A method for producing hydrogen by Clostridium pasteurii-rhodopseudomonas palustris coupling fermentation based on nitrogen limitation and application thereof

Abstract

The application discloses a method for producing hydrogen through Clostridium pasteurianum-Rhodobacter sphaeroides coupling fermentation based on nitrogen limitation and application thereof. In the method, activated Clostridium pasteurianum is subjected to dark fermentation for producing hydrogen in a nitrogen-free culture medium and in an argon environment; then, the pH value of the obtained culture solution is adjusted to 7.0-8.0, the activated Rhodobacter sphaeroides is inoculated after selective sterilization, and the light fermentation for producing hydrogen is carried out in the argon environment. In the application, the fermentation solution of Clostridium pasteurianum does not need to be subjected to centrifugal removal of bacterial bodies and the like, and only needs to be simply adjusted in pH value, so that the light fermentation for producing hydrogen of Rhodobacter sphaeroides can be directly carried out. In the application, the nitrogen limitation is used to inhibit excessive proliferation of bacterial bodies, save carbon source flow, induce high expression of nitrogen fixation enzyme, and open a second high-efficiency hydrogen production path in addition to hydrogenase, so that the hydrogen production yield is further improved while the cost of nitrogen source is reduced. The hydrogen production yield of the application reaches 9.81 mol H2 / mol glucose, which is the highest level at present.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological hydrogen production, and particularly relates to a Clostridium-rhodopseudomonas coupling fermentation hydrogen production method based on nitrogen limitation and application thereof. BACKGROUND

[0002] With the growth of global energy demand and the intensification of environmental problems, hydrogen energy as a clean energy has attracted much attention. Microbial fermentation hydrogen production has become a research hotspot due to its wide range of raw material sources (such as agricultural waste, organic wastewater) and environmental friendliness.

[0003] The current mainstream two-step fermentation hydrogen production system usually adopts dark fermentation hydrogen-producing bacteria (such as Clostridium) and photosynthetic hydrogen-producing bacteria (such as Rhodopseudomonas) to cooperate: in the dark fermentation stage, glucose is converted into hydrogen and organic acid through anaerobic metabolism, and in the light fermentation stage, photosynthetic bacteria are used to further convert organic acid into hydrogen. However, there are significant differences in the optimal hydrogen production conditions of the two types of microorganisms - dark fermentation bacteria usually produce hydrogen efficiently at 30-37 ℃, pH 5.5-6.5, and no light, while photosynthetic bacteria require 30-35 ℃, pH 7.0-8.0, and light (2000-10000 lux) environment; in addition, dark fermentation bacteria rely on organic nitrogen sources such as glutamate sodium to maintain metabolic activity, while photosynthetic bacteria can grow autotrophically through nitrogenase, and excess nitrogen source inhibits their hydrogen production capacity. The above contradictions lead to low hydrogen production efficiency (<4.0 mol H2 / mol glucose) in direct co-culture system, forcing the industry to adopt distributed two-step method. The maximum hydrogen production yield of traditional two-step method (such as Clostridium + Rhodobacter) is generally less than 7 mol H2 / mol glucose, which is far lower than the theoretical limit (12 mol H2 / mol glucose).

[0004] Currently, the highest hydrogen yield reported by two-step fermentation of glucose using dark fermentation hydrogen-producing bacteria and photosynthetic bacteria is 9.4 mol H2 / mol glucose. This method uses Thermotoga neapolitana DSM 4359 and Rhodopseudomonas palustris CGMCC 1.42OL. However, this hydrogen production system has many limitations, such as the need to maintain high temperature of 80℃ in the dark fermentation stage, and the need to continuously pass in carbon dioxide, resulting in huge energy consumption; and after the first step of dark fermentation is completed, multiple processes such as centrifugal separation, nutrient addition and sterilization are required, resulting in soaring costs and difficulties in large-scale application. In summary, it is urgent to develop a two-step hydrogen production process with high yield, low energy consumption and sterilization-free characteristics to break through the technical bottleneck and promote the industrialization process of biological hydrogen production. Rhodopseudomonas palustris 42OL. However, this hydrogen production system has many limitations, such as the need to maintain high temperature of 80℃ in the dark fermentation stage, and the need to continuously pass in carbon dioxide, resulting in huge energy consumption; and after the first step of dark fermentation is completed, multiple processes such as centrifugal separation, nutrient addition and sterilization are required, resulting in soaring costs and difficulties in large-scale application. In summary, it is urgent to develop a two-step hydrogen production process with high yield, low energy consumption and sterilization-free characteristics to break through the technical bottleneck and promote the industrialization process of biological hydrogen production. SUMMARY

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a nitrogen-limited method for hydrogen production by coupled fermentation of Clostridium pasteurellii and Rhodotorula globosum.

[0006] Another object of the present invention is to provide an application of the above-described nitrogen-limited Clostridium pasteurellium-Rhodophyton floccosum coupled fermentation method for hydrogen production.

[0007] The objective of this invention is achieved through the following technical solution: a nitrogen-limited Clostridium pasteurellii-Rhodophyton floccosum co-fermentation method for hydrogen production, comprising the following steps:

[0008] (1) Activated Clostridium pasteurellium was subjected to dark fermentation to produce hydrogen in a nitrogen-free medium and an argon atmosphere;

[0009] (2) Next, the pH of the culture medium obtained in step (1) is adjusted to 7.0-8.0, selectively sterilized, and then activated Rhodopseudomonas spp. are introduced to carry out photo-fermentation to produce hydrogen in an argon atmosphere.

[0010] The *Clostridium pasteurellum* mentioned in step (1) is preferably *Clostridium pasteurellum* (… Clostridium pasteurianum DSM525.

[0011] The activated Clostridium pasteurellium described in step (1) is preferably prepared by the following steps: inoculating Clostridium pasteurellium into a seed culture medium to obtain a culture solution, and then incubating it in a nitrogen atmosphere in the dark until the OD reaches 100°C. 600 The concentration was 1.0–1.6; then solid-liquid separation was performed, and the obtained bacterial cells were washed with a nitrogen-free solvent to obtain activated Clostridium pasteurellii.

[0012] The seed culture medium is a nitrogen-containing medium, including MS medium and RCVBN seed culture medium.

[0013] The MS medium is composed of the following: 2 g / L glucose monohydrate, 1 g / L sodium glutamate, 5 g / L NaCl, 2 g / L K2HPO4, 0.5 g / L KH2PO4, 10 mL / L trace element concentrate, pH=6.0, and water as the solvent.

[0014] The components of the trace element concentrate are as follows: CaCl2·2H2O 0.2 g / L, MgCl2·6H2O 2 g / L, FeCl2·4H2O 40 mg / L, ZnCl2 1 mg / L, MnCl2·4H2O 1 mg / L, CuCl2·2H2O 0.6 mg / L, Na2MoO4 1 mg / L, AlCl3 1 mg / L, CoCl2·6H2O 4 mg / L, 20 μL of saturated boric acid solution, 20 μL of 37%wt hydrochloric acid, biotin 0.04 mg / L, folic acid 0.04 mg / L, vitamin B6 0.2 mg / L, riboflavin 0.1 mg / L, vitamin B1 0.1 mg / L, niacin 0.1 mg / L, vitamin B2... 12 0.1 mg / L, p-aminobenzoic acid 0.1 mg / L, pantothenic acid 0.1 mg / L, solvent is water.

[0015] The composition of the RCVBN seed culture medium is as follows: 2 g / L glucose monohydrate, 5 g / L sodium glutamate, 50 mL / L basic salt solution, 20 mL / L 0.2 M PBS, 1 mL / L vitamin solution, pH=7.0, and water as the solvent;

[0016] The basic salt solution has the following composition: MgSO4·7H2O 4 g / L, CaCl2·H2O 1.5 g / L, FeSO4·7H2O 0.236 g / L, Na2EDTA 0.4 g / L, trace element solution 20 mL / L, and water as the solvent;

[0017] The composition of the trace element solution is as follows: MnSO4·H2O 0.21 g / L, H3BO3 0.28 g / L, CuSO4·5H2O 0.004 g / L, ZnSO4·7H2O 0.024 g / L, NaMoO4·2H2O 0.075 g / L, with water as the solvent;

[0018] The vitamin solution contains the following components: niacin 10 g / L, vitamin B1 5 g / L, biotin 0.1 g / L, and water as the solvent.

[0019] The preferred inoculation amount is 5-10% of the culture medium volume.

[0020] The nitrogen environment described refers to a headspace atmosphere in which nitrogen is present.

[0021] The preferred culture system is as follows: 20 mL culture medium / 120 mL vial.

[0022] The preferred culture temperature is 37±1 ℃.

[0023] The preferred culture time is 8 to 16 hours.

[0024] The preferred method for solid-liquid separation is centrifugation.

[0025] The nitrogen-free solvents mentioned refer to solvents that do not contain nitrogen, including but not limited to water, PBS, and nitrogen-free culture media.

[0026] The nitrogen-free culture medium mentioned in step (1) is a culture medium that does not contain a nitrogen source but contains a carbon source, preferably RN(-)-Ar culture medium.

[0027] The preferred composition of the RN(-)-Ar culture medium is as follows: 2 g / L glucose monohydrate, 50 mL / L basic salt solution, 20 mL / L 0.2 M PBS, 1 mL / L vitamin solution, pH = 5.0-6.5, and water as the solvent; the preferred pH is 5.5.

[0028] The basic salt solution has the following composition: MgSO4·7H2O 4 g / L, CaCl2·H2O 1.5 g / L, FeSO4·7H2O 0.236 g / L, Na2EDTA 0.4 g / L, trace element solution 20 mL / L, and water as the solvent;

[0029] The trace element solution is composed of the following components: MnSO4·H2O 0.21 g / L, H3BO3 0.28 g / L, CuSO4·5H2O 0.004 g / L, ZnSO4·7H2O 0.024 g / L, NaMoO4·2H2O 0.075 g / L, with water as the solvent;

[0030] The vitamin solution contains the following components: niacin 10 g / L, vitamin B1 5 g / L, biotin 0.1 g / L, and water as the solvent.

[0031] The argon environment described in step (1) is a headspace gas of argon.

[0032] The preferred conditions for dark fermentation in step (1) are 37±1 ℃ and static culture in the dark for 24 to 48 h.

[0033] The pH value mentioned in step (2) is preferably 7.5.

[0034] The selective sterilization mentioned in step (2) means that sterilization may or may not be performed.

[0035] The sterilization conditions are preferably 115-121℃ for 15-20 min; more preferably 115℃ for 20 min.

[0036] The Rhodococcus-like bacteria mentioned in step (2) are preferably Rhodococcus-like bacteria ( Rhodobacter sphaeroides ZX-5.

[0037] The activated Rhodopseudomonas spp. described in step (2) is preferably prepared by the following steps: inoculating Rhodopseudomonas spp. into a seed culture medium to obtain a culture solution, and culturing it under nitrogen atmosphere and light until OD reaches 100°C. 600 The concentration was 4.5–5.5; then solid-liquid separation was performed, and the obtained bacterial cells were washed with a nitrogen-free solvent to obtain activated Rhodopseudomonas stolonifera.

[0038] The seed culture medium is a nitrogen-containing medium, including RCVBN seed culture medium and RB-2Gm culture medium.

[0039] The composition of RB-2Gm is as follows: sodium butyrate 1.1 g / L, sodium glutamate 2 g / L, basic salt solution 50 mL / L, 0.2 M PBS 20 mL / L, vitamin solution 1 mL / L, pH=7.0, solvent is water;

[0040] The basic salt solution has the following composition: MgSO4·7H2O 4 g / L, CaCl2·H2O 1.5 g / L, FeSO4·7H2O 0.236 g / L, Na2EDTA 0.4 g / L, trace element solution 20 mL / L, and water as the solvent.

[0041] The composition of the trace element solution is as follows: MnSO4·H2O 0.21 g / L, H3BO3 0.28 g / L, CuSO4·5H2O 0.004 g / L, ZnSO4·7H2O 0.024 g / L, NaMoO4·2H2O 0.075 g / L, with water as the solvent;

[0042] The vitamin solution contains the following components: niacin 10 g / L, vitamin B1 5 g / L, biotin 0.1 g / L, and water as the solvent.

[0043] The preferred inoculation amount is 5-10% of the culture medium volume.

[0044] The nitrogen environment described refers to a headspace atmosphere in which nitrogen is present.

[0045] The preferred culture system is as follows: 20 mL culture medium / 120 mL vial.

[0046] The preferred culture conditions are 30±1 ℃ and 7500~8500 lux light for 24~48 h; more preferably, 30±1 ℃ and 8000 lux light for 48 h.

[0047] The preferred method for solid-liquid separation is centrifugation.

[0048] The nitrogen-free solvent refers to a solvent that does not contain nitrogen, including but not limited to water, PBS, and nitrogen-free culture medium; preferably RN(-)-Ar culture medium.

[0049] The inoculum amount of activated Rhodopseudomonas spp. described in step (2) is preferably based on its initial OD value in the culture medium. 600 =2.5~3.5; more preferably calculated based on its initial OD in the culture medium. 600 =3.0 calculation.

[0050] The preferred conditions for photo-fermentation hydrogen production in step (2) are 30±1 ℃ and 7500~8500 lux light irradiation for 2~4 days; more preferably, 30±1 ℃ and 8000 lux light irradiation for 3 days.

[0051] The water used in this invention is preferably deionized water.

[0052] The above-mentioned nitrogen-limited Clostridium pasteurellium-Rhodophyton floccosum coupled fermentation hydrogen production method is applied to hydrogen production.

[0053] The present invention has the following advantages and effects compared with the prior art:

[0054] 1. The hydrogen production system provided by this invention achieves a hydrogen production yield of 9.81 mol H2 / mol glucose, which is the highest level to date.

[0055] 2. In the hydrogen production system provided by this invention, the Clostridium pasteurella fermentation broth does not require centrifugation to remove bacteria or the addition of extra nutrient solution. It can be directly cultured for hydrogen production by photofermentation of Rhodotorula glutinis simply by adjusting the pH.

[0056] 3. This invention inhibits excessive bacterial proliferation and saves carbon source flow by limiting nitrogen, while simultaneously inducing high expression of nitrogenase, opening up a second efficient hydrogen production pathway besides hydrogenase, thereby further increasing hydrogen production while reducing nitrogen source costs.

[0057] 4. Traditional processes rely on high-temperature sterilization at 115 ℃ for more than 20 minutes to suppress contaminating bacteria, which is both energy-intensive and increases equipment complexity. This invention utilizes the dual selective pressure of "nitrogen deficiency + argon" to spontaneously construct a microecological barrier that is beneficial to hydrogen-producing Clostridium and inhibits exogenous hydrogenophilic bacteria. It can operate stably under completely sterilization-free conditions, with hydrogen production efficiency decreasing by only about 5%, significantly reducing energy consumption and operating costs, and laying the technological foundation for subsequent sterilization-free continuous fermentation and large-scale scale-up. Attached Figure Description

[0058] Figure 1 This is a graph showing the hydrogen production results of DSM525 and ZX-5 in different fermentation media using a two-step fermentation process.

[0059] Figure 2 This is a graph showing the hydrogen production yield of DSM525 and ZX-5 in RCVBN fermentation medium with different nitrogen source concentrations during a two-step fermentation process.

[0060] Figure 3 This is a graph showing the biomass and hydrogen yield of DSM525 in different fermentation media for hydrogen production.

[0061] Figure 4 The graph shows the hydrogen production results of DSM525 with different inoculum amounts in RN(-)-Ar fermentation medium.

[0062] Figure 5 This is a graph showing the hydrogen production results of DSM525 in RN(-)-Ar fermentation media with different initial pH values.

[0063] Figure 6 This is a graph showing the growth and hydrogen production of ZX-5 in RB seed culture medium.

[0064] Figure 7 This is a graph showing the hydrogen production results of ZX-5 at different cultivation stages.

[0065] Figure 8 This is a graph showing the hydrogen production yield of ZX-5 with different inoculum amounts in fermentation media containing different nitrogen sources.

[0066] Figure 9 The figure shows the effect of different initial pH values ​​on hydrogen production by ZX-5 in a nitrogen-free fermentation medium.

[0067] Figure 10 This is a graph showing the hydrogen production results of DSM525 and ZX-5 in different fermentation media using a two-step fermentation process.

[0068] Figure 11 The graph shows the effect of sterilization on the hydrogen production yield of the two-step method in different culture systems. Detailed Implementation

[0069] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0070] The culture medium used in this invention is shown below:

[0071] (1) The composition of MS medium is as follows: glucose monohydrate 2 g / L, sodium glutamate 1 g / L, NaCl 5 g / L, K2HPO4 2 g / L, KH2PO4 0.5 g / L, trace element concentrate 10 mL / L, pH=6.0, and deionized water as solvent.

[0072] The composition of the trace element concentrate (1L) is as follows: CaCl2·2H2O 0.2 g / L, MgCl2·6H2O 2 g / L, FeCl2·4H2O 40 mg / L, ZnCl2 1 mg / L, MnCl2·4H2O 1 mg / L, CuCl2·2H2O 0.6 mg / L, Na2MoO4 1 mg / L, AlCl3 1 mg / L, CoCl2·6H2O 4 mg / L, 20 μL of saturated boric acid solution, 20 μL of 37%wt hydrochloric acid, biotin 0.04 mg / L, folic acid 0.04 mg / L, vitamin B6 0.2 mg / L, riboflavin 0.1 mg / L, vitamin B1 0.1 mg / L, niacin 0.1 mg / L, vitamin B2 12 0.1 mg / L, p-aminobenzoic acid 0.1 mg / L, pantothenic acid 0.1 mg / L, solvent is deionized water.

[0073] (2) The composition of RCVBN seed culture medium is as follows: 2 g / L glucose monohydrate, 5 g / L sodium glutamate, 50 mL / L basic salt solution, 20 mL / L 0.2 M PBS, 1 mL / L vitamin solution, pH=7.0, and deionized water as solvent.

[0074] The basic salt solution has the following composition: MgSO4·7H2O 4 g / L, CaCl2·H2O 1.5 g / L, FeSO4·7H2O 0.236 g / L, Na2EDTA 0.4 g / L, trace element solution 20 mL / L, and deionized water as the solvent.

[0075] The composition of the trace element solution is as follows: MnSO4·H2O 0.21 g / L, H3BO3 0.28 g / L, CuSO4·5H2O 0.004 g / L, ZnSO4·7H2O 0.024 g / L, NaMoO4·2H2O 0.075 g / L, and the solvent is deionized water.

[0076] The vitamin solution contains the following components: niacin 10 g / L, vitamin B1 5 g / L, biotin 0.1 g / L, and deionized water as the solvent.

[0077] (3) The difference between RCVBN fermentation medium and RCVBN seed medium is only the content of sodium glutamate and the pH. The composition is as follows: 2 g / L glucose monohydrate, 1 g / L sodium glutamate, 50 mL / L basic salt solution, 20 mL / L 0.2 M PBS, 1 mL / L vitamin solution, pH=6.0, and deionized water as solvent.

[0078] (4) The components of RB-2Gm medium are as follows: sodium butyrate 1.1 g / L, sodium glutamate 2 g / L, basic salt solution (composition same as (2)) 50 mL / L, PBS with a concentration of 0.2 M 20 mL / L, vitamin solution (composition same as (2)) 1 mL / L, pH=7.0, and deionized water as the solvent.

[0079] (5) The formulation of RN(-)-Ar-pH5.5 medium is as follows:

[0080] 2 g / L glucose monohydrate, 50 mL / L basic salt solution (same composition as (2)), 20 mL / L 0.2 M PBS, 1 mL / L vitamin solution (same composition as (2)), pH=5.5, and deionized water as the solvent. Argon was used as the headspace gas during incubation.

[0081] (6) The formulation of R-Gm-N2-pH5.5 medium is as follows:

[0082] 2 g / L glucose monohydrate, 0.8 g / L sodium glutamate, 50 mL / L basic salt solution (same composition as (2)), 20 mL / L 0.2M PBS, 1 mL / L vitamin solution (same composition as (2)), pH=5.5, and deionized water as the solvent. Unless otherwise specified, nitrogen is used in the headspace during incubation.

[0083] Example 1

[0084] (1) Clostridium pasteurellum ( Clostridium pasteurianum DSM 525 (purchased from the German Culture Collection of Microorganisms, DSMZ) was used as the bacterial strain. A 5% (v / v) inoculum of DSM 525 glycerol preservation solution was inoculated into MS medium to obtain the culture medium. The culture system was 20 mL culture medium / 120 mL vial. The headspace gas was replaced with nitrogen. The culture conditions were 37°C, incubated in the dark, and allowed to stand for 12 h. The resulting DSM525 seed culture OD... 600 =1.2.

[0085] (2) Rhodopseudomonas aeruginosa ( Rhodobacter sphaeroidesZX-5 (disclosed in the literature "Tao Y, et al. Characteristics of a new photosynthetic bacterial strain for hydrogen production and its application in wastewater treatment[J]. International Journal of Hydrogen Energy, 2008, 33(3):963-973") was used as the bacterial strain. ZX-5 glycerol preservation solution was inoculated into RCVBN seed culture medium at an inoculation rate of 5% (v / v) to obtain the culture medium. The culture system was 20 mL culture medium / 120 mL vial. The headspace gas was replaced with nitrogen gas, and the culture was carried out at 30 ℃ and 8000 lux light for 48 h. The OD of the obtained ZX-5 seed culture was... 600 =5.0.

[0086] (3) The Clostridium seed culture was inoculated into MS medium and RCVBN fermentation medium at 5% of the culture volume. The culture system was 20 mL culture medium / 120 mL vial, the headspace was replaced with nitrogen, and fermentation was carried out at 37℃ in the dark.

[0087] (4) The amount of hydrogen produced by Clostridium was determined by gas chromatography.

[0088] Gas chromatography analysis method: 0.2 mL of gas was taken from a vial, and the H2 concentration was determined using a gas chromatograph (Agilent 7820). N2 was used as the carrier gas in the gas chromatograph at a flow rate of 10 mL / min. -1 The temperature settings for the TCD detector, injection port, and column oven are 150 ℃, 80 ℃, and 80 ℃, respectively.

[0089] Hydrogen production calculation method: Based on the ideal gas law PV=nRT (P: partial pressure of gas; V: gas volume; n: amount of substance of gas; R: universal gas constant; T: gas temperature), the data measured by gas chromatography are converted into the molar mass of hydrogen. The hydrogen yield (hydrogen production conversion rate) is then calculated as: molar mass of hydrogen produced / molar mass of initial glucose.

[0090] (5) Adjust the pH of the Clostridium fermentation broth obtained from step (3) after 2 days of fermentation to 7.0, replace the headspace gas with argon, sterilize at 115 ℃ for 20 min, inoculate ZX-5 seed liquid at 10% of the volume of Clostridium culture broth, and culture at 30 ℃ and 8000 lux light. Monitor and measure the hydrogen production.

[0091] (6) Results are as follows Figure 1 As shown:

[0092] After two-step fermentation using DSM525 and ZX-5, the hydrogen conversion rates in MS and RCVBN fermentation media were 5.87 and 7.15 mol H2 / mol glucose, respectively. This indicates that RCVBN fermentation medium is more suitable for hydrogen production via the two-step fermentation method using DSM525 and ZX-5. Subsequent optimization of fermentation conditions will be based on RCVBN fermentation medium.

[0093] Example 2

[0094] (1) Clostridium pasteurellum ( Clostridium pasteurianum DSM 525 was used as the bacterial strain. DSM 525 was inoculated into RCVBN seed culture medium at an inoculum volume of 5% (v / v) to obtain the culture medium. The culture system was 20 mL culture medium / 120 mL vial. The headspace gas was replaced with nitrogen, and the culture was incubated at 37 ℃ in the dark for 12 h. The OD of the obtained DSM525 seed culture was... 600 =1.4. The RCVBN seed culture medium formula is the same as above.

[0095] (2) Rhodopseudomonas aeruginosa ( Rhodobacter sphaeroides ZX-5 was used as the bacterial strain. It was inoculated into RCVBN seed culture medium at a 5% (v / v) inoculation rate to obtain the culture solution. The culture system consisted of 20 mL of culture solution per 120 mL vial. The headspace gas was replaced with nitrogen, and the culture was carried out at 30 ℃ and 8000 lux light for 48 h. The OD of the obtained ZX-5 seed culture was... 600 =5.0. The RCVBN seed culture medium formula is the same as above.

[0096] (3) The sodium glutamate content in the RCVBN fermentation medium was adjusted to 0.6 g / L, 0.8 g / L, 1 g / L, and 1.2 g / L, respectively, and Clostridium seed culture was inoculated at 5% of the culture medium volume. The culture system was 20 mL culture medium / 120 mL vial, with headspace replaced by nitrogen. The culture conditions were 37 ℃ in the dark and static, and the hydrogen production was monitored. The RCVBN fermentation medium formula was the same as above.

[0097] (4) After 2 days of culturing, the pH of the Clostridium fermentation broth was adjusted to 7.0, the headspace gas was replaced with argon gas, and sterilized at 115 °C for 20 min. Then, ZX-5 seed liquid was inoculated at 10% of the volume of Clostridium culture broth and cultured at 30 °C under 8000 lux light. The amount of hydrogen produced was monitored and measured.

[0098] The results are as follows Figure 2As shown, RCVBN fermentation media with monosodium glutamate (MSG) concentrations of 0.6 g / L, 0.8 g / L, 1.0 g / L, and 1.2 g / L, after two-step fermentation using DSM525 and ZX-5 methods, produced total hydrogen yields of 7.32, 7.79, 7.35, and 7.06 mol H2 / mol glucose, respectively. Therefore, the optimal nitrogen source concentration for RCVBN fermentation media is 0.8 g / L MSG.

[0099] Example 3

[0100] (1) Clostridium pasteurellum ( Clostridium pasteurianum DSM 525 was used as the bacterial strain. It was inoculated into RCVBN seed culture medium at a 5% (v / v) inoculation rate to obtain the culture medium. The culture system consisted of 20 mL of culture medium per 120 mL vial. The headspace was replaced with nitrogen gas, and the culture was incubated at 37°C in the dark for 12 h. The OD of the resulting DSM525 seed culture was... 600 =1.4. The RCVBN seed culture medium formula is the same as above.

[0101] (2) Adjust the nitrogen source and headspace gas composition in the RCVBN fermentation medium to prepare 0.8 g / L sodium glutamate + headspace pure nitrogen (R-Gm-N2 medium), 0 g / L sodium glutamate + headspace pure nitrogen (RN(-)-N2 medium), and 0 g / L sodium glutamate + headspace pure argon (RN(-)-Ar) medium, respectively. Replace 0.2 M PBS in the RCVBN fermentation medium with 0.2 M MES (2-(N-morpholino)ethanesulfonic acid, with a pH buffering capacity equivalent to 0.2 M PBS), while keeping the headspace nitrogen gas, and prepare phosphorus-deficient medium (R-Gm-P(-)-N2 medium). After centrifuging to collect the Clostridium cells from the seed culture, resuspend them in the same volume of ddH2O to wash away the nitrogen source in the original medium. Take 1 mL of the resuspended bacterial solution and inoculate it into 19 mL of fermentation medium to obtain the culture solution. The culture system consisted of 20 mL of culture medium per 120 mL vial. The culture conditions were 37 ℃ incubation at room temperature in the dark. Hydrogen production and OD of the culture medium were monitored and measured. 600 Value. The RCVBN fermentation medium formulation is the same as above.

[0102] (3) such as Figure 3 As shown, as the available nitrogen source in the culture system gradually decreases, the growth of Clostridium decreases, while hydrogen production increases. The nitrogen-free culture system (RN(-)-Ar) performed best. Under these conditions, Clostridium hardly grows, and the OD of the culture medium... 600The pH value only increased slightly from the initial 0.12 to 0.18, but the hydrogen production yield reached 2.71 mol H2 / mol glucose, an increase of 39.9% compared to the initial growth conditions. Furthermore, phosphorus-deficient medium also inhibited the growth of Clostridium (maximum OD...). 600 The value was only 0.22), but Clostridium that did not grow under phosphorus-deficient conditions not only failed to increase hydrogen production, but its hydrogen conversion rate decreased by 11.86% compared to the control group (from 1.94 to 1.71). This indicates that simply inhibiting growth cannot increase hydrogen production; it can only be achieved under the special condition of nitrogen limitation.

[0103] Traditional research generally holds that the growth and hydrogen production of Clostridium are synchronous, and efficient hydrogen production can only be achieved when Clostridium is in a suitable growth environment. Even under special conditions, the hydrogen production of Clostridium is enhanced under nitrogen-fixing growth conditions, but this still depends on the growth of Clostridium itself. This invention is the first to discover that Clostridium can produce hydrogen without growth, and the amount of hydrogen produced is significantly increased, under the dual pressure of nitrogen-deficient culture medium and nitrogen-deficient headspace gas. This indicates that Clostridium still possesses hydrogen production activity even under nitrogen-limited conditions. Nitrogen-limited conditions may not only promote hydrogen production by inhibiting growth and saving energy, thus promoting hydrogen production by hydrogen esterase, but may also induce nitrogenase expression, opening up a second pathway for efficient hydrogen production besides hydrogen esterase.

[0104] Example 4

[0105] (1) Clostridium pasteurellum ( Clostridium pasteurianum DSM 525 was used as the bacterial strain. DSM 525 was inoculated into RCVBN seed culture medium at a 5% (v / v) inoculation rate to obtain the culture medium. The culture system was 20 mL culture medium / 120 mL vial. The headspace gas was replaced with nitrogen, and the culture was incubated at 37 ℃ in the dark for 12 h. The OD of the obtained DSM525 seed culture was... 600 =1.4. The RCVBN seed culture medium formula is the same as above.

[0106] (2) Prepare RN(-)-Ar medium. After centrifuging to collect the Clostridium mycelium from the seed culture, resuspend it in the same volume of RN(-)-Ar medium to wash away the nitrogen source in the original medium. Then, inoculate 1 mL, 2 mL, and 4 mL of the medium into 19 mL, 18 mL, and 16 mL of RN(-)-Ar fermentation medium, respectively, at inoculation ratios of 5%, 10%, and 20% of the culture medium volume. The culture system is 20 mL of culture medium / 120 mL vial, with argon as the headspace gas. The culture conditions are 37 ℃ incubation in the dark and static incubation, and the hydrogen production is monitored and measured. The RN(-)-Ar medium formulation is the same as above.

[0107] (3) The results are as follows Figure 4As shown, the hydrogen production of DSM525 did not change significantly with increasing inoculum size. Subsequent experiments used an inoculum size of 10%.

[0108] Example 5

[0109] (1) Clostridium pasteurellum ( Clostridium pasteurianum DSM 525 was used as the bacterial strain. DSM 525 was inoculated into RCVBN seed culture medium at an inoculum volume of 5% (v / v) to obtain the culture medium. The culture system consisted of 20 mL of culture medium per 120 mL vial. The headspace gas was replaced with nitrogen, and the culture was incubated at 37°C in the dark for 12 hours. The OD of the resulting DSM525 seed culture was... 600 =1.4. The RCVBN seed culture medium formula is the same as above.

[0110] (2) Prepare RN(-)-Ar medium with initial pH values ​​of 5.0, 5.5, 6.0, and 6.5. After centrifuging to collect the seed culture Clostridium cells, resuspend them in the same volume of RN(-)-Ar medium to wash away the nitrogen source in the original medium. Then, inoculate 2 mL of the resuspended solution into 18 mL of RN(-)-Ar fermentation medium at an inoculation ratio of 10% of the culture medium volume. The culture system is 20 mL of culture medium / 120 mL vial. The culture conditions are 37 ℃ incubation in the dark with argon as the headspace gas, and hydrogen production is monitored. The RN(-)-Ar medium formulation is the same as above.

[0111] (3) The results are as follows Figure 5 As shown: at an initial pH of 5.5, DSM525 achieved a maximum hydrogen production yield of 2.88 mol H2 / mol glucose.

[0112] Example 6

[0113] As shown in Example 1, after inoculating ZX-5 using the two-step method, hydrogen production was very low on days 1-2 (total time 3-4 days). During this period, ZX-5 utilized the nitrogen source in the culture medium for growth. Only after the system became nitrogen-deficient did it express nitrogenase and utilize it to produce hydrogen. This nitrogen-source-dependent growth resulted in a decrease in both hydrogen yield and efficiency. Considering that ZX-5's hydrogen production conditions require nitrogenase expression and a nitrogen-free system, the following experiment was conducted to ensure that ZX-5 could rapidly enter the hydrogen production phase after inoculation.

[0114] (1) Rhodopseudomonas aeruginosa ( Rhodobacter sphaeroidesZX-5 was used as the bacterial strain. It was inoculated into RB-2Gm medium at a 5% (v / v) inoculation rate to obtain the culture medium. The culture system consisted of 20 mL of culture medium per 120 mL vial. The headspace gas was replaced with nitrogen, and the culture was carried out at 30 ℃ and 8000 lux light intensity. Hydrogen production and growth were measured. The results are shown in Figure 6. ZX-5 grew rapidly on the first day but did not produce hydrogen. Growth stopped on the second day, and hydrogen production began. Hydrogen production increased rapidly on the third day, and biomass decreased. ZX-5 cells were harvested on days 1, 2, and 3 of culture, yielding seed cells in the logarithmic growth phase, pre-hydrogen production phase, and hydrogen production phase, respectively.

[0115] (2) The glucose in the RCVBN fermentation medium was adjusted to 1.1 g / L sodium butyrate, the headspace was adjusted to argon, and the nitrogen source was adjusted to 0 g / L sodium glutamate to obtain RBN(-)-Ar medium. The culture system was 20 mL culture medium / 120 mL vial. ZX-5 cells cultured in RB-2Gm seed medium for 1, 2, and 3 days were collected by centrifugation and analyzed according to the final OD. 600 =4 resuspended and inoculated into RBN(-)-Ar medium to obtain 20 mL of culture medium. Argon was used as the headspace gas, and the culture conditions were 30 ℃ and 8000 lux light. Hydrogen production and OD of the culture medium were monitored and measured. 600 .

[0116] The results are as follows Figure 7 As shown, centrifuging ZX-5 cells and placing them in a nitrogen-deficient culture medium with a headspace nitrogen-deficient atmosphere allows ZX-5 to rapidly enter the hydrogen production phase. Compared to cells inoculated in the logarithmic growth phase, cells inoculated in the early hydrogen production phase (2 days of culture) and the hydrogen production phase (3 days of culture) achieve higher hydrogen production rates in the initial stage. This may be because cells in the logarithmic growth phase store a certain amount of nitrogen source, which can still be used for growth and metabolism when transferred to a nitrogen-deficient environment, thus leading to a decrease in both the hydrogen production rate and the total hydrogen production. Further comparison between cells inoculated in the early hydrogen production phase (2 days of culture) and those inoculated in the hydrogen production phase (3 days of culture) reveals that after inoculating cells in the hydrogen production phase, the hydrogen production rate declines significantly from the second day, ultimately resulting in a 10.33% decrease in hydrogen conversion rate compared to cells inoculated in the early hydrogen production phase. Based on the above experimental results, it can be seen that the growth stage of ZX-5 cells has a significant impact on hydrogen production under nitrogen-deficient conditions. Inoculating ZX-5 cells in the early stage of hydrogen production (2 days of culture) can achieve the highest hydrogen production yield and the highest hydrogen production rate.

[0117] Example 7

[0118] (1) Rhodopseudomonas aeruginosa ( Rhodobacter sphaeroidesZX-5 was used as the bacterial strain. It was inoculated into RB-2Gm medium at a 5% (v / v) inoculation rate to obtain the culture medium. The culture system consisted of 20 mL of culture medium per 120 mL vial. The headspace was replaced with nitrogen gas, and the culture was carried out at 30 ℃ and 8000 lux light for 48 h to obtain the ZX-5 seed culture OD. 600 =5.0. The RB-2Gm medium formulation is the same as above.

[0119] (2) Prepare RBN(-)-Ar medium, with a culture system of 20 mL culture medium / 120 mL vial. After centrifuging to collect ZX-5 cells in the early hydrogen production stage, adjust the culture medium according to the initial OD of the culture medium. 600 =0.5, 1.0, 2.0, 3.0, and 4.0 mg were resuspended in RBN(-)-Ar medium. Argon was used as the headspace gas, and the culture conditions were 30 °C and 8000 lux light intensity. Hydrogen production was monitored. The RBN(-)-Ar medium formulation was the same as above.

[0120] (3) The results are as follows Figure 8 As shown: When using 0.8 g / L sodium glutamate as the nitrogen source, there was no significant difference in the maximum hydrogen production of ZX-5 with different inoculum amounts, and the maximum hydrogen yield was 5.66 mol H2 / mol sodium butyrate; when the culture system had no nitrogen source, the initial OD of the culture medium was... 600 When the inoculum size is between 0.5 and 2.0, the hydrogen production of ZX-5 increases significantly with increasing inoculum size, and the initial OD... 600 When the value is greater than 3.0, there is no significant difference in hydrogen production of ZX-5; the maximum hydrogen yield of ZX-5 in the nitrogen-free system is the initial OD. 600 The 6.84 mol H2 / mol sodium butyrate at a nitrogen source ratio of 3.0 represents a 20.84% ​​increase compared to the system with a nitrogen source. This result indicates that nitrogen source limitation can increase the total hydrogen production of ZX-5 by inhibiting ZX-5 growth and saving energy, while preserving the stimulation of nitrogenase expression and hydrogen production.

[0121] Example 8

[0122] (1) Rhodopseudomonas aeruginosa ( Rhodobacter sphaeroides ZX-5 was used as the bacterial strain. It was inoculated into RB-2Gm medium at a 5% (v / v) inoculation rate, with a culture system of 20 mL culture medium per 120 mL vial. The headspace was replaced with nitrogen. The culture was carried out at 30 ℃ under 8000 lux light for 48 h to obtain the ZX-5 seed culture OD. 600 =5.0. The RB-2Gm medium formulation is the same as above.

[0123] (2) Prepare RBN(-)-Ar medium and adjust the initial pH to 6.5, 7.0, 7.5, and 8.0. The culture system is 20 mL culture medium / 120 mL vial. After centrifuging to collect the seed culture ZX-5 cells, adjust the initial OD of the culture medium according to the ZX-5 standard. 600 =3.0 was resuspended in RBN(-)-Ar medium. Argon was used as the headspace gas, and the culture conditions were 30 ℃ and 8000 lux light. Hydrogen production was monitored. The RBN(-)-Ar medium formulation was the same as above.

[0124] (3) The results are as follows Figure 9 As shown: at an initial pH of 7.5, ZX-5 achieved a maximum hydrogen production yield of 6.97 mol H2 / mol sodium butyrate.

[0125] Example 9

[0126] (1) Clostridium pasteurellum ( Clostridium pasteurianum DSM 525 was used as the bacterial strain. It was inoculated into RCVBN seed culture medium at a 5% (v / v) inoculation rate. The headspace gas was replaced with nitrogen. The culture system consisted of 20 mL of culture medium per 120 mL vial. The culture was incubated at 37 ℃ in the dark for 12 h to obtain the OD of the DSM525 seed culture. 600 =1.4. The RCVBN seed culture medium formula is the same as above.

[0127] (2) Rhodopseudomonas aeruginosa ( Rhodobacter sphaeroides ZX-5 was used as the bacterial strain. It was inoculated into RB-2Gm medium at a 5% (v / v) inoculation rate, with a culture system of 20 mL culture medium per 120 mL vial. The headspace was replaced with nitrogen. The culture was carried out at 30 ℃ under 8000 lux light for 48 h to obtain the ZX-5 seed culture OD. 600 =5.0. The RB-2Gm medium formulation is the same as above.

[0128] (3) Prepare RN(-)-Ar-pH5.5 medium and R-Gm-N2-pH5.5 medium. After centrifuging to collect the seed culture Clostridium cells, resuspend them in the same volume of RN(-)-Ar-pH5.5 medium to wash away the nitrogen source in the original medium. Then, inoculate 2 mL of the resuspended solution into 18 mL of RN(-)-Ar or R-Gm-N2-pH5.5 medium, respectively. The culture system is 20 mL of culture medium / 120 mL vial. The culture conditions are 37℃ and static incubation in the dark for 1 day. The hydrogen production is then measured.

[0129] (4) After culturing the Clostridium fermentation broth for 1 day, adjust the pH to 7.5, replace the headspace gas with argon, and sterilize at 115 ℃ for 20 min. After centrifuging to collect the ZX-5 cells in the seed culture, add ZX-5 cells to the culture medium at the initial OD. 600 =3.0 Resuspend the sterilized Clostridium fermentation broth for inoculation. Culture conditions were 30 °C and 8000 lux light for 4 days. Hydrogen production was monitored and recorded, with the maximum hydrogen production count recorded.

[0130] (5) Results are as follows Figure 10 As shown: In nitrogen-free RN(-)-Ar-pH5.5 medium, after 5 days of two-step fermentation using DSM525 and ZX-5, the total hydrogen production yield was 9.81 mol H2 / mol glucose, which is 22.17% higher than that in nitrogen-containing R-Gm-N2-pH5.5 medium.

[0131] Traditional two-step fermentation processes are generally conducted under conditions favorable to bacterial growth, meaning the initial fermentation medium contains a nitrogen source. After the first step of Clostridium fermentation, the residual nitrogen source in the medium inhibits the expression of nitrogenase in photosynthetic bacteria, thus delaying the initiation of hydrogen production. This invention is the first to discover that Clostridium and photosynthetic bacteria can produce hydrogen immediately and continuously in a culture system with nitrogen-deficient medium and headspace nitrogen-deficient atmosphere, and the hydrogen production yield is significantly improved compared to nitrogen-containing systems. Based on this discovery, a nitrogen-free two-step fermentation system for hydrogen production was established. The hydrogen production yield of this system (9.81 mol H2 / mol glucose) is the highest level currently achieved in the field of research.

[0132] While nitrogen restriction can inhibit the growth of Clostridium / Rhodococcus-like bacteria, thus allowing for some predictability in advancing their hydrogen production phase, the metabolic pathways for glucose in Clostridium and for organic acids in Rhodococcus-like bacteria are not singular. Furthermore, under normal non-growth conditions, Clostridium / Rhodococcus-like bacteria are highly likely to convert limited organic substrates into intracellular components such as polysaccharides, starch, and PHB for storage, rather than continuously investing in hydrogen production metabolism. This undoubtedly leads to a reduction in hydrogen production (e.g., Clostridium's cessation of growth under phosphorus-deficient conditions results in a decrease in total hydrogen production). The innovation of this invention lies in the discovery that under the specific non-growth conditions of nitrogen source and nitrogen gas deficiency, the expression of nitrogenase in Clostridium and Rhodococcus-like bacteria promotes the preferential metabolism of glucose / organic acid substrates to generate sufficient reducing power to drive nitrogenase fixation. However, due to the absence of nitrogen as a substrate for nitrogenase, a large amount of reducing power cannot be consumed through the nitrogen fixation pathway and is forced to turn to the hydrogen production pathway, thus achieving a counter-trend increase in hydrogen production. This discovery cleverly reverses the adverse effects of unfavorable growth environments on fermentation production under conventional understanding of microbial fermentation.

[0133] Example 10

[0134] (1) Clostridium pasteurellum ( Clostridium pasteurianum DSM 525 was used as the bacterial strain. It was inoculated into RCVBN seed culture medium at a 5% (v / v) inoculation rate to obtain the culture medium. The culture system consisted of 20 mL of culture medium per 120 mL vial. The headspace was replaced with nitrogen gas, and the culture was incubated at 37°C in the dark for 12 h. The OD of the resulting DSM525 seed culture was... 600 =1.4. The RCVBN seed culture medium formula is the same as above.

[0135] (2) Rhodopseudomonas aeruginosa ( Rhodobacter sphaeroides ZX-5 was used as the bacterial strain. It was inoculated into RB-2Gm medium at a 5% (v / v) inoculation rate to obtain the culture medium. The culture system consisted of 20 mL of culture medium per 120 mL vial. The headspace was replaced with nitrogen gas, and the culture was carried out at 30 ℃ and 8000 lux light for 48 h. The OD of the resulting ZX-5 seed culture was... 600 =5.0. The RB seed culture medium formula is the same as above.

[0136] (3) Prepare sterile / non-sterilized RN(-)-Ar-pH5.5 and R-Gm-N2-pH5.5 media. After centrifuging to collect the seed culture Clostridium cells, resuspend them in the same volume of RN(-)-Ar-pH5.5 media to wash away the nitrogen source in the original medium. Then, inoculate 2 mL of the resuspended solution into 18 mL of RN(-)-Ar-pH5.5 or R-Gm-N2-pH5.5 media respectively. The culture system is 20 mL of culture medium / 120 mL vial. The culture conditions are 37 ℃ and static incubation in the dark for 1 day. The hydrogen production is measured. The composition of RN(-)-Ar-pH5.5 and R-Gm-N2-pH5.5 media is the same as above.

[0137] (4) After 1 day of cultivation, adjust the pH of the Clostridium fermentation broth to 7.5, replace the headspace gas with argon, and sterilize the sterilization group at 115℃ for 20 min. The non-sterilization group is not sterilized. After centrifuging to collect ZX-5 cells from the seed culture, adjust the initial OD of the culture medium according to the ZX-5 ratio. 600 =3.0 resuspension inoculation. Culture conditions were 30 ℃ and 8000 lux light. Hydrogen production was monitored and recorded, with the maximum hydrogen production count recorded.

[0138] (5) Results are as follows Figure 11As shown, in nitrogen-free RN(-)-Ar-pH5.5 medium, even without sterilization, the total hydrogen yield after two-step fermentation was 9.41 mol H2 / mol glucose, only 4.08% lower than the sterilized group (9.81 mol H2 / mol glucose). However, in nitrogen-rich R-Gm-N2-pH5.5 medium, without sterilization, the maximum total hydrogen yield after two-step fermentation was only 6.47 mol H2 / mol glucose, and the hydrogen content in the system decreased significantly after 3 days of culture, possibly due to infection with certain hydrogen-loving bacteria. This result demonstrates that the nitrogen-free RN(-)-Ar-pH5.5 culture system not only pushes the hydrogen production potential to near the theoretical limit through nitrogen limitation, but also spontaneously constructs a microecological barrier that is beneficial to hydrogen-producing Clostridium and inhibits exogenous bacteria due to the dual selection pressure of "nitrogen deficiency + argon headspace". This makes the system have high yield, operational robustness and low energy consumption, laying the technological foundation for subsequent sterile continuous fermentation or large-scale scale-up.

[0139] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nitrogen-limited co-fermentation method for hydrogen production from Clostridium pasteurellii and Rhodophyton floccosum, characterized in that... Includes the following steps: (1) Activated Clostridium pasteurellium was subjected to dark fermentation to produce hydrogen in a nitrogen-free medium and an argon atmosphere; (2) Next, the pH of the culture medium obtained in step (1) is adjusted to 7.0-8.0, selectively sterilized, and then activated Rhodopseudomonas spp. are inoculated and photo-fermented to produce hydrogen in an argon atmosphere. The *Clostridium pasteurellum* mentioned in step (1) is *Clostridium pasteurellum* (… Clostridium pasteurianum DSM 525; The Rhodococcus mentioned in step (2) is Rhodococcus ( Rhodobacter sphaeroides ZX-5; The nitrogen-free culture medium mentioned in step (1) is RN(-)-Ar medium; The composition of the RN(-)-Ar medium is as follows: 2 g / L glucose monohydrate, 50 mL / L basic salt solution, 20 mL / L 0.2 M PBS, 1 mL / L vitamin solution, pH=5.0~6.5, and deionized water as the solvent; The basic salt solution has the following composition: MgSO4·7H2O 4 g / L, CaCl2·H2O 1.5 g / L, FeSO4·7H2O 0.236 g / L, Na2EDTA 0.4 g / L, trace element solution 20 mL / L, and deionized water as the solvent. The trace element solution is composed of the following components: MnSO4·H2O 0.21 g / L, H3BO3 0.28 g / L, CuSO4·5H2O 0.004 g / L, ZnSO4·7H2O 0.024 g / L, NaMoO4·2H2O 0.075 g / L, with deionized water as the solvent. The vitamin solution contains the following components: niacin 10 g / L, vitamin B1 5 g / L, biotin 0.1 g / L, and deionized water as the solvent.

2. The method for hydrogen production by coupled fermentation of *Clostridium pasteurellii* and *Rhodotorula glutinis* based on nitrogen limitation according to claim 1, characterized in that: The activated Clostridium pasteurellium described in step (1) was prepared by the following steps: Clostridium pasteurellium was inoculated into a seed culture medium to obtain a culture solution, which was then cultured statically in a nitrogen atmosphere in the dark until the OD reached. 600 The concentration was 1.0–1.6; then solid-liquid separation was performed, and the obtained bacterial cells were washed with a nitrogen-free solvent to obtain activated Clostridium pasteurellii. The activated Rhodopseudomonas spp. described in step (2) is prepared by the following steps: Rhodopseudomonas spp. is inoculated into a seed culture medium to obtain a culture solution, which is then cultured under nitrogen atmosphere and light until the OD reaches 100°C. 600 The concentration was 4.5–5.5; then solid-liquid separation was performed, and the obtained bacterial cells were washed with a nitrogen-free solvent to obtain activated Rhodopseudomonas stolonifera.

3. The method for hydrogen production by coupled fermentation of Clostridium pasteurellii and Rhodophyton floccosum based on nitrogen limitation according to claim 2, characterized in that: In the step of preparing activated Pasteurella multocida: The seed culture medium is a nitrogen-containing culture medium; The inoculation amount is 5-10% of the culture medium volume; The nitrogen environment described refers to a headspace gas of nitrogen. The culture system is as follows: 20 mL culture medium / 120 mL vial; The culture temperature was 37±1℃; The culture time is 8–16 hours; The solid-liquid separation method is centrifugation; The nitrogen-free solvent mentioned refers to a solvent that does not contain nitrogen. In the steps of preparing activated Rhodopseudomonas stolonifer: The seed culture medium is a nitrogen-containing culture medium; The inoculation amount is 5-10% of the culture medium volume; The nitrogen environment described refers to a headspace gas of nitrogen. The culture system is as follows: 20 mL culture medium / 120 mL vial; The culture conditions are 30±1℃, 7500~8500 lux light intensity, and culture for 24~48h; The solid-liquid separation method is centrifugation; The nitrogen-free solvent mentioned refers to a solvent that does not contain nitrogen.

4. The method for hydrogen production by coupled fermentation of *Clostridium pasteurellii* and *Rhodophyta globosum* based on nitrogen limitation according to claim 3, characterized in that: In the step of preparing activated Pasteurella multocida: The seed culture medium is MS medium or RCVBN seed culture medium; The MS medium is composed of the following: glucose monohydrate 2 g / L, sodium glutamate 1 g / L, NaCl 5 g / L, K2HPO4 2 g / L, KH2PO4 0.5 g / L, trace element concentrate 10 mL / L, pH=6.0, and deionized water as the solvent. The composition of the RCVBN seed culture medium is as follows: 2 g / L glucose monohydrate, 5 g / L sodium glutamate, 50 mL / L basic salt solution, 20 mL / L 0.2 M PBS, 1 mL / L vitamin solution, pH=7.0, and deionized water as the solvent; The basic salt solution has the following composition: MgSO4·7H2O 4 g / L, CaCl2·H2O 1.5 g / L, FeSO4·7H2O 0.236 g / L, Na2EDTA 0.4 g / L, trace element solution 20 mL / L, and deionized water as the solvent. The composition of the trace element solution is as follows: MnSO4·H2O 0.21 g / L, H3BO3 0.28 g / L, CuSO4·5H2O 0.004 g / L, ZnSO4·7H2O 0.024 g / L, NaMoO4·2H2O 0.075 g / L, with deionized water as the solvent; The vitamin solution contains the following components: niacin 10 g / L, vitamin B1 5 g / L, biotin 0.1 g / L, and deionized water as the solvent. The nitrogen-free solvent is water, PBS, or nitrogen-free culture medium; In the steps of preparing activated Rhodopseudomonas stolonifer: The seed culture medium is RCVBN seed culture medium or RB-2Gm medium; The composition of RB-2Gm is as follows: sodium butyrate 1.1 g / L, sodium glutamate 2 g / L, basic salt solution 50 mL / L, 0.2 M PBS 20 mL / L, vitamin solution 1 mL / L, pH=7.0; The basic salt solution has the following composition: MgSO4·7H2O 4 g / L, CaCl2·H2O 1.5 g / L, FeSO4·7H2O 0.236 g / L, Na2EDTA 0.4 g / L, trace element solution 20 mL / L, and deionized water as the solvent. The composition of the trace element solution is as follows: MnSO4·H2O 0.21 g / L, H3BO3 0.28 g / L, CuSO4·5H2O 0.004 g / L, ZnSO4·7H2O 0.024 g / L, NaMoO4·2H2O 0.075 g / L, with deionized water as the solvent; The vitamin solution contains the following components: niacin 10 g / L, vitamin B1 5 g / L, biotin 0.1 g / L, and deionized water as the solvent. The culture conditions were 30±1℃ and 8000 lux light for 48 hours. The nitrogen-free solvent is water, PBS, or nitrogen-free culture medium.

5. The method for hydrogen production by coupled fermentation of *Clostridium pasteurellii* and *Rhodotorula glutinis* based on nitrogen limitation according to claim 1, characterized in that: The argon environment described in step (1) is a headspace gas of argon. The conditions for dark fermentation described in step (1) are 37±1℃ and static culture in the dark for 24 to 48 hours.

6. The method for hydrogen production by coupled fermentation of Clostridium pasteurellii and Rhodophyton floccosum based on nitrogen limitation according to claim 1, characterized in that: The inoculum amount of activated Rhodopseudomonas spp. described in step (2) is based on its initial OD value in the culture medium. 600 =Calculated from 2.5 to 3.5; The conditions for photo-fermentation hydrogen production described in step (2) are 30±1℃ and 7500~8500 lux light for 2~4 days.

7. The method for hydrogen production by coupled fermentation of *Clostridium pasteurellii* and *Rhodotorula glutinis* based on nitrogen limitation according to claim 6, characterized in that: The pH value mentioned in step (2) is 7.5; The sterilization conditions described in step (2) are sterilization at 115–121 °C for 15–20 min; The inoculum amount of activated Rhodopseudomonas spp. described in step (2) is based on its initial OD value in the culture medium. 600 =3.0 calculation; The conditions for photo-fermentation hydrogen production described in step (2) are 30±1℃ and 8000 lux light for 3 days.

8. The application of the nitrogen-limited Clostridium pasteurellium-Rhodophyton floccosum coupled fermentation hydrogen production method according to any one of claims 1 to 7 in hydrogen production.

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