Method for efficient biosynthesis of semiconductor nanoparticles by using recombinant sulfate reducing bacteria and application of semiconductor nanoparticles in photocatalysis of industrial wastewater

Abstract

The invention discloses a method for efficiently biosynthesizing semiconductor nanoparticles by using recombinant sulfate reducing bacteria and application of the semiconductor nanoparticles in photocatalysis of industrial wastewater. The invention belongs to the field of wastewater treatment. The invention aims to solve the problems of high treatment cost, low efficiency and serious secondary pollution of wastewater rich in sulfate, heavy metals and organic matters. The method comprises the following steps: adding recombinant sulfate reducing bacteria into industrial wastewater containing sulfate, heavy metal ions and organic matters, carrying out a stirring reaction under an anaerobic condition, then starting illumination, and setting wastewater retention time to obtain the semiconductor nanoparticles. According to the method, the semiconductor nano-particles are prepared from a product of recombined sulfate reducing bacteria metabolism sulfate and heavy metal ions, organic matter oxidation photo-induced electrons are promoted to be transferred into microbial cells through coupled photocatalysis, the efficiency of reducing sulfate by microorganisms, synthesizing the semiconductor nano-particles and transferring the photo-induced electrons is improved, reduction of CO2 in a system is promoted, and the yield of the system is increased. Low-carbon treatment and sustainable biological green manufacturing of the industrial wastewater are realized.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment, specifically relating to a method for the efficient biosynthesis of semiconductor nanoparticles using recombinant sulfate-reducing bacteria and its application in photocatalytic industrial wastewater. Background Art

[0002] Industrial wastewater contains high concentrations of sulfates, heavy metals, and organic matter, making it difficult to treat. Improperly treated wastewater rich in sulfates, heavy metals, and organic matter poses a significant threat to soil, water bodies, and the human environment. High concentrations of these pollutants can form poorly soluble compounds in the environment and enter the human body through the food chain, seriously threatening human health. Therefore, removing pollutants from industrial wastewater rich in sulfates, heavy metals, and organic matter is crucial for preventing pollution at its source and effectively protecting the ecological environment and human health.

[0003] The importance and attention given to the treatment of electroplating wastewater containing heavy metals is constantly increasing. To achieve effective and efficient treatment of wastewater containing bisulfate, heavy metals, and organic matter, current treatment methods include chemical precipitation, adsorption, and biological methods. Chemical precipitation involves adjusting the pH value and adding precipitants or reducing agents to generate low-toxicity substances. However, chemical treatment is costly and prone to secondary pollution. Adsorption mainly utilizes adsorbents with strong adsorption capacity to adsorb and fix heavy metals, but this method requires large amounts of adsorbent and intensive operation. Biological methods mainly remove organic matter from mineral processing wastewater through biological oxidation. Due to the low biodegradability of mineral processing wastewater, the treatment effect is limited. Furthermore, none of the above methods can simultaneously remove organic matter, heavy metals, and sulfates from mineral processing wastewater, and they all face problems such as high operating costs, low pollutant removal efficiency, and serious secondary pollution. Therefore, it is of great significance to find an efficient method for treating wastewater rich in sulfate, heavy metals, and organic matter while simultaneously reducing carbon emissions. Summary of the Invention

[0004] To overcome the above-mentioned technical problems, the present invention provides a method for efficient biosynthesis of semiconductor nanoparticles using recombinant sulfate-reducing bacteria and its application in photocatalytic industrial wastewater.

[0005] The technical solution of the present invention is as follows: One objective of this invention is to provide a method for the efficient biosynthesis of semiconductor nanoparticles using recombinant sulfate-reducing bacteria, the method comprising the following steps: Expressing riboflavin ( rib Gene encoding) and outer membrane protein A ( OmpAThe recombinant sulfate-reducing bacteria (rSRB-RiO) encoded by the gene was amplified and added to industrial wastewater containing sulfate, heavy metal ions and organic matter. The reaction was carried out under anaerobic conditions with stirring, followed by light stimulation. The wastewater retention time was set to 24 h, and the liquid was collected every 24 h, centrifuged, and semiconductor nanoparticles were obtained.

[0006] Further specifying, the preparation method of the recombinant sulfate-reducing bacteria (rSRB-RiO) includes the following steps: (1) Synthesizing riboflavin gene using gene synthesis technology ( rib (GenBank: XM_018880422.1) and outer membrane protein A gene ( OmpA (GenBank: AF135263.1), and constructed the pUC-GW-RiO recombinant plasmid by ligating it with the pUC-GW vector, and transformed it into E. coli DH5α competent cells; (2) Screening for expression using PCR technology rib and OmpA The target strain was obtained by extracting the pUC-GW-RiO plasmid, which was then ligated into the pUC-GW vector using electroporation and recombined into a specific site in the Desulfovibrio vulgaris genome. (3) Screening for expression using PCR and gene sequencing technologies. rib and OmpA Recombinant sulfate-reducing bacteria rSRB-RiO; (4) The recombinant sulfate-reducing bacteria rSRB-RiO from step (3) is amplified using bacterial culture technology to obtain the recombinant sulfate-reducing bacteria (rSRB-RiO).

[0007] Furthermore, in the above-mentioned method for preparing recombinant sulfate-reducing bacteria (rSRB-RiO), the strain preservation number of Desulfovibriovulgaris is GMCC46396.

[0008] The concentration of recombinant sulfate-reducing bacteria (rSRB-RiO) in industrial wastewater is further specified to be 0.5 × 10⁻⁶. 5 ~1.5×10 5 / mL.

[0009] Further specifying, heavy metal ions include Zn 2+ Cr 3+ Cd 2+ Ni 2+ 、Al 3+ and Fe 2+ .

[0010] Further specified, the reaction was stirred for 24 hours.

[0011] Further specifying, the light source is sunlight or an illumination source.

[0012] To further define, organic compounds include long-chain hydrocarbons and benzene ring compounds.

[0013] The second objective of this invention is to provide a semiconductor nanoparticle prepared by the above method.

[0014] The resulting semiconductor nanoparticles are a mixture of various heavy metal ion sulfides in wastewater, such as a mixture of Cr2S3, CdS, NiS, Al2S3 and FeS.

[0015] The third objective of this invention is to provide an application of semiconductor nanoparticles prepared by the above method as a catalyst in photocatalytic oxidation.

[0016] The fourth objective of this invention is to provide an application of semiconductor nanoparticles prepared by the above method in photocatalytic low-carbon emission water treatment.

[0017] The fifth objective of this invention is to provide an application of the above-mentioned method in the efficient treatment of wastewater rich in sulfates, heavy metals and organic matter.

[0018] To further define, organic compounds include long-chain hydrocarbons and benzene ring compounds.

[0019] The advantages of this invention compared to existing technologies are: This invention prepares semiconductor nanoparticles by combining the products of sulfate-reducing bacteria's metabolism of sulfate with heavy metal ions. Coupled with photocatalysis, it promotes the transfer of photogenerated electrons from the oxidation of organic matter into the microbial cells, increasing microbial activity and the efficiency of semiconductor nanoparticle synthesis. This further promotes CO2 reduction in the system, achieving low-carbon treatment of industrial wastewater and sustainable bio-green manufacturing.

[0020] This invention utilizes extracellular polymers from microorganisms and nano-metal particles to construct a semiconductor biohybrid. Under light, this biohybrid degrades macromolecular organic matter, improving the biodegradability of wastewater. It absorbs light energy to generate electrons and converts them into bioenergy, providing ample energy for microbial metabolism. The sulfate-reducing bacteria's sulfate-semiconductor biohybrid photosynthetic system effectively integrates the superior light-harvesting capabilities of semiconductors with the synthetic capabilities of microorganisms, enabling efficient transfer of photogenerated electrons into microbial cells and promoting microbial metabolism. Furthermore, sulfate-reducing bacteria can synthesize organic matter using CO, CO2, or hydrogen as the sole electron donor, and can also utilize formate and CO2 as carbon sources for the dissimilatory reduction of sulfate. This photocatalytic semiconductor-microorganism approach achieves efficient utilization of organic matter, with photogenerated electrons or reducing power driving the metabolism of high-value-added products within the organism. Simultaneously, the self-assembled semiconductor biohybrid of photo-driven sulfate-reducing bacteria further promotes extracellular electron transfer and intracellular CO2 reduction, thereby achieving the goal of reducing carbon emissions in the treatment of industrial wastewater. Attached Figure Description

[0021] Figure 1 The change in semiconductor nanoparticle concentration in the anaerobic system of Example 1; Figure 2 The change in sulfate concentration in the anaerobic system of Example 1; Figure 3 The COD concentration change in the anaerobic system of Example 1; Figure 4 The change in heavy metal ion concentration in the anaerobic system of Example 1; Figure 5 The change in semiconductor nanoparticle concentration in the photocatalytic anaerobic system of Example 2; Figure 6 The efficiency of organic matter degradation and removal in the photocatalytic anaerobic system of Example 2; Figure 7 The change in sulfate concentration in the photocatalytic anaerobic system of Example 2; Figure 8 The COD removal rate in the photocatalytic anaerobic system of Example 2; Figure 9 The change in heavy metal ion concentration in the photocatalytic anaerobic system of Example 2; Figure 10 The change in CO2 content in the photocatalytic anaerobic system of Example 2; Figure 11 This is a SEM-EDS image of semiconductor bio-hybridization in Example 3; Figure 12 The image shows the UV-Vis spectrum of the semiconductor bio-hybrid in Example 3. Figure 13 The XRD pattern of the semiconductor-biohybrid in Example 3; Figure 14 The FT-IR spectrum of the biological hybrid in Example 3; Figure 15 The electrochemical impedance spectroscopy of the biohybrid in Example 3 is shown. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0024] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used in the following embodiments, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0025] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range. In this specification and claims, range definitions may be combined and / or interchanged, unless otherwise stated, these ranges include all sub-ranges contained therein.

[0026] The indefinite articles “a” and “an” preceding an element or component of this invention do not impose any limitation on the quantity (i.e., number of times) of the element or component. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers only to the singular form.

[0027] In this invention, "an embodiment" or "embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that excludes other embodiments.

[0028] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0029] The physicochemical parameters of the industrial wastewater described in the following examples are shown in Table 1.

[0030] Table 1 Characteristics of Industrial Wastewater

[0031] The preparation method of the recombinant sulfate-reducing bacteria (rSRB-RiO) described in the following examples includes the following steps: (1) Synthesizing riboflavin gene using gene synthesis technology ( rib (GenBank: XM_018880422.1) and outer membrane protein A gene ( OmpA (GenBank: AF135263.1), and constructed the pUC-GW-RiO recombinant plasmid by ligating it with the pUC-GW vector, and transformed it into E. coli DH5α competent cells; (2) Screening for expression using PCR technology rib and OmpA The target strain was obtained by extracting the pUC-GW-RiO plasmid, which was then ligated to the pUC-GW vector using electroporation and recombined into a specific site in the Desulfovibrio vulgaris (GMCC46396) genome. (3) Screening for expression using PCR and gene sequencing technologies. rib and OmpA Recombinant sulfate-reducing bacteria rSRB-RiO; (4) The recombinant sulfate-reducing bacteria rSRB-RiO from step (3) is amplified using bacterial culture technology to obtain the recombinant sulfate-reducing bacteria (rSRB-RiO).

[0032] Example 1: This example describes a method for the efficient biosynthesis of semiconductor nanoparticles using recombinant sulfate-reducing bacteria, which is carried out according to the following steps: Step (1): Industrial wastewater containing sulfate, heavy metals, and organic matter (parameters as shown in Table 1) is introduced into an anaerobic reactor (40L), and inoculated with the amplified recombinant sulfate-reducing bacteria (rSRB-RiO) at a concentration of 1.0 × 10⁻⁶. 5 / mL, nitrogen gas was introduced to remove oxygen from the reactor, and the mixture was treated for 24 hours under mechanical stirring; Step (2): Run the reactor continuously until the bacterial concentration in the reaction system reaches 3.4 × 10⁻⁶. 9 / mL, the wastewater retention time was set to 24 hours, utilizing anaerobic microorganisms to metabolize sulfate and form S 2- They covalently bind with heavy metal ions to form semiconductor nanoparticles, and every 24 hours, 20% of the reactor volume of liquid is discharged from the system. Step (3): Collect the liquid, use ultrasound and centrifugation to obtain semiconductor nanoparticles, and detect the yield of semiconductor nanoparticles.

[0033] Comparative Example 1: The difference between this comparative example and Example 1 is that the original sulfate-reducing bacterium SRB, namely Desulfovibrio vulgaris (GMCC46396), was used. Other steps and parameters were the same as in Example 1.

[0034] Detection 1: The concentration of semiconductor nanoparticles in the detection system was measured, and the results are as follows. Figure 1 As shown, from Figure 1 The results showed that, compared with the SRB control group (Comparative Example 1), rSRB-RiO increased the concentration of semiconductor nanoparticles in the anaerobic system, with an average concentration of 60.35 mg / L.

[0035] Test 2: Detection of changes in sulfate ion concentration in the treatment system, results are as follows. Figure 2 As shown. From Figure 2 The results show that, compared with the control group (Comparative Example 1), rSRB-RiO significantly improved the removal efficiency of sulfate by utilizing the characteristic of sulfate-reducing bacteria to specifically metabolize sulfate, with an average removal rate of 82.91%.

[0036] Detection 3: Changes in COD concentration in the detection and treatment system, results are as follows: Figure 3 As shown. From Figure 3 The results show that, compared with the control group (Comparative Example 1), rSRB-RiO improved the COD removal efficiency, with an average removal rate of 90.30%.

[0037] Detection 4: Changes in the total heavy metal ion concentration in the detection and treatment system; results are as follows. Figure 4 As shown. Compared with the control group (Comparative Example 1), rSRB-RiO produced S through the metabolism of sulfate.2- Covalent binding of heavy metal ions achieves highly efficient removal of heavy metal ions, with an average removal rate of 83.80%.

[0038] Example 2: This example describes a method for efficiently biosynthesizing semiconductor nanoparticles using recombinant sulfate-reducing bacteria, which is carried out according to the following steps: Step (1): Industrial wastewater containing sulfate, heavy metal (Cd), and organic matter (parameters shown in Table 1, where only Cd is a heavy metal and its concentration is shown in Table 1) was introduced into an anaerobic reactor (40L). The amplified recombinant sulfate-reducing bacteria (rSRB-RiO) was inoculated at a concentration of 1.0 × 10⁻⁶. 5 / mL, nitrogen gas was introduced to remove oxygen from the reactor, and the mixture was treated for 24 hours under mechanical stirring; Step (2): Run the reactor continuously until the bacterial concentration in the reaction system reaches 3.4 × 10⁻⁶. 9 / mL, turn on the light (LED), set the light and dark alternation operation (operation cycle: light 8min; dark 2min), the wastewater retention time is 24 hours, and every 24 hours, 20% of the liquid volume of the reactor is discharged from the system; Step (3): Collect the liquid, use ultrasound and centrifugation to obtain semiconductor nanoparticles, and calculate the efficiency of the system in generating semiconductor nanoparticles.

[0039] Comparative Example 2: The difference between this comparative example and Example 2 is that the original sulfate-reducing bacterium SRB, namely Desulfovibrio vulgaris (GMCC46396), was used. Other steps and parameters were the same as in Example 2.

[0040] Detection 5: The concentration of semiconductor nanoparticles in the detection system was measured, and the results are as follows: Figure 5 As shown, the results indicate that, compared with the dark period, Comparative Example 2 photocatalytic SRB (Light-SRB) increased the concentration of semiconductor nanoparticles (concentration 42.14 mg / L), while photocatalytic rSRB-RiO synthesis of semiconductor nanoparticles (Light-rSRB-RiO) significantly increased the concentration of semiconductor nanoparticles to 70.60 mg / L. Data analysis showed that the difference was extremely significant.

[0041] Test 6: Degradation efficiency of organic matter in the detection system. For example... Figure 6 As shown, compared with Comparative Example 2 Light-SRB, Light-rSRB-RiO significantly increased the degradation of organic matter in the anaerobic reactor (by 20.60%).

[0042] Test 7: Detect changes in sulfate concentration in the treatment system. For example... Figure 7As shown, compared with Comparative Example 2, Light-rSRB-RiO significantly improved the sulfate removal efficiency, with an average removal rate of 92.91%.

[0043] Detection 8: Changes in COD concentration in the detection and treatment system. From... Figure 8 The results show that, compared with Comparative Example 2, Light-rSRB-RiO improved the COD removal efficiency, with an average removal rate of 90.30%.

[0044] Detection 9: Changes in heavy metal ion concentration in the detection and processing system; results are as follows. Figure 9 As shown in the figure. Compared with Comparative Example 2, Light-rSRB-RiO improved the system's removal efficiency for heavy metal ions, with an average removal rate of 90.26%.

[0045] Test 10: Detect the CO2 content in the treatment system. For example... Figure 10 As shown, compared with Light-SRB, the Light-rSRB-RiO system reduces the content of free CO2 and promotes the fixation of CO2 by microorganisms.

[0046] Example 3: This example characterizes the rSRB-RiO self-assembled semiconductor bio-hybrid.

[0047] Step (1): The semiconductor bio-hybrid product collected in Example 2 was fixed with 2.5% glutaraldehyde and then stored overnight at 4°C. Subsequently, the fixed precipitate was sequentially immersed in a series of ethanol solutions of different concentrations (30%, 50%, 75%, 85%, and 95%) for dehydration, each concentration for 15 minutes; then immersed twice in 100% ethanol for 20 minutes each time; then immersed in a 1:1 mixture of ethanol and isoamyl isovalerate for 30 minutes; finally, immersed in isoamyl isovalerate for 1 hour. The sample was then dried. After drying, the sample was sputter-coated with gold, and imaging was performed using a scanning electron microscope combined with an energy dispersive X-ray detector. The surface elemental composition was analyzed using an energy dispersive X-ray detector. The results are as follows: Figure 11 As shown, the hybrid contains S and Cd.

[0048] Step (2): The semiconductor biohybrid sample was uniformly dispersed in a hexane solution and measured using a spectrophotometer to perform baseline correction. Then, the sample solution was placed in the sample cell for measurement, and the absorbance at different wavelengths was recorded to obtain the UV-Vis spectrum, as shown below. Figure 12 As shown, the ultraviolet-visible absorption spectrum of the hybrid shows a characteristic absorption sideband at 520 nm.

[0049] Step (3): Prepare the sample by X-ray irradiation, and measure and analyze the position, intensity, and width of the diffraction peaks to determine the composition of the sample. The results are as follows: Figure 13 As shown, the XRD pattern is a perfect match with the cubic CdS standard card (JCPDS 10-0454).

[0050] Step (4): The infrared spectrum of CdS-rSRB-RiO was determined using Fourier transform infrared spectroscopy (FT-IR) to analyze the functional groups in the semiconductor bio-hybrid. The results are as follows: Figure 14 As shown, CdS nanoparticles and functional groups in bacterial extracellular polymeric substances (EPS) achieve molecular-level self-assembly through coordination bonds and hydrophobic interactions.

[0051] Step (5): Determine the electron transport resistance using electrochemical impedance spectroscopy (EIS). By measuring the system's response signal and analyzing the impedance at different frequencies, the electrochemical information of the system is obtained, such as... Figure 15 As shown, compared with the control group, CdS-rSRB-RiO reduces the resistance to electron transfer into microbial cells (charge transfer impedance is reduced by 68%) and improves the efficiency of photogenerated electron transfer into cells.

[0052] The above description is merely a preferred embodiment of the present invention. These specific embodiments are different implementations based on the overall concept of the present invention, and the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for efficiently biosynthesizing semiconductor nanoparticles using recombinant sulfate-reducing bacteria, characterized in that, The method described: express rib Gene coding and OmpA The gene-encoded recombinant sulfate-reducing bacteria were amplified and added to industrial wastewater containing sulfate, heavy metal ions and organic matter. The mixture was stirred and reacted under anaerobic conditions, followed by illumination. The wastewater retention time was set to 24 hours, and the liquid was collected every 24 hours and centrifuged to obtain semiconductor nanoparticles.

2. The method according to claim 1, characterized in that, The concentration of recombinant sulfate-reducing bacteria in industrial wastewater was 0.5 × 10⁻⁶. 5 ~1.5×10 5 / mL.

3. The method according to claim 1, characterized in that, Heavy metal ions include Zn 2+ Cr 3+ Cd 2+ Ni 2+ 、Al 3+ and Fe 2+ .

4. The method according to claim 1, characterized in that, The reaction was stirred for 24 hours, with sunlight or artificial lighting as the light source.

5. The method according to claim 1, characterized in that, Organic compounds include long-chain hydrocarbons and benzene ring compounds.

6. Semiconductor nanoparticles prepared by the method according to any one of claims 1-5.

7. The application of the semiconductor nanoparticles as a catalyst in photocatalytic oxidation according to claim 6.

8. The application of the semiconductor nanoparticles according to claim 6 in photocatalytic low-carbon emission water treatment.

9. The application of the method according to any one of claims 1-5 in the efficient treatment of wastewater rich in sulfates, heavy metals and organic matter.

10. The application according to claim 9, characterized in that, Organic compounds include long-chain hydrocarbons and benzene ring compounds.

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