Metal-organic framework encapsulated laccase-bacterial consortium and its application in bioremediation of tetracycline-dehp combined pollutants

Abstract

This invention belongs to the field of environmental pollution remediation technology, specifically involving laccase-microbial communities encapsulated in metal-organic frameworks (MOFs) and their application in the bioremediation of tetracycline-DEHP complex pollutants. This invention uses *Rhodococcus australis* sp., *Pseudomonas* sp., and *Cupriavidus* sp. to construct a ternary microbial community. Then, using ZIF-8 biomimetic mineralization encapsulation technology, laccase and the microbial community are co-loaded into the MOF shell. Finally, BEPZ composite catalytic material is prepared through biochar immobilization. The three different strains synergistically interact to form a metabolic network for complete DEHP mineralization. The ZIF-8 shell effectively resists damage to microorganisms and laccase from external stresses, exhibiting highly efficient synergistic degradation capabilities, strong stress resistance, and cycling stability in the tetracycline-DEHP complex pollution system.

Description

Technical Field

[0001] This invention belongs to the field of environmental pollution remediation technology, specifically relating to metal-organic framework-encapsulated laccase-microbial community and its application in the bioremediation of tetracycline-DEHP complex pollutants. Background Technology

[0002] Phthalate esters (PAEs), as dialkyl / alkoxy-substituted derivatives of phthalic acid, are widely used in the plasticizing processes of plastics, polyvinyl chloride (PVC), and related products. Their environmental distribution exhibits significant media specificity: water bodies and agricultural soils constitute the main pollutant carriers. Notably, PAEs in these two media can enter the human food chain through multiple pathways, including edible crops, poultry, and livestock. The continued expansion of antibiotic consumption directly constitutes the core driving force of environmental antibiotic pollution. Drug abuse not only disrupts the balance of ecosystems but also poses a potential threat to human health due to its persistent / pseudo-persistent characteristics.

[0003] Phthalate esters (PAEs) and antibiotics widely coexist in the environment, forming complex pollution that threatens ecosystems and human health. Traditional bioremediation technologies are limited by low single-strain degradation efficiency and poor environmental adaptability, and antibiotic stress significantly inhibits microbial activity, resulting in poor remediation effects. Furthermore, the coexistence of antibiotics may weaken the degradation efficiency of microorganisms for organic pollutants through multiple mechanisms. Laccase, as a typical extracellular polycopper oxidoreductase, can achieve targeted degradation of various structurally complex antibiotics through an oxygen-mediated free radical chain reaction mechanism. However, free laccase is limited in practical applications by low stability, easy inactivation, and non-reusability, severely restricting its engineering application. In summary, there are few reports on bioremediation and synergistic degradation mechanisms for PAE-antibiotic complex pollution, highlighting the urgent need to develop efficient, stable, and safe bioremediation systems. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a metal-organic framework (MOF) encapsulated laccase-microbial community and its application in the bioremediation of tetracycline-DEHP complex pollutants. This invention uses *Rhodococcus australis* sp., *Pseudomonas* sp., and *Cupriavidus* sp. to construct a ternary microbial community. Then, using ZIF-8 biomimetic mineralization encapsulation technology, laccase and the microbial community are co-loaded into the MOF shell. Finally, BEPZ composite catalytic material is prepared through biochar immobilization. The three different strains synergistically interact to form a metabolic network for complete DEHP mineralization. The ZIF-8 shell effectively resists damage to microorganisms and laccase from external stresses, exhibiting highly efficient synergistic degradation capabilities, strong stress resistance, and cycling stability in the tetracycline-DEHP complex pollution system.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a method for preparing a metal-organic framework-encapsulated laccase-microbial community immobilized composite catalytic material, comprising the following steps: (1) Inoculate the bacterial suspension containing Rhodococcus sp., Pseudomonas sp. and Cupriavidus sp. into LB medium and incubate. Wash with PBS during the exponential phase, then suspend in PBS containing laccase, let stand to complete the coating, wash with deionized water and then suspend in 2-methylimidazole aqueous solution. (2) Add the zinc source to the 2-methylimidazole aqueous solution in step (1), shake and let stand, wash with deionized water and then resuspend in deionized water; then add it to biochar and shake to adsorb, to obtain the metal-organic framework encapsulated laccase-microbe immobilized composite catalytic material.

[0006] Further, in step (1), the ratio of Rhodococcus sp., Pseudomonas sp. and Cupriavidus sp. is (1-3):(1-3):(1-3).

[0007] Furthermore, in step (1), the ratio of Rhodococcus sp., Pseudomonas sp., and Cupriavidus sp. is 2:2:1.

[0008] Further, in step (2), the zinc source is an aqueous solution of zinc acetate.

[0009] Furthermore, in step (2), the settling time is 1 hour.

[0010] Further, in step (2), the method for preparing the biochar includes the following steps: After washing, the corn stalks are crushed into powder, calcined in an inert gas atmosphere, washed with pure water, ground into powder, and dried for later use.

[0011] A second aspect of the present invention provides a metal-organic framework-encapsulated laccase-microbial community immobilized composite catalytic material prepared by the above-described preparation method.

[0012] A third aspect of the present invention provides the application of the above-mentioned metal-organic framework-encapsulated laccase-microbial community immobilized composite catalytic material in the remediation of tetracycline-DEHP composite pollutants.

[0013] Compared with the prior art, the beneficial effects of the present invention are: (1) In this invention, Rhodococcus sp., Pseudomonas sp. and Cupriavidus sp. were selected to construct a ternary bacterial community, which breaks through the limitations of traditional single strains. Based on metabolic complementarity and potential interaction, the bacterial community showed good performance in cell growth and pollutant degradation after optimization, demonstrating the potential to completely mineralize pollutants.

[0014] (2) This invention encapsulates laccase and degrading bacteria using ZIF-8 as a scaffold, enhancing the bacteria’s tolerance to adverse environments, improving their degradation and adaptation capabilities in complex polluted environments, reducing ecological risks, and providing new ideas and methods for bacterial community construction and the application of microorganisms in complex environments.

[0015] (3) The metal-organic framework encapsulated laccase-bacterial community of the present invention is immobilized by biochar adsorption treatment to prepare an immobilized composite catalytic material that can efficiently and stably repair DEHP-tetracycline complex pollution, solve the difficulties of cell recycling and reuse, and provide a new strategy for the remediation of PAEs-antibiotic complex pollution in the actual environment. Attached Figure Description

[0016] Figure 1 This shows the growth status of each strain in the plate confrontation experiment.

[0017] Figure 2 Growth curves of Rhodococcus sp., Pseudomonas sp., and Cupriavidus sp. in different ratios are shown. Standard deviations are from three biological replicates. Smaller values ​​are not shown.

[0018] Figure 3 Degradation curves of DEHP by Rhodococcus sp., Pseudomonas sp., and Cupriavidus sp. in different ratios within 72 h are shown. The standard deviation is from three biological replicates, and smaller values ​​are not shown.

[0019] Figure 4 The degradation and growth curves of Rhodococcus sp., Pseudomonas sp., and Cupriavidus sp. in a 2:2:1 ratio in a system containing 400 mg / L DEHP were shown in (A) as (A) (B) (C ...

[0020] Figure 5Fluorescence microscopy imaging of laccase (EC1.10.3.2).

[0021] Figure 6 XRD patterns of ZIF-8 samples prepared by different synthesis methods: (A) Zinc nitrate source, standing time 1 h; (B) Zinc acetate source, standing time 1 h; (C) Zinc acetate source, standing time 10 min; (D) ZIF-8 standard spectrum.

[0022] Figure 7 SEM images of ZIF-8 samples prepared by different synthesis methods: (A) Zinc nitrate source, standing time 1 h; (B) Zinc acetate source, standing time 1 h; (C) Zinc acetate source, standing time 10 min.

[0023] Figure 8 The images are (A) merged images, (B) laccase (EC1.10.3.2) coating, (C) bright field, and (D) CLSM cross-sectional images of live bacterial cells.

[0024] Figure 9 Infrared spectra of laccase / microbial community@ZIF-8 composite catalytic material (EPZ), ZIF-8, and naked microbial community (PC1).

[0025] Figure 10 XRD patterns of laccase / microbial community@ZIF-8 composite catalytic material (EPZ), ZIF-8, and naked microbial community (PC1).

[0026] Figure 11 The images show scanning electron microscope (SEM) images of the laccase / microbial community@ZIF-8 composite catalytic material (EPZ) and the naked microbial community (PC1), where A represents PC1 and B represents EPZ.

[0027] Figure 12 EDS spectrum of ZIF-8.

[0028] Figure 13 EDS spectrum of laccase / microbial community@ZIF-8 composite catalytic material (EPZ).

[0029] Figure 14 The figures represent the distribution ratios of various elements on the surface of the laccase / microbial community@ZIF-8 composite catalytic material (EPZ) and ZIF-8, where a represents ZIF-8 and b represents EPZ.

[0030] Figure 15 The zeta potentials are for naked bacterial colonies (PC1) and laccase / colony@ZIF-8 composite catalytic material (EPZ). The standard deviation is from three biological replicates; smaller values ​​are not shown.

[0031] Figure 16The effect of ZIF-8 on PC1 growth is shown in the standard deviation from three biological replicates; smaller values ​​are not shown.

[0032] Figure 17 (A) After culturing for 7 days, the ZIF-8 shell was removed and the bacteria were coated for observation of normal growth. (B) Bacteria suspected of cell division were observed in the scanning electron microscope image.

[0033] Figure 18 Fluorescence imaging of bacteria under adverse conditions, where A and B are EPZ and PC1 under UV-C treatment, respectively, and C and D are EPZ and PC1 under lysozyme treatment, respectively.

[0034] Figure 19 The effect of adverse conditions on the fluorescence intensity of Hoechest 33342 cells in each group.

[0035] Figure 20 The effects of adverse conditions on the ability of exogenous laccase to degrade tetracycline (A) and the ability of the microbial community to degrade DEHP (B) are represented by different letters. The standard deviation is from three biological replicates, and smaller values ​​are not shown.

[0036] Figure 21 Scanning electron microscopy (SEM) images of biochar morphology (A) and BEPZ morphology (B).

[0037] Figure 22 The scanning electron microscope morphology of BEPZ after 5 cycles.

[0038] Figure 23 Degradation rates of DEHP and tetracycline by ZIF-8, PC1 and BEZP at different time points (A), and degradation rates of DEHP and tetracycline by the immobilized composite catalyst after five cycles (B) and (C), with standard deviations from three biological replicates; smaller values ​​are not shown.

[0039] Figure 24 The activities of superoxide dismutase (SOD) and catalase (CAT) of PC1 and BEPZ at 72 h are shown. Standard deviations are from three biological replicates; smaller values ​​are not shown, and different letters indicate significant differences.

[0040] Figure 25 The changes in the viability of PC1 and BEPZ cells during degradation.

[0041] Figure 26 The standard deviations for COD values ​​at 72 h in each group of simulated wastewater are from three biological replicates; smaller values ​​are not shown. Detailed Implementation

[0042] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0044] Rhodococcus sp. from Australia was purchased from Ningbo Taisto Biotechnology Co., Ltd., Pseudomonas sp. from Shanghai Ruichu Biotechnology Co., Ltd., and Cupriavidus sp. from Beina Chuanglian Biotechnology Co., Ltd.

[0045] The liquid basic salt medium (MSM) consists of: 1.5 g / L K2HPO4, 0.5 g / L KH2PO4, 0.5 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 1 g / L NaCl, 1000 ml double-distilled water, and a pH of 7.

[0046] Example 1: Optimization of Degrading Microbial Community Combinations 1. Antagonism test The study employed the plate confrontation method for antagonism testing, precisely transferring 100 μL of the target bacterial strain suspension (OD200). 600 =1) Spread the mixture evenly onto a sterile LB agar plate. Using sterile forceps, remove two sterile circular filter paper discs and place them on the spread LB agar plate, pressing them gently. Then, add 10 μL of the bacterial suspension of the test strain to the corresponding filter paper disc. After complete absorption, place the plate in a laminar flow hood to dry and incubate at 30 ℃ for 3 days, observing the formation of inhibition zones daily. Results are as follows: Figure 1 As shown, when a single strain is used as the substrate, the other two strains both exhibit normal colony growth on the plate, and no inhibition zones were detected, indicating that there is no significant antagonistic effect between the strains and that they have the potential to synergistically construct a functional microbial agent system.

[0047] 2. Optimization of bacterial community ratio Rhodococcus sp., Pseudomonas sp., and Cupriavidus sp. were activated and cultured in LB medium for 24 h. Afterwards, the bacterial cells were collected by centrifugation at 5000 × g for 8 min. The strains were washed twice with phosphate-buffered saline (PBS) at pH 7.2, and then prepared into OD240 solutions. 600 A bacterial suspension with an OD value of 0.5 was prepared. Next, the three bacterial suspensions were thoroughly mixed according to different ratios. Then, the above bacterial suspension was inoculated into 50 mL of sterilized MSM medium, and the OD value of the bacterial suspension in the medium was adjusted to 0.5. 600 The value reached 0.02. Simultaneously, DEHP working stock solution was added to ensure a final concentration of 400 mg / L. The inoculated culture medium was placed in a constant temperature incubator with shaking at 30℃ and a shaking speed of 160 r / min. Samples were taken at 0, 12, 24, 36, 48, and 72 h during the culture process. The residual amount of DEHP was determined by GC-MS, and the OD value of the bacterial culture was measured using a UV spectrophotometer. 600 Values. To set up a control experiment, MSM medium supplemented with heat-inactivated bacteria and the same concentration of substrate was used as a non-biological control, and three replicates were set up for each treatment. The growth curves of Rhodococcus sp., Pseudomonas sp., and Cupriavidus sp. under different ratios are shown in the figure. Figure 2 As shown in the figure. The degradation curves of DEHP by Rhodococcus sp., Pseudomonas sp., and Cupriavidus sp. under different ratios are shown in the figure. Figure 3 As shown.

[0048] from Figure 2 It can be seen that different bacterial community ratios affect cell density (OD) during the culture period. 600 The results showed that when the bacterial community ratio was 2:2:1, its OD... 600 The value reached a peak of 0.72 at 48 h; while the 1:1:2 ratio group maintained a lower level of 0.15 during the same period. The above differences indicate that the 2:2:1 combination showed a significant growth advantage in the middle of the culture, and its microbial community growth was significantly better than other ratio schemes.

[0049] from Figure 3 It can be seen that the degradation rate of each treatment group approached the level of complete degradation after 72 h of incubation. This result shows that each treatment group reached its maximum degradation efficiency in the later stage of incubation, confirming that different bacterial combinations have the ability to efficiently degrade DEHP at specific time points.

[0050] Further analysis was conducted on the growth characteristics and physicochemical parameter changes of the bacterial community during DEHP degradation when the ratio of Rhodococcus australis sp., Pseudomonas sp., and Cupriavidus sp. was 2:2:1. The degradation and growth curves of the bacterial community in a system containing 400 mg / L DEHP, the changes in pH value in the culture medium, and the changes in intermediate products are shown below. Figure 4 As shown in (A), (B), and (C), by Figure 4 As can be seen from (A), OD in the early stage of culture (0-12h) 600 The value showed a slow proliferation trend, followed by a logarithmic growth phase (24-48h) and continued to climb, reaching a maximum of 0.72 at 48h. This phenomenon was positively correlated with increased cell density and enhanced metabolic activity, indicating that the microbial community was in an active metabolic state in the DEHP degradation system. Notably, the microbial community exhibited an accelerated proliferation trend during the 36-48h stage, suggesting that environmental parameters such as nutrient supply and DEHP concentration in the culture medium reached their optimal thresholds at this time, effectively supporting rapid microbial reproduction. Regarding the DEHP degradation rate, the microbial community achieved a pollutant degradation rate of 95.92% within 72h. The degradation process showed a good correlation with the microbial community proliferation trend, specifically: in the early stage of culture (0-24h), the microbial community was in the adaptation phase, and the degradation rate was only maintained at 25.33%-34.85%; during the logarithmic growth phase (24-48h), the metabolic activity of the microbial community surged, and the degradation rate rapidly increased to 95.92%. This suggests that the rapid growth of the microbial community after optimization may have provided more metabolic activity for the effective degradation of DEHP, while the increased efficiency of extracellular enzyme secretion accelerated the co-metabolism of DEHP. The above results confirm that this microbial community ratio possesses both high degradation efficiency and stable proliferation characteristics. Figure 4 As shown in (B), the bacterial community's metabolic processes caused the culture medium pH to gradually decrease from an initial 7.32 to 6.94 after 48 hours, mainly attributed to the accumulation of acidic metabolites (such as phthalic acid). However, it is noteworthy that the pH rebounded (6.60→6.94) during the 36-48 hour period, suggesting possible further metabolism of some intermediate products or adaptive regulation of the bacterial community's physiological state. Figure 4As shown in (C), the content of intermediate product PCA reached a peak of 11.2 mg / L at 36 h, while the content of intermediate product PA gradually decreased after reaching 11.8 mg / L at 12 h, indicating that the bacterial community can rapidly mineralize DEHP and its intermediates. The study showed that the soil microbiome with a pH of 6.0-8.0 exhibited a high DEHP degradation efficiency of 39.1-43.7%, while the degradation efficiency in acidic and alkaline soils decreased to <25% and 8.6%, respectively. The microbial community could maintain a high DEHP degradation rate within a neutral pH range (6.0-8.0), and its metabolic function showed a certain biological tolerance to microenvironment pH fluctuations. Therefore, although the pH of the microbial community decreased slightly, it had little impact on its degradation efficiency.

[0051] Example 2: Preparation and testing of laccase / microbial community@ZIF-8 composite catalytic material 1. Preparation of fluorescently labeled proteins In the fluorophoretic labeling of laccase (EC1.10.3.2), FITC (green fluorescent protein) was selected for labeling to prepare the fluorophore-labeled protein. 7.56 g NaHCO3, 1.06 g Na2CO3, and 7.36 g NaCl were weighed and diluted with water to a final volume of 1 L to obtain the cross-linking reaction solution. Simultaneously, the protein to be cross-linked (with a concentration of 1 mg / mL or higher) was placed at 4°C for dialyzing. This dialyzing process was repeated three times until the pH of the solution reached 9.0. Additionally, FITC was dissolved in DMSO to a concentration of 1 mg / mL. It is crucial that the FITC used in each cross-linking reaction be freshly prepared and stored in the dark. During the specific cross-linking reaction, FITC was slowly added to the antibody solution at a P:F ratio of 1 mg:150 μg (protein to FITC). During the addition process, gently agitate the solution to ensure thorough mixing of FITC and antibody. Then, allow the reaction to continue for 8 hours in the dark at 4°C. Finally, add 5 mol / L NH4Cl to the reaction system to achieve a final concentration of 50 mmol / L, and allow the reaction to continue at 4°C for another 2 hours to terminate the cross-linking process. Observe the FITC-labeled laccase (EC1.10.3.2) using CLSM. Figure 5 The fluorescence signal indicates that laccase was successfully labeled with FITC and detected. The particles are evenly dispersed and small, exhibiting good overall contrast.

[0052] 2. Preparation and characterization of laccase / microbial community@ZIF-8 composite catalytic material To achieve the exponential growth phase, 2% (v / v) of bacteria were inoculated into LB medium and incubated at 30°C for 12 h. During the exponential phase, 10 mL of cells were washed three times with PBS to remove excess LB medium and then resuspended in 0.5 mL of 1x PBS containing laccase (EC1.10.3.2). After 10 min, the enzyme-coated bacteria were washed twice with deionized water. After washing, the cells were finally resuspended in 1 mL of 160 mM 2-methylimidazole aqueous solution. Next, 1 mL of 40 mM zinc acetate dihydrate aqueous solution was added to the 2-methylimidazole solution containing the bacteria. The resulting mixture was then placed on a shaker and shaken at 160 rpm for 15 min. After shaking, the mixture was allowed to stand for 1 h to promote the formation of the ZIF-8 coating. Finally, the coated cells were washed three times with deionized water to remove excess ZIF-8 precursor material. After the washing process is complete, the cells are finally suspended in deionized water.

[0053] The preparation of ZIF-8 coated bacteria is the same as described above, but without laccase coating.

[0054] XRD patterns of ZIF-8 samples prepared by different synthesis methods are shown below. Figure 6 As shown, (A) is a zinc nitrate source with a standing time of 1 h; (B) is a zinc acetate source with a standing time of 1 h; (C) is a zinc acetate source with a standing time of 10 min; and (D) is the ZIF-8 standard spectrum. All synthesized ZIF-8 samples (AC) and the standard card spectrum of the ZIF-8 structure (D) are basically consistent. Based on the presence of characteristic peaks (100), (022), (013), (222), (114), (233), and (134), it can be confirmed that ZIF-8 was successfully prepared in each group, but some diffraction peaks in group A show slight shifts, with slight deviations in intensity and width. Figure 7 Electron microscopy observations showed that different zinc sources and settling times significantly affected the crystal morphology of ZIF-8. (A) When zinc nitrate was used as the zinc source, the crystal surface exhibited obvious lattice distortion and porosity defects. The surface roughness of the spherical particles was high, and there were non-uniform protrusions and depressions. Compared with (A), (B) when zinc acetate was used as the zinc source, the crystal faces were fully developed and the surface roughness was significantly reduced. The crystals exhibited a regular geometric configuration, with a smooth surface and typical rhombic dodecahedral characteristics. (C) Due to the shortened settling time, the crystals did not reach a fully mature state, and their morphology showed a lamellar aggregate morphology.

[0055] The FITC-labeled laccase (EC1.10.3.2) coating was observed using CLSM. Figure 8Fluorescence microscopy revealed a continuous green fluorescent signal around each individual cell, indicating the formation of a uniform laccase coating on the individual cells. Cell viability assays using Hoechst 33342 as a fluorescent indicator showed that Hoechst 33342 is a blue fluorescent dye that can penetrate the cell membrane; normal bacterial cells exhibit some resistance to staining, thus displaying a dim blue fluorescence. The pale blue fluorescence indicates that the enzyme and MOF coating process have little impact on bacterial viability, validating the compatibility of the biomineralization process with cell function.

[0056] Infrared spectra of laccase / microbial community@ZIF-8 composite catalytic material (EPZ), ZIF-8, and naked microbial community (PC1) are shown below. Figure 9 As shown, the original bacterial cell surface possesses abundant functional groups such as -OH, R-COOH, -CO-NH, -NH2, and -NH. These results indicate that the surface of natural bacterial cells has multiple anchoring sites, providing potential binding sites for the directional fixation of surface metal ions, thus laying the chemical foundation for the subsequent construction of the ZIF-8 biomineralization coating. The metal ions anchored on the bacterial cell surface can serve as a key precursor reservoir for MOF nucleation and growth. Comparative analysis shows that the ZIF-8-coated cells still retain the original functional group characteristics. Due to the coating effect of ZIF-8, the relevant characteristic peaks show varying degrees of red shift. Further research revealed that the coated samples exhibited more complex peak characteristics in the FTIR spectrum, with a significantly increased number of peak positions compared to the untreated group. Specifically, ZIF-8-coated cells showed a higher peak size at 422 cm⁻¹. -1 The addition of characteristic peaks at isotope numbers is attributed to the aromatic and aliphatic CH extensions of imidazole. Furthermore, the structural integrity of ZIF-8 is maintained in the presence of bacteria, and the characteristic FTIR peaks used to indicate various bond stretching vibrations remain almost unchanged. For example, the CH stretching vibration (2927 cm⁻¹) remains consistent. -1 ), C = N (1569 cm) -1 ), CN (1146 cm) -1 995cm -1 ), and Zn-N (422 cm -1 ).

[0057] Analysis of the XRD diffraction patterns before and after ZIF-8 coating (e.g.) Figure 10As shown in the figure, no obvious crystal peaks were observed on the surface of the naked bacterial colony (PC1). However, the ZIF-8-coated laccase / bacterial colony@ZIF-8 composite catalytic material (EPZ) exhibited many significant crystal peaks. ZIF-8 showed characteristic peaks at 2θ = 7.36°, 10.38°, 12.72°, and 14.7°, exhibiting a tetragonal sodalite topology, consistent with previous reports. The presence of characteristic peaks (100), (022), (013), (222), (114), (233), and (134) was highly consistent with the diffraction pattern of the pure ZIF-8 standard card, confirming the successful preparation of ZIF-8. Furthermore, the XRD patterns of EPZ and ZIF-8 were very close, indicating that the ZIF-8 coating was formed during a biomimetic mineralization process. This verifies that a ZIF-8 coating was successfully formed on the bacterial cell surface and possesses good crystallinity.

[0058] The morphological characteristics of naked bacteria and laccase / bacterial community@ZIF-8 composite catalytic material (EPZ) were evaluated by SEM (e.g., Figure 11 (As shown). SEM microscopic morphology analysis revealed that the bacterial surface exhibited a uniform ZIF-8 coating while maintaining the original rod-shaped morphology of the bacterial cells. These results confirm that the ZIF-8 protective shell can encapsulate the bacterial surface and achieve the observed biomimetic mineralization.

[0059] The spatial distribution characteristics of elements in ZIF-8 and laccase / microbial community@ZIF-8 composite catalytic materials (EPZ) were evaluated by EDS as follows: Figure 12 and Figure 13 As shown, ZIF-8 is mainly composed of carbon (C), oxygen (O), nitrogen (N), and zinc (Zn), with C, N, O, and Zn elements exhibiting a uniform distribution on the cell surface. This phenomenon not only confirms the uniform coating characteristics of the ZIF-8 protective shell on the cell surface, but also indicates that the stable presence of Zn element demonstrates that the ZIF-8 crystal structure maintains its framework integrity during the biomimetic mineralization process.

[0060] Laccase / microbial community@ZIF-8 composite catalytic material (EPZ) and the elemental distribution ratios on the ZIF-8 surface, for example. Figure 14 As shown, a represents ZIF-8, and b represents EPZ. Carbon (C) is the most abundant element in ZIF-8, accounting for 56.94%, demonstrating its excellent organic framework properties, while zinc (Zn) accounts for 6.46%, indicating its effectiveness as a MOF. Notably, the oxygen (O) content in the biomineralized coated bacteria significantly increased to 61.96%, endowing the composite material with excellent biocompatibility and functional activity, while the Zn content decreased to 3.73%. This difference stems from the dominant distribution of the bacterial organic components in the coating layer, leading to a reduced local enrichment of Zn at the interface.

[0061] Zeta potential (e.g.) Figure 15 As shown in the figure, the anisotropic attraction between ZIF-8 (positive) and bacterial cells (negative) drives the successful assembly of EPZ. After ZIF-8 is combined with the bacterial community, the zeta potential of the community changes significantly from negative (-1.38mV) to positive (+0.15mV). The bacteria exhibit a negative zeta potential, which corresponds to higher bacterial activity. When its zeta potential turns into a positive charge, it indicates that ZIF-8 has been successfully deposited on the cell surface.

[0062] 3. Performance testing of laccase / microbial community@ZIF-8 composite catalytic material (1) Cell viability test: After culturing the laccase / microbial community@ZIF-8 composite catalytic material for seven days, EDTA (0.5M, pH=7) was added to remove the ZIF-8 shell. A portion was then plated to observe cell growth. OD was measured during the process. 600 The changes were observed. The effect of ZIF-8 on PC1 growth was as follows: Figure 16 As shown, it can be seen that OD during the process 600 The growth was slow, leveling off after the fourth day, indicating that the coating treatment had little effect on bacterial proliferation. After 7 days of incubation, the ZIF-8 outer shell coating was removed, and the normal bacterial growth was observed. Scanning electron micrographs are shown below. Figure 17 As shown, the bacteria grow normally. The electron micrograph further reveals that the bacterial cells may still retain their ZIF-8 shell during cell division. This phenomenon suggests that MOF-based protective exoskeleton may be passed down across generations through cell division.

[0063] (2) Protection test in a compound pollution environment: To evaluate the survival potential of the laccase / microbial community@ZIF-8 composite catalytic material in extreme environments, experiments were conducted by simulating two typical stress conditions: Experimental group 1 was treated with lysozyme as a toxic compound in the culture medium, Experimental group 2 was treated with UV-C radiation (because UV-C can induce DNA and protein damage in organisms), and the control group was an untreated system. 400 mg / L DEHP and 1 mg / L tetracycline were added to MSM culture medium as a toxic environment, and 2000 U of lysozyme was added to it. Naked bacteria and laccase / microbial community@ZIF-8 composite catalytic material were used as mutual controls. The mixed solution was placed in a shaker (160 rpm) at 30 °C, and cell viability was monitored using a Hoechst 33342 / PI double staining kit. For the UV-C radiation test, each group was transferred to a flat-bottomed glass dish (capacity 15 ml) with the lid off and placed 10 cm away from a 15W UV lamp (λ: 254 nm) in a closed room for 30 s exposure. Cell viability changes in each group were dynamically monitored using a double staining system of Hoechst 33342 (blue fluorescence) and propidium iodide (PI, red fluorescence). Hoechst 33342 can generate a weak blue light signal through intact cell membranes, while PI can only penetrate damaged cell membranes to achieve nuclear staining. Fluorescence imaging of bacteria under adverse conditions was obtained as follows: Figure 18 As shown, A and B represent EPZ and PC1 under UV-C treatment, respectively, while C and D represent EPZ and PC1 under lysozyme treatment, respectively. It can be seen that compared to the uncoated naked bacterial colony PC1, the ZIF-8 coated bacterial colony (EPZ) exhibits significantly enhanced survival under dual stress conditions. In both the UV-C exposure and lysozyme treatment groups, the PI fluorescence intensity and Hoechst 33342 fluorescence intensity of EPZ are lower than those of the PC1 group. This phenomenon suggests that the ZIF-8 coating has multiple protective effects on bacteria, with the UV protection function possibly stemming from the characteristic absorption of ZIF-8 material in the UV spectral region. Further combining... Figure 19 Analysis revealed that under adverse environmental stress, the Hoechst 33342 fluorescence intensity in the EPZ group was significantly lower than that in the PC1 group, while the signal of this dye was enhanced in apoptotic cells, thus confirming that the ZIF-8 coating layer effectively alleviated cell damage caused by external pressure.

[0064] (3) Degradation performance test of laccase / microbial community@ZIF-8 composite catalytic material: 400 mg / L DEHP and 1 mg / L tetracycline were added to MSM medium as a toxic environment, and 5 U of protease was added to it. Naked laccase, naked bacteria (PC1), naked bacteria + laccase (PC1 + lac), ZIF-8 coated bacteria (ZIF-8@PC1), and laccase / microbial community@ZIF-8 composite catalytic material (EPZ) were used as mutual controls. The mixed solution was placed in a shaker (160 rpm) at 30℃ and cultured. The concentrations of tetracycline and DEHP in each group were detected by HPLC-MS / MS and GC-MS, respectively.

[0065] First, the degradation of tetracycline in cells in the presence of proteases was assessed. Proteases, as catalysts for peptide bond hydrolysis, can influence biocatalytic activity by cleaving proteins. Experimental data are as follows: Figure 20 As shown in (A), after 24 h of protease exposure, the degradation rate of tetracycline in the uncoated group plummeted to 37.07%, while the ZIF-8 coated group (EPZ) maintained a degradation efficiency of 94.27%. This result clearly confirms that the ZIF-8 coating exhibits significant protective efficacy against laccase, effectively resisting proteolytic activity.

[0066] Furthermore, under tetracycline stress, the degradation dynamics of DEHP showed significant differences among the experimental groups (e.g., Figure 20(As shown in B): Within 48 hours, the degradation rate of the EPZ group increased from 68.33% to 84.62%, the ZIF-8@PC1 group from 62.51% to 80.02%, the PC1+lac group reached 60.57%-80.04%, while the PC1 group only increased from 46.94% to 56.48%. Tetracycline inhibits bacterial physiological activity through multiple pathways, including blocking protein synthesis pathways, inducing oxidative stress responses, inhibiting bacterial proliferation rates, and reconstructing metabolic networks. The combined effects of these factors lead to a severe decline in the bacterial DEHP degradation capacity. These factors work together to significantly affect the physiological state of naked bacteria under tetracycline stress. It is worth noting that although ZIF-8 coating did not completely eliminate the inhibitory effect of tetracycline, its protective effect increased the degradation efficiency of the ZIF-8@PC1 group by approximately 23.54% compared to the PC1 group at 48 hours, fully verifying the feasibility of the ZIF-8 coating strategy in maintaining bacterial functional activity under antibiotic stress.

[0067] Example 3: Preparation of immobilized composite catalytic materials and their application in the remediation of DEHP-tetracycline contaminated wastewater 1. Preparation and characterization of immobilized composite catalytic materials Cleaned and dried corn stalks were pulverized into powder, placed in a crucible, and covered. The mixture was then calcined at 500°C in a muffle furnace for 4 hours under a nitrogen atmosphere. After calcination, the obtained biochar was washed with pure water to remove surface ash. Finally, the biochar was ground into a fine powder, passed through a 100-mesh sieve, and stored in a dry environment for later use. The prepared biochar was weighed and placed in an Erlenmeyer flask, sterilized, and then cooled. Subsequently, 50 mL of a uniformly mixed laccase / microbial community@ZIF-8 composite catalytic material was poured into the biochar, and the Erlenmeyer flask was placed on a shaker at 30°C for 2 hours to adsorb the material, resulting in immobilized laccase / PC1@ZIF-8 composite catalytic material (BEPZ). Scanning electron micrographs of the biochar and BEPZ are shown below. Figure 21 As shown, the surface of the biochar exhibits a distinct porous structure with large pores and no obvious collapse. This structural feature provides a favorable growth environment for microorganisms, promoting their colonization on its surface. Simultaneously, this porosity increases the specific surface area of ​​the biochar, providing more active sites for pollutant adsorption. Furthermore, the laccase / microbial community@ZIF-8 composite catalytic material (EPZ) was successfully immobilized on the biochar surface and distributed within the pores.

[0068] 2. Repair Experiments Using Immobilized Composite Catalytic Materials Batch experiments were conducted in 250 mL Erlenmeyer flasks used as bioreactors in an incubator shaker (30°C and 160 rpm). ZIF-8 (1 g), BEPZ (1 g), and free bacterial flora (1 mL cell suspension, OD) were used.600 =1.0) was added to simulated wastewater (100 mL). The contents of DEHP and tetracycline in the solution were measured at different reaction time points. The bacterial cell survival at 24 h, 72 h, and 120 h was recorded using a flow cytometer. The specific parameters are shown in Table 1 below.

[0069] Table 1 To investigate the effect of multiple recycling on the degradation efficiency of immobilized composite catalytic materials, DEHP and tetracycline were added to 100 mL of MSM medium with an initial pH of 7. The final concentration of DEHP was 400 mg / L, and the final concentration of tetracycline was 1 mg / L. 1 g of immobilized composite catalytic material was weighed and added to the medium. The sealed Erlenmeyer flask was then placed in a 30°C shaker (160 rpm) for 48 h, and the residual concentrations of DEHP and tetracycline were recorded. The immobilized composite catalytic material was collected by filtration, washed three times with sterile physiological saline, and then transferred to a new culture medium. The culture was repeated under the same conditions, for a total of five reuses. This process was repeated to evaluate its reusability.

[0070] The scanning electron microscope morphology of BEPZ after 5 cycles is as follows: Figure 22 As shown, after five cycles of use, the BEPZ composite carrier gradually breaks down, its mesoporous structure partially collapses, the carrier becomes loose, and the amount of microorganisms attached decreases, but it can still adsorb some microorganisms.

[0071] The degradation rates of DEHP and tetracycline by ZIF-8, PC1, and BEZP at different time points are as follows: Figure 23As shown in (A), experimental data indicate that the BEPZ system achieved a DEHP removal rate of 86.21% and a tetracycline degradation efficiency of 95.14% within 72 hours. This synergistic effect is mainly attributed to the effective protection of laccase activity by the enzyme-coated metal-organic framework shell. This protective mechanism not only inhibits the toxic inhibitory effect of tetracycline on bacterial metabolic activities but also maintains the catalytic stability of laccase in complex environments, thereby improving the pollutant removal efficiency under complex pollution systems. Notably, ZIF-8 significantly enhanced the environmental adaptability of the strain by constructing a physical barrier and simultaneously regulated the functional stability of laccase at the molecular level, enabling it to maintain catalytic activity in toxic matrices. In contrast, the naked bacterial colony PC1 exhibited significant physiological inhibition under tetracycline stress, with a DEHP degradation rate of only 59.62% (72 hours). Tetracycline inhibits bacterial protein synthesis, leading to weakened bacterial growth and metabolic activities, thus affecting their ability to degrade pollutants. Furthermore, the presence of antibiotics may trigger a stress response in bacteria, consuming their energy and resources, thereby exacerbating the loss of bioremediation efficiency. The above results confirm that, compared with BEPZ, unprotected naked bacteria exhibit significant deficiencies in environmental adaptability and functional durability in the DEHP-tetracycline coexistence system. ZIF-8 alone primarily plays an adsorption and enrichment role in this process.

[0072] The degradation rates of DEHP and tetracycline by the immobilized composite catalyst material after five cycles are as follows: Figure 23 As shown in (B) and (C), repeated use may lead to saturation of the biochar surface due to the accumulation of pollutants or metabolites, and the occupation of active sites, resulting in decreased adsorption capacity and clogged pores. This reduces the opportunity for bacteria to come into contact with pollutants and also reduces the number of adsorbed bacteria. Therefore, with the increase in the number of cycles, the degradation rate that the immobilized composite catalytic material can achieve in the same amount of time will also decrease. However, after five repeated runs, the DEHP removal rate still remained above 50%.

[0073] The activities of two essential antioxidant enzymes, SOD and CAT, were assessed during the degradation process. The activities of superoxide dismutase (SOD) and catalase (CAT) in the treated bacteria were measured using an activity assay kit purchased from Beijing Solarbio Science & Technology Co., Ltd. The results are as follows: Figure 24 As shown, the inhibitory effect of DEHP-induced oxidative stress on biodegradation processes has been confirmed in the literature. SOD and CAT provide crucial defenses, converting superoxide radicals and breaking down hydrogen peroxide, respectively. Experimental data show that the activities of both enzymes in BEPZ are significantly upregulated compared to PC1 without MOF protection. This phenomenon suggests that the enzyme-coated metal-organic framework shell may minimize oxidative damage to cells by reducing DEHP-induced free radical production.

[0074] The dynamic changes in cell viability of PC1 and BEPZ at 24, 72, and 120 h were quantitatively analyzed by flow cytometry. Results are as follows: Figure 25 As shown, experimental data revealed that the percentage of viable cells in the BEPZ group reached 73.70% at 72 hours, a significant increase of 19.11% compared to the naked bacterial control group (PC1); even at 120 hours, its survival advantage remained at an increase of 14.24%. Mechanistic analysis indicated that laccase in the BEPZ system significantly reduced the accumulation of toxic substances by efficiently catalyzing tetracycline degradation, while biochar and ZIF-8 maintained the homeostasis of the enzyme's active site microenvironment and reduced the impact of external stressors (such as oxidative stress or toxic substances) on cells by constructing a physical confinement barrier. This biomimetic mineralization strategy reduced free radical production and cell damage under stress conditions through a dual protection mechanism (physical barrier and synergistic chemical catalysis). The above tests confirm that the biochar-immobilized ZIF-8 / laccase composite system significantly enhanced the bacterial environmental adaptability in a complex pollution system and maintained its metabolic activity through a synergistic protection mechanism, providing a theoretical basis for the development of novel environmental remediation biomaterials.

[0075] The COD content of the treated wastewater was analyzed using the potassium dichromate oxidation method. The test results are as follows: Figure 26 As shown in the experimental data, the COD value of the BEPZ group was 9.39 mg / L, significantly lower than that of the PC1 group (13.87 mg / L), the ZIF-8 group (18.64 mg / L), and the CK group (20.40 mg / L). This result confirms that the BEPZ system significantly reduces the organic pollution load in water bodies by enhancing the DEHP degradation efficiency. Its mechanism of action stems from the synergistic effect of the ZIF-8 coating structure and the laccase catalytic activity, which enhances the stability of bacteria in degrading DEHP, thereby effectively reducing the concentration of organic pollutants in water bodies. Furthermore, it verifies the synergistic effect of the enzyme-coated metal-organic framework shell. Overall, both BEPZ and PC1 can remove COD from DEHP-containing wastewater to a certain extent, but BEPZ exhibits the highest removal rate, indicating its potential application value in the degradation of complex pollution.

[0076] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for preparing a metal-organic framework encapsulated laccase-bacterial consortium immobilized composite catalytic material, characterized in that, Includes the following steps: (1) Inoculate the bacterial suspension containing Rhodococcus sp., Pseudomonas sp. and Cupriavidus sp. into LB medium and incubate. Wash with PBS during the exponential phase, then suspend in PBS containing laccase, let stand to complete the coating, wash with deionized water and then suspend in 2-methylimidazole aqueous solution. (2) Add the zinc source to the 2-methylimidazole aqueous solution in step (1), shake and let stand, wash with deionized water and then resuspend in deionized water; then add it to biochar and shake to adsorb, to obtain the metal-organic framework encapsulated laccase-microbe immobilized composite catalytic material.

2. The method for preparing a metal-organic framework-encapsulated laccase-microbial community immobilized composite catalytic material according to claim 1, characterized in that, In step (1), the ratio of Rhodococcus sp., Pseudomonas sp. and Cupriavidus sp. is (1-3):(1-3):(1-3).

3. The method for preparing a metal-organic framework-encapsulated laccase-microbial community immobilized composite catalytic material according to claim 2, characterized in that, In step (1), the ratio of Rhodococcus sp., Pseudomonas sp., and Cupriavidus sp. is 2:2:

1.

4. The method for preparing a metal-organic framework-encapsulated laccase-microbial community immobilized composite catalytic material according to claim 1, characterized in that, In step (2), the zinc source is an aqueous solution of zinc acetate.

5. The method for preparing a metal-organic framework-encapsulated laccase-microbial community immobilized composite catalytic material according to claim 4, characterized in that, In step (2), the settling time is 1 hour.

6. The method for preparing a metal-organic framework-encapsulated laccase-microbial community immobilized composite catalytic material according to claim 1, characterized in that, In step (2), the method for preparing the biochar includes the following steps: After washing, the corn stalks are crushed into powder, calcined in an inert gas atmosphere, washed with pure water, ground into powder, and dried for later use.

7. A metal-organic framework-encapsulated laccase-microbial community immobilized composite catalytic material prepared by the preparation method according to any one of claims 1-6.

8. The application of a metal-organic framework-encapsulated laccase-microbial community immobilized composite catalytic material as described in claim 7 in the remediation of tetracycline-DEHP composite pollutants.

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