Hydrogen-generating composition, method for producing hydrogen, and hydrogen production apparatus

Abstract

An object of the present invention is to provide a hydrogen-generating composition capable of suppressing a decrease in apparent quantum yield and increasing the total amount of hydrogen generated, and a method and an apparatus for producing hydrogen using the hydrogen-generating composition.SOLUTION: The hydrogen generating composition contains a pigment having a photocatalytic function, a sacrificial agent, an electron transfer agent having an oxidation-reduction potential higher than that of the pigment and lower than a hydrogen generating potential, and a biocatalyst.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a hydrogen generation composition containing a dye having photocatalytic function, a sacrificial agent, an electron transfer agent, and a biocatalyst, and a hydrogen production method and hydrogen production device using the hydrogen generation composition. [Background technology]

[0002] A method for producing hydrogen (H2) using a photocatalyst, TiO2, and a biocatalyst has been proposed (Non-Patent Documents 1 and 2). Specifically, excited electrons generated by irradiating TiO2 with light are transferred to a biocatalyst (a genetically modified Escherichia coli that mass-produces a hydrogen-generating enzyme) via an electron transfer agent (methyl viologen, hereafter also referred to as "MV"), and the biocatalyst generates hydrogen. Meanwhile, positive holes generated by irradiating TiO2 with light are eliminated by the oxidation of water molecules and sacrificial agents (see Figure 1). However, ultraviolet light is required to generate excited electrons and positive holes in TiO2.

[0003] Furthermore, in order to make effective use of limitless sunlight, a hydrogen production method (see Figure 2) has been proposed that uses a dye (Eosin Y, hereinafter also referred to as "EY") that exhibits photocatalytic function under visible light, a biocatalyst (genetically modified Escherichia coli that mass-produces a hydrogen-generating enzyme), and a sacrificial agent (triethanolamine, hereinafter also referred to as "TEOA") (Non-Patent Document 3). Because EY-based systems can utilize visible light, they have the advantage of having a higher solar conversion efficiency than systems that use TiO2. However, they also have the disadvantage of reduced sustainability of hydrogen production due to EY discoloration. Non-Patent Document 3 describes that adding an electron transfer agent, MV, to an EY-based system can suppress EY discoloration and improve the sustainability of hydrogen production. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Yuki Honda, Hidehisa Hagiwara, Shintaro Ida, and Tatsumi Ishihara, Angew. Chem. Int. Ed. 2016, 55, 8045-8048 [Non-patent document 2] Yuki Honda, Motonori Watanabe, Hidehisa Hagiwara, Shintaro Ida, Tatsumi Ishihara, Applied Catalysis B: Environmental 210 (2017) 400-406 [Non-patent document 3] Yuki Honda, Yuka Shinohara and Hiroshi Fujii, Catal. Sci. Technol., 2020, 10, 6006-6012 Summary of the Invention [Problem to be solved by the invention]

[0005] However, although the addition of the electron transfer agent MV to a system using EY can suppress the fading of EY and improve the sustainability of hydrogen production, there is a problem in that the apparent quantum yield AQY (amount of hydrogen produced per photon irradiated to the reaction system), calculated using the following formula, decreases. AQY (%) = 100 × [(2 × mol of hydrogen produced) / (mol of photons irradiated to the system)]

[0006] An object of the present invention is to provide a hydrogen generation composition that can suppress a decrease in apparent quantum yield and increase the total amount of hydrogen generated, and a hydrogen production method and hydrogen production device that use the hydrogen generation composition. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above problems and have found that adding an electron transfer agent having a specific redox potential to a hydrogen generating composition containing a dye having photocatalytic function, a sacrificial agent, and a biocatalyst can suppress the decrease in apparent quantum yield and increase the total amount of hydrogen generated. The present invention was completed through further research based on this finding.

[0008] That is, the present invention provides the following aspects. <1> A hydrogen generating composition comprising a dye having photocatalytic function, a sacrificial agent, an electron transfer agent having a redox potential higher than the redox potential of the dye and lower than the hydrogen generating potential, and a biocatalyst. <2> The dye is an organic dye. <1> The hydrogen generating composition according to claim 1. <3> the organic dye is at least one selected from the group consisting of eosin Y, eosin B, 5′-carboxyeosin Y, fluorescein, 2,7-dichloro-5′-carboxyfluorescein, proflavine, and rose bengal; <2> The hydrogen generating composition according to claim 1. <4> The electron transfer agent is a bipyridinium salt. <1> ~ <3> The hydrogen generating composition according to any one of the preceding claims. <5> The biocatalyst comprises a hydrogenase. <1> ~ <4> The hydrogen generating composition according to any one of the preceding claims. <6> The biocatalyst is a genetically modified Escherichia coli into which a hydrogenase gene and a protein gene required for its maturation have been introduced. <1> ~ <4> The hydrogen generating composition according to any one of the preceding claims. <7> <1> ~ <6> 10. A method for producing hydrogen, comprising the step of irradiating the hydrogen generation composition according to any one of claims 1 to 9 with light. <8> <1> ~ <6> A hydrogen production device comprising the hydrogen generation composition according to any one of the preceding items. [Effects of the Invention]

[0009] Use of the hydrogen generation composition of the present invention can suppress a decrease in apparent quantum yield and increase the total amount of hydrogen generated, thereby improving hydrogen production efficiency. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing the mechanism of hydrogen production using TiO2 and a biocatalyst. [Figure 2] FIG. 1 is a schematic diagram showing the mechanism of hydrogen production using dye EY and a biocatalyst. [Figure 3] FIG. 1 is a schematic diagram showing a hydrogen production mechanism in the present invention. [Figure 4] 1 is a schematic diagram illustrating an example of a hydrogen production device according to the present invention. [Figure 5] FIG. 1 shows the redox potential of EY2- / EY3-·, the hydrogen production potential, and the redox potential of each electron transfer agent at pH 8. [Figure 6] 1 is a graph showing the relationship between light irradiation time and the total amount of hydrogen produced when hydrogen was produced using the hydrogen generating compositions of Examples 1 and 2 and Comparative Examples 1 to 4. DETAILED DESCRIPTION OF THE INVENTION

[0011] 1. Hydrogen generating composition The hydrogen generation composition of the present invention is characterized by containing a dye having photocatalytic function, a sacrificial agent, an electron transfer agent having a redox potential higher than that of the dye and lower than that of hydrogen generation, and a biocatalyst. The hydrogen generation composition of the present invention is described in detail below.

[0012] [Pigment] The hydrogen generation composition of the present invention contains a dye as a photocatalyst.

[0013] The dye having a photocatalytic function is not particularly limited as long as it is a dye that is excited by receiving light, but is preferably a dye that is excited by receiving visible light (wavelength 360 to 830 nm). The dye may be an organic dye or a metal complex dye, but is preferably an organic dye.

[0014] Examples of organic dyes include azo dyes, quinone dyes, quinoneimine dyes, quinacridone dyes, squarylium dyes, cyanine dyes, merocyanine dyes, triphenylmethane dyes, xanthene dyes, porphyrin dyes, perylene dyes, indigo dyes, and naphthalocyanine dyes. These may be used alone or in combination of two or more.

[0015] As the organic dye, it is preferable to use at least one selected from the group consisting of eosin Y, eosin B, 5'-carboxyeosin Y, fluorescein, 2,7-dichloro-5'-carboxyfluorescein, proflavine, and rose bengal, more preferably at least one selected from the group consisting of eosin Y, eosin B, and 5'-carboxyeosin Y, and even more preferably eosin Y.

[0016] Metal complex dyes are dyes having a structure in which a metal is coordinately bonded to a molecule. Examples of the molecule include bipyridines, porphyrins, phthalocyanines, and naphthalocyanines. Examples of the metal include at least one selected from the group consisting of Cu, Ni, Fe, Co, V, Sn, Si, Ti, Ge, Cr, Zn, Ru, Mg, Al, Pb, Mn, In, Mo, Y, Zr, Nb, Sb, La, W, Pt, TA, Ir, Pd, Os, Ga, Tb, Eu, Rb, Bi, Se, As, Sc, Ag, Cd, Hf, Re, Au, Ac, Tc, Te, and Rh. Among these, metal complex dyes in which a metal is coordinated to a bipyridine-based molecule are preferred, and metal complex dyes in which Ru is coordinated to a bipyridine-based molecule are more preferred. As an example of a metal complex dye in which Ru is coordinated to a bipyridine molecule, there is tris(2,2'-bipyridine)ruthenium(II) complex ion ([Ru(bpy)3] 2+ , bpy: 2,2'-bipyridine), and [Ru(bpy)2(4,4-(PO3H2)2bpy)] 2+ Examples include:

[0017] [Sacrificial Agents] The hydrogen generation composition of the present invention contains a sacrificial agent (reducing agent) for reducing a dye excited by exposure to light to generate a reduced dye. The sacrificial agent is not particularly limited as long as it can generate a reduced dye, and examples thereof include triethanolamine, cysteine, 2-morpholinoethanesulfonic acid, 3-morpholinopropanesulfonic acid, ethylenediaminetetraacetic acid, and ascorbic acid. These may be used alone or in combination of two or more. Of these, triethanolamine is preferably used.

[0018] [Electron transfer agent] The hydrogen generation composition of the present invention contains an electron transfer agent having a redox potential higher than that of the dye and lower than that of the hydrogen generation potential. The electron transfer agent of the present invention has the function of suppressing dye fading by accepting electrons from the reduced dye reduced by the sacrificial agent, and also has the function of transferring electrons accepted from the reduced dye to the biocatalyst. Furthermore, the electron transfer agent of the present invention has the characteristics of easily accepting electrons from the reduced dye because it has a redox potential higher than that of the dye, and easily transferring electrons to the biocatalyst because it has a redox potential lower than the hydrogen generation potential. The characteristics of the electron transfer agent can suppress a decrease in apparent quantum yield and increase the total amount of hydrogen generated.

[0019] The electron transfer agent is not particularly limited as long as it has a redox potential higher than that of the dye used and lower than the hydrogen generation potential, but is preferably a bipyridinium salt. Examples of the salt include halide salts such as chlorides, bromides, and iodides, and chlorides are preferred. Specific examples of bipyridinium include bipyridiniums A to G below. [ka]

[0020] For example, when eosin Y is used as the dye, examples of electron transfer agents having a redox potential higher than that of eosin Y at pH 8 (−0.82 V vs. SHE) and lower than the hydrogen generation potential at pH 8 (−0.47 (units omitted, hereinafter)) include bipyridinium D (redox potential at pH 8: −0.62), bipyridinium E (redox potential at pH 8: −0.53), bipyridinium F (redox potential at pH 8: −0.55), and bipyridinium G (redox potential at pH 8: −0.66). Note that the redox potential of bipyridinium A at pH 8 is −0.99, that of bipyridinium B at pH 8 is −0.92, and that of bipyridinium C at pH 8 is −0.90. Therefore, when eosin Y is used as the dye, bipyridiniums A to C cannot be used as electron transfer agents in the present invention. However, as a dye, [Ru(bpy)3] 2+ ([Ru(bpy)3] at pH 8 + / [Ru(bpy)3] 2+ When a bipyridinium having a redox potential of -1.3 is used, not only bipyridiniums D to G but also bipyridiniums A to C can be used as the electron transfer agent of the present invention. Furthermore, methyl viologen having a bipyridinium of the following structure has a redox potential of -0.44 at pH 8, which is higher than the hydrogen generation potential at pH 8, and therefore does not fall under the category of electron transfer agents of the present invention. [ka]

[0021] [Biocatalysis] The hydrogen generating composition of the present invention contains a biocatalyst capable of generating hydrogen (H) through electron transfer from a reduced dye or an electron transfer agent. The biocatalyst is not particularly limited as long as it is capable of generating hydrogen (H) through electron transfer, and known biocatalysts may be used. Examples of the biocatalyst include hydrogenase (a hydrogen-generating enzyme) and bacteria containing hydrogenase. Examples of hydrogenase include [Fe] hydrogenase, [FeFe] hydrogenase, [NiFe] hydrogenase, and [NiFeSe] hydrogenase, with [FeFe] hydrogenase being preferred. Furthermore, to mass-produce hydrogenase, it is preferable to use recombinant Escherichia coli introduced with a hydrogenase gene and a protein gene involved in its maturation. The recombinant Escherichia coli can be produced by known recombinant DNA techniques, for example, by the methods described in Non-Patent Document 1 (Angew. Chem. Int. Ed. 2016, 55, 8045-8048) and ChemSusChem 2024, 17, e202300958 (Photo-Electro-Biochemical H2 Production Using the Carbon Material-Based Cathode Combined with Genetically Engineered Escherichia coli Whole-Cell Biocatalysis). Specific methods for producing the recombinant Escherichia coli are described in the Examples.

[0022] [Form of hydrogen generating composition] The hydrogen generating composition of the present invention is usually a suspension containing the above-mentioned components and water, and may contain an organic solvent and various additives as necessary. The contents of the above-mentioned components may be appropriately adjusted taking into consideration the hydrogen production efficiency, the total amount of hydrogen generated, etc.

[0023] The pH of the hydrogen generation composition of the present invention is usually 6 to 10, and from the viewpoint of the protonation state of triethanolamine, which is preferable as a sacrificial agent, is preferably 7 to 9, and more preferably 7.5 to 8.5.

[0024] 2. Hydrogen generation mechanism FIG. 3 is a schematic diagram showing the hydrogen production mechanism in the present invention. In FIG. 3, as a specific example, eosin Y (EY 2- ), triethanolamine (TEOA) as a sacrificial agent, and bipyridinium D (hereinafter referred to as "tmMV") as an electron transfer agent. 2+ The hydrogen production mechanism is shown when a hydrogen generating composition containing a biocatalyst and a catalyst (also referred to as "a biocatalyst") is used. However, this hydrogen production mechanism is estimated from the results of various spectroscopic analyses, and the present invention is not limited to this mechanism.

[0025] First, when the hydrogen generating composition is irradiated with visible light or sunlight, the ground state EY 2- is excited to the excited state EY * And, EY * is reduced with TEOA to form reduced EY 3-· Next, EY 3-· EY 3-· EY 2- The electrons are transferred back to the biocatalyst, which then catalyzes the synthesis of hydrogen (H2), producing hydrogen. In theory, hydrogen is produced continuously through this reaction cycle. However, in reality, hydrogen is not produced continuously. The reason for this is that EY 3-· Not only electron transfer from the enzyme to the biocatalyst, but also EY 3-· This is thought to be due to the fact that the discoloration of EY occurs due to undesirable electron transfer from the EY. As described in Non-Patent Document 3, the addition of methyl viologen to a system using EY can suppress the discoloration of EY and improve the sustainability of hydrogen production. However, when methyl viologen is added to a system using EY, the apparent quantum yield AQY decreases. This is because the EY 3-· Electron transfer from the enzyme to the biocatalyst and EY 3-· This is because the electron transfer from EY to methyl viologen competes with the electron transfer from EY to methyl viologen. Methyl viologen is widely used as an electron transfer agent for biocatalysts. 3-·Efficient hydrogen production would be possible if reduced methylviologen, which has accepted electrons from methylviologen, transferred the electrons to the biocatalyst. However, the redox potential of methylviologen is higher than the hydrogen production potential, making electron transfer from reduced methylviologen to the biocatalyst unfavorable.

[0026] Therefore, in the present invention, as shown in FIG. 3-· In order to suppress the bleaching reaction of EY by making it easier to accept electrons from EY, 2- / EY 3-· and EY 3-· To efficiently transfer electrons received from the biocatalyst, the electron transfer agent tmMV, which has a redox potential lower than the hydrogen production potential, was used. 2+ This suppresses the fading of EY and improves the sustainability of hydrogen production, while also suppressing the decrease in apparent quantum yield and increasing the total amount of hydrogen produced.

[0027] 3. Hydrogen production method and hydrogen production device The hydrogen production method of the present invention includes a step of irradiating the hydrogen generation composition with light. Figure 4 is a schematic diagram showing an example of the hydrogen production apparatus of the present invention. Hereinafter, the hydrogen production method of the present invention will be described with reference to Figure 4.

[0028] The hydrogen production device includes a sealed container containing a hydrogen generation composition in a container at least partially formed of a light-transmitting material, and a gas circulation system. The gas circulation system is composed of, for example, stainless steel piping or glass tubing. A sealed container having at least a light-transmitting transparent window (e.g., a quartz window) in its at least part is connected to the gas circulation system, and the hydrogen generation composition (reaction suspension) in the sealed container is irradiated with light through the transparent window. The reaction suspension in the sealed container is prepared, for example, in a glove box under a nitrogen atmosphere, and the sealed container is connected to the gas circulation system in a sealed state. The light to be irradiated needs to be selected depending on the dye to be used, but is preferably visible light, more preferably sunlight.

[0029] The gas circulation system circulates gas in one direction, for example, using an electromagnetic pump. By opening valves 2 and 3, the air is evacuated using a vacuum pump, and by opening valves 1 and 2, an inert gas such as Ar is introduced. By repeating this process, the gas inside the sealed container is replaced, and finally the pressure is reduced (for example, about -70 kPa) with an inert gas atmosphere. With valves 2 and 4 closed, the gas is circulated for a certain period of time without light irradiation, and after checking for leaks, the reaction is initiated by light irradiation.

[0030] The six-way valve, valves 4, and 5 may be used to sample from the gas circulation system at any time and analyze the gas composition. A gas chromatography (GC) device equipped with a TCD detector can be used for analysis. When the six-way valve is set to position 1 and valve 5 is opened, a vacuum is created in the sample loop. When valve 5 is then closed and valve 4 is opened, some gas is introduced from the gas circulation system into the sample loop. When valve 4 is closed and the six-way valve is set to position 2, the gas in the sample loop is introduced into the GC-TCD, and the gas composition can be analyzed.

[0031] The hydrogen production method of the present invention uses the hydrogen generation composition, which can effectively suppress dye fading and increase the total amount of hydrogen produced while maintaining a high quantum yield, thereby enabling hydrogen to be produced with high efficiency. [Example]

[0032] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0033] <Synthesis of electron transfer agents> Synthesis Example 1: Bipyridinium D (tmMV 2+ ) dichloride synthesis Under an Ar atmosphere, zinc powder (140 mg, 2.1 mmol) and bis(triphenylphosphine)nickel(II) dichloride (1.1 g, 1.7 mmol) were suspended in dry DMF (30 mL). The mixture was stirred under an Ar atmosphere at 50 °C until the mixture turned brown. To this mixture, a solution of 4-bromo-2,6-dimethylpyridine (630 mg, 3.4 mmol) in dry DMF (5 mL) was added dropwise over 10 min at 50 °C under an Ar atmosphere, and the reaction mixture was further stirred at 50 °C for 20 h. The solvent was evaporated to dryness, and then 50 mL of an aqueous solution of ethylenediamine (10% v / v) was added. The crude compound was extracted three times with chloroform (50 mL). The combined organic layers were extracted three times with 1 M aqueous hydrochloric acid (50 mL). The combined aqueous solution was neutralized with saturated aqueous Na2CO3. The resulting white precipitate was extracted three times with chloroform (50 mL). The combined organic layers were dried over Na2SO4 and evaporated to dryness. The resulting pale yellow solid (2,2',6,6'-tetramethyl-4,4'-bipyridine) was dissolved in acetonitrile (50 mL), and iodomethane (approximately 1 mL, 16 mmol) was added to the solution. The mixture was refluxed for 17 hours. The yellow precipitate was filtered, washed with chloroform, acetone, and hexane, and then dried in vacuo. The resulting solid (tmMV(PF6)2) was dissolved in a minimum amount of water, and then a minimum amount of saturated aqueous NaPF6 solution was added to precipitate the hexafluorophosphate salt (tmMV(PF6)2). The precipitate was filtered, dried, and then dissolved in a minimum amount of acetonitrile. A minimum amount of saturated tetrabutylammonium acetonitrile solution was added to the solution, and the precipitate was filtered and purified to give tmMV. 2+ Dichloride (tmMVCl2) was obtained.

[0034] Synthesis Example 2: Bipyridinium E (dmMV 2+ ) dichloride synthesis dmMV was synthesized in the same manner as in Synthesis Example 1, except that 4-bromo-2-methylpyridine was used instead of 4-bromo-2,6-pyridine. 2+ Dichloride (dmMVCl2) was obtained.

[0035] Synthesis Example 3: Bipyridinium C (2,2'-MV 2+ ) dichloride synthesis 2,2'-MV was synthesized in the same manner as in Synthesis Example 1, except that commercially available 2,2'-bipyridine was used instead of the 2,2',6,6'-tetramethyl-4,4'-bipyridine synthesized in Synthesis Example 1. 2+ Dichloride (2,2'-MVCl2) was obtained.

[0036] Benzyl viologen (BVCl2) was a commercially available product.

[0037] <Preparation of biocatalysts> Construction of genetically engineered E. coli cells carrying the hydrogenase gene and the protein gene required for its maturation The commercially available Escherichia coli BL21(DE3) (Novagen) was used as the host. A strain was used in which the [FeFe]-hydrogenase (HydA) gene and three modifying enzyme (HydE, HydF, HydG) genes derived from Clostridium acetobutylicum NBRC 13948 (=ATCC 824) strain were introduced. HydA was produced as a protein with a Strep II tag sequence (SAWSHPQFEK) attached to its C-terminus. The hydrogen production reaction of the present invention can be carried out using either the recombinant E. coli as is or HydA purified by affinity chromatography using the Strep II tag sequence.

[0038] (Genetically modified E. coli used) Plasmids were used to introduce the [FeFe]-hydrogenase gene into E. coli BL21(DE3). The plasmids used were pEHydEA, obtained by inserting the HydE gene into multicloning site (MCS) 1 of commercially available pETDuet-1 (Novagen) and the HydA gene with a Strep II tag sequence into MCS2, and pCHydFG, obtained by inserting the HydF gene into MCS1 and the HydG gene into MCS2 of commercially available pCDFDuet-1 (Novagen). E. coli BL21(DE3) / pEHydEA+pCHydFG (constructed using the method described in Non-Patent Document 1) simultaneously harbors two plasmids. Gene expression is controlled by the T7 promoter and is induced by adding isopropyl-β-thiogalactopyranoside (IPTG) to the medium. We also used pEHydEFG-A, which was obtained by inserting an artificial operon sequence encoding the three proteins HydE, HydF, and HydG into MCS1 of pETDuet-1 and a Strep II-tagged HydA gene into MCS2 (constructed using the method described in ChemSusChem 2024, 17, e202300958). E. coli BL21(DE3) / pEHydEFG-A, which harbors this construct, can also be used in the same way as E. coli BL21(DE3) / pEHydEA+pCHydFG.

[0039] (Plasmid construction) pEHydEA was prepared by the method described in Non-Patent Document 1. Specifically, pEHydEA was prepared by the following method. The genome solution of C. acetobutylicum NBRC 13948 strain was obtained from the NITE Biotechnology Center (NBRC), a strain collection. Using this genome solution as a template, DNA fragment A encoding HydA-StrepII was obtained using primers 1 and 2 (Table 1), and DNA fragment B encoding HydE was obtained using primers 3 and 4. The primers were designed to add restriction enzyme sequences to the fragments obtained by PCR. pETDuet-1 and DNA fragment A were digested with NdeI and XhoI and then ligated to obtain pEHydA, in which the sequence encoding HydA-StrepII was inserted into MCS2. pEHydA and DNA fragment B were then digested with restriction enzymes NcoI and BamHI and ligated to obtain pEHydEA, in which the sequence encoding HydE was inserted between NcoI and BamHI in MCS1. The nucleotide sequence and amino acid sequence of the produced protein are shown in Table 2. Similarly, pCHydFG was generated by PCR using the genome solution of the C. acetobutylicum NBRC 13948 strain as a template and primers 5 and 6 and primers 7 and 8 shown in Table 1 to obtain DNA fragment C encoding HydF and DNA fragment D encoding HydG, each containing restriction enzyme sequences. pCDFDuet-1 and DNA fragment C were digested with NcoI and BamH I and then ligated to obtain pCHydF. Subsequently, pCHydF and DNA fragment D were digested with NdeI and BglII and then ligated to obtain pCHydFG.

[0040] pEHydEFG-A was prepared by the method described in ChemSusChem 2024, 17, e202300958. Specifically, pEHydEFG-A was prepared by the following method. An artificial operon designed to enable production of HydE, F, and G using a single messenger RNA was constructed by inserting DNA fragment E, encoding HydF and HydG, into pEHydEA using the In-Fusion Cloning Kit (Clontech). The sequences encoding HydF and HydG were amplified by PCR using primers 11 and 12 and primers 13 and 14, respectively, with pCHydFG as the template. The mixture of the resulting two DNA fragments was then subjected to PCR with primers 11 and 14 as the template to amplify DNA fragment F, which combines the sequences encoding HydF and HydG. A linear vector backbone was then prepared by PCR using primers 9 and 10 with pEHydEA as the template. Subsequently, In-Fusion cloning was performed using DNA fragment F and the linear vector backbone of pEHydEA to obtain pEHydEFG-A.

[0041] (Culture method) E. coli BL21(DE3) / pEHydEA+pCHydFG or E. coli BL21(DE3) / pEHydEFG-A was cultured in 4 ml of Luria-Bertani medium (hereafter simply referred to as "medium") (Nacalai Tesque) at 37°C for 16 hours. The medium was supplemented with 100 μg / ml ampicillin when carrying pEHydEA and pEHydEFG-A, and 40 μg / ml streptomycin when carrying pCHydFG. 400 μl of the resulting culture was inoculated into 400 ml of medium supplemented with the appropriate antibiotic, ammonium iron citrate (250 μg / mL), and 100 mM 3-morpholinopropanesulfonic acid (pH 7.4, adjusted with NaOH). The E. coli was grown aerobically in a 2 L Erlenmeyer flask at 37°C and 150 rpm until the OD600 reached 0.4. The culture was then transferred to a centrifuge bottle (Nalgene). TMThe cells were transferred to PPCO Centrifuge Bottles with a Sealing Closure (Product No. 3141-0500PK) and cysteine ​​(final concentration: 2 mM) and disodium fumarate (final concentration: 20 mM) were added. The centrifuge bottles containing the culture medium were transferred to a glove box (nitrogen atmosphere, oxygen concentration: 1 ppm or less), and 0.1 mM IPTG was added for gene induction. The culture was continued at room temperature (25°C) with stirring for 20 hours. After gene induction, the centrifuge bottles were removed from the glove box and centrifuged (3000 × g, 10 minutes, 4°C) to recover the cells. After centrifugation, the centrifuge bottles were returned to the glove box and the supernatant was removed. The cell pellet was suspended in phosphate-buffered saline (PBS) (0.2 g / L KH2PO4, 0.2 g / L KCl, 1.15 g / L Na2HPO4, 8 g / L NaCl), and the resulting suspension was used for hydrogen production. After centrifugation, the supernatant was removed, and the wet cell weight was measured, and a predetermined amount was used for each reaction.

[0042] [Table 1]

[0043] [Table 2] TIFF2026028314000006.tif228156TIFF2026028314000007.tif228156TIFF20260283140 00008.tif228156TIFF2026028314000009.tif228156TIFF2026028314000010.tif128156

[0044] Example 1 In a glove box (N atmosphere, O2 1 ppm or less), eosin Y (30 ml, 0.1 mM) was used as a photocatalyst, and tmMV synthesized in Synthesis Example 1 was used as an electron transfer agent. 2+The reaction solution was mixed with 0.2 mM tmMVCl2, 100 mM TEOA as a sacrificial agent, and 0.1 g wet cell weight E. coli BL21(DE3) / pEHydEFG-A (biocatalyst). The pH was adjusted to 8 with HCl, and the resulting solution was filled into a sealed vessel equipped with a quartz window. The resulting sealed vessel was connected to the hydrogen production apparatus shown in Figure 4, and the gas inside the vessel was replaced with Ar. The internal pressure was adjusted to -70 kPa, and the gas was circulated for a certain period of time to check for leaks. Hydrogen was then produced by irradiating the vessel with light from an LED light source (470 nm) through the quartz window on the top of the sealed vessel. The amount of hydrogen produced was measured by gas chromatography (detector: TCD).

[0045] Example 2 tmMV 2+ Instead of dichloride (tmMVCl2), dmMV synthesized in Synthesis Example 2 was used. 2+ Hydrogen was produced in the same manner as in Example 1, except that dichloride (dmMVCl2) was used.

[0046] Comparative Example 1 Hydrogen was produced in the same manner as in Example 1, except that no electron transfer agent was added to the reaction solution.

[0047] Comparative Example 2 tmMV 2+ Hydrogen was produced in the same manner as in Example 1, except that methyl viologen (MV) was used instead of methyl MV dichloride (tmMVCl2).

[0048] Comparative Example 3 tmMV 2+ Instead of dichloride (tmMVCl2), 2,2'-MV synthesized in Synthesis Example 3 was used. 2+ Hydrogen was produced in the same manner as in Example 1, except that dichloride (2,2'-MVCl2) was used.

[0049] Comparative Example 4 tmMV 2+ Hydrogen was produced in the same manner as in Example 1, except that benzyl viologen (BVCl2) was used instead of tmMVCl2.

[0050] Figure 5 shows the EY at pH 8. 2- / EY 3-· 1 shows the oxidation-reduction potential of tmMV, the hydrogen generation potential, and the oxidation-reduction potential of each electron transfer agent. 2+ and dmMV 2+ corresponds to the electron transfer agent of the present invention.

[0051] 6 is a graph showing the relationship between light irradiation time and the total amount of hydrogen produced when hydrogen was produced using the hydrogen generating compositions of Examples 1 and 2 and Comparative Examples 1 to 4. The hydrogen generating compositions of Examples 1 and 2 were prepared using tmMV, which has a redox potential higher than that of Eosin Y and a redox potential lower than that of Eosin Y. 2+ or dmMV 2+ The hydrogen generating composition of Comparative Example 1, which does not contain an electron transfer agent, and the hydrogen generating composition of Comparative Example 2, which contains 2,2'-MV 2+ a hydrogen generating composition of Comparative Example 3 containing MV 2+ or BV 2+ It can be seen that the total amount of hydrogen generated can be increased compared to the hydrogen generating compositions of Comparative Examples 2 and 4 containing the above.

[0052] Table 3 below shows the redox potentials of the electron transfer agents used in the Examples and Comparative Examples, as well as the measurement results of AQY and TON when the hydrogen generation compositions of the Examples and Comparative Examples were used. The hydrogen generation compositions of Examples 1 and 2 exhibited a suppressed decrease in apparent quantum yield AQY compared to the hydrogen generation composition of Comparative Example 1, which did not contain an electron transfer agent, and exhibited significantly improved hydrogen generation amounts TON per mole of dye, confirming their excellent hydrogen production efficiency. Meanwhile, the hydrogen generation composition of Comparative Example 2 exhibited a significantly decreased AQY compared to the hydrogen generation composition of Comparative Example 1, which did not contain an electron transfer agent. Furthermore, the hydrogen generation composition of Comparative Example 3 exhibited an equivalent AQY to the hydrogen generation composition of Comparative Example 1, which did not contain an electron transfer agent, but did not exhibit an improved TON. Furthermore, the hydrogen generation composition of Comparative Example 4 exhibited a significantly decreased AQY and did not exhibit an improved TON compared to the hydrogen generation composition of Comparative Example 1, which did not contain an electron transfer agent. These results demonstrate that a hydrogen generating composition containing an electron transfer agent with a redox potential higher than that of the dye used and lower than the hydrogen generation potential can effectively suppress dye fading and increase TON while maintaining a high AQY, thereby enabling highly efficient hydrogen production. While E. coli BL21(DE3) / pEHydEFG-A was used as the biocatalyst in the above examples and comparative examples, similar results were obtained when E. coli BL21(DE3) / pEHydEA+pCHydFG was used as the biocatalyst. [Table 3]

Claims

1. A hydrogen generating composition comprising a dye having photocatalytic function, a sacrificial agent, an electron transfer agent having a redox potential higher than the redox potential of the dye and lower than the hydrogen generating potential, and a biocatalyst.

2. The hydrogen generation composition of claim 1 , wherein the dye is an organic dye.

3. 3. The hydrogen generating composition according to claim 2, wherein the organic dye is at least one selected from the group consisting of eosin Y, eosin B, 5′-carboxyeosin Y, fluorescein, 2,7-dichloro-5′-carboxyfluorescein, proflavine, and rose bengal.

4. The hydrogen generation composition according to claim 1 , wherein the electron transfer agent is a bipyridinium salt.

5. The hydrogen generating composition of claim 1 , wherein the biocatalyst comprises a hydrogenase.

6. The hydrogen generating composition according to claim 1 , wherein the biocatalyst is a genetically modified Escherichia coli into which a hydrogenase gene and a protein gene involved in its maturation have been introduced.

7. A method for producing hydrogen, comprising the step of irradiating the hydrogen generation composition according to any one of claims 1 to 6 with light.

8. A hydrogen production device comprising the hydrogen generation composition according to any one of claims 1 to 6.

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