Clostridium pasteurianum biological / non-biological hydrogen production system based on hydrogen production mechanism

Abstract

The invention belongs to the technical field of microbial hydrogen production optimization, and particularly relates to a clostridium pasteurianum biological / non-biological hydrogen production system based on a hydrogen production mechanism. According to the system, an endogenous hydrogenase gene of clostridium pasteurianum is over-expressed in the clostridium pasteurianum, or a hydrogen production element is added in the culture process of the clostridium pasteurianum, and the hydrogen production element comprises one or more of Fe < 2 + >, Ni < 2 + >, riboflavin, humic acid or magnetic nanoparticles. According to the invention, the effects of different types of hydrogenase on hydrogen production of clostridium pasteurianum are explored, and the hydrogen production mechanism of clostridium pasteurianum is defined; by adding a new hydrogen production element externally, the hydrogen yield of the clostridium pasteurianum is further improved, the development of a biological hydrogen production technology can be promoted, and a feasible biological solution is provided for production of clean energy.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial hydrogen production optimization, and in particular relates to a Clostridium pasteurianum biological / non-biological hydrogen production system based on a hydrogen production mechanism. Background Art

[0002] In the process of global response to climate change and energy transformation, hydrogen energy plays an important role as one of the various green energy sources including solar energy, wind energy, and hydropower due to its advantages of high energy density, zero pollution, and simple storage and transportation. In recent years, my country has continuously increased its attention to the hydrogen energy industry. In 2022, the "Medium- and Long-Term Plan for the Development of the Hydrogen Energy Industry" clearly pointed out that hydrogen energy is an important component of the future national energy system and an important carrier for the green and low-carbon transformation of energy-consuming terminals.

[0003] There are currently a variety of hydrogen production technologies. As a traditional method, hydrogen production from fossil fuels uses natural gas as raw material, including steps such as desulfurization, steam reforming and conversion. Although this technology is mature and low-cost, it is limited by natural gas supply, relies on fossil fuels, and emits greenhouse gases; industrial by-product hydrogen production uses by-products generated in industrial production processes (such as coke oven gas) to extract pure hydrogen through a pressure swing adsorption process. Although it reduces resource waste, it is limited by the availability and processing technology of industrial by-products. Hydrogen production from water electrolysis produces hydrogen by electrolyzing water, but electricity prices are an important factor limiting its widespread application.

[0004] At present, some new hydrogen production methods such as biomass hydrogen production, biological hydrogen production and thermochemical hydrogen production are still in the experimental and development stage and have not yet reached the requirements of industrial hydrogen production scale. Compared with other hydrogen production methods, biological hydrogen production uses renewable energy as raw materials and produces hydrogen at ambient temperature and atmospheric pressure. It has the advantages of sustainability, environmental protection, low energy consumption and extensive resources. The research boom in the field of biological hydrogen production broke out during the energy crisis in the 1970s, and gradually formed hydrogen production technology routes including photohydrolysis, photofermentation, dark fermentation and light-dark combined fermentation. The microbial groups involved in these hydrogen production processes include green algae, cyanobacteria, photofermentation bacteria and dark fermentation bacteria. Among them, fermentation-type hydrogen-producing bacteria have higher hydrogen production efficiency and capacity. Considering the hydrogen production and substrate conversion rate, Clostridium pasteurianum DSM 525 is a more suitable hydrogen-producing microorganism. As a Gram-positive, strictly anaerobic bacterium that can form endospores, it exhibits high substrate concentration tolerance, inhibitor resistance and sustainability under adverse conditions in the hydrogen production process.

[0005] In the hydrogen production pathway of Clostridium pasteurianum, hydrogenase is a key enzyme responsible for catalyzing the hydrogen production reaction. The expression and regulation of genes are crucial to the hydrogen production ability of Clostridium pasteurianum. Studying these genes will help improve the efficiency of biohydrogen production. By further exploring the regulatory mechanism and metabolic network of hydrogenase genes, the molecular basis of the metabolic mechanism of Clostridium pasteurianum was revealed, and new elements that can increase the hydrogen production of Clostridium were explored. Clostridium was promoted to produce hydrogen by increasing electron transfer in the hydrogen production pathway or enhancing the catalytic activity of hydrogenase or expressing exogenous hydrogenase. By integrating the hydrogen production element culture system to promote hydrogen production in genetically modified Clostridium pasteurianum, it is expected to achieve efficient and stable hydrogen production in Clostridium pasteurianum, making an important contribution to the development and utilization of clean energy. Summary of the invention

[0006] In view of the contents in the prior art, in order to promote the ability of Clostridium pasteurianum to produce hydrogen, the present invention provides a Clostridium pasteurianum biological / non-biological hydrogen production system based on the hydrogen production mechanism.

[0007] The technical solution of the present invention is: A biological / non-biological hydrogen production system of Clostridium pasteurianum based on hydrogen production mechanism, in which the endogenous hydrogenase gene of Clostridium pasteurianum is overexpressed in Clostridium pasteurianum, or a hydrogen production element is added during the cultivation of Clostridium pasteurianum, wherein the hydrogen production element includes Fe 2+ 、Ni 2+ , riboflavin, humic acid or magnetic nanoparticles.

[0008] Furthermore, in the above-mentioned biological / non-biological hydrogen production system of Clostridium pasteurianum based on hydrogen production mechanism, the method for overexpressing the endogenous hydrogenase gene of Clostridium pasteurianum is to construct a dual-promoter inducible plasmid suitable for Clostridium pasteurianum, integrate the amplified four Clostridium pasteurian hydrogenase genes C00280, C37830, C07060-70, and RS16520 into the plasmid, and transfer them into wild-type Clostridium pasteurianum respectively, to obtain Clostridium pasteurianum / overexpression C00280 strain, Clostridium pasteurianum / overexpression C37830 strain, Clostridium pasteurianum / overexpression C07060-70 strain, and Clostridium pasteurianum / overexpression RS16520 strain.

[0009] Furthermore, in the above-mentioned biological / non-biological hydrogen production system of Clostridium pasteurianum based on hydrogen production mechanism, the magnetic nanoparticles are magnetic nanoparticles Fe3O4, with a particle size of 50-100nm and a concentration of 0-200ppm; riboflavin, humic acid, Fe 2+ 、Ni 2+ The concentration is 0-200ppm.

[0010] Furthermore, the above-mentioned biological / non-biological hydrogen production system of Clostridium pasteurianum based on hydrogen production mechanism, and the hydrogen production method of the hydrogen production system based on overexpression of the endogenous hydrogenase gene of Clostridium pasteurianum in Clostridium pasteurianum comprises the following steps: ① Culture: Take out the endogenous hydrogenase gene overexpressing Clostridium pasteurii strain from a -80℃ refrigerator, transfer the strain to 5 mL 2×YTG or 2×YTG medium with chloramphenicol resistance, and culture it overnight at 37℃ for 14 h; ② Transfer: Transfer the obtained strain to an anaerobic bottle containing 10 mL of 2×YTG or 2×YTG fresh medium with chloramphenicol resistance at a ratio of 1:20, and culture for 12 h until OD600 = 0.8; ③ Induction: Add 1 mL of 1 mg / mL anhydrotetracycline to a final concentration of 100 ng / mL and induce culture for 12 h; ④ Measure hydrogen: Use gas chromatograph to measure hydrogen production.

[0011] Furthermore, the above-mentioned biological / non-biological hydrogen production system of Clostridium pasteurianum based on hydrogen production mechanism, and the method for producing hydrogen based on the hydrogen production system by adding hydrogen production elements during the cultivation process of Clostridium pasteurianum, comprises the following steps: ① Culture: Take out Clostridium pasteurianum from -80 ℃ refrigerator and transfer it to 5 mL 2×YTG medium, and culture it at 37 ℃ overnight for 14 h; ② Prepare the culture medium: weigh 10-50 mg of hydrogen production element, sterilize it under ultraviolet conditions for 30 min, add it to 2×YTG culture medium, and perform gradient dilution to make the final concentration of hydrogen production element culture medium between 10-200 ppm; ③ Transfer: Transfer Clostridium pasteurianum to the diluted hydrogen production element culture medium at a ratio of 1:20 and culture for 12 hours; ④ Hydrogen measurement: Use gas chromatograph to measure hydrogen production.

[0012] Furthermore, in the above-mentioned biological / non-biological hydrogen production system of Clostridium pasteurianum based on hydrogen production mechanism, the components of 2×YTG culture medium are: 16 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl, 5 g / L glucose and 0.2 g / L cysteine. Advantages and beneficial effects of the present invention: The Clostridium pasteurianum biological / non-biological hydrogen production system of the present invention overexpresses the endogenous hydrogenase gene of Clostridium pasteurianum to explore the effects of different types of hydrogenases on hydrogen production by Clostridium pasteurianum and clarify the hydrogen production mechanism of Clostridium pasteurianum; by adding new hydrogen production elements exogenously, the hydrogen production of Clostridium pasteurianum is further improved, which can promote the development of biological hydrogen production technology and provide a feasible biological solution for the production of clean energy; after optimizing the hydrogen production conditions, the hydrogen production is greatly improved, which helps to reduce the cost of biological hydrogen production and improve its economic feasibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram of the effect of hydrogenase overexpression on hydrogen production by Clostridium pasteurianum in Example 1; Figure 2 This is a schematic diagram of the effect of adding magnetic nanoparticles on hydrogen production by Clostridium pasteurianum in Example 2; Figure 3 This is a schematic diagram of the effect of adding riboflavin on hydrogen production by Clostridium pasteurianum in Example 3; Figure 4 This is a schematic diagram of the effect of adding humic acid on hydrogen production by Clostridium pasteurianum in Example 4; Figure 5 This is a schematic diagram of the effect of adding metal ions on hydrogen production by Clostridium pasteurianum in Example 5; Figure 6 This is a schematic diagram of the effect of multiple hydrogen production elements working together on hydrogen production by Clostridium pasteurianum in Example 6. DETAILED DESCRIPTION

[0014] The specific implementation modes of the present invention are described in further detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention but cannot be used to limit the scope of the present invention.

[0015] In the following embodiments, The wild-type Clostridium pasteurianum (WT) was selected. The composition of 2×YTG medium was: 16 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl, 5 g / L glucose, and 0.2 g / L cysteine; The method for overexpressing the endogenous hydrogenase gene of Clostridium pasteurianum is to construct a dual-promoter inducible plasmid suitable for Clostridium pasteurianum, integrate the amplified four Clostridium pasteurian hydrogenase genes C00280, C37830, C07060-70, and RS16520 into the plasmid, and transfer them into the wild-type Clostridium pasteurianum respectively, to obtain Clostridium pasteurianum / overexpression C00280 strain, Clostridium pasteurianum / overexpression C37830 strain, Clostridium pasteurianum / overexpression C07060-70 strain, and Clostridium pasteurianum / overexpression RS16520 strain. Example 1

[0016] This embodiment provides a biological / non-biological hydrogen production system of Clostridium pasteurianum based on hydrogen production mechanism, in which the endogenous hydrogenase gene of Clostridium pasteurianum is overexpressed in Clostridium pasteurianum. The specific hydrogen production method includes the following steps: ① Culture: Take out wild-type Clostridium pasteurianum (WT), Clostridium pasteurianum / overexpression C00280 strain, Clostridium pasteurianum / overexpression C37830 strain, Clostridium pasteurianum / overexpression C07060-70 strain, and Clostridium pasteurianum / overexpression RS16520 strain from a -80℃ refrigerator, transfer the strains to 5 mL 2×YTG medium, and culture them overnight at 37℃ for 14 h; ② Transfer: Transfer the obtained strain to an anaerobic bottle containing 10 mL of 2×YTG fresh medium at a ratio of 1:20 and culture for 12 h until OD600 = 0.8; ③ Induction: Add 1 mL of 1 mg / mL anhydrotetracycline to a final concentration of 100 ng / mL and induce culture for 12 h; ④ Measure hydrogen: Use gas chromatograph to measure hydrogen production.

[0017] Hydrogen relative production (HPC) Figure 1 The experimental results showed that overexpression of the four endogenous hydrogenase genes increased the hydrogen production capacity of Clostridium pasteurianum by 3 times, 2.6 times, 2.5 times, and 3.5 times, respectively, indicating that the four hydrogenases play a key role in the hydrogen production process of Clostridium pasteurianum. Example 2

[0018] This embodiment is a biological / non-biological hydrogen production system of Clostridium pasteurianum based on hydrogen production mechanism. Magnetic nanoparticles of hydrogen production element are added during the cultivation process of Clostridium pasteurianum. The magnetic nanoparticles are purchased from McLean, nano-iron tetroxide, purity 99.5%, particle size 50 / 100Φ, and the specific hydrogen production method includes the following steps: ① Culture: Take out Clostridium pasteurianum from -80 ℃ refrigerator and transfer it to 5 mL 2×YTG medium, and culture it at 37 ℃ overnight for 14 h; ② Prepare the culture medium: weigh 10 mg of magnetic nanoparticles, sterilize under UV conditions for 30 min, add 50 mL of 2×YTG at a concentration of 200 ppm, and dilute the concentration of the magnetic nanoparticle culture medium to 10 ppm, 25 ppm, 50 ppm, and 100 ppm respectively; ③ Transfer: Wild-type Clostridium pasteurianum (WT) was transferred to medium containing different concentrations of magnetic nanoparticles at a ratio of 1:20, 0, 10, 25, 50, 100, and 200 ppm, and cultured for 12 h; ④ Hydrogen measurement: Use gas chromatograph to measure hydrogen production.

[0019] In this example, magnetic nanoparticles Fe3O4 of different particle sizes were added to the culture medium, and the hydrogen production capacity of Clostridium pasteurianum was tested after co-culturing with Clostridium pasteurianum. Figure 2 The experimental results show that Fe3O4 nanoparticles with a particle size of 100 nm can improve the hydrogen production capacity of the strain, and the hydrogen production capacity of Clostridium pasteurianum shows a trend of first increasing and then decreasing with the increase of the concentration of magnetic nanoparticles. The nanoparticle concentration when the highest hydrogen production is obtained is 25 ppm, and the amount of hydrogen produced is 1.25 times that of the wild-type Clostridium pasteurianum; while Fe3O4 nanoparticles with a particle size of 50 nm have no obvious effect on improving the hydrogen production capacity of Clostridium pasteurianum. Example 3

[0020] This embodiment is a biological / non-biological hydrogen production system of Clostridium pasteurianum based on hydrogen production mechanism. During the cultivation of Clostridium pasteurianum, riboflavin, a hydrogen production element, is added. The selected riboflavin is purchased from BD Company of the United States. The specific hydrogen production method includes the following steps: ① Culture: Take out Clostridium pasteurianum from -80 ℃ refrigerator and transfer it to 5 mL 2×YTG medium, and culture it at 37 ℃ overnight for 14 h; ② Prepare the culture medium: Weigh 10 mg of riboflavin, sterilize it under ultraviolet light for 30 min, add it to 2×YTG culture medium at a concentration of 200 ppm, and dilute the riboflavin culture medium to 10 ppm, 25 ppm, 50 ppm, and 100 ppm respectively; ③ Transfer: Transfer Clostridium pasteurianum to riboflavin culture medium with different concentrations of 0, 10, 25, 50, 100, and 200 ppm at a ratio of 1:20 and culture for 12 h; ④ Hydrogen measurement: Use gas chromatograph to measure hydrogen production.

[0021] In this example, different concentrations of riboflavin were added to the culture medium, and the hydrogen production capacity of Clostridium pasteurianum was tested after co-culturing with Clostridium pasteurianum. Figure 3 As shown. The experimental results show that riboflavin can improve the hydrogen production capacity of Clostridium, and the hydrogen production capacity shows a trend of first increasing and then decreasing with the increase of riboflavin concentration. The riboflavin concentration of 50 ppm when the highest hydrogen production is obtained is 1.45 times that of wild-type Clostridium pasteurianum. The reason why the high concentration of riboflavin weakens the effect of promoting hydrogen production may be that it has a toxic effect on Clostridium and inhibits the growth of bacteria. Example 4

[0022] This embodiment is a biological / non-biological hydrogen production system based on the hydrogen production mechanism of Clostridium pasteurianum. Humic acid, a hydrogen production element, is added during the cultivation of Clostridium pasteurianum. The humic acid selected is purchased from BD Company of the United States. The specific hydrogen production method includes the following steps: ① Culture: Take out Clostridium pasteurianum from -80 ℃ refrigerator and transfer it to 5 mL 2×YTG medium, and culture it at 37 ℃ overnight for 14 h; ② Prepare the culture medium: Weigh 10 mg of humic acid, sterilize it under ultraviolet light for 30 min, add it to 2×YTG culture medium at a concentration of 200 ppm, and dilute the humic acid culture medium to 10 ppm, 25 ppm, 50 ppm, and 100 ppm respectively; ③ Transfer: Transfer Clostridium pasteurianum to humic acid culture medium with different concentrations of 0, 10, 25, 50, 100, and 200 ppm at a ratio of 1:20 and culture for 12 h; ④ Hydrogen measurement: Use gas chromatograph to measure hydrogen production.

[0023] In this example, humic acid of different concentrations was added to the culture medium, and after co-culturing with Clostridium pasteurianum, the hydrogen production capacity of Clostridium pasteurianum was tested. Figure 4 The experimental results show that humic acid can improve the hydrogen production capacity of Clostridium, and the hydrogen production capacity shows a trend of first increasing and then decreasing with the increase of humic acid concentration. The humic acid concentration at which the highest hydrogen production is obtained is 25 ppm, and the amount of hydrogen produced is 1.14 times that of wild-type Clostridium pasteurianum. The reason why the high concentration of humic acid weakens the effect of promoting hydrogen production may be that it has a toxic effect on Clostridium and inhibits the growth of bacteria. Example 5

[0024] This embodiment is a biological / non-biological hydrogen production system based on the hydrogen production mechanism of Clostridium pasteurianum. The hydrogen production element Ni is added during the cultivation process of Clostridium pasteurianum. 2+ and Fe 2+ , the specific hydrogen production method comprises the following steps: ① Culture: Take out Clostridium pasteurianum from -80 ℃ refrigerator and transfer it to 5 mL 2×YTG medium, and culture it at 37 ℃ overnight for 14 h; ② Prepare the culture medium: weigh 10 mg NiCl2 and FeCl2 respectively, sterilize under UV conditions for 30 min, add 50 mL 2×YTG (concentration is 200 ppm), dilute Ni 2+ and Fe 2+ The culture medium concentrations were 10 ppm, 25 ppm, 50 ppm, and 100 ppm; ③ Transfer: Wild-type Clostridium pasteurianum (WT) was transferred to culture medium containing 0, 10, 25, 50, 100, and 200 ppm Ni at a ratio of 1:20. 2+ and Fe 2+ culture medium, culture for 12 h; ④ Hydrogen measurement: Use gas chromatograph to measure hydrogen production.

[0025] In this example, different concentrations of Ni were added to the culture medium. 2+ and Fe 2+ After co-culture with Clostridium pasteurianum, the hydrogen production capacity of Clostridium pasteurianum was tested. Figure 5 The experimental results show that Ni 2+ The hydrogen production capacity showed a trend of first increasing and then decreasing with the increase of concentration. The ion concentration when the highest hydrogen production was obtained was 50 ppm, and the amount of hydrogen produced was 1.48 times that of the wild-type Clostridium pasteurianum. 2+ The hydrogen production capacity showed a trend of first increasing and then decreasing with the increase of concentration. The ion concentration when the highest hydrogen production was obtained was 50 ppm, and the amount of hydrogen produced was 1.50 times that of the wild-type Clostridium pasteurianum. Example 6

[0026] This embodiment provides a biological / non-biological hydrogen production system of Clostridium pasteurianum based on hydrogen production mechanism. The endogenous hydrogenase genes C00280, C37830, C07060-70, and RS16520 of Clostridium pasteurianum are overexpressed in Clostridium pasteurianum, and multiple hydrogen production elements are added during the culture process: magnetic nanoparticles, riboflavin, humic acid, and metal ions Ni 2+ , Fe 2+ , the specific hydrogen production method comprises the following steps: ① Culture: Take out Clostridium pasteurianum / overexpression C00280 strain, Clostridium pasteurianum / overexpression C37830 strain, Clostridium pasteurianum / overexpression C07060-70 strain, and Clostridium pasteurianum / overexpression RS16520 strain from a -80 ℃ refrigerator, transfer them to 5 mL 2×YTG medium, and culture them overnight at 37 ℃ for 14 h; ② Prepare the culture medium: weigh 25 mg magnetic nanoparticles, 50 mg riboflavin, 25 mg humic acid, 25 mg NiCl2, and 50 mg FeCl2, sterilize under ultraviolet irradiation for 30 min, and add to 1 L 2×YTG culture medium; ③ Transfer: transfer the wild type Clostridium pasteurianum (WT) into the culture medium prepared in ② at a ratio of 1:20 and culture for 12 hours until OD600 = 0.8; ④ Induction: Add 1 mL of 1 mg / mL anhydrotetracycline to a final concentration of 100 ng / mL and induce culture for 12 h; ⑤ Hydrogen measurement: Use gas chromatograph to measure hydrogen production.

[0027] In this embodiment, the optimal culture system in which multiple elements in the above embodiments work together is added to the culture medium of Clostridium pasteurianum that overexpresses hydrogenase, and the hydrogen production of the genetically modified Clostridium pasteurianum is tested. The results are as follows: Figure 6The experimental results showed that under the optimal conditions, the hydrogen production capacity of Clostridium pasteurianum overexpressing hydrogenase was 3.78 times, 3.13 times, 3.06 times, and 4.18 times higher than that of WT, and the hydrogen production could reach 75763 ppm, 62826 ppm, 61350 ppm, and 83796 ppm. Among them, the two strains with the strongest hydrogen production capacity were C00280 and RS16520.

Claims

1. A biological / non-biological hydrogen production system based on the hydrogen production mechanism of Clostridium pasteurianum, characterized in that: Overexpressing the endogenous hydrogenase gene of Clostridium pasteurianum in Clostridium pasteurianum, or adding hydrogen production elements during the cultivation of Clostridium pasteurianum, wherein the hydrogen production elements include Fe 2+ 、Ni 2+ , riboflavin, humic acid or magnetic nanoparticles.

2. The biological / non-biological hydrogen production system based on the hydrogen production mechanism of Clostridium pasteurianum according to claim 1, characterized in that: The method for overexpressing the endogenous hydrogenase gene of Clostridium pasteurianum is to construct a dual-promoter inducible plasmid suitable for Clostridium pasteurianum, integrate the amplified four Clostridium pasteurian hydrogenase genes C00280, C37830, C07060-70, and RS16520 into the plasmid, and transfer them into the wild-type Clostridium pasteurianum respectively, to obtain Clostridium pasteurianum / overexpression C00280 strain, Clostridium pasteurianum / overexpression C37830 strain, Clostridium pasteurianum / overexpression C07060-70 strain, and Clostridium pasteurianum / overexpression RS16520 strain.

3. The biological / non-biological hydrogen production system based on the hydrogen production mechanism of Clostridium pasteurianum according to claim 1, characterized in that: The magnetic nanoparticles are magnetic nanoparticles Fe3O4, with a particle size of 50-100nm and a concentration of 0-200ppm; riboflavin, humic acid, Fe 2+ 、Ni 2+ The concentration is 0-200ppm.

4. The biological / non-biological hydrogen production system based on the hydrogen production mechanism of Clostridium pasteurianum according to claim 1, characterized in that: The method for producing hydrogen based on a hydrogen production system in which a hydrogenase gene endogenous to Clostridium pasteurianum is overexpressed in Clostridium pasteurianum comprises the following steps: ① Culture: Take out the endogenous hydrogenase gene overexpressing Clostridium pasteurii strain from a -80℃ refrigerator, transfer the strain to 5 mL 2×YTG or 2×YTG medium with chloramphenicol resistance, and culture it overnight at 37℃ for 14 h; ② Transfer: Transfer the obtained strain to an anaerobic bottle containing 10 mL of 2×YTG or 2×YTG fresh medium with chloramphenicol resistance at a ratio of 1:20, and culture for 12 h until OD600 = 0.8; ③ Induction: Add 1 mL of 1 mg / mL anhydrotetracycline to a final concentration of 100 ng / mL and induce culture for 12 h; ④ Measure hydrogen: Use gas chromatograph to measure hydrogen production.

5. The biological / non-biological hydrogen production system based on the hydrogen production mechanism of Clostridium pasteurianum according to claim 1, characterized in that: The method for producing hydrogen based on a hydrogen production system in which a hydrogen production element is added during the cultivation of Clostridium pasteurianum comprises the following steps: ① Culture: Take out Clostridium pasteurianum from -80 ℃ refrigerator and transfer it to 5 mL 2×YTG medium, and culture it at 37 ℃ overnight for 14 h; ② Prepare the culture medium: weigh 10-50 mg of hydrogen production element, sterilize it under ultraviolet conditions for 30 min, add it to 2×YTG culture medium, and perform gradient dilution to make the final concentration of hydrogen production element culture medium between 10-200 ppm; ③ Transfer: Transfer Clostridium pasteurianum to the diluted hydrogen production element culture medium at a ratio of 1:20 and culture for 12 hours; ④ Hydrogen measurement: Use gas chromatograph to measure hydrogen production.

6. A biological / non-biological hydrogen production system based on the hydrogen production mechanism of Clostridium pasteurianum according to claim 4 or 5, characterized in that: The composition of 2×YTG medium is: 16 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl, 5 g / L glucose and 0.2 g / L cysteine.

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