Artificial photosynthetic bacterium, construction method and application of artificial photosynthetic bacterium in photocatalytic hydrogen production

Abstract

The invention belongs to the technical field of biological photocatalytic hydrogen production, and provides an artificial photosynthetic bacterium, a construction method and application of the artificial photosynthetic bacterium in photocatalytic hydrogen production. According to the invention, Escherichia coli (Escherichia coli) is added into a culture medium, and the culture medium is prepared by the following steps of: adding the Escherichia coli into the culture medium; according to the present invention, the Chalmers and the natural thylakoid are combined through the PDA to form the heterozygote, such that the hydrogen production metabolism path is clear, and the energy cofactor level required by the hydrogen production path is improved; in addition, microorganisms have self-replication and self-repairing capabilities, and the biological energy module natural capsule-like body is good in biocompatibility and free of toxic effects on the microorganisms. The method provided by the invention meets the concept of green and environmental protection, has mild reaction conditions, can obtain very high conversion efficiency at room temperature, and has very high hydrogen selectivity.

Description

Technical Field

[0001] The invention belongs to the technical field of biological photocatalytic hydrogen production, and specifically relates to a construction method of photosynthetic bacteria and the production of hydrogen by photosynthetic bacteria. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Hydrogen energy is a clean, recyclable and efficient energy carrier. Its combustion calorific value is higher than that of gasoline, and the combustion process does not produce pollutants. It is a potential candidate for sustainable energy. However, more than 95% of hydrogen is produced by consuming fossil energy, which has aggravated the energy crisis and environmental pollution problems. In recent years, photocatalysis and photoelectrocatalysis technologies for solar hydrogen production have been developed and received widespread attention. At present, the reaction conditions for photocatalytic hydrogen production are mild and renewable clean solar energy is used. However, the catalyst cost is high and the efficiency of hydrogen production is relatively low. Microbial hydrogen production uses the advantages of microorganisms such as self-replication, self-repair ability and low cost, but the hydrogen production efficiency is low. Therefore, it is very important to develop a clean, pollution-free and efficient hydrogen production technology.

[0004] Combining semiconductor materials with microorganisms to achieve photosynthetic biohydrogen production is a hydrogen production strategy that is currently attracting much attention.

[0005] Methods for preparing hydrogen from hybrids have been reported. Wang Bo et al. constructed a whole-cell Escherichia coli-I-HTCC hybrid, in which negatively charged iodine-doped hydrothermal carbonized carbon (I-HTCC) was interfaced with surface-modified E. coli cells through a simple "attachment" mode via electrostatic interactions. Due to the photoexcited electrons, I-HTCC@E.coli was self-assembled. Under illumination, the hydrogen production efficiency of E. coli was improved, with a quantum efficiency of 9.11%. However, the electrons generated by the I-HTCC nanomaterials located on the bacterial surface under photoexcitation were hindered by the bacterial membrane, resulting in a low utilization rate of electrons involved in the bacterial hydrogen production pathway. Therefore, it failed to significantly promote the conversion of glucose to pyruvate, thereby affecting the hydrogen generation process. Wang Bo et al. also combined metal organic frameworks (MOFs) with industrial microbial cell factories to construct a hybrid system that integrated engineered Escherichia coli (E. coli) to study light-driven hydrogen and lysine production. However, the electron utilization rate was also low, limiting the hydrogen yield.

[0006] Whole-cell inorganic-biohybrid systems that integrate inorganic photosensitizers with intact living cells have shown great potential for solar hydrogen production. However, typical whole-cell biohybrid systems often suffer from sluggish electron transfer kinetics during transmembrane diffusion, which severely limits their photocatalytic activity. Zhang et al. explored the photointeractive activity of Escherichia coli, a non-photosynthetic bacterium, and developed it into a living inorganic-microbial biohybrid for efficient biomass conversion. An intracellular inorganic-biohybrid system C 3 N 4 QDs / E.coli to improve the hydrogen production rate.

[0007] Although some progress has been made in the method of preparing hydrogen from hybrids, there are still problems. Researchers generally use CdS, C 3 N 4 Semiconductor materials such as quantum dots and PCN are combined with microorganisms, but there are still defects such as complex system construction process, poor biocompatibility, and low charge transfer efficiency. Affecting the internal redox balance of bacteria. Semiconductor materials absorb light energy to produce electrons, and the energy used by bacteria is bioenergy (ATP and NAD(P)H). Due to the insulating nature of the cell membrane phospholipid bilayer, it is extremely difficult for photogenerated electrons from semiconductor materials to enter cells, and the efficiency of converting electrons into bioenergy available to bacteria is low, limiting the hydrogen yield. Summary of the invention

[0008] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a method for producing hydrogen by microorganisms powered by natural energy modules. Under mild light conditions, hydrogen-producing bacteria Escherichia coli and natural energy module thylakoids are assembled through polydopamine with good conductivity and strong adhesion to form photosynthetic bacteria to produce hydrogen. The natural energy module can produce the energy cofactor required for hydrogen production, the yield of hydrogen is very high, and the exogenously introduced natural energy module has good biocompatibility.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] The first aspect of the present invention provides a method for constructing artificial photosynthetic bacteria, characterized in that it comprises:

[0011] E. coli was first cultured aerobically and then anaerobically, and the bacterial solution was collected;

[0012] The bacterial solution is resuspended and cultured in an anaerobic culture medium, and then coated with PDA. The PDA-coated Escherichia coli is mixed with natural thylakoids and incubated under anaerobic conditions to form natural thylakoid-microorganism hybrids, namely, artificial photosynthetic bacteria.

[0013] The reaction mechanism of the natural thylakoid-microorganism hybrid of the present invention for preparing hydrogen is that energy cofactors such as NADH, NADPH and ATP in the microorganism play a vital role in the hydrogen production process of Escherichia coli. The process directly depends on these bioenergy molecules (NADH, NADPH and ATP) rather than electrons. The natural energy module, thylakoid, as the core site of the photosynthetic light reaction, can efficiently generate these energy cofactors (NADH, NADPH and ATP) under light conditions. Therefore, the construction of natural thylakoid-microorganism hybrids provides a very potential approach for microbial hydrogen production. Natural thylakoids are distributed inside and on the surface of Escherichia coli, and the electrons generated under light can be directly or indirectly transferred to Escherichia coli through electron transfer media such as nicotinamide adenine dinucleotide phosphate (FAD) and riboflavin, thereby promoting the generation of energy cofactors. This process further promotes Escherichia coli to convert glucose into pyruvate and ultimately into formic acid. Under the catalytic action of formate dehydrogenase, formic acid is further decomposed, which significantly promotes the generation of hydrogen.

[0014] Furthermore, the natural thylakoids are evenly distributed on the surface and inside of the E. coli, providing electrons and energy cofactors.

[0015] In some embodiments, the Escherichia coli is Escherichia coli bio-52502.

[0016] In some embodiments, the aerobic culture medium is LB broth, and the aerobic culture time is 3-4 hours;

[0017] In some embodiments, the anaerobic culture solution consists of LB broth, glucose and cysteine, and the anaerobic culture time is 2-3 hours.

[0018] In some embodiments, during the incubation, the anaerobic culture medium consists of LB broth, glucose and cysteine.

[0019] In some embodiments, the OD600 value of the bacterial solution obtained after resuspending the bacterial solution in anaerobic culture medium is 1.5-1.8.

[0020] The natural thylakoid concentration was 80 μg chl / mL and the volume was 2 mL.

[0021] The concentration of dopamine is 0.2-0.3 mg / μL.

[0022] In some embodiments, the incubation time is 14 hours or more.

[0023] The second aspect of the present invention provides artificial photosynthetic bacteria constructed by the above method.

[0024] The second aspect of the present invention provides the use of the above-mentioned artificial photosynthetic bacteria in photocatalytic hydrogen production.

[0025] Specifically, they include:

[0026] The artificial photosynthetic bacteria are dispersed into a light reaction container containing an anaerobic culture medium, sealed and filled with inert gas, stirred, and the reaction temperature is 20°C to 35°C. Under light conditions, the reaction time is 2-6h, and hydrogen is produced by the reaction.

[0027] More specifically, the method includes: centrifuging the natural thylakoid-microorganism hybrid, dispersing the precipitate into a photoreaction container containing an anaerobic culture medium, sealing and filling with inert gas, stirring, reacting at a temperature of 20°C to 35°C, under light conditions, for a reaction time of 2-6 hours, and producing hydrogen.

[0028] In some embodiments, the centrifugation speed is 6500-7000 rcf, and the time is 5-10 min;

[0029] In some embodiments, the OD in the anaerobic medium is 600 The value is 1.6-1.9;

[0030] In some embodiments, the anaerobic medium consists of LB broth, glucose, cysteine, and sodium sulfite;

[0031] In some embodiments, the inert gas is nitrogen.

[0032] Beneficial effects of the present invention

[0033] (1) The present invention utilizes hydrogen-producing bacteria Escherichia coli and natural energy module thylakoids to form a hybrid through polydopamine, and the hydrogen production metabolic pathway is clear, which increases the level of energy cofactors required for the hydrogen production pathway; and the microorganisms have the ability to self-repair and self-repair, and the natural thylakoids of the bioenergy module have good biocompatibility and are non-toxic to microorganisms. It conforms to the concept of green environmental protection, the reaction conditions are mild, and a very high conversion efficiency can be achieved at room temperature, and the selectivity of hydrogen is very high.

[0034] (2) The present invention uses glucose, which is abundant in resources and cheap and easily available, as a carbon source, and does not add expensive semiconductor materials, which greatly reduces the cost and provides a new green method for the preparation of hydrogen;

[0035] (3) The hydrogen production method of the present invention is simple, and the hydrogen production rate of the natural thylakoid-microorganism hybrid is 1.997 times that of pure Escherichia coli, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 This is a scanning electron microscope image of Escherichia coli-native thylakoids used in this application.

[0038] Figure 2 This is a 3D slice confocal image of the scanning electron microscope image of the Escherichia coli-natural thylakoid synthesized in this application. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0040] As introduced in the background technology, although some progress has been made in the preparation of hydrogen, there are still problems such as harsh reaction conditions, high raw material prices, easy recombination of electrons and holes in semiconductors under light excitation, and low hydrogen yield. Therefore, the present invention proposes a natural thylakoid-microorganism hybrid and its preparation method and application.

[0041] The present invention provides a method for preparing a natural thylakoid-microorganism hybrid, comprising the following steps:

[0042] (1) First, Escherichia coli is cultured aerobically under aerobic conditions for 3-4 hours, and then the aerobic cultured bacterial solution is cultured anaerobically under anaerobic conditions for 2-3 hours;

[0043] The Escherichia coli 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.

[0044] (2) Resuspend the bacterial solution in 50 mL of anaerobic culture medium to obtain a bacterial solution, then add 160 μg of chl natural thylakoids and incubate under anaerobic atmosphere for 14 h.

[0045] 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite;

[0046] The bacterial solution was resuspended in anaerobic medium to obtain the OD value of the bacterial solution. 600 The value is 1.4-1.6;

[0047] The preferred concentration of native thylakoids is 160 μg chl, and the preferred incubation time is 14 h.

[0048] The present invention combines Escherichia coli and natural thylakoids through polydopamine PDA to form a hybrid, the hydrogen production metabolic pathway is clear, and the energy cofactor level required for the hydrogen production pathway is increased; the microorganisms have self-replication and self-repair capabilities, and the natural thylakoids of the bioenergy module have good biocompatibility and are non-toxic to microorganisms. It conforms to the concept of green environmental protection, the reaction conditions are mild, and a very high conversion efficiency can be achieved at room temperature, and the selectivity of hydrogen is very high.

[0049] The present invention also provides a method for photocatalytic hydrogen production by the above-mentioned natural thylakoid-microorganism hybrid, comprising the following steps:

[0050] The natural thylakoid-microorganism hybrid is centrifuged, and then the precipitate is dispersed in a photoreaction container containing 50 mL of anaerobic culture medium, sealed and filled with inert gas, stirred, and the reaction temperature is 20°C to 35°C, under the illumination of a xenon lamp (λ>420nm), the reaction time is 2-6h, and the reaction produces hydrogen;

[0051] Wherein, the centrifugal speed is 6500-7000rcf, and the time is 6-8min;

[0052] The OD of the nanomaterial-microorganism hybrid dispersed into the anaerobic culture medium 600 The value is 1.6-1.9;

[0053] 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite; the inert gas was nitrogen.

[0054] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0055] In the following examples, Escherichia coli (ATCC-25922) is a commercially available strain.

[0056] Preparation of thylakoids: 1 g of fresh spinach leaves was mixed with 330 mM sorbitol, 50 mM HEPES-KOH pH 7.6, 5 mM MgCl 2The pellet was mixed with 5 mL of buffer A containing 0.1% (w / v) bovine serum albumin, crushed with a blender for 10 min, filtered through 8 layers of nylon cloth, and centrifuged at 3000 × g for 10 min at 4°C. The pellet was resuspended in buffer B (300 mM sorbitol, 50 mM HEPES-KOH pH 7.6, 5 mM MgCl 2 and 10 mM sodium L-ascorbate), then slowly add 80% / 40% Percoll gradient, centrifuge at 3000×g for 10 min at 4°C, collect the obtained natural thylakoid precipitate, and resuspend the obtained thylakoid precipitate in cold buffer C (10 mM HEPES-KOH, 10 mM MgCl2, 10 mM sodium L-ascorbate). Finally, the natural thylakoids were quickly frozen with 10% DMSO as an osmotic protectant and stored in a dark environment at -80°C until use.

[0057] Example 1

[0058] First, Escherichia coli bio-52502 was cultured aerobically for 3 h under aerobic conditions (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7)), and then the aerobic cultured bacterial solution was cultured anaerobically for 2 h under anaerobic conditions (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), and the bacterial solution was resuspended in 50 mL anaerobic culture medium (the anaerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, 0.5 g / L cysteine ​​and 10 mM sodium sulfite), and the OD of the bacterial solution was obtained. 600 The value is 1.6. After anaerobic culture, E. coli was centrifuged at 7000rpm for 10min and resuspended in 20mL Tris buffer (10mM, pH=8.5). Then, 100μL DMSO in which 20mg dopamine (DA) was dissolved was added dropwise to the Tris buffer, and the mixture was gently shaken at 37°C for 2h to coat the E. coli with PDA. Subsequently, the E. coli coated with PDA was collected by centrifugation, washed three times with Tris buffer (10mM, pH=8.5), and named E. coli@PDA. Then, 160μg chl natural thylakoids were added to E. coli@PDA, and incubated for 14h under anaerobic atmosphere to form a hybrid.

[0059] The natural thylakoid-microorganism hybrid was centrifuged at 7000 rcf for 5 min, and then the precipitate was dispersed into a photoreactor containing 50 mL of anaerobic 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite). The OD of the natural thylakoid-microorganism hybrid dispersed into the anaerobic medium was 2.34 %. 600 The value was 1.8. After nitrogen replacement for 30 minutes, the product was irradiated with a xenon lamp (λ>420nm) for 2 hours. The product was detected by gas chromatography. The hydrogen production rate was 15.1437mmol / h / g dcw .

[0060] Example 2

[0061] First, Escherichia coli bio-52502 was cultured aerobically for 3 h under aerobic conditions (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7)), and then the aerobic cultured bacterial solution was cultured anaerobically for 2 h under anaerobic conditions (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), and the bacterial solution was resuspended in 50 mL anaerobic culture medium (the anaerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, 0.5 g / L cysteine ​​and 10 mM sodium sulfite), and the OD of the bacterial solution was obtained. 600 The value is 1.6. After anaerobic culture, E. coli was centrifuged at 7000rpm for 10min and resuspended in 20mL Tris buffer (10mM, pH=8.5). Then, 100μL DMSO in which 20mg dopamine (DA) was dissolved was added dropwise to the Tris buffer, and the mixture was gently shaken at 37°C for 2h to coat the E. coli with PDA. Subsequently, the E. coli coated with PDA was collected by centrifugation, washed three times with Tris buffer (10mM, pH=8.5), and named E. coli@PDA. Then, 160μg chl natural thylakoids were added to E. coli@PDA and incubated for 10h under anaerobic atmosphere to form a hybrid.

[0062] The natural thylakoid-microorganism hybrid was centrifuged at 7000 rcf for 5 min, and then the precipitate was dispersed into a photoreactor containing 50 mL of anaerobic 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite). The OD of the natural thylakoid-microorganism hybrid dispersed into the anaerobic medium was 2.34 %. 600 The value was 1.8. After nitrogen replacement for 30 minutes, the product was irradiated with a xenon lamp (λ>420nm) for 2 hours. The product was detected by gas chromatography. The hydrogen production rate was 13.268mmol / h / g dcw .

[0063] Example 3

[0064] First, Escherichia coli bio-52502 was cultured aerobically for 3 h under aerobic conditions (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7)), and then the aerobic cultured bacterial solution was cultured anaerobically for 2 h under anaerobic conditions (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), and the bacterial solution was resuspended in 50 mL anaerobic culture medium (the anaerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, 0.5 g / L cysteine ​​and 10 mM sodium sulfite), and the OD of the bacterial solution was obtained. 600 The value is 1.6. After anaerobic culture, E. coli was centrifuged at 7000rpm for 10min and resuspended in 20mL Tris buffer (10mM, pH=8.5). Then, 100μL DMSO in which 20mg dopamine (DA) was dissolved was added dropwise to the Tris buffer, and the mixture was gently shaken at 37°C for 2h to coat the E. coli with PDA. Subsequently, the E. coli coated with PDA was collected by centrifugation, washed three times with Tris buffer (10mM, pH=8.5), and named E. coli@PDA. Then, 160μg chl natural thylakoids were added to E. coli@PDA, and incubated for 8h under anaerobic atmosphere to form a hybrid.

[0065] The natural thylakoid-microorganism hybrid was centrifuged at 7000 rcf for 5 min, and then the precipitate was dispersed into a photoreactor containing 50 mL of anaerobic 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite). The OD of the natural thylakoid-microorganism hybrid dispersed into the anaerobic medium was 2.34 %. 600 The value was 1.8. After nitrogen replacement for 30 minutes, the product was illuminated by a xenon lamp (λ>420nm) for 2 hours. The product was detected by gas chromatography. The hydrogen production rate was 12.289mmol / h / g dcw .

[0066] Example 4

[0067] First, Escherichia coli bio-52502 was cultured aerobically for 3 h under aerobic conditions (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7)), and then the aerobic cultured bacterial solution was cultured anaerobically for 2 h under anaerobic conditions (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), and the bacterial solution was resuspended in 50 mL anaerobic culture medium (the anaerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, 0.5 g / L cysteine ​​and 10 mM sodium sulfite), and the OD of the bacterial solution was obtained. 600 The value is 1.6. After anaerobic culture, E. coli was centrifuged at 7000rpm for 10min and resuspended in 20mL Tris buffer (10mM, pH=8.5). Then, 100μL DMSO in which 20mg dopamine (DA) was dissolved was added dropwise to the Tris buffer and gently shaken at 37°C for 2h to coat the E. coli with PDA. Subsequently, the E. coli coated with PDA was collected by centrifugation and washed 3 times with Tris buffer (10mM, pH=8.5) and named E.coli@PDA. Then, 160μg chl natural thylakoids were added to E.coli@PDA and incubated for 5h under anaerobic atmosphere to form a hybrid.

[0068] The natural thylakoid-microorganism hybrid was centrifuged at 7000 rcf for 5 min, and then the precipitate was dispersed into a photoreactor containing 50 mL of anaerobic 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite). The OD of the natural thylakoid-microorganism hybrid dispersed into the anaerobic medium was 2.34 %. 600 The value was 1.8. After nitrogen replacement for 30 minutes, the product was irradiated with a xenon lamp (λ>420nm) for 2 hours. The product was detected by gas chromatography. The hydrogen production rate was 10.583mmol / h / g dcw .

[0069] Example 5

[0070] First, Escherichia coli bio-52502 was cultured aerobically for 3 h under aerobic conditions (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7)), and then the aerobic cultured bacterial solution was cultured anaerobically for 2 h under anaerobic conditions (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), and the bacterial solution was resuspended in 50 mL anaerobic culture medium (the anaerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, 0.5 g / L cysteine ​​and 10 mM sodium sulfite), and the OD of the bacterial solution was obtained. 600 The value is 1.6. After anaerobic culture, E. coli was centrifuged at 7000rpm for 10min and resuspended in 20mL Tris buffer (10mM, pH=8.5). Then, 100μL DMSO in which 20mg dopamine (DA) was dissolved was added dropwise to the Tris buffer and gently shaken at 37°C for 2h to coat the E. coli with PDA. Subsequently, the E. coli coated with PDA was collected by centrifugation, washed 3 times with Tris buffer (10mM, pH=8.5), and named E. coli@PDA. Then, 80μg chl natural thylakoids were added to E. coli@PDA and incubated for 14h under anaerobic atmosphere to form a hybrid.

[0071] The natural thylakoid-microorganism hybrid was centrifuged at 7000 rcf for 5 min, and then the precipitate was dispersed into a photoreactor containing 50 mL of anaerobic 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite). The OD of the natural thylakoid-microorganism hybrid dispersed into the anaerobic medium was 2.34 %. 600 The value was 1.8. After nitrogen replacement for 30 minutes, the product was irradiated with a xenon lamp (λ>420nm) for 2 hours. The product was detected by gas chromatography, and the hydrogen production rate was 9.269mmol / h / g dcw .

[0072] Example 6

[0073] First, Escherichia coli bio-52502 was cultured aerobically for 3 h under aerobic conditions (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7)), and then the aerobic cultured bacterial solution was cultured anaerobically for 2 h under anaerobic conditions (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), and the bacterial solution was resuspended in 50 mL anaerobic culture medium (the anaerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, 0.5 g / L cysteine ​​and 10 mM sodium sulfite), and the OD of the bacterial solution was obtained. 600 The value is 1.6. After anaerobic culture, E. coli was centrifuged at 7000rpm for 10min and resuspended in 20mL Tris buffer (10mM, pH=8.5). Then, 100μL DMSO in which 20mg dopamine (DA) was dissolved was added dropwise to the Tris buffer and gently shaken at 37°C for 2h to coat the E. coli with PDA. Subsequently, the E. coli coated with PDA was collected by centrifugation, washed 3 times with Tris buffer (10mM, pH=8.5), and named E.coli@PDA. Then, 40μg chl natural thylakoids were added to E.coli@PDA and incubated for 14h under anaerobic atmosphere to form a hybrid.

[0074] The natural thylakoid-microorganism hybrid was centrifuged at 7000 rcf for 5 min, and then the precipitate was dispersed into a photoreactor containing 50 mL of anaerobic 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite). The OD of the natural thylakoid-microorganism hybrid dispersed into the anaerobic medium was 2.34 %. 600 The value was 1.8. After nitrogen replacement for 30 minutes, the product was illuminated by a xenon lamp (λ>420nm) for 2 hours. The product was detected by gas chromatography, and the hydrogen production rate was 4.986mmol / h / g dcw .

[0075] Example 7

[0076] First, Escherichia coli bio-52502 was cultured aerobically for 3 h under aerobic conditions (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7)), and then the aerobic cultured bacterial solution was cultured anaerobically for 2 h under anaerobic conditions (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), and the bacterial solution was resuspended in 50 mL anaerobic culture medium (the anaerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, 0.5 g / L cysteine ​​and 10 mM sodium sulfite), and the OD of the bacterial solution was obtained. 600 The value is 1.6. After anaerobic culture, E. coli was centrifuged at 7000rpm for 10min and resuspended in 20mL Tris buffer (10mM, pH=8.5). Then, 100μL DMSO in which 20mg dopamine (DA) was dissolved was added dropwise to the Tris buffer, and the mixture was gently shaken at 37°C for 2h to coat the E. coli with PDA. Subsequently, the E. coli coated with PDA was collected by centrifugation, washed three times with Tris buffer (10mM, pH=8.5), and named E. coli@PDA. Then, 160μg chl natural thylakoids were added to E. coli@PDA, and incubated for 14h under anaerobic atmosphere to form a hybrid.

[0077] The natural thylakoid-microorganism hybrid was centrifuged at 7000 rcf for 5 min, and then the precipitate was dispersed into a photoreactor containing 50 mL of anaerobic 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite). The OD of the natural thylakoid-microorganism hybrid dispersed into the anaerobic medium was 2.34 %. 600 The value was 1.8. After nitrogen replacement for 30 minutes, the product was illuminated with a xenon lamp (λ>420nm) for 3 hours. The product was detected by gas chromatography and the hydrogen production was 40.5439mmol / gdcw.

[0078] Example 8

[0079] First, Escherichia coli bio-52502 was cultured aerobically for 3 h under aerobic conditions (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7)), and then the aerobic cultured bacterial solution was cultured anaerobically for 2 h under anaerobic conditions (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), and the bacterial solution was resuspended in 50 mL anaerobic culture medium (the anaerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, 0.5 g / L cysteine ​​and 10 mM sodium sulfite), and the OD of the bacterial solution was obtained. 600 The value is 1.6. After anaerobic culture, E. coli was centrifuged at 7000rpm for 10min and resuspended in 20mL Tris buffer (10mM, pH=8.5). Then, 100μL DMSO in which 20mg dopamine (DA) was dissolved was added dropwise to the Tris buffer, and the mixture was gently shaken at 37°C for 2h to coat the E. coli with PDA. Subsequently, the E. coli coated with PDA was collected by centrifugation, washed three times with Tris buffer (10mM, pH=8.5), and named E. coli@PDA. Then, 160μg chl natural thylakoids were added to E. coli@PDA, and incubated for 14h under anaerobic atmosphere to form a hybrid.

[0080] The natural thylakoid-microorganism hybrid was centrifuged at 7000 rcf for 5 min, and then the precipitate was dispersed into a photoreactor containing 50 mL of anaerobic 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite). The OD of the natural thylakoid-microorganism hybrid dispersed into the anaerobic medium was 2.34 %.600 The value was 1.8. After nitrogen replacement for 30 minutes, the product was illuminated by a xenon lamp (λ>420nm) for 4 hours. The product was detected by gas chromatography, and the hydrogen production was 46.96193mmol / g dcw .

[0081] Example 9

[0082] First, Escherichia coli bio-52502 was cultured aerobically for 3 h under aerobic conditions (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7)), and then the aerobic cultured bacterial solution was cultured anaerobically for 2 h under anaerobic conditions (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), and the bacterial solution was resuspended in 50 mL anaerobic culture medium (the anaerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, 0.5 g / L cysteine ​​and 10 mM sodium sulfite), and the OD of the bacterial solution was obtained. 600 The value is 1.6. After anaerobic culture, E. coli was centrifuged at 7000rpm for 10min and resuspended in 20mL Tris buffer (10mM, pH=8.5). Then, 100μL DMSO in which 20mg dopamine (DA) was dissolved was added dropwise to the Tris buffer, and the mixture was gently shaken at 37°C for 2h to coat the E. coli with PDA. Subsequently, the E. coli coated with PDA was collected by centrifugation, washed three times with Tris buffer (10mM, pH=8.5), and named E. coli@PDA. Then, 160μg chl natural thylakoids were added to E. coli@PDA, and incubated for 14h under anaerobic atmosphere to form a hybrid.

[0083] The natural thylakoid-microorganism hybrid was centrifuged at 7000 rcf for 5 min, and then the precipitate was dispersed into a photoreactor containing 50 mL of anaerobic 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite). The OD of the natural thylakoid-microorganism hybrid dispersed into the anaerobic medium was 2.34 %. 600 The value was 1.8. After nitrogen replacement for 30 minutes, the product was illuminated by a xenon lamp (λ>420nm) for 5 hours. The product was detected by gas chromatography and the hydrogen production was 53.9996mmol / g dcw .

[0084] Example 10

[0085] First, Escherichia coli bio-52502 was cultured aerobically for 3 h under aerobic conditions (the aerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7)), and then the aerobic cultured bacterial solution was cultured anaerobically for 2 h under anaerobic conditions (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), and the bacterial solution was resuspended in 50 mL anaerobic culture medium (the anaerobic culture medium was LB broth (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride, pH = 7), 5 g / L glucose, 0.5 g / L cysteine ​​and 10 mM sodium sulfite), and the OD of the bacterial solution was obtained. 600 The value is 1.6. After anaerobic culture, E. coli was centrifuged at 7000rpm for 10min and resuspended in 20mL Tris buffer (10mM, pH=8.5). Then, 100μL DMSO in which 20mg dopamine (DA) was dissolved was added dropwise to the Tris buffer, and the mixture was gently shaken at 37°C for 2h to coat the E. coli with PDA. Subsequently, the E. coli coated with PDA was collected by centrifugation, washed three times with Tris buffer (10mM, pH=8.5), and named E. coli@PDA. Then, 160μg chl natural thylakoids were added to E. coli@PDA, and incubated for 14h under anaerobic atmosphere to form a hybrid.

[0086] The natural thylakoid-microorganism hybrid was centrifuged at 7000 rcf for 5 min, and then the precipitate was dispersed into a photoreactor containing 50 mL of anaerobic 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, 0.5 g / L cysteine ​​and 10 mM sodium sulfite). The OD of the natural thylakoid-microorganism hybrid dispersed into the anaerobic medium was 2.34 %. 600 The value was 1.8. After nitrogen replacement for 30 minutes, the product was illuminated by a xenon lamp (λ>420nm) for 6 hours. The product was detected by gas chromatography and the hydrogen production was 58.90403mmol / g dcw .

[0087] Comparative Example 1

[0088] The difference from Example 1 is that the photocatalyst is only Escherichia coli bio-52502, and the OD of the bacterial solution is 600 The value is 1.8, and the hydrogen production rate is 7.58174mmol / h / g dcw .

[0089] Comparative Example 2

[0090] The difference from Example 1 is that the photocatalyst is only natural thylakoid, and its hydrogen production rate is 0mmol / h / g dcw .

[0091] By comparing Example 1 with Comparative Examples 1 and 2, it can be seen that the natural thylakoid-microorganism hybrid of the present invention is advanced: compared with Escherichia coli bio-52502 (Comparative Example 1) and natural thylakoids (Comparative Example 2), the hydrogen production rate of the hybrid of the present invention is significantly improved.

[0092] By comparing Examples 1-4, it can be seen that the incubation time in anaerobic atmosphere when Escherichia coli bio-52502 is combined with natural thylakoids affects the hydrogen production rate of the hybrid. The hybrid of the present invention has a higher hydrogen production rate. The optimal incubation time of the present invention is 14 hours.

[0093] By comparing Examples 1 and 5-6, it can be seen that the amount of natural thylakoids added when Escherichia coli bio-52502 is combined with natural thylakoids affects the hydrogen production rate of the hybrid. The hybrid of the present invention has a higher hydrogen production rate. The optimal natural thylakoid content of the present invention is 160 μg / chl.

[0094] By comparing Example 5 and Examples 7-9, it can be seen that under the optimal incubation time of 14 h and the optimal natural thylakoid amount of 160 μg / chl, the hydrogen production gradually increases with the extension of the light reaction time.

[0095] In summary, the present invention utilizes hydrogen-producing bacteria Escherichia coli and natural thylakoids to form a hybrid through polydopamine PDA. The natural thylakoids generate photoelectrons under light conditions, which are directly or indirectly transferred to microorganisms to produce energy cofactors (NADPH, NADH and ATP), thereby increasing the energy cofactors required for the hydrogen production pathway in the microorganisms and increasing the hydrogen production efficiency. The advantages of the hybrid system in photocatalytic reduction hydrogen production are brought into play. In addition, the natural thylakoids are easy to extract and have good biocompatibility, and are green and pollution-free.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing artificial photosynthetic bacteria, characterized in that: include: E. coli was first cultured aerobically and then anaerobically, and the bacterial solution was collected; The bacterial solution is resuspended and cultured in an anaerobic culture medium, and then coated with PDA. The PDA-coated Escherichia coli is mixed with natural thylakoids and incubated under anaerobic conditions to form natural thylakoid-microorganism hybrids, namely, artificial photosynthetic bacteria.

2. The method for constructing artificial photosynthetic bacteria according to claim 1, characterized in that: The Escherichia coli is Escherichia coli bio-52502.

3. The method for constructing artificial photosynthetic bacteria according to claim 1, characterized in that: The aerobic culture medium was LB broth, and the aerobic culture time was 3-4h; Alternatively, the anaerobic culture medium consists of LB broth, glucose and cysteine, and the anaerobic culture time is 2-3 hours.

4. The method for constructing artificial photosynthetic bacteria according to claim 1, characterized in that: During the incubation, the anaerobic culture medium consisted of LB broth, glucose and cysteine.

5. The method for constructing artificial photosynthetic bacteria according to claim 1, characterized in that: The bacterial solution was resuspended in anaerobic medium to obtain an OD600 value of 1.5-1.

8.

6. The method for constructing artificial photosynthetic bacteria according to claim 1, characterized in that: The natural thylakoid concentration is 80 μg chl / mL or 160 μg chl / mL; The concentration of dopamine is 0.2-0.3 mg / μL; The incubation time is 14 hours or more.

7. Artificial photosynthetic bacteria constructed by the method described in any one of claims 1 to 6.

8. Use of the artificial photosynthetic bacteria according to claim 7 in photocatalytic hydrogen production.

9. The use of artificial photosynthetic bacteria in photocatalytic hydrogen production according to claim 8, characterized in that: include: The artificial photosynthetic bacteria of claim 7 are dispersed in a light reaction container containing an anaerobic culture medium, sealed and filled with inert gas, stirred, the reaction temperature is 20° C. to 35° C., the reaction time is 2-6 hours under light conditions, and hydrogen is produced by the reaction.

10. The use of artificial photosynthetic bacteria in photocatalytic hydrogen production according to claim 8, characterized in that: The OD of the anaerobic medium 600 The value is 1.6-1.9; Alternatively, anaerobic medium consisted of LB broth, glucose, cysteine, and sodium sulfite; Alternatively, the inert gas is nitrogen.

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