A lignin-degrading microorganism gradient screening and fungal ball in-situ induction monitoring integrated microfluidic chip system and method

Abstract

The present application belongs to the cross field of environmental microorganism technology, microfluidic chip technology and Raman spectrum detection technology, and discloses a lignin-degrading microorganism gradient screening and fungal ball in-situ induction monitoring integrated microfluidic chip system and method. The chip system is sequentially stacked from bottom to top with an intrinsic low-noise sensing layer, a gradient screening and capturing layer, a fungal ball morphology regulating layer, a chemical microenvironment constructing layer and a selective gas-permeable packaging layer. The gradient screening and capturing layer is provided with three groups of through-hole arrays with gradually increasing pore diameters, and size screening and in-situ capturing of bacteria, fungal spores and mycelium segments can be realized through sample driving operation. The present application realizes integrated operation of lignin-degrading microorganism screening, fungal ball culture and in-situ monitoring, has the advantages of high screening efficiency, lossless in-situ monitoring and the like, and is suitable for functional fungus screening and culture process optimization.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of environmental microbiology technology, microfluidic chip technology and Raman spectroscopy detection technology, and specifically relates to a microfluidic chip system and method for integrating gradient sieving of lignin-degrading microorganisms and in-situ induction monitoring of fungal pellets. Background Technology

[0002] Lignin is the most abundant aromatic natural polymer in nature, widely found in agricultural and forestry waste. Its green and efficient degradation and high-value transformation are core research directions in the fields of biomass resource utilization and environmental remediation. Filamentous fungi, such as white-rot fungi, are the core functional microbial communities for lignin degradation. They can break down the phenylpropane skeleton of lignin under normal temperature and pressure by secreting extracellular oxidase systems such as lignin peroxidase, manganese peroxidase, and laccase. They possess extremely high engineering application value in scenarios such as biomass refining, treatment of recalcitrant organic wastewater, and in-situ remediation of humic substances. In practical engineering applications, filamentous fungi are often added in the form of fungal balls. The particle size distribution, density, cell wall structure, and extracellular metabolic activity of the fungal balls directly determine the degradation efficiency and operational stability. Therefore, efficiently identifying functional strains in the environment and precisely optimizing the culture conditions of the fungal balls are key prerequisites for promoting the practical application of lignin biodegradation technology.

[0003] Currently, the isolation and screening of lignin-degrading filamentous fungi still mainly relies on traditional plate isolation methods. This requires steps such as sample dilution, plating culture, single colony picking, multiple subculturing purifications, and shake-flask activity verification, resulting in an overall screening cycle of several weeks and easily leading to the loss of rare functional strains from the environment. Lignin-degrading bacteria in environmental samples include both bacteria and fungi, with fungi existing in various forms such as spores and hyphae. These three types exhibit a large size range and varying degradation potentials, making it difficult for existing screening methods to achieve graded capture and precise sorting of bacteria of different sizes, hindering the targeted discovery of functional strains. While microfluidic chips have been gradually applied in the field of microbial sorting, existing structures are mostly designed for single-celled bacteria and are difficult to adapt to the morphological characteristics and size range of filamentous fungal spores and hyphal segments, failing to balance screening efficiency with the preservation of bacterial physiological activity.

[0004] At the level of regulating mycelial pellet culture, existing studies mostly rely on shake-flask batch systems for condition optimization. This requires examining multiple influencing factors such as carbon source, nitrogen source, pH, surfactant, and trace elements one by one, resulting in high reagent consumption, poor parallelism, and low screening throughput. More importantly, the shake-flask system is a macroscopic mixed environment, which cannot precisely control the microenvironment of individual mycelial pellet growth and makes it difficult to continuously track the formation process of individual mycelial pellets, thus failing to elucidate the dynamic regulatory mechanisms of hyphal aggregation and pellet densification. Furthermore, traditional detection methods require offline analysis after sampling the mycelial pellets, which easily damages the three-dimensional structure and physiological state of the pellets, making it difficult to obtain in-situ, accurate growth and metabolic data.

[0005] Raman spectroscopy is characterized by its non-destructive, label-free nature and rich fingerprint information. It can simultaneously acquire multi-dimensional structural and compositional information of fungal cell wall skeletons, extracellular polymers, and intracellular metabolites, enabling in-situ dynamic detection at the single-cell and single-spore level, making it highly suitable for non-invasive monitoring of the growth and metabolism of filamentous fungi. However, current Raman detection is independent of fungal screening and culture processes, lacking an integrated microfluidic chip platform, making it impossible to achieve a seamless operation of "gradient screening and capture—in-situ induction of spore-forming cells—Raman dynamic monitoring." Furthermore, conventional detection substrates tend to generate background peaks in the fingerprint region, interfering with the weak characteristic signals of filamentous fungi and failing to meet the requirements for high-sensitivity detection, further limiting the in-depth application of Raman technology in the screening and spore regulation of lignin-degrading fungi.

[0006] In summary, existing technologies for the research and application of lignin-degrading microorganisms suffer from three core shortcomings: First, the lack of gradient screening methods adapted to the morphological characteristics of bacteria, filamentous fungal spores, and hyphal segments makes it difficult to achieve efficient hierarchical capture and targeted discovery of functional strains in environmental samples; second, the lack of in-situ induction culture and multi-factor parallel screening platforms at the single-strain level makes it impossible to accurately analyze the dynamic regulatory mechanisms of strain formation; and third, the lack of an integrated system combining screening, culture, and detection makes it difficult to achieve a seamless operation from resource discovery to process optimization of functional strains. These problems collectively restrict the efficiency of lignin-degrading microbial resource discovery and the accuracy of strain culture process optimization, necessitating the development of a microfluidic technology platform integrating gradient screening, in-situ induction, and Raman monitoring. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, the present invention aims to provide an integrated microfluidic chip system and method for gradient sieving of lignin-degrading microorganisms and in-situ induction and monitoring of fungal pellets. This system constructs an integrated technology system that adapts to the morphological characteristics of bacterial and filamentous fungal spores and hyphal segments, encompassing gradient sieving capture, in-situ induction of pellets, and Raman dynamic monitoring. This addresses the problems of mismatch between sieving methods and the morphology of lignin-degrading bacteria, insufficient precision in pellet control, and fragmented sieving, culture, and detection processes in existing technologies. Ultimately, it improves the discovery efficiency of lignin-degrading functional strains in the environment and the optimization precision of pellet culture processes.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A microfluidic chip system integrating lignin-degrading microbial gradient sieving and in-situ induction monitoring of fungal pellets is provided. The system consists of an intrinsic low-noise sensing layer, a gradient sieving and capture layer, a pellet morphology regulation layer, a chemical microenvironment construction layer, and a selectively breathable encapsulation layer, stacked sequentially from bottom to top. Adjacent layers are bonded together with biocompatible adhesive to form a vertically stacked integrated chip body.

[0009] The gradient sieving and capturing layer has three sets of through-hole arrays arranged in sections along the plane of the layer. The diameter of the through-holes in each set increases sequentially along the arrangement direction, forming a three-level gradient sieving structure. This structure can achieve graded retention and in-situ fixation of fungal spores and hyphal segments of different particle sizes through a single sample-driven operation. The sample-driven operation includes, but is not limited to, scraping, laminar flow pushing, and rolling.

[0010] The mycelial morphology control layer has a three-dimensional cavity array. The number and arrangement of the three-dimensional cavity array correspond one-to-one with the three sets of through-hole arrays in the gradient sieving and capture layer, providing a restricted three-dimensional growth space for the fungi captured by sieving and inducing the mycelium to spontaneously aggregate and form mycelial balls.

[0011] The chemical microenvironment construction layer has an array of through holes. The number and arrangement of the through holes correspond one-to-one with the three-dimensional cavity array of the bacterial pellet morphology regulation layer. By introducing culture media of different components into each well, multiple sets of parallel gradient chemical microenvironments can be constructed to achieve high-throughput parallel screening of bacterial pellet culture conditions.

[0012] The intrinsic low-noise sensing layer is the bottom substrate of the chip system. It has the characteristics of low background interference, high reflectivity and low absorption in the Raman fingerprint spectrum region, which can effectively reduce the interference of substrate noise peaks on fungal characteristic signals and optimize the Raman signal acquisition quality.

[0013] The selectively breathable encapsulation layer is the topmost cap of the chip system. It has a continuous breathable film structure and can maintain gas exchange within the closed system to meet the oxygen requirements for the growth and metabolism of aerobic filamentous fungi.

[0014] Furthermore, in the three sets of through-hole arrays of the gradient sieving capture layer, the pore size is 8~12μm, the medium pore size is 45~55μm, and the macropore size is 90~110μm. The three-level pore size matches the size distribution characteristics of bacterial cells, filamentous fungal spores, and hyphal segments of different lengths.

[0015] Furthermore, the three-dimensional cavity of the bacterial sphere morphology regulation layer has a cubic structure, with each cavity having a length, width, and height of 0.8~1.2mm, combining the limited space effect for bacterial sphere formation with sufficient culture medium capacity.

[0016] Furthermore, the aspect ratio of the through-pores in the chemical microenvironment construction layer is 1.5~1.8, which is suitable for the operational requirements of culture medium loading and reagent gradient setting.

[0017] Furthermore, the intrinsic low-noise sensing layer is a glass substrate surface coated with a thin metal film, wherein the metal is selected from any one of gold, silver, aluminum, chromium, and copper.

[0018] Furthermore, the selectively breathable encapsulation layer is made of PDMS with a thickness of 50-200 μm; the gradient sieving capture layer, the bacterial morphology regulation layer, and the chemical microenvironment construction layer are made of any one of PMMA, SU-8 epoxy resin, and PDMS. The overall size of the chip system is compatible with a conventional Raman microscope stage, allowing it to be directly placed under a Raman microscope for in-situ non-destructive testing.

[0019] This invention also provides a method for screening and regulating lignin-degrading fungi based on the above-mentioned microfluidic chip system, comprising the following steps: S1. Sample pretreatment: Microbial samples were collected from in-situ environments containing lignin-degrading bacteria, such as decaying wood and humus soil, and pre-cultured in liquid culture medium supplemented with lignin inducers. Metabolic labeling was carried out simultaneously to enable metabolically active microorganisms to carry detectable characteristic signals.

[0020] S2. Gradient pore size sieving and capture: The pre-cultured bacterial suspension is dropped onto one side of the gradient sieving and capture layer. A single continuous sample driving operation is performed along the gradient pore size direction using a sample driving component, so that the bacterial suspension is evenly spread on the surface of the layer. Relying on the size sieving effect of the three-level gradient pore size, bacterial cells, fungal spores and hyphal fragments of different sizes are trapped in the pores of the corresponding sizes, completing size classification and in-situ capture in one step.

[0021] S3. Raman spectroscopy identification of germplasm: The entire chip that has been screened and captured is placed on the stage of a Raman microscope, and Raman spectra of the microorganisms captured in each well are collected one by one; the metabolic activity of microorganisms is determined by the characteristic peaks of metabolic markers, and the target strains with lignin degradation function are identified by comparing and analyzing the fingerprint peaks related to lignin degradation with the standard database, the corresponding wells are marked and fresh culture medium is added.

[0022] S4. Three-dimensional induction culture of fungal balls: A fungal ball morphology regulation layer is bonded above the gradient sieving capture layer, fresh culture medium is introduced, and then a chemical microenvironment construction layer is bonded. Culture mediums with different components and concentrations are introduced into each corresponding well to construct a multi-dimensional gradient chemical induction environment covering surfactants, pH, substrate concentration, carbon source, nitrogen source, and trace elements. The captured fungal spores or hyphal segments grow upward into the cavity space under the supply of nutrients. In the confined space, they spontaneously aggregate by combining electrostatic, hydrophobic interactions and spore wall polysaccharide salt bridging mechanisms, and gradually form structurally complete hyphal balls.

[0023] S5. In-situ monitoring of bacterial pellets: During the cultivation process, Raman spectroscopy is used to conduct in-situ detection of bacterial pellets in each cavity at regular intervals to dynamically track the growth status and metabolic characteristics of bacterial pellets, comprehensively evaluate the bacterial pellet formation effect and degradation activity under different cultivation conditions, and screen to obtain the optimal bacterial pellet cultivation conditions.

[0024] Furthermore, the sample driving operation described in step S2 includes, but is not limited to, scraping, pushing, and rolling.

[0025] Furthermore, the in-situ Raman spectroscopy monitoring indicators in step S5 include three categories: Indicators of bacterial coccus cell wall skeletal structure, including dynamic changes in chitin, mannan, melanin, etc. Indicators of spatial distribution of extracellular polymeric substances (EPS), including proteins, nuclei, lipids, etc., as well as indicators of dynamic changes in intracellular metabolites, including carotenoids, lipid droplets, organic acids, etc.

[0026] Furthermore, the metabolic labeling in step S1 uses heavy water labeling, and the metabolic activity of microorganisms is characterized by detecting CD characteristic peaks.

[0027] Compared with the prior art, the beneficial effects of the present invention include: 1. The size screening, activity identification, induction culture and condition optimization of lignin-degrading microorganisms are integrated into a single chip platform, realizing the whole process of screening and application condition optimization of lignin-degrading microorganisms, which greatly shortens the research and development cycle.

[0028] 2. By employing a gradient aperture array in conjunction with single-sample driven operation, it achieves graded capture of bacterial cells, fungal spores and hyphal segments of different sizes in one step. The operation is simple, the sorting efficiency is high, and the in-situ physiological state of the strain can be preserved.

[0029] 3. By combining a low-noise Raman sensing substrate with Raman spectroscopy, multi-indicator, in-situ, and dynamic monitoring of single bacterial balls can be achieved without damaging the bacterial ball structure, enabling precise analysis of the structural and metabolic changes during bacterial ball formation.

[0030] 4. By constructing a chemical microenvironment layer, multiple sets of culture condition optimization experiments can be carried out in parallel, significantly improving the screening throughput of bacterial pellet culture conditions and reducing reagent consumption. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the layered structure of the microfluidic chip system described in this invention; Figure 2 This is a cross-sectional view of the microfluidic chip system described in this invention; Figure 3 This is a top view of the gradient sieving and capture layer of the microfluidic chip system described in this invention; Figure 4 This is a flowchart of the operation method of the microfluidic chip system described in this invention; Figure 5 This is a schematic diagram of the sample scraping driving operation of the microfluidic chip system described in this invention; Figure 6 This is a schematic diagram of the sample pushing driving operation of the microfluidic chip system described in this invention; Figure 7 This is a schematic diagram of the sample rolling driving operation of the microfluidic chip system described in this invention.

[0032] In the figure: 1-Intrinsic low-noise sensing layer, which includes 11-glass substrate and 12-metal coating; 2-Gradient sieving and capture layer, which includes 21-small pore capture area, 22-medium pore capture area, and 23-large pore capture area; 3-Bacterial ball morphology regulation layer; 4-Chemical microenvironment construction layer; 5-Selective breathable encapsulation layer; 6-Bacterial ball; 7-Roller; 8-Scraper; 9-Push rod. Detailed Implementation

[0033] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] Example 1: Specific Structure of the Microfluidic Chip System like Figure 1 As shown, this embodiment provides an integrated microfluidic chip system for gradient sieving and in-situ induction monitoring of lignin-degrading fungi. The system has a vertically stacked structure, consisting of an intrinsic low-noise sensing layer 1, a gradient sieving and capture layer 2, a fungal ball morphology regulation layer 3, a chemical microenvironment construction layer 4, and a selectively breathable encapsulation layer 5 from bottom to top. Adjacent layers are bonded together with biocompatible epoxy resin. The overall chip size is 75mm × 25mm, consistent with the standard slide size, and can be directly adapted to a conventional Raman microscope stage.

[0036] The intrinsic low-noise sensing layer 1 is the bottommost substrate of the chip, employing a glass substrate with a thin metal film deposited on its surface. The metal is chromium, and the film thickness is approximately 50 nm. This substrate has a density of 600~1800 cm⁻¹. -1 The Raman fingerprint region has extremely low background spectral interference, high reflectivity and low absorbance, which can effectively reduce the interference of substrate impurities on weak fungal characteristic signals and improve the detection signal-to-noise ratio.

[0037] The gradient sieving capture layer 2 is made of SU-8 epoxy resin with a thickness of 100 μm. The layer is divided into three rows of through-hole arrays along the horizontal direction, with each row containing 20 pores. The pores in the small pore row have a diameter of 10 μm, the medium pore row has a diameter of 50 μm, and the large pore row has a diameter of 100 μm. The three rows of pores are arranged in parallel with equal spacing, and the pore diameter increases sequentially along the sample driving direction to match the size distribution of filamentous fungal spores and hyphal segments of different lengths.

[0038] The morphology regulation layer 3 is made of PMMA with a thickness of 1 mm. The layer contains a cubic cavity array that corresponds one-to-one with the through-hole array in the lower layer. The length, width and height of each cavity are 1 mm, and the effective volume is about 1 μL. This provides a restricted three-dimensional growth space for fungi and induces the spontaneous aggregation of hyphae to form fungal balls.

[0039] The chemical microenvironment construction layer 4 is made of PMMA with a thickness of 500μm. Rectangular through-holes are set in the layer, corresponding one-to-one with the lower cavity array. Each hole is 1mm long and 0.6mm wide, with an aspect ratio of approximately 1.67, which is suitable for loading culture media and setting up multiple gradient reagents in parallel.

[0040] The selectively breathable encapsulation layer 5 is made of PDMS with a thickness of 100μm. It has a continuous and homogeneous film structure and has good oxygen permeability. It can maintain gas exchange in a closed system, meet the growth and metabolic needs of aerobic filamentous fungi, and block external contamination.

[0041] Example 2: Screening and regulation of lignin-degrading fungi and mycelial pellets Using the microfluidic chip system described in Example 1, lignin-degrading microorganisms in forest humus soil were screened and the culture conditions for mycelial pellets were optimized. The sample driving method employed a sterile scraping operation, and the specific operation steps are as follows: S1 Sample Pretreatment: Forest humus soil samples were collected and inoculated into a liquid culture medium supplemented with lignin. The samples were then pre-cultured at a constant temperature of 30℃ for 48 hours. During the culture period, heavy water was added for metabolic labeling, which enabled metabolically active microorganisms to synthesize cell components containing CD bonds and carry detectable characteristic signals.

[0042] S2 Gradient Pore Size Sieving and Capturing: Take a sieving substrate composed of an intrinsic low-noise sensing layer and a gradient sieving and capturing layer, and drop the pre-cultured bacterial suspension onto one end of the small pore row of the gradient sieving and capturing layer. Using a sterile scraper, starting from one side of the small pore row, scrape the suspension horizontally and uniformly to the other side of the large pore row in one pass, so that the bacterial suspension is evenly coated on the surface of the layer. Relying on the size sieving effect of the three-level gradient pore size, bacterial cells, fungal spores and hyphal fragments of different sizes are trapped in the pores of the corresponding sizes, completing size classification and in-situ capture in one step.

[0043] S3 Raman Spectroscopy Germplasm Identification: The entire chip, after screening and capture, was placed on the stage of a Raman microscope. Raman spectra were acquired for each well using a 532 nm excitation wavelength. The metabolic activity of the strains was determined by identifying CD characteristic peaks. Combined with lignin degradation-related fingerprint peaks, the results were compared with a standard database to screen for target fungi with lignin degradation activity. The corresponding wells were marked and scraped into fresh culture medium. The isolated bacteria were collected in independent functional areas for subsequent analysis, detection, strain preservation, or other experimental research, and were not used in the cultivation of target bacterial balls.

[0044] S4 Mycelial Ball Three-Dimensional Induction Culture: A mycelial ball morphology regulation layer is bonded above the gradient sieving and capture layer. Fresh culture medium is scraped into each cubic cavity, and then a chemical microenvironment construction layer is bonded. This layer introduces culture medium with different carbon and nitrogen source concentrations, pH values, and trace element ratios into each corresponding well to construct a multi-dimensional gradient chemical induction environment. Finally, a selectively permeable encapsulation layer is bonded, and the culture is statically incubated at a constant temperature of 30°C. Fungal spores and hyphal segments in the marked wells grow upward into the cavity space of the mycelial ball morphology regulation layer under nutrient supply. In the confined space, they spontaneously aggregate due to electrostatic, hydrophobic interactions, and spore wall polysaccharide-salt bridging mechanisms, gradually forming mycelial balls with a diameter of 500~800μm.

[0045] S5 gradient chemical regulation and in-situ monitoring: Every 12 hours, the chip was placed under a Raman microscope for in-situ detection to dynamically track the skeletal structure of the bacterial cocoon cell wall: information on chitin, β-glucan and mannan, and melanin. In-situ spatial distribution of extracellular polymeric substances (EPS) was also monitored: changes in proteins, extracellular polysaccharides, lipids, and eDNA. Dynamic changes in intracellular metabolites were also observed: including carotenoids and lipid droplets, purine and pyrimidine nucleic acid peaks, and organic acids. After 72 hours of continuous monitoring, the morphological integrity and degradation metabolic activity of the bacterial cocoons under different culture conditions were comprehensively evaluated to screen for the optimal culture conditions.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microfluidic chip system integrating lignin-degrading microbial gradient sieving and in-situ induction monitoring of fungal pellets, characterized in that, The following layers are stacked sequentially from bottom to top: an intrinsic low-noise sensing layer (1), a gradient sieving and capturing layer (2), a fungal ball morphology regulation layer (3), a chemical microenvironment construction layer (4), and a selectively breathable encapsulation layer (5). Adjacent layers are bonded together with biocompatible adhesive to form a vertically stacked integrated chip body. The gradient sieving and capturing layer (2) has three sets of through-hole arrays arranged along the plane of the layer body. The pore size of each set of through-holes increases sequentially along the arrangement direction to form a three-level gradient sieving structure. It can achieve graded interception and in-situ fixation of fungal spores and hyphal segments of different particle sizes through a single sample-driven operation. The fungal ball morphology regulation layer (3) has a three-dimensional cavity array. The number and arrangement of the three-dimensional cavity array are related to the gradient sieving and capturing layer. The three sets of through-hole arrays in layer (2) correspond one-to-one, providing a restricted three-dimensional growth space for the fungi captured by sieving, and inducing the hyphae to spontaneously aggregate to form hyphal balls (6); the chemical microenvironment construction layer (4) has through-hole arrays, and the number and arrangement of the through-hole arrays correspond one-to-one with the three-dimensional cavity array of the fungal ball morphology regulation layer (3). By introducing culture media of different components into each pore, multiple sets of parallel gradient chemical microenvironments are constructed; the intrinsic low-noise sensing layer (1) is the bottom substrate of the chip system, and has the characteristics of low background interference, high reflectivity and low absorptivity in the Raman fingerprint spectrum region; the selectively breathable encapsulation layer (5) is the top cap of the chip system, and has a continuous breathable film structure, which can maintain gas exchange in the closed system.

2. The integrated microfluidic chip system for lignin-degrading microbial gradient sieving and in-situ induction monitoring of fungal pellets according to claim 1, characterized in that, In the three sets of through-hole arrays of the gradient sieving capture layer (2), the pore size is 8~12μm, the medium pore size is 45~55μm, and the macropore size is 90~110μm. The three-level pore size matches the size distribution characteristics of bacterial cells, filamentous fungal spores and hyphal segments of different lengths.

3. The integrated microfluidic chip system for lignin-degrading microbial gradient sieving and in-situ induction monitoring of fungal pellets according to claim 1, characterized in that, The three-dimensional cavity of the bacterial morphology regulation layer (3) is a cubic structure, with each cavity having a length, width, and height of 0.8~1.2 mm.

4. The integrated microfluidic chip system for lignin-degrading microbial gradient sieving and in-situ induction monitoring of fungal pellets according to claim 1, characterized in that, The aspect ratio of the through holes in the chemical microenvironment construction layer (4) is 1.5~1.

8.

5. The integrated microfluidic chip system for lignin-degrading microbial gradient sieving and in-situ induction monitoring of fungal pellets according to claim 1, characterized in that, The intrinsic low-noise sensing layer (1) is a structure with a metal thin film (12) deposited on the surface of a glass substrate (11), wherein the metal is selected from any one of gold, silver, aluminum, chromium and copper.

6. The integrated microfluidic chip system for lignin-degrading microbial gradient sieving and in-situ induction monitoring of fungal pellets according to claim 1, characterized in that, The selectively breathable encapsulation layer (5) is made of PDMS and has a thickness of 50~200μm; the gradient sieving capture layer (2), the bacterial morphology regulation layer (3) and the chemical microenvironment construction layer (4) are made of any one of PMMA, SU-8 epoxy resin and PDMS.

7. The integrated microfluidic chip system for lignin-degrading microbial gradient sieving and in-situ induction monitoring of fungal pellets according to claim 1, characterized in that, The overall size of the chip system is compatible with the stage of a conventional Raman microscope.

8. A method for screening and regulating lignin-degrading fungi based on a microfluidic chip system according to any one of claims 1 to 7, characterized in that, Includes the following steps: S1. Sample pretreatment: Collect microbial samples containing lignin-degrading bacteria, inoculate them into liquid culture medium containing lignin inducers for pre-culture, and simultaneously carry out metabolic labeling to enable metabolically active microorganisms to carry detectable characteristic signals. S2. Gradient pore size sieving and capture: The pre-cultured bacterial suspension is dropped onto one side of the gradient sieving and capture layer (2). The sample driving component is used to perform a single continuous sample driving operation along the gradient pore size direction so that the bacterial suspension is evenly spread on the surface of the layer. Relying on the size sieving effect of the three-level gradient pore size, bacterial cells, fungal spores and hyphal fragments of different sizes are respectively trapped in the pores of the corresponding pore sizes, and size classification and in-situ capture are completed in one step. S3. Raman spectroscopy identification of germplasm: The entire chip that has been screened and captured is placed on the stage of a Raman microscope, and Raman spectra of the microorganisms captured in each well are collected one by one; the metabolic activity of microorganisms is determined by the characteristic peaks of metabolic markers, and the target strains with lignin degradation function are identified by comparing and analyzing the fingerprint peaks related to lignin degradation with the standard database, the corresponding wells are marked and fresh culture medium is added. S4. Three-dimensional induction culture of fungal balls: A fungal ball morphology regulation layer (3) is bonded above the gradient sieving capture layer (2), fresh culture medium is introduced, and then a chemical microenvironment construction layer (4) is bonded. Culture medium with different components and concentrations is introduced into each corresponding well to construct a multi-dimensional gradient chemical induction environment covering surfactants, pH, substrate concentration, carbon source, nitrogen source and trace elements. The captured fungal spores or hyphal segments grow upward into the cavity space under the nutrient supply and spontaneously aggregate in the confined space to gradually form hyphal balls (6). S5. In-situ monitoring of mycelial balls: During the cultivation process, Raman spectroscopy is used to conduct in-situ detection of mycelial balls (6) in each cavity at regular intervals, dynamically track the growth status and metabolic characteristics of mycelial balls, comprehensively evaluate the formation effect and degradation activity of mycelial balls under different cultivation conditions, and screen to obtain the optimal mycelial ball cultivation conditions.

9. The method according to claim 8, characterized in that, The sample driving operation described in step S2 includes any one of scraping, laminar flow pushing, and rolling.

10. The method according to claim 8, characterized in that, The in-situ Raman spectroscopy monitoring indicators mentioned in step S5 include: bacterial cell wall skeleton structure indicators, including chitin, mannan, and melanin; extracellular polymeric substances (EPS) spatial distribution indicators, including proteins, nucleic acids, and lipids; and intracellular metabolite dynamic change indicators, including carotenoids, lipid droplets, and organic acids.

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