A nanomaterial-microbial hybrid, its preparation method and hydrogen production application

Abstract

This invention relates to the field of green hydrogen production technology, specifically to a nanomaterial-microorganism hybrid, its preparation method, and its hydrogen production application. The nanomaterial-microorganism hybrid is composed of *Escherichia coli* and Au@CeO2 nanomaterials electrostatically bonded together. The preparation method includes the following steps: first, *E. coli* is aerobically cultured under aerobic conditions; then, the aerobic culture is anaerobically cultured to obtain a bacterial solution; the bacterial solution is resuspended in an anaerobic culture medium to obtain another bacterial solution; finally, Au@CeO2 is added, and the mixture is incubated under an anaerobic atmosphere to obtain the nanomaterial-microorganism hybrid. This invention utilizes the electrostatic bonding between the hydrogen-producing bacterium *E. coli* and the nanomaterial Au@CeO2 to form a hybrid. Under relatively mild light conditions, the hydrogen yield is very high, and the catalyst is easily separated and can be recycled multiple times.

Description

Technical Field

[0001] This invention relates to the field of green hydrogen production technology, and more specifically to a nanomaterial-microbial hybrid, its preparation method, and its hydrogen production application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Hydrogen, as an important reducing agent, has significant applications in production and daily life. For example, it is used as fuel in hydrogen-powered vehicles, for cleaning and reduction in petroleum processing and metal production, for welding and cutting in industrial production, and for producing ammonia, methanol, and other chemicals. These methods of hydrogen production typically suffer from drawbacks such as complex processes, high energy consumption, and environmental pollution. Photocatalytic hydrogen production can utilize clean solar energy, reduce dependence on fossil fuels, and achieve energy sustainability. Photocatalytic hydrogen production is a green, environmentally friendly, and safe method. During photocatalytic hydrogen production, the catalyst generates electrons and holes under light irradiation. These electrons and holes participate in reduction-oxidation reactions, but the rapid recombination of electron-hole pairs within the photocatalyst presents a challenge, making efficient hydrogen production a key objective. Hydrogen-producing organisms possess self-replication and self-repair capabilities, contain enzymes related to hydrogen production, and exhibit high selectivity for hydrogen. Therefore, combining photocatalysts with hydrogen-producing organisms to prepare hybrid systems, utilizing the photocatalyst to provide energy to the hydrogen-producing organisms, is of great significance for increasing hydrogen yield and realizing the resource utilization of hydrogen.

[0004] Methods for producing hydrogen from hybrids have been reported. Wang B, Zeng C, Chu KH, et al. Enhanced biological hydrogen production from Escherichia coli with surface-precipitated cadmium sulfide nanoparticles[J]. Advanced Energy Materials, 2017, 7(20): 1700-611. constructed a whole-cell Escherichia coli-CdS hybrid and optimized the CdS concentration through a series of experiments, thus constructing and characterizing the hybrid. After successful construction, under anaerobic conditions, E. coli could easily induce endogenous [Ni-Fe] hydrogenase, thereby avoiding the costs of complex gene modification, bacterial screening, and enzyme purification. The authors investigated the effects of pyruvate production, lactic acid fermentation, formic acid concentration, hydrogenase activity stimulation, and NADH / NAD ratio on the hybrid. +Is the ratio the primary reason for promoting biological hydrogen production? Under illumination, the hybrid *E. coli* produced 400 μmol more hydrogen gas than the pure phase within 3 hours, with quantum efficiencies of 7.93% and 9.59% under 470 nm and 620 nm monochromatic xenon lamp irradiation, respectively. However, the electrons generated by the CdS nanomaterials on the bacterial surface under photoexcitation were hindered by the bacterial membrane, resulting in low electron utilization in the bacterial hydrogen production pathway. Therefore, it did not strongly promote the conversion of glucose to pyruvate, further generating hydrogen gas. Secondly, the CdS nanoparticles formed through in-situ mineralization on the surface of *E. coli* have a certain impact on the bioactivity of *E. coli*. Yuan Shouqi et al. (Jiang Z, Wang B, Jimmy CY, et al. AglnS2 / In2S3 heterostructure sensitization of Escherichia coli for sustainable hydrogen production[J]. NanoEnergy, 2018, 46: 234-240.) prepared a heterojunction of AglnS2 and In2S3, which formed an inorganic / whole-cell hybrid with Escherichia coli. The AglnS2 / In2S3 junction is an excellent light-absorbing material with faster conductivity than In2S3. In this study, the authors added an appropriate amount of In... 3+In2S3 nanoparticles were biocultured on the surface of *E. coli* with cysteine, and AglinS2 nanoparticles were immobilized on the In2S3 surface under mild conditions via in-situ ion exchange. When the hybrid was illuminated, both AglinS2 and In2S3 generated photoelectrons and holes. Photoelectrons from the conduction band of AglinS2 rapidly transferred to the conduction band of In2S3, and ultimately to *E. coli* for microbial hydrogen production. Simultaneously, photogenerated holes from the valence band of In2S3 transferred to the valence band of AglinS2. This heterojunction can improve the utilization rate of photogenerated electrons in microbial hydrogen production by inhibiting the recombination of photoelectrons and holes, and by enabling rapid transfer of photogenerated electrons into the microorganism without the need for electron transfer agents. However, the efficiency of electrons generated by this heterojunction on the bacterial surface under photoexcitation to penetrate the bacterial membrane and enter the bacterial interior is low, resulting in a relatively low improvement in hydrogen production. Wang Shaojie et al. (Wang Y, Zhao Y, Wang S, et al. Visible-Light-Driven Enhanced Biohydrogen Production by Photo-Biohybrid System Based on Photoelectron Transfer between Intracellular Photosensitizer Gold Nanoparticles and Clostridium butyricum[J].ACS Sustainable Chemistry & Engineering, 2022, 11(1):300-311.) constructed a hybrid that efficiently produces hydrogen under visible light by combining gold nanoparticles as an intracellular photosensitizer with Clostridium butyricum, achieving a quantum yield as high as 19.31%. Compared with dark-fermented Clostridium butyricum, the hybrid's biohydrogen production increased by 88.74%. The formed gold nanoparticle-clostridium butyricum hybrid has the advantages of being simple, efficient, and not requiring complex and costly genetic modification. In addition, it avoids the problem of photogenerated electrons shuttling between cell membranes and consuming a large amount of energy. Compared with Clostridium butyricum, the hybrid's biohydrogen production rate increased by 88.74%. Under light, Au NPs can promote the expression of hydrogenases and key enzymes in the pyruvate-formate lyase hydrogen production pathway. Furthermore, photoelectrons from intracellular Au NPs can be transferred via electron transfer flavoproteins and FAD to ferroredoxin-coupled hydrogenases, thereby promoting biohydrogen production. This achieves efficient synergistic catalysis of biohydrogen production between inorganic Au NPs based on photogenerated electron transfer and Clostridium butyricum.

[0005] Although some progress has been made in the methods of preparing hydrogen from hybrids, problems still exist. For example, electrons generated by extracellular nanomaterials under light are difficult to enter the bacteria, resulting in a small number of electrons participating in the bacterial hydrogen production pathway. Under light, the holes generated by nanomaterials undergo oxidation reactions, promoting the production of reactive oxygen species and affecting the normal physiological activities of bacteria. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a nanomaterial-microbial hybrid, its preparation method, and its hydrogen production application. The hybrid is formed by electrostatic bonding of the hydrogen-producing bacterium Escherichia coli and the nanomaterial Au@CeO2. Under relatively mild light conditions, the hydrogen yield is very high, and the catalyst is easy to separate and can be recycled multiple times.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a nanomaterial-microbial hybrid composed of Escherichia coli and Au@CeO2 nanomaterials electrostatically bonded together.

[0009] Furthermore, the phosphate groups of the *E. coli* and the Au@CeO2 nanomaterials are covalently bonded to form a cerium-phosphate group electron bridge;

[0010] Preferably, the Escherichia coli is Escherichia coli bio-52502.

[0011] In a second aspect, the present invention provides a method for preparing the above-mentioned nanomaterial-microbial hybrid, comprising the following steps:

[0012] (1) First, Escherichia coli was cultured aerobically under aerobic conditions, and then the aerobic culture was cultured anaerobically under anaerobic conditions to obtain the bacterial culture.

[0013] (2) Resuspend the bacterial solution in an anaerobic culture medium to obtain the bacterial solution, then add Au@CeO2 and incubate under an anaerobic atmosphere to obtain the final product.

[0014] Furthermore, in step (1), the Escherichia coli is Escherichia coli bio-52502;

[0015] The aerobic culture medium was LB broth, and the aerobic culture time was 3-4 hours; the anaerobic culture medium was LB broth, glucose and cysteine, and the anaerobic culture time was 2-3 hours.

[0016] Furthermore, in step (2), the anaerobic culture medium is LB broth, glucose, and cysteine.

[0017] Furthermore, in step (2), the OD of the bacterial suspension is obtained after resuspending the bacterial solution in an anaerobic culture medium. 600The value is 1.6-1.8;

[0018] The Au@CeO2 content is 27.89-55.79 nmol, preferably 41.84 nmol.

[0019] Furthermore, in step (2), the incubation time is 10-15 hours, preferably 10 hours.

[0020] A third aspect of the present invention provides a photocatalytic hydrogen production method using the above-mentioned nanomaterial-microbial hybrid, comprising the following steps:

[0021] The nanomaterial-microbial hybrid was centrifuged, and the precipitate was dispersed in a photoreactor containing anaerobic culture medium. The container was sealed and filled with inert gas. The mixture was stirred and reacted at a temperature of 20℃ to 35℃ under light conditions for 3 to 36 hours to produce hydrogen.

[0022] Furthermore, the centrifugation speed is 6500-7000 rpm, and the time is 3-4 min;

[0023] The nanomaterial-microbial hybrid dispersed in anaerobic culture medium OD 600 The value is 1.8-2.0;

[0024] The anaerobic culture medium contains LB broth, glucose, and cysteine;

[0025] The inert gas is nitrogen.

[0026] In a fourth aspect, the present invention provides the application of the above-mentioned nanomaterial-microbial hybrid in photocatalytic hydrogen production.

[0027] One or more embodiments of the present invention have at least the following beneficial effects:

[0028] (1) This invention utilizes the hydrogen-producing bacteria *Escherichia coli* and the nanomaterial *Au@CeO2* to form a hybrid through electrostatic bonding. The hydrogen production metabolic pathway is clear, reducing the rapid recombination of electron-hole pairs in semiconductor nanomaterials. Furthermore, the microorganisms possess self-replication and self-repair capabilities, and the resulting catalyst is easy to separate and can be recycled multiple times. This aligns with the concept of green environmental protection, with mild reaction conditions, achieving very high conversion efficiency even at room temperature, and exhibiting very high hydrogen selectivity.

[0029] (2) This invention uses glucose, which is abundant and inexpensive, as a carbon source and does not add expensive photocatalysts, which greatly reduces costs and provides a green new method for hydrogen production.

[0030] (3) The hydrogen production method in this invention is simple, and the hydrogen production rate of the nanomaterial-microbial hybrid is 5.173 times that of pure Escherichia coli, which is beneficial to industrial production. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0032] Figure 1 This is a TEM image of the nanomaterial-microorganism hybrid prepared in Example 1 of this application.

[0033] Figure 2 This is a confocal image of the nanomaterial-microbial hybrid prepared in Example 1 of this application. Detailed Implementation

[0034] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0035] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0036] As described in the background section, while some progress has been made in hydrogen production methods, problems remain, including demanding reaction conditions, high raw material prices, easy recombination of electrons and holes in semiconductors under photoexcitation, and low hydrogen yield. Therefore, this invention proposes a nanomaterial-microbial hybrid, its preparation method, and its application in hydrogen production. To enable those skilled in the art to better understand the technical solution of this invention, the following detailed description, in conjunction with specific embodiments, will illustrate the technical solution of this invention.

[0037] This invention provides a method for preparing nanomaterial-microbial hybrids, comprising the following steps:

[0038] (1) First, culture Escherichia coli aerobically for 3-4 hours under aerobic conditions, and then culture the aerobic culture solution anaerobically for 2-3 hours under anaerobic conditions.

[0039] The *Escherichia coli* strain is *Escherichia coli* bio-52502; the aerobic culture medium is LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH=7); the anaerobic culture medium is LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH=7), 5 g / L glucose and 0.5 g / L cysteine.

[0040] (2) Resuspend the bacterial culture in 50 mL of anaerobic culture medium to obtain the bacterial culture, then add 27.89-55.79 nmol of Au@CeO2 and incubate under an anaerobic atmosphere for 10-15 h to obtain the final product.

[0041] The anaerobic culture medium consists of LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH=7), 5 g / L glucose and 0.5 g / L cysteine.

[0042] The OD of the bacterial suspension was obtained after being suspended in an anaerobic culture medium. 600 The value is 1.6-1.8;

[0043] The preferred concentration of Au@CeO2 is 41.84 nmol, and the preferred incubation time is 10 h.

[0044] This invention combines Escherichia coli (Casellani & Chalmers) and Au@CeO2 nanomaterials through electrostatic bonding to form a hybrid. The phosphate groups of E. coli and Au@CeO2 nanomaterials are covalently bonded to form cerium-phosphate group electron bridges, resulting in a clear hydrogen production metabolic pathway and reducing the rapid recombination of electron-hole pairs in semiconductor nanomaterials. Furthermore, the organism has self-replication and self-repair capabilities, and the resulting catalyst is easy to separate and can be recycled multiple times.

[0045] This invention also provides a photocatalytic hydrogen production method for the above-mentioned nanomaterial-microbial hybrid, comprising the following steps:

[0046] The nanomaterial-microbial hybrid was centrifuged, and the precipitate was dispersed into a photoreaction vessel containing 50 ml of anaerobic culture medium. The vessel was sealed and filled with inert gas. The mixture was stirred, and the reaction temperature was 20℃~35℃. Under xenon lamp (λ>420nm) illumination, the reaction time was 3-12 h, and hydrogen was produced.

[0047] The centrifugation speed is 6500-7000 rpm, and the time is 3-4 min;

[0048] The nanomaterial-microbial hybrid dispersed in anaerobic culture medium OD 600 The value is 1.8-2.0;

[0049] The anaerobic culture medium contained LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH=7), 5 g / L glucose and 0.5 g / L cysteine; the inert gas was nitrogen.

[0050] The reaction mechanism for hydrogen production from nanomaterial-microbial hybrids in this invention lies in the oxidized nicotinamide adenine dinucleotide (NAD) within the microorganisms. + Au@CeO2 nanomaterials, including flavin mononucleotide (FMN) and nicotinamide adenine dinucleotide phosphate (FAD), act as electron acceptors. These electron acceptors participate in intracellular redox reactions in microorganisms and are crucial for promoting the transfer of photogenerated electrons from the nanomaterial surface to microorganisms and improving the efficiency of hybrid systems for photocatalytic hydrogen production. Au@CeO2 nanomaterials can covalently bind to the phosphate groups of the electron acceptors. Under illumination, Au@CeO2 nanomaterials generate electrons, which are then transferred to the electron acceptor NAD. + FMN and FAD promote the formation of NADH, FMNH2, and FADH2, and the conduction band position of Au@CeO2 nanomaterials satisfies the photogenerated electrons' orientation towards NAD. + The transfer between FMN and FAD promotes the conversion of glucose into pyruvate and then into formic acid in E. coli. Under the action of formic acid dehydrogenase, hydrogen production is promoted.

[0051] Example 1

[0052] First, *Escherichia coli* bio-52502 was cultured aerobically for 3 hours (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7)). Then, the aerobic culture was anaerobically cultured for 2 hours (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine). The culture was then resuspended in 50 mL of anaerobic medium (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine) to obtain the OD of the culture. 600 The value was 1.6. 41.84 nmol Au@CeO2 was added and incubated under an anaerobic atmosphere for 15 h to form a hybrid.

[0053] The nanomaterial-microbial hybrid was centrifuged at 6000 rpm for 3 minutes. The precipitate was then dispersed into a photoreaction vessel containing 50 ml of anaerobic culture medium (the anaerobic medium contained LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, and 0.5 g / L cysteine). The OD of the nanomaterial-microbial hybrid dispersed in the anaerobic medium was... 600 The value was 1.8. After purging with nitrogen for 30 min, the product was irradiated with a xenon lamp (λ > 420 nm) for 3 h. The product was detected by gas chromatography, and the hydrogen production rate was 24.74 mmol / h / g. dcw .

[0054] Example 2

[0055] First, *Escherichia coli* bio-52502 was cultured aerobically for 3 hours (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7)). Then, the aerobic culture was anaerobically cultured for 2 hours (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine). The culture was then resuspended in 50 mL of anaerobic medium (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine) to obtain the OD of the culture. 600 The value was 1.6. 55.79 nmol Au@CeO2 was added and incubated under an anaerobic atmosphere for 15 h to form a hybrid.

[0056] The nanomaterial-microbial hybrid was centrifuged at 6000 rpm for 3 minutes. The precipitate was then dispersed into a photoreaction vessel containing 50 ml of anaerobic culture medium (the anaerobic medium contained LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, and 0.5 g / L cysteine). The OD of the nanomaterial-microbial hybrid dispersed in the anaerobic medium was... 600 The value was 1.8. After purging with nitrogen for 30 min, the product was irradiated with a xenon lamp (λ > 420 nm) for 3 h. The product was detected by gas chromatography, and the hydrogen production rate was 17.76 mmol / h / g. dcw .

[0057] Example 3

[0058] First, *Escherichia coli* bio-52502 was cultured aerobically for 3 hours (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7)). Then, the aerobic culture was anaerobically cultured for 2 hours (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine). The culture was then resuspended in 50 mL of anaerobic medium (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine) to obtain the OD of the culture. 600 The value was 1.6. 27.89 nmol Au@CeO2 was added and incubated under an anaerobic atmosphere for 15 h to form a hybrid.

[0059] The nanomaterial-microbial hybrid was centrifuged at 6000 rpm for 3 minutes. The precipitate was then dispersed into a photoreaction vessel containing 50 ml of anaerobic culture medium (the anaerobic medium contained LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, and 0.5 g / L cysteine). The OD of the nanomaterial-microbial hybrid dispersed in the anaerobic medium was... 600 The value was 1.8. After purging with nitrogen for 30 min, the product was irradiated with a xenon lamp (λ > 420 nm) for 3 h. The product was detected by gas chromatography, and the hydrogen production rate was 9.79 mmol / h / g. dcw .

[0060] Example 4

[0061] First, *Escherichia coli* bio-52502 was cultured aerobically for 3 hours (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7)). Then, the aerobic culture was anaerobically cultured for 2 hours (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine). The culture was then resuspended in 50 mL of anaerobic medium (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine) to obtain the OD of the culture. 600 The value was 1.6. 41.84 nmol Au@CeO2 was added, and the mixture was incubated under an anaerobic atmosphere for 10 h to form a hybrid.

[0062] The nanomaterial-microbial hybrid was centrifuged at 6000 rpm for 3 minutes. The precipitate was then dispersed into a photoreaction vessel containing 50 ml of anaerobic culture medium (the anaerobic medium contained LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, and 0.5 g / L cysteine). The OD of the nanomaterial-microbial hybrid dispersed in the anaerobic medium was... 600 The value was 1.8. After purging with nitrogen for 30 min, the product was irradiated with a xenon lamp (λ > 420 nm) for 3 h. The product was detected by gas chromatography, and the hydrogen production rate was 32.96 mmol / h / g. dcw .

[0063] Example 5

[0064] First, *Escherichia coli* bio-52502 was cultured aerobically for 3 hours (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7)). Then, the aerobic culture was anaerobically cultured for 2 hours (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine). The culture was then resuspended in 50 mL of anaerobic medium (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine) to obtain the OD of the culture. 600 The value was 1.6. 41.84 nmol Au@CeO2 was added, and the mixture was incubated under an anaerobic atmosphere for 10 h to form a hybrid.

[0065] The nanomaterial-microbial hybrid was centrifuged at 6000 rpm for 3 minutes. The precipitate was then dispersed into a photoreaction vessel containing 50 ml of anaerobic culture medium (the anaerobic medium contained LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, and 0.5 g / L cysteine). The OD of the nanomaterial-microbial hybrid dispersed in the anaerobic medium was... 600 The value was 1.8. After purging with nitrogen for 30 min, the product was irradiated with a xenon lamp (λ > 420 nm) for 6 h. The product was detected by gas chromatography, and the hydrogen production rate was 35.14 mmol / h / g. dcw .

[0066] Example 6

[0067] First, *Escherichia coli* bio-52502 was cultured aerobically for 3 hours (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7)). Then, the aerobic culture was anaerobically cultured for 2 hours (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine). The culture was then resuspended in 50 mL of anaerobic medium (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine) to obtain the OD of the culture. 600 The value was 1.6. 41.84 nmol Au@CeO2 was added, and the mixture was incubated under an anaerobic atmosphere for 10 h to form a hybrid.

[0068] The nanomaterial-microbial hybrid was centrifuged at 6000 rpm for 3 minutes. The precipitate was then dispersed into a photoreaction vessel containing 50 ml of anaerobic culture medium (the anaerobic medium contained LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, and 0.5 g / L cysteine). The OD of the nanomaterial-microbial hybrid dispersed in the anaerobic medium was... 600 The value was 1.8. After purging with nitrogen for 30 min, the product was irradiated with a xenon lamp (λ > 420 nm) for 9 h. The product was detected by gas chromatography, and the hydrogen production rate was 40.43 mmol / h / g. dcw .

[0069] Example 7

[0070] First, *Escherichia coli* bio-52502 was cultured aerobically for 3 hours (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7)). Then, the aerobic culture was anaerobically cultured for 2 hours (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine). The culture was then resuspended in 50 mL of anaerobic medium (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine) to obtain the OD of the culture. 600 The value was 1.6. 41.84 nmol Au@CeO2 was added, and the mixture was incubated under an anaerobic atmosphere for 10 h to form a hybrid.

[0071] The nanomaterial-microbial hybrid was centrifuged at 6000 rpm for 3 minutes. The precipitate was then dispersed into a photoreaction vessel containing 50 ml of anaerobic culture medium (the anaerobic medium contained LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, and 0.5 g / L cysteine). The OD of the nanomaterial-microbial hybrid dispersed in the anaerobic medium was... 600 The value was 1.8. After purging with nitrogen for 30 min, the product was irradiated with a xenon lamp (λ > 420 nm) for 12 h. The product was detected by gas chromatography, and the hydrogen production rate was 42.06 mmol / h / g. dcw .

[0072] Comparative Example 1

[0073] The difference from Example 1 is that the photocatalyst is only Escherichia coli bio-52502, with a hydrogen production rate of 0.42 mmol / h (the hydrogen production rate was not divided by dry weight in the calculation process).

[0074] Comparative Example 2

[0075] The difference from Example 1 is that the photocatalyst is only Au@CeO2, with a hydrogen production rate of 8.7 mmol / h / g. dcw .

[0076] Comparative Example 3

[0077] First, *Escherichia coli* bio-52502 was cultured aerobically for 3 hours (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7)). Then, the aerobic culture was anaerobically cultured for 2 hours (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine). The culture was then resuspended in 50 mL of anaerobic medium (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine) to obtain the OD of the culture. 600 The value was 1.6. 41.84 nmol Au@CeO2 was added, and the mixture was incubated under an anaerobic atmosphere for 5 hours to form a hybrid.

[0078] The nanomaterial-microbial hybrid was centrifuged at 6000 rpm for 3 minutes. The precipitate was then dispersed into a photoreaction vessel containing 50 ml of anaerobic culture medium (the anaerobic medium contained LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, and 0.5 g / L cysteine). The OD of the nanomaterial-microbial hybrid dispersed in the anaerobic medium was... 600 The value was 1.8. After purging with nitrogen for 30 min, the product was irradiated with a xenon lamp (λ > 420 nm) for 3 h. The product was detected by gas chromatography, and the hydrogen production rate was 8.58 mmol / h / g. dcw .

[0079] Comparative Example 4

[0080] First, *Escherichia coli* bio-52502 was cultured aerobically for 3 hours (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7)). Then, the aerobic culture was anaerobically cultured for 2 hours (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine). The culture was then resuspended in 50 mL of anaerobic medium (LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH=7), 5 g / L glucose, and 0.5 g / L cysteine) to obtain the OD of the culture. 600 The value was 1.6. 41.84 nmol Au@CeO2 was added, and the mixture was incubated under an anaerobic atmosphere for 20 h to form a hybrid.

[0081] The nanomaterial-microbial hybrid was centrifuged at 6000 rpm for 3 minutes. The precipitate was then dispersed into a photoreaction vessel containing 50 ml of anaerobic culture medium (the anaerobic medium contained LB broth (5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, and 0.5 g / L cysteine). The OD of the nanomaterial-microbial hybrid dispersed in the anaerobic medium was... 600 The value was 1.8. After purging with nitrogen for 30 min, the product was irradiated with a xenon lamp (λ > 420 nm) for 3 h. The product was detected by gas chromatography, and the hydrogen production rate was 8.49 mmol / h / g. dcw .

[0082] By comparing Examples 1-7 with Comparative Examples 1 and 2, we can see the superiority of the nanomaterial-microbial hybrid of the present invention: compared with Escherichia coli bio-52502 (Comparative Example 1) and Au@CeO2 (Comparative Example 2), the hydrogen production rate of the hybrid of the present invention is significantly improved.

[0083] A comparison of Examples 1 and 5 with Comparative Examples 3 and 4 shows that the anaerobic incubation time of Escherichia coli bio-52502 and Au@CeO2 affects the hydrogen production rate of the hybrid. Compared with 5 h of anaerobic incubation (Comparative Example 3) and 20 h of anaerobic incubation (Comparative Example 4), the hybrid of the present invention has a higher hydrogen production rate when the incubation time is 10 h and 15 h. Further comparison of Examples 1 and 5 shows that the optimal incubation time of the present invention is 10 h.

[0084] The comparison of Examples 1-3 shows that the amount of Au@CeO2 added affects the hydrogen production rate of the hybrid. When the amount of Au@CeO2 added is 41.84 nmol, the hybrid has a higher hydrogen production rate. Therefore, the optimal amount of Au@CeO2 added in this invention is 41.84 nmol.

[0085] A comparison of Examples 5 and 7-9 shows that, under the optimal incubation time of 10 hours and the optimal amount of nanomaterials of 41.84 nmol, the hydrogen production rate gradually increases with the extension of the photo-reaction time.

[0086] In summary, this invention utilizes the electrostatic bonding between hydrogen-producing bacteria Escherichia coli and nanomaterial Au@CeO2 to form a hybrid, thereby improving the efficiency of photogenerated electron transfer from the nanomaterial surface to the microorganism, shortening the transfer distance between photogenerated electrons and electron acceptors NAD+, FAD, and FMN within the microbial cell, increasing hydrogen production efficiency, and leveraging the advantages of the hybrid system in photocatalytic reduction of hydrogen. Furthermore, the catalyst is easy to separate and can be recycled multiple times.

[0087] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not a limitation on the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a nanomaterial-microbial hybrid, wherein the nanomaterial-microbial hybrid is composed of *Escherichia coli* and Au@CeO2 nanomaterials electrostatically bonded together, characterized in that... Includes the following steps: (1) First, Escherichia coli was cultured aerobically under aerobic conditions, and then the cultured bacterial solution was cultured anaerobically under anaerobic conditions to obtain the bacterial solution; (2) Resuspend the bacterial culture in an anaerobic culture medium to obtain the bacterial culture, then add Au@CeO2 nanomaterials and incubate under an anaerobic atmosphere to obtain the final product; In step (2), the bacterial suspension is resuspended in an anaerobic culture medium to obtain the OD of the bacterial suspension. 600 The value is 1.6-1.8; The Au@CeO2 content is 27.89 -55.79 nmol; In step (2), the incubation time is 10-15 hours.

2. The preparation method according to claim 1, characterized in that, In step (1), the Escherichia coli is Escherichia coli bio-52502; The aerobic culture medium was LB broth, and the aerobic culture time was 3-4 hours; the anaerobic culture medium was LB broth, glucose and cysteine, and the anaerobic culture time was 2-3 hours.

3. The preparation method according to claim 1, characterized in that, In step (2), the anaerobic culture medium is LB broth, glucose and cysteine.

4. The preparation method according to claim 1, characterized in that, In step (2), the Au@CeO2 is 41.84 nmol.

5. The preparation method according to claim 1, characterized in that, In step (2), the incubation time is 10 hours.

6. A nanomaterial-microorganism hybrid prepared by the preparation method of claim 1, characterized in that, It is composed of Escherichia coli and Au@CeO2 nanomaterials electrostatically bonded together.

7. The nanomaterial-microorganism hybrid as described in claim 6, characterized in that, The phosphate groups of the Escherichia coli are covalently bonded to the Au@CeO2 nanomaterial to form a cerium-phosphate group electron bridge; The Escherichia coli was identified as Escherichia coli bio-52502.

8. A method for photocatalytic hydrogen production using the nanomaterial-microbial hybrid as described in claim 6, characterized in that, Includes the following steps: The nanomaterial-microbial hybrid was centrifuged, and the precipitate was dispersed in a photoreactor containing anaerobic culture medium. The container was sealed and filled with inert gas. The mixture was stirred and reacted at a temperature of 20℃~35℃ under light conditions for 3-36 hours to produce hydrogen.

9. The photocatalytic hydrogen production method of nanomaterial-microbial hybrid as described in claim 8, characterized in that, The centrifugation speed is 6500-7000 rpm, and the time is 3-4 min; The nanomaterial-microbial hybrid dispersed in anaerobic culture medium OD 600 The value is 1.8-2.0; The anaerobic culture medium contains LB broth, glucose, and cysteine; The inert gas is nitrogen.

10. The application of the nanomaterial-microbial hybrid as described in claim 6 in photocatalytic hydrogen production.

Images