A Clostridium pasteurianum biological / abiological hydrogen production system based on the hydrogen production mechanism

Abstract

The present invention belongs to the technical field of optimization of microbial hydrogen production, and particularly relates to a Clostridium pasteurianum biological / abiological hydrogen production system based on a hydrogen production mechanism. In this system, 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, and the hydrogen production element includes one or more of Fe<supgt;2+< / supgt>, Ni<supgt;2+< / supgt>, riboflavin, humic acid or magnetic nanoparticles. The present invention explores the effects of different types of hydrogenases on hydrogen production by Clostridium pasteurianum, and clarifies the hydrogen production mechanism of Clostridium pasteurianum; by externally adding a new hydrogen production element, the hydrogen production yield of Clostridium pasteurianum is further increased, which can promote the development of biological hydrogen production technology and provide a feasible biological solution for the production of clean energy.

Description

Technical Field

[0001] The present invention belongs to the technical field of optimizing microbial hydrogen production, and particularly relates to a Clostridium pasteurianum biological / abiological hydrogen production system based on a hydrogen production mechanism. Background Art

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

[0003] Currently, there are various hydrogen production technical means. Hydrogen production from fossil fuels, as a traditional method, uses natural gas as the raw material, including steps such as desulfurization, steam reforming, and water-gas shift. Although this technology is mature and has a low cost, it is restricted by the natural gas supply, relies on fossil fuels, and emits greenhouse gases; Hydrogen production from industrial by-products utilizes by-products generated in the industrial production process (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 of industrial by-products and treatment technologies. Hydrogen production by electrolyzing water produces hydrogen by electrolyzing water, but the electricity price is an important factor restricting its wide application.

[0004] Currently, some new hydrogen production methods such as hydrogen production from biomass, biological hydrogen production, and thermochemical hydrogen production are still in the experimental and development stage and have not yet met the requirements of industrial hydrogen production scale. Compared with other hydrogen production methods, the biological hydrogen production method uses renewable energy as the raw material, produces hydrogen at ambient temperature and atmospheric pressure, and has advantages such as sustainability, environmental protection, low energy consumption, and wide resources. The research boom in the field of biological hydrogen production broke out during the energy crisis in the 1970s, and gradually formed a hydrogen production technical route including photo-hydrolysis, photo-fermentation, dark fermentation, and photo-dark co-fermentation. The microbial groups involved in these hydrogen production processes include green algae, cyanobacteria, photo-fermentative bacteria, and dark-fermentative bacteria. Among them, fermentative hydrogen-producing bacteria have relatively high hydrogen production efficiency and capacity. Considering the hydrogen production yield and substrate conversion rate comprehensively, Clostridium pasteurianum DSM 525 is a more suitable hydrogen-producing microorganism. As a Gram-positive, strictly anaerobic bacterium that can form endospores, it shows high tolerance to high substrate concentrations, inhibitor resistance, and sustainability to adverse conditions during the hydrogen production process.

[0005] In the hydrogen production pathway of Clostridium pasteurianum, hydrogenase acts as a key enzyme responsible for catalyzing the hydrogen production reaction. The expression and regulation of genes are crucial for the hydrogen production ability of Clostridium pasteurianum. Studying these genes helps improve the efficiency of biological hydrogen production. By further exploring the regulation mechanism and metabolic network of hydrogenase genes, the molecular basis of the metabolic mechanism of Clostridium pasteurianum is revealed, and new components that can increase the hydrogen production of Clostridium are explored. Hydrogen production by Clostridium is promoted by increasing the electron transfer of the hydrogen production pathway, enhancing the catalytic activity of hydrogenase, or expressing exogenous hydrogenase. The hydrogen production of genetically modified Clostridium pasteurianum is promoted by integrating the hydrogen production component culture system, which is expected to achieve efficient and stable hydrogen production by Clostridium pasteurianum and make important contributions to the development and utilization of clean energy. Summary of the Invention

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

[0007] The technical solution of the present invention is as follows:

[0008] A biological / abiological hydrogen production system of Clostridium pasteurianum based on the hydrogen production mechanism, overexpressing the endogenous hydrogenase gene of Clostridium pasteurianum in Clostridium pasteurianum, or adding a hydrogen production component during the cultivation of Clostridium pasteurianum, and the hydrogen production component includes Fe 2+ , Ni 2+ , riboflavin, humic acid or one or more of magnetic nanoparticles.

[0009] Furthermore, for the above-mentioned biological / abiological hydrogen production system of Clostridium pasteurianum based on the hydrogen production mechanism, the method of overexpressing the endogenous hydrogenase gene of Clostridium pasteurianum is to construct a dual-promoter inducible plasmid suitable for Clostridium pasteurianum, integrate the amplified 4 Clostridium pasteurianum hydrogenase genes C00280, C37830, C07060-70, and RS16520 into the plasmid, and transfer them into wild-type Clostridium pasteurianum respectively to obtain Clostridium pasteurianum / overexpressing C00280 strain, Clostridium pasteurianum / overexpressing C37830 strain, Clostridium pasteurianum / overexpressing C07060-70 strain, and Clostridium pasteurianum / overexpressing RS16520 strain.

[0010] Furthermore, for the above-mentioned biological / abiological hydrogen production system of Clostridium pasteurianum based on the hydrogen production mechanism, the magnetic nanoparticles are magnetic nanoparticles Fe3O4, with a particle size of 50-100 nm and a concentration of 0-200 ppm; the concentrations of riboflavin, humic acid, Fe 2+ , Ni 2+ are 0-200 ppm.

[0011] Further, for the above Clostridium pasteurianum biological / abiological hydrogen production system based on the hydrogen production mechanism, the hydrogen production method of the hydrogen production system based on overexpressing the endogenous hydrogenase gene of Clostridium pasteurianum in Clostridium pasteurianum includes the following steps:

[0012] ① Bacteria cultivation: Take out the Clostridium pasteurianum strain after overexpressing the endogenous hydrogenase gene from the -80 °C refrigerator, and transfer the strain to 5 mL of 2×YTG or 2×YTG medium with chloramphenicol resistance, and incubate statically overnight at 37 °C for 14 h;

[0013] ② Transfer: Transfer the obtained strain to an anaerobic bottle containing 10 mL of fresh 2×YTG or 2×YTG medium with chloramphenicol resistance at a ratio of 1:20, and culture for 12 h until OD600 = 0.8;

[0014] ③ Induction: Add 1 mL of 1 mg / mL anhydrous tetracycline to a final concentration of 100 ng / mL, and induce culture for 12 h;

[0015] ④ Hydrogen measurement: Measure the hydrogen production with a gas chromatograph.

[0016] Further, for the above Clostridium pasteurianum biological / abiological hydrogen production system based on the hydrogen production mechanism, the hydrogen production method of the hydrogen production system based on adding a hydrogen production component during the cultivation of Clostridium pasteurianum includes the following steps:

[0017] ① Bacteria cultivation: Take out Clostridium pasteurianum from the -80 °C refrigerator and transfer it to 5 mL of 2×YTG medium, and incubate statically overnight at 37 °C for 14 h;

[0018] ② Medium preparation: Weigh 10 - 50 mg of the hydrogen production component, sterilize it by irradiating under ultraviolet light for 30 min, add it to the 2×YTG medium, and perform gradient dilution so that the final concentration of the hydrogen production component medium is between 10 - 200 ppm;

[0019] ③ Transfer: Transfer Clostridium pasteurianum to the diluted hydrogen production component medium at a ratio of 1:20, and culture for 12 h;

[0020] ④ Hydrogen measurement: Measure the hydrogen production with a gas chromatograph.

[0021] Further, for the above Clostridium pasteurianum biological / abiological hydrogen production system based on the hydrogen production mechanism, the components of the 2×YTG 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:

[0022] The Clostridium pasteurianum biological / abiological hydrogen production system of the present invention overexpresses the endogenous hydrogenase gene of Clostridium pasteurianum, explores the effects of different types of hydrogenases on hydrogen production by Clostridium pasteurianum, and clarifies the hydrogen production mechanism of Clostridium pasteurianum; by adding new hydrogen production components externally, the hydrogen production of Clostridium pasteurianum can be further increased, 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 increased, which helps to reduce the cost of biological hydrogen production and improve its economic feasibility. Description of the Drawings

[0023] Figure 1 Schematic diagram of the effect of hydrogenase overexpression on hydrogen production by Clostridium pasteurianum in Example 1;

[0024] Figure 2 Schematic diagram of the effect of adding magnetic nanoparticles on hydrogen production by Clostridium pasteurianum in Example 2;

[0025] Figure 3 Schematic diagram of the effect of adding riboflavin on hydrogen production by Clostridium pasteurianum in Example 3;

[0026] Figure 4 Schematic diagram of the effect of adding humic acid on hydrogen production by Clostridium pasteurianum in Example 4;

[0027] Figure 5 Schematic diagram of the effect of adding metal ions on hydrogen production by Clostridium pasteurianum in Example 5;

[0028] Figure 6 Schematic diagram of the effect of the combined action of multiple hydrogen production components on hydrogen production by Clostridium pasteurianum in Example 6. Detailed Description of the Invention

[0029] Next, the specific implementation manners of the present invention will be further described in detail with reference to the accompanying drawings of the specification and examples. The following examples are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0030] In the following examples,

[0031] The selected Clostridium pasteurianum is the wild-type Clostridium pasteurianum (WT);

[0032] The composition of the 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;

[0033] The method for overexpressing the hydrogenase gene endogenous to Clostridium pasteurianum is to construct a dual-promoter inducible plasmid suitable for Clostridium pasteurianum, integrate the amplified 4 kinds of Clostridium pasteurianum hydrogenase genes C00280, C37830, C07060-70, and RS16520 into the plasmid, and transfer them into wild-type Clostridium pasteurianum respectively to obtain Clostridium pasteurianum / overexpressing C00280 strain, Clostridium pasteurianum / overexpressing C37830 strain, Clostridium pasteurianum / overexpressing C07060-70 strain, and Clostridium pasteurianum / overexpressing RS16520 strain. Example 1

[0034] In this example, a biological / abiological hydrogen production system of Clostridium pasteurianum based on the hydrogen production mechanism overexpresses the hydrogenase gene endogenous to Clostridium pasteurianum. The specific hydrogen production method includes the following steps:

[0035] ① Bacterial cultivation: Take out wild-type Clostridium pasteurianum (WT), Clostridium pasteurianum / overexpressing C00280 strain, Clostridium pasteurianum / overexpressing C37830 strain, Clostridium pasteurianum / overexpressing C07060-70 strain, and Clostridium pasteurianum / overexpressing RS16520 strain from the -80 °C refrigerator, and transfer the strains to 5 mL of 2×YTG medium respectively, and culture statically overnight at 37 °C for 14 h;

[0036] ② Transfer: Transfer the obtained strains to an anaerobic bottle containing 10 mL of fresh 2×YTG medium at a ratio of 1:20, and culture for 12 h until OD600 = 0.8;

[0037] ③ Induction: Add 1 mL of anhydrous tetracycline at 1 mg / mL to a final concentration of 100 ng / mL, and induce culture for 12 h;

[0038] ④ Hydrogen measurement: Measure the hydrogen production with a gas chromatograph.

[0039] The relative hydrogen production amount (HPC) is as Figure 1 shown. The experimental results show that the overexpression of the 4 kinds of endogenous hydrogenase genes respectively increases the hydrogen production capacity of Clostridium pasteurianum by 3 times, 2.6 times, 2.5 times, and 3.5 times, indicating that these four hydrogenases all play a key role in the hydrogen production process of Clostridium pasteurianum. Example 2

[0040] In this example, a biological / abiological hydrogen production system of Clostridium pasteurianum based on the hydrogen production mechanism adds hydrogen production element magnetic nanoparticles during the cultivation of Clostridium pasteurianum. The magnetic nanoparticles are purchased from Macklin, nano-ferroferric oxide, with a purity of 99.5% and a particle size of 50 / 100Φ. The specific hydrogen production method includes the following steps:

[0041] ① Bacteria cultivation: Take Clostridium pasteurianum out of the -80 °C refrigerator and transfer it to 5 mL of 2×YTG medium, and incubate it statically overnight at 37 °C for 14 h;

[0042] ② Medium preparation: Weigh 10 mg of magnetic nanoparticles, sterilize them by irradiating under ultraviolet conditions for 30 min, add 50 mL of 2×YTG, with a concentration of 200 ppm, and dilute the concentration of the magnetic nanoparticle medium to 10 ppm, 25 ppm, 50 ppm, and 100 ppm respectively;

[0043] ③ Transfer: Transfer the wild-type Clostridium pasteurianum (WT) at a ratio of 1:20 to the medium containing magnetic nanoparticles with different concentrations of 0, 10, 25, 50, 100, and 200 ppm, and culture for 12 h;

[0044] ④ Hydrogen measurement: Measure the hydrogen production using a gas chromatograph.

[0045] In this example, magnetic nanoparticles Fe3O4 with different particle sizes were selected to be added to the medium. After co-culturing with Clostridium pasteurianum, the hydrogen production ability of Clostridium pasteurianum was tested. The results are as Figure 2 shown. The experimental results show that Fe3O4 nanoparticles with a particle size of 100 nm can improve the hydrogen production ability of the strain, and the hydrogen production ability of Clostridium pasteurianum shows a trend of first increasing and then decreasing with the increase in the concentration of magnetic nanoparticles. The nanoparticle concentration at the highest hydrogen production 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 ability of Clostridium pasteurianum. Example 3

[0046] In this example, a biological / abiological hydrogen production system of Clostridium pasteurianum based on the hydrogen production mechanism. Riboflavin, a hydrogen production component, is added during the cultivation of Clostridium pasteurianum. The riboflavin selected is purchased from BD Company, USA. The specific hydrogen production method includes the following steps:

[0047] ① Bacteria cultivation: Take Clostridium pasteurianum out of the -80 °C refrigerator and transfer it to 5 mL of 2×YTG medium, and incubate it statically overnight at 37 °C for 14 h;

[0048] ② Medium preparation: Weigh 10 mg of riboflavin, sterilize it by irradiating under ultraviolet conditions for 30 min, and add it to the 2×YTG medium, with a concentration of 200 ppm. Dilute the concentration of the riboflavin medium to 10 ppm, 25 ppm, 50 ppm, and 100 ppm respectively;

[0049] ③ Transfer: Transfer Clostridium pasteurianum at a ratio of 1:20 to the medium with different concentrations of 0, 10, 25, 50, 100, and 200 ppm of riboflavin, and culture for 12 h;

[0050] ④ Measuring hydrogen: Measure the hydrogen production using a gas chromatograph.

[0051] In this example, different concentrations of riboflavin were added to the culture medium, and after co-culturing with Clostridium pasteurianum, the hydrogen production ability of Clostridium pasteurianum was tested. The results are as Figure 3 shown. The experimental results show that riboflavin can improve the hydrogen production ability of Clostridium, and the hydrogen production ability first increases and then decreases with the increase of riboflavin concentration. The riboflavin concentration at the highest hydrogen production is 50 ppm, and the amount of hydrogen produced is 1.45 times that of wild-type Clostridium pasteurianum. The reason for the weakened promotion effect of high-concentration riboflavin on hydrogen production may be its toxic effect on Clostridium, inhibiting the growth of bacteria. Example 4

[0052] In this example, a biological / abiological hydrogen production system of Clostridium pasteurianum based on the hydrogen production mechanism was used. Humic acid, a hydrogen production component, was added during the cultivation of Clostridium pasteurianum. The humic acid used was purchased from BD Company, USA. The specific hydrogen production method includes the following steps:

[0053] ① Bacteria cultivation: Take Clostridium pasteurianum out of the -80 °C refrigerator and transfer it to 5 mL of 2×YTG medium, and incubate it statically at 37 °C overnight for 14 h;

[0054] ② Preparation of culture medium: Weigh 10 mg of humic acid, sterilize it by irradiating under ultraviolet light for 30 min, and add it to 2×YTG medium with a concentration of 200 ppm. Dilute the humic acid medium concentrations to 10 ppm, 25 ppm, 50 ppm, and 100 ppm respectively;

[0055] ③ Transfer: Transfer Clostridium pasteurianum to different concentrations of humic acid media with concentrations of 0, 10, 25, 50, 100, and 200 ppm at a ratio of 1:20, and culture for 12 h;

[0056] ④ Measuring hydrogen: Measure the hydrogen production using a gas chromatograph.

[0057] In this example, different concentrations of humic acid were added to the culture medium, and after co-culturing with Clostridium pasteurianum, the hydrogen production ability of Clostridium pasteurianum was tested. The results are as Figure 4 shown. The experimental results show that humic acid can improve the hydrogen production ability of Clostridium, and the hydrogen production ability first increases and then decreases with the increase of humic acid concentration. The humic acid concentration at the highest hydrogen production is 25 ppm, and the amount of hydrogen produced is 1.14 times that of wild-type Clostridium pasteurianum. The reason for the weakened promotion effect of high-concentration humic acid on hydrogen production may be its toxic effect on Clostridium, inhibiting the growth of bacteria. Example 5

[0058] In this embodiment, a Clostridium pasteurianum biological / abiological hydrogen production system based on the hydrogen production mechanism adds the hydrogen production element Ni during the cultivation of Clostridium pasteurianum. 2+ and Fe 2+ , and its specific hydrogen production method includes the following steps:

[0059] ① Bacteria cultivation: Take Clostridium pasteurianum out of the -80 °C refrigerator and transfer it to 5 mL of 2×YTG medium, and cultivate it statically overnight at 37 °C for 14 h;

[0060] ② Medium preparation: Weigh 10 mg of NiCl2 and FeCl2 respectively, sterilize them by irradiating under ultraviolet conditions for 30 min, add 50 mL of 2×YTG, with a concentration of 200 ppm, and dilute the concentrations of Ni 2+ and Fe 2+ media to 10 ppm, 25 ppm, 50 ppm, and 100 ppm respectively;

[0061] ③ Transfer: Transfer the wild-type Clostridium pasteurianum (WT) at a ratio of 1:20 to media containing different concentrations of Ni 2+ and Fe 2+ of 0, 10, 25, 50, 100, 200 ppm, and cultivate for 12 h;

[0062] ④ Hydrogen measurement: Measure the hydrogen production with a gas chromatograph.

[0063] In this embodiment, different concentrations of Ni 2+ and Fe 2+ are selected to be added to the medium. After co-cultivation with Clostridium pasteurianum, the hydrogen production ability of Clostridium pasteurianum is tested. The results are as Figure 5 shown. The experimental results show that the hydrogen production ability of Ni 2+ shows a trend of first increasing and then decreasing with the increase of concentration. The ionic concentration at the highest hydrogen production is 50 ppm, and the amount of hydrogen produced is 1.48 times that of the wild-type Clostridium pasteurianum; the hydrogen production ability of Fe 2+ shows a trend of first increasing and then decreasing with the increase of concentration. The ionic concentration at the highest hydrogen production is 50 ppm, and the amount of hydrogen produced is 1.50 times that of the wild-type Clostridium pasteurianum. Example 6

[0064] In this embodiment, a Clostridium pasteurianum biological / abiological hydrogen production system based on the hydrogen production mechanism overexpresses the endogenous hydrogenase genes C00280, C37830, C07060-70, and RS16520 of Clostridium pasteurianum, and adds multiple hydrogen production elements during the cultivation process: magnetic nanoparticles, riboflavin, humic acid, and metal ions Ni 2+ , Fe 2+ , and its specific hydrogen production method includes the following steps:

[0065] ① Incubate bacteria: Take Clostridium pasteurianum / overexpressing C00280 strain, Clostridium pasteurianum / overexpressing C37830 strain, Clostridium pasteurianum / overexpressing C07060-70 strain, and Clostridium pasteurianum / overexpressing RS16520 strain out of the -80 °C refrigerator and transfer them to 5 mL of 2×YTG medium. Incubate statically overnight at 37 °C for 14 h.

[0066] ② Prepare the medium: Weigh 25 mg of magnetic nanoparticles, 50 mg of riboflavin, 25 mg of humic acid, 25 mg of NiCl2, and 50 mg of FeCl2. Sterilize them by irradiating under ultraviolet light for 30 min, and then add them to 1 L of 2×YTG medium.

[0067] ③ Transfer: Transfer the wild-type Clostridium pasteurianum (WT) to the medium prepared in ② at a ratio of 1:20 and culture for 12 h until OD600 = 0.8.

[0068] ④ Induce: Add 1 mL of anhydrous tetracycline at a concentration of 1 mg / mL to a final concentration of 100 ng / mL and induce for 12 h.

[0069] ⑤ Measure hydrogen: Measure the hydrogen production using a gas chromatograph.

[0070] In this example, the optimal culture system in which multiple components in the above example act together was selected and added to the medium of Clostridium pasteurianum overexpressing hydrogenase for co-culture. The hydrogen production of the genetically modified Clostridium pasteurianum was tested, and the results are as Figure 6 shown. The experimental results show that under the optimal conditions, the hydrogen production capacity of Clostridium pasteurianum overexpressing hydrogenase was increased by 3.78 times, 3.13 times, 3.06 times, and 4.18 times compared with that of WT, respectively. The hydrogen production can reach 75763 ppm, 62826 ppm, 61350 ppm, and 83796 ppm. The two strains with the strongest hydrogen production capacity are C00280 and RS16520.

Claims

1. A Clostridium pasteurianum biological / abiological hydrogen production system based on a hydrogen production mechanism, characterized in that, Overexpress the endogenous hydrogenase gene of Clostridium pasteurianum in Clostridium pasteurianum, and the hydrogenase gene is selected from C00280, C37830, C07060-70. During the cultivation of Clostridium pasteurianum, add 50 mg / L FeCl2, 25 mg / L NiCl2, 50 mg / L riboflavin, 25 mg / L humic acid and 25 mg / L magnetic nanoparticles, and the magnetic nanoparticles are magnetic nanoparticles Fe3O4 with a particle size of 100 nm.

2. A Clostridium pasteurianum biological / abiological hydrogen production system based on a hydrogen production mechanism according to claim 1, characterized in that The specific hydrogen production method includes the following steps: ① Bacteria cultivation: Take out Clostridium pasteurianum / overexpressing C00280 strain, Clostridium pasteurianum / overexpressing C37830 strain, and Clostridium pasteurianum / overexpressing C07060-70 strain from the -80 °C refrigerator and transfer them to 5 mL of 2×YTG medium, and culture them statically overnight at 37 °C for 14 h; ② Medium preparation: Weigh 25 mg of magnetic nanoparticles, 50 mg of riboflavin, 25 mg of humic acid, 25 mg of NiCl2, and 50 mg of FeCl2, sterilize them by irradiating under ultraviolet light for 30 min, and add them to 1 L of 2×YTG medium; ③ Transfer: Transfer the bacteria transfected with the hydrogenase above to the medium prepared in ② at a ratio of 1:20, and culture for 12 h until OD600 = 0.8; ④ Induction: Add 1 mL of anhydrous tetracycline at 1 mg / mL to a final concentration of 100 ng / mL, and induce culture for 12 h; ⑤ Hydrogen measurement: Measure the hydrogen production with a gas chromatograph.

3. A Clostridium pasteurianum biological / abiological hydrogen production system based on a hydrogen production mechanism according to claim 2, characterized in that, The components of the 2×YTG 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.

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