Method for producing hydrogen by dark fermentation of pericarp biomass

Abstract

The invention belongs to the technical field of biological fermentation hydrogen production. The invention provides a method for producing hydrogen by dark fermentation of pericarp biomass, which comprises the following steps: drying pericarp, crushing, and sieving to obtain powdery pericarp particles; performing acid heat pretreatment on the powdery fruit peel particles to obtain fruit peel pretreatment liquid; and inoculating the clostridium butyricum solution into the pericarp pretreatment liquid, and carrying out dark fermentation under an anaerobic condition to obtain a fermentation gas containing hydrogen. According to the method, the pericarp byproducts are used for dark fermentation for hydrogen production, so that high-value recycling utilization of agricultural wastes is realized; lignin in the pericarp is effectively removed through acid heat pretreatment, the availability of cellulose and hemicellulose is remarkably improved, conditions such as acid concentration, temperature and time are optimized, the inhibition effect of by-products is avoided, and the hydrogen production efficiency is remarkably improved; moreover, the method has the advantages of good stability and high repeatability, and provides a new technical approach for efficient production of clean energy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological fermentation hydrogen production, and particularly relates to a method for dark fermentation hydrogen production by using peel biomass. BACKGROUND

[0002] Hydrogen has the advantages of high combustion heat value, combustion product being water, and almost no emission of pollutants, and is considered as an important way to realize energy structure transformation. In the fields of transportation, energy storage, electricity and chemical industry, hydrogen energy has shown great application potential. With the increasing of energy crisis and environmental problems, how to produce hydrogen on a large scale, economically and sustainably has become a key problem to be solved in the field of energy science and engineering.

[0003] At present, the preparation methods of hydrogen mainly include fossil energy hydrogen production, water electrolysis hydrogen production and biomass hydrogen production. The fossil energy hydrogen production process is mature and has large output, but it emits a large amount of carbon dioxide, which has heavy environmental burden and is contrary to the development direction of green and low carbon. Water electrolysis hydrogen production can obtain high-purity hydrogen, especially when combined with renewable energy, but it has high energy consumption and large cost, and still has the problem of insufficient economy. In comparison, biomass hydrogen production has attracted more and more attention in recent years because of its wide range of raw materials, low price and strong renewability, and can realize the resource utilization of waste, and is considered as one of the most potential hydrogen production methods.

[0004] Among the many available biomass resources, agricultural and food processing by-products have become the focus of biomass hydrogen production research because of their huge output, stable source and low value processing. For example, fruit and vegetable processing, fruit juice beverage and food industry will produce a large amount of peel by-products in the production process. If these peel resources are not properly treated, not only will they cause waste, but also may cause environmental pollution. The peel is rich in organic carbon sources, including a certain proportion of soluble sugars and a high content of cellulose and hemicellulose, which has great energy conversion potential in theory. However, at the same time, the peel has complex tissue structure, high lignin content, and often contains essential oils, flavonoids and other bioactive or antibacterial ingredients. These characteristics make it difficult for peel biomass to be directly and efficiently utilized by microorganisms, resulting in low resource utilization efficiency.

[0005] In the prior art, the processing and utilization of fruit peel by-products have been studied to some extent. For example, common methods include direct composting as organic fertilizer, or producing biogas through anaerobic digestion, and serving as a feed additive, etc. These methods achieve the utilization of waste to some extent, but have problems such as low added value, insufficient energy conversion efficiency, and single utilization mode. In particular, in terms of energy utilization, fruit peel biomass has not yet formed a mature and effective conversion path. Compared with direct composting or biogasification, if fruit peel can be converted into hydrogen, a high-value clean energy, not only the economic benefits can be improved, but also the development of clean energy industry can be further promoted.

[0006] Studies have shown that crop straw, food waste, fruit and vegetable residues and other raw materials can be used for hydrogen production, but the overall hydrogen production efficiency and stability are still not ideal. The reasons mainly lie in the following aspects: first, the structure of raw materials is complex. Cellulose and hemicellulose of fruit peel biomass are mostly embedded in lignin, which is difficult for microorganisms to directly degrade and utilize. If the treatment condition is too light, lignin removal is insufficient, substrate utilization rate is low, and hydrogen production is low. If the condition is too heavy, by-products such as furfural and hydroxymethyl furfural are easily produced, which inhibits the growth of microorganisms, further limiting the hydrogen production efficiency. Second, the process conditions are difficult to control stably. The process of biomass hydrogen production involves the coupling regulation of pH, temperature, substrate concentration, stirring rate and other factors. These conditions are interrelated and highly sensitive. Once they deviate from the appropriate range, the metabolic pathway efficiency will be reduced or hydrogen will be consumed again. In addition, the adaptability and activity of microbial strains are also important factors that restrict the hydrogen production efficiency of biomass. Different types of microorganisms have differences in substrate utilization ability, environmental tolerance and hydrogen production metabolic pathways. When encountering complex substrates such as fruit peel, the hydrogen production performance of some strains is limited, and even the activity will be affected by the inhibitory components in the raw materials.

[0007] In summary, although biomass hydrogen production has received widespread attention in academia and industry, and has made some progress in raw material utilization and process optimization, there are still some problems in the hydrogen production of fruit peel biomass, such as obvious limitation of raw material structure, insufficient release of available carbon sources, low hydrogen production rate, poor stability of hydrogen production process, and difficulty in long-term maintenance of fermentation conditions and microbial activity.

[0008] Therefore, the development of a method for dark fermentation hydrogen production using fruit peel biomass can not only improve the added value of fruit peel by-products and promote resource recycling, but also provide a new technical approach for clean energy production. SUMMARY

[0009] The present application aims at the problems of low direct utilization rate of fruit peel biomass, insufficient hydrogen production rate and poor process stability in the prior art, and provides a new method for hydrogen production by dark fermentation of fruit peel biomass.

[0010] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions. The present application provides a method for hydrogen production by dark fermentation of fruit peel biomass, comprising the following steps: 1) drying the fruit peel, crushing and sieving the dried fruit peel to obtain powder-like fruit peel particles; 2) acid-heat pretreating the powder-like fruit peel particles to obtain a fruit peel pretreatment solution; 3) inoculating a Clostridium butyricum solution into the fruit peel pretreatment solution and performing dark fermentation under anaerobic conditions, to obtain a hydrogen-containing fermentation gas.

[0011] Preferably, the fruit peel in step 1) is citrus peel. The drying temperature is 50-70 DEG C, and the drying time is 40-60 h. The mesh number of the sieving is 50-70.

[0012] Preferably, the acid-heat pretreatment in step 2) uses hydrochloric acid or sulfuric acid, the concentration of the acid is 0.5-1.5 mol / L, and the mass-volume ratio of the powder-like fruit peel particles to the acid is 1-2 g:18-25 mL.

[0013] Preferably, the temperature of the acid-heat pretreatment in step 2) is 120-140 DEG C, and the acid-heat pretreatment time is 30-60 min.

[0014] Preferably, the preparation method of the Clostridium butyricum solution in step 3) comprises the following steps: (1) activating Clostridium butyricum in a PYG culture medium to obtain an activated Clostridium butyricum solution; (2) inoculating the activated Clostridium butyricum solution into a new PYG culture medium, culturing under anaerobic conditions until the cells reach the middle logarithmic phase, then removing the supernatant by centrifugation, and resuspending the bacterial precipitate with a salt solution to obtain a Clostridium butyricum solution.

[0015] Preferably, the activation temperature in step (1) is 30-40 DEG C, and the activation time is 20-30 h. In step (2), the inoculation amount of the activated Clostridium butyricum solution is 2-4% (volume fraction) of the volume of the PYG culture medium, the culture temperature is 30-40 DEG C, and the culture time is 10-14 h.

[0016] Preferably, the anaerobic conditions in step (2) are anaerobic environments established by inert gas replacement, wherein the inert gas is nitrogen and the replacement time is 5 to 10 minutes. The centrifugation speed is 4500~5500 r / min, and the centrifugation time is 8~12 min.

[0017] Preferably, the volume ratio of the Clostridium butyricum solution to the pretreatment solution in step 3) is 4~5:10~12.

[0018] Preferably, the anaerobic conditions in step 3) are anaerobic environments established by inert gas replacement, wherein the inert gas is nitrogen, and the replacement time is 5-10 minutes.

[0019] Preferably, the dark fermentation in step 3) is carried out under the conditions of a water bath temperature of 35~38℃ and a stirring speed of 50~80r / min.

[0020] The beneficial effects of this invention include the following: 1) This invention utilizes fruit peel by-products for dark fermentation to produce hydrogen, realizing the high-value utilization of agricultural waste, increasing the added value of fruit peel resources, and promoting resource recycling.

[0021] 2) This invention effectively removes lignin from the pericarp through acid-heat pretreatment, improving the availability of cellulose and hemicellulose, thereby significantly enhancing the microbial decomposition of substrates and hydrogen production capacity.

[0022] 3) This invention optimizes key parameters such as acid concentration, pretreatment temperature and time, avoiding the problems of low substrate utilization due to excessively light conditions or the generation of inhibitory byproducts due to excessively heavy conditions, thus ensuring a high hydrogen yield.

[0023] 4) The hydrogen production yield obtained by this invention reaches 35.1~39.3 mL / g TS, which is significantly higher than that of the comparative example, with an improvement of more than 50%, and is significantly better than the prior art.

[0024] 5) The method described in this invention maintains a high and stable hydrogen production level under different conditions, and has good repeatability and process stability, providing a new technical approach for the efficient production of clean energy. Detailed Implementation

[0025] This invention provides a method for producing hydrogen through dark fermentation of fruit peel biomass, comprising the following steps: 1) After drying the fruit peel, crush and sieve it to obtain powdered fruit peel particles; 2) The powdered fruit peel particles are subjected to acid and heat pretreatment to obtain a fruit peel pretreatment solution; 3) The Clostridium butyricum solution was inoculated into the pretreated fruit peel solution and subjected to dark fermentation under anaerobic conditions to obtain fermentation gas containing hydrogen.

[0026] In this invention, the peel mentioned in step 1) is preferably the peel of citrus fruits, and more preferably the peel of navel oranges; The drying temperature is preferably 50~70℃, more preferably 55~65℃, and even more preferably 60℃; the drying time is preferably 40~60h, more preferably 45~55h, and even more preferably 50h. The preferred mesh size for sieving is 50-70 mesh, and more preferably 60 mesh.

[0027] In this invention, the acid used in the acid-heat pretreatment in step 2) is preferably hydrochloric acid or sulfuric acid; the concentration of the acid is preferably 0.5~1.5 mol / L, more preferably 0.8~1.2 mol / L, and even more preferably 1 mol / L; the mass-volume ratio of powdered fruit peel particles to acid is preferably 1~2 g: 18~25 mL, more preferably 1.4~1.6 g: 20~22 mL, and even more preferably 1.5 g: 21 mL.

[0028] In this invention, the temperature of the acid heat pretreatment in step 2) is preferably 120~140℃, more preferably 125~135℃, and even more preferably 130℃; the time of the acid heat pretreatment is preferably 30~60min, more preferably 40~50min, and even more preferably 45min.

[0029] In this invention, the method for preparing the Clostridium butyricum solution in step 3) preferably includes the following steps: (1) The frozen Clostridium butyricum was activated in PYG medium to obtain activated Clostridium butyricum solution; (2) The activated Clostridium butyricum solution was inoculated into a new PYG medium and cultured under anaerobic conditions until the cells reached the mid-log phase. The supernatant was then removed by centrifugation, and the bacterial precipitate was resuspended in a salt solution to obtain the Clostridium butyricum solution.

[0030] In this invention, the activation temperature in step (1) is preferably 30~40℃, more preferably 32~38℃, and even more preferably 34~36℃; the activation time is preferably 20~30h, more preferably 22~26h, and even more preferably 24h.

[0031] In this invention, the cryopreservation temperature in step (1) is preferably -90~-70℃, and more preferably -80℃; the inoculation amount of the cryopreserved Clostridium butyricum is preferably 1~3% (volume fraction) of the PYG culture medium volume, and more preferably 2% (volume fraction).

[0032] In this invention, the inoculation amount of the activated Clostridium butyricum solution in step (2) is preferably 2-4% (volume fraction) of the PYG culture medium, more preferably 3% (volume fraction); the culture temperature is preferably 30-40℃, more preferably 32-36℃, and more preferably 34℃; the culture time is preferably 10-14h, more preferably 11-13h, and more preferably 12h.

[0033] In this invention, the anaerobic conditions in step (2) are preferably an anaerobic environment established by inert gas replacement, wherein the inert gas is preferably nitrogen, and the replacement time is preferably 5-10 min, more preferably 6-8 min, and more preferably 7 min; The centrifugation speed is preferably 4500~5500 r / min, more preferably 4800~5200 r / min, and even more preferably 5000 r / min; the centrifugation time is preferably 8~12 min, more preferably 9~11 min, and even more preferably 10 min.

[0034] In this invention, the mid-log phase in step (2) refers to the rapid growth phase of Clostridium butyricum. The middle stage of the logarithmic phase, which is when both cell proliferation rate and metabolic activity are at a high level, is usually judged by the optical density at a wavelength of 600 nm (OD600). Generally, when the OD600 value of the bacterial culture reaches 0.8~1.2, it can be regarded as being in the middle of the logarithmic phase.

[0035] In this invention, the salt solution in step (2) is preferably a sodium chloride solution with a mass concentration of 0.7-0.9%, more preferably a sodium chloride solution with a mass concentration of 0.85%, and the volume of the salt solution is preferably 5-8 times the volume of the precipitate, more preferably 6-7 times.

[0036] In this invention, the volume ratio of the Clostridium butyricum solution and the fruit peel pretreatment solution in step 3) is preferably 4~5:10~12, and more preferably 4.5:11.

[0037] In this invention, after inoculating the Clostridium butyricum solution into the pretreatment solution in step 3), it is preferable to adjust the pH of the mixture to 6-8 using hydrochloric acid solution or sodium hydroxide powder.

[0038] In this invention, the anaerobic conditions in step 3) are preferably an anaerobic environment established by inert gas replacement, wherein the inert gas is preferably nitrogen, and the replacement time is preferably 5-10 min, more preferably 6-8 min, and even more preferably 7 min.

[0039] In this invention, the dark fermentation in step 3) is preferably carried out at a water bath temperature of 35~38℃ and a stirring speed of 50~80r / min, and more preferably at a water bath temperature of 37℃ and a stirring speed of 60r / min.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] The Clostridium butyricum used in this invention ( Clostridium butyricum The sample originated from the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC 1.5205. The culture medium used in this invention is the modified PYG liquid medium recommended by CGMCC (number 0264), and its composition is shown in Table 1. The main body of the culture medium is purged with nitrogen for 30 minutes to fully remove oxygen, and then sterilized at 121°C for 20 minutes. After cooling, heme chloride solution and vitamin K1 solution are added. Table 1 Composition of PYG medium

[0042] Example 1 Wash the fresh navel orange peels thoroughly and dry them at 60℃ for 48 hours. After drying, crush the peels and pass them through a 60-mesh sieve to obtain powdered peel granules.

[0043] 15g of powdered fruit peel particles were placed in a 500mL reaction vessel with a polytetrafluoroethylene liner, and then 210mL of 1mol / L hydrochloric acid solution was added. The mixture was kept in an oil bath at 140℃ for 30min to complete the acid-heat pretreatment. The mixture was then cooled to room temperature to obtain the fruit peel pretreatment solution.

[0044] Remove the frozen Clostridium butyricum from the -80°C freezer, thaw it rapidly at room temperature, and then... The inoculum was 2% (volume fraction) of the culture medium volume, inoculated into 10 mL of PYG medium and activated in a 37℃ incubator for 24 h to obtain activated Clostridium butyricum solution. Subsequently, the activated Clostridium butyricum solution was inoculated into a new 500 mL PYG medium at an inoculum volume of 3% (volume fraction) and cultured under anaerobic conditions (an anaerobic environment was established by nitrogen purging for 8 min) at 37℃ for 12 h until the OD600 value of the bacterial solution reached 0.9, corresponding to the mid-log phase of cell growth. After the culture was completed, the bacterial solution was centrifuged at 5000 r / min for 10 min, the supernatant was discarded, and the bacterial precipitate was resuspended in a 0.85% sodium chloride solution with a mass concentration equivalent to 6 times the volume of the precipitate to obtain Clostridium butyricum solution.

[0045] 90 mL of Clostridium butyricum solution and 210 mL of fruit peel pretreatment solution were added to a 500 mL sealed glass fermentation flask to form a fermentation system. The pH of the system was then adjusted to 7 using a 1 mol / L hydrochloric acid solution. Nitrogen gas was then purged for 5 min to displace the air in the flask and establish an anaerobic environment. The fermentation flask was then placed in a BPC microbial reactor (water bath temperature 37℃, stirring speed 60 r / min) for dark fermentation until the gas production rate decreased to a stable level. The resulting gas was mainly composed of hydrogen, with a certain amount of carbon dioxide.

[0046] Example 2 Wash the fresh navel orange peels thoroughly and dry them at 50℃ for 60 hours. After drying, crush the peels and pass them through a 50-mesh sieve to obtain powdered peel granules.

[0047] 10g of powdered fruit peel particles were placed in a 500mL reaction vessel with a polytetrafluoroethylene liner, and then 180mL of 1.5mol / L sulfuric acid solution was added. The mixture was kept at 120℃ in an oil bath for 60min to complete the acid-heat pretreatment. The mixture was then cooled to room temperature to obtain the fruit peel pretreated solution.

[0048] Remove the frozen Clostridium butyricum from the -90°C freezer, thaw it rapidly at room temperature, and then... The inoculum volume is 1% (volume fraction) of the culture medium. Inoculate into 10 mL of PYG medium. The activated Clostridium butyricum solution was obtained by activating the bacteria in a 30℃ constant temperature incubator for 30 hours; subsequently, the activated Clostridium butyricum solution was... Inoculate 2% (volume fraction) of the culture medium into a fresh 500 mL PYG medium and culture under anaerobic conditions (an anaerobic environment established by nitrogen purging for 5 min) at 30 °C for 14 h until the OD600 value of the bacterial solution reaches 0.8, corresponding to the mid-log phase of cell growth. After the culture is completed, centrifuge the bacterial solution at 4500 r / min for 12 min, discard the supernatant, and resuspend the bacterial precipitate in a 0.7% sodium chloride solution with a mass concentration equivalent to 5 times the volume of the precipitate to obtain the Clostridium butyricum solution.

[0049] 80 mL of Clostridium butyricum solution and 200 mL of fruit peel pretreatment solution were added to a 500 mL sealed glass fermentation flask to form a fermentation system. The pH of the system was then adjusted to 6 using sodium hydroxide powder, followed by purging with nitrogen gas for 8 minutes to displace the air in the flask and establish an anaerobic environment. The fermentation flask was then placed in a BPC microbial reactor (water bath temperature 35℃, stirring speed 80 r / min) for dark fermentation until the gas production rate decreased to a stable level. The resulting gas was mainly composed of hydrogen, with a certain amount of carbon dioxide.

[0050] Example 3 Wash the fresh navel orange peels thoroughly and dry them at 70℃ for 40 hours. After drying, crush the peels and pass them through a 70-mesh sieve to obtain powdered peel granules.

[0051] 20g of powdered fruit peel particles were placed in a 500mL reaction vessel with a polytetrafluoroethylene liner, and then 250mL of 0.5mol / L sulfuric acid solution was added. The mixture was kept in an oil bath at 130℃ for 50min to complete the acid-heat pretreatment. The mixture was then cooled to room temperature to obtain the fruit peel pretreated solution.

[0052] Remove the frozen Clostridium butyricum from the -70°C freezer, thaw it rapidly at room temperature, and then... The inoculum was 3% (volume fraction) of the culture medium volume, inoculated into 10 mL of PYG medium and activated in a constant temperature incubator at 40℃ for 20 h to obtain activated Clostridium butyricum solution. Subsequently, the activated Clostridium butyricum solution was inoculated into a new 500 mL PYG medium at an inoculum volume of 4% (volume fraction) and cultured under anaerobic conditions (an anaerobic environment was established by nitrogen purging for 10 min) at 40℃ for 10 h until the OD600 value of the bacterial solution reached 1.2, corresponding to the mid-log phase of cell growth. After the culture was completed, the bacterial solution was centrifuged at 5500 r / min for 8 min, the supernatant was discarded, and the bacterial precipitate was resuspended in a 0.9% sodium chloride solution with a mass concentration equivalent to 8 times the volume of the precipitate to obtain Clostridium butyricum solution.

[0053] Add 100 mL of Clostridium butyricum solution and 240 mL of fruit peel pretreatment solution to a 500 mL sealed glass incubator. In the fermentation flask, the mixture is combined to form a fermentation system, and then the pH of the system is adjusted to 8 using sodium hydroxide powder. Nitrogen gas was then introduced for 10 minutes to replace the air in the bottle and establish an anaerobic environment. The fermentation bottle was then placed in a BPC microbial reactor (water bath temperature 38℃, stirring speed 50 r / min) for dark fermentation until the gas production rate decreased to a stable level. The resulting gas was mainly composed of hydrogen, with a certain amount of carbon dioxide.

[0054] Example 4 Wash the fresh navel orange peels thoroughly and dry them at 65℃ for 55 hours. After drying, crush the peels. The granules were then passed through a 60-mesh sieve to obtain powdered fruit peel particles.

[0055] 18g of powdered fruit peel particles were placed in a 500mL reaction vessel with a polytetrafluoroethylene liner, and then 220mL of 1mol / L hydrochloric acid solution was added. The mixture was kept in an oil bath at 125℃ for 45min to complete the acid-heat pretreatment. The mixture was then cooled to room temperature to obtain the fruit peel pretreatment solution.

[0056] Remove the frozen Clostridium butyricum from the -80°C freezer, thaw it rapidly at room temperature, and then... The inoculum was 3% (volume fraction) of the culture medium volume, inoculated into 10 mL of PYG medium, and activated in a 35℃ incubator for 25 h to obtain activated Clostridium butyricum solution. Subsequently, the activated Clostridium butyricum solution was inoculated into a new 500 mL PYG medium at the same inoculum volume. The medium was then cultured under anaerobic conditions (an anaerobic environment was established by nitrogen purging for 6 min) at 37℃ for 11 h until the OD600 value of the bacterial solution reached 1, corresponding to the mid-log phase of cell growth. After the culture was completed, the bacterial solution was centrifuged at 4500 r / min for 10 min, the supernatant was discarded, and the bacterial precipitate was resuspended in a 0.85% sodium chloride solution with a mass concentration equivalent to 7 times the volume of the precipitate to obtain Clostridium butyricum solution.

[0057] 90 mL of Clostridium butyricum solution and 220 mL of fruit peel pretreatment solution were added to a 500 mL sealed glass fermentation flask to form a fermentation system. The pH of the system was then adjusted to 7 using sodium hydroxide powder, followed by purging with nitrogen gas for 8 minutes to displace the air in the flask and establish an anaerobic environment. The fermentation flask was then placed in a BPC microbial reactor (water bath temperature 37℃, stirring speed 70 r / min) for dark fermentation until the gas production rate decreased to a stable level. The resulting gas was mainly composed of hydrogen, with a certain amount of carbon dioxide.

[0058] Comparative Example 1 It is basically the same as Example 1, except that the preparation process of the fruit peel pretreatment liquid is omitted and powdered fruit peel particles are used directly.

[0059] Comparative Example 2 The process is basically the same as in Example 1, except that the concentration of hydrochloric acid used in the preparation of the peel pretreatment solution is changed to 0.1 mol / L.

[0060] Comparative Example 3 It is basically the same as Example 1, except that the concentration of hydrochloric acid used in the preparation of the peel pretreatment solution is changed to 2 mol / L.

[0061] Comparative Example 4 It is basically the same as Example 1, except that the temperature of the oil bath in the preparation of the peel pretreatment solution is changed to 60°C.

[0062] Comparative Example 4 It is basically the same as Example 1, except that the temperature of the oil bath in the preparation of the peel pretreatment solution is changed to 60°C.

[0063] Comparative Example 6 It is basically the same as Example 1, except that the temperature of the oil bath in the preparation of the peel pretreatment solution is changed to 160°C.

[0064] Comparative Example 7 It is basically the same as Example 1, except that the heat preservation time in the preparation process of the fruit peel pretreatment solution is modified to 15 min.

[0065] Comparative Example 8 It is basically the same as Example 1, except that the heat preservation time in the preparation process of the peel pretreatment solution is modified to 120 min.

[0066] Comparative Example 9 The process is basically the same as in Example 1, except that the heat preservation time during the preparation of the fruit peel pretreatment solution is changed to 240 min.

[0067] The results of cellulose content, lignin removal rate and hydrogen production of navel orange peel under different conditions in Examples 1-4 and Comparative Examples 1-9 are shown in Table 2.

[0068] Table 2. Results of cellulose content, lignin removal rate and hydrogen production of navel orange peel under different conditions.

[0069] As shown in Table 1, the present invention can significantly improve the hydrogen production of fruit peel biomass. Compared with Comparative Example 1 (which did not undergo pretreatment and had a hydrogen production of only 21.6 mL / g TS), the hydrogen production of Examples 1-4 was increased to 35.1-39.3 mL / g TS, an increase of more than 50%, indicating that the method of the present invention can effectively improve the utilization efficiency and hydrogen production performance of fruit peel biomass.

[0070] This invention can also improve the structure of the peel substrate and promote the utilization of carbon sources by microorganisms. Under the conditions of the examples, the lignin removal rate of navel orange peel reached more than 75%, which was significantly higher than that of comparative examples 2-9 (63.5-70.1%). At the same time, the cellulose content decreased significantly, indicating that acid-heat pretreatment can effectively break down the lignin barrier, making it easier for Clostridium butyricum to utilize the cellulose and hemicellulose in the peel for hydrogen production.

[0071] This invention has a significant advantage in the selection of process parameters. When the acid concentration is too low (e.g., Comparative Example 2, 0.1 mol / L HCl) or too high (e.g., Comparative Example 3, 2 mol / L HCl), the hydrogen production is only 29.2~29.6 mL / g TS, which is significantly lower than that of Example 1 (35.1 mL / g TS), indicating that the acid concentration needs to be controlled within a suitable range to obtain a high hydrogen production effect.

[0072] This invention also demonstrates superiority in the control of temperature and time during acid-heat pretreatment. Comparative Examples 4, 5, and 6, treated at excessively low (60°C), excessively high (160°C), or unsuitable (100°C) temperatures, respectively, resulted in hydrogen production decreasing to 25.6–32.7 mL / g TS, while the hydrogen production under the conditions of the examples remained above 35 mL / g TS. Similarly, when the pretreatment time was too short (e.g., 15 min in Comparative Example 7) or too long (e.g., 120–240 min in Comparative Examples 8 and 9), the hydrogen production decreased to 19.7–26.4 mL / g TS, while the results under the conditions of this invention remained stable above 35 mL / g TS. Therefore, this invention is significantly superior to the comparative examples in the selection of temperature and time windows.

[0073] In summary, the overall process of this invention exhibits good stability and repeatability. Examples 1-4 maintained high hydrogen production levels (35.1-39.3 mL / g TS) under different conditions, with minimal differences in data. In contrast, the results of the comparative samples showed significant fluctuations, even substantial decreases. The method of this invention not only significantly increases hydrogen production but also ensures process stability and reliability, demonstrating excellent potential for industrial application.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for producing hydrogen through dark fermentation of fruit peel biomass, characterized in that, Includes the following steps: 1) After drying the fruit peel, crush and sieve it to obtain powdered fruit peel particles; 2) The powdered fruit peel particles are subjected to acid and heat pretreatment to obtain a fruit peel pretreatment solution; 3) The Clostridium butyricum solution was inoculated into the pretreated fruit peel solution and subjected to dark fermentation under anaerobic conditions to obtain fermentation gas containing hydrogen.

2. The method according to claim 1, characterized in that, Step 1) The peel is a citrus peel; The drying temperature is 50~70℃, and the drying time is 40~60h; The sieve mesh size is 50-70 mesh.

3. The method according to claim 1 or 2, characterized in that, Step 2) The acid used in the acid heat pretreatment is hydrochloric acid or sulfuric acid, with an acid concentration of 0.5~1.5mol / L, and the mass-volume ratio of powdered fruit peel particles to acid is 1~2g:18~25mL.

4. The method according to claim 3, characterized in that, Step 2) The acid heat pretreatment temperature is 120~140℃, and the acid heat pretreatment time is 30~60min.

5. The method according to claim 4, characterized in that, Step 3) The preparation method of the Clostridium butyricum solution includes the following steps: (1) Clostridium butyricum was activated in PYG medium to obtain activated Clostridium butyricum solution; (2) The activated Clostridium butyricum solution was inoculated into a new PYG medium and cultured under anaerobic conditions until the cells reached the mid-log phase. The supernatant was then removed by centrifugation, and the bacterial precipitate was resuspended in a salt solution to obtain the Clostridium butyricum solution.

6. The method according to claim 4 or 5, characterized in that, The activation temperature in step (1) is 30~40℃, and the activation time is 20~30h; The inoculation amount of the activated Clostridium butyricum solution in step (2) is 2-4% (volume fraction) of the PYG medium volume, the culture temperature is 30-40℃, and the culture time is 10-14h.

7. The method according to claim 6, characterized in that, The anaerobic conditions described in step (2) are an anaerobic environment established by inert gas replacement, wherein the inert gas is nitrogen, and the replacement time is 5 to 10 minutes; The centrifugation speed is 4500~5500 r / min, and the centrifugation time is 8~12 min.

8. The method according to claim 1, characterized in that, Step 3) Clostridium butyricum lysate The volume ratio of liquid to fruit peel pretreatment liquid is 4~5:10~12.

9. The method according to claim 8, characterized in that, Step 3) The anaerobic conditions are an anaerobic environment established by inert gas replacement, wherein the inert gas is nitrogen, and the replacement time is 5~10 minutes.

10. The preparation method according to claim 9, characterized in that, Step 3) The dark fermentation is carried out under the conditions of water bath temperature of 35~38℃ and stirring speed of 50~80r / min.

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