Hydrogen production e. coli-bso bio-complex system and preparation method thereof

Abstract

The application prepares an E.coli-BSO biological composite system for biological hydrogen production. The biological composite system breaks through the limitation of traditional single microorganism and material hydrogen production system, combines bismuth stannate (BSO) nanoparticles and E.coli through electrostatic interaction, and constructs a new composite system with the hydrogen production ability of microbial metabolism and the photocatalytic performance of nanomaterials. The system integrates the inherent hydrogen production metabolic pathway in E.coli cells and the excellent visible light absorption ability and photocatalytic electron transfer performance of BSO nanomaterials, solves the problems of low light energy utilization rate of pure microorganisms and hydrogen production of pure photocatalytic materials depending on external electron donors. Experimental verification shows that under visible light irradiation, the hydrogen production of the composite system reaches 0.8 mmol within 4 hours, which is 1.5 times higher than that of pure E.coli, and significantly breaks through the efficiency bottleneck of existing biological hydrogen production technology.

Description

TECHNICAL FIELD

[0001] The present application focuses on the field of biological hydrogen production technology, and particularly relates to a hydrogen-producing E. coli and BSO biological complex system and a preparation method thereof. BACKGROUND

[0002] Hydrogen energy has become a new star with great potential due to its high energy density, cleanliness, and renewable advantages. It is widely expected to become an ideal alternative to traditional energy sources such as oil and natural gas because of its wide range of sources and no secondary pollution after combustion, thus attracting extensive attention and in-depth research worldwide. Currently, the main production methods of hydrogen include extraction from fossil fuels and electrolysis of water. However, both traditional methods have obvious drawbacks: hydrogen production from fossil fuels produces a large amount of carbon dioxide, exacerbating environmental burden; hydrogen production by water electrolysis is costly and relies on a large amount of electricity supply.

[0003] Biological hybrid-mediated semi-artificial photosynthesis system is an innovative technology path that ingeniously combines the excellent light trapping performance of photosensitizer and the high catalytic activity of biological catalyst, opening up new ideas for constructing more efficient whole-cell biological complex systems. In the semi-artificial photosynthesis system, sunlight irradiates the semiconductor to generate photoelectrons, which are transferred to adjacent organisms through a specific transfer pathway, thereby driving the physiological metabolism and product synthesis in the organisms. The key to achieving efficient biological hybridization reaction lies in ensuring good compatibility of the interface between photosensitizer and biological catalyst, which depends on the effective combination of photosensitizer active site and biological catalyst, directly affecting the generation, capture and utilization efficiency of photoelectrons. SUMMARY

[0004] Based on the above technical problems, the present application designs a material bismuth stannate with strong visible light absorption, which is combined with E. coli through electrostatic interaction to construct a semi-artificial photosynthesis system. The research selects a nano material bismuth stannate with wide spectral absorption characteristics as the basic material, and at the same time, selects the widely used E. coli as the biological carrier of the biological hybrid system. The experimental results show that the E.coli-BSO system significantly improves the hydrogen production.

[0005] The development of biological complex system (E.coli-BSO) for hydrogen production covers three key links: first, the design and characterization of BSO nanomaterials; second, the exploration of the biocompatibility of BSO; third, the comprehensive testing of the hydrogen production performance of E.coli-BSO biological complex system. The purpose is to improve the hydrogen production efficiency through innovative technical means.

[0006] The present application is dedicated to solving the technical problems of low catalytic efficiency of photosynthesis, high cost and low efficiency of hydrogen production caused by the dependence of artificial photosynthesis on high-purity semiconductors. The technical scheme provided by the present application can produce a large amount of nanomaterial BSO, which has wide-range visible light absorption capacity and good catalytic performance. Through adsorption on the surface of E. coli, a self-assembled E. coli-BSO biological composite system is formed, which effectively improves the hydrogen production efficiency of the system. The preparation process of the E. coli-BSO biological composite system for hydrogen production of the present application is carried out around three core steps of design and characterization of TBSO nanomaterial, exploration of biocompatibility of BSO and hydrogen production performance test of the composite system.

[0007] Specifically, the present application provides an E. coli-BSO biological composite system for hydrogen production and a preparation method thereof, comprising the following steps:

[0008] Step one: prepare LB culture solution for culturing E. coli according to a specific formula, and after preparation, place the culture solution in a high-pressure steam sterilization pot at 121 DEG C for sterilization treatment for 20 minutes to ensure that the culture solution is sterile.

[0009] Step two: under the sterile environment of a super-clean workbench, inoculate E. coli into the sterilized culture solution. After inoculation, place the container containing the bacterial culture solution in a shaking bed at 37 DEG C for culture.

[0010] Step three: weigh bismuth nitrate [Bi (NO3) 3·5H2O] and stannous chloride (SnCl2·2H2O) according to the mass ratio of Bi to Sn of 1:1.

[0011] Step four: place the weighed raw materials into two clean beakers respectively. Prepare an appropriate amount of solvent, an ethanol-water mixed solution. Slowly add bismuth nitrate to the solvent, stirring while adding, until completely dissolved. Since bismuth nitrate is easily hydrolyzed, add a small amount of dilute nitric acid to the solution to inhibit hydrolysis, so that the solution remains clear and transparent. Similarly, add stannous chloride to the other part of the solvent and stir to dissolve.

[0012] Step five: mix the two solutions uniformly, and under stirring conditions, slowly add NaOH solution until white precipitate is produced. Then, place the solution in a reaction kettle and react in a constant-temperature drying box, with a temperature of 180 DEG C and a reaction time of 12 hours. During the reaction, the metal ions in the solution react with the precipitant to gradually form bismuth stannate nanocrystal nuclei and continuously grow.

[0013] Step six: After the reactor is naturally cooled to room temperature, the supernatant is discarded, and the precipitate is collected. The precipitate is washed with deionized water and anhydrous ethanol alternately for three times to remove the surface adsorbed impurity ions and solvent molecules. The washed precipitate is transferred to a clean culture dish and placed in a vacuum drying oven, and the temperature is set to 60 DEG C, and dried for 12 hours to obtain bismuth stannate nanoparticles.

[0014] Step seven: The cultured E. coli bacterial liquid is transferred to a centrifuge tube, and the E. coli bacterial body is collected.

[0015] Step eight: A certain amount of bismuth stannate nanoparticles is weighed and placed in a clean beaker. The dispersed bismuth stannate nanoparticle suspension is slowly added to the E. coli bacterial liquid, and stirring is performed to fully mix the two.

[0016] Step nine: 200 μg / ml of bismuth stannate nanoparticles are added to 100 ml of E. coli culture solution, and the mixture is incubated at a shaking speed of 100 rpm for 2 hours. The bismuth stannate nanoparticles are attached to the surface of the E. coli by electrostatic interaction. Sampling is performed, and the combination of the nanoparticles and the E. coli is observed under a microscope.

[0017] Step ten: After the end, the mixture is centrifuged at a speed of 6000 rpm for 10 min at 4 DEG C, the supernatant is discarded, and the precipitate is collected to obtain an E. coli - bismuth stannate (E. coli - BSO) biological composite system.

[0018] The present application has the following beneficial effects:

[0019] The E. coli - BSO material is prepared by a simple synthesis process, and the material not only has a wide light absorption range, but also has excellent electron transfer performance.

[0020] The constructed E. coli - BSO biological composite system perfectly combines the excellent light trapping characteristics of inorganic semiconductor materials and the high catalytic ability of biological catalysts. It is verified by experiments that under the condition of visible light irradiation with a wavelength of 420 - 780 nm and an intensity of 2000 W·m -2 The hydrogen production amount of the biological composite system within 4 hours can reach 0.8 mmol. Compared with the hydrogen production amount of pure E. coli, the hydrogen production amount is increased by 1.5 times. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the XRD pattern of BSO.

[0022] Figure 2 is the Zeta pattern of E. coli + BSO.

[0023] Figure 3 is the ultraviolet-visible absorption spectrum of BSO.

[0024] Figure 4 OD600 values of E. coli and E. coli + BSO at different time periods.

[0025] Figure 5 Hydrogen production graph of E. coli - BSO. DETAILED DESCRIPTION

[0026] Example 1

[0027] Step 1: Prepare the LB culture solution for cultivating E. coli according to the specific formula. After preparation, place the culture solution in a high-pressure steam sterilization pot at 121°C for 20 minutes to ensure sterility.

[0028] Step 2: In a sterile environment on the clean bench, inoculate the E. coli into the sterilized culture solution. After inoculation, place the container with the bacterial culture solution in a 37°C shaking bed for cultivation.

[0029] Step 3: Weigh bismuth nitrate [Bi(NO3)3·5H2O] and stannous chloride (SnCl2·2H2O) according to the ratio of Bi to Sn by mole.

[0030] Step 4: Place the weighed raw materials into two clean beakers respectively. Prepare an appropriate amount of solvent, an ethanol-water mixed solution. Slowly add bismuth nitrate to the solvent, stirring while adding, until completely dissolved. Since bismuth nitrate is prone to hydrolysis, add a small amount of dilute nitric acid to the solution to inhibit hydrolysis and keep the solution clear and transparent. Similarly, add stannous chloride to the other part of the solvent and stir to dissolve.

[0031] Step 5: Mix the two solutions uniformly, and slowly add NaOH solution under stirring conditions until a white precipitate is formed. Then, place the solution in a reaction kettle and react in a constant-temperature drying oven at a temperature of 180°C for 12 hours. During the reaction, metal ions in the solution react with the precipitant to gradually form bismuth stannate nanocrystal nuclei and continuously grow.

[0032] Step 6: After the reaction kettle is naturally cooled to room temperature, discard the supernatant and collect the precipitate. Wash the precipitate with deionized water and anhydrous ethanol alternately for 3 times to remove surface-adsorbed impurity ions and solvent molecules. Transfer the washed precipitate to a clean culture dish and place it in a vacuum drying oven, set the temperature to 60°C, and dry for 12 hours to obtain bismuth stannate nanoparticles.

[0033] Step 7: Transfer the cultivated E. coli bacterial solution to a centrifuge tube and collect the E. coli bacterial bodies.

[0034] Step eight: weigh a certain amount of bismuth stannate nanoparticles, put it into a clean beaker, slowly add the dispersed bismuth stannate nanoparticle suspension into the E. coli bacterial solution, stir, and mix them thoroughly.

[0035] Step nine: add 200 μg / ml of bismuth stannate nanoparticles to 100 ml of E. coli culture solution and incubate for 2 hours at a shaking speed of 100 rpm. The bismuth stannate nanoparticles are attached to the surface of the E. coli by electrostatic interaction. Take samples and observe the combination of the nanoparticles and the E. coli under a microscope.

[0036] Step ten: after the end, centrifuge the mixture at 4°C at a speed of 6000 rpm for 10 min, discard the supernatant, collect the precipitate, and obtain the E. coli - bismuth stannate (E. coli - BSO) biological complex system.

[0037] The following experiments are used to verify the effect of the application:

[0038] 1. Figure 1 The XRD pattern shows that BSO has multiple diffraction peaks within the 2 - Theta angle from 20° to 80°. The successful preparation of BSO nanoparticles is verified by comparison with the standard card.

[0039] 2. Figure 2 is a Zeta potential distribution graph, which is used to analyze the surface charge and other characteristics of the BSO sample. It is found that the Zeta potential peak of the BSO sample is concentrated at about 25.7 mV, and the peak shape is relatively narrow, indicating that the Zeta potential of the sample particles is relatively uniform, the surface is positively charged, and the dispersion is stable.

[0040] 3. Figure 3 is the ultraviolet-visible absorption spectrum of BSO. BSO has strong visible light absorption ability, which provides a theoretical basis for the enhancement of hydrogen production by the biological complex system.

[0041] 4. Figure 4 The growth curve of OD600 of E. coli under the condition of with and without BSO shows that with the increase of time, OD600 first rises, and the growth rate slows down at about 15 - 25 h, and then tends to be stable, reflecting the growth law of E. coli itself. The overall trend of E. coli + BSO is the same as that of E. coli, indicating that the addition of BSO does not significantly inhibit the growth of E. coli.

[0042] 5. Figure 5The hydrogen production experiment comparison results can obtain that the single BSO basically does not produce hydrogen and is irrelevant to time. This is mainly because pure BSO cannot decompose the substrate glucose, resulting in no hydrogen production. E. coli can produce hydrogen by itself, but the hydrogen production efficiency is limited, with an average of 0.2 mol per hour. The E. coli-BSO biological complex system produces 0.5 mol of hydrogen per hour, which is 1.5 times higher than the hydrogen production efficiency of pure E. coli. At the same time point, the hydrogen production amount of the E. coli + BSO combination is higher than that of BSO and E. coli, the hydrogen production amount of single BSO is relatively low and the growth rate is small; the hydrogen production of single E. coli has growth but the growth rate is not as good as that of E. coli + BSO.

[0043] The E. coli-BSO semi-artificial photosynthetic system constructed by the application deeply integrates the hydrogen production capacity of E. coli and the light absorption and photocatalytic performance of BSO material by means of the physical adsorption characteristics of E. coli, accelerates the preparation of hydrogen, and E. coli-BSO produces 0.5 mol of hydrogen per hour, which is 1.5 times higher than the hydrogen production efficiency of pure E. coli. The system can produce hydrogen at low cost without additional energy input, and there is no greenhouse gas emission in the whole process, which opens up a new path for sustainable energy production. At the same time, the microorganism used in the system is E. coli, which is widely distributed in nature and has strong reproductive ability, and has key value for industrial application from the aspects of environmental friendliness and economic feasibility.

Claims

1. A method for preparing a hydrogen-producing Escherichia coli-BSO biocomplex system, characterized in that, Includes the following steps: 200 μg / ml bismuth stannate nanoparticles were added to 100ml of E. coli culture medium and mixed. The mixture was then shaken and incubated to allow the two to self-assemble into an E. coli-BSO biocomposite system. The bismuth stannate nanoparticles described above have a positively charged surface and are obtained by a method comprising the following steps: 1) Mix bismuth nitrate solution and stannous chloride solution evenly according to the molar ratio of Bi to Sn of 1:1, and slowly add NaOH solution dropwise under stirring to make the pH value of the solution 8; 2) Place the mixture obtained in step 1) at 180°C for a complete solvothermal reaction; 3) After the solvothermal reaction is completed, cool to room temperature, discard the supernatant, collect the precipitate, wash and dry to obtain bismuth stannate nanoparticles.

2. The method as described in claim 1, characterized in that, Includes the following steps: 1) Under aseptic conditions, Escherichia coli was inoculated into sterile culture medium and cultured in a shaker at 37°C; 2) Slowly add the bismuth stannate nanoparticle suspension to the E. coli bacterial culture and stir until homogeneous; 3) Add 200 μg / ml bismuth stannate nanoparticles to 100 ml of Escherichia coli culture medium and incubate at 100 rpm for 2 hours to allow the bismuth stannate nanoparticles and Escherichia coli to self assemble and obtain a biocomposite system.

3. The method as described in claim 1, characterized in that, Includes the following steps: 1) Prepare LB culture medium for culturing Escherichia coli. After preparation, place the culture medium in a high-pressure steam sterilizer at 121°C and sterilize for 20 minutes to ensure that the culture medium is sterile. 2) In the sterile environment of the laminar flow hood, inoculate E. coli into the sterilized culture medium; after inoculation, place the container containing the bacterial culture medium in a shaker at 37°C for incubation. 3) Accurately weigh bismuth nitrate Bi(NO3)3·5H2O and stannous chloride SnCl2·2H2O, according to a molar ratio of Bi to Sn of 1:

1. 4) Place the weighed raw materials into two clean beakers respectively; use a 1:1 volume ratio ethanol-water mixture as the solvent; add 1 mL of dilute nitric acid to one part of the solvent, and slowly add bismuth nitrate to the solvent while stirring until completely dissolved; add stannous chloride to the other part of the solvent, stir to dissolve, and add a small amount of tin granules. 5) Mix the bismuth nitrate solution and stannous chloride solution prepared in step 4) evenly. Under stirring, slowly add NaOH solution dropwise until the pH value of the solution reaches 8 and a white precipitate is produced. Transfer the mixed solution into a reaction vessel, seal the reaction vessel, and place it in a constant temperature drying oven. Set the reaction temperature to 180℃ and the reaction time to 12 hours. 6) After the reaction vessel has cooled to room temperature, discard the supernatant and collect the precipitate; wash the precipitate three times alternately with deionized water and anhydrous ethanol; transfer the washed precipitate to a clean petri dish, place it in a vacuum drying oven, set the temperature to 60℃, and dry for 12 hours to obtain bismuth stannate nanoparticles. 7) Transfer the cultured E. coli solution to a centrifuge tube and collect the E. coli cells; 8) Weigh a certain amount of bismuth stannate nanoparticles and put them into a clean beaker to prepare a suspension; slowly add the well dispersed bismuth stannate nanoparticle suspension to the E. coli bacterial solution while stirring to ensure that the two are fully mixed. 9) Place the mixture obtained in step 8) on a shaker and incubate at 100 rpm for 2 hours at room temperature to perform self-assembly; take a sample and observe the binding of nanoparticles with E. coli under a microscope. 10) After completion, the mixture was centrifuged at 5000 rpm for 10 min at 4℃, the supernatant was discarded, and the precipitate was collected to obtain the E. coli-BSO biocomplex system. 11) Wash the composite system three times with an appropriate amount of PBS to remove unbound nanoparticles and set aside. Throughout the entire preparation process, aseptic techniques must be strictly followed to avoid contamination by other microorganisms.

4. The method as described in claim 3, characterized in that, The self-assembly in step 9) produces bismuth stannate nanoparticles with a positively charged surface, which can bind to Escherichia coli through electrostatic interaction.

5. The method as described in claim 3, characterized in that, The self-assembly of the nanoparticles in step 9) was analyzed by measuring OD600 using ultraviolet-visible absorption spectroscopy to determine the biocompatibility of the nanoparticles with Escherichia coli.

6. The method as described in claim 1, characterized in that, The E. coli-BSO biocomplex system was placed in a culture medium containing a carbon source and subjected to a hydrogen production reaction under visible light irradiation. The wavelength range of the visible light was 400-760 nm, and the hydrogen production reaction temperature was 30-37 °C. Moreover, the hydrogen production in this process does not consume any non-renewable energy sources, but rather utilizes solar energy to drive E. coli to convert glucose into hydrogen through metabolism.

7. The E. coli-BSO biocomplex system for hydrogen production obtained by the method of any one of claims 1-6.

8. The application of the system according to claim 7 in photocatalytic hydrogen production.

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