Co-fermentation of bituminous coal and corn straw to produce hydrogen

Co-fermentation of bituminous coal and corn straw to produce hydrogen | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Co-fermentation of bituminous coal and corn straw to produce hydrogen Xin Guo, Xiaobo Xu, Litong Ma, Jun Li, Yanan Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7222272/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Nov, 2025 Read the published version in Waste and Biomass Valorization → Version 1 posted 5 You are reading this latest preprint version Abstract In recent years, research and development of new energy sources has gained attention. Coal is a combustible organic biological rock comprising polymer hydrocarbons and traces of inorganic minerals. Notably, bituminous coal has the largest coal reserve. Corn stalk is an agricultural waste and a renewable resource. In this experiment, bituminous coal and corn straw were used to prepare clean hydrogen using microbial conversion, to achieve zero-carbon energy. The results showed that adding different amounts of bituminous coal affected hydrogen production during co-fermentation with corn stalk. On the 1st day of fermentation, the hydrogen fermentation group containing bituminous coal showed a peak. The gas production of the fermentation group with 5% bituminous coal was 102.4 mL at maximum, and that of the fermentation group without bituminous coal was 77.4 mL. The gas production of the fermentation group with 5% bituminous coal was 25 mL higher than that of the group without bituminous coal. The pH and humic acid content of the fermentation group supplemented with 5% bituminous coal were lower than those of the other fermentation systems; the benzoic acid, pyruvate, and glucose contents were higher than those of the other fermentation groups. The specific surface area of the blank control group without bituminous coal was 1.99765 m2/g, total pore volume was 0.008372 m3/g, and average pore diameter was 16.05975 nm. The specific surface area of the blank control group with 5% bituminous coal was 1.6375 m2/g and the average pore diameter was 16.4875 nm. In conclusion, 5% bituminous coal supplementation to the fermentation of corn straw was beneficial to produce hydrogen; however, further addition of bituminous coal inhibited the hydrogen production. bituminous coal Corn stalk Microbial transformation Hydrogen Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1 Introduction Corn stalks are the main agricultural waste in northern China. Corn stalks have poor palatability and digestion, and cannot be used as livestock feed. Because corn stalk is not commonly used as feed, combustion, or agricultural slag fertilizer, most of corn stalk waste is incinerated, resulting in air pollution [ 1 ]. Corn straw primarily comprises cellulose, hemicellulose, and lignin that have a high potential for microbial hydrogen production [ 2 ]. Coal is the dominant mineral resource in China, ranging from lignite and bituminous coal to anthracite. Bituminous coal reserves amount to approximately 405.8 billion tons, but their usage as a fuel causes several environmental problems. Compared to traditional bituminous coal utilization methods, bituminous coal bio-gasification can substantially reduce carbon dioxide emissions, and is also more environment-friendly [ 3 ]. Recently, attempts have been made to produce hydrogen gas from corn straw; however, the mechanism of co-fermentation of bituminous coal and corn straw to produce hydrogen gas remain unclear. To realize low-carbon transformation of high-carbon resources, bituminous coal and corn straw, this study conducted an experiment co-fermentation of bituminous coal and corn straw to produce hydrogen gas. Changes in humic acid, benzoic acid, pyruvate, glucose, and pH during the co-fermentation process, as well as changes in the pore size and structure characteristics of the co-fermentation residue was evaluated. The results lay a foundation for exploring new raw materials for microbial hydrogen production. 2 Materials and methods Bituminous coal was sourced from the Yangchangwan Coal Mine, China Energy Group Ningxia Coal Industry Company. Corn stalks were sourced from Machi Town, Kundulun District, Baotou City. Activated sludge was sourced from the Baotou Nanjiao sewage treatment plant. 2.1 Experimental method The anaerobically activated sludge was boiled in a water bath for 30 min and cooled. Different proportions of bituminous coal and corn straw were added to a 500 mL fermentation bottle for a total of 40 g. The contents of the added corn straw and bituminous coal are listed in Table 2 − 1, and three parallels were set in each group. Distilled water (400 mL) was added, and the initial pH was adjusted to 7. The HH-8 digital display constant temperature water bath was set at 50°C, and the fermentation bottle was placed in the water bath and shaken every 2 h. Daily gas output was recorded, and samples were taken once every 3 d to determine the concentration of pyruvate, humic acid, glucose, benzoic acid, and pH. The physical adsorption capacity of the activated sludge after co-fermentation was determined. Table 2.1 Contents of corn stalk and bituminous coal Experimental group Corn stalk mass /g Bituminous coal mass /g 0% 40 0 5% 38 2 10% 36 4 20% 32 8 40% 24 16 2.2 Determination of humic acid concentration The fermentation liquid (10 mL) was added to a centrifuge tube and subjected to high speed centrifugation. After centrifugation, (0.1 mL) of supernatant was collected and diluted 100 times. The absorbance in the range of 256–400 nm was determined using a 765 ultraviolet spectrophotometer, and the humic acid concentration was calculated using the standard curve method [ 4 ]. 2.3 Determination of benzoic acid concentration The fermentation liquid (5 mL) was centrifuged, and 1 mL of the supernatant was filtered through a 0.22 µm nylon injection filter. The concentration of benzoic acid was determined by high-performance liquid chromatography (HPLC) using a C18 column, and the mobile phase was ammonium acetate: methanol (92:8). The ammonium acetate concentration was 0.02 mol/L. 2.4 Determination of pyruvate concentration After centrifugation, 2.5 mL of the fermentation solution was diluted 100 times, the pH was adjusted to 11 with sodium hydroxide, and the absorbance at the range of 320 nm was measured using a 765 ultraviolet spectrophotometer. The pyruvate concentration was obtained using the standard curve method [ 4 ]. 2.5 Determination of glucose concentration To 1 mL of supernatant, 0.25 mL 12% trichloroacetic acid solution was added to remove proteins. Thereafter, trichloroacetic acid was added, shaken, forested for 5 min, and centrifuged again for 15 min. The supernatant was then passed through a 0.22 µm filter head and infused into a liquid vial. To determine the glucose concentration by HPLC (AGILENT1260), it was necessary to replace the Hi-Plex H column with a flow rate of 0.1 mL/min. After 30 min, the column temperature box was opened and the column temperature was set to 70°C. Then, the flow rate was gradually increased from 0.2 to 0.5 mL/min. When the flow rate reached 0.5 mL/min, the column temperature box was washed for 3 h, the washing valve was closed, and the recovery was started. 2.6 Determination of pH A pH meter [PHS-3C] was used to determine the pH of the fermentation solution. 2.7 Determination of aperture structure The fermented residue was dried and weighed (0.2 g), and the pore structure was determined using a Micromeritics ASAP 2460 physical adsorption instrument. The average pore diameter, specific surface area, and total pore volume of the residues were measured. 3 Results and discussion 3.1 Effect of co-fermentation of bituminous coal and corn stalk on total hydrogen gas production As shown in Fig. 3.1 , the total gas production of the bituminous coal and corn straw co-fermentation reaction system with bituminous coal contents of 0%, 5%, 10%, 20%, and 40% was 237.2, 253.6, 134.1, 129.5, and 60.0 mL, respectively. The results should that the total hydrogen gas production with the supplementation of 5% bituminous coal was the highest, and was 16.4 mL more than the co-fermentation system without bituminous coal. However, supplementation of 10%, 20%, and 40% bituminous coal to the co-fermentation system, the total hydrogen gas production was lower than that of the co-fermentation system without bituminous coal by 103.1, 107.7, and 177.2 mL, respectively. The results showed that the addition of 5% bituminous coal promoted corn stalk fermentation. Because the organic matter in bituminous coal is difficult to degrade, the hydrogen gas production was lower with 10%, 20%, and 40% bituminous coal. Bituminous coal contains soluble organic matter, and adding 5% bituminous coal provides a nutrient carbon source for microorganisms and improves their activity in the process of hydrogen gas production during co-fermentation with corn stalks. The addition of 5% bituminous coal to corn straw in the co-fermentation reaction system degrades the soluble organic matter of bituminous coal to produce pyruvate, which decarboxylates hydrogen gas and carbon dioxide under the action of enzymes [ 5 ]. Thus, the co-fermentation group supplemented with 5% bituminous coal produces more hydrogen gas than the fermentation group without bituminous coal supplementation. 3.2 Effect of co-fermentation of bituminous coal and corn stalk on daily production of hydrogen gas Figure 3.2 shows the effects of adding different concentrations of bituminous coal to corn stalks. The co-fermentation system with bituminous coal contents of 5%, 10%, 20%, and 40% all reached their peak values on the 1st day, and then gradually stabilized to zero. The fermentation system without the addition of bituminous coal reached its peak value on the 2nd day, and then gradually stabilized at zero. On the 1st day, the hydrogen gas production of the co-fermentation system with 0% bituminous coal was 77.4 mL; and the gas production of the fermentation systems with 5%, 10%, 20%, and 40% bituminous coal was 102.4, 55.1, 59.4, and 11.3 mL, respectively. The hydrogen gas production of fermentation system with 5% bituminous coal was 25 mL more than that of the fermentation group with 0% bituminous coal. The hydrogen gas production in the fermentation groups supplemented with 10%, 20%, and 30% was 22.3, 18.0, and 66.1 mL less than that of the fermentation group with 0% bituminous coal, respectively. In the fermentation process, the hydrogen gas production of the 0% and 5% fermentation systems reached 89% of the total gas production in the first 5 d. Adding 10% bituminous coal to the co-fermentation system in the first 5 d, the hydrogen gas production reached 84% of the total gas production, whereas addition of 20% bituminous coal, the hydrogen gas production reached 79% of the total gas production. The addition of 40% of the fermentation system obtained 58% of the total gas production in the first 5 days. These results show that co-fermentation with 5% bituminous coal could promote hydrogen gas production from corn stalks. Because bituminous coal contains a certain amount of nitrogen [ 6 ], it can regulate the carbon-nitrogen ratio of corn straw to meet the needs of microbial metabolism. When the carbon to nitrogen ratio of 5% bituminous coal and corn straw co-fermentation, the hydrogen production reaction system, is appropriate, microorganisms can make full use of the substrate bituminous coal and corn straw for growth and metabolism, thus producing more hydrogen [ 7 – 9 ]. 3.3 Effect of co-fermentation of bituminous coal and corn straw on humic acid content The humic acid content in the corn straw co-fermentation system was affected by adding different concentrations of bituminous coal. The humic acid content in the fermentation system without bituminous coal and in the fermentation system with 5%, 10%, 20%, and 40% bituminous coal was the highest on the 1st day at 6.03, 5.10, 5.4, 6.95, and 7.77 g/L, respectively. Subsequently, the humic acid content of the system gradually decreased. The humic acid content in the fermentation system with 5% bituminous coal was the lowest in the 1st d, and the gas production in the reaction system was the highest in the 1st d. The fermentation system supplemented with 40% bituminous coal had the highest humic acid concentration, which was consistent with the lowest gas production of 40% bituminous coal after 1 d. The results showed that the addition of 5% bituminous coal promoted the production of hydrogen, while the addition of 10%, 20%, and 40% bituminous coal inhibited the production of hydrogen. This is because a high concentration of humic acid affects anaerobic microbial cells and hydrolases in the fermentation process, resulting in the inactivity of microbial cells and hydrolases [ 10 ]. Thus, the total gas production in the fermentation system will increase or decrease. It also limits the decomposition of organic matter in bituminous coal and corn straw, and then slows down or speeds up the fermentation rate, affecting the increase or decrease in total gas production. 3.4 Effect of co-fermentation of bituminous coal and corn stalk on benzoic acid content Figure 3.4 shows the effect of different bituminous coal contents on the benzoic acid content of the corn stalk. The addition of different bituminous coal contents affected the benzoic acid content in the fermentation system. On the 1st d of fermentation, the benzoic acid contents of 0%, 5%, 10%, 20%, and 40% bituminous coal were 8.75, 9.85, 8.61, 7.68, and 5.89 mg/L. The highest benzoic acid concentration of 5% bituminous coal is consistent with the highest hydrogen production of 5% bituminous coal on the 1st d. The results showed that humic acid in the co-fermentation group supplemented with 5% bituminous coal was more easily degraded into benzoic acid by microorganisms than in the other co-fermentation groups; therefore, the promotion effect on the co-fermentation of bituminous coal and corn straw to produce hydrogen gas was stronger. Low concentrations of humic acid in the fermentation system can trigger electron transfer through the microbial cell membrane, thus enhancing hydrolase activity and promoting benzoic acid degradation, increasing hydrogen production [ 11 – 13 ]. As the benzoic acid content in the fermentation groups with 10%, 20%, and 40% bituminous coal was lower than that without bituminous coal, the total hydrogen production in the fermentation groups with 10%, 20%, and 40% bituminous coal was lower than that without bituminous coal. Although the concentration of benzoic acid in the fermentation group supplemented with 5% bituminous coal was also lower than that in the fermentation group without bituminous coal, the content of benzoic acid on the 1st d was higher in the fermentation group supplemented with 5% bituminous coal than in the fermentation group without bituminous coal. the hydrogen gas production on the 1st d was the highest in the co-fermentation process, and the influence of benzoic acid concentration on hydrogen production was not observed after that. 3.5 Effect of co-fermentation of bituminous coal and corn straw on pyruvate content Figure 3.5 shows the effects of different bituminous coal contents on the pyruvate content in corn stalk fermentation, which is an important intermediate product in the fermentation process. Decarboxylation produces hydrogen gas, and hydrogen-producing bacteria directly decarboxylate glucose and transfer electrons to ferridoxin, which is then catalyzed by hydrogenase to reduce protons into H 2 molecules [ 14 ]. Formic acid is formed after decarboxylation of pyruvate and then by hydrogenase. Splitting formic acid entirely or partially into H 2 [ 15 ]. As shown in Fig. 3.5 , the pyruvate concentrations in the corn straw fermentation system with 0%, 5%, 10%, 20%, and 40% bituminous coal added on the first day of fermentation were 105.8, 136.0, 124.0, 99.1, and 64.8 g/L, respectively. On 21st d of fermentation, the pyruvate concentrations of the 0%, 5%, 10%, and 20% fermentation groups reached the lowest values of 53.5, 57.3, 54.2, and 40.2 g/L, respectively. The fermentation group supplemented with 40% bituminous coal reached its lowest value on the 18th d. In the fermentation group with 5% bituminous coal, the pyruvate concentration decreased the most, and the pyruvate degradation was up to 78.7 g/L. The greater the degree of pyruvate degradation, the greater the amount of hydrogen produced. Therefore, the co-fermentation group with 5% bituminous coal exhibited the highest total gas production. 3.6 Effect of co-fermentation of bituminous coal and corn stalk on glucose content Figure 3.6 shows the changes in glucose concentration in the fermentation group with different bituminous coal contents. The glucose concentration in the corn straw fermentation system with 0%, 5%, 10%, 20%, and 40% bituminous coal added reached the maximum value at the 3rd d, which were 2.45, 2.58, 2.41, 2.44, and 2.29 g/L, respectively. The glucose content in the first 2-d reaction system was close to 0. As shown in the figure, the fermentation group with 5% bituminous coal had the highest glucose concentration at the 3rd d, which was 0.13 g/L more than the fermentation group without bituminous coal. As corn straw is rich in cellulose, the glucoside bonds of cellulose break down to produce glucose [ 16 ], which can be converted and degraded into hydrogen. The glucose concentration of the other fermentation groups was lower than that of the fermentation group without bituminous coal, indicating that adding too much bituminous coal reduces the microbial activity, degree of corn stalk degradation, and hydrogen production. 3.7 Effect of co-fermentation of bituminous coal and corn straw on pH Figure 3.7 shows the effects of adding different amounts of bituminous coal to the fermentation system. pH is one of the factors affecting microbial fermentation and the activity of anaerobic microorganisms in the fermentation system, and has a certain effect on the amount of gas produced. With and without the addition of bituminous coal, the initial pH of the fermentation system showed a downward trend with the increase of fermentation days, and reached the lowest value on the 21st d due to the production of volatile fatty acids such as butyric acid and propionic acid [ 17 ], resulting in a decrease in pH. The pH values of the fermentation groups containing 0%, 5%, 10%, 20%, and 40% bituminous coal were 4.83, 4.26, 5.53, 5.25, and 5.23, respectively, on the 21st d. The pH of the fermentation system with 5% bituminous coal was lower than that of the system without bituminous coal, and the total gas production was the highest. The pH of the fermentation systems with 10%, 20%, and 40% bituminous coal was higher than that without bituminous coal, and the total gas production was lower than that without bituminous coal. 3.8 Effect of co-fermentation of bituminous coal and corn straw on structure characteristics of residual pores As shown in Fig. 3.8 , the isotherms of N 2 adsorption and desorption of the fermentation residue with and without bituminous coal were similar to those of type IV, and the upward convex curve in the low P/P o region was similar to that of type II isotherms [ 18 ]. Capillary condensation occurred in the higher P/P o region, and the isotherm increased rapidly. When all the pores are condensed, adsorption occurs only on the outer surface, which is much smaller than the inner surface area, and the curve is flat. When the relative pressure is close to 1, it adsorbs onto the large pores, and the curve rises. Capillary condensation resulted in hysteresis in this region, i.e., the isotherm obtained during desorption did not coincide with that obtained during adsorption. The desorption isotherm was above the adsorption isotherm, resulting in adsorption hysteresis and an H3-type hysteresis loop, indicating that the pore shape of bituminite coal was dominated by slit pores [ 18 – 19 ]. Figure 3.9 shows the structural analysis of the residual pores after hydrogen production by fermentation. Table () shows that the residual pore structure is different after hydrogen fermentation in fermentation groups with different concentrations of bituminous coal. The specific surface area of the blank control group without bituminous coal was 1.99765 m 2 /g, total pore volume was 0.008372 m 3 /g, and average pore diameter was 16.05975 nm. The specific surface area of the 5% bituminous coal was the smallest at 1.6375 m 2 /g. The small specific surface area may be due to the destruction of the pore collapse structure of the fermentation raw material due to the microbial degradation of bituminous coal. However, the maximum average pore size was 16.4875 nm, indicating that more microorganisms entered the pore structure of the raw fermentation material, and the degradation of organic matter resulted in an increase in the pore size, which is consistent with the maximum total gas production of 5% bituminous coal. 4 Conclusion Bituminous coal and corn straw fermentation produce hydrogen, and a moderate concentration of bituminous coal is conducive to corn straw fermentation to produce hydrogen. (1) In the reaction system of the co-fermentation of bituminous coal and corn straw to produce hydrogen, the addition of 5% bituminous coal increased the total hydrogen production, and the gas production was 6.91% higher than that of the fermentation system without bituminous coal. The excessive addition of bituminous coal can inhibit hydrogen production in corn stalks. (2) On the 1st day of fermentation, the hydrogen fermentation group with bituminous coal reached its peak value, and the fermentation group with 5% bituminous coal produced up to 102.4 mL of gas. The gas production in the fermentation group without bituminous coal was 77.4 mL on the 1st day, and the gas production in the fermentation group with 5% bituminous coal was 32.30% more than that in the fermentation group without bituminous coal. Their results showed that the addition of 5% bituminous coal promoted daily hydrogen production from corn straw. (3) The contents of benzoic acid, pyruvate, and glucose in the reaction system for the co-fermentation of bituminous coal and corn straw were higher than those in the other fermentation groups. The results showed that the addition of 5% bituminous coal was more conducive to the fermentation of corn stalks to produce hydrogen. (4) In the reaction system of co-fermentation of bituminous coal and corn straw to produce hydrogen, the specific surface area of residue in the blank control group without bituminous coal was 1.99765 m 2 /g, total pore volume was 0.008372 m 3 /g, and average pore size was 16.05975 nm. The specific surface area of the residue with 5% bituminous coal was the smallest at 1.6375 m 2 /g. The average pore size was 16.4875 nm, indicating that the pore size of the residue increased because of the degradation of organic matter. Declarations Author contributions All authors have contributed to the research concept and design. The first draft of this manuscript was written by Xin Guo. Data collection is the responsibility of Yanan Yu. Jun Li & Litong Ma are responsible for language polishing. The material preparation work is the responsibility of Xiaobo Xu. And all authors commented on the previous version of the manuscript. All authors have read and approved the final manuscript. Funding This work is financially supported by the Science and Technology Plan Project of Inner Mongolia Autonomous Region (2025YFHH0127); Natural Science Foundation of Inner Mongolia (2025MS03024). Declaration of competing interest The authors declare that they have no conflicts of interest. References Han, R., Yong, F., ,Fang, X., et al.: Influences of Fermented Corn Straw Fiber on Performance and Nutrient Utilization in Different Breeds of Finishing Pigs.Animals,2024, 14 (23):3393–3393 Fu, Y., Zhang, J., ,Guan, T.: High-Value Utilization of Corn Straw: From Waste to Wealth. Sustainability, 2023,15(19). Zhao, S., Guo, H., ,Klitzsch, N., et al.: Revealing the mechanisms of rhamnolipid enhancing methane production from anaerobic digestion of bituminous coal.Biomass and Bioenergy,2025,194107619-107619 Yanfeng, Z.: Hanmei Q, Dehua Liao. 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1","display":"","copyAsset":false,"role":"figure","size":45463,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.1 Effect of co-fermentation of bituminous coal and corn stalk on total gas production\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7222272/v1/f8d32fe10e3e3fb898dc00b4.png"},{"id":90201297,"identity":"b14f541f-0d09-4d05-94cb-266237cb0cfb","added_by":"auto","created_at":"2025-08-29 18:38:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68363,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.2 Effect of co-fermentation of bituminous coal and corn stalk on daily gas production\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7222272/v1/798a6f9e5d7f62a54623732b.png"},{"id":90201469,"identity":"4cde4abd-0dc1-42c1-a23e-4c22f40035d2","added_by":"auto","created_at":"2025-08-29 18:46:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82042,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.3 Effect of co-fermentation of bituminous coal and corn straw on humic acid content in reaction system\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7222272/v1/dac58269c1f5216106aa6e10.png"},{"id":90200704,"identity":"e0a5f0f8-4bb5-4f72-83cf-9d3d2dd49f34","added_by":"auto","created_at":"2025-08-29 18:30:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":102339,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.4 Effect of co-fermentation of bituminous coal and corn stalk on benzoic acid content in reaction system\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7222272/v1/be3758159bf06f6d7b938046.png"},{"id":90201298,"identity":"ec8c67dd-ee2d-4e9b-a1f0-b15c61e7d801","added_by":"auto","created_at":"2025-08-29 18:38:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":131852,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.5 Effect of co-fermentation of bituminous coal and corn straw on pyruvate content in reaction system\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7222272/v1/887b2e8f24bc6c2d55669b70.png"},{"id":90200708,"identity":"e488ef4b-8aed-4e65-862e-4c759625a537","added_by":"auto","created_at":"2025-08-29 18:30:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":101506,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.6 Effect of co-fermentation of bituminous coal and corn straw on glucose content in reaction system\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7222272/v1/6e41a99bbf1bce3aa69bbf65.png"},{"id":90200706,"identity":"89c1379b-3095-46f5-889a-71a12e864ea5","added_by":"auto","created_at":"2025-08-29 18:30:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":105375,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.7 Effect of co-fermentation of bituminous coal and corn straw on pH of reaction system\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7222272/v1/1385765f6f3a47390b224adf.png"},{"id":90201301,"identity":"ded650d0-56e0-4131-b173-cb906551a1fd","added_by":"auto","created_at":"2025-08-29 18:38:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":201632,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.8 N\u003csub\u003e2\u003c/sub\u003e- adsorption desorption isothermal curve of bituminous coal and corn straw co-fermentation residue\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7222272/v1/cd612f1ffb214901cf1218fb.png"},{"id":90200723,"identity":"2715e1bd-06f0-4941-b47e-dcee94827112","added_by":"auto","created_at":"2025-08-29 18:30:35","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":148101,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.9 Structure analysis of residual pore after hydrogen production by fermentation\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7222272/v1/32132de445d7f263453e5055.png"},{"id":97179297,"identity":"99b22911-2923-43ef-98f8-662a71864c08","added_by":"auto","created_at":"2025-12-01 16:14:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1718399,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7222272/v1/220aa63f-255b-4c30-99e7-2ad7e75793e2.pdf"}],"financialInterests":"","formattedTitle":"Co-fermentation of bituminous coal and corn straw to produce hydrogen","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eCorn stalks are the main agricultural waste in northern China. Corn stalks have poor palatability and digestion, and cannot be used as livestock feed. Because corn stalk is not commonly used as feed, combustion, or agricultural slag fertilizer, most of corn stalk waste is incinerated, resulting in air pollution [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Corn straw primarily comprises cellulose, hemicellulose, and lignin that have a high potential for microbial hydrogen production [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCoal is the dominant mineral resource in China, ranging from lignite and bituminous coal to anthracite. Bituminous coal reserves amount to approximately 405.8\u0026nbsp;billion tons, but their usage as a fuel causes several environmental problems. Compared to traditional bituminous coal utilization methods, bituminous coal bio-gasification can substantially reduce carbon dioxide emissions, and is also more environment-friendly [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecently, attempts have been made to produce hydrogen gas from corn straw; however, the mechanism of co-fermentation of bituminous coal and corn straw to produce hydrogen gas remain unclear. To realize low-carbon transformation of high-carbon resources, bituminous coal and corn straw, this study conducted an experiment co-fermentation of bituminous coal and corn straw to produce hydrogen gas. Changes in humic acid, benzoic acid, pyruvate, glucose, and pH during the co-fermentation process, as well as changes in the pore size and structure characteristics of the co-fermentation residue was evaluated. The results lay a foundation for exploring new raw materials for microbial hydrogen production.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cp\u003eBituminous coal was sourced from the Yangchangwan Coal Mine, China Energy Group Ningxia Coal Industry Company.\u003c/p\u003e\u003cp\u003eCorn stalks were sourced from Machi Town, Kundulun District, Baotou City.\u003c/p\u003e\u003cp\u003eActivated sludge was sourced from the Baotou Nanjiao sewage treatment plant.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental method\u003c/h2\u003e\u003cp\u003eThe anaerobically activated sludge was boiled in a water bath for 30 min and cooled. Different proportions of bituminous coal and corn straw were added to a 500 mL fermentation bottle for a total of 40 g. The contents of the added corn straw and bituminous coal are listed in Table\u0026nbsp;2\u0026thinsp;\u0026minus;\u0026thinsp;1, and three parallels were set in each group. Distilled water (400 mL) was added, and the initial pH was adjusted to 7. The HH-8 digital display constant temperature water bath was set at 50\u0026deg;C, and the fermentation bottle was placed in the water bath and shaken every 2 h. Daily gas output was recorded, and samples were taken once every 3 d to determine the concentration of pyruvate, humic acid, glucose, benzoic acid, and pH. The physical adsorption capacity of the activated sludge after co-fermentation was determined.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2.1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eContents of corn stalk and bituminous coal\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperimental group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCorn stalk mass /g\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBituminous coal mass /g\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Determination of humic acid concentration\u003c/h2\u003e\u003cp\u003eThe fermentation liquid (10 mL) was added to a centrifuge tube and subjected to high speed centrifugation. After centrifugation, (0.1 mL) of supernatant was collected and diluted 100 times. The absorbance in the range of 256\u0026ndash;400 nm was determined using a 765 ultraviolet spectrophotometer, and the humic acid concentration was calculated using the standard curve method [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Determination of benzoic acid concentration\u003c/h2\u003e\u003cp\u003eThe fermentation liquid (5 mL) was centrifuged, and 1 mL of the supernatant was filtered through a 0.22 \u0026micro;m nylon injection filter. The concentration of benzoic acid was determined by high-performance liquid chromatography (HPLC) using a C18 column, and the mobile phase was ammonium acetate: methanol (92:8). The ammonium acetate concentration was 0.02 mol/L.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Determination of pyruvate concentration\u003c/h2\u003e\u003cp\u003eAfter centrifugation, 2.5 mL of the fermentation solution was diluted 100 times, the pH was adjusted to 11 with sodium hydroxide, and the absorbance at the range of 320 nm was measured using a 765 ultraviolet spectrophotometer. The pyruvate concentration was obtained using the standard curve method [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Determination of glucose concentration\u003c/h2\u003e\u003cp\u003eTo 1 mL of supernatant, 0.25 mL 12% trichloroacetic acid solution was added to remove proteins. Thereafter, trichloroacetic acid was added, shaken, forested for 5 min, and centrifuged again for 15 min. The supernatant was then passed through a 0.22 \u0026micro;m filter head and infused into a liquid vial. To determine the glucose concentration by HPLC (AGILENT1260), it was necessary to replace the Hi-Plex H column with a flow rate of 0.1 mL/min. After 30 min, the column temperature box was opened and the column temperature was set to 70\u0026deg;C. Then, the flow rate was gradually increased from 0.2 to 0.5 mL/min. When the flow rate reached 0.5 mL/min, the column temperature box was washed for 3 h, the washing valve was closed, and the recovery was started.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Determination of pH\u003c/h2\u003e\u003cp\u003eA pH meter [PHS-3C] was used to determine the pH of the fermentation solution.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Determination of aperture structure\u003c/h2\u003e\u003cp\u003eThe fermented residue was dried and weighed (0.2 g), and the pore structure was determined using a Micromeritics ASAP 2460 physical adsorption instrument. The average pore diameter, specific surface area, and total pore volume of the residues were measured.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Effect of co-fermentation of bituminous coal and corn stalk on total hydrogen gas production\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3.1\u003c/span\u003e, the total gas production of the bituminous coal and corn straw co-fermentation reaction system with bituminous coal contents of 0%, 5%, 10%, 20%, and 40% was 237.2, 253.6, 134.1, 129.5, and 60.0 mL, respectively. The results should that the total hydrogen gas production with the supplementation of 5% bituminous coal was the highest, and was 16.4 mL more than the co-fermentation system without bituminous coal. However, supplementation of 10%, 20%, and 40% bituminous coal to the co-fermentation system, the total hydrogen gas production was lower than that of the co-fermentation system without bituminous coal by 103.1, 107.7, and 177.2 mL, respectively. The results showed that the addition of 5% bituminous coal promoted corn stalk fermentation. Because the organic matter in bituminous coal is difficult to degrade, the hydrogen gas production was lower with 10%, 20%, and 40% bituminous coal. Bituminous coal contains soluble organic matter, and adding 5% bituminous coal provides a nutrient carbon source for microorganisms and improves their activity in the process of hydrogen gas production during co-fermentation with corn stalks. The addition of 5% bituminous coal to corn straw in the co-fermentation reaction system degrades the soluble organic matter of bituminous coal to produce pyruvate, which decarboxylates hydrogen gas and carbon dioxide under the action of enzymes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Thus, the co-fermentation group supplemented with 5% bituminous coal produces more hydrogen gas than the fermentation group without bituminous coal supplementation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Effect of co-fermentation of bituminous coal and corn stalk on daily production of hydrogen gas\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e shows the effects of adding different concentrations of bituminous coal to corn stalks. The co-fermentation system with bituminous coal contents of 5%, 10%, 20%, and 40% all reached their peak values on the 1st day, and then gradually stabilized to zero. The fermentation system without the addition of bituminous coal reached its peak value on the 2nd day, and then gradually stabilized at zero. On the 1st day, the hydrogen gas production of the co-fermentation system with 0% bituminous coal was 77.4 mL; and the gas production of the fermentation systems with 5%, 10%, 20%, and 40% bituminous coal was 102.4, 55.1, 59.4, and 11.3 mL, respectively. The hydrogen gas production of fermentation system with 5% bituminous coal was 25 mL more than that of the fermentation group with 0% bituminous coal. The hydrogen gas production in the fermentation groups supplemented with 10%, 20%, and 30% was 22.3, 18.0, and 66.1 mL less than that of the fermentation group with 0% bituminous coal, respectively. In the fermentation process, the hydrogen gas production of the 0% and 5% fermentation systems reached 89% of the total gas production in the first 5 d. Adding 10% bituminous coal to the co-fermentation system in the first 5 d, the hydrogen gas production reached 84% of the total gas production, whereas addition of 20% bituminous coal, the hydrogen gas production reached 79% of the total gas production. The addition of 40% of the fermentation system obtained 58% of the total gas production in the first 5 days. These results show that co-fermentation with 5% bituminous coal could promote hydrogen gas production from corn stalks. Because bituminous coal contains a certain amount of nitrogen [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], it can regulate the carbon-nitrogen ratio of corn straw to meet the needs of microbial metabolism. When the carbon to nitrogen ratio of 5% bituminous coal and corn straw co-fermentation, the hydrogen production reaction system, is appropriate, microorganisms can make full use of the substrate bituminous coal and corn straw for growth and metabolism, thus producing more hydrogen [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Effect of co-fermentation of bituminous coal and corn straw on humic acid content\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe humic acid content in the corn straw co-fermentation system was affected by adding different concentrations of bituminous coal. The humic acid content in the fermentation system without bituminous coal and in the fermentation system with 5%, 10%, 20%, and 40% bituminous coal was the highest on the 1st day at 6.03, 5.10, 5.4, 6.95, and 7.77 g/L, respectively. Subsequently, the humic acid content of the system gradually decreased. The humic acid content in the fermentation system with 5% bituminous coal was the lowest in the 1st d, and the gas production in the reaction system was the highest in the 1st d. The fermentation system supplemented with 40% bituminous coal had the highest humic acid concentration, which was consistent with the lowest gas production of 40% bituminous coal after 1 d. The results showed that the addition of 5% bituminous coal promoted the production of hydrogen, while the addition of 10%, 20%, and 40% bituminous coal inhibited the production of hydrogen. This is because a high concentration of humic acid affects anaerobic microbial cells and hydrolases in the fermentation process, resulting in the inactivity of microbial cells and hydrolases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Thus, the total gas production in the fermentation system will increase or decrease. It also limits the decomposition of organic matter in bituminous coal and corn straw, and then slows down or speeds up the fermentation rate, affecting the increase or decrease in total gas production.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Effect of co-fermentation of bituminous coal and corn stalk on benzoic acid content\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3.4\u003c/span\u003e shows the effect of different bituminous coal contents on the benzoic acid content of the corn stalk. The addition of different bituminous coal contents affected the benzoic acid content in the fermentation system. On the 1st d of fermentation, the benzoic acid contents of 0%, 5%, 10%, 20%, and 40% bituminous coal were 8.75, 9.85, 8.61, 7.68, and 5.89 mg/L. The highest benzoic acid concentration of 5% bituminous coal is consistent with the highest hydrogen production of 5% bituminous coal on the 1st d. The results showed that humic acid in the co-fermentation group supplemented with 5% bituminous coal was more easily degraded into benzoic acid by microorganisms than in the other co-fermentation groups; therefore, the promotion effect on the co-fermentation of bituminous coal and corn straw to produce hydrogen gas was stronger. Low concentrations of humic acid in the fermentation system can trigger electron transfer through the microbial cell membrane, thus enhancing hydrolase activity and promoting benzoic acid degradation, increasing hydrogen production [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. As the benzoic acid content in the fermentation groups with 10%, 20%, and 40% bituminous coal was lower than that without bituminous coal, the total hydrogen production in the fermentation groups with 10%, 20%, and 40% bituminous coal was lower than that without bituminous coal. Although the concentration of benzoic acid in the fermentation group supplemented with 5% bituminous coal was also lower than that in the fermentation group without bituminous coal, the content of benzoic acid on the 1st d was higher in the fermentation group supplemented with 5% bituminous coal than in the fermentation group without bituminous coal. the hydrogen gas production on the 1st d was the highest in the co-fermentation process, and the influence of benzoic acid concentration on hydrogen production was not observed after that.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Effect of co-fermentation of bituminous coal and corn straw on pyruvate content\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3.5\u003c/span\u003e shows the effects of different bituminous coal contents on the pyruvate content in corn stalk fermentation, which is an important intermediate product in the fermentation process. Decarboxylation produces hydrogen gas, and hydrogen-producing bacteria directly decarboxylate glucose and transfer electrons to ferridoxin, which is then catalyzed by hydrogenase to reduce protons into H\u003csub\u003e2\u003c/sub\u003e molecules [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Formic acid is formed after decarboxylation of pyruvate and then by hydrogenase. Splitting formic acid entirely or partially into H\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3.5\u003c/span\u003e, the pyruvate concentrations in the corn straw fermentation system with 0%, 5%, 10%, 20%, and 40% bituminous coal added on the first day of fermentation were 105.8, 136.0, 124.0, 99.1, and 64.8 g/L, respectively. On 21st d of fermentation, the pyruvate concentrations of the 0%, 5%, 10%, and 20% fermentation groups reached the lowest values of 53.5, 57.3, 54.2, and 40.2 g/L, respectively. The fermentation group supplemented with 40% bituminous coal reached its lowest value on the 18th d. In the fermentation group with 5% bituminous coal, the pyruvate concentration decreased the most, and the pyruvate degradation was up to 78.7 g/L. The greater the degree of pyruvate degradation, the greater the amount of hydrogen produced. Therefore, the co-fermentation group with 5% bituminous coal exhibited the highest total gas production.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Effect of co-fermentation of bituminous coal and corn stalk on glucose content\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3.6\u003c/span\u003e shows the changes in glucose concentration in the fermentation group with different bituminous coal contents. The glucose concentration in the corn straw fermentation system with 0%, 5%, 10%, 20%, and 40% bituminous coal added reached the maximum value at the 3rd d, which were 2.45, 2.58, 2.41, 2.44, and 2.29 g/L, respectively. The glucose content in the first 2-d reaction system was close to 0. As shown in the figure, the fermentation group with 5% bituminous coal had the highest glucose concentration at the 3rd d, which was 0.13 g/L more than the fermentation group without bituminous coal. As corn straw is rich in cellulose, the glucoside bonds of cellulose break down to produce glucose [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], which can be converted and degraded into hydrogen. The glucose concentration of the other fermentation groups was lower than that of the fermentation group without bituminous coal, indicating that adding too much bituminous coal reduces the microbial activity, degree of corn stalk degradation, and hydrogen production.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Effect of co-fermentation of bituminous coal and corn straw on pH\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3.7\u003c/span\u003e shows the effects of adding different amounts of bituminous coal to the fermentation system. pH is one of the factors affecting microbial fermentation and the activity of anaerobic microorganisms in the fermentation system, and has a certain effect on the amount of gas produced. With and without the addition of bituminous coal, the initial pH of the fermentation system showed a downward trend with the increase of fermentation days, and reached the lowest value on the 21st d due to the production of volatile fatty acids such as butyric acid and propionic acid [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], resulting in a decrease in pH. The pH values of the fermentation groups containing 0%, 5%, 10%, 20%, and 40% bituminous coal were 4.83, 4.26, 5.53, 5.25, and 5.23, respectively, on the 21st d. The pH of the fermentation system with 5% bituminous coal was lower than that of the system without bituminous coal, and the total gas production was the highest. The pH of the fermentation systems with 10%, 20%, and 40% bituminous coal was higher than that without bituminous coal, and the total gas production was lower than that without bituminous coal.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Effect of co-fermentation of bituminous coal and corn straw on structure characteristics of residual pores\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3.8\u003c/span\u003e, the isotherms of N\u003csub\u003e2\u003c/sub\u003e adsorption and desorption of the fermentation residue with and without bituminous coal were similar to those of type IV, and the upward convex curve in the low P/P\u003csup\u003eo\u003c/sup\u003e region was similar to that of type II isotherms [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Capillary condensation occurred in the higher P/P\u003csup\u003eo\u003c/sup\u003e region, and the isotherm increased rapidly. When all the pores are condensed, adsorption occurs only on the outer surface, which is much smaller than the inner surface area, and the curve is flat. When the relative pressure is close to 1, it adsorbs onto the large pores, and the curve rises. Capillary condensation resulted in hysteresis in this region, i.e., the isotherm obtained during desorption did not coincide with that obtained during adsorption. The desorption isotherm was above the adsorption isotherm, resulting in adsorption hysteresis and an H3-type hysteresis loop, indicating that the pore shape of bituminite coal was dominated by slit pores [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3.9\u003c/span\u003e shows the structural analysis of the residual pores after hydrogen production by fermentation. Table () shows that the residual pore structure is different after hydrogen fermentation in fermentation groups with different concentrations of bituminous coal. The specific surface area of the blank control group without bituminous coal was 1.99765 m\u003csup\u003e2\u003c/sup\u003e/g, total pore volume was 0.008372 m\u003csup\u003e3\u003c/sup\u003e/g, and average pore diameter was 16.05975 nm. The specific surface area of the 5% bituminous coal was the smallest at 1.6375 m\u003csup\u003e2\u003c/sup\u003e/g. The small specific surface area may be due to the destruction of the pore collapse structure of the fermentation raw material due to the microbial degradation of bituminous coal. However, the maximum average pore size was 16.4875 nm, indicating that more microorganisms entered the pore structure of the raw fermentation material, and the degradation of organic matter resulted in an increase in the pore size, which is consistent with the maximum total gas production of 5% bituminous coal.\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eBituminous coal and corn straw fermentation produce hydrogen, and a moderate concentration of bituminous coal is conducive to corn straw fermentation to produce hydrogen.\u003c/p\u003e\u003cp\u003e(1) In the reaction system of the co-fermentation of bituminous coal and corn straw to produce hydrogen, the addition of 5% bituminous coal increased the total hydrogen production, and the gas production was 6.91% higher than that of the fermentation system without bituminous coal. The excessive addition of bituminous coal can inhibit hydrogen production in corn stalks.\u003c/p\u003e\u003cp\u003e(2) On the 1st day of fermentation, the hydrogen fermentation group with bituminous coal reached its peak value, and the fermentation group with 5% bituminous coal produced up to 102.4 mL of gas. The gas production in the fermentation group without bituminous coal was 77.4 mL on the 1st day, and the gas production in the fermentation group with 5% bituminous coal was 32.30% more than that in the fermentation group without bituminous coal. Their results showed that the addition of 5% bituminous coal promoted daily hydrogen production from corn straw.\u003c/p\u003e\u003cp\u003e(3) The contents of benzoic acid, pyruvate, and glucose in the reaction system for the co-fermentation of bituminous coal and corn straw were higher than those in the other fermentation groups. The results showed that the addition of 5% bituminous coal was more conducive to the fermentation of corn stalks to produce hydrogen.\u003c/p\u003e\u003cp\u003e(4) In the reaction system of co-fermentation of bituminous coal and corn straw to produce hydrogen, the specific surface area of residue in the blank control group without bituminous coal was 1.99765 m\u003csup\u003e2\u003c/sup\u003e/g, total pore volume was 0.008372 m\u003csup\u003e3\u003c/sup\u003e/g, and average pore size was 16.05975 nm. The specific surface area of the residue with 5% bituminous coal was the smallest at 1.6375 m\u003csup\u003e2\u003c/sup\u003e/g. The average pore size was 16.4875 nm, indicating that the pore size of the residue increased because of the degradation of organic matter.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have contributed to the research concept and design. The first draft of this manuscript was written by Xin Guo. Data collection is the responsibility of Yanan Yu. Jun Li \u0026amp; Litong Ma are responsible for language polishing. The material preparation work is the responsibility of Xiaobo Xu. And all authors commented on the previous version of the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is financially supported by the Science and Technology Plan Project of Inner Mongolia Autonomous Region (2025YFHH0127); Natural Science Foundation of Inner Mongolia (2025MS03024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHan, R., Yong, F., ,Fang, X., et al.: Influences of Fermented Corn Straw Fiber on Performance and Nutrient Utilization in Different Breeds of Finishing Pigs.Animals,2024,\u003cb\u003e14\u003c/b\u003e(23):3393\u0026ndash;3393\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFu, Y., Zhang, J., ,Guan, T.: High-Value Utilization of Corn Straw: From Waste to Wealth. 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Coal Mine Saf. 2020,\u003cb\u003e51\u003c/b\u003e(11):14\u0026ndash;18\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"bituminous coal, Corn stalk, Microbial transformation, Hydrogen","lastPublishedDoi":"10.21203/rs.3.rs-7222272/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7222272/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn recent years, research and development of new energy sources has gained attention. Coal is a combustible organic biological rock comprising polymer hydrocarbons and traces of inorganic minerals. Notably, bituminous coal has the largest coal reserve. Corn stalk is an agricultural waste and a renewable resource. In this experiment, bituminous coal and corn straw were used to prepare clean hydrogen using microbial conversion, to achieve zero-carbon energy. The results showed that adding different amounts of bituminous coal affected hydrogen production during co-fermentation with corn stalk. On the 1st day of fermentation, the hydrogen fermentation group containing bituminous coal showed a peak. The gas production of the fermentation group with 5% bituminous coal was 102.4 mL at maximum, and that of the fermentation group without bituminous coal was 77.4 mL. The gas production of the fermentation group with 5% bituminous coal was 25 mL higher than that of the group without bituminous coal. The pH and humic acid content of the fermentation group supplemented with 5% bituminous coal were lower than those of the other fermentation systems; the benzoic acid, pyruvate, and glucose contents were higher than those of the other fermentation groups. The specific surface area of the blank control group without bituminous coal was 1.99765 m2/g, total pore volume was 0.008372 m3/g, and average pore diameter was 16.05975 nm. The specific surface area of the blank control group with 5% bituminous coal was 1.6375 m2/g and the average pore diameter was 16.4875 nm. In conclusion, 5% bituminous coal supplementation to the fermentation of corn straw was beneficial to produce hydrogen; however, further addition of bituminous coal inhibited the hydrogen production.\u003c/p\u003e","manuscriptTitle":"Co-fermentation of bituminous coal and corn straw to produce hydrogen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-29 18:30:30","doi":"10.21203/rs.3.rs-7222272/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-08-21T18:38:38+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-21T16:43:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Waste and Biomass Valorization","date":"2025-08-16T19:24:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-26T18:32:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Waste and Biomass Valorization","date":"2025-07-26T12:04:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"24b7b42e-6d14-4af1-a0e0-c89ff87bb0d0","owner":[],"postedDate":"August 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:09:13+00:00","versionOfRecord":{"articleIdentity":"rs-7222272","link":"https://doi.org/10.1007/s12649-025-03412-y","journal":{"identity":"waste-and-biomass-valorization","isVorOnly":false,"title":"Waste and Biomass Valorization"},"publishedOn":"2025-11-25 15:58:47","publishedOnDateReadable":"November 25th, 2025"},"versionCreatedAt":"2025-08-29 18:30:30","video":"","vorDoi":"10.1007/s12649-025-03412-y","vorDoiUrl":"https://doi.org/10.1007/s12649-025-03412-y","workflowStages":[]},"version":"v1","identity":"rs-7222272","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7222272","identity":"rs-7222272","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}