Semi-artificial photosynthetic system for hydrogen production based on escherichia coli, preparation method and application

Abstract

The invention provides a preparation method of a Cu-Bp / escherichia coli (Cu-BP / E. coli) semi-artificial photosynthetic system for efficient biological hydrogen production. The preparation method comprises the following steps: designing black phosphorus nanosheets doped with copper quantum dots, testing the catalytic performance of Cu-Bp, and testing the hydrogen production performance of the Cu-BP / E. coli semi-artificial photosynthetic system. The system combines Escherichia coli capable of effectively catalyzing hydrogen generation and excellent light absorption performance of inorganic materials, and energy in sunlight is converted into chemical energy. Under the irradiation of visible light with the wavelength range of 420-780 nm and the light intensity of 2000 Wm, the system shows excellent hydrogen production performance, the hydrogen production amount within 5 hours reaches up to 2.12 mmol, and the hydrogen production amount is 2.07 times that of a BP / E. coli system and 2.32 times that of pure escherichia coli. A new path is opened up for the development of the field of biological hydrogen production.

Description

Technical Field

[0001] The present invention belongs to the technical field of renewable energy, and mainly relates to a preparation method of a semi-artificial photosynthetic system based on hydrogen production by Escherichia coli. Background Art

[0002] As a clean, sustainable and renewable energy, hydrogen energy has become a key direction for global energy transformation. However, current hydrogen production methods, which rely on fossil fuel combustion and electrolysis of water to produce hydrogen, have problems such as low efficiency and the easy generation of greenhouse gases during the preparation process, exacerbating global warming. In recent years, the combination of photosensitizers and biological materials has brought new research ideas for the field of solar hydrogen production. For a society pursuing sustainable development, light is an extremely abundant energy source. Photosynthetic organisms ingeniously utilize solar radiation to synthesize energy-rich compounds from water and carbon dioxide. Generally, semi-artificial photosynthetic hydrogen production systems can be divided into enzyme-based (cell-free) and whole-cell types. In cell-free systems, the process of purifying enzymes is costly and technically difficult, so its application is restricted to a certain extent. In contrast, whole-cell semi-artificial photosynthetic systems use intact cells as biocatalysts, and raw materials are extensive and can be mass-produced by low-cost methods such as microbial fermentation.

[0003] Traditional semi-artificial photosynthetic system systems have problems such as poor biocompatibility, weak visible light absorption, and insufficient electron transfer efficiency. To address these problems, this technology selects the chemoheterotrophic microorganism Escherichia coli BL21(DE3), which is widely present and rapidly reproducing in nature, as the microbial component of the semi-artificial photosynthetic system. Escherichia coli BL21(DE3) is selected as the microbial part of this system mainly because of its wide adaptability, rapid reproduction rate, mature gene manipulation technology, and clear metabolic pathway, which helps to deeply study the energy and electron migration problems at the material-microorganism interface. Summary of the Invention

[0004] What the present invention aims to solve is that traditional hydrogen production methods often require expensive equipment and a large amount of energy consumption. For example, relying on fossil fuel combustion and electrolysis of water to produce hydrogen has problems such as too high hydrogen production cost and the easy generation of greenhouse gases during the preparation process, exacerbating global warming. To this end, the present invention combines bacteria with black phosphorus nanosheets doped with copper quantum dots to prepare an Escherichia coli / Cu-BP semi-artificial photosynthetic system. Under visible light irradiation, the Escherichia coli / Cu-BP semi-artificial photosynthetic system can convert solar energy into hydrogen.

[0005] The method of the present invention realizes the mass production of black phosphorus nanosheets doped with copper quantum dots to achieve low-cost and high-efficiency hydrogen production. By combining black phosphorus nanosheets doped with copper quantum dots with Escherichia coli and self-assembling them into a Cu-BP / E. coli semi-artificial photosynthetic system, this system exhibits excellent hydrogen production ability.

[0006] The present invention relates to the preparation of Cu - BP for hydrogen production, mainly involving the design and characterization of black phosphorus nanosheets doped with copper quantum dots, the catalytic performance test of Cu - Bp, E. coli and the hydrogen production performance test of the / Cu - Bp semi - artificial photosynthetic system. Specifically, it is carried out according to the following steps: E. coli / Cu - Bp semi - artificial photosynthetic system. Specifically, it is carried out according to the following steps: Step 1: Add 50.0 mg of black phosphorus nanosheets to a solution containing 0.94 mL of 0.1 M copper sulfate and 1.0 mL of 1.0 M ammonia water solution, and mix well; Step 2: Under the ice - bath condition of 0 - 4 °C, slowly drop the liquid obtained in Step 1 into 4.0 mL of 500 mM sodium borohydride solution; Step 3: Continuously stir for 1 h, wash three times using a centrifuge, and vacuum dry to obtain Cu - BP powder doped with copper quantum dots; Step 4: Weigh 10.0 g / L peptone, 5.0 g / L yeast extract, and 10.0 g / L sodium chloride respectively. Dissolve these components in 100 mL of deionized water and adjust the pH value to 7.4. This is the Luria - Bertani culture medium required for culturing Escherichia coli.

[0007] Step 5: Sterilize the Luria - Bertani culture medium prepared in Step 4 in an autoclave at 121 °C for 20 min; Step 6: In a laminar flow hood, we first turn on the ultraviolet lamp for 30 - minute sterilization to ensure the aseptic state of the operating environment. Under aseptic conditions, inoculate 100 μL of Escherichia coli into the sterilized Luria - Bertani culture medium, and place the inoculated bacteria in a shaker at 37 °C for cultivation; Step 7: Use ultraviolet - visible absorption spectroscopy to measure the growth density of bacteria. When the OD600 value is observed to reach the range of 0.5 to 0.8, add Cu - BP and culture for 12 hours to prepare Cu-BP / E. coli a semi - artificial photosynthetic system; Step 8: Transfer the prepared Cu - BP / E. coli semi - artificial photosynthetic system into an anaerobic bottle and replace the fresh culture medium. Place the anaerobic bottle in a shaker for overnight cultivation to allow the bacteria to gradually adapt to the anaerobic environment and promote the enhancement of the activity of hydrogenase; Step 9: After 12 hours, replace the fresh culture medium for the Cu - BP / E. coli semi - artificial photosynthetic system again and prepare for the test of hydrogen production; Step 10: Start measuring the Cu - BP / E. coliThe hydrogen production of the semi-artificial photosynthetic system was measured starting from the 0th hour, once every hour for 5 consecutive hours.

[0008] The beneficial effects of the present invention are as follows: The semi-artificial photosynthetic system provided by the present invention combines the ability of Escherichia coli to effectively catalyze hydrogen production and the excellent light absorption performance of inorganic materials, converting the energy in sunlight into chemical energy.

[0009] The Cu-BP synthesized in the present invention not only has the characteristics of low-cost mass production but also realizes high-efficiency hydrogen production. After doping with copper quantum dots, the visible light absorption ability of Cu-BP is significantly improved.

[0010] The Cu-BP in the present invention has strong visible light absorption ability and more efficient electron transfer efficiency. After doping with copper quantum dots, the resistance of electron transfer of Cu-BP is significantly reduced.

[0011] The Cu-BP in the present invention / E. coli The semi-artificial photosynthetic system combines the excellent visible light capture characteristics of Cu-BP and the specific catalytic ability of Escherichia coli. The hydrogen production of this system does not rely on resources such as petroleum, gas, and electric energy, but relies on Escherichia coli to convert glucose into hydrogen through in vivo metabolism.

[0012] The Cu-BP in the present invention / E. coli Under visible light irradiation, the hydrogen production of the semi-artificial photosynthetic system can reach 2.12 mmol in 5 hours, and the hydrogen production is 2.07 times that of the BP / E. coli system and 2.32 times that of pure Escherichia coli. Brief Description of the Drawings

[0013] Figure 1 are the TEM and EDS spectra of Cu-BP.

[0014] Figure 2 are the UV-Vis diffuse reflectance absorption spectra of BP and Cu-BP.

[0015] Figure 3 are the LSV diagrams of BP and Cu-BP.

[0016] Figure 4 are the electrochemical impedance (EIS) spectra of BP and Cu-BP.

[0017] Figure 5 is the growth curve of bacteria.

[0018] Figure 6 is the hydrogen production of the semi-artificial photosynthetic system. Detailed Description of the Invention Examples

[0019] Step 1: Add 50.0 mg of black phosphorus nanosheets to a solution containing 0.94 mL of 0.1 M copper sulfate and 1.0 mL of 1.0 M ammonia water, and mix well. Step 2: Under an ice bath condition of 0 - 4 °C, slowly drop the liquid obtained in Step 1 into 4.0 mL of 500 mM sodium borohydride solution. Step 3: Stir continuously for 1 h, wash three times using a centrifuge, and vacuum dry to obtain Cu - BP powder doped with copper quantum dots. Step 4: Weigh 10.0 g / L peptone, 5.0 g / L yeast extract, and 10.0 g / L sodium chloride respectively. Dissolve these components in 100 mL of deionized water and adjust the pH value to 7.4. This is the Luria - Bertani culture medium required for culturing Escherichia coli.

[0020] Step 5: Sterilize the Luria - Bertani culture medium prepared in Step 4 in an autoclave at 121 °C for 20 min. Step 6: In a laminar flow hood, we first turn on the ultraviolet lamp for 30 - minute sterilization treatment to ensure the aseptic state of the operating environment. Under aseptic conditions, inoculate 100 μL of Escherichia coli into the sterilized Luria - Bertani culture medium, and place the inoculated bacteria in a shaker at 37 °C for cultivation. Step 7: Use ultraviolet - visible absorption spectroscopy to measure the growth density of the bacteria. When the OD600 value is observed to reach the range of 0.5 to 0.8, add Cu - BP and culture for 12 hours to prepare Cu-BP / E. coli a semi - artificial photosynthetic system; Step 8: Transfer the prepared Cu - BP / E. coli semi - artificial photosynthetic system into an anaerobic bottle and replace the fresh culture medium. Place the anaerobic bottle in a shaker for overnight cultivation to allow the bacteria to gradually adapt to the anaerobic environment and promote the enhancement of hydrogenase activity. Step 9: After 12 hours, replace the fresh culture medium for the Cu - BP / E. coli semi - artificial photosynthetic system again to prepare for the test of hydrogen production. Step 10: Start measuring the hydrogen production of the Cu - BP / E. coli semi - artificial photosynthetic system. The test starts from the 0th hour and is measured every hour for 5 consecutive hours.

[0021] The following experiments are used to verify the invention effect: 1. Design and characterization of black phosphorus nanosheets doped with copper quantum dots From the TEM images (such as Figure 1As can be seen (as shown), the synthesized black phosphorus nanomaterials are uniformly distributed and show a flaky structure. The EDS image further shows that in addition to P and O elements, Cu elements are also uniformly distributed on the surface of the black phosphorus nanosheets, which confirms the successful preparation of Cu-BP.

[0022] Figure 2 Figure 4 shows the UV-visible diffuse reflectance absorption spectra of BP and Cu-BP. It is found that when copper quantum dots are doped into black phosphorus, the absorption intensity of the formed Cu-BP material in the visible light region is significantly enhanced.

[0023] 2. Catalytic performance test of Cu-Bp Figure 3 Figure 5 shows the linear sweep voltammetry (LSV) curves of BP and Cu-BP. The LSV curve is a commonly used electrochemical characterization method for evaluating the catalytic activity of materials at a specific potential. As Figure 3 shown, at the same potential, Cu-BP shows a higher current density than BP. This indicates that under photocatalytic conditions, Cu-BP has better electrocatalytic hydrogen evolution activity.

[0024] Figure 4 Figure 6 is the electrochemical impedance (EIS) spectra of BP and Cu-BP. Comparing the experimental results, it is observed that the charge transfer resistance of Cu-BP is significantly lower than that of BP. This finding reveals that the doping of copper quantum dots greatly promotes the charge transfer efficiency in the system.

[0025] E. coli Hydrogen production performance test of the / Cu-Bp semi-artificial photosynthetic system Before the hydrogen production test, the biocompatibility of Cu-BP was investigated. Figure 5 Figure 7 monitors the OD E. coli values of and Cu-BP / E. coli at different growth time periods for 52 hours. Through detection, it is found that Cu-BP has almost no effect on bacterial growth, indicating its good biocompatibility. 600 The hydrogen production performance of the three semi-artificial photosynthetic systems of Cu-BP /

[0026] BP / E. coli, BP / E. coli and E. coli was detected using a gas chromatograph. The hydrogen production test was carried out once every hour starting from the 0th hour for 5 hours. As Figure 6 can be seen, pure Escherichia coli produces 0.29 mmol of H2 within the initial 1 hour and reaches 0.91 mmol after 5 hours of irradiation. The BP / E. coli system can produce 1.02 mmol of H2 in 5 hours. While the Cu-BP / E. coliThe maximum hydrogen production of the semi-artificial photosynthetic system can reach 2.12 mmol in 5 hours.

[0027] In addition, the BP group, Cu-BP group, E. coli (dead) + BP group, and E. coli (dead) + Cu-BP group were set up, and the hydrogen production performance of the artificial photosynthetic system was tested under the same conditions. The test results showed that almost no hydrogen was produced in the above groups, which means that living bacteria played an important role as catalysts in the hydrogen production process.

[0028] In the Cu-BP / E. coli semi-artificial photosynthetic system prepared in the present invention, E. coli, as a biocatalyst, is responsible for converting glucose into hydrogen through metabolic conversion, while Cu-BP promotes the transfer of electrons under visible light irradiation to accelerate hydrogen production. This semi-artificial photosynthetic system can produce hydrogen at low cost without using any energy, and the whole system does not produce any greenhouse gases, providing a new way for sustainable energy production. In addition, the microorganism for developing the Cu-BP / E. coli semi-artificial photosynthetic system is also E. coli, which is widely present in nature and has strong reproductive ability. From the environmental and economic perspectives, it is of great significance for industrial applications.

Claims

1. A Cu-BP / E. coli semi-artificial photosynthetic system for hydrogen production, characterized in that, It is composed of the self-assembly combination of E. coli and black phosphorus nanosheets doped with copper quantum dots Cu-BP.

2. The preparation method of the system according to claim 1, characterized in that, It includes the following steps: Under sterile conditions, inoculate E. coli in sterilized Luria-Bertani culture medium for cultivation; When the OD of the culture medium 600 reaches the range of 0.5 to 0.8, add black phosphorus nanosheets doped with copper quantum dots Cu-BP, and culture until the self-assembly of black phosphorus nanosheets doped with copper quantum dots Cu-BP and Escherichia coli is completed, and a Cu-BP / E. coli semi-artificial photosynthetic system is prepared.

3. The method according to claim 2, characterized in that, The black phosphorus nanosheets Cu-BP doped with copper quantum dots are obtained by a method including the following steps: Under ice bath conditions, slowly drop the sodium borohydride solution into the precursor containing copper ions, ammonia water and black phosphorus nanosheets, and stir and react to obtain the black phosphorus nanosheets Cu-BP powder doped with copper quantum dots.

4. The method according to claim 3, wherein The black phosphorus nanosheets Cu-BP doped with copper quantum dots are obtained by a method including the following steps: 1) Add 50.0 mg of black phosphorus nanosheets to 0.94 mL of copper sulfate with a concentration of 0.1 M and 1.0 mL of ammonia water with a concentration of 1.0 M, and mix evenly; 2) Under ice bath conditions at 0-4 °C, slowly drop the liquid obtained in step 1) into 4.0 mL of 500 mM sodium borohydride solution; 3) Continuously stir for 1 h, wash three times with a centrifuge, and vacuum dry to obtain the black phosphorus nanosheets Cu-BP powder doped with copper quantum dots.

5. The method according to claim 2, characterized in that, The Luria-Bertani culture medium contains 10.0 g / L of peptone, 5.0 g / L of yeast extract and 10.0 g / L of sodium chloride, and the pH value is 7.4; Before using the Luria-Bertani culture medium, place it in an autoclave and sterilize at 121 °C for 20 min; Before inoculating E. coli in the sterilized Luria-Bertani culture medium, turn on the ultraviolet lamp on the workbench for 30 minutes of sterilization treatment to ensure that the operating environment is in a sterile state; During cultivation, place the Luria-Bertani culture medium inoculated with E. coli in a shaker at 37 °C for cultivation.

6. The method according to claim 2, wherein After adding the black phosphorus nanosheets Cu-BP doped with copper quantum dots, continue to cultivate for 12 hours.

7. Application of the system described in claim 1 in photosynthetic hydrogen production.

8. The application according to claim 7, wherein After the black phosphorus nanosheets Cu-BP doped with copper quantum dots are doped with copper quantum dots, the resistance of electron transfer is significantly reduced, and the visible light absorption ability is significantly improved.

9. The application according to claim 7, wherein The system produces hydrogen independently of petroleum, gas, and electric energy. Instead, it relies on E. coli to metabolize glucose in the body into hydrogen.

10. The application according to claim 7, wherein Under visible light irradiation, the hydrogen production of the described system is 2.12 mmol in 5 hours, and the hydrogen production is 2.07 times that of the black phosphorus nanosheet modified Escherichia coli BP / E. coli system and 2.32 times that of pure Escherichia coli.

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