Method for producing hydrogen by using straw-like biomass microorganism

Abstract

The application discloses a method for hydrogen production by using straw biomass microorganisms. The method comprises the following steps: crushing straw, and then sequentially performing hydrothermal acidification and hydrothermal alkalization pretreatment to obtain straw pretreatment liquid and alkalization residual solid; performing pH adjustment, adsorption detoxification and solid-liquid separation on the pretreatment liquid to obtain regulated pretreatment liquid; preparing the alkalization residual solid into a straw-based immobilized carrier, and immobilizing Clostridium butyricum which is gradiently domesticated by the regulated pretreatment liquid on the carrier to form an immobilized hydrogen production bacteria system; and mixing the immobilized hydrogen production bacteria system with the regulated pretreatment liquid to produce hydrogen by dark fermentation under anaerobic conditions. The application can improve the accessibility of straw substrate, reduce fermentation inhibition, and improve the hydrogen production stability of Clostridium butyricum and the hydrogen production amount per unit of straw.

Description

Technical Field

[0001] This invention relates to the field of biomass energy conversion technology, and in particular to a method for producing hydrogen using straw-based biomass microorganisms. Background Technology

[0002] Hydrogen energy, with its high calorific value and clean combustion products, is an important direction for renewable energy development and the construction of a low-carbon energy system. Microbial dark fermentation technology, which converts lignocellulosic biomass such as agricultural waste into hydrogen, offers advantages such as a wide range of raw material sources, mild reaction conditions, and high resource utilization. Crop straws such as corn straw, wheat straw, and rice straw contain a high proportion of cellulose and hemicellulose, theoretically serving as fermentation substrates for hydrogen-producing microorganisms. However, the cellulose, hemicellulose, and lignin in straw are intertwined, forming a dense and stable lignocellulosic composite structure. This makes it difficult for microorganisms to directly access and utilize the polysaccharide components, resulting in low substrate hydrolysis efficiency, slow fermentation start-up, and limited hydrogen production per unit of straw. Therefore, effectively breaking down the lignocellulosic barrier and increasing the release of fermentable sugars are key issues affecting hydrogen production efficiency in the straw microbial hydrogen production process.

[0003] Existing straw pretreatment methods mainly include acid treatment, alkali treatment, hydrothermal treatment, enzymatic hydrolysis, and combinations thereof. Acid treatment is beneficial for hemicellulose hydrolysis and partial sugar release, while alkali treatment is beneficial for lignin dissolution and improved cellulose accessibility. However, acid treatment alone often results in insufficient lignin removal and inadequate utilization of substrates for subsequent fermentation, while alkali treatment alone may lead to insufficient sugar release, high alkali consumption, and poor compatibility of the treated solution with fermentation. Furthermore, acid-alkali pretreatment may generate fermentation inhibitors such as furfural, 5-hydroxymethylfurfural, and phenolic substances, which adversely affect the growth and metabolic activity of hydrogen-producing microorganisms such as Clostridium butyricum. Traditional processes typically focus on the release of fermentable sugars in the pretreatment solution, while neglecting the control of pretreatment inhibitors, the adaptability of hydrogen-producing bacteria to the pretreatment solution, and the resource utilization of remaining solids. This results in room for improvement in the stability of the hydrogen production process and the efficiency of substrate conversion. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes a method for producing hydrogen using straw-based biomass microorganisms. The straw is pretreated by hydrothermal acidification and hydrothermal alkalization to obtain a regulated pretreatment solution and alkalized residual solids, respectively. The pretreatment solution is detoxified and regulated, and the residual solids are made into a straw-based immobilization carrier. Gradient domestication of Clostridium butyricum is used for immobilized fermentation to produce hydrogen.

[0005] This invention can be achieved through the following technical solutions: A method for producing hydrogen using straw-based biomass microorganisms includes the following steps: S1. After crushing the straw biomass, mix it with an acid solution for hydrothermal acidification pretreatment. After solid-liquid separation, collect the acid-treated solid. Mix the acid-treated solid with an alkaline solution for hydrothermal alkalization pretreatment. After solid-liquid separation, obtain the straw pretreatment liquid and the alkalization residue solid, respectively. S2. The straw pretreatment liquid is subjected to pH adjustment, adsorption detoxification and solid-liquid separation to obtain a regulated pretreatment liquid suitable for Clostridium butyricum fermentation; and the remaining solid after alkalization is washed, neutralized, dried and thermally activated to obtain a straw-based immobilization carrier. S3. The butyric acid bacteria were subjected to gradient acclimatization culture in an acclimatization medium containing a pretreatment solution to obtain an acclimatized butyric acid bacteria solution; the acclimatized butyric acid bacteria solution was then cultured in contact with a straw-based immobilization carrier to allow the butyric acid bacteria to attach to the surface and pores of the straw-based immobilization carrier, thus obtaining an immobilized hydrogen-producing bacteria system. S4. Mix the immobilized hydrogen-producing bacteria system, the pretreatment solution, and water, adjust the pH of the fermentation system to 6.0-7.0, carry out dark fermentation to produce hydrogen under anaerobic conditions, and collect the hydrogen produced during fermentation.

[0006] Preferably, the straw biomass in S1 is one or more of corn straw, wheat straw, rice straw, and sorghum straw; the straw biomass is dried at 60-85℃ before being crushed and then passed through a 40-80 mesh sieve.

[0007] Preferably, the acid solution in S1 is a hydrochloric acid solution with a concentration of 0.5–1.5 mol / L; the temperature of the hydrothermal acidification pretreatment is 100–130℃, the time is 3–4 h, and the solid-liquid ratio of straw biomass to acid solution is 1 g: (8–12) mL.

[0008] Preferably, the alkaline solution in S1 is a sodium hydroxide solution with a concentration of 0.05–0.3 mol / L; the temperature of the hydrothermal alkalization pretreatment is 110–130°C, the time is 3–4 h, and the solid-liquid ratio of the acid-treated solid to the alkaline solution is 1 g: (8–12) mL.

[0009] Preferably, the preparation of the straw-based immobilization carrier in step S2 includes: washing the remaining solid after alkalization to a pH of 6.5–7.5, drying it at 60–90°C, and then treating it at 120–180°C for 0.5–2 h to form a straw-based immobilization carrier with a porous structure.

[0010] Preferably, the gradient acclimatization culture in S3 includes sequentially inoculating Clostridium butyricum into acclimatization culture media containing 10%, 30%, 50%, and 70% volume fraction pretreatment solutions, and transferring the bacteria to the next acclimatization culture media after each stage of culture until the bacterial cells reach the logarithmic growth phase.

[0011] Preferably, in S3, the domesticated Clostridium butyricum bacterial solution and the straw-based immobilization carrier are cultured in contact at a volume-to-mass ratio of (5-20) mL:1 g, the contact culture temperature is 35-38℃, and the time is 12-24 h.

[0012] Preferably, the temperature for hydrogen production during dark fermentation in S4 is 35-38°C, and the stirring speed is 50-80 r / min; high-purity nitrogen gas is introduced into the fermentation system for 5-15 min before fermentation to establish an anaerobic environment, and the immobilized hydrogen-producing bacteria system, the pretreatment liquid, and water are mixed at a mass-volume ratio of 1 g: (20-60) mL: (10-40) mL.

[0013] The beneficial effects of this invention are: This invention pretreats straw biomass sequentially with hydrothermal acidification and hydrothermal alkalization, allowing hemicellulose hydrolysis and lignin removal to occur stepwise. This effectively disrupts the straw's lignocellulose complex structure, improving the release and accessibility of fermentable substrates. Simultaneously, the pretreated straw solution undergoes pH adjustment, adsorption detoxification, and solid-liquid separation to reduce the impact of fermentation inhibitors such as furfural, 5-hydroxymethylfurfural, and phenolic substances on Clostridium butyricum. The remaining solids from alkalization are washed, dried, and thermally activated to create a straw-based immobilized carrier, enabling the resource utilization of pretreatment byproducts. Through gradient acclimatization and immobilization, Clostridium butyricum can stably attach to the carrier surface and pores, improving its adaptability to the straw pretreated solution, cell density, and fermentation stability. The cumulative hydrogen production of this invention is at a high level overall, showing a significant improvement compared to untreated, single acid treatment, and only acid-base two-step pretreatment. This indicates that the invention can simultaneously address straw structure breakdown, fermentation inhibitor control, microbial cell adaptability enhancement, and hydrogen production efficiency improvement. It has the advantages of mild process conditions, wide availability of raw materials, high degree of waste resource utilization, and good stability of microbial hydrogen production. Attached Figure Description

[0014] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 The study investigated the cellulose content, lignin removal rate, and hydrogen production of straw under different pretreatment conditions. Detailed Implementation

[0015] The following provides a detailed description of the embodiments of the present invention: These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and processes. However, the scope of protection of the present invention is not limited to the following embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions.

[0016] Example 1: A method for producing hydrogen using straw-based biomass microorganisms, characterized by comprising the following steps: S1. Take corn stalks and dry them in an 80℃ constant temperature oven for 72 h. Then, pulverize them using a high-speed pulverizer and pass them through a 60-mesh sieve to obtain stalk powder. Weigh 100 g of stalk powder and add it to a 1.0 mol / L hydrochloric acid solution at a solid-liquid ratio of 1 g: 10 mL. After stirring and mixing evenly, place the mixture in a hydrothermal reactor and perform hydrothermal acidification pretreatment at 120℃ for 4 h. After the reaction, cool to room temperature, separate the solid and liquid, collect the acid-treated solid, and wash it with deionized water until the washing liquid is nearly neutral. Add the obtained acid-treated solid to a 0.1 mol / L sodium hydroxide solution at a solid-liquid ratio of 1 g: 10 mL, stir and mix evenly, and place the mixture in a hydrothermal reactor for hydrothermal alkalization pretreatment at 120℃ for 4 h. After the treatment, cool to room temperature, separate the solid and liquid, and obtain the stalk pretreatment liquid and the remaining alkalized solid. Adjust the pH of the stalk pretreatment liquid to 6.5, add activated carbon for adsorption and detoxification treatment (1.5% of the stalk pretreatment liquid mass), and stir at room temperature for 45 minutes. After min, solid-liquid separation was performed to obtain the conditioning pretreatment solution; the remaining solid after alkalization was washed with deionized water until the pH was 7.0, dried at 75℃ to constant weight, and then thermally activated at 150℃ for 1 h to obtain the straw-based immobilized carrier. S2. The overnight seed culture of Clostridium butyricum was inoculated into PYG liquid medium at an inoculation rate of 3% and cultured anaerobically at 37°C until the logarithmic phase. Subsequently, Clostridium butyricum was sequentially inoculated into acclimatization medium containing 10%, 30%, 50%, and 70% volume fraction pretreatment solution for gradient acclimatization culture. After each stage of culture reached the logarithmic phase, the culture was transferred to the next stage of acclimatization medium to obtain acclimatized Clostridium butyricum bacterial solution. The acclimatized Clostridium butyricum bacterial solution was mixed with straw-based immobilization carrier at a volume-to-mass ratio of 10 mL: 1 g and cultured in contact at 37°C for 18 h to allow Clostridium butyricum to attach to the surface and pores of the straw-based immobilization carrier, thus obtaining an immobilized hydrogen-producing bacteria system. S3. The immobilized hydrogen-producing bacteria system, the pretreatment solution, and deionized water were added to a glass fermenter at a mass-to-volume ratio of 1 g: 40 mL: 25 mL. The pH of the fermentation system was adjusted to 6.5. High-purity nitrogen gas was introduced into the fermenter for 10 min to replace oxygen and establish an anaerobic environment. After sealing, the fermentation was carried out in the dark at 37℃ and 60 r / min to produce hydrogen. During the fermentation process, the fermentation gas was collected by water displacement and gas chromatography was used to detect the hydrogen content in the gas. The cumulative hydrogen production per unit of straw was calculated.

[0017] Example 2: A method for producing hydrogen using straw-based biomass microorganisms, characterized by comprising the following steps: S1. Take corn stalks, dry them at 60℃ to constant weight, pulverize them using a high-speed pulverizer, and pass them through a 40-mesh sieve to obtain stalk powder. Weigh 100 g of stalk powder and add it to 0.5 mol / L hydrochloric acid solution at a solid-liquid ratio of 1 g: 8 mL. After stirring and mixing evenly, place the mixture in a hydrothermal reactor and perform hydrothermal acidification pretreatment at 110℃ for 3 h. After the reaction, cool to room temperature, separate the solid and liquid, collect the acid-treated solid, and wash it with deionized water until the washing liquid is close to neutral. Add the obtained acid-treated solid to 0.05 mol / L sodium hydroxide solution at a solid-liquid ratio of 1 g: 8 mL, stir and mix evenly, and place the mixture in a hydrothermal reactor and perform hydrothermal alkalization pretreatment at 110℃ for 3 h. After the treatment, cool to room temperature, separate the solid and liquid, and obtain stalk pretreatment liquid and alkalization residue solid, respectively. Adjust the pH of the stalk pretreatment liquid to 6.0, add activated carbon for adsorption and detoxification treatment, with the amount of activated carbon added being 1.0% of the mass of the stalk pretreatment liquid. Stir at room temperature for 30 minutes. After min, solid-liquid separation was performed to obtain the pretreated solution. The remaining solid after alkalization was washed with deionized water until the pH was 6.5, dried at 60℃ to constant weight, and then thermally activated at 120℃ for 0.5 h to obtain the straw-based immobilized carrier. S2. The overnight seed culture of Clostridium butyricum was inoculated into PYG liquid medium at an inoculation rate of 3% and cultured anaerobically at 35°C until the logarithmic phase. Subsequently, Clostridium butyricum was sequentially inoculated into acclimatization medium containing 10%, 30%, 50%, and 70% volume fraction pretreatment solution for gradient acclimatization culture. After each stage of culture reached the logarithmic phase, the culture was transferred to the next stage of acclimatization medium to obtain acclimatized Clostridium butyricum bacterial culture. The acclimatized Clostridium butyricum bacterial culture was mixed with straw-based immobilization carrier at a volume-to-mass ratio of 5 mL:1 g and cultured in contact at 35°C for 12 h to allow Clostridium butyricum to attach to the surface and pores of the straw-based immobilization carrier, thus obtaining an immobilized hydrogen-producing bacteria system. S3. The immobilized hydrogen-producing bacteria system, the pretreatment solution, and deionized water were added to a glass fermenter at a mass-to-volume ratio of 1 g: 20 mL: 10 mL. The pH of the fermentation system was adjusted to 6.0. High-purity nitrogen was introduced into the fermenter for 5 min to replace oxygen and establish an anaerobic environment. After sealing, the fermentation was carried out in the dark at 35℃ and 50 r / min to produce hydrogen. During the fermentation process, the fermentation gas was collected by water displacement and gas chromatography was used to detect the hydrogen content in the gas. The cumulative hydrogen production per unit of straw was calculated.

[0018] Example 3: A method for producing hydrogen using straw-based biomass microorganisms, characterized by comprising the following steps: S1. Take corn stalks, dry them at 85℃ to constant weight, pulverize them using a high-speed pulverizer, and pass them through an 80-mesh sieve to obtain stalk powder. Weigh 100 g of stalk powder and add it to 1.5 mol / L hydrochloric acid solution at a solid-liquid ratio of 1 g: 12 mL. After stirring and mixing evenly, place the mixture in a hydrothermal reactor and perform hydrothermal acidification pretreatment at 130℃ for 4 h. After the reaction, cool to room temperature, separate the solid and liquid, collect the acid-treated solid, and wash it with deionized water until the washing liquid is close to neutral. Add the obtained acid-treated solid to 0.3 mol / L sodium hydroxide solution at a solid-liquid ratio of 1 g: 12 mL, stir and mix evenly, and place the mixture in a hydrothermal reactor and perform hydrothermal alkalization pretreatment at 130℃ for 4 h. After the treatment, cool to room temperature, separate the solid and liquid, and obtain stalk pretreatment liquid and alkalization residue solid, respectively. Adjust the pH of the stalk pretreatment liquid to 7.0, add activated carbon for adsorption and detoxification treatment, with the amount of activated carbon added being 2.0% of the mass of the stalk pretreatment liquid. Stir at room temperature for 60 minutes. After min, solid-liquid separation was performed to obtain the pretreated solution. The remaining solid after alkalization was washed with deionized water until the pH was 7.5, dried at 90℃ to constant weight, and then thermally activated at 180℃ for 2 h to obtain the straw-based immobilized carrier. S2. The overnight seed culture of Clostridium butyricum was inoculated into PYG liquid medium at an inoculation rate of 3% and cultured anaerobically at 38°C until the logarithmic phase. Subsequently, Clostridium butyricum was sequentially inoculated into acclimatization medium containing 10%, 30%, 50%, and 70% volume fraction pretreatment solution for gradient acclimatization culture. After each stage of culture reached the logarithmic phase, the culture was transferred to the next stage of acclimatization medium to obtain acclimatized Clostridium butyricum bacterial culture. The acclimatized Clostridium butyricum bacterial culture was mixed with straw-based immobilization carrier at a volume-to-mass ratio of 20 mL: 1 g and cultured in contact at 38°C for 24 h to allow Clostridium butyricum to attach to the surface and pores of the straw-based immobilization carrier, thus obtaining an immobilized hydrogen-producing bacteria system. S3. The immobilized hydrogen-producing bacteria system, the pretreatment solution, and deionized water were added to a glass fermenter at a mass-to-volume ratio of 1 g: 60 mL: 40 mL. The pH of the fermentation system was adjusted to 7.0. High-purity nitrogen gas was introduced into the fermenter for 15 min to replace oxygen and establish an anaerobic environment. After sealing, the fermentation was carried out in the dark at 38℃ and 80 r / min to produce hydrogen. During the fermentation process, the fermentation gas was collected by water displacement and gas chromatography was used to detect the hydrogen content in the gas. The cumulative hydrogen production per unit of straw was calculated.

[0019] Comparative Example 1: The difference between this comparative example and Example 1 is that untreated straw powder was directly used for dark fermentation to produce hydrogen.

[0020] This comparative method for hydrogen production using straw-based biomass microorganisms includes the following steps: S1. Take corn stalks and dry them in an 80℃ constant temperature oven for 72 h. After crushing them with a high-speed pulverizer, pass them through a 60-mesh sieve to obtain stalk powder. Weigh 100 g of stalk powder, add deionized water to prepare a stalk suspension, and adjust the pH to 6.5 as a fermentation substrate. S2. The overnight seed culture of Clostridium butyricum was inoculated into PYG liquid medium at an inoculum rate of 3% and cultured anaerobically at 37°C to the logarithmic phase to obtain Clostridium butyricum culture solution. The Clostridium butyricum culture solution was inoculated into the straw suspension obtained in step S1. High-purity nitrogen gas was introduced into the fermentation system for 10 min to replace oxygen and establish an anaerobic environment. After sealing, the system was subjected to dark fermentation at 37°C and 60 r / min to produce hydrogen. During the fermentation process, the fermentation gas was collected by water displacement and gas chromatography was used to detect the hydrogen content in the gas. The cumulative hydrogen production per unit straw was calculated.

[0021] Comparative Example 2: The difference between this comparative example and Example 1 is that only hydrothermal acidification pretreatment was performed.

[0022] This comparative method for hydrogen production using straw-based biomass microorganisms includes the following steps: S1. Take corn stalks and dry them in an 80℃ constant temperature oven for 72 h. After pulverizing them with a high-speed pulverizer, pass them through a 60-mesh sieve to obtain stalk powder. Weigh 100 g of stalk powder and add 1.0 mol / L hydrochloric acid solution at a solid-liquid ratio of 1 g: 10 mL. Stir and mix evenly, then place the mixture in a hydrothermal reactor and perform hydrothermal acidification pretreatment at 120℃ for 4 h. After the reaction is completed, cool to room temperature, separate the solid and liquid, collect the acid treatment liquid, and adjust the pH of the acid treatment liquid to 6.5 as the fermentation substrate. S2. The overnight seed culture of Clostridium butyricum was inoculated into PYG liquid medium at an inoculum rate of 3% and cultured anaerobically at 37°C to the logarithmic phase to obtain Clostridium butyricum culture. The Clostridium butyricum culture was then inoculated into the fermentation substrate obtained in step S1. High-purity nitrogen gas was introduced into the fermentation system for 10 min to replace oxygen and establish an anaerobic environment. After sealing, the system was subjected to dark fermentation at 37°C and 60 r / min to produce hydrogen. During the fermentation process, the fermentation gas was collected by water displacement and the hydrogen content in the gas was detected by gas chromatography. The cumulative hydrogen production per unit of straw was calculated.

[0023] Comparative Example 3: The difference between this comparative example and Example 1 is that the acid-base two-step pretreatment solution is used to directly carry out the dark fermentation of free Clostridium butyricum to produce hydrogen.

[0024] This comparative method for hydrogen production using straw-based biomass microorganisms includes the following steps: S1. Take corn stalks and dry them in an 80℃ constant temperature oven for 72 h. After pulverizing them with a high-speed pulverizer, pass them through a 60-mesh sieve to obtain stalk powder. Weigh 100 g of stalk powder and add 1.0 mol / L hydrochloric acid solution at a solid-liquid ratio of 1 g: 10 mL. Stir and mix evenly, then place the mixture in a hydrothermal reactor and perform hydrothermal acidification pretreatment at 120℃ for 4 h. After the reaction, cool to room temperature, separate the solid and liquid, collect the acid-treated solid, and wash it with deionized water until the washing liquid is close to neutral. Add the obtained acid-treated solid to 0.1 mol / L sodium hydroxide solution at a solid-liquid ratio of 1 g: 10 mL, stir and mix evenly, then place the mixture in a hydrothermal reactor and perform hydrothermal alkalization pretreatment at 120℃ for 4 h. After the treatment, cool to room temperature, separate the solid and liquid, collect the stalk pretreatment liquid, adjust the pH of the stalk pretreatment liquid to 6.5, and use it as a fermentation substrate. The remaining solid after alkalization is not used to prepare the immobilization carrier. S2. The overnight seed culture of Clostridium butyricum was inoculated into PYG liquid medium at an inoculum rate of 3% and cultured anaerobically at 37°C to the logarithmic phase to obtain Clostridium butyricum culture. The Clostridium butyricum culture was then inoculated into the fermentation substrate obtained in step S1. High-purity nitrogen gas was introduced into the fermentation system for 10 min to replace oxygen and establish an anaerobic environment. After sealing, the system was subjected to dark fermentation at 37°C and 60 r / min to produce hydrogen. During the fermentation process, the fermentation gas was collected by water displacement and the hydrogen content in the gas was detected by gas chromatography. The cumulative hydrogen production per unit of straw was calculated.

[0025] Performance testing 1. Hemicellulose content The determination was carried out in accordance with the NY / T3494-2019 standard.

[0026] 2. Lignin removal rate The determination was carried out in accordance with the NY / T3494-2019 standard.

[0027] 3. Hydrogen production Hydrogen production was determined using a combination of water displacement and gas chromatography. Fermentation systems obtained in Examples 1-3 and Comparative Examples 1-3 were used for hydrogen production experiments under their respective anaerobic dark fermentation conditions. During fermentation, the gas produced was collected periodically using a water displacement device, and the gas volume at each time point was recorded. Fermentation was terminated when the gas production did not change significantly over two consecutive recording periods. The collected gas volume was converted to standard gas volume based on the ambient temperature and atmospheric pressure at the time of measurement. Gas samples were taken from the collection device and injected into the gas chromatograph using a gas-tight syringe. The hydrogen integral was detected using a thermal conductivity detector. A molecular sieve 5A column was used, with high-purity nitrogen as the carrier gas. A standard curve was established based on hydrogen standard gases, and the hydrogen integral in the fermentation gas was calculated. The cumulative hydrogen production per unit of straw was calculated using the formula H = Σ(Vi × Ci) / M, where H is the cumulative hydrogen production per unit of straw (mL / g); Vi is the gas production volume of the i-th sampling and converted to standard conditions (mL); Ci is the hydrogen gas fraction in the i-th gas sample; and M is the dry weight of straw in the corresponding fermentation system (g). Three parallel samples were set up for each group, and the results were averaged.

[0028] Table 1. Hemicellulose content, lignin removal rate, and hydrogen production in straw.

[0029] As shown in Table 1, the present invention exhibits good straw structure breaking and microbial hydrogen production effects under different pretreatment intensities, detoxification conditions, immobilization carrier preparation conditions, and fermentation parameters. The cumulative hydrogen production of the three sets of examples reached 36.8 mL / g, 28.7 mL / g, and 33.5 mL / g, respectively, which are at a relatively high level, indicating that the process of the present invention has good applicability and stability. In Examples 1-3, the hemicellulose content was reduced to 8.32%, 11.05%, and 7.94%, respectively, and the lignin removal rates reached 45.16%, 34.82%, and 51.37%, respectively. This indicates that hydrothermal acidification and hydrothermal alkalization treatments can effectively destroy the lignocellulose complex structure of straw and improve substrate accessibility. Simultaneously, adsorption and detoxification of the straw pretreatment solution can reduce the adverse effects of inhibitory substances generated during pretreatment on the growth and metabolism of Clostridium butyricum. By preparing the alkalization residue as a straw-based immobilization carrier and combining it with gradient acclimatization and immobilization culture of Clostridium butyricum, the adaptability, attachment stability, and fermentation utilization efficiency of the bacteria to the straw pretreatment solution can be improved. Therefore, Examples 1-3 can all achieve high cumulative hydrogen production per unit of straw under different process conditions, demonstrating a good synergistic effect between the acid-alkali two-step pretreatment, pretreatment solution detoxification, residue solid carrier formation, and Clostridium butyricum acclimatization and immobilization of the present invention.

[0030] Compared to Example 1, Comparative Example 1, which used untreated straw for direct fermentation, showed a significant decrease in cumulative hydrogen production to 1.7 mL / g. This was primarily because the untreated straw retained a dense lignocellulose structure with a hemicellulose content as high as 21.89%, resulting in a lignin removal rate of 0%. Clostridium butyricum struggled to directly access and utilize the fermentable sugar components, leading to extremely low hydrogen production efficiency. Comparative Example 2, which underwent only hydrothermal acidification pretreatment, achieved a cumulative hydrogen production of 11.2 mL / g, a significant decrease compared to Example 1. This was because while acid treatment promoted hemicellulose hydrolysis, reducing the hemicellulose content to 10.27%, the lignin removal rate was only 11.23%. The lignin barrier in the straw was not sufficiently disrupted, limiting the utilization of the fermentation substrate and thus limiting the increase in hydrogen production. Comparative Example 3 employed a two-step acid-alkali pretreatment, reducing the hemicellulose content to 8.36%, increasing the lignin removal rate to 44.73%, and achieving a cumulative hydrogen production of 26.3 mL / g. However, this was still lower than that of Example 1. This was because it did not undergo straw pretreatment liquid adsorption and detoxification treatment. Residual furfural, 5-hydroxymethylfurfural, and phenolic substances in the pretreatment liquid may inhibit the growth and hydrogen production metabolism of Clostridium butyricum. In addition, Comparative Example 3 did not prepare the remaining solids from alkalization into a straw-based immobilization carrier, nor did it conduct gradient acclimatization and immobilization culture of Clostridium butyricum. Therefore, the adaptability of the bacteria to the pretreatment liquid, its attachment stability, and the sustainability of fermentation were all inferior to those of Example 1.

[0031] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for producing hydrogen using straw-based biomass microorganisms, characterized in that, Includes the following steps: S1. After crushing the straw biomass, mix it with an acid solution for hydrothermal acidification pretreatment. After solid-liquid separation, collect the acid-treated solid. Mix the acid-treated solid with an alkaline solution for hydrothermal alkalization pretreatment. After solid-liquid separation, obtain the straw pretreatment liquid and the alkalization residue solid, respectively. S2. The straw pretreatment liquid is subjected to pH adjustment, adsorption detoxification and solid-liquid separation to obtain a regulated pretreatment liquid suitable for Clostridium butyricum fermentation; and the remaining solid after alkalization is washed, neutralized, dried and thermally activated to obtain a straw-based immobilization carrier. S3. The butyric acid bacteria were subjected to gradient acclimatization culture in an acclimatization medium containing a pretreatment solution to obtain an acclimatized butyric acid bacteria solution; the acclimatized butyric acid bacteria solution was then cultured in contact with a straw-based immobilization carrier to allow the butyric acid bacteria to attach to the surface and pores of the straw-based immobilization carrier, thus obtaining an immobilized hydrogen-producing bacteria system. S4. Mix the immobilized hydrogen-producing bacteria system, the pretreatment solution, and water, adjust the pH of the fermentation system to 6.0-7.0, carry out dark fermentation to produce hydrogen under anaerobic conditions, and collect the hydrogen produced during fermentation.

2. The method for producing hydrogen from straw-based biomass microorganisms according to claim 1, characterized in that, The straw biomass in S1 is one or more of corn straw, wheat straw, rice straw, and sorghum straw; the straw biomass is dried at 60-85℃ before being crushed and then passed through a 40-80 mesh sieve.

3. The method for producing hydrogen from straw-based biomass microorganisms according to claim 1, characterized in that, The acid solution in S1 is a hydrochloric acid solution with a concentration of 0.5–1.5 mol / L; the temperature of the hydrothermal acidification pretreatment is 100–130℃, the time is 3–4 h, and the solid-liquid ratio of straw biomass to acid solution is 1 g: (8–12) mL.

4. The method for producing hydrogen from straw-based biomass microorganisms according to claim 1, characterized in that, The alkaline solution in S1 is a sodium hydroxide solution with a concentration of 0.05–0.3 mol / L; the hydrothermal alkalization pretreatment is carried out at a temperature of 110–130℃ for 3–4 h, and the solid-liquid ratio of the acid-treated solid to the alkaline solution is 1 g: (8–12) mL.

5. The method for producing hydrogen from straw-based biomass microorganisms according to claim 1, characterized in that, The preparation of the straw-based immobilized carrier in S2 includes: washing the remaining solid after alkalization to a pH of 6.5–7.5, drying it at 60–90°C, and then treating it at 120–180°C for 0.5–2 h to form a straw-based immobilized carrier with a porous structure.

6. The method for producing hydrogen from straw-based biomass microorganisms according to claim 1, characterized in that, The gradient acclimatization culture in S3 includes sequentially inoculating Clostridium butyricum into acclimatization culture media containing 10%, 30%, 50%, and 70% volume fraction pretreatment solutions, and transferring the bacteria to the next acclimatization culture media after each stage of culture until the bacterial growth reaches the logarithmic phase.

7. The method for producing hydrogen from straw-based biomass microorganisms according to claim 1, characterized in that, In the S3, the domesticated Clostridium butyricum bacterial solution and the straw-based immobilization carrier are cultured in contact at a volume-to-mass ratio of (5-20) mL:1 g, with a contact culture temperature of 35-38℃ and a time of 12-24 h.

8. The method for producing hydrogen from straw-based biomass microorganisms according to claim 1, characterized in that, The temperature for hydrogen production during dark fermentation in S4 is 35-38℃, and the stirring speed is 50-80 r / min. Before fermentation, high-purity nitrogen gas is introduced into the fermentation system for 5-15 min to establish an anaerobic environment. The immobilized hydrogen-producing bacteria system, the pretreatment liquid, and water are mixed at a mass-volume ratio of 1 g: (20-60) mL: (10-40) mL.

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