Hydrogen production reaction system with formaldehyde and water as co-substrate and hydrogen production method

Abstract

The invention provides a hydrogen production reaction system taking formaldehyde and water as co-substrates, which comprises a buffer solution, maltodextrin, formaldehyde, magnesium chloride, manganese chloride, NAD (Nicotinamide Adenine Dinucleotide), benzyl viologen, sodium phosphate, alpha-glucan phosphorylase, phosphoglucomutase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, 6-phosphoglucolactonase, diaphorase and hydrogenase. The enzyme is prepared from 1, 6-hexanediol phosphate synthase, hexanediol phosphate isomerase, glucose phosphate isomerase and 2, 6-hexanediol phosphate synthase. The invention also provides a corresponding hydrogen production method. Formaldehyde and water are utilized to generate hydrogen, so that excessive dependence on agricultural grain resources is avoided; meanwhile, high-energy formaldehyde is used for replacing traditional carbohydrates as a substrate, so that the energy barrier of co-substrate water is reduced, and the process of small molecular utilization of carbohydrates and hydrogen production is simplified; in addition, the method has the advantages of being not harsh in water quality condition and low in cost.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogen production, and particularly to a hydrogen production reaction system and a hydrogen production method using formaldehyde and water as co-substrates. Background Art

[0002] Fossil fuels (oil, natural gas, and coal) meet most of the world's current energy demands, but they are non-renewable resources that are being rapidly depleted. In addition, their combustion products are causing global problems such as the greenhouse effect, ozone layer depletion, acid rain, and pollution, which pose a great threat to our environment. Hydrogen, a well-known carbon-free gaseous fuel, is considered a future energy carrier to solve the above dilemmas. Hydrogen is a very efficient and reliable fuel whose combustion does not produce greenhouse gases, does not produce ozone-depleting chemicals, and has little or no acid rain components and pollution. Nowadays, hydrogen production still mainly relies on fossil technologies, which is considered undesirable due to low conversion efficiency and greenhouse gas emissions. Therefore, it is necessary to find a green and sustainable hydrogen production route with low energy consumption and low cost.

[0003] Hydrogen production methods mainly include hydrogen production from fossil raw materials, water electrolysis for hydrogen production, and biological hydrogen production: 1) Currently, hydrogen production from fossil fuels is the most widely used method, producing approximately 96% of the hydrogen. It is usually based on cracking, gasification, and catalytic reforming processes, mostly using natural gas as the main raw material. In this technology, methane is first extracted with steam, carbon dioxide, and hydrogen. After reforming, carbon monoxide further reacts with steam through the water-gas shift reaction to produce hydrogen and carbon monoxide. This method has a relatively high hydrogen production efficiency, and the hydrogen concentration can reach up to 74%. However, it produces serious carbon dioxide pollution. Producing 1 ton of hydrogen using this technology will release approximately 14 tons of carbon dioxide. Similarly, coal liquids can be used as raw materials for hydrogen production. Coal, oxygen, and steam react under high temperature and pressure to produce a gas mixture composed of carbon monoxide, carbon dioxide, and hydrogen, which further reacts to form a mixture of hydrogen and carbon dioxide. It can be seen that hydrogen production from fossil fuels has problems such as high cost, low energy conversion rate, and serious environmental pollution, and cannot fundamentally meet the needs of energy sustainable development. 2) Water electrolysis for hydrogen production is another mature hydrogen production technology. By passing direct current through an aqueous solution containing electrolytes (such as sodium hydroxide, potassium hydroxide, etc.), water is electrolyzed to produce hydrogen. This technology does not cause environmental pollution, and the purity of the produced hydrogen can reach over 99%. It is generally considered the most efficient hydrogen production method. However, this method requires a large amount of electricity. Producing 1 cubic meter of hydrogen requires consuming 4.5 - 5.5 kW*h of electricity, which is equivalent to 80% of the hydrogen energy output. In addition, problems such as expensive electrode materials and the need for high-quality water in the electrolysis process also limit the large-scale application of water electrolysis for hydrogen production. 3) Biological hydrogen production is based on biomass raw materials produced by photosynthesis. Since the carbon in biomass does not come from fossil energy but from carbon dioxide captured by plants in the atmosphere, the carbon dioxide produced during biological hydrogen production is part of the natural carbon element recycling. Therefore, compared with traditional hydrogen production methods, biological hydrogen production is basically carbon-neutral. However, due to the catalytic activity problem of enzymes, common biological hydrogen production has relatively high quality requirements for biomass substrates. In addition, the supply of biomass raw materials is closely related to seasons, climate, and geographical conditions, which may lead to unstable production. More importantly, using high-quality biomass raw materials for substrate input may exacerbate the dilemma of competing with people for food.

[0004] Formaldehyde is an excellent hydrogen storage and release carrier. First, compared with carbohydrates such as starch, formaldehyde (CH2O) and glucose (C6H 12O6) has the same hydrogen proportion, so it is also a high-hydrogen-density carrier. Secondly, macromolecular carbohydrates have complex types of glycosidic bonds and are usually insoluble in water, which is not conducive to the efficient progress of the reaction. Enzymes responsible for hydrolyzing different glycosidic bonds further complicate the process of biomass utilization at the small molecule level and hydrogen production. The molecule of formaldehyde is simpler. As a gas molecule soluble in water, it is more suitable for the aqueous phase system of multi-enzyme catalysis. Moreover, the stable property of formaldehyde being soluble in water also contributes to the portability and safety of this hydrogen storage material. Coincidentally, water is also an excellent hydrogen storage and release carrier. First of all, water (H2O) has a higher hydrogen proportion than carbohydrates. Secondly, the process of hydrogen storage and release in water completely avoids the generation of carbon dioxide. However, it must be admitted that producing hydrogen from water requires a large amount of energy. The high energy barrier limits the practice of hydrogen production from water resources and requires a high-energy cosubstrate to assist in hydrogen production from water resources. Secondly, although the source of water is very abundant, 96.5% of the water resources globally are seawater, and human society continuously discharges waste sewage every day, but this does not provide a huge direct raw material for hydrogen production from water resources. The reason is that hydrogen production equipment usually has clear requirements for water quality.

[0005] All in all, combining the current status of biological hydrogen production technology, the problems to be solved include: how to avoid the excessive dependence of biological hydrogen production on agricultural and food resources; how to tap the potential of formaldehyde, an excellent hydrogen storage and release carrier, as much as possible; how to accept different water quality resources to the greatest extent; how to lower the thermodynamic barrier of hydrogen production from water resources. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a hydrogen production reaction system and a hydrogen production method using formaldehyde and water as cosubstrates. This system uses formaldehyde and water to generate hydrogen, avoiding excessive dependence on agricultural and food resources; at the same time, replacing traditional carbohydrates with high-energy formaldehyde as the substrate, reducing the energy barrier of the cosubstrate water, and simplifying the process of biomass utilization at the small molecule level and hydrogen production; in addition, it also has the advantages of not being demanding on water quality conditions and low cost.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A hydrogen production reaction system using formaldehyde and water as co-substrates, comprising a buffer solution, maltodextrin, formaldehyde, magnesium chloride, manganese chloride, NAD, benzyl viologen (BV), sodium phosphate, α-glucan phosphorylase (α-GP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), 6-phosphogluconolactonase (6PGL), diaphorase (DI), hydrogenase (SHI), 3-hexulose-6-phosphate synthase (HPS)-6-phospho-3-hexuloisomerase (PHI) fusion protein, and phosphoglucose isomerase (PGI).

[0009] As one of the preferred embodiments of the present invention, the nucleotide sequences of the α-glucan phosphorylase, phosphoglucomutase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, 6-phosphogluconolactonase, diaphorase, hydrogenase, 3-hexulose-6-phosphate synthase-6-phospho-3-hexuloisomerase fusion protein, and phosphoglucose isomerase are respectively shown in SEQ ID NO.1 to SEQ ID NO.9, and the corresponding amino acid sequences are shown in SEQ ID NO.10 to SEQ ID NO.18.

[0010] As one of the preferred embodiments of the present invention, in the reaction system, the pH of the buffer solution is 5 to 10.

[0011] As one of the preferred embodiments of the present invention, in the reaction system, maltodextrin serves as a cofactor donor, with a glucose equivalent of 4 to 7 and a concentration of 1 to 1000 mM.

[0012] As one of the preferred embodiments of the present invention, in the reaction system, formaldehyde is one of the co-substrates, with an initial concentration of 10 to 20 mM, and then a 200 mM formaldehyde mother liquor is added dropwise at a rate of 0 to 5 μL / min; more preferably, it is added dropwise at a rate of 5 μL / min.

[0013] As one of the preferred embodiments of the present invention, in the reaction system, the concentration of NAD is 1 to 10 mM.

[0014] As one of the preferred embodiments of the present invention, in the reaction system, the concentration of benzyl viologen is 0.5 to 5 mM.

[0015] As one of the preferred embodiments of the present invention, the reaction system comprises the following specific components:

[0016] 100 mM of buffer solution with pH 7.5, 20 mM of maltodextrin with a glucose equivalent of 4 to 7, 5 mM of magnesium chloride, 0.5 mM of manganese chloride, 8 mM of NAD, 2 mM of benzyl viologen, 5 mM of sodium phosphate, 0.25 mg / mL of α-glucan phosphorylase, 0.05 mg / mL of phosphoglucomutase, 2.65 mg / mL of glucose 6-phosphate dehydrogenase, 0.53 mg / mL of 6-phosphogluconate dehydrogenase, 0.08 mg / mL of 6-phosphogluconolactonase, 5.13 mg / mL of diaphorase, 1.5 mg / mL of hydrogenase, 0.49 mg / mL of 6-phosphohexose synthase-phosphohexose isomerase fusion protein, 1.12 mg / mL of phosphoglucose isomerase, formaldehyde; the initial concentration of the formaldehyde is 10 to 20 mM, and then a 200 mM formaldehyde mother liquor is added dropwise at a rate of 0 to 5 μL / min.

[0017] A hydrogen production method using formaldehyde and water as co-substrates, comprising the following steps:

[0018] (1) Construct the above reaction system in a reactor;

[0019] (2) React the reaction system constructed in step (1) at a temperature of 10 to 90 °C, and add a 200 mM formaldehyde mother liquor dropwise at a rate of 0 to 5 μL / min, and use formaldehyde and water as co-substrates to produce hydrogen.

[0020] As one of the preferred embodiments of the present invention, the reaction temperature is specifically 70 °C.

[0021] Reaction principle:

[0022] In the present invention, by directly constructing a reaction system in a reaction vessel, hydrogen is generated by the action of formaldehyde and water under the action of a variety of thermophilic enzymes. Among them, a small amount of maltodextrin, in the presence of sodium phosphate, generates glucose 6-phosphate by using α-glucan phosphorylase and phosphoglucomutase, and then, through glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, and diaphorase, electrons are respectively transferred to NAD and BV ox , and finally, through hydrogenase, the electrons are transferred to H generated by the hydrolysis of the co-substrate water + , and finally hydrogen is generated. The 6-phosphohexose synthase-phosphohexose isomerase fusion protein and phosphoglucose isomerase are responsible for the regeneration of the cofactor glucose 6-phosphate to initiate the next cycle (see Figure 1)。

[0023] The advantages of the present invention compared with the prior art are as follows:

[0024] The present invention provides a brand-new technology for preparing hydrogen with formaldehyde and water as co-substrates. The hydrogen production reaction without the addition of exogenous ATP can be achieved by using a multi-enzyme reaction system. This system uses easily storable formaldehyde and easily accessible water as co-substrates for the reaction to synthesize hydrogen. During the reaction process, formaldehyde as a co-substrate provides a reducing hydrogen, which combines with one hydrogen of water to form hydrogen, while the hydrogen of another formaldehyde is used for the cyclic regeneration of glucose 6-phosphate. Therefore, this system can generate hydrogen by using formaldehyde and water only with a small amount of sugar molecules as cofactors for the cycle, greatly avoiding the dilemma of competing with humans for food.

[0025] In addition, replacing carbohydrates with high-energy formaldehyde as the substrate reduces the energy barrier of co-substrate water and simplifies the process of small-molecule utilization of carbohydrates and hydrogen production.

[0026] In addition, the enzymes required by this system are not harsh on water quality conditions, and excellent hydrogen production effects can be achieved with seawater or sewage. Therefore, the present invention provides a new way for the rational utilization of waste water resources.

[0027] In addition, each thermophilic enzyme used in this system can be obtained only by heat purification, and the heating condition also helps to further improve the reaction rate.

[0028] In addition, the present invention only needs to add a small amount of NAD to transfer the reducing hydrogen of the substrate, and does not need to add expensive energy molecules such as ATP or NADP, which can greatly reduce the cost and facilitate large-scale hydrogen production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a schematic diagram of the molecular cycle principle of the reaction system of the present invention;

[0030] Figure 2 is a recombinant plasmid map of α-glucan phosphorylase (α-GP) of the present invention;

[0031] Figure 3 is a recombinant plasmid map of phosphoglucomutase (PGM) of the present invention;

[0032] Figure 4 is a recombinant plasmid map of glucose 6-phosphate dehydrogenase (G6PDH) of the present invention;

[0033] Figure 5 is a recombinant plasmid map of 6-phosphogluconate dehydrogenase (6PGDH) of the present invention;

[0034] Figure 6 is a recombinant plasmid map of 6-phosphogluconolactonase (6PGL) of the present invention;

[0035] Figure 7 It is the recombinant plasmid map of the diaphorase (DI) of the present invention;

[0036] Figure 8 It is the recombinant plasmid map of the hydrogenase (SHI) of the present invention;

[0037] Figure 9 It is the recombinant plasmid map of the phosphoketolase-phosphohexose isomerase fusion protein (HPS-PHI) of the present invention;

[0038] Figure 10 It is the recombinant plasmid map of the phosphoglucose isomerase (PGI) of the present invention;

[0039] Figure 11 It is the hydrogen production result of adding formaldehyde and HPS-PHI enzyme in the later stage of the hydrogen production pathway of the present invention;

[0040] Figure 12 It is the hydrogen production result of the high-temperature enzyme system of the present invention at 60 and 80 °C;

[0041] Figure 13 It is the hydrogen production result at 30 °C in the normal-temperature enzyme system;

[0042] Figure 14 It is the hydrogen production result of the present invention under different NAD concentrations, BV concentrations and buffer solution pH values (in the figure, Figure A is the result of different NAD concentrations, Figure B is the result of different BV concentrations, and Figure C is the result of different buffer solution pH values);

[0043] Figure 15 It is the hydrogen production result of the system of the present invention at different reaction temperatures;

[0044] Figure 16 It is the comparison of hydrogen production amounts at different formaldehyde concentrations of the present invention;

[0045] Figure 17 It is the comparison of hydrogen production amounts of the system of the present invention in different water qualities. Detailed implementation manners

[0046] The embodiments of the present invention will be described in detail below. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. At the same time, unless otherwise specified, the culture media, carriers, plasmids, and reagents used in the present invention are conventional culture media, carriers, plasmids, and reagents in the technical field; unless otherwise specified, the methods and equipment used in the present invention are conventional methods and equipment in the technical field. <> <>

[0047] The culture medium formulas involved in the following embodiments are as follows:

[0048] LB medium: general commercial medium, LB broth Miller.

[0049] ASW-YT medium: Sodium chloride 3 g / L, sodium citrate 20 g / L, magnesium chloride hexahydrate 3 g / L, ammonium sulfate 1 g / L, potassium chloride 0.5 g / L, potassium dihydrogen phosphate 0.42 g / L, sodium bromide 0.05 g / L, strontium chloride hexahydrate 0.02 g / L, ferrous ammonium sulfate hexahydrate 0.01 g / L, cysteine 0.25 g / L, cysteine hydrochloride monohydrate 0.25 g / L, MOPS 2 g / L, yeast extract 5 g / L, peptone 5 g / L, resazurin 0.001 g / L, magnesium sulfate heptahydrate 6 g / L, sodium bicarbonate 0.2 g / L, calcium chloride dihydrate 0.3 g / L, niacin 2 mg / L, biotin 0.8 mg / L, pantothenate 2 mg / L, lipoic acid 2 mg / L, folic acid acid0.8mg / L, Aminobenzoic acid 2mg / L, Thiamine 2mg / L, Riboflavin 2mg / L, Pyriodoxine 2mg / L, Cobalamin 2mg / L, Manganese sulfate monohydrate 2.5mg / L, Cobalt chloride hexahydrate 0.5mg / L, Zinc sulfate heptahydrate 0.5mg / L, Copper sulfate pentahydrate 0.05mg / L, Potassium aluminum sulfate dodecahydrate 0.05mg / L, Boric acid 0.05mg / L, Sodium molybdate dihydrate 0.05mg / L, Nickel chloride hexahydrate 0.05mg / L.

[0050] The buffers involved in the following embodiments are:

[0051] Elution Buffer: 100 mM PBS, NaCl 17.532 g / L, imidazole 34.04 g / L, pH 7.5.

[0052] Wash Buffer: 100 mM PBS, NaCl 17.532 g / L, imidazole 3.404 g / L, pH 7.5.

[0053] Lysis Buffer: 100 mM PBS, NaCl 17.532 g / L, imidazole 0.68 g / L, pH 7.5.

[0054] Example 1, obtaining of high temperature enzyme:

[0055] The high-temperature enzymes of the present invention include α-glucan phosphorylase (α-GP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), 6-phosphogluconolactonase (6PGL), diaphorase (DI), hydrogenase (SHI), 6-phosphohexulose synthase-phosphohexulose isomerase fusion protein (HPS-PHI), and phosphoglucose isomerase (PGI). Among them, the 6-phosphohexulose synthase-phosphohexulose isomerase fusion protein (HPS-PHI) is obtained by fusing 6-phosphohexulose synthase (HPS) and phosphohexulose isomerase (PHI).

[0056] The nucleotide sequences of the above-mentioned α-GP, PGM, G6PDH, 6PGDH, 6PGL, DI, SHI, HPS-PHI, and PGI are shown in SEQ ID NO.1 to SEQ ID NO.9 respectively, and the corresponding amino acid sequences are shown in SEQ ID NO.10 to SEQ ID NO.18 respectively.

[0057] The high-temperature enzymes in this example are all existing enzyme preparations. For example, α-GP, PGM, G6PDH, 6PGDH, and 6PGL are reported in "Advanced water splitting for green hydrogen gas production through complete oxidation of starch by in vitro metabolic engineering", DI, SHI, and PGI are reported in the article "Ultra-Rapid Rates of Water Splitting for Biohydrogen Gas Production through in vitro Artificial Enzymatic Pathways", and HPS-PHI is reported in the article "The Ribulose Monophosphate Pathway Substitutes for the Missing Pentose Phosphate Pathway in the Archaeon Thermococcus kodakaraensis".

[0058] The above high-temperature enzymes can also be obtained by the following method:

[0059] (1) Referring to Table 1, insert the target enzyme gene into the corresponding vector to construct a recombinant plasmid; and transfer the recombinant plasmid into the host bacterium to construct a recombinant engineering bacterium (this step is entrusted to a biological company).

[0060] Table 1 Construction of Recombinant Engineered Bacteria

[0061]

[0062] (2) Expression and purification of PGM, G6PDH, 6PGDH, 6PGL, DI, PHI - PGI, and PGI proteins: Culture the corresponding recombinant engineered bacteria in LB medium respectively, then collect the bacterial cells, resuspend them with Lysis Buffer and break the cells (by ultrasonic disruption / high - pressure homogenization); centrifuge at 12,000 rpm for 1 h; pour the supernatant into a nickel column, and recycle the liquid and repeat the loading twice; add WashBuffer to wash away the impurity proteins, then add a small amount of Elution Buffer to wash and collect. Generally, two or three elutions can wash away most of the proteins; collect these protein solutions into an ultrafiltration tube for desalting; ultrafiltrate to reduce the imidazole and sodium chloride concentrations to below 1 mM; add glycerol with a final concentration of 10% to the obtained enzyme solution, detect the protein concentration, and then store it in a - 80 °C refrigerator for later use.

[0063] Expression and purification of SHI protein: Culture the corresponding recombinant engineered bacteria in ASW - YT medium, then collect the bacterial cells, and carry out purification in an anaerobic chamber. The purification process is the same as that of the above - mentioned other enzyme proteins; add glycerol with a final concentration of 10% to the obtained enzyme solution, detect the protein concentration, and then store it in a - 80 °C refrigerator for later use.

[0064] Example 2 Construction of Hydrogen - producing Reaction System:

[0065] The hydrogen - producing reaction system of this example includes the following components and contents:

[0066] HEPES buffer (pH 7.2, prepared with ultrapure water) 100 mM, maltodextrin (glucose equivalent 4 - 7) 20 mM, formaldehyde 10 mM (initial concentration), magnesium chloride 5 mM, manganese chloride 0.5 mM, NAD 2 mM, benzyl viologen (BV) 0.5 mM, sodium phosphate 5 mM, α - glucan phosphorylase (α - GP) 0.25 mg / mL, phosphoglucomutase (PGM) 0.05 mg / mL, glucose 6 - phosphate dehydrogenase (G6PDH) 2.65 mg / mL, 6 - phosphogluconate dehydrogenase (6PGDH) 0.53 mg / mL, 6 - phosphogluconolactonase (6PGL) 0.08 mg / mL, diaphorase (DI) 5.13 mg / mL, hydrogenase (SHI) 1.5 mg / mL, 6 - phosphohexulose synthase - phosphohexose isomerase fusion protein (HPS - PHI) 0.49 mg / mL, phosphoglucose isomerase (PGI) 1.12 mg / mL.

[0067] Among them, formaldehyde, as one of the cosubstrates, has an initial concentration of 10 mM as described above. However, after the start of the reaction, a 200 mM formaldehyde stock solution will be added dropwise at a rate of 5 μL / min.

[0068] Example 3: Construction of a hydrogen production reaction system:

[0069] The hydrogen production reaction system of this example includes the following components and contents:

[0070] HEPES buffer (pH 7.5, prepared with ultrapure water) 100 mM, maltodextrin (glucose equivalent 4 - 7) 20 mM, formaldehyde 10 mM (initial concentration), magnesium chloride 5 mM, manganese chloride 0.5 mM, NAD 8 mM, benzyl viologen (BV) 2 mM, sodium phosphate 5 mM, α-glucan phosphorylase (α-GP) 0.25 mg / mL, phosphoglucomutase (PGM) 0.05 mg / mL, glucose 6-phosphate dehydrogenase (G6PDH) 2.65 mg / mL, 6-phosphogluconate dehydrogenase (6PGDH) 0.53 mg / mL, 6-phosphogluconolactonase (6PGL) 0.08 mg / mL, diaphorase (DI) 5.13 mg / mL, hydrogenase (SHI) 1.5 mg / mL, 6-phosphohexulose synthase-phosphohexose isomerase fusion protein (HPS-PHI) 0.49 mg / mL, phosphoglucose isomerase (PGI) 1.12 mg / mL.

[0071] Among them, formaldehyde, as one of the cosubstrates, has an initial concentration of 10 mM as described above. However, after the start of the reaction, a 200 mM formaldehyde stock solution will be added dropwise at a rate of 5 μL / min.

[0072] Example 4: Construction of a hydrogen production reaction system:

[0073] The hydrogen production reaction system of this example includes the following components and contents:

[0074] HEPES buffer (pH 7.5, prepared with ultrapure water) 100 mM, maltodextrin (glucose equivalent 4 - 7) 100 mM, formaldehyde 20 mM (initial concentration), magnesium chloride 5 mM, manganese chloride 0.5 mM, NAD 10 mM, benzyl viologen (BV) 2 mM, sodium phosphate 5 mM, α - glucan phosphorylase (α - GP) 0.25 mg / mL, phosphoglucomutase (PGM) 0.05 mg / mL, glucose 6 - phosphate dehydrogenase (G6PDH) 2.65 mg / mL, 6 - phosphogluconate dehydrogenase (6PGDH) 0.53 mg / mL, 6 - phosphogluconolactonase (6PGL) 0.08 mg / mL, diaphorase (DI) 5.13 mg / mL, hydrogenase (SHI) 1.5 mg / mL, 6 - phosphohexulose synthase - phosphohexulose isomerase fusion protein (HPS - PHI) 0.49 mg / mL, phosphoglucose isomerase (PGI) 1.12 mg / mL.

[0075] Among them, formaldehyde, as one of the cosubstrates, has an initial concentration of 20 mM as above, but after the start of the reaction, a 200 mM formaldehyde mother liquor will be added dropwise at a rate of 5 μL / min.

[0076] Example 5: Construction of a hydrogen - producing reaction system:

[0077] The hydrogen - producing reaction system in this example includes the following components and contents:

[0078] HEPES buffer (pH 8.1, prepared with ultrapure water) 100 mM, maltodextrin (glucose equivalent 4 - 7) 400 mM, formaldehyde 15 mM (initial concentration), magnesium chloride 5 mM, manganese chloride 0.5 mM, NAD 10 mM, benzyl viologen (BV) 5 mM, sodium phosphate 5 mM, α - glucan phosphorylase (α - GP) 0.25 mg / mL, phosphoglucomutase (PGM) 0.05 mg / mL, glucose 6 - phosphate dehydrogenase (G6PDH) 2.65 mg / mL, 6 - phosphogluconate dehydrogenase (6PGDH) 0.53 mg / mL, 6 - phosphogluconolactonase (6PGL) 0.08 mg / mL, diaphorase (DI) 5.13 mg / mL, hydrogenase (SHI) 1.5 mg / mL, 6 - phosphohexulose synthase - phosphohexulose isomerase fusion protein (HPS - PHI) 0.49 mg / mL, phosphoglucose isomerase (PGI) 1.12 mg / mL.

[0079] Among them, formaldehyde, as one of the cosubstrates, has an initial concentration of 15 mM as above, but after the start of the reaction, a 200 mM formaldehyde mother liquor will be added dropwise at a rate of 5 μL / min.

[0080] Example 6: Construction of a hydrogen - producing reaction system:

[0081] The hydrogen production reaction system of this example includes the following components and contents:

[0082] HEPES buffer (pH 5.0, prepared with ultrapure water) 100 mM, maltodextrin (glucose equivalent 4 - 7) 1 mM, formaldehyde 10 mM (initial concentration), magnesium chloride 5 mM, manganese chloride 0.5 mM, NAD 1 mM, benzyl viologen (BV) 2 mM, sodium phosphate 5 mM, α - glucan phosphorylase (α - GP) 0.25 mg / mL, phosphoglucomutase (PGM) 0.05 mg / mL, glucose 6 - phosphate dehydrogenase (G6PDH) 2.65 mg / mL, 6 - phosphogluconate dehydrogenase (6PGDH) 0.53 mg / mL, 6 - phosphogluconolactonase (6PGL) 0.08 mg / mL, diaphorase (DI) 5.13 mg / mL, hydrogenase (SHI) 1.5 mg / mL, 6 - phosphohexulose synthase - phosphohexulose isomerase fusion protein (HPS - PHI) 0.49 mg / mL, phosphoglucose isomerase (PGI) 1.12 mg / mL.

[0083] Among them, formaldehyde, as one of the cosubstrates, has an initial concentration of 10 mM as above, but after the start of the reaction, a 200 mM formaldehyde stock solution will be added dropwise at a rate of 5 μL / min.

[0084] Example 7. Construction of the hydrogen production reaction system:

[0085] The hydrogen production reaction system of this example includes the following components and contents:

[0086] HEPES buffer (pH 10, prepared with ultrapure water) 100 mM, maltodextrin (glucose equivalent 4 - 7) 1000 mM, formaldehyde 10 mM (initial concentration), magnesium chloride 5 mM, manganese chloride 0.5 mM, NAD 10 mM, benzyl viologen (BV) 2 mM, sodium phosphate 5 mM, α - glucan phosphorylase (α - GP) 0.25 mg / mL, phosphoglucomutase (PGM) 0.05 mg / mL, glucose 6 - phosphate dehydrogenase (G6PDH) 2.65 mg / mL, 6 - phosphogluconate dehydrogenase (6PGDH) 0.53 mg / mL, 6 - phosphogluconolactonase (6PGL) 0.08 mg / mL, diaphorase (DI) 5.13 mg / mL, hydrogenase (SHI) 1.5 mg / mL, 6 - phosphohexulose synthase - phosphohexulose isomerase fusion protein (HPS - PHI) 0.49 mg / mL, phosphoglucose isomerase (PGI) 1.12 mg / mL.

[0087] Among them, formaldehyde is one of the co-substrates, and its initial concentration is 10 mM as described above. However, after the reaction starts, a 200 mM formaldehyde mother liquor will be added dropwise at a rate of 5 μL / min.

[0088] Example 8: A hydrogen production method using formaldehyde and water as co-substrates:

[0089] (1) Construct the reaction system shown in the above examples in a reactor.

[0090] (2) React the reaction system constructed in step (1) at a temperature of 10 °C, and add a 200 mM formaldehyde mother liquor at a rate of 5 μL / min. Use nitrogen as the carrier gas with a flow rate of 50 ml / min to carry the reaction gas out of the reaction vessel and introduce it into a hydrogen analyzer for detection.

[0091] Example 9: A hydrogen production method using formaldehyde and water as co-substrates:

[0092] (1) Construct the reaction system shown in the above examples in a reactor.

[0093] (2) React the reaction system constructed in step (1) at a temperature of 50 °C, and add a 200 mM formaldehyde mother liquor at a rate of 5 μL / min. Use nitrogen as the carrier gas with a flow rate of 50 ml / min to carry the reaction gas out of the reaction vessel and introduce it into a hydrogen analyzer for detection.

[0094] Example 10: A hydrogen production method using formaldehyde and water as co-substrates:

[0095] (1) Construct the reaction system shown in the above examples in a reactor.

[0096] (2) React the reaction system constructed in step (1) at a temperature of 70 °C, and add a 200 mM formaldehyde mother liquor at a rate of 5 μL / min. Use nitrogen as the carrier gas with a flow rate of 50 ml / min to carry the reaction gas out of the reaction vessel and introduce it into a hydrogen analyzer for detection.

[0097] Example 11: A hydrogen production method using formaldehyde and water as co-substrates:

[0098] (1) Construct the reaction system shown in the above examples in a reactor.

[0099] (2) React the reaction system constructed in step (1) at a temperature of 80 °C, and add a 200 mM formaldehyde mother liquor at a rate of 5 μL / min. Use nitrogen as the carrier gas with a flow rate of 50 ml / min to carry the reaction gas out of the reaction vessel and introduce it into a hydrogen analyzer for detection.

[0100] Example 12: A hydrogen production method using formaldehyde and water as co-substrates:

[0101] (1) Construct the reaction system shown in the above embodiments in the reactor.

[0102] (2) React the reaction system constructed in step (1) at a temperature of 90 °C, and flow in a 200 mM formaldehyde mother liquor at a rate of 5 μL / min. Use nitrogen gas with a flow rate of 50 ml / min as the carrier gas to carry the reaction gas out of the reaction vessel and introduce it into a hydrogen analyzer for detection.

[0103] Experimental Example 1

[0104] This experimental example is used to verify the feasibility of hydrogen production using formaldehyde and water as co-substrates in the present invention.

[0105] Take the reaction system of Example 4 as an example for hydrogen production reaction. However, at the beginning of the reaction, do not add the HPS-PHI fusion protein and formaldehyde. After reacting for 1 hour (reacting at a temperature of 80 °C), then add HPS-PHI and formaldehyde.

[0106] Figure 11 The results of hydrogen production after adding formaldehyde and HPS-PHI enzyme in the later stage of the hydrogen production path are shown. It can be seen that at the beginning, the reaction system consumes maltodextrin to produce hydrogen, but because the cycle is not established, the hydrogen production rate rapidly decreases; subsequently, HPS-PHI and formaldehyde are added, and the hydrogen production rate gradually increases and returns to the highest value, and continuous hydrogen production lasts for nearly 2 hours. This indicates that the formaldehyde cycle system is successfully constructed, and hydrogen is synthesized using formaldehyde and water as co-substrates.

[0107] Experimental Example 2

[0108] This experimental example is used to verify the advantages of the present invention compared with the normal temperature enzyme system.

[0109] Take the reaction system of Example 4 of the present invention as an example to construct a corresponding high-temperature enzyme reaction system; and react the reaction system at temperatures of 60 °C and 80 °C respectively. Use nitrogen gas with a flow rate of 50 ml / min as the carrier gas to carry the reaction gas out of the reaction vessel and introduce it into a hydrogen analyzer for detection.

[0110] At the same time, replace each enzyme in the reaction system of Example 4 with the corresponding normal temperature enzyme in the art (shown in Table 2) to construct a corresponding normal temperature enzyme reaction system; then react the reaction system at a temperature of 30 °C, and use nitrogen gas with a flow rate of 50 ml / min as the carrier gas to carry the reaction gas out of the reaction vessel and introduce it into a hydrogen analyzer for detection.

[0111] Table 2 Information of normal temperature enzymes

[0112]

[0113]

[0114] Figure 12Hydrogen production results of the high-temperature enzyme system of the present invention at 60 and 80 °C Figure 13 Hydrogen production results of the room-temperature enzyme system at 3 °C. It can be seen that the room-temperature enzyme system can also produce hydrogen, but the production rate is not as good as that of the high-temperature enzyme system and high-temperature conditions of the present invention.

[0115] Experimental Example 3

[0116] This experimental example is used to verify the selection of the optimal parameters of the reaction system and reaction conditions of the present invention.

[0117] I. NAD concentration, BV concentration, and buffer pH:

[0118] Referring to the system of Example 4, different NAD concentrations (0, 2, 4, 8, 10 mM), BV concentrations (0, 0.5, 1, 2, 5 mM), and buffer pH values (7.2, 7.5, 7.8, 8.1) were set respectively to carry out hydrogen production reactions. Among them, when different NAD concentrations, BV concentrations, and buffer pH values were set respectively, the dosages of other components in the reaction system were kept the same as those in Example 4.

[0119] The results are as Figure 14 shown. It can be seen that the optimal selection is: 8 mM NAD, 2 mM BV, pH 7.5.

[0120] II. Reaction temperature:

[0121] Referring to the system of Example 4, different temperatures were set to carry out hydrogen production reactions:

[0122] (1) Construct a reaction system in the reactor.

[0123] (2) React the reaction system constructed in step (1) at different temperatures (50 °C, 60 °C, 70 °C, 80 °C) respectively. Nitrogen with a flow rate of 50 ml / min was used as the carrier gas to carry the reaction gas out of the reaction vessel and introduced into a hydrogen analyzer for detection.

[0124] The results are as Figure 15 shown. It can be seen that the optimal reaction temperature is: 70 °C.

[0125] III. Substrate formaldehyde concentration:

[0126] Based on the optimal parameters of NAD, BV, pH, and reaction temperature obtained above, the optimal feeding concentration of the substrate formaldehyde was further optimized. That is, while setting the NAD concentration in the reaction system to 8 mM, the BV concentration to 2 mM, the buffer pH to 7.5, and the reaction temperature to 70 °C (the contents of other components in the reaction system are the same as those in Example 4), different formaldehyde concentrations (0 mM, 10 mM, and 20 mM) were set to carry out hydrogen production reactions.

[0127] The results are as Figure 16 shown. It can be seen that when the formaldehyde concentration is 0 mM, the highest hydrogen production rate cannot be sustained. When the formaldehyde concentration is increased to 20 mM, the highest hydrogen production rate can be maintained for a longer time. Therefore, increasing the formaldehyde concentration to a certain extent can increase the concentration of reaction substrate regeneration and the total hydrogen production. At the same time, the hydrogen production rates of 10 mM formaldehyde and 20 mM formaldehyde are close. Considering the saving of feedstock, the optimal feed concentration of formaldehyde in the high-temperature reaction system is 10 mM.

[0128] Experimental Example 4

[0129] This experimental example is used to verify the applicability of the present invention to different water quality conditions.

[0130] Based on Experimental Example 3, the following optimal reaction system is obtained:

[0131] HEPES buffer (pH 7.5) 100 mM, maltodextrin (glucose equivalent 4 - 7) 20 mM, formaldehyde 10 mM, magnesium chloride 5 mM, manganese chloride 0.5 mM, NAD 8 mM, benzyl viologen (BV) 2 mM, sodium phosphate 5 mM, α-glucan phosphorylase (α-GP) 0.25 mg / mL, phosphoglucomutase (PGM) 0.05 mg / mL, glucose 6-phosphate dehydrogenase (G6PDH) 2.65 mg / mL, 6-phosphogluconate dehydrogenase (6PGDH) 0.53 mg / mL, 6-phosphogluconolactonase (6PGL) 0.08 mg / mL, diaphorase (DI) 5.13 mg / mL, hydrogenase (SHI) 1.5 mg / mL, 6-phosphohexulose synthase-phosphohexose isomerase fusion protein (HPS-PHI) 0.49 mg / mL, phosphoglucose isomerase (PGI) 1.12 mg / mL.

[0132] Prepare HEPES buffer using ultrapure water, seawater, and sewage respectively, and construct the corresponding reaction systems according to the above "optimal reaction system"; then react the constructed reaction systems at 70 °C, use nitrogen as the carrier gas at a flow rate of 50 ml / min, and carry the reaction gas out of the reaction vessel and introduce it into a hydrogen analyzer for detection.

[0133] The results are as Figure 17 shown. It can be seen that the system of the present invention has relatively loose requirements for water quality and is applicable to various types of water resources.

[0134] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hydrogen production reaction system using formaldehyde and water as co-substrates, characterized in that, It includes buffer, maltodextrin, formaldehyde, magnesium chloride, manganese chloride, NAD, benzyl viologen, sodium phosphate, α-glucan phosphorylase, phosphoglucomutase, glucose 6-phosphate dehydrogenase, 6-phosphogluconic acid dehydrogenase, 6-phosphogluconolactonase, diaphorase, hydrogenase, 6-phosphohexulose synthase-phosphohexulose isomerase fusion protein and phosphoglucose isomerase.

2. The hydrogen production reaction system using formaldehyde and water as co-substrates according to claim 1, characterized in that, The nucleotide sequences of the α-glucan phosphorylase, phosphoglucomutase, glucose 6-phosphate dehydrogenase, 6-phosphogluconic acid dehydrogenase, 6-phosphogluconolactonase, diaphorase, hydrogenase, 6-phosphohexulose synthase-phosphohexulose isomerase fusion protein and phosphoglucose isomerase are respectively shown as SEQ ID NO.1 to SEQ ID NO.9, and the corresponding amino acid sequences are shown as SEQ ID NO.10 to SEQ ID NO.

18.

3. The hydrogen production reaction system using formaldehyde and water as co-substrates according to claim 1, characterized in that, In the reaction system, the pH of the buffer is 5 to 10.

4. The hydrogen production reaction system using formaldehyde and water as co-substrates according to claim 1, wherein, In the reaction system, maltodextrin is used as a cofactor donor, with a glucose equivalent of 4 to 7 and a concentration of 1 to 1000 mM.

5. The hydrogen production reaction system using formaldehyde and water as co-substrates according to claim 1, characterized in that, In the reaction system, formaldehyde is used as one of the cosubstrates, with an initial concentration of 10 to 20 mM, and then a 200 mM formaldehyde mother liquor is added dropwise at a rate of 0 to 5 μL / min.

6. The hydrogen production reaction system using formaldehyde and water as co-substrates according to claim 1, characterized in that, In the reaction system, the concentration of NAD is 1 to 10 mM.

7. The hydrogen production reaction system using formaldehyde and water as co-substrates according to claim 1, characterized in that, In the reaction system, the concentration of benzyl viologen is 0.5 to 5 mM.

8. The hydrogen production reaction system using formaldehyde and water as co-substrates according to any one of claims 1 to 7, characterized in that, The reaction system includes the following specific components: 100 mM buffer at pH 7.5, 20 mM maltodextrin with a glucose equivalent of 4 to 7, 5 mM magnesium chloride, 0.5 mM manganese chloride, 8 mM NAD, 2 mM benzyl viologen, 5 mM sodium phosphate, 0.25 mg / mL α-glucan phosphorylase, 0.05 mg / mL phosphoglucomutase, 2.65 mg / mL glucose 6-phosphate dehydrogenase, 0.53 mg / mL 6-phosphogluconic acid dehydrogenase, 0.08 mg / mL 6-phosphogluconolactonase, 5.13 mg / mL diaphorase, 1.5 mg / mL hydrogenase, 0.49 mg / mL 6-phosphohexulose synthase-phosphohexulose isomerase fusion protein, 1.12 mg / mL phosphoglucose isomerase, formaldehyde; the initial concentration of the formaldehyde is 10 to 20 mM, and then a 200 mM formaldehyde mother liquor is added dropwise at a rate of 0 to 5 μL / min.

9. A hydrogen production method using formaldehyde and water as co-substrates, characterized in that, It includes the following steps: (1) Construct a reaction system in a reactor, and the reaction system is as shown in the reaction system described in any one of claims 1 to 8; (2) React the reaction system constructed in step (1) at a temperature of 10 to 90 °C, and add a 200 mM formaldehyde mother liquor at a rate of 0 to 5 μL / min, and use formaldehyde and water as cosubstrates to produce hydrogen.

10. The hydrogen production method using formaldehyde and water as co-substrates according to claim 9, characterized in that, The specific reaction temperature is 70 °C.

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