A sulfur-copper nanobiological hybrid, and a preparation method and application thereof

Abstract

The application discloses a kind of sulfur copper nanobiological hybrid and its preparation method and application;The application is by electroactive bacteria inoculation to the LB liquid medium containing copper salt, anaerobic culture, obtain sulfur copper nanobiological hybrid;The LB liquid medium containing copper salt includes copper salt, lactic acid salt, yeast extract, trypsin and sodium chloride;The concentration of copper salt in the LB liquid medium containing copper salt is 0.25-0.5mM.The preparation process of the application does not depend on exogenous addition or endogenous production of toxic, easily dispersed H2S, improves the operation safety and environmental friendliness.No need to add cysteine and other expensive reagents as sulfur source or detoxification agent, greatly reduces the cost of raw materials;The hybrid obtained shows excellent performance in nitrogen recovery, and the overall nitrogen recovery efficiency is significantly higher than that of single bacteria or Cu 2‑x S material, with good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of water treatment technology, specifically relating to a copper-sulfur nanobio-hybrid, its preparation method, and its application. Background Technology

[0002] Industrial activities, such as mining, metallurgy, electroplating, and electronics manufacturing, generate large amounts of copper-containing wastewater, which contains copper ions (Cu). 2+ Concentrations range from a few milligrams per liter to over ten thousand milligrams per liter. Direct discharge of such wastewater without effective treatment poses a serious threat to ecosystems and human health. Therefore, developing technologies that can efficiently remove copper ions while also enabling copper resource recovery and high-value utilization has significant environmental and economic benefits, and is expected to partially offset wastewater treatment costs while supplementing copper resources.

[0003] Currently, the mainstream technologies for recovering copper from wastewater include physicochemical methods such as electrowinning and adsorption. While these technologies are mature, they generally suffer from drawbacks such as high energy consumption, large chemical reagent consumption, and high operating costs. In contrast, biological recycling, as an emerging green technology, has attracted much attention due to its mild reaction conditions, environmental friendliness, and the elimination of the need for large amounts of harmful chemical reagents. Particularly noteworthy is that during the transformation of metal ions, microorganisms often tightly bind with the generated metal particles (such as sulfides and oxides), forming natural "material-microorganism" hybrids in situ. Studies have shown that such hybrids may exhibit unique properties beyond those of a single component. For example, ferrous sulfide (FeS) can enhance the extracellular electron transport capacity of microorganisms, and cadmium sulfide (CdS) can even endow non-photosynthetic bacteria with light-harvesting properties. This means that if the recycling process can be combined with the construction of functional hybrids while maintaining microbial activity, potentially valuable biomaterials can be directly obtained, achieving "waste treatment of waste, turning waste into treasure."

[0004] In existing technologies, the method of using microbial reduction of sulfate to generate hydrogen sulfide (H2S) to precipitate copper ions is considered promising, as it can produce copper sulfide (CuS or Cu2S) nanomaterials with stable structures and diverse catalytic properties. However, this method has significant technical bottlenecks: firstly, H2S itself is highly toxic and easily dispersed, posing risks to operational safety and the environment; secondly, the process usually requires the addition of expensive cysteine ​​as a detoxifying agent and sulfur source, further increasing operating costs. It is worth noting that studies have found that *Geobacterium thioreductoides* (… Geobacter sulfurreducens Cu can be directly reduced and precipitated without relying on H2S. 2+ (Kimber RL, Bagshaw H, Smith K, et al. Biomineralization of Cu2S Nanoparticles by Geobacter sulfurreducens[J]. Applied and Environmental Microbiology, 2020, 86:e00967-20. However, the conversion efficiency of this technology is low (in the initial Cu...). 2+ At a concentration of 50 μM, the maximum conversion rate is only 63%, and the recovery cycle is too long (up to 72 hours), making it difficult to meet the efficiency and economic requirements of practical wastewater treatment. Therefore, developing a new biotechnology that can overcome the above-mentioned shortcomings and integrate efficient removal, resource recovery, and material utilization has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a copper-sulfur nanobiohybrid, its preparation method, and its applications. The preparation method of this invention does not rely on H2S and can rapidly prepare the copper-sulfur nanobiohybrid. This method is well-suited for practical copper-containing wastewater and can directly utilize actual wastewater as a reaction medium to achieve copper resource recovery. The copper-sulfur nanobiohybrid prepared by this invention can efficiently recover nitrogen resources (NO3) from wastewater. - ).

[0006] The technical solution adopted by this invention to solve its technical problem is: This invention provides a method for preparing copper-sulfur nanobiohybrids, comprising the following steps: Electroactive bacteria were inoculated into LB liquid medium containing copper salts and cultured anaerobically to obtain copper-sulfur nanobio-hybrids; the LB liquid medium containing copper salts included copper salts, lactate, yeast extract, tryptone and sodium chloride; the concentration of copper salts in the LB liquid medium containing copper salts was 0.25-0.5 mM.

[0007] Preferably, the electroactive bacteria are Oneida Shewanella or other bacteria.

[0008] Preferably, the initial OD600 value of the electroactive bacteria inoculated in LB liquid medium containing copper salt is 1.5 to 2.0.

[0009] More preferably, the initial OD600 optimal value is 2.0.

[0010] Preferably, the copper salt is at least one of copper nitrate, copper chloride, and copper sulfate.

[0011] More preferably, the copper salt is copper nitrate.

[0012] Preferably, the concentration of copper salt in the copper-containing LB liquid culture medium is 0.5 mM.

[0013] Preferably, the lactate is sodium lactate.

[0014] Preferably, the concentration of lactate in the copper-containing LB liquid culture medium is 15–25 mM.

[0015] More preferably, the concentration of lactate in the copper-containing LB liquid culture medium is 20 mM.

[0016] Preferably, the concentration of yeast extract in the copper-containing LB liquid culture medium is 3-8 g / L, the concentration of tryptone is 5-15 g / L, and the concentration of sodium chloride is 0.05-0.15 g / L.

[0017] More preferably, the concentration of yeast extract in the copper-containing LB liquid culture medium is 5 g / L, the concentration of tryptone is 10 g / L, and the concentration of sodium chloride is 0.1 g / L.

[0018] Preferably, the LB liquid culture medium containing copper salts may also contain other components that do not affect the activity of electroactive bacteria and Cu. 2+ The interaction with electroactive bacterial cells, and NO3 - With Cu 2+ The molar ratio is 0.5~3.

[0019] More preferably, the LB liquid culture medium containing copper salt contains NO3 - With Cu 2+ The molar ratio is 2.

[0020] More preferably, the copper-containing LB liquid culture medium may also contain citrate.

[0021] More preferably, the citrate is sodium citrate.

[0022] More preferably, the concentration of the citrate is 0~2mM.

[0023] More preferably, the copper-containing LB liquid culture medium may also contain nickel ions.

[0024] More preferably, the concentration of nickel ions is 0~0.2mM.

[0025] More preferably, the copper-containing LB liquid culture medium does not contain ethylenediaminetetraacetic acid dichloride.

[0026] More preferably, the ethylenediaminetetraacetic acid disalt is disodium ethylenediaminetetraacetic acid.

[0027] Preferably, the anaerobic culture is subjected to nitrogen aeration treatment beforehand.

[0028] More preferably, the nitrogen aeration treatment time is 15 to 20 minutes.

[0029] Preferably, the anaerobic culture is a closed static culture, the temperature of the anaerobic culture is 25-35℃, and the culture time is 48-96h.

[0030] More preferably, the anaerobic culture temperature is 30°C.

[0031] More preferably, the optimal anaerobic culture time is 48 hours.

[0032] Preferably, the solution of the sulfur-containing copper nanobio-hybrid obtained after anaerobic culture is centrifuged; the centrifugation conditions are 3000g and 5min.

[0033] This invention provides a copper-sulfur nanobiohybrid prepared by the above-described preparation method.

[0034] This invention provides an application of the above-mentioned copper-sulfur nanobiohybrid in nitrogen recovery.

[0035] Preferably, the copper-sulfur nanobiohybrid is suspended in a solution containing NO3. - Anaerobic treatment is carried out on the wastewater.

[0036] More preferably, the NO3-containing - The carbon-to-nitrogen ratio (COD / TN) in the wastewater is 3 to 8.

[0037] More preferably, the NO3-containing - NO3 in wastewater - The concentration is 2–6 mM.

[0038] More preferably, the NO3-containing - The wastewater contains 8–24 mM of carbon source.

[0039] More preferably, the temperature of the anaerobic treatment is 25–35°C.

[0040] More preferably, the temperature of the anaerobic treatment is 30°C.

[0041] More preferably, the anaerobic treatment is carried out in a closed environment.

[0042] More preferably, the anaerobic treatment is carried out under oscillation at 150 rpm for a reaction time of 48 h.

[0043] More preferably, the NO3-containing - The wastewater is also treated with denitrifying bacteria or anaerobic activated sludge with denitrification function.

[0044] More preferably, the NO3-containing - The wastewater is aerated with high-purity nitrogen for 15-20 minutes before anaerobic treatment.

[0045] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The entire preparation process of the copper-sulfur nanobiohybrid provided by this invention does not rely on exogenous addition or endogenous generation of toxic and easily dissipated H2S, significantly improving operational safety and environmental friendliness. It eliminates the need for expensive reagents such as cysteine ​​as sulfur sources or detoxifying agents, greatly reducing raw material costs. Under optimal conditions, Cu... 2+ The conversion cycle was shortened from 4 days to 2 days. Furthermore, this method can directly utilize copper-containing wastewater as the preparation medium to remove the pollutant Cu. 2+ The transformation of waste into high-value-added functional nanomaterials—microbial hybrids—achieved high-value resource recycling.

[0046] (2) The copper-sulfur nanoclusters (Cu(I)-S) generated in this invention are mainly coordinated by Cu(I)-S and have a long-range disordered structure. 2-x S), and mainly selectively deposited in the periplasmic space of bacteria, constructing a structurally stable bacterial-nanostructure hybrid. This hybrid system effectively controls NO3 in wastewater. - Reduced to NH4 + (i.e., nitrogen recovery) exhibits significantly enhanced performance. Its synergistic mechanism lies in the Cu located in the periplasm. 2-x S nanoclusters can serve as efficient electron transport channels, directly delivering intracellular electrons to nitrogen recovery-related functional enzymes, effectively reducing electron transmembrane transport resistance; on the other hand, Cu 2-x S nanoclusters themselves can generate NO3 - Reduced to NO2 - The synergistic effect of the two interactions between microorganisms and nanomaterials results in a significantly higher overall nitrogen recovery efficiency for the hybrid compared to bacteria or Cu alone. 2-x S material. Attached Figure Description

[0047] Figure 1 The graphs show the kinetics of copper removal in Example 1 and Comparative Examples 1 and 2 of this invention.

[0048] Figure 2 These are transmission electron microscopy (TEM) characterization images of the transformation products of Example 1 and Comparative Example 1 of the present invention. In the images, a and c represent TEM images of ultrathin bacterial sections, high-angle annular dark-field scanning TEM images, and energy-dispersive X-ray spectroscopy elemental distribution maps; b and d represent selected area electron diffraction (SID) patterns.

[0049] Figure 3The figures show the material science characterization of the conversion products of Example 1 and Comparative Example 1 of this invention. a represents the X-ray diffraction pattern; b represents the full X-ray photoelectron spectroscopy spectrum; c represents the S2p high-resolution X-ray photoelectron spectroscopy spectrum of pure Shewanella bacteria; e represents the Cu2p and S2p high-resolution X-ray photoelectron spectroscopy spectra of the products of Example 1 and Comparative Example 1.

[0050] Figure 4 These are the X-ray fine spectral correlation characterization diagrams of the conversion products of Example 1 and Comparative Example 1 of the present invention. a represents the Cu K-edge X-ray absorption near-edge structure spectrum; b represents the first derivative spectrum of the X-ray absorption near-edge structure spectrum; c represents k... 3 Weighted Fourier transform X-ray absorption fine structure spectrum; d represents the fitting curve of the corresponding X-ray absorption fine structure; e represents the corresponding wavelet transform contour plot.

[0051] Figure 5 The graphs show the kinetics of copper removal in Comparative Examples 3, 4, 5, and 6 of this invention.

[0052] Figure 6 This is a graph showing the kinetics of copper removal in Comparative Example 7 of the present invention.

[0053] Figure 7 This is a graph showing the kinetics of copper removal in Comparative Example 8 of the present invention.

[0054] Figure 8 The graphs show the kinetics of copper removal in Comparative Example 9 and Example 2 of this invention.

[0055] Figure 9 This is a graph showing the kinetics of copper removal in Comparative Example 10 of the present invention.

[0056] Figure 10 This is the X-ray diffraction pattern of the sample obtained in Comparative Example 10 of the present invention.

[0057] Figure 11 This is a material characterization diagram of the transformation product in Example 2 of the present invention. a represents a transmission electron microscope image of an ultrathin bacterial section, a high-angle annular dark-field scanning transmission electron microscope image, and an energy-dispersive X-ray spectroscopy elemental distribution map; b represents a selected area electron diffraction pattern; c represents an X-ray diffraction pattern; d represents the Cu K-edge X-ray absorption near-edge structure pattern; e represents the first derivative of the X-ray absorption near-edge structure pattern; f represents k... 3 Weighted Fourier transform X-ray absorption fine structure spectrum.

[0058] Figure 12 The bar chart shows the Cu content in the hybrids prepared in Example 1 and Comparative Example 1 of this invention, as well as the Cu and Ni content in the hybrid prepared in Example 2.

[0059] Figure 13 This is a graph showing the dynamic changes in substance concentration during the copper biotransformation process in each group of Example 3 of the present invention. Wherein, a represents Cu. + Concentration versus time curve; b represents Cu 2+ The graph shows the concentration of NO3 over time; c represents the concentration of NO3 under the condition that Cu(NO3)2 is the copper source. - NO2 - and NH4 + Concentration change over time.

[0060] Figure 14 This is a graph showing the change of S(-II) concentration over time in Example 3 of the present invention.

[0061] Figure 15 The pure Shewanella and N- bacteria in Example 4 of this invention So -Cu 2-x S and Cl- So -Cu 2-x S under high C / N ratio conditions NO3 - NO2 - and NH4 + A graph showing how concentration changes over time.

[0062] Figure 16 The pure Shewanella and N- in Comparative Example 11 of this invention So -Cu 2-x S and Cl- So -Cu 2-x S under low C / N ratio conditions NO3 - NO2 - and NH4 + A graph showing how concentration changes over time.

[0063] Figure 17 Pure Cu in Comparative Example 12 of this invention 2-x S material under high C / N ratio conditions NO3 - NO2 - and NH4 + A graph showing how concentration changes over time.

[0064] Figure 18 Cys- in Comparative Example 13 of this invention So -CuNC hybrid NO3 under high C / N ratio conditions - NO2 - and NH4 + A graph showing how concentration changes over time.

[0065] Figure 19 In Example 5 of this invention, AnS and So -Cu 2-xS's sequencing batch anaerobic reactor, over 22 cycles, reduced NO3 in the influent. - Concentration of NO3 in effluent - NO2 - NH4 + The concentration of NH4 + The trend chart of recovery rate changes.

[0066] Figure 20 For the present invention So -Cu 2-x A schematic diagram of nitrogen recovery. Detailed Implementation

[0067] The present invention will be further described below with reference to embodiments.

[0068] The following will clearly and completely describe the concept, specific solutions, and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. The various technical features in the present invention can be combined interactively without contradicting each other.

[0069] Example 1: A method for preparing a copper-sulfur nanobiohybrid, the specific steps of which are as follows: (1) Preparation of Shewanella seeds: Shewanella oneidensis MR-1 strain (ATCC® 700550) TM Shewanella (hereinafter referred to as *Shewanella*) was purchased from the American Type Culture Collection. The bacterial culture was preserved in glycerol at -80°C. Before the experiment, a loop was used to pick up the glycerol-preserved bacterial culture and streak it onto a solid agar plate for revival, then incubated at 30°C for 24-36 hours. Subsequently, single colonies were picked from the plate and inoculated into 100 mL of LB broth (containing 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride) and incubated at a constant temperature and shaking (30°C, 150 rpm) for 12 hours to obtain the bacterial culture. After centrifugation, the precipitate was resuspended in sterile physiological saline, and the process was repeated twice. (2) Preparation of anaerobic working medium: Dissolve 2.28g sodium lactate and 0.094g (about 0.5mM) copper nitrate in 1L of adjusted LB liquid medium (containing 5g / L yeast extract, 10g / L tryptone and 0.1g / L sodium chloride) and sterilize (121℃, 20min).

[0070] (3) Shewanella-Cu 2-xPreparation of S-nanocluster hybrids: The bacterial sludge from step (1) was resuspended in the anaerobic working medium of step (2), and the OD600 was adjusted to 2.0. The inoculated anaerobic working medium was dispensed into 50 mL serum bottles (25 mL per bottle), and the bottles were thoroughly aerated with nitrogen for 20 min. After sealing, the bottles were incubated at a constant temperature of 30℃ for 96 hours to obtain the hybrid solution. The supernatant was removed by centrifugation at 3000 g for 5 min to obtain the hybrid (denoted as N-). So .-CuNC).

[0071] Comparative Example 1: In Example 1, step (2) of the anaerobic working medium was replaced with 0.067 g (approximately 0.5 mM) of copper chloride, with all other parameters remaining unchanged. The anaerobic working medium inoculated with Shewanella was dispensed into 50 mL serum bottles (25 mL per bottle), thoroughly aerated with nitrogen for 20 min, sealed, and incubated at a constant temperature of 30°C for 96 h to obtain a heterozygous solution. The supernatant was removed by centrifugation at 3000 g for 5 min to obtain the heterozygote (denoted as Cl-). So .-CuNC).

[0072] Comparative Example 2: In Example 1, step (2) of the anaerobic working medium was replaced with 0.080 g (approximately 0.5 mM) of copper sulfate, with all other components remaining unchanged. The anaerobic working medium inoculated with Shewanella was dispensed into 50 mL serum bottles (25 mL per bottle), thoroughly aerated with nitrogen for 20 min, sealed, and incubated at a constant temperature of 30°C for 96 h to obtain a heterozygous solution. The supernatant was removed by centrifugation at 3000 g for 5 min to obtain the heterozygotes.

[0073] The kinetics of Cu removal from solution in Example 1 and Comparative Examples 1 and 2 are as follows: Figure 1 As shown in the figure. The results show that Example 1, i.e., the Cu(NO3)2 group, achieved a copper ion removal rate of 97.23% within 48 hours, and its removal kinetics were significantly faster than those of Comparative Example 1 and Comparative Example 2, which required approximately 96 hours to achieve a similar removal rate. It should be noted that in Comparative Example 2, Shewanella itself does not possess the ability to dissimilate SO42-. 2- With the ability to generate S(-II).

[0074] To confirm that the transformation product is a bacterial-copper nanostructure hybrid, Cu 2+ The distribution characteristics, structure, and chemical features of the biotransformation products in bacteria were systematically analyzed. The hybrids obtained in Example 1 and Comparative Example 1 were observed to be brownish-red (the original Shewanella was pinkish-red). Figure 2In examples a and c, both in Example 1 and Comparative Example 1, a large number of high-electron-density nanoparticle aggregates were observed in the region between the inner and outer membranes (i.e., the periplasmic space) within the bacterial cells. These nanoaggregates are tightly bound to the bacteria, indicating that they form in situ within the periplasm, thus constructing a structurally stable bacterial-nanostructure hybrid. Further elemental distribution analysis of this region was performed using energy-dispersive X-ray spectroscopy (EDS). The results showed that copper (Cu) and sulfur (S) signals were highly enriched in the nanoaggregate region, and their spatial distributions highly overlapped. Figure 2 a and c in the text). Selected area electron diffraction analysis of a single Cu / S aggregate shows a diffuse halo pattern in its diffraction pattern. Figure 2 (b and d) No clear crystal diffraction spots were detected, indicating that the mineral structure has amorphous or short-range ordered characteristics, and its basic building blocks are nanoscale atomic clusters.

[0075] like Figure 3 As shown in a, Example 1 and Comparative Example 1 (i.e., N- So -CuNC and Cl- So The X-ray diffraction pattern of Cu / S (-CuNC) showed no obvious crystalline diffraction peaks throughout the entire scanning range, with only a broad peak observed near 2θ≈20°, which can be attributed to the amorphous carbon characteristic peak of bacterial organic matter. This result further confirms that the Cu / S nanostructure formed in the permamaterial space mainly exists in the form of long-range disordered amorphous nanoclusters. To clarify the chemical valence states of Cu and S in the sample, X-ray photoelectron spectroscopy analysis was performed. The Cu2p spectrum shows that N- So -CuNC and Cl -So -CuNC sample Cu 2p 1 / 2 and Cu 2p 3 / 2 The binding energies are located at 932.1 eV and 952.1 eV, respectively. Figure 3 The binding energy positions in the S2p spectrum are consistent with those of Cu(I) or Cu(0) species, and no characteristic satellite peaks of Cu(II) at 940–945 eV and 962 eV were observed. This indicates that the initially added Cu(II) was almost completely reduced to Cu(I) and / or Cu(0). Peak fitting of the S2p spectrum shows that in N- So -CuNC and Cl -So In all CuNC samples, a concentration at approximately 161.3 eV (S2p) can be observed. 3 / 2 ) and 163.0 eV (S 2p 1 / 2 A pair of characteristic peaks () Figure 3 The *e* group, belonging to the S(-II) species, accounted for approximately 73% and 68% of the total sulfur signal, respectively. In contrast, only CSC-form organic sulfur species were detected in *Shewanella*. Figure 3c) indicates that sulfur exists primarily in the form of reduced sulfides in biomass hybrids.

[0076] In the near-edge X-ray absorption spectrum of Cu K-side, N- So -CuNC and Cl- So The absorption edge position of the CuNC sample is basically consistent with that of the standard Cu2S reference (~8982 eV). Figure 4 (a) After converting the spectrum to its first derivative, the correspondence between the absorbing edges becomes clearer. Figure 4 (b) further confirms that the valence state of copper is predominantly Cu(I), consistent with the above X-ray photoelectron spectroscopy analysis results. The Fourier transform of the extended X-ray absorption fine structure further reveals the nearest-neighbor coordination structure of copper. Both samples exhibit a main peak at a radial distance of approximately 1.8 Å without phase correction, corresponding to the Cu-S scattering path of the first coordination layer (…). Figure 4 The c in the figure confirms the formation of Cu-S coordination bonds. Furthermore, wavelet transform simultaneously confirmed the existence of Cu-S coordination signals in both k-space and R-space. Figure 4 The term "e" eliminates interference from similarly spaced scattering paths such as Cu-O or Cu-Cu. Fitting results from extended X-ray absorption fine structure data show that copper atoms mainly exist in the Cu-S coordination form (…). Figure 4 (d) Based on the above analysis results, it can be determined that Cu 2+ Under the action of Shewanella, it was biotransformed into a long-range disordered copper-sulfur nanocluster mainly composed of Cu(I)-S coordination bonds. Its non-stoichiometric and disordered structural characteristics were analyzed using Cu... 2-x S is represented by this.

[0077] Comparative Example 3: Prepare anaerobic working medium according to step (2) in Example 1. Do not inoculate with Shewanella. Directly dispense into 50mL serum bottles (25mL per bottle). After fully aerating with nitrogen for 20min, seal and let stand under sealed conditions at a constant temperature of 30℃ for 96h to obtain the heterozygous solution.

[0078] Comparative Example 4: Based on the anaerobic working medium described in step (2) of Example 1, sodium lactate was removed, while other contents remained unchanged. The anaerobic working medium inoculated with Shewanella was dispensed into 50 mL serum bottles (25 mL per bottle), thoroughly aerated with nitrogen for 20 min to remove oxygen, then sealed and incubated at a constant temperature of 30°C for 96 h to obtain the heterozygous solution.

[0079] Comparative Example 5: Based on the anaerobic working medium described in step (2) of Example 1, the yeast extract was removed, while other aspects remained unchanged. The anaerobic working medium inoculated with Shewanella was dispensed into 50 mL serum bottles (25 mL per bottle), thoroughly aerated with nitrogen for 20 min to remove oxygen, then sealed and incubated at a constant temperature of 30°C for 96 h to obtain the heterozygous solution.

[0080] Comparative Example 6: Based on the anaerobic working medium described in step (2) of Example 1, remove tryptone, and keep everything else the same. Dispense the anaerobic working medium inoculated with Shewanella into 50 mL serum bottles (25 mL per bottle), aerate thoroughly with nitrogen for 20 min, seal, and incubate at a constant temperature of 30°C for 96 h to obtain the heterozygous solution.

[0081] Comparative Example 7: Replace the copper nitrate in the anaerobic working medium described in step (2) of Example 1 with 0.188 g (approximately 1 mM) or 0.376 g (approximately 2 mM) of copper nitrate, keeping everything else unchanged. Dispense the anaerobic working medium inoculated with Shewanella into 50 mL serum bottles (25 mL per bottle), aerate thoroughly with nitrogen for 20 min, seal, and incubate at a constant temperature of 30°C for 96 h to obtain the heterozygous solution.

[0082] Comparative Example 8: Add 1 mM or 3 mM sodium nitrate to the anaerobic working medium described in step (2) of Example 1, keeping everything else unchanged. Dispense the anaerobic working medium inoculated with Shewanella into 50 mL serum bottles (25 mL per bottle), aerate thoroughly with nitrogen for 20 min to remove oxygen, seal, and incubate at a constant temperature of 30°C for 96 h to obtain the heterozygous solution.

[0083] Comparative Example 9: Add 0.5 mM sodium citrate (Cit) or disodium ethylenediaminetetraacetate (EDTANa2) to the anaerobic working medium described in step (2) of Example 1, keeping everything else unchanged. Dispense the anaerobic working medium inoculated with Shewanella into 50 mL serum bottles (25 mL per bottle), aerate thoroughly with nitrogen for 20 min, seal, and incubate at a constant temperature of 30°C for 96 h to obtain the heterozygous solution.

[0084] Add an additional 0.5 mM sodium citrate (Cit) to the anaerobic working medium described in step (2) of Comparative Example 1, while keeping everything else unchanged. Dispense the anaerobic working medium inoculated with Shewanella into 50 mL serum bottles (25 mL per bottle), aerate thoroughly with nitrogen for 20 min, seal, and incubate at a constant temperature of 30 °C for 96 h to obtain the heterozygous solution.

[0085] In Comparative Example 3, no significant decrease in dissolved copper concentration was detected when Shewanella was not inoculated. Figure 5 In Comparative Example 4, when sodium lactate is lacking as an electron donor in the system, Cu 2+ The biological removal process was almost completely suppressed. Figure 5 This indicates that the process depends on S. oneidensis The metabolic activity of Cu is likely closely related to its extracellular electron transport mechanism. In Comparative Example 5, when yeast extract was absent from the system, Cu... 2+ The removal of [something] was almost completely suppressed. Figure 5 This indicates that the copper biotransformation process may depend on short peptides provided by tryptone. In Comparative Example 6, when tryptone was absent from the system, Cu... 2+ The removal of [something] was almost completely suppressed. Figure 5 This indicates that the copper biotransformation process may depend on the complex nutrients contained in the yeast extract. In Comparative Example 7, when the concentration of copper nitrate in the anaerobic working medium was 1 or 2 mM, Cu... 2+ Not completely removed, approximately 25% ( Figure 6 The resulting hybrid was pinkish-red in color, showing little change from the initial Shewanella. In Comparative Example 8, when an additional 1 mM sodium nitrate, i.e., Cu, was added to the anaerobic working medium... 2+ With NO3 - When the molar ratio is increased to 4, Cu 2+ The removal of [the substance] was actually significantly inhibited (the removal rate was only 20% after 96 hours, see [reference]). Figure 7 In Comparative Example 9, the presence of sodium citrate affected Cu. 2+ Biological removal has a relatively small impact, Cu 2+ The removal rate remains above 95%. Figure 8 In contrast, the introduction of EDTA almost completely suppressed Cu. 2+ The biological removal process, within 96 hours, Cu 2+ The removal rate was only about 10%. This difference is mainly due to the extremely high thermodynamic stability of the Cu(II)-EDTA complex (log0.1 = 0.1 M ionic strength). K =18.8), its stability is much higher than that of Cu(II)-Cit complex (at an ionic strength of 0.1 M, log K =5.9), thus severely limiting Cu 2+ Interactions between bacteria and subsequent biotransformation processes.

[0086] Comparative Example 10: The anaerobic working medium described in step (2) of Example 1 was replaced with an inorganic salt medium containing 20 mM sodium lactate, 0.221 g / L Na2HPO4, 0.099 g / L NaH2PO4, 0.168 g / L NaHCO3, 1.189 g / L (NH4)2SO4, 7.305 g / L NaCl, 0.094 g / L MgCl2, and 0.0713 g / L CaCl2, with all other components remaining unchanged. The anaerobic working medium inoculated with Shewanella was dispensed into 50 mL serum bottles (25 mL per bottle), thoroughly aerated with nitrogen for 20 min, sealed, and incubated at a constant temperature of 30°C for 96 h. After incubation, a heterozygous solution was obtained. The culture was centrifuged at 3000 g for 5 min, the supernatant was discarded, and the precipitate was collected to obtain the heterozygote (denoted as Cys-). So -CuNC).

[0087] The results showed that Cu in the culture system 2+ Completely removed within 36 hours. Figure 9 X-ray diffraction analysis of the obtained hybrid sample showed that its diffraction pattern contained obvious CuS characteristic crystal form peaks, accompanied by a small amount of Cu2S crystal form peaks. Figure 10 The phase composition indicates that under cysteine-containing inorganic salt culture conditions, Shewanella's metabolic activity generates a large amount of divalent sulfur, which then reacts with copper and cuprous ions in the solution to form a sulfide precipitate mainly composed of CuS. This result contrasts sharply with Example 1 on LB liquid medium, confirming the decisive influence of the presence or absence of divalent sulfur on the product composition.

[0088] Example 2: In step (2) of Example 1, the copper nitrate in the anaerobic working medium was replaced with the adjusted actual copper-containing wastewater, while other aspects remained unchanged. (The actual copper-containing wastewater sample was first filtered and sterilized using a 0.22 μm filter membrane, then diluted 3 times with sterile deionized water, and the pH of the system was adjusted to 6.5 using NaOH solution. Based on this, tryptone (10 g / L) was added to the system.) -1 ), yeast extract (5 g L) -1 ), NaCl (0.1 g L) -1 The anaerobic working medium inoculated with Shewanella was dispensed into 50 mL serum bottles (25 mL per bottle), and thoroughly aerated with nitrogen for 20 min. After being cultured in a sealed container at a constant temperature of 30 °C for 96 h, a heterozygous solution was obtained. The supernatant was removed by centrifugation at 3000 g for 5 min to obtain the heterozygotes.

[0089] The copper in the wastewater mainly exists in the form of Cu(II)-Cit (diluted to a concentration of 0.51 mM) complex, and also contains a certain concentration of NO3. - and Ni 2+ (The diluted concentrations were 0.44 and 0.05 mM, respectively). The results showed that the system achieved approximately 90% copper ion removal efficiency within 72 h. Figure 8 It is worth noting that although Ni is present in the wastewater 2+ (The concentration after dilution is approximately 3.1 mg / L), but Cu 2+ The removal efficiency was not significantly affected.

[0090] Further systematic materials science characterization was performed on the copper-containing materials recovered from the actual wastewater system. Ultrathin section TEM images showed that the generated Cu / S hybrids were mainly distributed within the periplasmic space, exhibiting a high degree of coupling with the bacterial cells. Figure 11 (a) X-ray diffraction analysis showed that no obvious crystal diffraction peaks appeared in the sample throughout the scanning range, except for a broad diffraction band at 2θ≈20°. Figure 11 c). The SAED results also showed diffuse halos, with no clear crystal diffraction spots detected. Figure 11 (b) further confirms the amorphous characteristics of the material. The absorption edge position of the sample in the X-ray absorption near-edge structure spectrum is similar to that of the standard Cu2S sample. Figure 11 The results of the Fourier transform of the extended X-ray absorption fine structure show a significant main peak at approximately 1.8 Å, confirming the formation of the Cu-S coordination structure. Figure 11 (f) Elemental analysis of the precipitate product by inductively coupled plasma atomic emission spectrometry (ICP-AES) showed that the nickel content was below the detection limit (1 mg / kg), while the copper content was approximately 4.4% (43984.27 mg / kg), comparable to the copper content of the materials prepared in Example 1 and Comparative Example 1. Figure 12 In summary, these results preliminarily demonstrate that Shewanella can efficiently recover Cu under complex real-world wastewater conditions. 2+ and in situ preparation of Shewanella-Cu 2-x The S-biohybrid highlights the application potential of this bio-driven strategy in the resource recovery of copper-containing wastewater and the green preparation of functional materials.

[0091] Example 3: This example is used to illustrate the investigation of NO3. - Promoting the preparation of Cu by Shewanella 2-x The promoting effect of S nanoclusters and the characteristics of related processes. Specific steps are as follows: The *Shewanella* seed culture prepared according to step (1) of Example 1 was inoculated into the anaerobic working medium prepared according to step (2) of Example (1) or Comparative Example 2. The copper conversion process in different experimental groups was monitored using copper-containing disulfonic acid. + Dynamic changes in concentration. Cu 2+ Concentration is measured by the total dissolved copper concentration and Cu at the same time point. + It was calculated from the difference in concentration. NO3 - NO2 - and NH4 + The concentration was determined by ion chromatography. The concentration of S(-II) was determined by methylene blue spectrophotometry.

[0092] The results showed that both systems rapidly generated approximately 0.3–0.35 mM Cu after inoculation. + ( Figure 13 (a) In the uninoculated culture medium, approximately 0.1 mM Cu was also detected after the addition of copper salt. + It is speculated that this originates from the reducing effect of substances such as proteins in the culture medium. In the Cu(NO3)2 group, Cu 2+ It was completely reduced to Cu within 12 hours. + ( Figure 13 (b) in the middle, then Cu + The concentration gradually decreased, and by 48 hours it had fallen below the detection limit. This was in conjunction with total free copper (including Cu). 2+ and Cu + Analysis of the removal kinetic curves of Cu can be used to infer... + The decrease in concentration is due to its conversion to Cu. 2-x S. In comparison, Cu in the CuCl2 group 2+ Complete reduction is delayed to 24 hours, while Cu + Completely converted to Cu 2-x S requires 96 hours. The above results indicate that Shewanella bacteria are resistant to Cu. 2+ The biomineralization process is divided into two stages: Cu 2 + Reduction stage and Cu + During the mineralization stage, NO3... - The promoting effect is mainly manifested in accelerating the second stage. Further synchronous monitoring of nitrogen species concentrations accompanying the mineralization process in the Cu(NO3)2 group revealed that NO3 was the dominant nitrogen species in the system during the first 12 hours. - Reduced to NO2 - The dominant process; thereafter NO3 - Reduction stops, NO2 accumulates in the system - Start reducing to NH4 + ( Figure 13 (c) It is worth noting that the above nitrogen conversion process is related to Cu.2+ The mineralization processes roughly overlapped in time, suggesting a possible intrinsic coupling between them. For example, NO3... - Reduction process and Cu 2+ The overlap of the reduction phases may mean NO3 - It may act as an electron acceptor to promote extracellular electron transfer in Shewanella, thereby accelerating Cu. 2+ Reduction; while NO2 - Reduction process and Cu 2 The overlap of transformation stages may be related to the expression of relevant proteins or the formation of intermediate products affecting Cu. + The fixation and sulfur coordination processes are related to the promotion. Furthermore, H2S was not detected in the copper mineralization process of different copper salt treatment groups. Figure 14 This further rules out the possibility that Shewanella produces H2S via cysteine ​​desulfurization to drive Cu production. + The possibility of mineralization.

[0093] Example 4: Assessment of Shewanella-Cu 2-x The nitrogen recovery performance of the S hybrid under high carbon-to-nitrogen ratio conditions is demonstrated by the following steps: The hybrid solutions prepared according to the methods of Example 1 and Comparative Example 1 were centrifuged at 3000g for 10 min, then resuspended in an equal volume of anaerobic working medium (containing 4 mM sodium nitrate and 20 mM sodium formate), sealed, and incubated in a shaker at 30°C and 150 rpm. All operations were performed in an anaerobic glove box. Samples were taken using a sterile syringe at 0, 1, 3, 6, 9, 12, 24, 36, and 48 h, and NO3 was analyzed by ion chromatography. - NO2 - and NH4 + Quantitative analysis is performed.

[0094] Comparative Example 11: Assessment of Shewanella-Cu 2-x The nitrogen recovery performance of the S-hybrid under low carbon-to-nitrogen ratio conditions is demonstrated by the following steps: The hybrid solutions prepared according to the methods of Example 1 and Comparative Example 1 were centrifuged at 3000g for 10 min, then resuspended in an equal volume of anaerobic working medium (containing 4 mM sodium nitrate and 12.5 mM sodium formate, with a C / N ratio (COD / TN) of 3.6), sealed, and incubated in a shaker at 30°C and 150 rpm. All operations were performed in an anaerobic glove box. Samples were taken using a sterile syringe at 0, 1, 3, 6, 9, 12, 24, 36, and 48 h, and NO3 was analyzed by ion chromatography. - NO2 - and NH4 + Quantitative analysis is performed.

[0095] The bio-hybrids prepared in Example 1 and Comparative Example 1 (denoted as N-) were evaluated. So -Cu 2-x S and Cl- So -Cu 2-x S) NO3 - Reduced to NH4 + (i.e., nitrogen recovery, such as) Figure 20 The results showed that, under conditions of sufficient electron donors, both hybrids completely removed NO3 within 12 hours. - ( Figure 15 The apparent first-order rate constant of the reduction process was 1.94 times higher than that of the pure bacterial control group. This indicates that the biological heterozygote can significantly increase NO3-. - The rate of reduction. Simultaneously, the NH4 in the biohybrid system... + The formation rate is significantly accelerated, with up to 88.9% of nitrates being converted into ammonium ( Figure 15 It is worth noting that in the early stages of the nitrogen recovery process, NO2, as an intermediate product in the hybrid system, - The accumulation was more obvious, and then it was quickly consumed. Figure 15 ).

[0096] Considering the common carbon source limitations in actual wastewater (e.g., COD / TN ratio less than 5), the nitrogen recovery performance of two biohybrids was investigated under the condition of limited electron donor (sodium formate) at a C / N ratio of 3.5. The results showed that even under limited electron donor conditions, both biohybrids maintained high NO3 levels. - Reduction and NH4 + The generation rate decreased, but the nitrogen recovery efficiency decreased from 89.7% and 89.9% to 59.2% and 59.3%, respectively. Figure 16 (), but still much higher than the control group. It is noteworthy that at the reaction endpoint, NO2 in the pure Shewanella group and the two heterozygous groups was... - The contents were 19.39, 18.65, and 18.60 mg / L, respectively. Figure 16 ).In summary, So- Cu 2-x The S-type hybrid system showed a significant improvement in nitrogen recovery performance compared to pure Shewanella, especially maintaining high reduction efficiency under electron donor-limited conditions. Furthermore, regardless of carbon source availability, different copper sources prepared... So- Cu 2-x The nitrogen recovery performance of the S biohybrids did not show significant differences.

[0097] Comparative Example 12: Evaluation of pure Cu 2-x The nitrogen recovery performance of material S under high carbon-to-nitrogen ratio conditions is demonstrated through the following steps: The hybrid solution prepared according to the method in Example 1 was placed in an autoclave and sterilized by moist heat at 121°C for 20 min. After the sample cooled to room temperature, it was centrifuged at 3000g for 10 min. The supernatant was discarded, and the sample was resuspended in an equal volume of anaerobic working medium (containing 4 mM sodium nitrate and 20 mM sodium formate), sealed, and incubated in a shaker at 30°C and 150 rpm. All operations were performed in an anaerobic glove box. Samples were taken using a sterile syringe at 0, 1, 3, 6, 9, 12, 24, 36, and 48 h, and NO3 was analyzed by ion chromatography. - NO2 - and NH4 + Quantitative analysis is performed.

[0098] Comparative Example 13: Evaluation of Cys- So The nitrogen recovery performance of CuNC hybrids under high carbon-to-nitrogen ratio conditions is demonstrated through the following steps: The heterozygous solution prepared according to the method of Comparative Example 10 was centrifuged at 3000g for 10 min. The supernatant was discarded, and the solution was resuspended in an equal volume of anaerobic working medium (containing 4 mM sodium nitrate and 20 mM sodium formate), sealed, and incubated in a shaker at 30°C and 150 rpm. All operations were performed in an anaerobic glove box. Samples were taken using a sterile syringe at 0, 1, 3, 6, 9, 12, 24, 36, and 48 h, and NO3 was analyzed by ion chromatography. - NO2 - and NH4 + Quantitative analysis is performed.

[0099] The results showed that the nitrogen conversion efficiency of Comparative Example 12 was almost completely suppressed, with only 3.9% of NO3 being converted. - It was converted into NO2 - ( Figure 17 ), indicating pure Cu 2-x S component pair So -Cu 2-x The overall nitrogen transformation contribution of the S heterozygote is very limited. In Comparative Example 13, Cys- synthesized using an inorganic salt medium containing cysteine... So -Nitrogen recovery tests were conducted on the CuNC hybrid, and its nitrogen recovery rate was improved compared to the pure bacterial group without material loading. Figure 18 However, this rate is still slower than that of N- synthesized under conditions without exogenous sulfur. So -Cu 2-x S and Cl- So -Cu 2-x S-hemisphere hybrids. A possible mechanism for this phenomenon is: Cys- SoThe copper sulfide nanostructures in the CuNC hybrid exhibit a more stable crystal form (mainly manifested as the CuS phase), and the predominant valence state of copper is +2 [Cu(II)]. Compared to hybrids synthesized from exogenous sulfur, which may contain more low-valence or amorphous copper active sites, this highly crystalline Cu(II) species has relatively low electron transfer capacity in nitrate reduction reactions, resulting in limited enhancement of its catalytic activity.

[0100] Example 5: Low C / N conditions So -Cu 2-x The scale-up experiment for nitrogen recovery from S-hemisphere hybrids is as follows: To improve under low C / N ratio conditions So -Cu 2-x The S-hybrid produced NO2 in the effluent at the end of the DNRA reaction. - The problem of high residue levels will So -Cu 2-x S-type hybrids were mixed with anaerobic activated sludge (AnS) with denitrification function in an appropriate ratio and acclimated to construct a preparative microbial community ( So -Cu 2-x S+AnS), using AnS to remove part of NO2 - It is reduced to N2, thereby reducing NO2 in the effluent. - Content. The scale-up experiment was conducted using a laboratory-scale sequencing batch bioreactor (SBR). The reactor body was made of cylindrical plexiglass with an effective volume of 2.0 L. The entire operation was maintained at a constant temperature of 30 ± 0.5 °C via a constant-temperature water bath jacket. The AnS used for inoculation was collected from the anaerobic unit of the Guangzhou Datansha Wastewater Treatment Plant. So -Cu 2-x The S-biohybrid was prepared according to the method described in Example 1. At reactor startup, the two inoculums were mixed at a 1:1 mass ratio of volatile suspended solids, with the total inoculum amount accounting for 30% of the reactor's working volume.

[0101] The experiment ran for 22 complete cycles, including an 8-cycle start-up and acclimatization phase and a 14-cycle formal operation phase. The start-up phase employed a batch acclimatization model, with artificially prepared simulated wastewater as the influent. Its basic formulation (per 1L) contained: 0.607g NaNO3 (providing 100 mg / L NO3). -The initial chemical oxygen demand (COD) to total nitrogen (TN) ratio was set at 8.0, with the following components: 3.4 g sodium formate (as carbon source and electron donor), 0.197 g MgSO4·7H2O, 2 g KH2PO4, 0.02 g FeSO4·7H2O, 0.022 g CaCl2, 0.01 g yeast extract, and 1 mL trace element stock solution. Each operating cycle lasted 24 hours and consisted of: 5 min feed, 23 h anaerobic reaction, 50 min sludge settling, and 5 min effluent discharge. After entering the formal operation phase, the influent COD / TN ratio was adjusted to 3.5 (i.e., sodium formate dosage was reduced to 1.49 g / L), while the concentrations of all other components, hydraulic retention time, and operating procedures remained consistent with the start-up phase. To monitor the nitrogen recovery performance of the system, influent and effluent samples were collected at the beginning and end of each operating cycle and immediately stored at 4°C for subsequent NO3 analysis. - NO2 - and NH4 + Perform quantitative analysis.

[0102] The results show that ( Figure 19 During the startup phase (C / N=8), in the 8th cycle after system acclimatization, NO3... - Removal rate and NH4 + The recovery rates reached 99.93% and 57.87%, respectively. After entering the stable operation phase (C / N=3.5), NO3... - The removal rate can still be maintained at around 99%; in the first 10 cycles, NH4 + The recovery rate stabilized between 35.84% and 40.23% and then gradually decreased to below 30%, indicating that the system's NH4 recovery rate was low. + Recovery performance may decrease over time. No NO2 was detected in the effluent throughout the entire experiment. - and Cu 2+ .

[0103] The above is a detailed description of the preferred embodiments of the present invention. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of the present invention.

Claims

1. A method for preparing a copper-sulfur nanobiohybrid, characterized in that, Includes the following steps: Electroactive bacteria were inoculated into LB liquid medium containing copper salts and cultured anaerobically to obtain copper-sulfur nanobio-hybrids; the LB liquid medium containing copper salts included copper salts, lactate, yeast extract, tryptone and sodium chloride; the concentration of copper salts in the LB liquid medium containing copper salts was 0.25-0.5 mM.

2. The preparation method according to claim 1, characterized in that, The electroactive bacteria is *Schizium anisopliae*. The initial OD600 value of the electroactive bacteria inoculated in LB liquid medium containing copper salts was 1.5–2.

0.

3. The preparation method according to claim 1, characterized in that, The copper salt is at least one of copper nitrate, copper chloride, and copper sulfate; The lactate is sodium lactate.

4. The preparation method according to claim 1, characterized in that, The concentration of lactate in the copper-containing LB liquid medium is 15–25 mM; The concentration of yeast extract in the copper-containing LB liquid medium is 3–8 g / L, the concentration of tryptone is 5–15 g / L, and the concentration of sodium chloride is 0.05–0.15 g / L.

5. The preparation method according to claim 1, characterized in that, The copper-containing LB liquid culture medium may also contain other components that do not affect the activity of electroactive bacteria and Cu. 2+ The interaction with electroactive bacterial cells, and NO3 - With Cu 2 + The molar ratio is 0.5~3.

6. The preparation method according to claim 1, characterized in that, The copper-containing LB liquid culture medium may also contain citrate; The copper-containing LB liquid culture medium may also contain nickel ions; The copper-containing LB liquid culture medium does not contain ethylenediaminetetraacetic acid dichloride.

7. The preparation method according to claim 1, characterized in that, The anaerobic culture was subjected to nitrogen aeration treatment beforehand. The anaerobic culture was a closed, static culture, with a temperature of 25–35°C and a culture time of 48–96 hours.

8. The copper sulfate nanobiohybrid prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the copper-sulfur nanobiohybrid as described in claim 8 in nitrogen recovery.

10. The application according to claim 9, characterized in that, Sulfur copper nanobiohybrids were suspended in a NO3-containing atmosphere. - Anaerobic treatment is carried out on the wastewater; The NO3-containing - The carbon-to-nitrogen ratio in the wastewater is 3–8; The temperature for the anaerobic treatment is 25–35°C. The NO3-containing - The wastewater is also treated with denitrifying bacteria or anaerobic activated sludge with denitrification function.

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