A microfluidic visualization device and simulation method for microbial regulation of non-aqueous phase liquid interfacial behavior

Abstract

The present application relates to the technical field of soil and groundwater pollution control and remediation, and particularly relates to a microfluidic visualization device and simulation method for microbial regulation of non-aqueous phase liquid interface behavior. The visualization device comprises a microfluidic chip system for simulating the heterogeneous structure of porous media in the underground environment and an experimental device for observing the microbial regulation of non-aqueous phase interface behavior. The microfluidic chip system constructs a spatial structure simulating the surface heterogeneity of porous media, and combines a fluid injection control system, an optical visualization system, a pressure monitoring system, and a data acquisition and control system in the experimental device, thereby realizing controllable frequency, micron-level precision, and real-time observation of micron to centimeter level for the microbial regulation of non-aqueous phase liquid interface behavior. The multi-source data processing method proposed in the present application realizes quantitative characterization and response of the biological membrane regulation of non-aqueous phase liquid migration path, retention form and redistribution.

Description

Technical Field

[0001] This invention relates to the field of soil and groundwater pollution control and remediation technology, and in particular to a microfluidic visualization device and simulation method for microbial regulation of non-aqueous liquid interface behavior. Background Technology

[0002] With the continuous development of industrial and agricultural activities, a large amount of organic pollutants are constantly entering the soil-groundwater environment. Among them, non-aqueous liquids, due to their low solubility, large density differences, strong migration ability and long residence time, are prone to forming residual phases or deep pollution sources in porous media after entering the ground, becoming long-term and difficult-to-remediate sources of pollution in the groundwater environment, posing a continuous threat to the safety of the groundwater environment.

[0003] Microbial communities are prevalent in underground environments, often attaching to particle surfaces or multiphase interfaces in the form of biofilms. The formation and evolution of biofilms alter pore channel structure, interfacial wetting properties, and local flow resistance, thus significantly influencing the migration paths, interfacial morphology, and retention distribution of non-aqueous liquids. However, current research primarily focuses on the biodegradation of pollutants by microorganisms, with relatively little research on the physical regulation of non-aqueous liquid interfacial behavior by microorganisms through altering pore structure and interfacial conditions. Furthermore, the structural heterogeneity of porous media surfaces affects microbial attachment and biofilm formation processes, further influencing the degree of their regulation of non-aqueous liquid interfacial behavior; however, quantitative studies under controlled conditions are lacking on the underlying mechanisms of these influences.

[0004] Currently, most related experiments employ macroscopic devices such as sand columns or two-dimensional sand boxes. While these methods can obtain overall migration patterns, they struggle to directly observe interface morphology, biofilm structure, and local flow characteristics at the pore scale. Furthermore, they are difficult to simultaneously acquire and correlate microscopic structural evolution with macroscopic migration behavior. In addition, the complex and poorly controllable structure of natural porous media hinders systematic research into the coupling relationship between porous media structural characteristics, microbial growth processes, and non-aqueous liquid migration behavior.

[0005] Therefore, there is an urgent need for an experimental device and method that can construct controllable surface heterogeneous porous media structures, realize the visualization and observation of microbial growth and non-aqueous liquid migration processes, and simultaneously acquire information on structural evolution and flow response, so as to study the regulatory process of microorganisms regulating the behavior of non-aqueous liquid interfaces. Summary of the Invention

[0006] This invention provides a microfluidic visualization device and simulation method for microbial regulation of non-aqueous liquid interface behavior. The device enables the controllable construction of a fluid domain with a heterogeneous porous media structure on the surface of a microfluidic chip. Combined with an industrial camera and an inverted microscope, it allows for real-time observation of non-aqueous liquid interface behavior under microbial influence within a micrometer to centimeter scale under adjustable spatial resolution (0.66-4.8 μm / pixel) and image acquisition frequency (0.06-30 Hz). Simultaneously, through multi-source data processing, it achieves quantitative characterization of the migration paths, retention morphologies, and redistribution processes of non-aqueous liquids regulated by biofilms, thus solving problems existing in the prior art.

[0007] One of the technical solutions adopted in this invention is: A microfluidic visualization device for microbial regulation of non-aqueous liquid interface behavior is provided, including a microfluidic chip system for simulating the heterogeneous structure of porous media in underground environments. The microfluidic chip system includes a microfluidic chip, with a chip inlet and a chip outlet respectively provided at both ends of the microfluidic chip. A fluid domain is provided in the middle of the microfluidic chip cavity between the chip inlet and the chip outlet. The microfluidic chip cavities on both sides of the fluid domain are a liquid inlet cavity connected to the chip inlet and a liquid outlet cavity connected to the chip outlet, respectively. The fluid domain is a smooth surface porous medium structure fluid domain, a medium rough surface porous medium structure fluid domain, or a rough surface porous medium structure fluid domain; each fluid domain is formed by variable-sized, uniformly distributed column structures set in the middle of the microfluidic chip cavity; The column structure is arranged in a regular triangular lattice within the fluid domain, with an adjacent column spacing of 1.1 mm and a column radius uniformly distributed within the range of 0.25-0.5 mm; The column structure within the fluid domain of the medium-roughness porous medium structure and the column sidewalls within the fluid domain of the roughness porous medium structure both have a rough profile that varies in concavity and convexity along the circumferential direction of the column; the surface roughness of the column structure within the fluid domain of the medium-roughness porous medium structure is less than the surface roughness of the column structure within the fluid domain of the roughness porous medium structure.

[0008] Furthermore, the average column radius of the column structure within the fluid domain is 0.38 mm.

[0009] Furthermore, the microfluidic chip is a polydimethylsiloxane microfluidic chip.

[0010] Furthermore, the uneven and rough contour formed on the side wall of the column structure gives the column structure a certain roughness. The corresponding roughness is characterized by surface contour parameters: arithmetic mean height Ra, root mean square Rq, skewness Rsk, and kurtosis Rku. Based on the difference in the above parameter settings, porous media structures with two different roughness levels, namely medium roughness and roughness, are formed.

[0011] Furthermore, the length of the fluid domain is half the length of the microfluidic chip.

[0012] Furthermore, the aforementioned microfluidic visualization device for microbial regulation of non-aqueous liquid interface behavior includes: a fluid injection control system, a microfluidic chip system, an optical visualization system, a pressure monitoring system, and a data acquisition and control system. The fluid injection control system is connected to the microfluidic chip system and is used to inject fluid into the microfluidic chip and realize flow regulation and fluid switching; The optical visualization system and pressure monitoring system are respectively connected to the data acquisition and control system to realize synchronous monitoring and recording of the experimental process; the pressure monitoring system is used to collect pressure changes on both sides of the microfluidic chip system.

[0013] Furthermore, the fluid includes anhydrous ethanol, sterile water, nutrient solution, and microbial culture solution.

[0014] Furthermore, the aforementioned device also includes a dark box, in which the fluid injection control system, microfluidic chip system, optical visualization system, and pressure monitoring system are all located, while the data acquisition and control system is located outside the dark box.

[0015] Furthermore, the fluid injection control system includes an injection pump, a syringe, a main pipeline, a side pipeline, and a waste liquid tank; the syringe is connected to the main pipeline via an adapter; the microfluidic chip is connected and installed on the main pipeline; the side pipeline is connected to the main pipeline in front of the microfluidic chip via a multi-port valve, and the outlet of the side pipeline is connected to the waste liquid tank; a check valve and a switching valve are installed on both the main pipeline and the side pipeline behind the microfluidic chip, and another waste liquid tank is connected to the outlet of the main pipeline.

[0016] Furthermore, the microfluidic chip is fixed in position by a backplate.

[0017] Furthermore, the optical visualization system includes an imaging device and a light source; the imaging device includes an industrial camera and an inverted microscope; the microfluidic chip is disposed above the inverted microscope, and the industrial camera is disposed above the microfluidic chip; the light source includes an ultraviolet light source and a white light source.

[0018] Furthermore, the industrial camera is equipped with a macro lens and is fixed by a base and a support frame that is adjustable in both horizontal and vertical directions; the inverted microscope is equipped with an objective lens, an objective lens converter and a stage; a micron-thick shim is placed at the light-transmitting hole of the stage, and the microfluidic chip is fixed on the shim, with the gap in the middle of the shim being larger than the fluid domain.

[0019] Furthermore, the ultraviolet light source is positioned above the space inside the dark chamber to avoid creating shadow areas during illumination; the white light source is millimeter-thick and slides in, fixed, and slides out from both sides through the gap between the stage and the objective lens.

[0020] Furthermore, the pressure monitoring system includes a pressure sensor, which is connected by a pipe and placed outside the chip inlet front end and chip outlet rear end of the microfluidic chip.

[0021] Furthermore, the data acquisition and control system is a computer.

[0022] The second technical solution adopted in this invention is: A simulation method for microbial regulation of non-aqueous liquid interface behavior using the microfluidic visualization device described above is provided, comprising the following steps: Step 1: Construct a porous dielectric microfluidic chip with a heterogeneous surface structure and complete the microfluidic chip packaging; Step 2: Install the microfluidic chip into the experimental system within the microfluidic visualization device, and perform airtightness testing and operational stability debugging on the experimental system; Step 3: Pre-treat the connecting pipes of the microfluidic chip and experimental system by sequentially introducing fluids: anhydrous ethanol, sterile water and nutrient solution for rinsing, and gradually reducing the flow rate from high to experimental flow rate to remove residual air bubbles in the system and achieve stable pre-saturation; Step 4: During the liquid switching process, first close the switch valve at the rear end of the main pipeline of the fluid injection control system and then replace the liquid. Open the side pipeline of the fluid injection control system to quickly introduce new fluid at a high flow rate to remove residual gas. Then introduce liquid at a low flow rate of no less than 5 times the system volume, close the side pipeline to switch to the main pipeline and adjust to the experimental flow rate. Step 5: Inject the microbial suspension cultured to the exponential growth phase at a set flow rate. The injection volume is larger than the system volume to achieve complete replacement. Then stop the flow and let it stand for a preset time to allow the microorganisms to attach in the porous media structure fluid domain (porous media structure surface) of the microfluidic chip. Step 6: After the microorganisms attach, the nutrient solution is continuously introduced at a low flow rate to allow the biofilm to gradually form and reach a stable state. During the cultivation process, pressure data from the pressure monitoring system and image data from the optical visualization system are collected simultaneously. Step 7: After determining the formation of the biofilm by combining pressure data and image data, inject non-aqueous liquid into the experimental system at the set flow rate, and continue to collect images at the overall scale (images taken by an industrial camera) and pore scale (images acquired by an inverted microscope), while recording the pressure changes at the chip inlet and chip outlet of the microfluidic chip. Step 8: Use an industrial camera to capture the overall distribution and evolution process of non-aqueous liquid under the regulation of biofilm, use an inverted microscope to capture the interface morphology and structural changes of biofilm and non-aqueous phase in local areas, and simultaneously acquire pressure time series data. Perform quantitative analysis on the image data and pressure time series data to obtain the migration path, motion characteristics, retention morphology and redistribution parameters of biological and non-aqueous liquid. Step 9: After the experiment, ethanol and sterile water are passed through the experimental system in sequence to clean it and restore it to its initial state.

[0023] Furthermore, the experimental system described in the above method steps refers to the experimental system composed of a fluid injection control system, an optical visualization system, and a pressure monitoring system installed within the microfluidic visualization device. The airtightness test of the experimental system mainly involves testing the airtightness of the main pipe, side pipes, and the microfluidic chip connected to the main pipe of the fluid injection control system. The system volume mentioned in the above method refers to the total volume of the syringe, main pipe, side pipes, and the internal cavity of the microfluidic chip within the fluid injection control system.

[0024] Furthermore, the operational steps for constructing the porous dielectric structure fluid domain microfluidic chip with surface heterogeneity in step 1 are as follows: Step 1.1: Set the length and width range of the microfluidic fluid domain of the microfluidic chip, determine the column spacing of the column structure within the microfluidic fluid domain, and generate the column center coordinates using a regular triangular lattice structure; Step 1.2: Set the average column radius of the column structure and use it as a reference. Randomly disturb within the preset variation range of the column radius to make the column radius meet the uniform distribution within the specified size range (0.25-0.5mm), thereby forming a porous medium spatial structure in the microfluidic fluid domain. Step 1.3: Set the target parameters for the statistical roughness of the column sidewall surface of the column structure (arithmetic mean height Ra, root mean square Rq, skewness Rsk, kurtosis Rku) according to the experimental requirements, so as to characterize different roughness levels of porous media. Step 1.4: Construct the initial profile of the column surface based on the superposition of periodic functions and random noise, and establish a parameterized generation model of the surface morphology of the column sidewall by introducing local peak and valley disturbances and nonlinear modulation. Step 1.5: Use a global optimization algorithm to iteratively adjust the model parameters of the surface topography parameterization generation model so that the error between the calculated surface statistical roughness parameters and the statistical roughness target parameters meets the preset requirements, thereby obtaining a surface profile that conforms to the target statistical roughness characteristics. Step 1.6: Discretize the optimized surface profile into multi-point coordinates along the circumference of the cylinder and superimpose them onto the cylinder radius to generate a cylinder boundary with random roughness characteristics. Step 1.7: Perform boundary integrity checks on the generated columns, delete columns that intersect with the fluid domain boundary, and center the overall structure to ensure the complete distribution of the medium field in the porous medium region. Step 1.8: Output the coordinate data of the generated porous dielectric structure. The output results are used for subsequent photolithography mask design and microfluidic chip fabrication.

[0025] Furthermore, in step 8, the migration paths, motion characteristics, retention morphologies, and redistribution parameters of biological and non-aqueous phase liquids are obtained through multi-source data acquisition and processing. The "multi-source data acquisition and processing" includes microfluidic domain analysis, local region characterization, and pressure time series data analysis, followed by time-synchronous correlation analysis, comprising the following sub-steps: Step 8.1: Before the experiment, zero the reference of the pressure sensor of the pressure monitoring system, and continuously collect the chip inlet and chip outlet pressure data of the microfluidic chip at preset time intervals during the experiment, i.e., pressure time series data. Based on the collection results, obtain the process of the system pressure difference changing over time, which is used to characterize the response characteristics of the flow state during the changes in pore resistance caused by microbial growth, local blockage, and migration and redistribution of non-aqueous liquids. Step 8.2: Denoise, correct brightness, and crop regions of the overall field-of-view image of the microfluidic chip. Use threshold segmentation and color feature recognition methods to distinguish the distribution areas of aqueous phase, non-aqueous liquid and biological phase. Extract the spatial distribution information of each phase and the interface position changes to obtain the migration path, front evolution and redistribution characteristics of biological and non-aqueous liquid at the overall scale. Step 8.3: Denoise and enhance the microscopic image of the selected local area, and segment the biofilm structure and the non-aqueous phase interface to extract the biofilm attachment range, growth morphology and local morphological changes of the interface. This information is used to characterize the influence of microorganisms occupying the pore space on the morphological stability, contact state and local retention behavior of the non-aqueous phase liquid interface. Step 8.4: Perform time synchronization and correlation analysis on pressure time series data, overall migration distribution and local structural features to establish the correspondence between pressure difference changes, biological growth state, pore structure evolution and non-aqueous liquid interface migration and stability response.

[0026] The beneficial effects of this invention are: The device of this invention constructs a porous medium structure in the fluid domain of a microfluidic chip with a regular lattice structure, and uses surface profile parameters to quantitatively characterize the roughness of the pillars in the fluid domain of the porous medium structure of the microfluidic chip. This achieves the controllable construction and parameterized design of the heterogeneity of the porous medium surface structure, and improves the consistency and repeatability of experimental conditions.

[0027] This invention employs a combination of an industrial camera and an inverted microscope for imaging, enabling real-time observation of biofilm evolution and dynamic changes at the non-aqueous liquid interface within a micrometer to centimeter scale under adjustable spatial resolution (0.66-4.8 μm / pixel) and image acquisition frequency (0.06-30 Hz). A multi-source data collaborative simulation and analysis method combining pressure monitoring and optical imaging was established. By acquiring changes in system flow resistance through pressure sensors and combining this with time-synchronous analysis of image data, a quantitative correlation was achieved between biofilm growth, pore structure changes, and non-aqueous phase migration behavior. Based on image processing methods, parameters such as non-aqueous phase saturation, biofilm coverage, biomass distribution intensity, frontal migration distance, and velocity can be obtained, enabling detailed characterization of migration paths, retention morphology, and redistribution processes.

[0028] The device and method of this invention are applicable to the analysis of multiphase migration processes under different microbial types, different porous media surface structures, and different flow conditions. They are characterized by simple operation, high observation accuracy, complete data acquisition, and wide applicability. Attached Figure Description

[0029] Figure 1 This is a plan view of the visualization device of the present invention; Figure 2 for Figure 1 A schematic diagram of the fluid domain structure of a microfluidic chip; U in the lower left corner of the figure represents the average distribution within the interval, and λ represents the fluctuation coefficient / dispersion. Figure 3 This is a schematic diagram of the microfluidic chip for fluid domains with smooth, moderately rough, and rough surfaces and porous media structures according to the present invention. Figure 4 A 3D view of a microfluidic chip; Figure 5 This is a flowchart illustrating the construction of surface heterogeneous structures using porous media according to the present invention. Figure 6 This is a flowchart of the multi-source data processing and analysis process of the simulation method of the present invention; Figure 7 The following are the results of multi-source data processing in the example of this invention: (a) shows the evolution of biofilm coverage in the fluid domain of the microfluidic chip at different times; (b) shows the migration path of non-aqueous liquid in the fluid domain of the microfluidic chip at different times; and (c) shows the time-pressure difference correlation between biofilm evolution and overall migration of non-aqueous liquid.

[0030] The components are as follows: 1-Injection pump, 2-Injector, 3-Adapter, 4-Multi-port valve, 5-Check valve, 6-Switch valve, 7-Waste liquid tank, 8-Pressure sensor, 9-Inverted microscope, 10-Base, 11-Microfluidic chip, 12-Chip inlet, 13-Chip outlet, 14-Smooth surface porous media structure fluid domain, 15-Medium rough surface porous media structure fluid domain, 16-Rough surface porous media structure fluid domain, 17-Stainless steel gasket, 18-Stage, 19-White light source, 20-Objective lens, 21-Objective lens turret, 22-Horizontal support frame, 23-Vertical support frame, 24-Industrial camera, 25-Ultraviolet light source, 26-Dark box, 27-Computer, 28-Inlet chamber, 29-Outlet chamber. Detailed Implementation

[0031] To clearly illustrate the technical features of this solution, the invention will be described in detail below through specific embodiments and in conjunction with the accompanying drawings.

[0032] In the description of this invention, it should be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0034] Example 1

[0035] To simulate the behavior of microorganisms regulating non-aqueous liquid interfaces in underground environments at the pore scale, this invention provides a device that allows for intuitive observation of interface morphology, biofilm structure, and local flow characteristics. It also enables the simultaneous acquisition and correlation analysis of microstructural evolution and macroscopic migration behavior, thus systematically studying the coupling relationship between porous media structural characteristics, microbial growth processes, and non-aqueous liquid migration behavior. The device comprises a fluid injection control system, a microfluidic chip system, an optical visualization system, a pressure monitoring system, and a data acquisition and control system. The fluid injection control system is connected to the microfluidic chip system and is used to inject fluid into the microfluidic chip and achieve flow regulation and fluid switching. The optical visualization system and pressure monitoring system are respectively connected to the data acquisition and control system for synchronous monitoring and recording of the experimental process.

[0036] For details, see Figure 1 The fluid injection control system includes a syringe pump 1, a syringe 2, and a main pipeline and a bypass pipeline (i.e., a side pipeline) constructed by a multi-port valve 4, connected in sequence. The main pipeline and the bypass pipeline are each connected to a waste liquid pool 7. The syringe pump 1 and the syringe 2 are connected to the main pipeline via an adapter 3. A microfluidic chip 11 is installed on the main pipeline. After passing through the microfluidic chip, the main pipeline is equipped with a check valve 5 and a switching valve 6 and leads to another waste liquid pool 7 connected to its rear end. The bypass pipeline is located outside the main pipeline before the microfluidic chip and is also equipped with a check valve 5 and a switching valve 6, and then leads to another waste liquid pool 7 connected to its rear end. The multi-port valve 4, check valve 5, switching valve 6, main pipeline, bypass pipeline, and adapter 3 are all made of polytetrafluoroethylene (PTFE) to prevent non-aqueous liquids from causing swelling or corrosion to the overall system.

[0037] See Figure 3 The aforementioned microfluidic chip is made of polydimethylsiloxane material and includes an internal fluid domain 14, a chip inlet 12, and an outlet 13; the fluid domain 14 is located in the middle of the microfluidic chip cavity between the chip inlet 12 and the chip outlet 13. Figure 4 The microfluidic chip cavities on both sides of the fluid domain 14 are a liquid inlet cavity 28 connected to the chip inlet 12 and a liquid outlet cavity 29 connected to the chip outlet 13, respectively. The chip inlet 12 is connected to the front section of the main pipe via a connector (not shown in the figure) fixed to the upper left side of the microfluidic chip, and the chip outlet 13 is connected to the rear section of the main pipe via a connector (not shown in the figure) fixed to the lower right side of the microfluidic chip. The fluid domain 14 is formed by variable-sized, uniformly distributed column structures set in the middle of the microfluidic chip cavity. The column structures simulate a porous media structure fluid domain. The column dimensions of the column structures are variable and their surfaces have quantifiable contour parameters, thereby characterizing the porous media structure surfaces with different roughnesses within the microfluidic chip fluid domain. By using column structures with differentiated roughness, microfluidic chips can be fabricated into microfluidic chips with various specifications of fluid domains, including microfluidic chips with smooth surface porous media structure fluid domains, microfluidic chips with medium-roughness surface porous media structure fluid domains, and microfluidic chips with rough surface porous media structure fluid domains, such as... Figure 3 As shown, by replacing the microfluidic chip with the different fluid domains described above, the behavior and changes of microorganisms regulating non-aqueous liquid interfaces under various porous media structures can be simulated.

[0038] In one specific implementation, the microfluidic chip measures 10 mm × 40 mm. The fluid domain is located in the center of the microfluidic chip, with a length of 20 mm and a width slightly less than 10 mm. Several pillar structures are arranged within the fluid domain, employing a regular triangular lattice arrangement. The spacing between adjacent pillar structures (the horizontal distance between the axes of adjacent pillars) is 1.1 mm. The pillar radii follow a uniform distribution within the range of 0.25-0.5 mm, with an average radius of 0.38 mm, simulating the size of the fine-to-medium-grained porous media required for the experiment. To clarify the regulatory effect of roughness variations on microbial attachment and non-aqueous liquid redistribution, a rough profile with varying concavity and convexity is constructed at the pillar boundaries. Surface profile parameters (arithmetic mean height Ra, root mean square Rq, skewness Rsk, and kurtosis Rku) are used to quantitatively characterize the surface roughness of the pillars, forming porous media structures with different roughness levels. The heights of the fluid domain, inlet chamber, outlet chamber, chip inlet, and chip outlet are all 0.05 mm, and the overall thickness of the microfluidic chip is 3 mm.

[0039] The aforementioned microfluidic chip can be supported by a backplate, glass slide, or other support components in the entire system, and its position can be fixed by bonding, clamping, or limiting structures to ensure stability during the experiment.

[0040] The aforementioned optical visualization system includes an imaging device and a light source. The imaging device includes an industrial camera 24 and an inverted microscope 9. The industrial camera 24 is equipped with a macro lens, an image acquisition frame rate of 0.06-30 frames / s, a field of view of 10 mm-20 cm, and is fixed and height-adjustable via a base 10, a longitudinal support frame 23, and a transverse support frame 22. The inverted microscope 9 is equipped with a stage 18, an objective lens turret 21, and 5× and 20× objectives 20, achieving a resolution of up to 0.66 μm / pixel and a field of view of 1.6 mm × 1.6 mm. A stainless steel gasket 17 is placed at the light-transmitting aperture of the stage, and a microfluidic chip 11 is fixed to the stainless steel gasket, with the central gap of the stainless steel gasket larger than the fluid domain. The light source includes an ultraviolet light source 25 and a white light source 19. The ultraviolet light source 25 is positioned above the system to ensure uniform irradiation and reduce the influence of shadows, thereby achieving the sterilization or inhibition of microorganisms before the experiment; the white light source 19 is positioned between the stage and the objective lens, and can slide in or move out as needed for the experiment, and its brightness can be adjusted to meet the imaging requirements.

[0041] The stainless steel gasket 17 can be configured with the following dimensions: outer diameter 54 mm, inner diameter 34 mm, and thickness 0.05 mm. The ultraviolet light source has a power of 30 W, a lamp tube length of 894 mm, a diameter of 25 mm, and a corresponding bracket length of 920 mm, width of 32 mm, and height of 49 mm, achieving an ultraviolet intensity of 112 μW / cm². 2The white light source is 8 cm × 8 cm in size, with a light-emitting area of ​​6 cm × 6 cm and a thickness of 1 cm. It is covered with a 6 cm × 6 cm diffuse reflector on top and has an aluminum alloy shell around it. The light source has ten brightness levels that can match the camera aperture to meet the selection of the optimal light intensity for the experiment.

[0042] The aforementioned pressure monitoring system is connected by pipes and placed at the inlet front and outlet rear of the microfluidic chip. The microfluidic visualization device also includes a dark box 26, in which the aforementioned fluid injection control system, microfluidic chip system, optical visualization system, and pressure monitoring system are all located. The data acquisition and control system is a computer 27, which is located outside the dark box.

[0043] As one specific embodiment, the visualization device of the present invention employs the following equipment: a high-precision syringe pump (PhDUltra 70-3007, Harvard Apparatus), a 10 mL disposable sterile syringe, a Y-type multi-port valve (Hamilton, USA), a pressure sensor (Ups-250-T116, Labsmith, USA), an optical inverted microscope (Carl Zeiss, AxioVert.A1), an industrial camera (COMS, G3-XC30-C4095, DALSA), an LED white light source (24V), an ultraviolet light source (30W, PHILIPS), a microfluidic chip (polydimethylsiloxane), matching PTFE tubing, Luer adapters, check valves, on / off valves (1 / 16, Labsmith, USA), stainless steel gaskets, and a computer (DELL, Intel i7, 16 G).

[0044] Example 2

[0045] The visualization device described in Example 1 was used to simulate the behavior of microorganisms regulating non-aqueous liquid interfaces. The method and steps are as follows: I. Method for constructing heterogeneous structures on porous media surfaces, the process is as follows: Figure 5 The specific steps are as follows: Step 1: Set the length and width range of the fluid domain of the microfluidic chip (20 mm × 10 mm in this example), determine the arrangement of the pillars and the spacing between the pillars (a regular triangular lattice arrangement is used in this example), calculate the coordinate position of the pillar center, and form the basic structural framework.

[0046] Step 2: Using the set average column radius as a reference (0.38 mm in this example), randomly perturb the column radius within a preset range to make the column radius satisfy a uniform distribution (the radius range in this example is 0.25-0.50 mm), thereby simulating the spatial structure of fine and medium-grained porous media.

[0047] Step 3: To clarify the effect of roughness changes on microbial attachment-non-aqueous phase liquid redistribution, target parameters for statistical roughness of the column surface are set (in this example, the parameters are arithmetic mean height Ra, root mean square Rq, skewness Rsk, and kurtosis Rku) to characterize different roughness levels (in this example, three surface types are set: smooth group, medium rough group, and rough group. Smooth group Ra=0, Rq=0, Rsk=0, Rku=3; medium rough group Ra=0.01 mm, Rq=0.01 mm, Rsk=0.40, Rku=5.67; rough group Ra=0.02 mm, Rq=0.02 mm, Rsk=0.76, Rku=2.61).

[0048] Step 4: Construct the initial profile of the cylindrical surface based on the superposition of periodic functions and random noise. The periodic function is a multi-frequency sine function, superimposed with Gaussian random noise with a mean of 0 (amplitude 0.1-0.8). By introducing local peak-valley perturbations (the number of which is 5% to 10% of the number of discrete points, and the peak-valley height range is ±0.25) and nonlinear amplitude modulation (skewness modulation parameter range is ±3.0), a parameterized generation model of surface morphology is established to describe the random rough structure characteristics.

[0049] Step 5: Use a global optimization algorithm to iteratively adjust the surface morphology model parameters. By adjusting parameters such as surface amplitude coefficient (0.03-0.35), peak-valley disturbance intensity (0-1), and peak sharpness (0.5-3.5), the error between the calculated surface statistical parameters and the target roughness parameters is less than a preset threshold (5% in this example), thereby obtaining a cylindrical surface profile that meets the statistical characteristics requirements.

[0050] Step 6: Discretize the optimized surface profile into multi-point coordinates along the circumference of the cylinder. In this example, the circumference is evenly divided into 360 equal-angle sampling points. Calculate the surface height value at the corresponding angle position and superimpose it onto the base radius of the cylinder to generate a cylinder boundary with random roughness characteristics.

[0051] Step 7: Perform boundary integrity checks on the generated column structures, delete columns that intersect with the fluid domain boundary, and center and correct the boundaries of the overall structure to ensure the complete distribution of the porous media region. The result is as follows: Figure 2 As shown.

[0052] Step 8: Export the final generated porous dielectric structure coordinate data as a vector file for photolithography mask design, and fabricate the microfluidic chip 11 using photolithography. Complete chip packaging to form a porous dielectric fluid domain microfluidic chip with different roughness levels: a smooth surface porous dielectric fluid domain 14, a medium-roughness surface porous dielectric structure fluid domain 15, and a roughness surface porous dielectric structure fluid domain 16, as shown below. Figure 3 , Figure 4 .

[0053] II. The experimental procedure for microbial regulation of non-aqueous liquid interface behavior is as follows: Step 1: Inoculate the target strain (Bacillus subtilis in this example) into liquid culture medium, incubate at a constant temperature with shaking until the exponential growth phase, and measure the bacterial concentration (OD in this example) using a spectrophotometer. 600 = 0.1), for backup.

[0054] Step 2: Add staining agent according to the set mass concentration to label the aqueous phase liquid (in this example, the staining agent is brilliant blue, which is non-toxic to microorganisms, and its mass concentration in the aqueous phase is 0.03 g / L). The non-aqueous phase liquid (in this example, hydrofluoroether) is not stained to enhance the contrast of subsequent image recognition.

[0055] Step 3: Use a balance weighing method to calibrate the flow rate of injection pump 1 to achieve a repeatability of 0.05%, and set parameters such as injection flow rate, injection volume and running time according to the experimental plan.

[0056] Step 4: Adjust the height of the industrial camera 24 so that its field of view covers the fluid domain of the microfluidic chip 11; adjust the lens focal length to obtain a clear image, adjust the aperture and exposure parameters to obtain appropriate brightness, and set the image acquisition parameters according to the experimental plan, including exposure time and acquisition frame rate; at the same time, adjust the position of the inverted microscope 9 to coordinate and match it with the observation area of ​​the industrial camera 24.

[0057] Step 5: Install the microfluidic chip 11 on the stage 18 of the inverted microscope 9, and connect the chip inlet 12 of the microfluidic chip to the syringe 2 and the injection pump 1 through the adapter 3 and PTFE tubing. Connect the pressure sensor 8 to the main pipeline before the chip inlet of the microfluidic chip, and connect the pressure sensor 8 and the waste liquid tank 7 to the main pipeline connected to the chip outlet 13 in sequence. Install a multi-way valve 4, a check valve 5 and a switching valve 6 on the pipeline to realize fluid switching and flow direction control.

[0058] Step 6: Turn on the syringe pump 1 to introduce air into the system for airtightness testing. After confirming that there are no leaks at each interface, introduce ultrapure water into the system and run it stably for 10 minutes to check the system's operational stability.

[0059] Step 7: Pre-treat the microfluidic chip 11 and connecting tubing by sequentially passing anhydrous ethanol, sterile water and nutrient solution through it, and gradually reducing the flow rate from high to experimental flow rate (in this example, the flow rate is gradually reduced from 100 μL / min to 50 μL / min, and then to 10 μL / min) to remove residual air bubbles in the system and achieve a stable pre-saturated state.

[0060] Step 8: During the liquid switching process, first close the switch valve 6 at the rear end of the main pipeline. After changing the liquid, open the bypass pipeline and quickly introduce the new fluid at a high flow rate (100 μL / min in this example, for 5 min) to remove residual gas. Then introduce it into the main pipeline at a low flow rate (10 μL / min in this example) for a certain period of time (3 h in this example).

[0061] Step 9: Inject the microbial suspension in the exponential growth phase into the system (in this example, the injection flow rate is 10 μL / min and the injection volume is 200 μL) to completely replace the original liquid in the system with microorganisms; after injection, stop the flow and let it stand for 3 hours in this example to achieve the attachment of microorganisms on the surface of the porous medium.

[0062] Step 10: After the microorganisms attach, the nutrient solution is continuously introduced at a low flow rate (2 μL / min in this example, for 48 h) for cultivation, so that the biofilm gradually forms and reaches a stable state. The overall and local scale images are simultaneously acquired by the industrial camera 24 and the inverted microscope 9, and the changes in chip inlet and chip outlet pressure are continuously recorded by the pressure sensor 8.

[0063] Step 11: After the biofilm has stabilized, a non-aqueous liquid is injected into the system at a set flow rate (5 μL / min in this example, for 20 h). Images and pressure data are continuously acquired. The industrial camera 24 is used to capture the distribution and evolution of the non-aqueous liquid and the biofilm within the overall field of view of the microfluidic chip. The inverted microscope 9 uses objectives 20 at different magnifications to acquire the pore-scale interface morphology and biofilm structural changes. In this step, biofilm stability is generally determined after 48 h of growth, or by observing no significant changes over a long period through images, thus indicating stable biofilm formation.

[0064] Step 12: The image acquisition frequency is adjusted according to the experimental stage (0.06-30 Hz in this example), and the image spatial resolution is 0.66-4.8 μm / pixel. The image acquisition and pressure data are stored and synchronized in real time through computer 27.

[0065] Step 13: After the experiment, anhydrous ethanol and ultrapure water are injected into the system sequentially through syringe pump 1 for cleaning, so that the microfluidic chip 11 and the tubing can be restored to their initial state for subsequent repeated experiments.

[0066] III. Multi-source data processing methods, such as Figure 6 The multi-source data refers to pressure-time data, data collected from the fluid domain of the microfluidic chip, and data collected within local areas. Through the above data collection and analysis, the response relationship of the non-aqueous liquid interface during microbial regulation is obtained. The specific steps are as follows: Step 1: Before the experiment, zero the pressure sensor 8. During the experiment, continuously collect the pressure data of the microfluidic chip inlet 12 and chip outlet 13 at preset time intervals (the collection interval is 0.3 s in this example), and calculate the change of system pressure difference over time to characterize the flow response characteristics of pore resistance changes caused by microbial growth, local blockage, and non-aqueous liquid migration and redistribution.

[0067] As a specific embodiment, the method for calculating the pressure difference is as follows:

[0068] in, For microfluidic chip inlet pressure 12, For the microfluidic chip outlet pressure 13.

[0069] Step 2: Denoise the overall field-of-view image acquired by the industrial camera 24, perform brightness correction and region cropping, and distinguish the distribution areas of aqueous phase, non-aqueous liquid and biological phase by RGB threshold segmentation and color feature recognition method, and extract the spatial distribution information of each phase.

[0070] Step 3: Calculate the saturation, biomass coverage and intensity distribution of the non-aqueous liquid at the overall scale, and extract the interface edge to obtain the position, migration distance and migration direction of the biological and non-aqueous frontier, characterizing the overall migration and redistribution characteristics of the biological and non-aqueous liquid in the porous medium.

[0071] As a specific embodiment, the saturation of the non-aqueous phase liquid ( ) and biodiversity ( Calculated by the number of pixels in the image:

[0072] in, It represents the number of pixels across the entire flow channel. and These represent the number of pixels identified in the experiment for non-aqueous liquids and biological samples, respectively.

[0073] As a specific embodiment, the normalized biological intensity ( The calculation method for () is as follows:

[0074] in, This is the initial grayscale value of the selected area before the experiment, when it was not covered by organisms. It is the grayscale value after biological coverage of the selected area. It is the maximum gray value corresponding to the location with the highest biological coverage.

[0075] Step 4: Denoise and enhance the local area images acquired by the inverted microscope 9. Use the threshold segmentation method to extract the biofilm attachment range and non-aqueous phase interface morphology change information to analyze the impact of biological occupation on interface stability, contact state and retention behavior at the pore scale.

[0076] As a specific implementation, by performing connected component analysis and edge detection on the segmented binary image and combining it with the original fluid domain image, the position, migration distance and migration direction of the biological and non-aqueous phase front can be obtained well, and the attachment range of the biofilm and the morphology of the non-aqueous phase interface can be clearly defined.

[0077] Step 5: Perform time synchronization and correlation analysis on the above pressure time series data, overall migration characteristic parameters and local structural change parameters to establish the correspondence between pressure difference change, biological growth state, pore structure evolution, non-aqueous liquid interface migration and stability response.

[0078] See Figure 7 , Figure 7 (a) shows the evolution of biofilm coverage within the microfluidic chip fluid domain at 26h, 36h, and 48h after the target bacterial strain was introduced into the system. With increasing time, the bacterial solution, influenced by the columnar structure within the fluid domain, gradually forms a film and establishes distinct preferential channels. The migration of the non-aqueous liquid, measured at 7h, 15h, and 20h after continued introduction, shows that the non-aqueous liquid mainly advances and widens along the preferential channels, while simultaneously promoting the regional aggregation and dispersion of some microorganisms attached to the medium surface, such as... Figure 7 (b) Based on the relationship curves between the above changes and the pressure difference at the inlet and outlet of the microfluidic chip, it can be seen that the biofilm evolution process leads to an increase in the pressure fluctuation of the watershed. Subsequently, the migration of non-aqueous liquids changes the pressure difference caused by the biofilm evolution, causing the pressure change to drop rapidly.

[0079] In summary, by designing interface morphology at the pore scale and visually simulating the behavior of microorganisms regulating non-aqueous liquid interfaces, we achieved controllable frequency, micron-level precision, and real-time observation of micron- to centimeter-level microorganism regulation of non-aqueous liquid interface behavior. The above multi-source data processing methods enabled more effective synchronous acquisition and correlation analysis of microscopic structural evolution processes and macroscopic migration behaviors, and achieved quantitative characterization and response of biofilm-regulated non-aqueous liquid migration paths, retention morphologies, and redistribution.

[0080] The present invention has been described in detail above. The specific embodiments described above should not be construed as limiting the scope of protection of the present invention. Any alternative modifications or variations made to the embodiments of the present invention by those skilled in the art will fall within the scope of protection of the present invention.

[0081] Any aspects of this invention not described in detail are well-known to those skilled in the art.

Claims

1. A microfluidic visualization device for microbial regulation of non-aqueous liquid interface behavior, characterized in that, The system includes a microfluidic chip system for simulating the heterogeneous structure of porous media in underground environments. The microfluidic chip system includes a microfluidic chip with a chip inlet and a chip outlet at both ends. A fluid domain is provided in the middle of the microfluidic chip cavity between the chip inlet and the chip outlet. The microfluidic chip cavities on both sides of the fluid domain are a liquid inlet cavity connected to the chip inlet and a liquid outlet cavity connected to the chip outlet, respectively. The fluid domain is a smooth surface porous medium structure fluid domain, a medium rough surface porous medium structure fluid domain, or a rough surface porous medium structure fluid domain; each fluid domain is formed by variable-sized, uniformly distributed column structures set in the middle of the microfluidic chip cavity; The column structure is arranged in a regular triangular lattice within the fluid domain, with an adjacent column spacing of 1.1 mm and a column radius uniformly distributed within the range of 0.25-0.5 mm; The column structure within the fluid domain of the medium-roughness porous medium structure and the column sidewalls within the fluid domain of the roughness porous medium structure both have a rough profile that varies in concavity and convexity along the circumferential direction of the column; the surface roughness of the column structure within the fluid domain of the medium-roughness porous medium structure is less than the surface roughness of the column structure within the fluid domain of the roughness porous medium structure.

2. The microfluidic visualization device for microbial regulation of non-aqueous liquid interface behavior according to claim 1, characterized in that, The length of the fluid domain is half the length of the microfluidic chip.

3. The microfluidic visualization device for microbial regulation of non-aqueous liquid interface behavior according to claim 1 or 2, characterized in that, It also includes a fluid injection control system, an optical visualization system, a pressure monitoring system, and a data acquisition and control system; The fluid injection control system is connected to the microfluidic chip system and is used to inject fluid into the microfluidic chip and realize flow regulation and fluid switching; The optical visualization system and pressure monitoring system are respectively connected to the data acquisition and control system to realize synchronous monitoring and recording of the experimental process; the pressure monitoring system is used to collect pressure changes on both sides of the microfluidic chip system.

4. The microfluidic visualization device for microbial regulation of non-aqueous liquid interface behavior according to claim 3, characterized in that, It also includes a dark box, in which the fluid injection control system, microfluidic chip system, optical visualization system, and pressure monitoring system are all located, while the data acquisition and control system is located outside the dark box.

5. The microfluidic visualization device for microbial regulation of non-aqueous liquid interface behavior according to claim 3, characterized in that, The fluid injection control system includes an injection pump, a syringe, a main pipe, a side pipe, and a waste liquid tank. The syringe is connected to the main pipe via an adapter. The microfluidic chip is connected to the main pipe. The side pipe is connected to the main pipe in front of the microfluidic chip via a multi-port valve, and the outlet of the side pipe is connected to the waste liquid tank. Check valves and on / off valves are installed on both the main pipe and the side pipe behind the microfluidic chip, and the outlet of the main pipe is connected to the waste liquid tank.

6. The microfluidic visualization device for microbial regulation of non-aqueous liquid interface behavior according to claim 3, characterized in that, The optical visualization system includes an imaging device and a light source; the imaging device includes an industrial camera and an inverted microscope; the microfluidic chip is located above the inverted microscope, and the industrial camera is located above the microfluidic chip; the light source includes an ultraviolet light source and a white light source.

7. The microfluidic visualization device for microbial regulation of non-aqueous liquid interface behavior according to claim 3, characterized in that, The pressure monitoring system includes a pressure sensor, which is connected by a pipe and placed on the outside of the chip inlet front end and chip outlet rear end of the microfluidic chip.

8. A method for simulating the behavior of microorganisms regulating non-aqueous liquid interfaces using the microfluidic visualization device described in claim 3, characterized in that, Includes the following steps: Step 1: Construct a porous dielectric microfluidic chip with a surface heterogeneous structure and complete the microfluidic chip packaging; Step 2: Install the microfluidic chip into the experimental system within the microfluidic visualization device, and perform airtightness testing and operational stability debugging on the experimental system; Step 3: Pre-treat the connecting tubing of the microfluidic chip and experimental system by sequentially passing anhydrous ethanol, sterile water and nutrient solution through it, and gradually reducing the flow rate from high to experimental flow rate to remove residual air bubbles in the system and achieve stable pre-saturation. Step 4: During the liquid switching process, first close the main pipeline back-end switch valve of the fluid injection control system and then replace the liquid. Open the side pipeline of the fluid injection control system to quickly introduce new fluid at a high flow rate to remove residual gas. Then introduce liquid at a low flow rate of no less than 5 times the system volume, close the side pipeline to switch to the main pipeline and adjust to the experimental flow rate. Step 5: Inject the microbial suspension in the exponential growth phase at a set flow rate. The injection volume is larger than the system volume to achieve complete replacement. Then stop the flow and let it stand for a preset time to allow the microorganisms to attach in the porous media structure fluid domain of the microfluidic chip. Step 6: After the microorganisms attach, the nutrient solution is continuously introduced at a low flow rate to allow the biofilm to gradually form and reach a stable state. During the cultivation process, pressure data from the pressure monitoring system and image data from the optical visualization system are collected simultaneously. Step 7: After determining the formation of the biofilm by combining pressure data and image data, inject non-aqueous liquid into the experimental system at the set flow rate, and continuously acquire images at the overall scale and pore scale, while recording the pressure changes at the chip inlet and chip outlet of the microfluidic chip. Step 8: Use an industrial camera to capture the overall distribution and evolution process of non-aqueous liquid under the regulation of biofilm, use an inverted microscope to capture the interface morphology and structural changes of biofilm and non-aqueous phase in local areas, and simultaneously acquire pressure time series data. Perform quantitative analysis on the image data and pressure time series data to obtain the migration path, motion characteristics, retention morphology and redistribution parameters of biological and non-aqueous liquid. Step 9: After the experiment, ethanol and sterile water are passed through the experimental system in sequence to clean it and restore it to its initial state.

9. The simulation method according to claim 8, characterized in that, The steps for constructing the porous dielectric microfluidic chip with surface heterogeneity in step 1 are as follows: Step 1.1: Set the length and width range of the microfluidic fluid domain of the microfluidic chip, determine the column spacing of the column structure within the microfluidic fluid domain, and generate the column center coordinates using a regular triangular lattice structure; Step 1.2: Set the average column radius of the column structure and use it as a reference. Randomly perturb within the preset variation range of the column radius to make the column radius meet the uniform distribution within the specified size range, thereby forming a porous medium spatial structure in the microfluidic fluid domain. Step 1.3: Set the target parameters for the statistical roughness of the column sidewall surface of the column structure according to the experimental requirements, so as to characterize different roughness levels of porous media; Step 1.4: Construct the initial profile of the cylindrical surface based on the superposition of periodic functions and random noise, and establish a parameterized generation model of surface morphology by introducing local peak and valley disturbances and nonlinear modulation. Step 1.5: Use a global optimization algorithm to iteratively adjust the model parameters of the surface topography parameterization generation model so that the error between the calculated surface statistical roughness parameters and the statistical roughness target parameters meets the preset requirements, thereby obtaining a surface profile that conforms to the target statistical roughness characteristics. Step 1.6: Discretize the optimized surface profile into multi-point coordinates along the circumference of the cylinder and superimpose them onto the cylinder radius to generate a cylinder boundary with random roughness characteristics. Step 1.7: Perform boundary integrity checks on the generated columns, delete columns that intersect with the fluid domain boundary, and center the overall structure to ensure the complete distribution of the medium field; Step 1.8: Output the generated porous dielectric structure coordinate data for subsequent photolithography mask design and microfluidic chip fabrication.

10. The simulation method according to claim 8, characterized in that, In step 8, the migration paths, motion characteristics, retention morphologies, and redistribution parameters of biological and non-aqueous phase liquids are obtained through multi-source data acquisition and processing. This multi-source data acquisition and processing includes microfluidic domain analysis, local region characterization, and pressure time series data analysis, followed by time-synchronous correlation analysis, comprising the following sub-steps: Step 8.1: Before the experiment, zero the reference of the pressure sensor of the pressure monitoring system, and continuously collect the chip inlet and chip outlet pressure data of the microfluidic chip at preset time intervals during the experiment, i.e., pressure time series data. Based on the collection results, obtain the process of the system pressure difference changing over time, which is used to characterize the response characteristics of the flow state during the changes in pore resistance caused by microbial growth, local blockage, and migration and redistribution of non-aqueous liquids. Step 8.2: Denoise, correct brightness, and crop regions of the overall field-of-view image of the microfluidic chip. Use threshold segmentation and color feature recognition methods to distinguish the distribution areas of aqueous phase, non-aqueous liquid and biological phase. Extract the spatial distribution information of each phase and the interface position changes to obtain the migration path, front evolution and redistribution characteristics of biological and non-aqueous liquid at the overall scale. Step 8.3: Denoise and enhance the microscopic image of the selected local area, and segment the biofilm structure and the non-aqueous phase interface to extract the biofilm attachment range, growth morphology and local morphological changes of the interface. This information is used to characterize the influence of microorganisms occupying the pore space on the morphological stability, contact state and local retention behavior of the non-aqueous phase liquid interface. Step 8.4: Perform time synchronization and correlation analysis on pressure time series data, overall migration distribution and local structural features to establish the correspondence between pressure difference changes, biological growth state, pore structure evolution and non-aqueous liquid interface migration and stability response.

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