Preparation and application of carbon quantum dot-shewanella biological hybrid system for uranium removal

Abstract

The invention discloses a preparation method of a carbon quantum dot-shewanella biohybridization system for uranium removal, which comprises the following steps: step 1, inoculating shewanella into an LB liquid culture medium for aerobic pre-culture until the logarithmic phase, then adding a carbon quantum dot solution, and continuously culturing to obtain a bacterial solution; and 2, transferring the bacterial liquid obtained in the step 1 into an inorganic mineral culture medium, continuing aerobic culture, centrifuging, collecting precipitate, and then washing by using the inorganic mineral culture medium to obtain the carbon quantum dot-shewanella biohybridization system. According to the method, the carbon quantum dot-shewanella biological hybrid system is constructed in situ by using the carbon quantum dots with excellent conductivity and biocompatibility, so that the uranium removal capacity of the S.oneidensis MR-1 is effectively improved; and moreover, the introduction of the carbon quantum dots does not have an inhibition effect on the growth phenotype and the cell survival rate of S.oneidensis MR-1.

Description

Technical Field

[0001] The present invention belongs to the technical field of radioactive element processing, and more specifically, relates to the preparation and application of a carbon quantum dot-Shewanella biohybrid system for uranium removal. Background Art

[0002] Shewanella oneidensis MR-1, a typical dissimilatory metal-reducing bacterium, can reduce highly toxic soluble uranium (U(VI)) to less toxic insoluble U(IV) via an extracellular electron transfer pathway under anaerobic conditions. However, in practical applications, the uranium reduction efficiency of S. oneidensis MR-1 is often limited by fluctuations in environmental conditions (such as pH, temperature, and uranium concentration) and the strain's own metabolic capacity, which in turn severely hinders the normal operation of the extracellular electron transfer pathway. In recent years, many researchers have attempted to develop advanced catalytic materials to enhance the electron transfer efficiency in bioelectrocatalytic systems. By leveraging multidisciplinary approaches such as materials science, electroactive microbiology, and synthetic biology, efforts are underway to rationally optimize traditional inorganic catalytic materials and electroactive microorganisms, potentially enhancing electron transfer flux and efficiency.

[0003] Carbon quantum dots (CQDs), emerging zero-dimensional carbon nanomaterials, have attracted considerable attention due to their unique physicochemical properties. These materials exhibit outstanding electrical conductivity, excellent biocompatibility, and a particle size distribution ranging from 2 to 10 nanometers, demonstrating a typical nanosize effect. Studies have shown that CQDs can penetrate cell membranes and localize to regions enriched in cytochrome c. Notably, cytochrome c, a key electron transport protein, is primarily located in the inner mitochondrial membrane, suggesting a potential interaction between CQDs and the mitochondrial electron transport chain. Although material-microbe hybrids have been widely reported and play a key role in geochemical cycles, the detailed molecular mechanisms of their interactions remain largely unexplained. Therefore, constructing material-microbe hybrid systems and conducting in-depth studies of the transmembrane electron transfer mechanism at the microbial-material interface will provide new theoretical perspectives for understanding microbially mediated biogeochemical cycles. Due to the low efficiency of the extracellular electron transfer (EET) process of wild S. oneidensis MR-1, the efficiency of U(VI) reduction to U(IV) is unsatisfactory, which seriously limits the application of S. oneidensis MR-1 in the biogeochemical cycle of uranium. Summary of the Invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] To achieve these objects and other advantages of the present invention, a method for preparing a carbon quantum dot-Shewanella biohybrid system for uranium removal is provided, comprising the following steps:

[0006] Step 1: inoculate Shewanella into LB liquid medium for aerobic pre-culture until the logarithmic growth phase, then add the carbon quantum dot solution and continue culturing to obtain a bacterial solution;

[0007] Step 2: Transfer the bacterial solution obtained in step 1 to an inorganic mineral culture medium, continue aerobic culture, centrifuge, collect the precipitate, and then wash it with an inorganic mineral culture medium to obtain a carbon quantum dot-Shewanella biohybrid system for uranium removal.

[0008] Preferably, in the step 1, the Shewanella is Shewanella oneidensis MR-1.

[0009] Preferably, in the step 1, Shewanella is inoculated into the LB liquid culture medium at a volume percentage of 0.5-2.0%.

[0010] Preferably, in step 1, the aerobic pre-culture is carried out at 30° C. and 150 rpm.

[0011] Preferably, in step 1, the concentration of the carbon quantum dot solution is 0.05 to 0.2 mg / mL; and the amount of the carbon quantum dot solution added is 1.0 to 3.0% by volume.

[0012] Preferably, in step 1, the culture is continued for 1 to 3 hours.

[0013] Preferably, in step 2, the volume ratio of the bacterial liquid to the inorganic mineral culture medium is 0.5-2:98-99.5.

[0014] Preferably, in step 2, the composition ratio of the inorganic mineral culture medium is: 2.5 g / L NaHCO3, 0.25 g / L NH4Cl, 0.1 g / L KCl, 0.1 g / L NaCl, 0.04 g / L KHPO4, 0.2 g / L MgCl2·6H2O and 1.0 g / L yeast extract.

[0015] Preferably, in step 2, aerobic culture is continued at 30° C. and 150 rpm for 10 to 14 hours; centrifugation is performed at 6,000 to 10,000 rpm for 5 to 15 minutes; and washing is performed 1 to 3 times with an inorganic mineral culture medium.

[0016] Preferably, in step 1, modified carbon quantum dots are used to replace carbon quantum dots, and the preparation method of the modified carbon quantum dots is:

[0017] S11, adding carbon quantum dots and hyaluronic acid to deionized water, ultrasonically treating for 1 to 3 hours, standing at room temperature for 6 to 18 hours, filtering, and freeze-drying at -30 to -80°C to obtain pretreated carbon quantum dots;

[0018] S12. Add the pretreated carbon quantum dots to 10 mM Tris-HCl buffer (pH = 8.5), stir evenly, then add dopamine hydrochloride, stir at a constant temperature of 30-50 ° C for 2-5 hours, dialyze with a 1 kDa dialysis membrane for 2-5 hours, and freeze-dry the non-permeated liquid at -30-80 ° C to obtain modified carbon quantum dots.

[0019] Preferably, in S11, the molecular weight of hyaluronic acid is 100-8000 kDa; the mass-to-volume ratio of carbon quantum dots, hyaluronic acid and deionized water is 10 g:0.5-2 g:300-700 mL; the ultrasonic power is 200-500 W, and the frequency is 50-80 kHz.

[0020] Preferably, in S12, the mass volume ratio of the pretreated carbon quantum dots, dopamine hydrochloride and Tris-HCl buffer is 10 g: 1-3 g: 50-150 mL.

[0021] Application of the carbon quantum dot-Shewanella biohybrid system for uranium removal prepared by the preparation method described above in uranium removal.

[0022] The present invention includes at least the following beneficial effects: the present invention utilizes carbon quantum dots with excellent conductivity and biocompatibility to in situ construct a carbon quantum dot-Shewanella biohybrid system, which effectively improves the uranium removal ability of S. oneidensis MR-1; and the introduction of carbon quantum dots has no inhibitory effect on the growth phenotype and cell survival rate of S. oneidensis MR-1; in addition, based on non-targeted metabolomics analysis, it is found that carbon quantum dots may enhance the environmental adaptability of S. oneidensis MR-1 through three mechanisms: (1) promoting the secretion of extracellular polymers (EPS) and enhancing the ability to form biofilms; (2) increasing the activity of the antioxidant enzyme system; and (3) optimizing the energy metabolism pathway. The present invention provides a new theoretical basis and technical path for the bioremediation of radioactive contamination.

[0023] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Scanning electron micrographs of S. oneidensis MR-1 (a) and S. oneidensis MR-1-CQDs (b) prepared in Example 1;

[0025] Figure 2 Three-dimensional fluorescence spectra of S. oneidensis MR-1 (a) and S. oneidensis MR-1-CQDs prepared in Example 1 (b);

[0026] Figure 3 are the growth curves of S. oneidensis MR-1, S. oneidensis MR-1-CQDs of Example 1, and S. oneidensis MR-1-CQDs after reaction with uranium;

[0027] Figure 4 Fluorescence confocal microscopy of S. oneidensis MR-1, S. oneidensis MR-1-CQDs of Example 1, and S. oneidensis MR-1-CQDs after reaction with uranium;

[0028] Figure 5 Comparison of uranium removal performance of S. oneidensis MR-1 and S. oneidensis MR-1-CQDs prepared in Example 1;

[0029] Figure 6 FTIR spectra of S. oneidensis MR-1 and S. oneidensis MR-1-CQDs prepared in Example 1 after reaction with uranium;

[0030] Figure 7 XPS analysis of S. oneidensis MR-1 and S. oneidensis MR-1-CQDs prepared in Example 1 after reaction with uranium;

[0031] Figure 8 Cluster analysis (a) and OPLS-DA score graph (b) of differential metabolites between S. oneidensis MR-1 and S. oneidensis MR-1-CQDs prepared in Example 1;

[0032] Figure 9 Statistics of the number of differential metabolites (a) and volcano plot of differential metabolites (b) between S. oneidensis MR-1 and S. oneidensis MR-1-CQDs prepared in Example 1;

[0033] Figure 10 Correlation analysis of differential metabolites between S. oneidensis MR-1 and S. oneidensis MR-1-CQDs prepared in Example 1;

[0034] Figure 11Pie chart showing the differential metabolite classification of S. oneidensis MR-1 and S. oneidensis MR-1-CQDs prepared in Example 1;

[0035] Figure 12 VIP value statistics of differential metabolites of S. oneidensis MR-1 and S. oneidensis MR-1-CQDs prepared in Example 1;

[0036] Figure 13 The top 20 differential metabolic pathways of KEGG enrichment analysis for S. oneidensis MR-1 and S. oneidensis MR-1-CQDs prepared in Example 1;

[0037] Figure 14 This is a statistical diagram of the VIP values of differential metabolites in the D-amino acid metabolic pathway;

[0038] Figure 15 This is a statistical diagram of the VIP values of differential metabolites in the ABC transport metabolic pathway;

[0039] Figure 16 This is a statistical chart of VIP values of differential metabolites in the caffeine metabolic pathway;

[0040] Figure 17 It is the metabolic pathway interaction network diagram;

[0041] Figure 18 VIP value statistics of differential metabolites in cysteine and methionine metabolic pathways;

[0042] Figure 19 Statistical graph of VIP values of differential metabolites in glycine, serine and threonine metabolic pathways. DETAILED DESCRIPTION

[0043] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0044] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0045] In the following examples, the strain number of Shewanella oneidensis MR-1 used is ATCC700550;

[0046] The preparation method of LB liquid medium is as follows: 5g / L beef extract, 10g / L peptone, 5g / L NaCl, and the pH value is adjusted to 7.2. The medium is sterilized under high pressure for 20 minutes and then cooled. When preparing LB solid medium, 20g / L agar is added to the LB liquid medium and then sterilized. After sterilization, the medium is poured into the culture dish to ensure that it is handled before it solidifies. After completing the above steps, the medium is placed in a 37℃ constant temperature incubator for 24 hours to observe whether it has been successfully sterilized.

[0047] The composition ratio of the inorganic mineral medium is: 2.5g / L NaHCO3, 0.25g / L NH4Cl, 0.1g / L KCl, 0.1g / L NaCl, 0.04g / L KHPO4, 0.2g / L MgCl2·6H2O and 1.0g / L yeast extract;

[0048] Uranyl nitrate hexahydrate (≥99.9%) was provided by Hubei Chushengwei Chemical Co., Ltd.

[0049] RNA extraction kit, reverse transcription kit, and SYBR Green I were purchased from Novazom;

[0050] Carbon quantum dots (CQDs) were purchased from Jiangsu Xianfeng Nanotechnology Co., Ltd.

[0051] Example 1

[0052] A method for preparing a carbon quantum dot-Shewanella biohybrid system for uranium removal comprises the following steps:

[0053] Step 1: Inoculate S. oneidensis MR-1 into LB liquid medium at a 1% (v / v) inoculum and perform aerobic pre-culture (30°C, 150 rpm) until the logarithmic growth phase (OD 600 ≈0.6), and then added 2% (v / v) carbon quantum dot solution (concentration of 0.1 mg / mL), and continued to culture for 2 hours to achieve full interaction between CQDs and bacteria to obtain bacterial solution;

[0054] Step 2: The bacterial solution obtained in step 1 was transferred to an inorganic mineral culture medium with a volume ratio of 1:99. The culture was continued under aerobic conditions for 12 hours (30°C, 150 rpm), centrifuged at 8000 rpm for 10 minutes, and the precipitate was collected. The precipitate was then washed twice with an inorganic mineral culture medium to completely remove the LB liquid culture medium residue, thereby obtaining a carbon quantum dot-Shewanella biohybrid system for uranium removal, namely, S. oneidensis MR-1-CQDs.

[0055] Example 2

[0056] In this embodiment, modified carbon quantum dots are used to replace carbon quantum dots, and the remaining steps are the same as those in Example 1, including the following steps:

[0057] Step 1: Inoculate S. oneidensis MR-1 into LB liquid medium at a 1% (v / v) inoculum and perform aerobic pre-culture (30°C, 150 rpm) until the logarithmic growth phase (OD 600 ≈0.6), and then added 2% (v / v) modified carbon quantum dot solution (concentration of 0.1 mg / mL), and continued to culture for 2 hours to achieve full interaction between CQDs and bacteria to obtain bacterial solution;

[0058] Step 2: The bacterial solution obtained in step 1 was transferred to an inorganic mineral culture medium with a volume ratio of 1:99. The culture was continued under aerobic conditions for 12 hours (30°C, 150 rpm), centrifuged at 8000 rpm for 10 minutes, and the precipitate was collected. The precipitate was then washed twice with an inorganic mineral culture medium to completely remove the LB liquid culture medium residue, thereby preparing a carbon quantum dot-Shewanella biohybrid system for uranium removal, namely, S. oneidensis MR-1-CQDs-C.

[0059] Wherein, the preparation method of the modified carbon quantum dots is:

[0060] S11, adding 10 g of carbon quantum dots and 1 g of hyaluronic acid (2000 kDa) to 500 mL of deionized water, ultrasonically treating at 60 kHz and 300 W for 2 h, standing at room temperature for 12 h, filtering, and freeze-drying at -40 ° C to obtain pretreated carbon quantum dots;

[0061] S12. Add 10 g of pretreated carbon quantum dots to 100 mL of 10 mM Tris-HCl buffer (pH = 8.5), stir evenly, then add 1.5 g of dopamine hydrochloride, stir at a constant temperature of 40 ° C for 3 h, dialyze with a 1 kDa dialysis membrane for 3 h, and freeze-dry the non-permeated liquid at -40 ° C to obtain modified carbon quantum dots.

[0062] Example 3

[0063] In this embodiment, no pretreatment was performed when preparing the modified carbon quantum dots, and the remaining steps were the same as in Example 2 to prepare a carbon quantum dot-Shewanella biohybrid system for uranium removal, namely S. onei densis MR-1-CQDs-C1; wherein, the preparation method of the modified carbon quantum dots was as follows: 10 g of carbon quantum dots was added to 100 mL of 10 mM Tris-HCl buffer (pH = 8.5), stirred evenly, and then 1.5 g of dopamine hydrochloride was added. The mixture was stirred at a constant temperature of 40 ° C for 3 h, dialyzed with a 1 kDa dialysis membrane for 3 h, and the non-permeated liquid was freeze-dried at -40 ° C to obtain modified carbon quantum dots.

[0064] Example 4

[0065] In this embodiment, pretreated carbon quantum dots were used to replace carbon quantum dots, and the remaining steps were the same as in Example 1 to prepare a carbon quantum dot-Shewanella biohybrid system for uranium removal, namely S. oneid ensis MR-1-CQDs-C2; wherein, the preparation method of the pretreated carbon quantum dots was as follows: 10 g of carbon quantum dots and 1 g of hyaluronic acid (2000 kDa) were added to 500 mL of deionized water, ultrasonically treated at 60 kHz and 300 W for 2 h, allowed to stand at room temperature for 12 h, filtered, and freeze-dried at -40 ° C to obtain pretreated carbon quantum dots.

[0066] Uranium removal experiment: The carbon quantum dots-Shewanella biohybrid system was resuspended in fresh inorganic mineral culture medium and the OD 600 To 0.8, an inoculum of 1.0% (v / v) was inoculated into two culture media: an experimental group containing 100 mg / LU(VI) (added with 1 mL of a 1.0 g / L UO2(NO3)2·6H2O solution) and a control group without U(VI). Both groups were incubated anaerobically at 30°C for 12 days. The experiment involved three replicates of S. oneidensis MR-1-CQDs and S. oneidensis MR-1. After incubation, the bacterial pellet was collected for protein extraction. All operations were performed in an anaerobic glove box.

[0067] The morphology of the S. oneidensis MR-1-CQDs biohybrid system constructed in Example 1 was observed by SEM: First, the sample to be tested was evenly dispersed in anhydrous ethanol solvent to form a suspension. The suspension was accurately added to the surface of the pretreated aluminum foil substrate using a micropipette and allowed to stand at room temperature to allow the solvent to completely evaporate. Subsequently, a gold film was deposited on the sample surface using an ion sputtering instrument for 30 seconds. During electron microscopy observation, the system operating voltage was maintained at a constant 10kV. The results are shown in Figure 2. Figure 1 As shown in Figure 2, the surface of S. oneidensis MR-1 is relatively smooth, and no obvious particles are observed ( Figure 1a). Spherical protrusions and floccules were found on the surface of the carbon quantum dots-Shewanella biohybrid system ( Figure 1 b) The appearance of these structures may be related to the secretion of extracellular polymeric substances (EPS), which plays a key role in extracellular electron transfer (EET). Specifically, EPS is a key medium for extracellular electron transfer in S. oneidensis MR-1, and changes in its composition and structure may directly affect the efficiency of electron transfer. The spherical protrusions and floccules observed in the biohybrid system may be areas of EPS enrichment, and their formation may be related to the regulation of cellular metabolism and electron transfer processes by nanomaterials.

[0068] In order to further explore the changes in EPS components, the present invention used three-dimensional fluorescence spectroscopy (3D-EEM) for comparative analysis. Figure 2 As shown, Figure 2 a is the three-dimensional fluorescence spectrum of S. oneidensis MR-1, Figure 2 b shows the three-dimensional fluorescence spectrum of S. oneidensis MR-1-CQDs. Two major fluorescence peaks were detected at Ex / Em ratios of 260 / 325 nm and 220 / 325 nm, corresponding to aromatic protein-like substances (tyrosine-like) and soluble microbial metabolites (tryptophan-like), respectively. Two major fluorescence peaks at 260 / 325 nm and 220 / 325 nm were also observed in the S. oneidensis MR-1-CQDs biohybrid system. The addition of carbon quantum dots enhanced the fluorescence peaks at these locations. Furthermore, a new fluorescence peak at Ex / Em ratios of 245 / 380 nm was observed, which may be associated with humic-like substances. This fluorescence peak is typically associated with small molecular weight, high fluorescence efficiency organic compounds. These organic compounds are resistant to photolysis and contain carbonyl-, carboxyl-, and phenol-like structures. Their weak aromaticity promotes electron transfer, which in turn improves uranium reduction efficiency.

[0069] In order to explore the potential effects of carbon quantum dots on microbial metabolism, it is necessary to analyze the cell activity in the biohybrid system. The present invention evaluated the growth activity of S. oneidensis MR-1, S. oneidensis MR-1-CQDs biohybrid system and S. oneidensis MR-1-CQDs biohybrid system after reaction with uranium by measuring the growth curve in LB liquid culture medium. Figure 3As shown, the bacteria maintained high viability under all three conditions. Specifically, the exponential growth rate of S. oneidensis MR-1-CQDs was slightly higher than that of the original wild-type S. oneidensis MR-1 strain. However, the growth rate of S. oneidensis MR-1-CQDs+U slowed after reaction with uranium, but gradually increased after 20 hours. The introduction of carbon quantum dots did not cause significant cytotoxicity to S. oneidensis MR-1. In contrast, the biohybrid system maintained high metabolic activity under uranium stress.

[0070] In order to visually display the living state of bacteria, the present invention uses confocal laser scanning microscopy (CLSM) for observation. Syto 9 is used as a green fluorescent dye to stain living cells, and the fluorescence signal of bacteria is observed using a fluorescence microscope under 485nm excitation. PI is used as an orange-red fluorescent dye to stain dead cells, and the fluorescence signal of bacteria is observed using a fluorescence microscope under 535nm excitation. Figure 4 As shown, S. oneidensis MR-1-CQDs exhibited green fluorescence after staining, indicating that the construction process of the biohybrid cells had little effect on cell activity, maintaining high activity. Furthermore, a green signal was also observed after reaction with uranium, demonstrating the high activity of the hybrid system cells. This result demonstrates that the introduction of carbon quantum dots not only enhances bacterial tolerance to uranium but also maintains the metabolic activity of the cells.

[0071] Carbon quantum dots (CQDs) can significantly enhance the extracellular electron transfer efficiency of microorganisms. The construction of a S. oneidensis MR-1-CQDs biohybrid system is of great significance for understanding the electron transfer mechanism in biogeochemical cycles. In order to systematically evaluate the uranium removal performance of the S. oneidensis MR-1-CQDs biohybrid system, the present invention conducted a 12-day uranium removal experiment. Figure 5 As shown, the S. oneidensis MR-1-CQDs biohybrid system prepared in Example 1 exhibited excellent uranium removal performance: on the eighth day of treatment, the uranium removal rate reached 100%, while the S. oneidensis MR-1 group ultimately removed only 80%. Therefore, the constructed S. oneidensis MR-1-CQDs biohybrid system significantly improved the uranium removal performance of S. oneidensis MR-1.

[0072] In order to further improve the uranium removal ability of the carbon quantum dot-Shewanella biohybrid system, the present invention also modifies the carbon quantum dots. First, the carbon quantum dots are treated with hyaluronic acid to improve biological performance, and then modified with polydopamine to increase surface active sites. The modified carbon quantum dots have better dispersibility and better reaction activity, which is beneficial for the subsequent combination with Shewanella to construct a biohybrid system, which is beneficial for the adsorption and reduction of uranium and the growth and metabolism of Shewanella in a uranium stress environment, thereby further improving the uranium removal efficiency. The S. oneidensis MR-1-CQDs-C prepared in Example 2 achieved a uranium removal rate of 100% on the 4th day of treatment, the S. oneidensis MR-1-CQDs-C1 prepared in Example 3 achieved a uranium removal rate of 100% on the 6th day of treatment, and the S. oneidensis MR-1-CQDs-C2 prepared in Example 4 achieved a uranium removal rate of 100% on the 7th day of treatment.

[0073] In order to explore the uranium removal mechanism of the S. oneidensis MR-1-CQDs biohybrid system, we analyzed the changes in its surface chemical properties by FTIR spectroscopy. Figure 6 As shown, compared with the wild-type S. oneidensis MR-1, the S. oneidensis MR-1-CQDs biohybrid system has a higher fluorescence intensity at 910 cm -1 The enhanced UO characteristic peak at the α-Hybrid ion indicates that CQDs modification enhances uranium fixation on the bacterial surface. Simultaneously, the intensities of functional groups such as OH, -COOH, -PO, and -NH2 in the S. oneidensis MR-1-CQDs biohybrid system also changed, indicating that CQDs modification effectively modulated the chemical properties of the cell surface, ultimately achieving a significant improvement in uranium removal efficiency.

[0074] In order to further reveal the composition and valence state of the biohybrid system after reaction with uranium, the XPS spectra of S. oneidensis MR-1 and S. oneidensis MR-1-CQDs biohybrid system after reaction with uranium were analyzed. Figure 7 As shown in the high-resolution XPS spectrum of U 4f, the peaks at 392.6 and 382.5 eV belong to U(VI), while the peaks at 392.6 and 381.6 eV belong to U(IV). Notably, the U(IV) peak area of the S. oneidensis MR-1-CQDs biohybrid system after uranium removal (78%) is significantly higher than that before uranium removal (66%), indicating that the uranium reduction efficiency of the biohybrid system is significantly improved.

[0075] Untargeted metabolomics analysis

[0076] ① Sample extraction:

[0077] Weigh 100 mg of the sample to be tested and add 400 μL of a pre-chilled (4°C) methanol-acetonitrile-water ternary extract (4:4:2 volume ratio). Vortex and oscillate for 3 minutes to thoroughly mix. Freeze the mixture at -20°C for 1 hour to precipitate the protein, then centrifuge at 14,000 g for 20 minutes at 4°C. Carefully aspirate 380 μL of the clarified supernatant, freeze-dry it under vacuum, and store it in a -80°C ultra-low temperature freezer. Prior to mass spectrometry analysis, resuspend the lyophilized sample in 100 μL of acetonitrile-water (1:1, v / v) resuspending solution, vortex for 30 seconds, and centrifuge at 14,000 g for 15 minutes at 4°C. Collect the supernatant for LC-MS / MS analysis.

[0078] ②Chromatography-mass spectrometry analysis:

[0079] Chromatographic separation was performed using an Agilent 1290 Infinity II ultra-high performance liquid chromatography system equipped with a hydrophilic interaction chromatography (HILIC) column to achieve compound separation. Specific chromatographic parameters were set as follows: column temperature was maintained at 25°C, mobile phase flow rate was constant at 0.5 mL / min, and injection volume was 2 μL. Mobile phase composition: Phase A was an aqueous solution containing 25 mM ammonium acetate and 25 mM ammonia, and Phase B was acetonitrile. A multi-step gradient elution program was used: initial phase (0-0.5 min) maintained at 95% Phase B; Phase B was linearly reduced to 65% within 0.5-7 min; Phase B was rapidly reduced to 40% Phase B within 7-8 min; isocratic elution was performed from 8-9 min; Phase B was rapidly returned to the initial ratio from 9-9.1 min; and column equilibration was performed from 9.1-12 min. All samples were injected in a random sequence using a 4°C temperature-controlled autosampler, and quality control (QC) samples were inserted regularly to evaluate instrument performance and data quality.

[0080] Mass spectrometry analysis was performed on an AB Triple TOF 6600 mass spectrometer. ESI source parameters were set as follows: Gas1 / Gas2 ratio 60, CUR 30, ion source temperature 600°C, and ISVF ±5500 V (positive and negative modes). The TOF MS scan range was 60–1000 Da, and the product ion scan range was 25–1000 Da, with scan accumulation times of 0.20 s and 0.05 s, respectively. Secondary mass spectrometry was performed in IDA mode under high sensitivity conditions, with a DP of ±60 V. Isotopic signals within 4 Da were excluded in the IDA setting, and 10 candidate ions were monitored per cycle.

[0081] ③Data processing:

[0082] Raw data were converted to .mzML format using ProteoWizard (v3.0.6428). Peak alignment, retention time correction, and peak area extraction were then performed using XCMS software (online 3.7.1). XCMS parameters were set as follows: for peak extraction, centWave m / z = 10 ppm, peakwidth = c(10,60), and prefilter = c(10,100); for peak grouping, bw = 5, mzwid = 0.025, and minfrac = 0.5. After data extraction, metabolite structure identification was performed, followed by data preprocessing (including null filtering (removing ion peaks with >50% missing values), null filling (using the KNN method), and data filtering (removing features with an RSD >50%). Finally, data analysis was performed.

[0083] In order to further explore the effects of carbon quantum dots on bacterial internal metabolic changes and electron transfer mechanisms, the present invention conducted non-targeted metabolomics analysis. Figure 8 a) It was found that the two data sets were significantly separated, indicating the reliability of the data and the repeatability of the experiment. In order to eliminate interference factors unrelated to the classification information, we performed orthogonal partial least squares discriminant analysis (OPLS-DA) on the characteristic substances in the two ionization modes. The analysis results showed that the model can clearly distinguish between the two groups of samples A (S. oneidensis MR-1) and B (S. oneidensis MR-1-CQDs) without overfitting ( Figure 8 b), further verifying the validity and discriminative ability of the metabolomics data.

[0084] In order to explore the metabolic response and regulatory mechanism of S. oneidensis MR-1 under the action of carbon quantum dots, we screened out 497 significantly different metabolites between group B and group A based on strict statistical analysis criteria (p < 0.05, VIP > 1, |log2FC| > 2). Figure 9 ). Among them, 374 metabolites were significantly upregulated in group B, and 123 metabolites were significantly downregulated. This result suggests that the introduction of carbon quantum dots has a significant impact on the metabolic process of bacteria, possibly by activating or inhibiting certain key metabolic pathways, thereby affecting the metabolic function of bacteria.

[0085] Through correlation analysis, we further explored the relationship between metabolite expression between S. oneidensis MR-1 and S. oneidensis MR-1-CQDs samples. The color depth in the figure represents the strength of the correlation, and the higher the value, the stronger the correlation. The results show that ( Figure 10), metabolite expression between the two groups showed significant positive and negative correlations, indicating complex interactions and mutual regulation between these metabolites. This mutual influence may reflect the extensive regulatory effect of carbon quantum dots on bacterial metabolic networks.

[0086] In addition, we classified the screened differentially expressed metabolites and calculated the proportions of each type of metabolite ( Figure 11 Among the top 10 metabolite categories, the top three categories were mainly lipids and lipid-like molecules (6.0%), organic acids and their derivatives (4.0%), and organic heterocyclic compounds (3.1%), further indicating that carbon quantum dots may have an effect on bacterial secondary metabolites or signaling molecules.

[0087] In order to measure the importance of differential metabolites in the two groups of samples, we performed VIP plot analysis on the key differential metabolites. The higher the VIP value, the greater the role of the metabolite in distinguishing the two groups of samples. The results showed that ( Figure 12 ), the differential metabolites with higher VIP values are mainly enriched in metabolites such as hypoxanthine, betaine, and phosphocholine. Among them, hypoxanthine is an important purine metabolite, involved in the purine metabolic pathway, and plays a key role in bacterial anti-oxidative stress. The research of Chen Xiaonan et al. shows that purine metabolites (such as uric acid) can effectively scavenge reactive oxygen species (ROS), thereby significantly reducing oxidative stress. Betaine is an important nutrient widely present in microorganisms, plants and animal tissues. It has multiple physiological functions in microorganisms, especially as an organic osmotic regulator under environmental stress, which can protect cells from damage caused by osmotic pressure changes. Phosphocholine is an important component of the cell membrane. As a precursor of phosphatidylcholine, it participates in the structural construction and functional regulation of the cell membrane. In summary, these differential metabolites played an important role in the metabolic differences between S. oneidensis MR-1 and S. oneidensis MR-1-CQDs mainly by alleviating oxidative stress, regulating osmotic pressure, and regulating the formation and function of cell membranes.

[0088] In addition, we further performed KEGG annotation on the differential metabolites and displayed the top 20 enriched pathways. Figure 13As shown, the differentially expressed metabolites were significantly enriched in the D-amino acid metabolism (ko00470), ABC transporters (ko02010), and caffeine metabolism (ko00232) pathways.

[0089] D-amino acid metabolism plays a key role in bacterial growth, biofilm formation and dispersion, and the regulation of peptidoglycan metabolism, providing physical and chemical protection for bacteria. Studies have shown that in Bacillus subtilis, D-amino acid metabolism promotes the formation of microbial biofilms and helps bacteria survive in cadmium-containing (Cd 2+ ) environment. Geobacter forms conductive nanowires (pili) in the biofilm. These nanowires can transfer electrons over long distances, transferring electrons from the inside of the cell to external receptors. Figure 14 As shown, seven metabolites are involved in this metabolic pathway, one of which (Histidine) is significantly upregulated. Histidine can act as a signaling molecule for biofilm formation, promoting microbial aggregation and biofilm formation on host surfaces. Therefore, carbon quantum dots (CQDs) may enhance the biofilm formation ability of S. oneidensis MR-1 by upregulating the expression of histidine metabolites in the D-amino acid metabolic pathway, further improving the efficiency of electron transfer between bacteria and thus helping them adapt to the external uranium stress environment.

[0090] The ABC transport system is a transmembrane transport mechanism that relies on ATP hydrolysis energy. It provides power for the transmembrane transport of nutrients and other substances by binding and hydrolyzing ATP. As an important effector for regulating bacterial functions, this system plays a key role in maintaining cellular metabolic homeostasis. Figure 15It was shown that there were 11 metabolites involved in this pathway, of which 3 metabolites were significantly upregulated: arginine, histidine, and N-acetylglucosamine (NAG). Arginine plays an important role in the early stages of biofilm formation by regulating pH. Studies have found that the biofilm formation process of Bifidobacterium bifidum, Bifidobacterium pseudocatenulatum, and Bifidobacterium longum is significantly associated with the biosynthesis of tyrosine, arginine, phenylalanine, lysine, and the galactose metabolic pathway. It is worth noting that histidine, as a key metabolite regulating biofilm formation, is also significantly enriched in the D-amino acid metabolic pathway. NAG is an important component of the bacterial cell wall and is involved in the synthesis and modification of peptidoglycan. Its upregulation may enhance the structural stability of the cell wall and help bacteria resist the toxicity of metal ions. Based on the above findings, carbon quantum dots (CQDs) may enhance the cell wall integrity and biofilm formation ability of S. oneidensis MR-1 by upregulating arginine, histidine and N-acetylglucosamine metabolites in the ABC transport pathway, thereby helping the bacteria better adapt and survive in metal stress environments.

[0091] The caffeine metabolic pathway is mainly mediated by xanthine oxidase and cytochrome P450 enzymes, which play a key role in the degradation of caffeine and its analogs. Studies have shown that there are at least 71 bacterial strains in the world that can degrade caffeine, among which Pseudomonas is the most intensively studied caffeine-degrading strain. These bacteria convert caffeine into xanthine through a series of metabolic reactions, which is then further oxidized into uric acid and finally converted into allantoin. This metabolic process enables bacteria to use caffeine as a carbon and nitrogen source, allowing bacteria to maintain growth and metabolic activity in complex environments. In the caffeine metabolic pathway, a total of 4 metabolites are involved, among which theophylline metabolite is significantly downregulated ( Figure 16 Theophylline is an alkaloid that affects gene expression and metabolic activity by regulating intracellular cAMP levels. In summary, CQDs may optimize bacterial energy metabolism and survival under uranium stress by altering caffeine metabolic pathways and redistributing bacterial carbon and nitrogen source utilization strategies.

[0092] To further explore the overall impact of CQDs on the metabolic regulation of S. oneidensis MR-1, based on the pathway enrichment results of differential metabolites, we further integrated the interaction relationships between different metabolic pathways and constructed a metabolic pathway interaction network diagram to reveal the core metabolic pathways and their regulatory mechanisms. By performing interaction network analysis on the enriched KEGG pathways, we found that many differential metabolic pathways had significant interaction relationships, forming a complex metabolic regulation network ( Figure 17Among them, ko00270 (Cysteine and methionine metabolism) and ko01100 (Metabolic pathways) pathways showed significant core positions in the interaction network.

[0093] The ko00270 pathway is related to the metabolism of sulfur-containing amino acids, plays an important role in protein synthesis, and is involved in glutathione synthesis, antioxidant defense, and one-carbon metabolism. Nine differential metabolites were enriched in this metabolic pathway, but none of them were significantly enriched ( Figure 18 Of particular note is S-methyl-5'-thioadenosine (MTA), which has the highest VIP score. This intermediate in the methionine cycle is involved in regulating methylation reactions and antioxidant defenses within cells. For example, it regulates glutathione (GSH) synthesis, scavenges reactive oxygen species (ROS), maintains cellular redox balance, and helps bacteria cope with metal ion-induced oxidative stress. Therefore, CQDs may affect glutathione synthesis by regulating cysteine and methionine metabolism, thereby altering cellular redox status and energy metabolism.

[0094] The ko01100 pathway, as the core of global metabolism, encompasses multiple metabolic processes, including carbohydrates, lipids, and amino acids. It may alter cellular energy supply, biosynthesis, and catabolism by regulating multiple metabolic pathways. In summary, CQDs may influence the redox state, energy metabolism, and biosynthesis and catabolism of S. oneidensis MR-1 by regulating core metabolic pathways such as ko00270 and ko01100.

[0095] In addition, the interaction network analysis also revealed interactions between some other pathways, such as glycine, serine and threonine metabolism (ko00260), D-amino acid metabolism (ko00470), etc. These pathways formed a tight network connection with the core pathways (such as ko00270 and ko01100) through shared metabolites or enzyme reactions. The glycine, serine and threonine metabolic pathway (ko00260) is an important precursor source for the synthesis of glutathione (GSH). Glutathione is an important antioxidant in cells that can effectively remove reactive oxygen species (ROS) induced by metal ions, thereby alleviating the damage caused to cells by oxidative stress. In this metabolic pathway, a total of 4 differential metabolites were enriched, but none of them reached a significant enrichment level ( Figure 19Notably, the metabolite with the highest VIP value was betaine (VIP: 33.4), which was also highlighted in a previous analysis of differential metabolite VIP values. Betaine has significant antioxidant activity, and its antioxidant mechanisms primarily include the following three aspects: activating antioxidant signaling pathways, regulating related enzyme levels, scavenging or inhibiting the production of reactive oxygen species (ROS), and regulating the body's osmotic pressure balance. Therefore, CQDs may further optimize the antioxidant mechanism of S. oneidensis MR-1 by regulating intracellular osmotic pressure, helping it improve its adaptability and survival in uranium-stressed environments.

[0096] In summary, metabolomics analysis of differential metabolite changes and enrichment screening of differential metabolic pathways revealed the regulatory effects of metabolite changes on gene expression in the S. oneidensis MR-1-CQDs biohybrid system. VIP plot analysis revealed that differential metabolites of hypoxanthine, betaine, and phosphocholine played key roles. Core metabolic pathways of differential metabolites were primarily enriched in D-amino acid metabolism (ko00470), ABC transporters (ko02010), and caffeine metabolism (ko00232). Carbon quantum dots promote biofilm formation by upregulating histidine in D-amino acid metabolism. Upregulating metabolites such as arginine, histidine, and N-acetylglucosamine (NAG) in the ABC transporter pathway enhances cell wall stability and biofilm formation, helping bacteria resist metal toxicity. Downregulation of theophylline metabolites redistributes the bacterial carbon and nitrogen utilization strategies, thereby optimizing its energy metabolism and survival under uranium stress. Furthermore, metabolic pathway interaction network analysis revealed that core metabolic pathways (such as cysteine and methionine metabolism and global metabolic pathways) maintain redox balance by regulating glutathione synthesis and antioxidant defense, helping the bacteria cope with metal-induced oxidative stress. Glycine, serine, and threonine metabolic pathways further optimize antioxidant mechanisms through metabolites such as betaine. Carbon quantum dots (CQDs) may enhance biofilm formation, antioxidant capacity, and energy metabolism of S. oneidensis MR-1 by regulating these metabolic pathways, thereby improving its adaptability and survival in metal-stressed environments. Therefore, CQDs in the S. oneidensis MR-1-CQDs biohybrid system enhance the biofilm formation, antioxidant capacity, and energy metabolism of S. oneidensis MR-1, thereby improving its adaptability and survival in metal-stressed environments.

[0097] Based on the theoretical framework of biogeochemical cycles, this invention innovatively utilizes nanoscale carbon quantum dots (CQDs) to in situ construct a carbon quantum dot-Shewanella biohybrid system, aiming to effectively enhance the uranium reduction tolerance of S. oneidensis MR-1. To further analyze the charge transfer mechanism of the biohybrid system, a combination of characterization methods, including scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR), were used to observe the effects of carbon quantum dots on S. oneidensis MR-1 under uranium stress and the changes in its performance. This confirmed that the material significantly enhances the uranium removal capacity of S. oneidensis MR-1. In addition, to evaluate the potential impact of carbon quantum dots on microbial metabolic activity, a systematic biocompatibility assessment was conducted using microbial growth kinetic curve measurements and live-dead cell fluorescence staining experiments. The results showed that the introduction of carbon quantum dots had no inhibitory effect on the microbial growth phenotype or cell viability. Based on non-targeted metabolomics analysis, it was found that carbon quantum dots may enhance the environmental adaptability of S. oneidensis MR-1 through three mechanisms: (1) promoting the secretion of extracellular polymers (EPS) and enhancing the ability to form biofilms; (2) increasing the activity of the antioxidant enzyme system; and (3) optimizing the energy metabolism pathway.

[0098] The present invention further reveals the changes in the endogenous metabolic network of bacteria under the regulation of carbon quantum dots, clarifies the molecular mechanism of electron transfer at the microorganism-nanomaterial interface, lays an important foundation for the development of new bioremediation technologies based on nanomaterial-microorganism coupling, and is of great significance for promoting scientific and technological progress in the field of radioactive pollution control.

[0099] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing a carbon quantum dot-Shewanella biohybrid system for uranium removal, characterized in that: The following steps are involved: Step 1: inoculate Shewanella into LB liquid medium for aerobic pre-culture until the logarithmic growth phase, then add the carbon quantum dot solution and continue culturing to obtain a bacterial solution; Step 2: Transfer the bacterial solution obtained in step 1 to an inorganic mineral culture medium, continue aerobic culture, centrifuge, collect the precipitate, and then wash it with an inorganic mineral culture medium to obtain a carbon quantum dot-Shewanella biohybrid system for uranium removal.

2. The method for preparing the carbon quantum dot-Shewanella biohybrid system for uranium removal according to claim 1, wherein: In the step 1, the Shewanella is Shewanella oneidensis MR-1.

3. The method for preparing the carbon quantum dot-Shewanella biohybrid system for uranium removal according to claim 1, wherein: In the step 1, Shewanella is inoculated into LB liquid culture medium at a volume percentage of 0.5-2.0%.

4. The method for preparing the carbon quantum dot-Shewanella biohybrid system for uranium removal according to claim 1, wherein: In the step 1, the aerobic pre-culture is carried out at 30° C. and 150 rpm.

5. The method for preparing the carbon quantum dot-Shewanella biohybrid system for uranium removal according to claim 1, wherein: In the step 1, the concentration of the carbon quantum dot solution is 0.05 to 0.2 mg / mL; the amount of the carbon quantum dot solution added is 1.0 to 3.0% by volume; and the culture is continued for 1 to 3 hours.

6. The method for preparing the carbon quantum dot-Shewanella biohybrid system for uranium removal according to claim 1, wherein: In the step 2, the volume ratio of the bacterial liquid to the inorganic mineral culture medium is 0.5-2:98-99.5; aerobic culture is continued at 30° C. and 150 rpm for 10-14 hours; centrifugation is performed at 6000-10000 rpm for 5-15 minutes; and washing is performed 1-3 times with the inorganic mineral culture medium.

7. The method for preparing the carbon quantum dot-Shewanella biohybrid system for uranium removal according to claim 1, wherein: In the step 2, the composition ratio of the inorganic mineral culture medium is: 2.5 g / L NaHCO3, 0.25 g / L NH4Cl, 0.1 g / L KCl, 0.1 g / L NaCl, 0.04 g / L KHPO4, 0.2 g / L MgCl2·6H2O and 1.0 g / L yeast extract.

8. The method for preparing the carbon quantum dot-Shewanella biohybrid system for uranium removal according to claim 1, wherein: In the step 1, modified carbon quantum dots are used to replace carbon quantum dots, and the preparation method of the modified carbon quantum dots is: S11, adding carbon quantum dots and hyaluronic acid to deionized water, ultrasonically treating for 1 to 3 hours, standing at room temperature for 6 to 18 hours, filtering, and freeze-drying at -30 to -80°C to obtain pretreated carbon quantum dots; S12. Add the pretreated carbon quantum dots to 10 mM Tris-HCl buffer, stir evenly, then add dopamine hydrochloride, stir at a constant temperature of 30-50°C for 2-5 hours, dialyze with a 1 kDa dialysis membrane for 2-5 hours, and freeze-dry the non-permeated liquid at -30-80°C to obtain modified carbon quantum dots.

9. The method for preparing the carbon quantum dot-Shewanella biohybrid system for uranium removal according to claim 8, wherein: In the S11, the molecular weight of hyaluronic acid is 100-8000 kDa; the mass volume ratio of carbon quantum dots, hyaluronic acid and deionized water is 10 g:0.5-2 g:300-700 mL; the ultrasonic power is 200-500 W, and the frequency is 50-80 kHz; In the S12, the mass volume ratio of the pretreated carbon quantum dots, dopamine hydrochloride and Tris-HCl buffer is 10 g: 1-3 g: 50-150 mL.

10. Use of a carbon quantum dot-Shewanella biohybrid system for uranium removal prepared by the preparation method according to any one of claims 1 to 9 in uranium removal.

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