Copper sulfide nanoparticles-roseobacter hybrid system and applications thereof

Abstract

The application belongs to the technical field of organic pollutant treatment, and particularly relates to a copper sulfide nanoparticle-Roseobacter hybrid system and application thereof. The application is based on in-situ generation of copper sulfide nanoparticles induced by Roseobacter DMSP metabolism, and constructs a photocatalysis-microorganism interface coupling system. Under light conditions, the metabolism process of Roseobacter is enhanced through photo-generated electrons, so that the degradation rate of o-diphenol and the biomass of the bacteria are significantly improved, the synchronization of pollutant degradation and microbial proliferation is realized, and a new technical approach is provided for the biological-photocatalytic collaborative treatment of phenolic pollutants.

Description

Technical Field

[0001] This invention belongs to the field of organic pollutant treatment technology, specifically relating to a copper sulfide nanoparticle-Roseobacterium hybrid system and its application. Background Technology

[0002] Catechol (CAT) is an important class of aromatic organic compounds widely used in industrial production processes such as photography, dyes, rubber, plastics, and pharmaceuticals, and can enter the aquatic environment through industrial wastewater discharge. Studies have shown that CAT is toxic to various aquatic organisms, laboratory animals, and related human cells, and its continuous discharge may pose potential risks to aquatic ecosystems and human health. At the same time, CAT is also a key intermediate product in the biodegradation of many aromatic organic pollutants. In the microbial degradation of aromatic compounds such as benzene, toluene, phenol, and benzoic acid, pollutants are typically converted into CAT or protocatechuic acid through reactions such as hydroxylation, and then further enter downstream metabolic pathways through aromatic ring cleavage reactions. Therefore, improving the efficiency of CAT conversion is not only beneficial for its own pollution removal but also helps promote the further degradation of various aromatic pollutants.

[0003] Microbial degradation technology, due to its mild reaction conditions, good environmental compatibility, and selectivity for specific organic pollutants, has great potential for application in the treatment of aromatic pollutants. However, single microbial systems still have certain limitations in practical applications. On the one hand, the degradation of aromatic compounds by microorganisms usually relies on intracellular enzymatic oxidation and continuous metabolic processes. Limited by cellular metabolic activity and energy supply capacity, the pollutant conversion rate and treatment efficiency need to be improved. On the other hand, intracellular reducing forces such as NADH and NADPH are mainly produced by the microorganisms themselves, and their supply level is limited, making it difficult to continuously meet the electron and energy demands of high-intensity pollutant conversion processes. Therefore, how to enhance the electronic processes at the microbial interface and intracellular metabolism through exogenous functional materials is an important technical direction for improving the bioconversion efficiency of aromatic pollutants.

[0004] In recent years, semiconductor nanomaterial-microbe hybrid systems have provided a new technological approach to improve pollutant conversion efficiency by combining the light absorption and photogenerated carrier generation capabilities of semiconductor materials with the selective catalytic metabolism of microorganisms. Under illumination, semiconductor materials can generate photogenerated electrons and holes, and influence microbial metabolism through interfacial electron transfer, reactive oxygen species generation, or regulation of intracellular redox processes, thereby potentially enhancing the oxidative conversion and subsequent biodegradation of aromatic pollutants. Among these, metal sulfide-microbe hybrid systems exhibit good visible light responsiveness and photoelectrochemical activity, showing potential for application in constructing photoresponsive microbe hybrid systems.

[0005] However, current methods for preparing metal sulfides typically use inorganic or chemically sulfur-containing precursors such as sodium sulfide, sodium hydrosulfide, and cysteine ​​as sulfur sources. While the rapid release of reactive sulfur species upon addition to these sources promotes rapid metal sulfide formation, it can also lead to a sudden increase in local sulfide concentrations. This can cause rapid precipitation of metal ions in the culture medium or the formation of free particles, thereby reducing the in-situ formation and effective coupling of nanomaterials at the microbial interface. Furthermore, higher concentrations of reactive sulfides may inhibit microbial activity, hindering the balance between material synthesis and cell activity, and further impacting the conversion efficiency of organic pollutants. Summary of the Invention

[0006] To address the shortcomings of the existing technology, the purpose of this invention is to provide a copper sulfide nanoparticle-Roseobacterium hybrid system and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a copper sulfide nanoparticle-Roseobacterium hybrid system, wherein the copper sulfide nanoparticle-Roseobacterium hybrid system comprises copper sulfide nanoparticles and Roseobacterium, wherein the copper sulfide nanoparticles are generated in situ inside Roseobacterium cells.

[0008] A second aspect of the present invention provides a method for constructing the copper sulfide nanoparticle-Roseobacter hybrid system described in the first aspect, comprising the following steps: Rose Bacillus was inoculated into an activation medium and activated to obtain activated Rose Bacillus. The activated Rose Bacillus was inoculated into an acclimatization medium and acclimatized to obtain acclimatized Rose Bacillus; The domesticated Roseobacterium was inoculated into a hybrid culture medium and cultured to obtain a copper sulfide nanoparticle-Roseobacterium hybrid system; The activation medium is 2216E medium containing DMSP; The acclimatization medium was 2216E medium containing DMSP; The hybrid culture medium is MBM medium containing DMSP and CuCl2.

[0009] Furthermore, the *Roseobacterium* mentioned is *Roseobacterium marineum*.

[0010] Furthermore, the activation culture conditions are: 28~32℃, 170~190 rpm.

[0011] Furthermore, the conditions for the domestication and cultivation are: 28~32℃, 170~190 rpm.

[0012] Furthermore, the conditions for the hybrid culture are: 28~32℃, 70~90 rpm, incubation in the dark for 3~5 h.

[0013] Furthermore, the concentration of DMSP in the activation medium and acclimatization medium is 20 μM; the concentration of DMSP in the hybrid medium is 10 mM.

[0014] A third aspect of the present invention provides the application of the copper sulfide nanoparticle-Roseobacterium hybrid system described in the first aspect in the degradation of catechol.

[0015] A fourth aspect of the present invention provides a method for degrading catechol, comprising: adding catechol to the copper sulfide nanoparticle-Roseobacter hybrid system described in the first aspect, and incubating and culturing it under light conditions.

[0016] Furthermore, the light conditions were 2500 lux; the incubation conditions were 28~32℃ and 170~190 rpm.

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention utilizes DMSP metabolism to induce in situ generation of copper sulfide nanoparticles, constructing copper sulfide nanoparticles (CuS-NPs) and *Roseobacterium* (… Roseovarius nubinhibens In the ISM (Integrated Semiconductor Body of Sugar) hybrid system, under light irradiation, copper sulfide nanoparticles are photoexcited to generate photogenerated electron-hole pairs. These pairs enhance the metabolic activity of *Roseobacterium* and the ring-opening degradation of catechol through interfacial electron transfer. Catechol degradation is achieved via a meta-cleavage pathway, and the intermediate product is further converted into pyruvate, a small organic molecule that enters the tricarboxylic acid cycle. This results in a synergistic degradation of catechol, achieving a degradation rate of up to 98%. In contrast, the pure *Roseobacterium* system only achieves a 65% degradation rate of catechol. This demonstrates the significant advantages of this invention in the degradation of catechol.

[0018] This invention not only achieves efficient degradation of catechol but also promotes the growth of *Roseobacterium*. Experimental results show that the OD of the hybrid system... 600 Compared to the pure Rose Bacterium system OD 600 There was a significant increase, which was 1.5 to 1.8 times that of the pure Roseobacterium system. Attached Figure Description

[0019] Figure 1 The growth curves of Roseobacterium under different treatment conditions are shown in the range of 0–72 h.

[0020] Figure 2 OD of Roseobacterium cultured for 24 h under different treatment conditions 600 And the differences between groups; in the figure. Indicates the significant differences between each treatment group and the control group at the same time point: P <0.05, P <0.01.

[0021] Figure 3 For the control group and Cu 2+ +DMSP+Cys、Cu 2+ +DMSP、Zn 2+ +DMSP and Cd 2+ Under +DMSP treatment conditions, the change of catechol concentration over time from 0 to 72 h.

[0022] Figure 4 The figure shows the residual concentration of catechol after 24 h of culture under different treatment conditions and the differences between groups; Indicates the significant differences between each treatment group and the control group at the same time point: P <0.05, P <0.01.

[0023] Figure 5 The figure shows the fold change in the degradation of catechol to produce the intermediate 2-HMS. Indicates the significant differences between each treatment group and the control group at the same time point: P <0.05, P <0.01.

[0024] Figure 6 To verify the determination results of catechol degradation to pyruvic acid in the examples, the figure shows... Indicates the significant differences between each treatment group and the control group at the same time point: P <0.05, P <0.01.

[0025] Figure 7 The figure shows the gas phase results used in the example to verify whether DMSP was consumed. Indicates the significant differences between each treatment group and the control group at the same time point: P <0.05, P <0.01.

[0026] Figure 8 The TEM and mapping images used in this example to verify the synthesis of the hybrid are shown below. A is a transmission electron microscope (TEM) image of the hybrid system, with red arrows indicating high electron density nanoparticles within the bacteria. B is a high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image of the hybrid system. C is a surface distribution map of Cu, D is a surface distribution map of S, E is a superimposed surface distribution map of Cu and S, and F is a superimposed map of the HAADF-STEM image and the surface distribution of Cu and S, showing that Cu and S are spatially co-distributed in the bacterial cell region. Detailed Implementation

[0027] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0029] In a typical embodiment of the present invention, a copper sulfide nanoparticle-Roseobacterium hybrid system is provided, the copper sulfide nanoparticle-Roseobacterium hybrid system comprising copper sulfide nanoparticles and Roseobacterium, wherein the copper sulfide nanoparticles are generated in situ inside Roseobacterium cells.

[0030] The construction steps of the copper sulfide nanoparticle-Rosebacterium hybrid system include: Rose Bacillus was inoculated into an activation medium and activated to obtain activated Rose Bacillus. The activated Rose Bacillus was inoculated into an acclimatization medium and acclimatized to obtain acclimatized Rose Bacillus; The domesticated Roseobacterium was inoculated into a hybrid culture medium and cultured to obtain a copper sulfide nanoparticle-Roseobacterium hybrid system; The activation medium is 2216E medium containing DMSP; The acclimatization medium was 2216E medium containing DMSP; The hybrid culture medium is MBM medium containing DMSP and CuCl2.

[0031] The construction of metal sulfide-microbe hybrid systems depends not only on the type of metal ions and the characteristics of the microorganisms, but also on the selection of the sulfur source and its supply method, which are important factors affecting the nucleation site, particle formation process, biocompatibility and interfacial coupling effect of nanomaterials.

[0032] This invention hybridizes metal ions with Roseobacterium, which has the ability to metabolize dimethylsulfoniopropionate (DMSP). By using DMSP as a sulfur source, it avoids the rapid precipitation and insufficient biocompatibility caused by the instantaneous release of traditional exogenous sulfides. Furthermore, it drives the gradual generation of sulfur-containing species near the microbial interface through cellular metabolism, thereby increasing the local contact opportunities between metal ions and biologically derived sulfur-containing species and promoting the in-situ construction of metal sulfides at the cell interface.

[0033] Unlike the direct addition of inorganic sulfides, DMSP requires the microorganisms to metabolize and transform it into various sulfur-containing intermediates and reduced sulfur-containing species. Therefore, its sulfur source supply process can be coupled with the cellular metabolic process.

[0034] In the preparation method provided by this invention, DMSP, as an important sulfur-containing metabolic substrate of *Roseobacterium marineum*, not only serves to supply the sulfur element required for nanomaterial synthesis in the system, but also directly couples the microbial sulfur metabolism process with the metal nanoparticle formation process. Compared with the traditional method of pre-synthesizing nanomaterials and then mixing them with microorganisms or directly adding inorganic sulfides for chemical precipitation, this strategy makes the nanomaterial formation process more dependent on the metabolic state of the microorganisms themselves, thereby achieving a tight interfacial coupling between semiconductor materials and living cells, and enabling the metal nanoparticles to be generated in situ inside *Roseobacterium marineum* cells.

[0035] In this invention, the copper sulfide nanoparticle-Roseobacter hybrid system generates photogenerated electron-hole pairs under light irradiation. These pairs enhance the metabolic activity of Roseobacter and the ring-opening degradation reaction of catechol through interfacial electron transfer. The degradation of catechol is achieved through the meta-cleavage pathway, and the intermediate product is further converted into small molecule organic matter that enters the tricarboxylic acid cycle as pyruvate. This results in the synergistic degradation of catechol, significantly improving the degradation rate and bacterial biomass, and simultaneously enhancing pollutant degradation and microbial proliferation.

[0036] The method for constructing the copper sulfide nanoparticle-Roseobacter hybrid system is as follows: S1, Activation of Roseobacterium Inoculate at 1% (v / v) in activation medium to activate culture; the activation medium is 2216E medium containing 20 μM DMSP.

[0037] Furthermore, the activation culture conditions are: 28~32℃, 170~190 rpm.

[0038] S2, Domestication of Roseobacterium The activated Rose Bacillus was inoculated into the acclimatization medium at a ratio of 1% (v / v) to acclimatize and culture the Rose Bacillus to bring it into the DMSP metabolic activation state; the acclimatization medium was 2216E medium containing 20 μM DMSP.

[0039] Furthermore, the conditions for the domestication and cultivation are: 28~32℃, 170~190 rpm.

[0040] Construction of S3, copper sulfide nanoparticle-Rosebacterium hybrid system The domesticated Rose Bacillus was inoculated at a rate of 1% with an initial OD value. 600 The cells were inoculated at a density of 0.4 into MBM medium containing 10 mM MDMSP and 0.3 mM CuCl2 for hybrid culture. The sulfides produced by the DMSP metabolism of Roseobacterium sulfide were used to generate copper sulfide nanoparticles in situ inside the cells, thus constructing a copper sulfide nanoparticle-Roseobacterium hybrid system.

[0041] Furthermore, the conditions for hybrid culture are: 28~32℃, 70~90 rpm, incubation in the dark for 3~5 h.

[0042] This invention relates to the construction of a copper sulfide nanoparticle-Roseobacterium hybrid system. Without adding inorganic or chemical sulfur-containing precursors such as sodium sulfide, sodium hydrosulfide, or cysteine, it utilizes only DMSP as a biological sulfur precursor. Relying on the sulfur metabolism of Roseobacterium itself, copper sulfide nanoparticles are constructed in situ at the cell interface, balancing material synthesis and cell activity. The biodegradation efficiency of catechol is significantly better than that of hybrid systems with added cysteine, providing a new technical approach for the bio-photocatalytic synergistic treatment of phenolic pollutants.

[0043] In another specific embodiment of the present invention, the application of a copper sulfide nanoparticle-Roseobacterium hybrid system in the degradation of catechol is provided.

[0044] In another specific embodiment of the present invention, a method for degrading catechol is provided, comprising: adding catechol to a copper sulfide nanoparticle-Roseobacter hybrid system and incubating it under light conditions.

[0045] Furthermore, the light conditions were 2500 lux; the incubation conditions were 28~32℃ and 170~190 rpm.

[0046] The Roseobacterium used in the following examples is *Roseobacterium marineum*. Roseovarius nubinhibensISM was purchased from the China General Microbiological Culture Collection Center, with the number CGMCC 1.10669.

[0047] 2216E culture medium was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., product number: HB0132-1. The formulation of 2216E liquid culture medium is as follows: peptone 5.0 g / L, yeast extract 1.0 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, potassium bromide 0.08 g / L, strontium chloride 0.034 g / L, boric acid 0.022 g / L, sodium silicate 0.004 g / L, sodium fluoride 0.0024 g / L, ammonium nitrate 0.0016 g / L, and disodium hydrogen phosphate 0.008 g / L.

[0048] MBM medium (fresh mineral basal medium) is formulated based on previous literature with slight modifications (Wei et al., 2023): 11.68 g / L NaCl, 6.02 g / L MgSO4, 1 g / L yeast extract, 0.53 g / L NH4Cl, 0.75 g / L KCl, 1.11 g / L CaCl2, 0.17 g / L K2HPO4, 0.04 g / L FeEDTA, and 1 mg / L vitamins.

[0049] Dimethyl mercaptopropionic acid inner salt (DMSP), purchased from Aladdin, CAS No. 4337-33-1.

[0050] L-cysteine ​​hydrochloride, monohydrate, purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 7048-04-6.

[0051] Copper chloride (copper chloride dihydrate), purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 10125-13-0.

[0052] Zinc chloride, purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 7646-85-7.

[0053] Cadmium chloride, 2.5% water, purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 7790-78-5.

[0054] Catechol, purchased from Aladdin, CAS No. 120-80-9.

[0055] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0056] Example 1 This embodiment provides a method for preparing a copper sulfide nanoparticle-Roseobacterium hybrid system.

[0057] (1) Activation of Roseobacterium Remove the Rose Bacillus from the refrigerator and inoculate it into the activation medium at 1% (v / v). Place the inoculated culture in a constant temperature steam bath shaker at 30°C and 180 rpm for activation culture.

[0058] The activation medium was 2216E medium containing 20 μM DMSP.

[0059] (2) Domestication of Rosebacterium Take the activated Roseobacterium from step (1) and measure its OD at 600 nm absorbance. 600 When the concentration was 0.7, a bacterial suspension was inoculated into the acclimatization medium at a ratio of 1% (v / v). The inoculated culture was then placed in a constant temperature steam bath shaker and incubated at 180 rpm and 30°C. At OD... 600 The bacterial culture was harvested when the OD value was 1.2, and then purified by centrifugation: 600 The 1.2% Roseobacterium was aliquoted into 50 mL sterile centrifuge tubes and centrifuged at 8000 rpm for 5 min at 4°C. After discarding the supernatant, approximately 5 mL of MBM was added to each tube for resuspending. The mixture was then pipetted and stirred thoroughly. After resuspending twice, the bacterial suspensions from different centrifuge tubes were combined into a single 50 mL centrifuge tube and stirred thoroughly with a pipette for use in subsequent experiments.

[0060] The acclimatization medium was 2216E medium containing 20 μM DMSP.

[0061] (3) Preparation of copper sulfide nanoparticle-Rosebacterium hybrid system The purified Rose Bacillus from step (2) was inoculated at a rate of 1% with an initial OD value. 600 The cells were inoculated into the hybrid medium at an initial density of 0.4 and incubated in a constant temperature steam bath at 30°C and 80 rpm for 4 h in the dark to synthesize the hybrid.

[0062] The hybrid medium was MBM medium containing 10 mM DMSP and 0.3 mM CuCl2.

[0063] Comparative Example 1 This embodiment provides a method for preparing a copper sulfide nanoparticle-Roseobacterium hybrid system.

[0064] The experimental method is the same as in Example 1. The difference from Example 1 is that in step (3), the hybrid culture medium also contains 1 mM cysteine.

[0065] Comparative Example 2 This embodiment provides a method for preparing a zinc sulfide nanoparticle-Roseobacterium hybrid system.

[0066] The experimental method is the same as in Example 1. The difference from Example 1 is that in step (3), CuCl2 in the hybrid culture medium is replaced with ZnCl2.

[0067] Comparative Example 3 This embodiment provides a method for preparing a cadmium sulfide nanoparticle-Roseobacter hybrid system.

[0068] The experimental method is the same as in Example 1. The difference from Example 1 is that in step (3), CuCl2 in the hybrid culture medium is replaced with CdCl2.

[0069] Test case 1. Using OD 600 A 0.4 mM *Roseobacterium* bacterial culture was used as the control group. 250 μL of 2M catechol stock solution was added to the control group, the example, and the comparative example, bringing the final concentration to 5 mM. Incubation was then carried out under light at 30°C and 180 rpm in a 2500 lux incubator. Absorbance at 600 nm was measured every 24 hours, and growth curves of *Roseobacterium* under different conditions were plotted. Figure 1 As shown, all groups of bacteria entered a rapid growth phase in the first 24 hours of culture, after which the growth rate gradually decreased and stabilized. Among them, Cu... 2+ +DMSP group (Example 1) OD 600 The OD level remained high throughout the entire culture process, with a 24-hour OD value of [missing information]. 600 The value was approximately 4.7, and after 48 h it further increased to approximately 5.5 and remained stable, indicating that Cu 2+ The presence of Cu in conjunction with DMSP promotes the growth of Roseobacter. In contrast, Cu... 2+ The cell growth of the +DMSP+Cys group (Comparative Example 1) was significantly inhibited, and the OD at 24 h was [missing data]. 600 The concentration was approximately 1.7, significantly lower than the control group, and remained at a low level throughout subsequent culture. (Zn) 2+ The growth trend of the +DMSP group (Comparative Example 2) was similar to that of the control group, while the Cd group... 2+ +DMSP group (Comparative Example 3) OD 600 Slightly higher than the control group. Figure 2 As shown, after 24 h of cultivation, Cu 2++DMSP group OD 600 Significantly higher than the control group ( P <0.01), approximately 1.6 times that of the control group; Cu 2+ +DMSP+Cys group's OD 600 It was significantly lower than the control group ( P <0.01), approximately 0.6 times that of the control group. These results indicate that DMSP, as a sulfur source, reacts with Cu... 2+ When added together, they exhibit good biocompatibility and can maintain or even promote the growth of Roseobacterium. However, the addition of cysteine ​​did not produce the same effect; instead, it significantly inhibited bacterial growth.

[0070] 2. Metabolite analysis was performed on the CuS-Rosebacterium hybrid systems prepared in the examples and comparative examples.

[0071] With OD 600 A 0.4 mM Roseobacterium bacterial suspension served as the control group. 250 μL of 2M catechol was added to the control group, the examples, and the comparative examples to bring the final concentration to 5 mM. The mixture was then incubated under light at 30°C and 180 rpm in a 2500 lux incubator. Periodically, 1 mL of bacterial suspension was collected from each treatment group. The suspension was centrifuged at 10,000 rpm for 5 minutes at 4°C to obtain the supernatant, which was then filtered through a 0.22 μm membrane to obtain the filtrate supernatant.

[0072] (1) Determination of catechol The concentration of residual catechol in the filtrate supernatant was determined using an ultra-high performance liquid chromatography (UPLC, Agilent 1290 Infinity, USA) system equipped with a UV-Vis detector and a C18 column (Waters μ Bondapack C18 column (3.9 × 300 mm)). The mobile phase consisted of mobile phase A (10 mM KH₂PO₄ buffer, pH 3) and mobile phase B (methanol), with a mobile phase ratio of A:B = 8:2. The flow rate was 1 mL / min, the injection volume was 10.0 μL, the detection wavelength was 277 nm, and the detection time was 6.5 min. Figure 3 As shown, the overall concentration of catechol in all treatment groups decreased with increasing culture time, but the rate of decrease and residual levels varied significantly among different treatments. Specifically, Cu... 2+ The +DMSP group exhibited the fastest catechol removal rate, with catechol levels essentially dropping to near the detection limit after 24 h of incubation, and maintaining near-complete removal for 48–72 h, indicating that Cu... 2+ The combination with DMSP significantly enhanced the early transformation of catechol by *Roseobacterium*. In contrast, the control group and Zn... 2++DMSP group and Cd 2+ The +DMSP group still contained a certain amount of catechol at 24 h, exhibiting a relatively slow and continuous degradation process. It is noteworthy that Cu... 2+ The +DMSP+Cys group showed similarities to Cu. 2+ The +DMSP group exhibited significantly different degradation behavior. After 24 h of culture, the residual concentration of catechol in this group was approximately 1.9 mmol / L, significantly higher than that in the control group. Even at 72 h, approximately 1.1 mmol / L remained, indicating that the addition of cysteine ​​did not further promote catechol degradation. Combined with the aforementioned cell growth results, Cu... 2+ The +DMSP+Cys treatment also showed lower cell growth levels, indicating that Cys, as an exogenous sulfur source, may alter Cu. 2+ The presence, sulfidation rate, or cellular availability of certain substances within the system may hinder the formation of a suitable microbial-nanomaterial interface environment. In contrast, DMSP, as an organic sulfur precursor actively metabolized by *Roseobacterium*, has its sulfur source supply process coupled with cellular metabolism, making it more conducive to achieving Cu production while maintaining cell viability. 2+ Effective binding with sulfur-containing species of biological origin. Therefore, DMSP exhibits better sulfur source compatibility compared to Cys. Figure 4 As shown, Cu 2+ The synergistic effect of the +DMSP group is the most prominent, while Zn 2+ The residual levels of catechol in the +DMSP group at 24 h were similar to those in the control group, and Cd 2+ Although the +DMSP group showed some promoting effect, it was still significantly weaker than Cu. 2+ The +DMSP group indicates that the type of metal ion is also an important factor affecting the system's performance. Among them, Cu... 2+ The combination with DMSP shows optimal system compatibility.

[0073] (2) Determination of 2-hydroxy-mucosinate semialdehyde (2-HMS) 2-HMS in the filtrate supernatant was determined using the method disclosed in the literature (Further exploitation of metabolic potential for catecholbiodegradation of Klebsiella sp. CD33). Quantification was performed by measuring the UV absorbance of 2-HMS at 375 nm. An increase of 1.2 in absorbance at 375 nm indicated the formation of 1 mM 2-hydroxy-mucofuranose semialdehyde. The concentration of 2-hydroxy-mucofuranose semialdehyde was calculated using the following formula: C= Where A is UV absorbance and c0 is 1.0 mM.

[0074] The results are as follows Figure 5 As shown, different combinations of metal ions and sulfur sources had a significant impact on the accumulation of 2-HMS, an intermediate product of catechol meta-cleavage, and significant differences were observed between each treatment group and the control group. P <0.01). The control group's 2-HMS level was normalized to 1, Cu 2+ The relative level of 2-HMS in the +DMSP+Cys treatment group increased to approximately 1.5 times that of the control group. Combined with the aforementioned results showing higher catechol residues and somewhat inhibited cell growth in this treatment group, this indicates that the addition of Cys still allows for the cleavage of the catechol aromatic ring and the generation of 2-HMS, but its subsequent metabolic transformation may be somewhat limited, leading to the accumulation of 2-HMS relative to the control group. This suggests that Cys, as an exogenous sulfur source, did not effectively enhance the continuous transformation process of catechol from aromatic ring cleavage to downstream metabolism. 2+ The DMSP-treated group had the lowest relative level of 2-HMS, only about 0.65 times that of the control group, which was significantly lower than the control group. Combined with the aforementioned Cu... 2+ The near-complete removal of catechol within 24 h under DMSP treatment suggests that the low relative level of 2-HMS in this group does not necessarily indicate inhibition of meta-ring-opening of catechol. It is more likely that the generated 2-HMS can be rapidly converted into downstream metabolites, making it difficult for them to accumulate continuously in the system. Therefore, Cu 2+ +DMSP treatment likely increased the overall metabolic flux from catechol ring-opening to downstream intermediate conversion, rather than just promoting the single-step 2-HMS generation. Zn 2+ +DMSP group and Cd 2+ The relative levels of 2-HMS in the +DMSP group were approximately 2.05 times and 1.85 times that of the control group, respectively, both significantly higher than those in the control group. Among these, Zn... 2+ The +DMSP group showed the highest relative level of 2-HMS. This suggests that Zn... 2+ and Cd 2+ Although the system can promote or maintain the aromatic ring cleavage of catechol, the further conversion of the generated 2-HMS to downstream metabolites is relatively slow, resulting in the accumulation of intermediate products. 2+ The +DMSP+Cys group mainly exhibited restricted overall degradation of catechol accompanied by 2-HMS accumulation; Zn 2+ +DMSP and Cd 2+ The +DMSP group showed some degree of catechol ring-opening and relatively obvious 2-HMS accumulation; while Cu 2+The +DMSP group showed rapid consumption of catechol and the lowest accumulation of 2-HMS, indicating that it may have a more efficient continuous metabolic capacity, promoting the further conversion of 2-HMS to downstream products. Combined with the cell growth and catechol degradation results, this further demonstrates that DMSP and Cu... 2+ The combination exhibits better system adaptability in maintaining the activity of Roseobacterium, promoting rapid ring-opening of catechol, and accelerating downstream metabolism, providing further evidence for the optimal selection of DMSP as a sulfur source and the construction of a CuS-Roseobacterium hybrid system.

[0075] (3) Determination of pyruvate The concentration of pyruvate in the culture medium was determined using a high-performance liquid chromatograph (LC-20A, Shimadzu, Japan) equipped with a C18 column and a UV-VIS detector. During culture, 1 mL of cell suspension was collected periodically, centrifuged at 10,000 rpm for 5 minutes at 4°C to obtain the supernatant, and acidified at a ratio of supernatant volume to 0.1 M H₂SO₄ volume of 1:9. After acidification, the sample was filtered through a 0.22 μm filter, and 0.2 mL of the sample was used for pyruvate determination. The determination parameters were: column temperature 40°C, mobile phase 0.05 M: NH₄H₂PO₄ (pH adjusted to 2.60-2.65 with H₃PO₄), flow rate 1 mL / min, detection wavelength 210 nm, and run time 4.5 minutes. Results are as follows: Figure 6 As shown, different metal ions and sulfur sources had a certain impact on the concentration of pyruvate in the system. The pyruvate concentration in the control group was approximately 0.26 mmol / L, and Cu... 2+ The +DMSP+Cys group had a concentration of approximately 0.24 mmol / L, and Zn 2+ The +DMSP group had a concentration of approximately 0.23 mmol / L, and Cd... 2+ The +DMSP group had a concentration of approximately 0.26 mmol / L, with little overall difference between groups. In contrast, Cu... 2+ The pyruvate concentration in the +DMSP group decreased to approximately 0.18 mmol / L, which was significantly lower than that in the control group. P < 0.05), indicating that Cu 2+ The synergistic effect with DMSP can significantly alter the dynamic equilibrium of downstream carbon metabolites in catechol degradation. Combined with the aforementioned catechol and 2-HMS detection results, it can be found that Cu... 2+ In the +DMSP group, catechol was essentially removed within 24 hours, with the lowest accumulation of 2-HMS and a lower pyruvate concentration compared to the control group. Since both 2-HMS and pyruvate are intermediate metabolites in the meta-cleavage pathway of catechol, their instantaneous concentrations are determined by both their formation and further consumption. Therefore, Cu... 2+The lower pyruvate concentration in the +DMSP group may indicate that the intermediate product was rapidly utilized after its formation, making it difficult for it to accumulate continuously in the culture system. 2+ The pyruvate concentration in the +DMSP+Cys group was close to that in the control group, indicating that the overall conversion rate of catechol was limited after the addition of Cys, and the ability of the generated intermediate products to be continuously converted into downstream carbon metabolism pathways was relatively weak. 2+ +DMSP and Cd 2+ Although the +DMSP group showed a higher accumulation of 2-HMS, the pyruvate concentration did not increase synchronously, indicating that different metal ions may have differential effects on different metabolic steps after catechol ring-opening, and the accumulation of intermediate products does not necessarily correspond to a higher level of downstream metabolism. Combining the changes in catechol, 2-HMS, and pyruvate, it can be seen that Cu... 2+ The +DMSP system does not simply promote the accumulation of a single intermediate, but more likely improves the overall conversion efficiency of catechol from aromatic ring cleavage to subsequent central carbon metabolism, enabling the rapid utilization of intermediates such as 2-HMS and pyruvate after their formation. This result further illustrates the interaction between DMSP and Cu... 2+ The combination exhibits good system compatibility and is a preferred option for DMSP as a bio-based sulfur precursor and Cu. 2+ As a metal precursor, it provides metabolic support for constructing CuS-Rosebacterium hybrid systems.

[0076] (4) Determination of DMSP Samples from different groups were collected after 24 h of incubation following the addition of catechol. For DMSP analysis, 3.5 mL of liquid sample was centrifuged at 10,000 rpm at 4 °C, and then filtered through a 0.22 μm filter. 3 mL of the filtrate was transferred to a new 5 mL centrifuge tube, and 30 μL of 50% sulfuric acid was added to remove pre-existing DMS and maintain the stability of the DMSP. The mixture was incubated at room temperature for 1 h. Subsequently, 2.4 mL of the sample was transferred to a 50 mL serum vial, and 5 M sodium hydroxide solution was added. The sample and 5 M NaOH were mixed at a 3:1 ratio and immediately sealed to lyse the DMSP into DMS. Liquid DMSP was used as the standard for gas chromatography analysis at concentrations of 80 μM, 160 μM, 320 μM, 640 μM, and 1280 μM, prepared using a serial dilution method. The standard processing method was the same as that for the samples. The released DMS was quantified using a gas chromatograph equipped with a flame ionization detector (FID) and an HP-5 (30 m × 0.32 mm × 0.25 μm) capillary column (Shimadzu). The results are as follows: Figure 7 As shown, the remaining concentration of DMSP in the control group was approximately 4.3 mmol / L; Cu 2+The residual concentration of DMSP in the +DMSP+Cys group decreased to approximately 3.5 mmol / L, significantly lower than that in the control group. P < 0.05), indicating that in Cu 2+ When co-existing with Cys, *Roseobacterium* significantly increased its consumption of DMSP. 2+ The residual DMSP concentration in the +DMSP group was approximately 4.5 mmol / L, which was close to and slightly higher than that in the control group, indicating that the overall DMSP consumption in this system was low; Zn 2+ +DMSP group and Cd 2+ The remaining DMSP concentrations in the +DMSP group were approximately 4.1 mmol / L and 3.9 mmol / L, respectively, which are between Cu 2+ +DMSP group and Cu 2+ The presence of DMSP and Cys groups indicates that different metal ions have varying degrees of regulatory effects on DMSP metabolism in *Roseobacterium*. Notably, increased DMSP consumption does not equate to enhanced catechol degradation. Combined with the aforementioned catechol degradation results, Cu... 2+ Although the +DMSP+Cys group showed higher DMSP consumption, its catechol degradation efficiency was significantly lower than that of Cu. 2+ The +DMSP group indicates that the additional addition of Cys may have altered the allocation of cellular sulfur metabolism, causing more DMSP to be used for cellular sulfur metabolism, stress regulation, or other metabolic processes, rather than being effectively converted into functional outputs that promote catechol degradation. In contrast, Cu 2+ Although the +DMSP group had a lower overall DMSP consumption, it exhibited the highest catechol removal efficiency, indicating that this system may have a higher DMSP utilization efficiency. This means that less DMSP consumption is needed to meet the requirements for in-situ construction of CuS nanoparticles and the subsequent functionalization of the hybrid system. Further comparison of different metal ion treatments reveals that Zn... 2+ +DMSP and Cd 2+ Although the +DMSP group consumed DMSP to some extent, its catechol degradation capacity did not reach that of Cu. 2+ The +DMSP group level indicates that the performance of the hybrid system does not solely depend on the amount of DMSP consumed, but is closely related to the type of metal ions, the sulfur source conversion efficiency, and the interfacial coupling characteristics between the formed metal sulfides and the cells. 2+ The +DMSP system exhibits characteristics of low DMSP consumption and high catechol degradation, indicating that DMSP has high functional utilization efficiency in this system.

[0077] 3. TEM analysis was performed on the CuS-Rosebacterium hybrid system prepared in the examples.

[0078] Mapping of CuS-Rosebacterium heterozygotes yielded the following results: Figure 8 As shown. Figure 8 A in the image represents the TEM image of the hybrid system. It can be observed that *Roseobacterium* maintains a relatively complete cell outline, while multiple granular or aggregated structures with high electron density are distributed inside the cell. The red arrows in the image indicate the hybrid particles. Figure 8 B in the diagram represents the HAADF-STEM image of the same region. Figure 8 C in the figure represents the surface distribution of Cu. The Cu signal is mainly distributed in the cell region, with localized enrichment at the cell periphery and some internal areas, while the Cu signal in the background region outside the cell is relatively low. This result indicates that the addition of Cu to the system... 2+ A significant portion of them bind to bacterial cells or accumulate near the cell interface. Figure 8 D in the diagram represents the surface distribution of sulfur (S). The S signal shows significant enrichment throughout the bacterial cell, particularly concentrated at the cell periphery. This indicates that sulfur-containing compounds produced by the metabolism of DMSP by *Roseobacterium* can accumulate near the cell, providing a sulfur source for the in-situ formation of metal sulfides. The pronounced distribution of S around the cell also suggests a strong correlation between sulfur source supply and *Roseobacterium*'s own metabolism and cell-interface processes. Figure 8 E in the figure represents the superposition surface distribution of Cu and S elements. There is significant spatial overlap between Cu and S signals in the cell regions, particularly at the cell periphery, where the two elements exhibit good co-localization. The simultaneous enrichment of Cu and S in the same spatial region suggests that the two elements are not completely independent distributions and may form CuS particles. This result indicates that sulfur-containing species from DMSP participate in Cu... 2+ The sulfidation and formation of Cu-S nanoparticles provide important elemental evidence. Figure 8 F in the figure represents the overlay of the HAADF-STEM image with the surface distributions of Cu and S elements. It can be seen that the regions co-enriched by Cu and S have a good spatial correspondence with the highlighted areas in the HAADF image. This result further indicates that the cell-associated high electron density particles are mainly composed of inorganic components containing Cu and S. In summary, *Roseobacterium* can be enriched in DMSP and Cu... 2+ Under the condition of coexistence, Cu and S elements are enriched and spatially co-located near the cell interface, forming obvious high electron density nanoparticles.

[0079] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A copper sulfide nanoparticle-Roseobacterium hybrid system, characterized in that, The copper sulfide nanoparticle-Roseobacterium hybrid system includes copper sulfide nanoparticles and Roseobacterium, wherein the copper sulfide nanoparticles are generated in situ inside Roseobacterium cells.

2. The method for constructing the copper sulfide nanoparticle-Roseobacterium hybrid system according to claim 1, characterized in that, Includes the following steps: Rose Bacillus was inoculated into an activation medium and activated to obtain activated Rose Bacillus. The activated Rose Bacillus was inoculated into an acclimatization medium and acclimatized to obtain acclimatized Rose Bacillus; The domesticated Roseobacterium was inoculated into a hybrid culture medium and cultured to obtain a copper sulfide nanoparticle-Roseobacterium hybrid system; The activation and acclimatization medium was 2216E medium containing DMSP; The hybrid culture medium is MBM medium containing DMSP and CuCl2.

3. The construction method as described in claim 2, characterized in that, The Roseobacterium mentioned is marine Roseobacterium.

4. The construction method as described in claim 2, characterized in that, The activation culture conditions are: 28~32℃, 170~190 rpm.

5. The construction method as described in claim 2, characterized in that, The conditions for domestication and cultivation are: 28~32℃, 170~190 rpm.

6. The construction method as described in claim 2, characterized in that, The conditions for hybridization culture are: 28~32℃, 70~90 rpm, incubation in the dark for 3~5 h; the conditions for culture after the formation of the hybrid are 2500 lux, 28~32℃, 170~190 rpm.

7. The construction method as described in claim 2, characterized in that, The concentration of DMSP in the activation and acclimatization media is 20 μM; the concentration of DMSP in the hybrid medium is 10 mM.

8. The application of the copper sulfide nanoparticle-Roseobacterium hybrid system according to claim 1 in the degradation of catechol.

9. A method for degrading catechol, characterized in that, include: The catechol was added to the copper sulfide nanoparticle-Roseobacter hybrid system described in claim 1 and incubated under light conditions.

10. The method as described in claim 9, characterized in that, The light conditions were 2500 lux; the incubation conditions were 28~32℃ and 170~190 rpm.

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