Clostridium pasteurianum loaded with nano-copper and application of clostridium pasteurianum in catalytic degradation of methylene blue

Abstract

The invention provides clostridium pasteurianum loaded with nano-copper and application of the clostridium pasteurianum in catalytic degradation of methylene blue. The clostridium pasteurianum loaded with nano-copper is obtained by adsorbing a clostridium pasteurianum solution with a copper solution under an anaerobic condition and then introducing hydrogen as a reducing agent for reaction; according to the clostridium pasteurianum loaded with the nano-copper, the loading capacity of the nano-copper is 25.33 mmol / g to 27.33 mmol / g, and the average particle size of the nano-copper is 42.28 + / -12.14 nm. The functional clostridium pasteurianum with nano-copper loaded in cells is constructed, methylene blue in an aqueous solution is degraded to serve as a template for reaction to verify the catalytic capacity of the clostridium pasteurianum, and a certain supporting effect is expected to be provided for biological preparation of a nano-metal catalyst.

Description

Technical Field

[0001] The present invention relates to the technical field of biocatalysis, in particular to a nano-copper-loaded Clostridium pasteurianum and application of the nano-copper-loaded Clostridium pasteurianum in catalytic degradation of methylene blue. Background Art

[0002] Metal nanoparticles are widely used in organic synthesis as a replacement for traditional catalysts due to their high catalytic activity, long lifespan, and ease of recycling. Compared to traditional metal-catalyzed reactions, metal nanoparticle-catalyzed reactions offer advantages such as low loading, high conversion, enhanced atom economy, high yield, high catalytic efficiency, and recyclable catalysts. This is because they can provide empty orbitals or electrons in chemical reactions, thereby producing intermediates, lowering the activation energy, and facilitating subsequent reactions.

[0003] Methods for preparing nano-copper particles include chemical reduction, physical vapor deposition, microemulsion, and biological methods. Chemical reduction may introduce impurities during the reaction, as reducing agents and other additives may remain on the surface of the nano-copper particles. Furthermore, the reaction product requires complex separation and purification processes to obtain high-purity nano-copper. Furthermore, nano-copper particles prepared by chemical reduction sometimes have poor dispersibility in solution and are prone to agglomeration. Physical vapor deposition (PVD) requires expensive equipment and relatively low production efficiency, making it unsuitable for large-scale production of nano-silver-copper particles. Furthermore, PVD requires a high vacuum environment, consumes significant energy, and is costly. The preparation process for microemulsions is complex, requiring the use of multiple surfactants and co-surfactants, resulting in high costs. Furthermore, after the reaction, separating the nano-silver-copper particles from the microemulsion is difficult, which can affect product yield.

[0004] The biosynthesis of CuNPs utilizes biomolecules within organisms to reduce copper ions and form nanosized particles. This method does not require high temperatures, high pressures, or toxic solvents, making it a green synthesis method. Compared to chemical and physical methods, biosynthesis is an innovative technology with low cost, high biosafety, and good environmental biocompatibility. Ingredients for the biosynthesis of CuNPs include extracts from various plant parts, such as roots, stems, leaves, fruits, peels, and seeds, as well as biological methods for preparing nanoparticles using microorganisms (bacteria, actinomycetes, yeast, and algae) and viral particles.

[0005] The use of microorganisms to recycle and synthesize biological nano-copper catalysts can not only recycle metallic copper in the environment, but also prepare high-efficiency catalysts for the chemical industry. At the same time, the preparation process also has the characteristics of low energy consumption and environmental friendliness. Nano-copper particles have a large specific surface area and high surface activity, and are very easy to agglomerate. After agglomeration, the catalytic performance decreases. Loading them on the surface of bacteria can effectively reduce the occurrence of agglomeration. Nano-copper particles do not need to be separated from biomass, nor do they need to add additional supporting additives. Directly using bacteria as a supporting matrix can achieve a good catalytic effect. Moreover, this catalyst can be easily filtered from a centrifuge, so it can be reused. This method integrates nanomaterials into organisms to prepare biomaterials with catalytic activity.

[0006] The biosynthesis method is green, environmentally friendly, low-cost and biosafe, but it still has some shortcomings, such as slow synthesis speed, difficult to control reaction conditions, low purity of synthesized nanoparticles and relatively large particle size. Summary of the Invention

[0007] The present invention aims to develop a nano-copper-loaded Clostridium pasteurianum and its application in the catalytic degradation of methylene blue. This work constructs a functional Clostridium pasteurianum with intracellular nano-copper loading and verifies its catalytic ability using the degradation of methylene blue in aqueous solution as a template reaction. This work is expected to provide support for the bio-production of nano-metal catalysts.

[0008] The following is a specific implementation of the technical solution of the present invention:

[0009] The first object of the present invention is to provide a nano-copper-loaded Clostridium pasteurianum, which is obtained by adsorbing a Clostridium pasteurianum bacterial liquid with a copper liquid under anaerobic conditions, and then introducing hydrogen as a reducing agent to react; the nano-copper loading amount of the nano-copper-loaded Clostridium pasteurianum is 25.33-27.33 mmol / g bacterial body, and the average particle size of the nano-copper is 42.28±12.14 nm.

[0010] Preferably, the copper solution is a Cu2SO4 aqueous solution with a concentration of 0.02-0.06M.

[0011] More preferably, the concentration of the Cu2SO4 aqueous solution is 0.04M.

[0012] Preferably, the adsorption temperature is 20-40° C., the adsorption time is 20-40 min, and the hydrogen introduction time is 20-40 min.

[0013] More preferably, the adsorption temperature is 30° C., the adsorption time is 30 min, and the hydrogen introduction time is 30 min.

[0014] Preferably, the loading amount of the nano-copper is 26.33 mmol / g of bacteria, the average particle size of the nano-copper is 42.28 nm, and the nano-copper is zero-valent.

[0015] A second object of the present invention is to provide the use of the above-mentioned nano-copper-loaded Clostridium pasteurianum in catalytic degradation of methylene blue, comprising: adding the nano-copper-loaded Clostridium pasteurianum to an aqueous solution containing methylene blue, mixing, and then adding a sodium borohydride solution and stirring to remove the methylene blue in the aqueous solution.

[0016] Preferably, the stirring is carried out at room temperature and atmospheric pressure for 3-10 minutes.

[0017] Preferably, the concentration of the sodium borohydride solution is 0.015-0.020 μmol / L, and 1-3 mL of sodium borohydride solution is added to every 200 mL of methylene blue solution.

[0018] More preferably, the concentration of the sodium borohydride solution is 0.0164 μmol / L, and 2 mL of sodium borohydride solution is added to every 200 mL of methylene blue solution.

[0019] Preferably, the mixing ratio of the nano-copper-loaded Clostridium pasteurianum and the aqueous solution containing methylene blue is 0.0071-0.013 g:100 mL.

[0020] Preferably, the aqueous solution containing methylene blue has a methylene blue concentration of 0-10 μmol / L. When the methylene blue concentration reaches 20 μmol / L, the catalytic degradation effect after adding nano-copper-loaded Clostridium pasteurianum is not obvious within the same time.

[0021] The present invention uses Clostridium pasteurianum as the starting strain, which can adsorb copper ions and reduce them using hydrogen as an electron donor to form black bio-copper nanoparticles. Scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) characterization of the product showed that Clostridium pasteurianum can adsorb and reduce copper ions to form nanoparticles on the cell surface.

[0022] The present invention has the following beneficial effects:

[0023] Compared to existing copper synthesis methods, this method utilizes a mild hydrogen source instead of a traditional hazardous one, and achieves cell-surface-directed synthesis of copper nanoparticles. This method is expected to have significant application potential in metal nanoparticle preparation, environmental remediation, biomedicine, and the chemical industry. The present invention constructs functional Clostridium pasteurianum bacteria loaded with copper nanoparticles intracellularly, and its catalytic ability is demonstrated using the degradation of methylene blue in aqueous solution as a template reaction. This is expected to provide support for the bioproduction of metal nanocatalysts. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is an SEM image of cells to which copper sulfate solution was added in Example 2;

[0025] Figure 2 is an SEM image of cells without the addition of copper sulfate solution in Example 2;

[0026] Figure 3 This is the XRD pattern of the cells in Example 2. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0028] In the following examples, from among the many microorganisms that can synthesize nano-copper catalysts, we screened out Clostridium pasteurianum with the highest reduction efficiency (the Clostridium pasteurianum used in the following examples was purchased from Beijing Beina Chuanglian Biotechnology Research Institute) as the experimental strain. The reagents, instruments, materials and methods used in the following examples, unless otherwise specified, are all conventional reagents, instruments, materials and methods in the art and can be obtained through commercial channels.

[0029] 1. Culture medium formula:

[0030] Conventional MSG-MES medium: 0.5 g / L soy peptone, 0.5 g / L tryptone, 10.00 g / L glucose, 2.10 g / L K₂HPO₄, 0.544 g / L KH₂PO₄, 5.00 g / L NaCl, 0.5 g / L L-cysteine hydrochloride, and the remainder water. Sterilize at 121°C for 20 min. Glucose should be aliquoted and sterilized separately at 115°C for 30 min.

[0031] 2. Mixed solution A: composed of Mineral:Trace element:Vitamin = 48:1:1.

[0032] Mineral solution formula (Mineral): 6.0g / L NH4Cl, 6.0g / L NaCl, 0.2g / L CaCl2·2H2O, 2.0g / LMgCl2·6H2O, and the rest is water.

[0033] Trace element solution formula (Trace element): 2.00g / L FeCl2·4H2O, 0.05g / L ZnCl2, 0.05g / L MnCl2·4H2O, 0.03g / L CuCl2·2H2O, 0.05g / L (NH4)6Mo7O24·4H2O, 0.05g / L AlCl3, 1mL H3BO3 saturated solution, 1.0mL concentrated HCl, and the rest is water.

[0034] Vitamin solution formula (Vitamin): 0.002g / L biotin, 0.002g / L folic acid, 0.010g / L vitamin B6, 0.005g / L riboflavin, 0.005g / L vitamin B1, 0.005g / L niacin, 0.005g / L vitamin B12, 0.005g / L p-aminobenzoic acid, 0.005g / L pantothenic acid, and the rest is water.

[0035] 3. Mixed solution B: consists of L-cysteine hydrochloride solution and Na2S·9H2O solution.

[0036] Formula of L-cysteine hydrochloride solution: 80.0 g / L NaHCO3, 1.0 g / L L-cysteine hydrochloride, and the rest is water.

[0037] Na2S·9H2O solution formula: 242g / L Na2S·9H2O, the rest is water.

[0038] Example 1 Preparation and inoculation of MSG-MES culture medium

[0039] 1. Preparation and treatment of MSG-MES medium

[0040] 1) Prepare the culture medium by adding the above formula to a beaker and adjusting the pH to 6.64 with 1 mol / L HCl. Place 20 mL of culture medium in a 50 mL vial. For sterilization, place only 19 mL of culture medium (excluding glucose) in the vial, seal with a cloth stopper, and sterilize at 121°C for 20 minutes. Sterilize the glucose solution at 115°C for 30 minutes and add the vial to the vial in a clean hood.

[0041] 2) After preparation, store Mixed Solution A in a refrigerator at 4°C. Prepare Mixed Solution B immediately before use. Also prepare the following: rubber stoppers and aluminum caps, nitrogen manifolds, vent needles, centrifuge tubes, and 0.22 μm microfilters. Sterilize all at 121°C for 20 minutes and transfer to a laminar flow hood.

[0042] 3) In a laminar flow hood, add 1 mL of glucose solution, 200 μL of mixed solution A, and 200 μL of mixed solution B to a 50 mL vial. Filter-sterilize mixed solutions A and B before adding them to the vial. After the culture medium is prepared, nitrogen should be introduced to maintain an oxygen-free environment within the vial. Use a stainless steel needle to puncture the bottom of the vial for 10 minutes to complete the nitrogen filling process.

[0043] 2. Conventional activation inoculation and passaging

[0044] Thaw the seed culture in a water bath. In a cleanroom, draw 1 mL of the culture into a vial. Incubate with shaking in a 37°C incubator for 12 hours. When the culture becomes turbid, draw 1 mL of the 5% culture into fresh minimal medium. Repeat 1-2 times to resuscitate the seed culture.

[0045] Example 2 Preparation of Bionano-Copper Catalysts from Gradient Concentration Copper Sulfate Solutions

[0046] Weigh the appropriate amount of CuSO4·5H2O, dissolve it, and adjust the volume to prepare copper sulfate solutions of 0.40 mol / L, 0.22 mol / L, 0.40 mol / L, and 0.58 mol / L, respectively. Add 5 mL of each copper sulfate solution to the inoculated bacterial solution. Incubate the solution in a 30°C incubator with shaking for 30 minutes. Then, introduce hydrogen gas for 20 minutes using a hydrogen generator. A color change of varying degrees will be observed in the vial, indicating the presence of nano-copper-loaded Clostridium bacteria. After the reaction, centrifuge the solution at 10,000 rpm for 10 minutes at 4°C. Discard the supernatant and repeat washing three times. The remaining solid precipitate is dried and pulverized in an oven to obtain the nano-copper-loaded Clostridium solid catalyst.

[0047] 30 mL of each nano-copper-loaded fermentation broth prepared by copper sulfate solution of different concentrations after the reaction was taken, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was discarded. The culture was resuspended with PBS solution and washed three times by centrifugation. The supernatant was removed, and 4% glutaraldehyde solution was added to cover the bacterial pellet. After fixation for 2 hours, the glutaraldehyde was poured out, and the culture was dehydrated in a gradient manner with 25%, 50%, and 75% ethanol and anhydrous ethanol, respectively. The culture was dehydrated for 15 minutes at each concentration, and dried in a constant temperature forced air drying oven at 60°C. The morphological changes of the clostridium were observed using a scanning electron microscope.

[0048] Figure 1 and Figure 2 The cell morphology of the bio-nano copper catalyst prepared by adsorption under 0.40 mol / L copper sulfate solution and without adding copper sulfate solution is shown in Figure 2. Compared with the bio-nano copper catalyst without adsorption, the bio-nano copper catalyst prepared by adsorption under 0.40 mol / L copper sulfate solution and without adding copper sulfate solution is shown in Figure 2. Figure 2 From the above, it can be seen that there is no obvious damage or breakage in the appearance of Clostridium, indicating that the prepared bacteria has a certain load capacity.

[0049] To characterize the generated cell-loaded nano-copper ions, XRD analysis was performed on the samples. A certain amount of dried bio-copper nanopowder was ground into powder and tested under SmartLab X-ray diffraction. The test conditions were: 40kV / 100mA (Cu target, Kα radiation, ), step size 0.02°, scanning range 5° to 90°, scanning mode 2Theta / Theta (continuous scanning), the results are as follows Figure 3 shown.

[0050] Within the scanning range (5° to 90°), distinct diffraction peaks appeared at 2θ of 43.34°, 50.48°, and 74.12°, corresponding to the characteristic diffraction peaks of the (111), (200), and (220) crystal planes of face-centered cubic copper, respectively, and were completely consistent with the copper standard card (PDF NO.04-0836). At the same time, a distinct diffraction peak also appeared at 2θ of 36.48°, corresponding to the (111) crystal plane of Cu2O, consistent with the standard card (PDF NO.05-0667). This proves that some nano-copper is attached to the Clostridium cells, and some of the copper may be oxidized to Cu2O. No other significant impurity peaks were detected, and no peaks appeared in the low-angle region, indicating that the influence of bacterial surface proteins is minimal.

[0051] According to the Debye-Scherrer formula, the average particle size of the nano-copper particles can be calculated to be 42.28 nm.

[0052] Example 3 Verification of the fading of methylene blue by bio-copper nanocatalyst

[0053] Using NaBH4 as a reducing agent, the catalytic performance of a bio-copper nanopowder catalyst in reducing and fading methylene blue dye was investigated. To a 200 mL 0.6575 μmol / L methylene blue solution as the target pollutant, 0.01 g of the bio-copper nanocatalyst, prepared from copper sulfate solutions of varying concentrations, was added, along with 2 mL 0.0164 mol / L NaBH4. The reaction was initiated with stirring. A methylene blue solution of the same concentration and volume, but without the bio-copper nanocatalyst, served as a blank control. Samples were collected from each group before, 3 minutes after, and 20 minutes after the addition of NaBH4. The samples were monitored using a UV spectrophotometer at the methylene blue's maximum absorption wavelength of 664 nm. It was observed that the blue methylene blue solution in each group exhibited significant fading at varying rates after the reaction began.

[0054] The verification effect of the bio-copper nanocatalyst on the fading of methylene blue in Example 3 was tested. In addition to the four groups of bio-nano-copper catalysts prepared by adsorption of copper sulfate at four concentration gradients in Example 2, a methylene blue solution without the bio-copper nanocatalyst was introduced as a blank control group, and the initial OD value of the target pollutant methylene blue was tested. The results are shown in Table 1.

[0055] Table 1 Initial OD value test of target pollutant methylene blue

[0056]

[0057] Lambert-Beer law can be used to calculate the concentration of target residues in the control group and experimental group after 3 minutes and 20 minutes of reaction, and further obtain the removal rate. The residual pollutant concentration of the control group is calculated to be 0.60 mol / L. At the same time, the residual pollutant concentration of each experimental group is calculated, and the removal rate is calculated. The results are shown in Table 2.

[0058] Removal rate (%) = (initial concentration of target pollutant - concentration after reaction) / initial concentration of target pollutant × 100%

[0059] Take the residual concentration of target pollutants and pollutant removal rate after 20 minutes of reaction with 0.040 mol / L copper sulfate as an example:

[0060] According to the Lambert-Beer law A=εlc, the residual concentration of the target pollutant after 20 minutes of reaction can be obtained:

[0061]

[0062] Pollutant removal rate after 20 minutes of reaction:

[0063]

[0064] Table 2 Residual pollutant concentrations and pollutant removal rates in each experimental group

[0065]

[0066] As can be seen from Tables 1 and 2, the bio-copper nanopowder catalyst exhibited a moderate degradation effect on methylene blue. After 20 minutes of reaction, the OD values of all groups treated with the bio-copper nanocatalyst were significantly lower than those of the uncatalyzed control group. The catalyst prepared by adsorption at a copper sulfate concentration of 0.040 mol / L exhibited the best catalytic degradation effect, achieving a pollutant removal rate of 64.26%, a significant improvement over the 9.50% achieved by the uncatalyzed control group.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nano-copper loaded Clostridium pasteurianum, characterized by: The nano-copper-loaded Clostridium pasteurianum is obtained by adsorbing a Clostridium pasteurianum bacterial liquid with a copper liquid under anaerobic conditions, and then introducing hydrogen as a reducing agent to react; the nano-copper loading amount of the nano-copper-loaded Clostridium pasteurianum is 25.33-27.33 mmol / g bacterial body, and the average particle size of the nano-copper is 42.28±12.14 nm.

2. The nano-copper-loaded Clostridium pasteurianum according to claim 1, characterized in that: The copper solution is a Cu2SO4 aqueous solution with a concentration of 0.02-0.06M.

3. The nano-copper-loaded Clostridium pasteurianum according to claim 2, characterized in that: The concentration of the Cu2SO4 aqueous solution is 0.04M.

4. The nano-copper-loaded Clostridium pasteurianum according to claim 1, characterized in that: The adsorption temperature is 20-40° C., the adsorption time is 20-40 min, and the hydrogen introduction time is 20-40 min.

5. The nano-copper-loaded Clostridium pasteurianum according to claim 1, characterized in that: The loading amount of the nano-copper is 26.33 mmol / g of bacteria, the average particle size of the nano-copper is 42.28 nm, and the nano-copper is zero-valent.

6. Use of the nano-copper-loaded Clostridium pasteurianum in catalytic degradation of methylene blue according to any one of claims 1 to 5, characterized in that: include: The methylene blue in the aqueous solution can be removed by adding the nano-copper-loaded Clostridium pasteurianum into the aqueous solution containing methylene blue and mixing the mixture, then adding a sodium borohydride solution and stirring.

7. The use according to claim 6, characterized in that: The stirring is carried out at room temperature and atmospheric pressure for 3-10 minutes.

8. The use according to claim 6, characterized in that: The concentration of the sodium borohydride solution is 0.015-0.020 μmol / L, and 1-3 mL of the sodium borohydride solution is added to every 200 mL of the methylene blue solution.

9. The use according to claim 6, characterized in that: The mixing ratio of the nano-copper-loaded Clostridium pasteurianum and the aqueous solution containing methylene blue is 0.0071-0.013 g:100 ml.

10. The use according to claim 6, characterized in that: The aqueous solution containing methylene blue has a methylene blue concentration of 0-10 μmol / L.

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