corrosion simulation under single dynamic and static load conditions, and cannot meet the needs of stress corrosion testing under complex load environments. Therefore, these existing technologies cannot effectively meet the research needs of microbial stress corrosion.

[0005] To address the shortcomings of existing technologies in studying the coupling of microbial corrosion and stress corrosion, such as the relatively fragmented methods, difficulty in maintaining stable experimental environmental parameters over long periods, challenges in simultaneously constructing microbial culture conditions and mechanical loading conditions, and the lack of continuous in-situ monitoring of the corrosion process, there is an urgent need to design a novel in-situ monitoring and detection device suitable for microbial-stress coupled corrosion research. This device should be able to regulate and maintain key environmental parameters such as temperature, atmosphere, dissolved oxygen, pH, nutrients, and corrosive media in a controlled experimental environment to meet the directional culture needs of different types of microorganisms, including aerobic, anaerobic, microaerophilic, and facultative anaerobic microorganisms. Simultaneously, it should be able to apply different forms of mechanical action to the test sample, such as constant load, constant strain, constant displacement, or slow strain rate tension, to simulate the stress state of the material during actual service. Based on this, by introducing in-situ monitoring methods such as electrochemical testing, bright-field reflection observation, fluorescence observation, and image acquisition, long-term, continuous, and in-situ observation and detection can be carried out on the material surface under the combined effects of microorganisms, corrosive media, and applied stress, to observe and detect microbial adhesion, biofilm formation, corrosion product generation, local corrosion morphology evolution, and damage initiation and propagation processes. This provides more comprehensive and reliable data support for elucidating the synergistic mechanism between microbial activity, corrosion, and stress, and provides an experimental basis for evaluating microbial stress corrosion behavior and developing related corrosion-resistant materials. Summary of the Invention

[0006] To address the problems in existing technologies, the present invention aims to provide an in-situ monitoring and detection device for microbial stress corrosion environments. By installing corrugated pipe clamps on both sides of the experimental vessel, the sample is completely sealed within the vessel body and can be individually sterilized under high temperature and high pressure. Multiple liquid inlet and outlet holes are provided on the experimental vessel, and combined with the heating plate at the bottom of the experimental vessel, precise control of the microbial culture environment is achieved. The experimental vessel has an in-situ observation window and an electrochemical electrode port. Combined with a microscope and an electrochemical workstation, in-situ observation and electrochemical monitoring of microbial stress-coupled corrosion are realized.

[0007] This invention provides an in-situ monitoring and detection device for microbial stress corrosion environments, comprising:

[0008] The main frame, servo motor, planetary gear reducer, drive pulley, belt, first ball screw, second ball screw, ball nut, first loading beam, second loading beam, force sensor, first connecting clamp, second connecting clamp, first adapter clamp, second adapter clamp, first connecting pin, second connecting pin, experimental vessel, first bellows clamp, second bellows clamp, in-situ bellows observation window, auxiliary observation window, sample, peristaltic pump, electrochemical workstation, and laser confocal microscope are structured as follows:

[0009] A motor is horizontally fixed on one side of the main frame and connected to the first and second ball screws in sequence through a planetary gear reducer, a drive wheel, and a belt. The output end of the servo motor is coaxially fixedly mounted with the planetary gear reducer, and the output end of the planetary gear reducer is mounted with the drive wheel. The drive wheel drives the first and second ball screws simultaneously through a closed-loop belt. The first and second ball screws are arranged parallel to each other, and the ends of the two ball screws away from the drive wheel are mounted on the side wall of the main frame through bearings to achieve stable rotational support.

[0010] Both the first and second ball screws are fitted with ball nuts. The first and second loading beams are fixedly mounted on the ball nuts on both sides and can rotate with the ball screws to achieve horizontal displacement. A through hole is opened in the center of the first loading beam, and a force sensor is fixedly installed in the through hole. The force sensor is set by a nut on the inner side of the assembly formed by the first loading beam, the first connecting fixture, the first adapter fixture, and the first connecting pin. After assembly, it is blocked by the outer fixture structure. The force detection end of the force sensor faces the test vessel and is fixedly mounted on the first connecting fixture. The first connecting fixture is set on the force sensor by a nut. The second loading beam is set on the ball screw by a ball nut. The second loading beam is set parallel to the first loading beam. A second connecting fixture is set in the center of the second loading beam. The second connecting fixture is set on the second loading beam by a nut. The servo motor drives the two ball screws to rotate synchronously in opposite directions through reduction and belt drive, causing the first loading beam and the second loading beam to move synchronously towards or away from each other, ensuring that the gauge length of the sample is always aligned with the center of the in-situ observation window of the bellows.

[0011] The experimental vessel has symmetrically arranged first and second through holes on its left and right sides, respectively, and a first and second bellows clamp is installed thereon. The first and second bellows clamps are locked to the side wall of the experimental vessel with nuts. The first and second bellows clamps are sealed to the experimental vessel with O-rings to ensure a sealed and sterile environment inside the vessel. The sample is horizontally mounted through the middle of the experimental vessel. The sample has pin through holes at both ends. The two sections of the sample are clamped and fixed between the first and second bellows clamps by the first and second fixing pins. Insulating gaskets and insulating sleeves are set between the sample, the bellows clamps, and the fixing pins to achieve complete insulation between the sample and the metal clamps and avoid short-circuit interference of electrochemical signals.

[0012] The first corrugated pipe clamp is threaded to the first adapter clamp on the outside, and the second corrugated pipe clamp is threaded to the second adapter clamp on the outside. The first corrugated pipe clamp is connected to the first connecting clamp via a first connecting pin, and the second corrugated pipe clamp is connected to the second connecting clamp via a second connecting pin. The first corrugated pipe clamp and the second corrugated pipe clamp can be flexibly replaced according to the test type to meet the requirements of different forms of stress corrosion.

[0013] The experimental vessel has a corrugated in-situ observation window at the center of its upper cover, and an auxiliary observation window on its front side wall. The left and right side walls of the vessel are respectively equipped with a liquid inlet thread, a liquid outlet thread, an air inlet thread, and an air outlet thread. The air outlet and liquid outlet thread are located on the right side wall of the vessel perpendicular to the sample direction, while the liquid inlet and air inlet thread are located on the left side wall of the vessel perpendicular to the sample direction. Each gas-liquid interface is connected to a gas peristaltic pump and a liquid peristaltic pump via pipelines. A 0.22 μm needle filter and a one-way check valve are connected in series on the pipelines to achieve precise delivery and backflow prevention of sterile gas and sterile culture medium. An adjustable temperature silicone rubber heating plate is installed at the bottom of the experimental vessel to create an environment suitable for the growth of different microorganisms by adjusting the gas, liquid, and temperature.

[0014] The upper end cover of the experimental vessel has a corrugated in-situ observation window at its center. The corrugated in-situ observation window can be equipped with the objective lens of a laser confocal microscope. The microscopic imaging mode can be flexibly adjusted according to the experimental requirements, including bright field, metallographic, differential interference and fluorescence modes, so as to realize in-situ observation of the corrosion morphology and biofilm on the sample surface. The corrugated in-situ observation window can be directly immersed in the liquid in the experimental vessel, or a quartz glass slide can be installed on the surface of the window for non-invasive observation. The corrugated in-situ observation window can be moved within an appropriate range parallel or perpendicular to the surface of the main frame as the objective lens moves.

[0015] A working electrode hole with internal threads is opened on the left side wall of the experimental vessel, and a reference electrode thread hole and a counter electrode thread hole are opened on the rear side wall of the experimental vessel respectively. The inner wall of the working electrode hole is machined with sealing internal threads, and the wire can be matched with a threaded sealing plug to lock and seal after passing through. Flexible conductive leads are pre-welded to the sample surface as working electrode leads. The flexible conductive leads extend from inside the vessel through the working electrode hole on the left side wall to the outside of the vessel. The threaded assembly positions of the reference electrode thread hole, the counter electrode thread hole, and the working electrode hole are all provided with annular sealing grooves, and O-rings are placed in the grooves. After the reference electrode rod, the counter electrode rod, and the working electrode sealing plug are tightened, the O-rings are axially compressed to achieve pressure-resistant and sterile sealing at each electrode installation position.

[0016] The reference electrode is installed in the threaded hole of the reference electrode via a salt bridge, and the counter electrode is fixedly installed in the threaded hole of the counter electrode. The electrode rod of the counter electrode passes through the threaded hole of the counter electrode, and an O-ring is placed between the outer wall of the electrode rod and the threaded hole. Tightening the locking nut compresses the sealing ring to achieve a seal, so that the counter electrode is stably fixed in the designated position in the inner cavity of the reactor. The working electrode, reference electrode, and counter electrode are respectively connected to the electrochemical workstation. According to the experimental requirements, the electrochemical workstation can realize open circuit potential detection, electrochemical impedance spectroscopy analysis, electrochemical noise, and constant potential / constant current polarization to adapt to the study of corrosion behavior under different stress, corrosive environments, or microorganisms. The electrochemical workstation, force sensor, and laser confocal microscope acquire signals synchronously in real time without interference, realizing the synchronous monitoring and analysis of electrochemical parameters, mechanical load, corrosion morphology, and biofilm.

[0017] The in-situ observation window of the bellows is a movable and sealed observation structure. The objective lens of the laser confocal microscope is set in the in-situ observation window of the bellows. The in-situ observation window of the bellows can be directly immersed in the liquid environment inside the experimental vessel, or a quartz glass plate can be installed on the surface of the window to achieve non-invasive observation. The in-situ observation window of the bellows can be moved parallel or perpendicular to the surface of the main frame as the objective lens moves, adapting to different observation fields and focusing requirements.

[0018] The device as a whole supports separate sterilization: After disassembling the first connecting pin, the second connecting pin, the first adapter clamp, and the second adapter clamp, and sealing the inlet, outlet, inlet, and outlet threads with plugs, the experimental vessel, the first bellows clamp, the second bellows clamp, the first fixing pin, the second fixing pin, the sample, the insulating gasket, the insulating sleeve, the in-situ observation window of the bellows, and the auxiliary observation window can be sterilized as a sealed whole separately; after sterilization, the first connecting pin, the second connecting pin, the first adapter clamp, and the second adapter clamp are reinstalled, and the inlet / outlet / air ports are connected to the gas / liquid peristaltic pumps through pipelines to inject sterile culture medium and sterile gas; alternatively, the culture medium and gas can be injected into the experimental vessel before sterilization and sterilized together with the experimental vessel.

[0019] A method for simulating and in-situ monitoring microbial stress corrosion, using the aforementioned apparatus, includes the following steps:

[0020] S1. Assemble the sample, insulating gasket, and insulating sleeve into place. Fix the sample between the two bellows clamps using the first and second fixing pins. Install the auxiliary observation window, counter electrode, and reference electrode. Pass the flexible working electrode lead wire, pre-welded to the sample, through the working electrode hole on the left side wall and lead it out of the test vessel. Tighten the threaded plug matching the working electrode hole. Seal the gap between the plug and the lead wire with an O-ring to seal all windows, forming a closed system inside the test vessel. Use a liquid peristaltic pump to transport microorganisms and culture medium into the test vessel through the inlet and outlet threaded holes. Use a gas peristaltic pump to transport and discharge gas through the inlet and outlet threaded holes to maintain a specific gas environment. Turn on the bottom adjustable temperature silicone rubber heating plate to heat the vessel body, set the microbial culture temperature, and maintain the system for a certain period of time.

[0021] S2. After the culture is completed, the servo motor is started. The first loading beam and the second loading beam are controlled to move synchronously in opposite directions through the electrode. The servo motor rotates and drives the beam to move. The sample is subjected to tensile load through the force transmission of the clamp and pin, that is, the sample is in the loading state. The sample is subjected to three loading modes: constant load, constant strain, or slow strain rate tension through closed-loop control of the force sensor.

[0022] S3. Connect the test leads of the electrochemical workstation to the working electrode leads, the counter electrode, and the reference electrode respectively; apply a voltage of a specific frequency to the electrodes and collect the electrochemical measurement parameters of the sample under different stress and microbial environments.

[0023] S4. Insert the microscope objective into the experimental vessel through the in-situ observation window of the bellows, and use a bright field white light source to observe the corrosion morphology of the sample 23 surface, such as microcracks and pitting. Combine the differential interferometer to analyze the morphology of the sample surface. For biofilms, deliver fluorescent dye to the vessel through the liquid inlet threaded hole, or use endogenous fluorescent microorganisms to excite cells with a laser light source and observe the biofilm on the sample surface.

[0024] Compared with existing technologies, the beneficial effects of this invention are as follows: The device described in this invention should be able to regulate and maintain key environmental parameters such as temperature, atmosphere, dissolved oxygen, pH, nutrients, and corrosive media in a controllable experimental environment to meet the directional culture requirements of different types of microorganisms, including aerobic, anaerobic, microaerophilic, and facultative anaerobic microorganisms. Simultaneously, it should be able to apply different forms of mechanical action to the test sample, such as constant load, constant strain, constant displacement, or slow strain rate tension, to simulate the stress state of the material during actual service. Based on this, by introducing in-situ monitoring methods such as electrochemical testing, bright-field reflection observation, fluorescence observation, and image acquisition, long-term, continuous, and in-situ observation and detection can be conducted on the microbial adhesion, biofilm formation, corrosion product generation, local corrosion morphology evolution, and damage initiation and propagation processes on the material surface under the combined effects of microorganisms, corrosive media, and applied stress. This provides more comprehensive and reliable data support for elucidating the synergistic mechanism between microbial activity, corrosion, and stress, and provides an experimental basis for evaluating microbial stress corrosion behavior and developing related corrosion-resistant materials. Attached Figure Description

[0025] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0026] Figure 1 This is a schematic diagram of the main structure of an in-situ monitoring and detection device applied to microbial stress corrosion environments.

[0027] Figure 2 This is a schematic diagram of the experimental vessel.

[0028] Figure 3 The stress-strain curve of the sample in Example 1 of this invention;

[0029] Figure 4 The linear polarization resistance values ​​of the sample in Example 1 of this invention at different stages;

[0030] Figure 5 The images show the surface morphology and fluorescence imaging of the sample at different stages in Example 1 of this invention. Figures (a)-(e) show the corrosion morphology of the material surface, and (f)-(j) show the images of the biofilm captured in fluorescence mode.

[0031] In the picture:

[0032] 1-Main frame; 2-Ball nut; 3-First ball screw; 4-First loading beam; 5-Corrugated pipe in-situ observation window; 6-First bellows clamp; 7-First connecting clamp; 8-First adapter clamp; 9-First connecting pin; 10-Second ball screw; 11-Drive wheel; 12-Servo motor; 13-Second loading beam; 14-Belt; 15-Second connecting clamp; 16-Second adapter clamp; 17-Second connecting pin; 18-Second bellows clamp; 19-Experimental vessel; 20-Gas outlet threaded port; 21-Liquid outlet threaded port; 22-Reference electrode; 23-Sample; 24-Counter electrode; 25-Working electrode hole; 26-First fixing pin; 27-Insulating gasket; 28-Auxiliary observation window; 29-Second fixing pin; 30-Gas inlet threaded port; 31-Liquid inlet threaded port. Detailed Implementation

[0033] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0034] Reference Figure 1 , Figure 2 The device shown is used for microbial stress corrosion simulation and in-situ monitoring. The device includes a main frame 1, a servo motor 12, a planetary gear reducer, a drive wheel 11, a belt 14, a first ball screw 3, a second ball screw 10, a ball nut 2, a first loading beam 4, a second loading beam 13, a force sensor, a first connecting clamp 7, a second connecting clamp 15, a first adapter clamp 8, a second adapter clamp 16, a first connecting pin 9, a second connecting pin 17, an experimental vessel 19, a first bellows clamp 6, a second bellows clamp 18, a bellows in-situ observation window 5, an auxiliary observation window 28, a peristaltic pump, an electrochemical workstation, and a laser confocal microscope.

[0035] A servo motor 12 is horizontally fixedly installed on one side of the main frame 1. The output end of the servo motor 12 is sequentially connected to the first ball screw 3 and the second ball screw 10 through a planetary gear reducer, a drive wheel 11, and a belt 14. The output end of the servo motor 12 is coaxially fixedly installed with the planetary gear reducer, and the output end of the planetary gear reducer is installed with the drive wheel 11. The drive wheel 11 drives the first ball screw 3 and the second ball screw 10 simultaneously through the closed-loop belt 14. The first ball screw 3 and the second ball screw 10 are arranged parallel to each other. The ends of the two ball screws away from the drive wheel 11 are mounted on the side wall of the main frame 1 through bearings to achieve stable rotational support.

[0036] Both the first ball screw 3 and the second ball screw 10 are fitted with ball nuts 2; the first loading beam 4 and the second loading beam 13 are fixedly mounted on the ball nuts 2 on both sides, and can achieve horizontal displacement by rotating with the ball screws. A through hole is opened in the center of the first loading beam 4, and a force sensor is fixedly installed in the through hole. The force sensor is set by a nut on the inner side of the assembly formed by the first loading beam 4, the first connecting clamp 7, the first adapter clamp 8, and the first connecting pin 9. After assembly, it is covered by the outer clamp structure. The force detection end of the force sensor faces the experimental vessel 19 and is fixedly mounted on the first connecting clamp 7. A connecting clamp 7 is mounted on the force sensor via a nut; the second loading beam 13 is mounted on the ball screw via a ball nut 2, and the second loading beam 13 and the first loading beam 4 are arranged parallel to each other. A second connecting clamp 15 is set at the center of the second loading beam 13, and the second connecting clamp 15 is mounted on the second loading beam 13 via a nut; the servo motor 12 drives the two ball screws to rotate synchronously in opposite directions through reduction and belt drive, causing the first loading beam 4 and the second loading beam 13 to move synchronously towards or away from each other, ensuring that the gauge length of the sample is always aligned with the center of the in-situ observation window 5 of the bellows.

[0037] The experimental vessel 19 has symmetrically opened first and second through holes on its left and right sides, respectively, and first and second bellows clamps 6 and 18 are respectively installed. The first and second bellows clamps 6 and 18 are locked to the side wall of the experimental vessel 19 by nuts. The first and second bellows clamps 6 and 18 are sealed to the experimental vessel 19 by O-rings to ensure a sealed and sterile environment inside the vessel. The experimental vessel 19 is equipped with a sample 23 transversely through the middle. The sample 23 is made of SUS316 plate with a gauge length of 50 mm and a width of 12.5 mm. After the sample 23 is polished with 240, 400, 600, 800 and 1000 grit sandpaper, it is ultrasonically cleaned in a mixture of ethanol and acetone for 20 min-40 min, and then dried in a laminar flow in a biosafety cabinet. The non-gauge section is carefully wrapped with polytetrafluoroethylene tape to ensure that only the gauge section is exposed to the environment during the test and is stored for later use. The sample 23 has pin through holes at both ends. The two ends of the sample 23 are clamped and fixed between the first corrugated pipe clamp 6 and the second corrugated pipe clamp 18 by the first fixing pin 26 and the second fixing pin 29. An insulating gasket 27 and an insulating sleeve are provided between the sample 23, the corrugated pipe clamp and the fixing pin to achieve complete insulation between the sample 23 and the metal clamp and avoid short circuit interference of electrochemical signal.

[0038] The first corrugated pipe clamp 6 is threaded to the first adapter clamp 8 on the outside, and the second corrugated pipe clamp 18 is threaded to the second adapter clamp 16 on the outside. The first corrugated pipe clamp 6 is connected to the first adapter clamp 7 through the first connecting pin 9, and the second corrugated pipe clamp 18 is connected to the second adapter clamp 15 through the second connecting pin 17.

[0039] The experimental vessel 19 has a corrugated in-situ observation window 5 at the center of its upper cover. An auxiliary observation window 28 is provided on the front side wall of the experimental vessel 19. The left and right side walls of the experimental vessel 19 are respectively provided with a liquid inlet thread 31, a liquid outlet thread 21, an air inlet thread 30, and an air outlet thread 20. The air outlet thread 20 and liquid outlet thread 21 are on the right side wall of the experimental vessel 19 perpendicular to the sample direction, while the liquid inlet thread 31 and air inlet thread 30 are on the left side wall of the experimental vessel 19 perpendicular to the sample direction. Each gas-liquid interface is connected to a gas peristaltic pump and a liquid peristaltic pump via pipelines. A 0.22 μm needle filter and a one-way check valve are connected in series on the pipelines to achieve precise delivery and backflow prevention of sterile gas and sterile culture medium. An adjustable temperature silicone rubber heating plate is provided at the bottom of the experimental vessel 19 to create an environment suitable for the growth of different microorganisms by adjusting the gas, liquid, and temperature.

[0040] The upper end cover of the experimental vessel 19 has a corrugated in-situ observation window 5 at its center. The corrugated in-situ observation window 5 can be equipped with the objective lens of a laser confocal microscope. The microscopic imaging mode can be flexibly adjusted according to the experimental requirements, including bright field, metallographic, differential interference and fluorescence modes, so as to realize the in-situ observation of the corrosion morphology and biological film on the sample surface. The corrugated in-situ observation window 5 can be directly immersed in the liquid inside the experimental vessel 19, or a quartz glass slide can be installed on the surface of the window for non-invasive observation. The corrugated in-situ observation window 5 can be moved within an appropriate range with the objective lens, parallel or perpendicular to the surface of the main frame 1.

[0041] The experimental vessel 19 has a working electrode hole 25 with internal threads on its left side wall, and a reference electrode threaded hole and a counter electrode threaded hole are respectively opened on the rear side wall of the experimental vessel 19. The inner wall of the working electrode hole 25 is machined with sealing internal threads, and the wire can be matched with a threaded sealing plug to lock and seal after passing through. The surface of the sample 23 is pre-welded with a flexible conductive lead as a working electrode lead. The flexible conductive lead extends from inside the vessel through the working electrode hole 25 on the left side wall to the outside of the vessel. The threaded assembly positions of the reference electrode threaded hole, the counter electrode threaded hole, and the working electrode hole 25 are all provided with annular sealing grooves, and O-rings are placed in the grooves. After the reference electrode rod, the counter electrode rod, and the working electrode sealing plug are tightened, the O-rings are axially compressed to achieve pressure-resistant and sterile sealing at each electrode installation position.

[0042] The reference electrode 22 is installed in the threaded hole of the reference electrode via a salt bridge, and the counter electrode 24 is fixedly installed in the threaded hole of the counter electrode. The electrode rod of the counter electrode 24 passes through the threaded hole of the counter electrode, and an O-ring is placed between the outer wall of the electrode rod and the threaded hole. Tightening the locking nut compresses the sealing ring to achieve a seal, so that the counter electrode 24 is stably fixed in the designated position in the inner cavity of the reactor. The working electrode, reference electrode 22, and counter electrode 24 are respectively connected to the electrochemical workstation. According to the experimental requirements, the electrochemical workstation can realize open circuit potential detection, electrochemical impedance spectroscopy analysis, electrochemical noise, and constant potential / constant current polarization to adapt to the study of corrosion behavior under different stress, corrosion environment, or microorganisms. The electrochemical workstation, force sensor, and laser confocal microscope acquire signals synchronously in real time without interference, realizing the synchronous monitoring and analysis of electrochemical parameters, mechanical load, corrosion morphology, and biofilm.

[0043] The in-situ observation window 5 of the bellows is a movable and sealed observation structure. The in-situ observation window 5 of the bellows is equipped with the objective lens of the laser confocal microscope. The in-situ observation window 5 of the bellows can be directly immersed in the liquid environment inside the experimental vessel 19, or a quartz glass plate can be installed on the surface of the window to achieve non-invasive observation. The in-situ observation window 5 of the bellows can be moved parallel or perpendicular to the surface of the main frame 1 as the objective lens moves, adapting to different observation fields and focusing requirements.

[0044] The first corrugated pipe clamp 6 and the second corrugated pipe clamp 18 can be flexibly replaced according to the test type to meet the requirements of different forms of stress corrosion.

[0045] The device as a whole supports separate sterilization: after disassembling the first connecting pin 9, the second connecting pin 17, the first adapter clamp 8, and the second adapter clamp 16, and sealing the liquid inlet thread 31, liquid outlet thread 21, air inlet thread 30, and air outlet thread 20 with plugs, the experimental vessel 19, the first bellows clamp 6, the second bellows clamp 18, the first fixing pin 26, the second fixing pin 29, the sample 23, the insulating gasket 27, the insulating sleeve, the in-situ observation window 5 of the bellows, and the auxiliary... The observation window 28 can be sterilized separately as a sealed unit. After sterilizing the entire unit at 121°C for 20 minutes using a steam sterilizer, the first connecting pin 9, the second connecting pin 17, the first adapter clamp 8, and the second adapter clamp 16 are reinstalled. The inlet / outlet liquid / gas ports are then connected to gas / liquid peristaltic pumps via pipelines to inject sterile culture medium and sterile gas. Alternatively, the culture medium and gas can be injected into the experimental vessel 19 before sterilization and sterilized together with the experimental vessel 19.

[0046] A method for using a microbial stress corrosion simulation and in-situ monitoring device, characterized by comprising the following steps:

[0047] S1. Assemble the sample 23, insulating gasket 27, and insulating sleeve into place. Fix the sample between the two bellows clamps using the first fixing pin 26 and the second fixing pin 29. Install the auxiliary observation window 28, counter electrode 24, and reference electrode 22. Lead the flexible working electrode wire, which is pre-welded to the sample 23, through the working electrode hole 25 on the left side wall and out of the test vessel. Tighten the threaded plug matching the working electrode hole. Seal the gap between the plug and the lead wire with an O-ring to seal all windows, making the internal space of the test vessel 19 a closed system. Use a liquid peristaltic pump to transport microorganisms and culture medium into the test vessel 19 through the liquid inlet thread 31 and the liquid outlet thread 21. Use a gas peristaltic pump to transport and discharge gas through the gas inlet thread 30 and the gas outlet thread 20 to maintain a specific gas environment. Turn on the bottom adjustable temperature silicone rubber heating plate to heat the vessel body, set the microbial culture temperature, and maintain the system for a certain period of time.

[0048] S2. After the culture is completed, the servo motor 12 is started. The first loading beam 4 and the second loading beam 13 are controlled by the electrode to move synchronously in opposite directions. The servo motor rotates and drives the beam to move. The sample 23 is subjected to tensile load through the force transmission of the clamp and the pin, that is, the sample 23 is in the loading state. The sample is subjected to three loading modes: constant load, constant strain, or slow strain rate tension, through the closed-loop control of the force sensor.

[0049] S3. Connect the test leads of the electrochemical workstation to the working electrode lead, the counter electrode, and the reference electrode respectively; apply a voltage of a specific frequency to the electrodes and collect the electrochemical measurement parameters of sample 23 under different stress and microbial environments.

[0050] S4. Insert the microscope objective into the experimental vessel through the in-situ observation window of the bellows, and use a bright field white light source to observe the corrosion morphology of the sample 23 surface, such as microcracks and pitting. Combine the differential interferometer to analyze the morphology of the sample surface. For biofilms, deliver fluorescent dye to the vessel through the liquid inlet thread 31, or use endogenous fluorescent microorganisms to excite cells with a laser light source and observe the biofilm on the sample 23 surface.

[0051] Example 1

[0052] In this example, the microbial stress corrosion behavior of SUS316 stainless steel in a simulated marine environment was studied. *Shewanella onenei* with fluorescent labeling was selected as the model microorganism and cultured in Luria-Bertani medium to the logarithmic growth phase. Subsequently, the inlet thread 31, outlet thread 21, inlet thread 30, and outlet thread 20 were connected to a liquid peristaltic pump and a gas peristaltic pump respectively via pipelines. Sterile Luria-Bertani medium, *Shewanella onenei* bacterial suspension in the logarithmic growth phase, and sterile gas were injected into the sterilized experimental vessel 19. During the experiment, the flow rate of the liquid peristaltic pump was set to 100 mL / h, the flow rate of the gas peristaltic pump was set to 10 L / h, and compressed air was introduced. Simultaneously, the rubber heating plate located below the experimental vessel 19 was turned on to control the experimental temperature at 30℃, and the vessel was cultured under these conditions for 48 hours.

[0053] After cultivation, a slow strain rate tensile test was conducted on specimen 23 in this embodiment. Before the test, a preload of 300 N was applied to specimen 23 to eliminate any gaps that might exist during clamping and transmission, and to keep the specimen in a stable stress state; subsequently, the strain rate of the loading beam was set to 5 × 10⁻⁶. -4 s -1 During the stretching process, the electrochemical activity, surface morphology changes, and biofilm status of sample 23 were simultaneously monitored by combining in-situ electrochemical testing, bright-field reflection observation, and fluorescence microscopy.

[0054] Electrochemical testing employed the linear polarization resistance method. A perturbation potential of -20mV to +20mV relative to the open-circuit potential was applied to sample 23. The scan rate was set to 0.5mV / s, and tests were performed every 3 minutes. Microscopic observation utilized two modes: bright-field reflection and fluorescence. In bright-field reflection mode, a halogen lamp was used as the illumination source without any additional filters. In fluorescence mode, a 488nm laser was used as the excitation source, with the laser power adjusted to 1W, and a narrow-band filter with a center wavelength of 505nm and a full width at half maximum (FWHM) of 10nm was used for fluorescence signal acquisition. During observation, the microscope was fixed at the center of the gauge length of sample 23, and the exposure time was controlled to approximately 500ms in both observation modes to ensure consistency in image acquisition conditions at different stretching stages.

[0055] The servo motor 12 was started to conduct a tensile test, and the stress-strain curve of the specimen 23 was simultaneously recorded during the tensile process. The results are as follows: Figure 3 As shown. According to Figure 3The stress-strain curves shown were subjected to dynamic in-situ linear polarization resistance testing and in-situ microscopic observation at different tensile stages. These different tensile stages included the initial unloaded stage, yield stage, work hardening stage, ultimate tensile strength stage, and necking stage. The sampling or observation points were located at... Figure 3 The values ​​are marked with orange dots. The in-situ electrochemical test results and in-situ observation results corresponding to different stretching stages are shown below. Figure 4 and Figure 5 As shown.

[0056] In the initial stage of the tensile test, specimen 23 had not yet undergone significant plastic deformation, and the original scratches on its gauge length surface were parallel and clearly visible, such as... Figure 5 (a) After switching to fluorescence observation mode, the fluorescence signal of *Shewanella onychomycosis dolichospasm* on the surface of sample 23 was clear and showed a localized clustered distribution, indicating that the microorganisms were tightly adhered to the sample surface and had formed a relatively obvious aggregated structure. It is speculated that the biofilm on the sample surface had entered a relatively mature stage at this time. Figure 5 (f). In this initial state, the linear polarization resistance of sample 23 is 85.9 kΩ·cm. 2 .

[0057] As the stretching process continues, such as Figure 5 (b) Figure 5 (c) Figure 5 (d) Figure 5 As shown in (e), significant deformation occurred in the gauge length section of sample 23. Under bright-field observation mode, the original polishing scratches on the sample surface gradually became distorted, and more obvious slip bands and surface undulations were gradually observed, indicating that strain within the gauge length section continuously accumulated and tended to concentrate during plastic deformation, reducing the deformation coordination between grains and increasing the surface roughness of the sample. Meanwhile, as... Figure 5 (g) Figure 5 (h) Figure 5 (i) Figure 5 As shown in (j), under fluorescence observation mode, the *Shewanella onychomycosis* bacteria that were originally aggregated on the sample surface gradually dispersed, and some fluorescence signals changed from clear punctate or aggregated to a blurred diffuse state. This indicates that the substrate surface underwent continuous deformation, and some microorganisms detached from the sample surface and entered the culture medium, deviating from the original focal plane of the sample surface. This suggests that the mechanical stress generated during the stretching process has a destructive effect on the biofilm formed by microorganisms on the sample surface. During the above process, the linear polarization resistance of the sample changed from 85.9 kΩ·cm before stretching. 2 It slowly decreased to 65.5 kΩ·cm 2The surface corrosion resistance of the sample decreased, while the electrochemical activity increased. This phenomenon may be related to the following factors: on the one hand, the applied stress and surface deformation caused the biofilm attached to the sample surface to be destroyed and detached, weakening the hindering effect of the biofilm on the electrochemical mass transfer process; on the other hand, continuous stretching caused the passivation film on the sample surface to be destroyed, accompanied by plastic deformation, surface roughening and an increase in local active sites, thereby promoting the corrosion reaction and increasing the sample corrosion rate.

[0058] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. An in-situ monitoring and detection device for microbial stress corrosion environments, characterized in that, include: The main frame, servo motor, planetary gear reducer, drive pulley, belt, first ball screw, second ball screw, ball nut, first loading beam, second loading beam, force sensor, first connecting clamp, second connecting clamp, first adapter clamp, second adapter clamp, first connecting pin, second connecting pin, experimental vessel, first bellows clamp, second bellows clamp, in-situ bellows observation window, auxiliary observation window, sample, peristaltic pump, electrochemical workstation, and laser confocal microscope are structured as follows: A motor is horizontally fixed on one side of the main frame and connected to the first and second ball screws in sequence through a planetary gear reducer, a drive wheel, and a belt. The output end of the servo motor is coaxially fixedly mounted with the planetary gear reducer, and the output end of the planetary gear reducer is mounted with the drive wheel. The drive wheel drives the first and second ball screws simultaneously through a closed-loop belt. The first and second ball screws are arranged parallel to each other, and the ends of the two ball screws away from the drive wheel are mounted on the side wall of the main frame through bearings to achieve stable rotational support. Both the first and second ball screws are fitted with ball nuts. The first and second loading beams are fixedly mounted on the ball nuts on both sides and can rotate with the ball screws to achieve horizontal displacement. A through hole is opened in the center of the first loading beam, and a force sensor is fixedly installed in the through hole. The force sensor is set by a nut on the inner side of the assembly formed by the first loading beam, the first connecting fixture, the first adapter fixture, and the first connecting pin. After assembly, it is blocked by the outer fixture structure. The force detection end of the force sensor faces the test vessel and is fixedly mounted on the first connecting fixture. The first connecting fixture is set on the force sensor by a nut. The second loading beam is set on the ball screw by a ball nut. The second loading beam is set parallel to the first loading beam. A second connecting fixture is set in the center of the second loading beam. The second connecting fixture is set on the second loading beam by a nut. The servo motor drives the two ball screws to rotate synchronously in opposite directions through reduction and belt drive, causing the first loading beam and the second loading beam to move synchronously towards or away from each other, ensuring that the gauge length of the sample is always aligned with the center of the in-situ observation window of the bellows.

2. The in-situ monitoring and detection device for microbial stress corrosion environment according to claim 1, characterized in that, The experimental vessel has symmetrically arranged first and second through holes on its left and right sides, respectively, and a first and second bellows clamp is installed thereon. The first and second bellows clamps are locked to the side wall of the experimental vessel with nuts. The first and second bellows clamps are sealed to the experimental vessel with O-rings to ensure a sealed and sterile environment inside the vessel. The sample is horizontally mounted through the middle of the experimental vessel. The sample has pin through holes at both ends. The two sections of the sample are clamped and fixed between the first and second bellows clamps by the first and second fixing pins. Insulating gaskets and insulating sleeves are set between the sample, the bellows clamps, and the fixing pins to achieve complete insulation between the sample and the metal clamps and avoid short-circuit interference of electrochemical signals. The first corrugated pipe clamp is threaded to the first adapter clamp on the outside, and the second corrugated pipe clamp is threaded to the second adapter clamp on the outside. The first corrugated pipe clamp is connected to the first connecting clamp via a first connecting pin, and the second corrugated pipe clamp is connected to the second connecting clamp via a second connecting pin. The first corrugated pipe clamp and the second corrugated pipe clamp can be flexibly replaced according to the test type to meet the requirements of different forms of stress corrosion.

3. The in-situ monitoring and detection device for microbial stress corrosion environment according to claim 1, characterized in that, The experimental vessel has a corrugated in-situ observation window at the center of its upper cover, and an auxiliary observation window on its front side wall. The left and right side walls of the vessel are respectively equipped with a liquid inlet thread, a liquid outlet thread, an air inlet thread, and an air outlet thread. The air outlet and liquid outlet thread are located on the right side wall of the vessel perpendicular to the sample direction, while the liquid inlet and air inlet thread are located on the left side wall of the vessel perpendicular to the sample direction. Each gas-liquid interface is connected to a gas peristaltic pump and a liquid peristaltic pump via pipelines. A 0.22 μm needle filter and a one-way check valve are connected in series on the pipelines to achieve precise delivery and backflow prevention of sterile gas and sterile culture medium. An adjustable temperature silicone rubber heating plate is installed at the bottom of the experimental vessel to create an environment suitable for the growth of different microorganisms by adjusting the gas, liquid, and temperature.

4. The in-situ monitoring and detection device for microbial stress corrosion environment according to claim 1, characterized in that, The upper end cover of the experimental vessel has a corrugated in-situ observation window at its center. The corrugated in-situ observation window can be equipped with the objective lens of a laser confocal microscope. The microscopic imaging mode can be flexibly adjusted according to the experimental requirements, including bright field, metallographic, differential interference and fluorescence modes, so as to realize in-situ observation of the corrosion morphology and biofilm on the sample surface. The corrugated in-situ observation window can be directly immersed in the liquid in the experimental vessel, or a quartz glass slide can be installed on the surface of the window for non-invasive observation. The corrugated in-situ observation window can be moved within an appropriate range parallel or perpendicular to the surface of the main frame as the objective lens moves.

5. The in-situ monitoring and detection device for microbial stress corrosion environment according to claim 1, characterized in that, A working electrode hole with internal threads is opened on the left side wall of the experimental vessel, and a reference electrode thread hole and a counter electrode thread hole are opened on the rear side wall of the experimental vessel respectively. The inner wall of the working electrode hole is machined with sealing internal threads, and the wire can be matched with a threaded sealing plug to lock and seal after passing through. Flexible conductive leads are pre-welded to the sample surface as working electrode leads. The flexible conductive leads extend from inside the vessel through the working electrode hole on the left side wall to the outside of the vessel. The threaded assembly positions of the reference electrode thread hole, the counter electrode thread hole, and the working electrode hole are all provided with annular sealing grooves, and O-rings are placed in the grooves. After the reference electrode rod, the counter electrode rod, and the working electrode sealing plug are tightened, the O-rings are axially compressed to achieve pressure-resistant and sterile sealing at each electrode installation position. The reference electrode is installed in the threaded hole of the reference electrode via a salt bridge, and the counter electrode is fixedly installed in the threaded hole of the counter electrode. The electrode rod of the counter electrode passes through the threaded hole of the counter electrode, and an O-ring is placed between the outer wall of the electrode rod and the threaded hole. Tightening the locking nut compresses the sealing ring to achieve a seal, so that the counter electrode is stably fixed in the designated position in the inner cavity of the reactor. The working electrode, reference electrode, and counter electrode are respectively connected to the electrochemical workstation. According to the experimental requirements, the electrochemical workstation can realize open circuit potential detection, electrochemical impedance spectroscopy analysis, electrochemical noise, and constant potential / constant current polarization to adapt to the study of corrosion behavior under different stress, corrosive environments, or microorganisms. The electrochemical workstation, force sensor, and laser confocal microscope acquire signals synchronously in real time without interference, realizing the synchronous monitoring and analysis of electrochemical parameters, mechanical load, corrosion morphology, and biofilm.

6. The in-situ monitoring and detection device for microbial stress corrosion environment according to claim 1, characterized in that, The in-situ observation window of the bellows is a movable and sealed observation structure. The objective lens of the laser confocal microscope is set in the in-situ observation window of the bellows. The in-situ observation window of the bellows can be directly immersed in the liquid environment inside the experimental vessel, or a quartz glass plate can be installed on the surface of the window to achieve non-invasive observation. The in-situ observation window of the bellows can be moved parallel or perpendicular to the surface of the main frame as the objective lens moves, adapting to different observation fields and focusing requirements.

7. The in-situ monitoring and detection device for microbial stress corrosion environment according to claim 1, characterized in that, The device as a whole supports separate sterilization: After disassembling the first connecting pin, the second connecting pin, the first adapter clamp, and the second adapter clamp, and sealing the inlet, outlet, inlet, and outlet threads with plugs, the experimental vessel, the first bellows clamp, the second bellows clamp, the first fixing pin, the second fixing pin, the sample, the insulating gasket, the insulating sleeve, the in-situ observation window of the bellows, and the auxiliary observation window can be sterilized as a sealed whole separately; after sterilization, the first connecting pin, the second connecting pin, the first adapter clamp, and the second adapter clamp are reinstalled, and the inlet / outlet / air ports are connected to the gas / liquid peristaltic pumps through pipelines to inject sterile culture medium and sterile gas; alternatively, the culture medium and gas can be injected into the experimental vessel before sterilization and sterilized together with the experimental vessel.

8. A method for simulating and in-situ monitoring microbial stress corrosion, using the apparatus described in any one of claims 1-7, characterized in that, Includes the following steps: S1. Assemble the sample, insulating gasket, and insulating sleeve into place. Fix the sample between the two bellows clamps using the first and second fixing pins. Install the auxiliary observation window, counter electrode, and reference electrode. Pass the flexible working electrode lead wire, pre-welded to the sample, through the working electrode hole on the left side wall and lead it out of the test vessel. Tighten the threaded plug matching the working electrode hole. Seal the gap between the plug and the lead wire with an O-ring to seal all windows, forming a closed system inside the test vessel. Use a liquid peristaltic pump to transport microorganisms and culture medium into the test vessel through the inlet and outlet threaded holes. Use a gas peristaltic pump to transport and discharge gas through the inlet and outlet threaded holes to maintain a specific gas environment. Turn on the bottom adjustable temperature silicone rubber heating plate to heat the vessel body, set the microbial culture temperature, and maintain the system for a certain period of time. S2. After the culture is completed, the servo motor is started. The first loading beam and the second loading beam are controlled to move synchronously in opposite directions through the electrode. The servo motor rotates and drives the beam to move. The sample is subjected to tensile load through the force transmission of the clamp and pin, that is, the sample is in the loading state. The sample is subjected to three loading modes: constant load, constant strain, or slow strain rate tension through closed-loop control of the force sensor. S3. Connect the test leads of the electrochemical workstation to the working electrode leads, the counter electrode, and the reference electrode respectively; apply a voltage of a specific frequency to the electrodes and collect the electrochemical measurement parameters of the sample under different stress and microbial environments. S4. Insert the microscope objective into the experimental vessel through the in-situ observation window of the bellows, and use a bright field white light source to observe the corrosion morphology of the sample 23 surface, such as microcracks and pitting. Combine the differential interferometer to analyze the morphology of the sample surface. For biofilms, deliver fluorescent dye to the vessel through the liquid inlet threaded hole, or use endogenous fluorescent microorganisms to excite cells with a laser light source and observe the biofilm on the sample surface.

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