LDHs-biological membrane solid-liquid interface culture method, LDHs-biological membrane complex and application

Abstract

The invention belongs to the crossing field of environmental functional materials and microbial technologies, and particularly relates to an LDHs-biological membrane solid-liquid interface culture method which comprises the following steps: S1, preparing an LDHs material; s2, constructing a standardized LDHs mineral membrane; s3, carrying out sterile treatment on the LDHs mineral membrane; and S4, carrying out solid-liquid interface culture on the LDHs-biological membrane. According to the method, the forming ability, metabolic activity, structural stability and pollutant removal characteristics of the LDHs material surface biological membrane can be effectively evaluated, and accurate and reliable evaluation is provided for deep exploration of a material-biological membrane interaction mechanism and promotion of application of the material-biological membrane interaction mechanism in environmental restoration.

Description

A method for solid-liquid interface culture of LDHs-biomembrane, an LDHs-biomembrane complex and its applications Technical Field

[0001] This invention belongs to the interdisciplinary field of environmental functional materials and microbial technology, specifically involving an LDHs-biofilm solid-liquid interface culture method, an LDHs-biofilm complex and its applications. Background Technology

[0002] LDHs, also known as hydrotalcite-like materials, are a class of artificial clay mineral materials with tunable structures, interlayer anion exchange properties, good biocompatibility, and high thermal stability. They have significant application value in environmental remediation, electrochemistry, photochemistry, and biomedicine. The preparation process of LDHs is relatively simple, requiring only basic co-precipitation of divalent and trivalent metal salts under controlled conditions. The resulting materials have controllable particle sizes (50-100 nm) and high specific surface areas (10-200 m²). 2 The LDHs interlayer anion exchange capacity reaches 2-5 mmol / g, and the exchange process does not damage the main layer structure. Currently, the LDHs-microorganism co-process has shown superior comprehensive efficiency compared to single biological or adsorption methods in the synergistic removal of hydrophilic inorganic anions (nitrates, phosphates, chromates, etc.) and hydrophobic organic pollutants (petroleum hydrocarbons, antibiotics, pesticides, etc.). However, existing applied research is mostly limited to macroscopic effect verification, and its underlying mechanism remains unclear.

[0003] Biofilms, as aggregates of extracellular polymeric substances (EPS) secreted by microorganisms on the substrate surface, have become key functional units for pollutant degradation due to their emergent properties such as nutrient retention, enhanced environmental resistance, and improved pollutant metabolism. Currently, the regulatory mechanisms of surface properties of natural clay minerals (such as montmorillonite, illite, and kaolinite) on biofilm formation and ecological functions have been systematically studied, and rich knowledge has been accumulated regarding the synergistic mediation of pollutant migration and transformation by minerals and biofilms. However, for artificial LDHs materials, by altering key artificially controllable properties (such as the metal composition of the laminations, the types of interlayer anions, the interlayer spacing, and surface hydrophilicity / hydrophobicity), a series of artificial clay mineral materials with different interfacial properties can be obtained (the number and types of which far exceed those of natural clay minerals). How these materials directionally regulate the microbial film-forming behavior, metabolic pathways, and community functions remains a significant challenge. Traditional evaluation methods based on mineral particle suspension systems have obvious limitations; the dynamic process of homogeneous mixing leads to distortion of the solid-liquid interface microenvironment, failing to simulate the stable adhesion state of biofilms on material surfaces. Moreover, microorganisms in suspension systems exist in a planktonic state or in a mixed form with a small amount of loose attachment, making it difficult to form a mature biofilm with a specific three-dimensional structure and a clear division of functions, which leads to inaccurate evaluation of biofilm community structure and function. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an LDHs-biofilm solid-liquid interface culture method, an LDHs-biofilm complex and its application.

[0005] This invention provides a method for cultivating LDHs-biofilm solid-liquid interface, comprising the following steps: S1, preparing LDHs materials; S2, constructing standardized LDHs mineral membranes: ultrasonically dispersing the LDHs materials prepared in step S1 to obtain an LDHs material suspension with a concentration of 1 g / L, and then quantitatively and uniformly loading the suspension onto an inert carrier to obtain LDHs mineral membranes; S3, sterilizing the LDHs mineral membranes; S4, cultivating LDHs-biofilm solid-liquid interface: selecting model biofilm strains and inoculating them onto the surface of the LDHs mineral membranes using a gradient inoculation method for directional cultivation until the biofilm matures and forms an LDHs-biofilm solid-liquid interface.

[0006] Furthermore, the preparation method of LDHs material is as follows: after mixing and dissolving divalent metal salt and trivalent metal salt evenly to obtain a mixed metal solution, the pH of the reaction system is adjusted, excess water in the reaction system is removed, and then an aging reaction is carried out; after the reaction is completed, the reaction system is washed and dried in sequence to obtain LDHs material.

[0007] Furthermore, the molar ratio between the divalent metal cations in the divalent metal salt and the trivalent metal cations in the trivalent metal salt is 1:1 to 1:3; and the total metal ion concentration in the mixed metal solution is preferably 0.5 to 2.0 mol / L.

[0008] Furthermore, the inert support is a glass slide or a polycarbonate film.

[0009] Furthermore, the model biofilm strain is Bacillus subtilis or Pseudomonas stutzeri.

[0010] The LDHs-biofilm complex obtained by the above culture method.

[0011] The above-mentioned LDHs-biofilm complexes are used in the degradation of hydrophilic and hydrophobic pollutants.

[0012] Furthermore, in both divalent and trivalent metal salts, the divalent metal cation can be Mn. 2+ Mg 2+ Co 2+ One of the trivalent metal cations is Fe. 3+ The anion is CO3. 2-or Cl - .

[0013] Furthermore, the hydrophilic pollutant is inorganic phosphorus, and the hydrophobic pollutant is BDE-47.

[0014] The beneficial effects of this invention are as follows: The LDHs-biofilm solid-liquid interface culture method proposed in this invention has a simple and easy preparation process for LDHs mineral membranes, and has significant advantages over mineral suspension systems: First, it has better biocompatibility, and its immobilized structure can effectively reduce the physical damage of LDHs particles to microorganisms; second, this method is conducive to the rapid colonization of microorganisms and biofilm accumulation, and can quickly obtain sufficient biofilm samples in a short period of time, fully meeting the analytical needs of multiple characterizations; third, it has higher simulation realism, and the membrane morphology is closer to the "coated state" application scenario of LDHs in actual environmental remediation, which can realistically simulate the interaction mechanism between LDHs coating and biofilm at the solid-liquid interface (such as static adsorption-biodegradation synergy), overcoming the problems of uneven particle dispersion and distortion of the action mechanism in suspension systems. Figure 1 shows the XRD images of the LDHs materials prepared in Examples 1-6 of this invention; Figure 2 shows the SEM images of the LDHs materials prepared in Examples 1-6 of this invention; wherein, (a) is MgFe-Cl-LDHs material; (b) is CoFe-Cl-LDHs material; (c) is MnFe-Cl-LDHs material; (d) is MgFe-CO3-LDHs material; (e) is CoFe-CO3-LDHs material; (f) is MnFe-CO3-LDHs material; Figure 3 shows the effect of the LDHs-biofilm composite system prepared in Examples 1-12 of this invention on Bacillus subtilis (GP-B) and Pseudomonas. The effect of stutzeri (GN-P) electron transfer activity (ETS); Figure 4 shows the dynamic phosphorus adsorption process of the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system prepared in this invention, and the LDHs mineral membrane-biofilm 72. Figure 5 shows the growth curve of the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system prepared in this invention during the dynamic phosphorus adsorption process of LDHs mineral membrane-biofilm biomass loading performance; where (a) is the biofilm mass change curve; (b) is the viable cell count change curve; (c) is the extracellular polymer (EPS) and its extracellular protein (PN) and extracellular polysaccharide (PS) component content change curve; (d) is the biofilm hydrophobicity index PN / PS ratio change curve; Figure 6 shows the phosphate distribution in the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system prepared in this invention; where (a) is the biofilm mass loading performance of the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system prepared in this invention. (a) Phosphate content in the biofilm of the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system obtained in this invention; (b) Phosphate content variation in LDHs of the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system obtained in this invention; (c) Phosphate content variation in the effluent of the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system obtained in this invention; (d) Contribution rate of the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system obtained in this invention to phosphate removal.

[0016] Figure 7 shows the dynamic adsorption of BDE-47 in the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system prepared by the invention, and the LDHs mineral membrane-biofilm 72 process. Figure 8 shows the growth curve of the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system prepared in this invention during the dynamic adsorption of BDE-47. (a) shows the biomass loading performance of the LDHs mineral membrane-biofilm during the dynamic adsorption of BDE-47. (b) shows the biofilm mass change; (c) shows the viable cell count change; (d) shows the content change of extracellular polymeric substances (EPS) and their extracellular proteins (PN) and extracellular polysaccharides (PS); (d) shows the change of the PN / PS ratio, a hydrophobicity index of the biofilm. Figure 9 shows the content distribution and removal trend of BDE-47 in the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system prepared in this invention. (a) shows the biofilm mass change; (b) shows the viable cell count change; (c) shows the content change of extracellular polymeric substances (EPS) and their extracellular proteins (PN) and extracellular polysaccharides (PS); (d) shows the content change of the PN / PS ratio, a hydrophobicity index of the biofilm. (a) The content of BDE-47 in the biofilm of the prepared MnFe-Cl-LDHs-GN-P composite system and CoFe-CO3-LDHs-GN-P composite system; (b) The change of BDE-47 content in LDHs of the prepared MnFe-Cl-LDHs-GN-P composite system and CoFe-CO3-LDHs-GN-P composite system; (c) The change of BDE-47 content in the effluent of the prepared MnFe-Cl-LDHs-GN-P composite system and CoFe-CO3-LDHs-GN-P composite system; (d) The contribution rate of the prepared MnFe-Cl-LDHs-GN-P composite system and CoFe-CO3-LDHs-GN-P composite system to the removal of BDE-47. Detailed Implementation

[0017] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0018] The core of this method lies in first processing LDHs into a mineral membrane coating through a standardized preparation process, then inoculating it with model microorganisms or targeted enrichment microbial communities for directed biofilm culture. Subsequently, key characteristic parameters of the biofilm are systematically measured and quantified for different hydrophilic and hydrophobic pollutants. This method can effectively evaluate the biofilm formation ability, metabolic activity, structural stability, and pollutant removal characteristics of LDH materials, providing accurate and reliable evaluation for in-depth research into the material-biofilm interaction mechanism and promoting its application in environmental remediation.

[0019] The primary objective of this invention is to protect a method for cultivating LDHs-biofilm solid-liquid interfaces, comprising the following steps: S1, preparing LDHs materials; specifically, in this invention, a series of LDHs materials with different physicochemical properties are prepared as the basis for subsequent biofilm cultivation and pollutant removal evaluation. The LDHs materials are prepared using an alkaline co-precipitation method to prepare LDHs composed of multiple metals. The selected metal ions should have the potential to serve as cofactors for microbial enzymes. By regulating interlayer anions, the interlayer spacing, surface charge, and hydrophobicity of LDHs are directionally adjusted. Specifically, the method includes the following steps: mixing and dissolving divalent and trivalent metal salts uniformly to obtain a mixed metal solution; adjusting the pH of the reaction system; removing excess water from the reaction system; and then carrying out an aging reaction; after the reaction is completed, the reaction system is washed and dried sequentially to obtain the LDHs materials; wherein, in the divalent and trivalent metal salts, the molar ratio between divalent and trivalent metal cations is 1:1 to 1:3, and the divalent metal cation can be Ca... 2+ Mg 2+ Zn 2+ Co 2+ One of the trivalent metal cations is Al. 3+ Fe 3+ One of them; the anion is CO3. 2- NO3 - Cl - One of them. In the mixed metal solution, the total metal ion concentration is preferably 0.5 ~ 2.0 mol / L; the specific operation for adjusting the pH of the reaction system is as follows: the pH of the reaction system is adjusted to 10±0.2~12±0.2 using an alkaline solution; if the anion is CO3... 2- At that time, the alkaline solution was adjusted to a mixed solution of 1 mol / L NaOH and 0.25 mol / L Na2CO3; the aging reaction was carried out at a temperature of 65~80℃ for 18 h.

[0020] After the LDHs materials were prepared, X-ray diffraction (XRD), specific surface area and porosity analyzer (BET), zeta potential and particle size analyzer were used to determine the interlayer spacing, specific surface area, pore size distribution and surface potential of the materials.

[0021] S2. Constructing standardized LDHs mineral membranes: The LDHs material prepared in step S1 is ultrasonically dispersed for 15 min to obtain an LDHs material suspension with a concentration of 1 g / L. The suspension is then quantitatively and uniformly loaded onto an inert carrier to obtain LDHs mineral membranes.

[0022] In this invention, the standardized LDHs mineral membrane serves as a carrier to simulate the water-solid interface between LDHs materials and biofilms, providing repeatability assurance for subsequent directed biofilm culture and contaminant removal tests. Preferably, the inert carrier is a circular glass slide with a diameter of 14 mm or a polycarbonate membrane measuring 25.4 mm × 76.2 mm. Preferably, before use, the inert carrier can be acid-washed (H2SO4:H2O2 = 7:3) and then pretreated with gelatin-potassium chromium sulfate (0.75 g gelatin and 0.075 g KCr(SO4)2 added to 100 mL distilled water, 56°C). During loading, the 100 μL suspension coating amount and 60°C drying conditions are precisely controlled.

[0023] S3. Aseptic treatment of LDHs mineral membrane; In this invention, the specific aseptic treatment steps of LDHs mineral membrane are as follows: sterilize LDHs mineral membrane with high-pressure steam at 121℃ for 15 min to ensure that LDHs mineral membrane is in a sterile state before specific operation.

[0024] S4. LDHs-Biofilm Solid-Liquid Interface Culture: Select model biofilm strains and inoculate them onto the surface of LDHs mineral membranes in microplates using a gradient inoculation method, directionally culturing until the biofilm matures and forms an LDHs-biofilm solid-liquid interface, thus obtaining the LDHs-biofilm matrix material. This means the biofilm can resist mild physical rinsing and remains stable at the interface.

[0025] In this invention, mature and stable biofilms are cultured on standardized LDHs membranes, and their formation process and key interfacial characteristics are monitored in real time. After the biofilm is loaded onto the surface of the LDHs mineral membrane, the viable cell count, biomass, and electron transport activity during the biofilm formation process are dynamically monitored to assess the initial colonization efficiency of microorganisms on the LDHs surface. Optimal, the directional culture period for the biofilm is 36 hours.

[0026] In this invention, as an embodiment, the model biofilm strain is Bacillus subtilis or Pseudomonas stutzeri. It should be noted that the Bacillus subtilis or Pseudomonas stutzeri strains of this invention are not newly screened strains obtained by the inventors. Bacillus subtilis or Pseudomonas stutzeri strains described in the prior art can also be used as technical effects of this invention.

[0027] For the LDHs-biofilm complex obtained by the above culture method, the surface hydrophobicity changes of the LDHs-biofilm complex were quantified using a contact angle meter. Combined with confocal microscopy (CLSM) and 16S rRNA high-throughput sequencing, the three-dimensional structure of the biofilm, the spatiotemporal distribution of hydrophilic / hydrophobic components in EPS, and the community succession pattern were analyzed. The above analysis results can verify the synergistic effect between LDHs materials and model biofilm strains, and further serve to explain the differences in subsequent pollutant removal.

[0028] The second thing this invention aims to protect is the LDHs-biofilm complex obtained by the above-mentioned culture method.

[0029] The third aspect of this invention is the application of the LDHs-biofilm complex in the degradation of hydrophilic and hydrophobic pollutants. Specifically, the LDHs-biofilm complex is loaded onto a substrate in the form of a coating. Depending on the degradation requirements, it is chosen whether to load a target functional strain onto the substrate to improve degradation efficiency. If a target functional strain is selected, and the target functional strain is a Gram-positive strain, the model biofilm strain is Bacillus subtilis. If the target functional strain is a Gram-negative strain, the model biofilm strain is Pseudomonas asstutzeri. It should be noted that the Bacillus subtilis and Pseudomonas stutzeri used in this invention are not newly screened strains obtained by the inventors. Bacillus subtilis and Pseudomonas stutzeri and their related species disclosed in the prior art can all be used as specific implementation strains of Bacillus subtilis and Pseudomonas stutzeri in this invention.

[0030] In this invention, the molar ratio of divalent metal cations to trivalent metal cations in divalent and trivalent metal salts is 1:1 to 1:3, and the divalent metal cation can be Ca. 2+ Mg 2+ Zn 2+ Co 2+ One of the trivalent metal cations is Al. 3+ Fe 3+ One of them, the anion is CO3. 2- NO3 - Cl -One of them. The selection of target functional strains depends on the type of pollutants in the specific application scenario, and strains with corresponding degradation functions are selected. Their source can be in-situ indigenous microbial communities or commercially available functional strains, or single strains or communities. This invention only provides guidance on the type of model biofilm strain based on the Gram-negative / positive characteristics of the target functional strain.

[0031] Furthermore, as an embodiment of the present invention, the matrix carrier can be quartz sand.

[0032] In the above applications, the hydrophilic pollutant is phosphorus, and the hydrophobic pollutant is BDE-47.

[0033] <Example 1> A method for cultivating LDHs-biofilm solid-liquid interfaces includes the following steps: S1, Mixing 0.2 mol / L MgCl solution and 0.1 mol / L FeCl3 solution at a 1:1 volume ratio and heating to 80°C; adjusting the pH to 10±0.2 by adding 1 mol / L NaOH solution dropwise; stirring continuously for about 3 h until almost all the water evaporates, then aging the resulting solid-liquid mixture in an 80°C oven for 18 h; subsequently adjusting the pH of the mixed solution to neutral with deionized water, then drying in a 70°C oven, and grinding through a 200-mesh sieve to obtain MgFe-Cl-LDHs; S2, Washing a glass slide (25.4 mm × 76.2 mm) with acid (H2SO4:H2O2=7:3) and then drying; Pre-treating with gelatin-potassium chromium sulfate (0.75 g gelatin and 0.075 g potassium chromium sulfate in 100 mL distilled water). KCr(SO4)2 (56℃) was prepared for later use; MgFe-Cl-LDHs material was ultrasonically dispersed for 15 min to obtain a MgFe-Cl-LDHs material suspension with a concentration of 1 g / L. 100 mL of the 1 g / L MgFe-Cl-LDHs suspension was then coated onto an inert carrier and dried at 60℃ to obtain a MgFe-Cl-LDHs mineral membrane; S3, Construction of LDHs-biofilm culture system: Bacillus subtilis (GP-B) was selected. In microplates, the model biofilm strain was inoculated onto the surface of the MgFe-Cl-LDHs mineral membrane and cultured directionally until the biofilm matured to obtain the MgFe-Cl-LDHs-GP-B composite system.

[0034] The construction methods of the LDHs-biofilm composite system in Examples 2-6 are the same as those in Example 1, except that the conditions in the table below are different.

[0035]

[0036] In Examples 4-6, the alkaline solution was adjusted to a mixed solution of 1 mol / L NaOH and 0.25 mol / L Na2CO3, the aging temperature was 80°C, the drying temperature was 70°C, and all other reaction conditions were the same as in Example 1.

[0037] The viable cell count was determined using the MPN method; the number of planktonic cells was characterized using OD600; the biofilm was quantified using crystal violet staining; the surface zeta potential and particle size distribution were determined using a Malvern Zetasizer Nano ZS900; the contact angle of the biofilm samples at 36 h was determined using a Dataphysics OCA 20 contact angle meter; and the metabolic activity of the biofilm was characterized by measuring electron transport activity (ETS).

[0038] The properties of the six LDHs materials obtained in Examples 1-6 were tested. Experimental results showed that, based on the XRD and SEM characterization results of Examples 1-6, the synthesis of the six layered LDHs structures was confirmed (Figures 1 and 2). Further comparison of the surface properties of the six iron-based LDHs materials revealed that different combinations of metal ions and anions significantly affected the surface potential and particle size distribution of the materials (Table 1). Among them, CoFe-Cl-LDHs exhibited the highest positive potential (19.7 mV), followed by MgFe-CO3-LDHs (15.3 mV) and CoFe-CO3-LDHs (13.2 mV), while the potentials of the other three LDHs were similar (approximately 10 mV). Particle size analysis showed that CoFe-Cl-LDHs had the smallest nanoscale particle size (196 nm), while MnFe-Cl-LDHs and MgFe-Cl-LDHs exhibited micrometer-scale particle sizes, with MnFe-CO3-LDHs having the largest particle size (6-12 times that of CoFe-LDHs). These results indicate that Co modification can significantly reduce the particle size of LDHs and increase the surface positive potential, while Mn modification leads to a significant increase in particle size.

[0039] Table 1. Surface and pore structure characteristics of particles from six types of LDHs materials

[0040] <Example 7> The difference between this example and Example 1 is that the model biofilm strain used for loading is Pseudomonas stutzeri (denoted as GN-P), resulting in a MgFe-Cl-LDHs-GN-P composite system.

[0041] <Example 8> The difference between this example and Example 2 is that the model biofilm strain used for loading is Pseudomonas stutzeri (denoted as GN-P), resulting in a MnFe-Cl-LDHs-GN-P composite system.

[0042] <Example 9> The difference between this example and Example 3 is that the model biofilm strain used for loading is Pseudomonas stutzeri (denoted as GN-P), resulting in a CoFe-Cl-LDHs-GN-P composite system.

[0043] <Example 10> The difference between this example and Example 4 is that the model biofilm strain used for loading is Pseudomonas stutzeri (denoted as GN-P), resulting in a MgFe-CO3-LDHs-GN-P composite system.

[0044] <Example 11> The difference between this example and Example 5 is that the model biofilm strain used for loading is Pseudomonas stutzeri (denoted as GN-P), resulting in a MnFe-CO3-LDHs-GN-P composite system.

[0045] <Example 12> The difference between this example and Example 6 is that the model biofilm strain used for loading is Pseudomonas stutzeri (denoted as GN-P), resulting in a CoFe-CO3-LDHs-GN-P composite system.

[0046] To test the hydrophobic properties of different materials, the contact angles of each material in Table 2 were measured, and the results are shown in Table 2.

[0047] Table 2 Contact Angle Testing of Different Materials

[0048] As shown in Table 2, the chloride-based iron-based LDHs materials are more hydrophilic than the carbonate-based materials. The introduction of iron-based LDHs materials affects the hydrophilicity of both bacterial biofilms. Among them, the three materials with chloride ion intercalation have the most significant effect on improving the hydrophilicity of the biofilm.

[0049] The performance of the six composite systems obtained in Examples 1-6 was tested. The model biofilm strains were cultured in a mineral membrane system and kept in a constant temperature of 28℃ in the dark. A dynamic monitoring system with seven time gradients (8, 12, 24, 36, 48, 60, 72 h) was set up, with the GP-B strain as the control group. The results are shown in 3a. The performance of the six composite systems obtained in Examples 7-12 was tested. The model biofilm strains were cultured in a mineral membrane system and kept in a constant temperature of 28℃ in the dark. A dynamic monitoring system with seven time gradients (8, 12, 24, 36, 48, 60, 72 h) was set up, with the GN-P strain as the control group. The results are shown in 3b.

[0050] The results showed that the presence of LDHs significantly prolonged the growth cycle of both model biofilm strains, which is beneficial for cultivating more mature biofilms in practice. Compared with iron-based LDHs with different metal ion combinations, different anion intercalation had a smaller impact on the biomass and bioactivity of the two model biofilm strains in the mineral membrane system. Chloride-based iron-based LDHs were more hydrophilic than carbonate-based LDHs, and the introduction of iron-based LDHs affected the hydrophilicity of both bacterial biofilms. Among them, the three materials with chloride ion intercalation showed the most significant improvement in biofilm hydrophilicity.

[0051] Phosphorus adsorption experiment for removing hydrophilic pollutants: The biomass change during loading of strain GN-P on MnFe-Cl-LDHs and CoFe-CO3-LDHs mineral membranes was tested. First, GN-P biofilms were incubated on MnFe-Cl-LDHs and CoFe-CO3-LDHs mineral membranes using a trickle-flow biofilm reactor (DFR), with the reactor and piping sterilized. The reactor was placed horizontally, and sterilized MgFe-Cl-LDHs or CoFe-CO3-LDHs mineral membranes were placed in each channel. 1 mL of logarithmic-phase GN-P bacterial culture and 15 mL of LB medium were added to each membrane, and the mixture was incubated at 28°C for 12 h. After incubation, the sterilized piping system was connected (10° tilt), and a peristaltic pump was used to control the flow rate at 40 mL / h. Fresh, artificially prepared phosphorus-containing mineral salt medium (containing (NH4)2SO4, 2.0 mg / L) was replaced every 24 h. The following were used in a dynamic continuous phosphorus adsorption experiment: CaCl2·12H2O, 0.01 g; FeSO4·7H2O, 0.001 g; MgSO4·7H2O, 0.2 g; glucose, 1.8 g; yeast extract, 1 g; pH 7.2~7.4; initial phosphorus concentration 6.5 mg / L).

[0052] One hour before sampling, a 50 mL centrifuge tube was attached to the effluent outlet, and the effluent sample was refrigerated for determination of total phosphorus and phosphate content. In a clean bench, slides were removed at 0, 8, 24, 32, 48, 56, 68, and 72 hours, dried in sterile petri dishes, and weighed. The biofilm was then scraped onto a 50 mL centrifuge tube containing 5 mL of sterile water using a sterile spatula, and the slide was rinsed with 5 mL of sterile water. The slide containing LDHs was then transferred to another 50 mL centrifuge tube containing 5 mL of sterile water, and the LDHs were scraped off using a spatula. Biofilm and LDH samples were obtained separately for the determination of biofilm indicators and phosphorus content. Phosphate content was determined using the national standard molybdenum-antimony spectrophotometric method.

[0053] Figure 4 shows the growth status of GN-P biofilms on the surfaces of the two mineral membranes (MnFe-Cl-LDHs and CoFe-CO3-LDHs) during phosphorus adsorption in the DFR reactor over 72 h (Figure 4a shows the process of GN-P biofilm loading on the surface of MnFe-Cl-LDHs, and Figure 4b shows the process of GN-P biofilm loading on the surface of CoFe-CO3-LDHs). During the continuous culture phase, key indicators of the biofilm were systematically characterized: the total biomass of the biofilm was quantified by gravimetric method, the number of culturable viable bacteria was assessed by MPN method, and the core components of extracellular polymeric substances (EPS), namely protein-PN and polysaccharide-PS, were quantitatively analyzed using commercially available kits. The results are shown in Figure 5. As shown in the figure, after 12 h of static incubation, the initial biofilm mass and subsequent growth of the MnFe-Cl-LDHs mineral membrane were significantly higher than those of the CoFe-CO3-LDHs group. Notably, although the initial viable bacterial count on the CoFe-CO3-LDHs surface was higher, the viable bacterial count in the MnFe-Cl-LDHs group increased rapidly within 8 h and exceeded that of the CoFe-CO3-LDHs group by 1–3 orders of magnitude after 32 h, consistent with the increasing trend in biofilm mass. Therefore, in the dynamic mineral membrane-biofilm phosphorus removal system, MnFe-Cl-LDHs significantly promoted biofilm growth, while CoFe-CO3-LDHs had a weaker promoting effect on biofilm accumulation.

[0054] The performance of the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system in removing hydrophilic pollutant phosphorus was tested. The contribution of each component in the LDHs-biofilm composite system to phosphorus removal was determined by measuring the solid biofilm components, LDHs components, and phosphate content in the effluent. The results are shown in Figure 6. The adsorption capacity of the LDHs mineral membrane itself is limited and not significantly different. The maximum adsorption capacities of CoFe-CO3-LDHs and MnFe-Cl-LDHs for phosphate were 4.8 mg / g and 2.7 mg / g, respectively, with low overall adsorption capacity, indicating that the mineral membrane itself has a limited contribution to phosphorus adsorption. The two composite systems showed opposite trends in biofilm phosphorus removal contribution: 1) In the CoFe-CO3-LDHs-GN-P system, the biofilm's contribution to phosphorus adsorption decreased continuously over time, from an initial 84% to 0% at 68 h, indicating that this mineral membrane is not conducive to the long-term maintenance of phosphorus adsorption capacity by the biofilm. After reaching its maximum adsorption capacity at 8 h, the system exhibited phosphate desorption, with some phosphorus being flushed into the effluent. 2) In the MnFe-Cl-LDHs-GN-P system, the biofilm's contribution to phosphorus adsorption showed a trend of first decreasing and then increasing, with the contribution rate significantly increasing from 34% to 96% during the period of 32 h–68 h. At 68 h, the phosphate content in the biofilm reached approximately 25 μg, nearly ten times that of the CoFe group at the same time, demonstrating excellent biofilm synergistic phosphorus removal capacity. These results indicate that the biofilm growth stage and activity are key factors affecting phosphorus removal efficiency. The CoFe group's biofilm entered the maturity stage earlier, with EPS reaching a high concentration at 24 h, thus exhibiting better phosphorus adsorption capacity in the early stage (0 h–32 h). Although the total biofilm growth in the MnFe group was slower, the number of viable bacteria and EPS concentration were higher during the period of 68 h–72 h, resulting in stronger phosphorus adsorption activity and giving it a significant advantage in the later stage. It is noteworthy that the surface hydrophilicity characterization (Table 3) is highly consistent with this: the surface hydrophilicity of the MnFe-Cl-LDHs mineral membrane (contact angle 23.6°) is significantly stronger than that of CoFe-CO3-LDHs (contact angle 56.2°). This stronger hydrophilic surface may be more conducive to the transport of hydrophilic phosphate pollutants and subsequent biofilm growth, thus creating a favorable microenvironment for the biofilm to exert efficient and continuous phosphorus removal in the later stages. In summary, although the CoFe mineral membrane itself has a slightly higher adsorption capacity, it is not conducive to the long-term adsorption of phosphorus by the biofilm; the MnFe mineral membrane promotes efficient and continuous phosphorus removal by the biofilm after adaptation, making the overall system more advantageous in long-term operation. Therefore, MnFe-Cl-LDHs-GN-P is superior to CoFe-CO3-LDHs-GN-P.

[0055] Adsorption test of BDE-47 for removing hydrophobic pollutants: The biomass change during the loading of strain GN-P on MnFe-Cl-LDHs and CoFe-CO3-LDHs mineral membranes was tested. First, the biofilm of strain GN-P was incubated on MnFe-Cl-LDHs and CoFe-CO3-LDHs mineral membranes respectively. A trickle flow biofilm reactor (DFR) was selected, and the reactor and pipeline were sterilized as a whole. The reactor was placed horizontally. Before placing the MnFe-Cl-LDHs and CoFe-CO3-LDHs mineral membranes into the reactor, 3 μL of 500 mg / L BDE-47 stock solution (dissolved in n-hexane) was added dropwise to the MnFe-Cl-LDHs and CoFe-CO3-LDHs mineral membranes in a clean bench using a 10 μL micro-syringe. After the organic solvent evaporated, the bacterial solution and culture medium were added. Add 1 mL of logarithmic GN-P bacterial culture and 15 mL of LB medium to each tablet, and incubate at 28°C for 12 h. After incubation, connect the sterile tubing system (10° tilt), and use a peristaltic pump to control the flow rate at 40 mL / h. Replace the medium with fresh, artificially prepared mineral salt medium (containing (NH4)2SO4, 2.0 g; CaCl2·12H2O, 0.01 g; FeSO4·7H2O, 0.001 g; MgSO4·7H2O, 0.2 g; glucose, 1.8 g; yeast extract, 1 g; trace elements: MnCl2·4H2O, 0.005 g; ZnCl2, 0.0005 g; H3BO3, 0.0005 g; NiCl2·6H2O, 0.0005 g; CoCl2·6H2O, 0.0005 g; CuCl2, 0.0003 g). g; Na2MoO4·2H2O, 0.0001 g; selenium / tungstate: NaOH, 0.005 g; Na2WO4·2H2O, 0.00004 g; Na2O4Se·5H2O, 0.00003 g, pH 7.2~7.4), dynamic continuous BDE-47 adsorption experiments were conducted. The effluent was filtered through an SPE column (SBEQ-CA1555, CNW) and then discharged into a waste liquid tank.

[0056] Samples were taken at predetermined time points in a clean bench at 0, 6, 12, 24, 48, 60, and 72 h of continuous culture. After drying and weighing the slides, the biofilm was scraped into PTFE centrifuge tubes containing 10 mL of sterile water. The scraped slides were then dried and stored in 50 mL centrifuge tubes. Simultaneously, the SPE column was replaced and eluted to collect BDE-47 from the aqueous phase. Finally, all samples from the biofilm, LDHs mineral membrane, and aqueous phase were pretreated, and the BDE-47 content was determined by gas chromatography-ECD.

[0057] Figure 7 shows the growth status of GN-P biofilms on the surfaces of two mineral membranes (MnFe-Cl-LDHs and CoFe-CO3-LDHs) during the removal process in the BDE-47 DFR reactor over 60 h (Figure 6a shows the process of loading GN-P biofilms onto the surface of MnFe-Cl-LDHs, and Figure 6b shows the process of loading GN-P biofilms onto the surface of CoFe-CO3-LDHs). Figure 8 shows the changes in key biofilm indicators during the continuous culture phase. The results indicate that, under the BDE-47 microdroplet environment, the growth kinetics and EPS secretion patterns of the biofilms on the surfaces of the two LDHs differed significantly: the CoFe group showed rapid biofilm formation, with the viable cell count being an order of magnitude higher than the MnFe group even at 0 h; the biofilm mass and viable cell count entered the logarithmic growth phase from 12 to 24 h, reaching a peak at 48 h before entering the decline phase; while the MnFe group showed slow biofilm growth, with the biofilm mass and viable cell count increasing slowly from 0 to 24 h, only significantly increasing at 60 h, and no decline phase was observed, indicating a longer growth cycle. Correspondingly, the EPS concentration in the CoFe group was higher in the early stage of cultivation (0-12 h), while the EPS in the MnFe group only accumulated significantly in the later stage of cultivation (24-60 h) and far exceeded that of the CoFe group. In the BDE-47 environment, the CoFe group, which showed faster biofilm growth, consistently had lower biofilm quality and viable cell count than the MnFe group during phosphorus removal. This indicates that the physicochemical properties of pollutants (such as hydrophobicity) are the key factors determining which mineral membrane is more conducive to biofilm growth.

[0058] The performance of the MnFe-Cl-LDHs-GN-P composite system and the CoFe-CO3-LDHs-GN-P composite system in removing the hydrophobic pollutant BDE-47 was tested, as shown in Figure 9. The fate and main removal mechanism of BDE47 in the continuous culture system were determined by measuring the BDE47 content in the LDHs mineral membrane (corresponding to the matrix in the figure), biofilm (corresponding to the biofilm in the figure), and effluent (corresponding to the effluent in the figure). During the static stage (0 h): the BDE-47 removal value (0.466 μg) in the CoFe group due to biofilm action was higher than that in the MnFe group (0.121 μg), indicating that the biofilm in the CoFe group had a better removal effect on BDE47 in the initial attachment stage. During the later stage of continuous culture (24-60 h): the residual amount of BDE47 on the slides in the CoFe group was higher than that in the MnFe group, which is consistent with the characterization result that the CoFe-CO3-LDHs surface is more hydrophobic, proving that its physical adsorption of BDE47 is stronger than that of MnFe-Cl-LDHs. The intracellular BDE47 content in the biofilm exhibited a time-specific peak, reaching its highest levels at 48 h in the MnFe group and 12 h in the CoFe group. These time points coincided with previously observed EPS secretion peaks, indicating that higher EPS concentrations resulted in better BDE47 adsorption by the biofilm. Notably, during the continuous culture phase, water flow impact was the primary cause of BDE-47 attenuation on the LDHs membrane, with 50%-79% of BDE-47 translocating to the effluent within 12-60 h. The biofilm's contribution to BDE-47 removal was relatively limited, with the MnFe group showing the highest contribution (34%) at 48 h, while the CoFe group's contribution was only 11% throughout the entire process.

[0059] The specific raw materials listed in this invention, as well as the upper and lower limits and ranges of values ​​for each raw material and process parameter, can all achieve this invention. Examples are not listed individually here. Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present 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 method for cultivating LDHs-biomembrane solid-liquid interface, characterized in that, Includes the following steps: S1. Preparation of LDHs materials; S2. Construction of standardized LDHs mineral membranes: The LDHs materials prepared in step S1 are ultrasonically dispersed to obtain an LDHs material suspension with a concentration of 1 g / L. The suspension is then quantitatively and uniformly loaded onto an inert carrier to obtain LDHs mineral membranes; S3. The LDHs mineral membranes are sterilized; S4. LDHs-biofilm solid-liquid interface culture: A model biofilm strain is selected and inoculated onto the surface of the LDHs mineral membrane using a gradient inoculation method for directional culture until the biofilm matures and forms an LDHs-biofilm solid-liquid interface.

2. The method for LDHs-biofilm solid-liquid interface culture according to claim 1, characterized in that, In S1, the preparation method of LDHs material is as follows: after mixing and dissolving divalent metal salt and trivalent metal salt evenly to obtain a mixed metal solution, the pH of the reaction system is adjusted, excess water in the reaction system is removed, and then an aging reaction is carried out. After the reaction is complete, the reaction system is washed and dried sequentially to obtain LDHs materials.

3. The method for LDHs-biomembrane solid-liquid interface culture according to claim 2, characterized in that, The molar ratio between the divalent metal cations in the divalent metal salt and the trivalent metal cations in the trivalent metal salt is 1:1 to 1:3; and the total metal ion concentration in the mixed metal solution is preferably 0.5 to 2.0 mol / L.

4. The method for LDHs-biofilm solid-liquid interface culture according to claim 2, characterized in that, The inert support is a glass slide or a polycarbonate film.

5. The method for LDHs-biofilm solid-liquid interface culture according to claim 2, characterized in that, The model biofilm strains are Bacillus subtilis or Pseudomonasstutzeri.

6. The LDHs-biofilm complex obtained by the culture method according to any one of claims 1-5.

7. The application of the LDHs-biofilm complex as described in claim 6 in the degradation of hydrophilic and hydrophobic pollutants.

8. The application according to claim 7, characterized in that, In divalent and trivalent metal salts, the divalent metal cation can be Mn. 2+ Mg 2+ Co 2+ One of the trivalent metal cations is Fe. 3+ The anion is CO3. 2- or Cl - .

9. The application according to claim 8, characterized in that, The hydrophilic pollutant is inorganic phosphorus, and the hydrophobic pollutant is BDE-47.

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