Biological hydrogen production method for coupling coal seam microbial community and beet pulp waste

Abstract

The invention discloses a biological hydrogen production method for coupling a coal seam microbial community and beet pulp waste. The biological hydrogen production method comprises the following steps: step 1, collecting coal seam microorganisms; step 2, sample pretreatment; 3, preparing a culture solution; step 4, flora enrichment culture; and 5, constructing a biological hydrogen production system to obtain hydrogen-rich gas. Microorganisms adopted in the method are original microorganisms in underground in-situ coal seam or ground coal-bed gas well drainage and mining water, sugar industry waste beet pulp is adopted as a hydrogen production substrate, and aiming at the bottlenecks of high strain cost, limited substrate applicability and the like in an existing biological hydrogen production technology, the coal seam microorganisms and sugar industry waste residues are coupled, so that the yield of hydrogen is increased. A low-cost hydrogen production system is constructed, so that the economic cost in the microbial hydrogen production process is reduced, and the beet pulp has the advantages of being safe, easy to obtain, low in cost and high in gas production rate, has the advantages of being easy and convenient to operate, economical, efficient, environmentally friendly and the like, and has a good industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioenergy, and in particular relates to a method for producing biohydrogen by coupling coal seam microbial communities with beet pulp waste. Background Art

[0002] Biohydrogen production technology has attracted widespread attention due to its advantages such as mild reaction conditions and renewable substrates. Existing technologies mainly rely on purified strains (such as Clostridium butyricum and Clostridium beijerinckii) to produce hydrogen in a single substrate system (such as glucose and agricultural waste). For example, patents such as application number CN202310065968 use iron-sulfur minerals to enhance the metabolic efficiency of Clostridia, while patent application number CN201410386834 uses polysaccharide substrates coupled with specific Clostridia. However, such solutions have problems such as high strain acquisition costs and complex substrate pretreatment.

[0003] A complete hydrogen production metabolic chain consisting of fermentative bacteria, hydrogen-producing bacteria and methanogens naturally exists in the coal seam ecosystem (the patent with application number CN201510289236 mentions a biomass conversion system), but due to the competitive metabolism of methanogens, the actual hydrogen production efficiency is limited. It is worth noting that beet pulp, a by-product of the sugar industry, is rich in degradable components such as hemicellulose and can theoretically be used as a high-quality hydrogen production substrate, but its high lignin content restricts the utilization efficiency of conventional strains. The present invention has made a breakthrough discovery: the in-situ microbial community in the coal seam has a strong decomposition ability for complex carbon sources, and by specifically inhibiting the activity of methanogens, beet pulp can be efficiently converted into hydrogen.

[0004] Current results show that biological hydrogen production is mainly produced by degrading sugars such as glucose and sucrose or agricultural waste through one or more bacterial strains. The Chinese patent document with application number CN202310065968 discloses a method for fermenting hydrogen-producing bacteria, directional screening and enhanced hydrogen production. By pre-treating estuary sediments, it is possible to directional enrich and screen fermentation hydrogen-producing bacteria-Clostridium butyricum, and then add iron-sulfur minerals to promote the utilization of glucose by Clostridium, thereby increasing the metabolic rate and hydrogen production. The Chinese patent document with application number CN201410386834 discloses a Clostridium beijerinckii for fermentation and hydrogen production, as well as its fermentation method and application. Clostridium beijerinckii is used to ferment monosaccharides, disaccharides and / or polysaccharides to produce ethanol, butanol, acetic acid, butyric acid and hydrogen. Chinese patent application number CN202111110985 discloses a hydrogen-producing bacterium and a method for producing hydrogen therefrom. The method uses a byproduct of a crop or a secondary processed product of the byproduct as a substrate and ferments hydrogen with hydrogen-producing Bacillus sp. and / or hydrogen-producing Clostridium sp. Chinese patent application number CN202210626920 discloses a method for improving the efficiency of hydrogen production by clostridial fermentation and its application. The method involves inoculating Clostridium pasteurianum (DSM 525) and / or Clostridium bacterium (BY-1) into a dark fermentation medium with glucose as the substrate and performing light-proof fermentation in an environment with nitrogen in the headspace to produce hydrogen. The Chinese patent document, application number CN201510289236, discloses a biomass conversion system that can convert corn, sugarcane, sugar beets, trees, shrubs, grasses, phytoplankton, zooplankton, algae, macroalgae, seaweed, wood waste, pulp, cotton, wool, flax, paper, agricultural waste, and mixtures thereof into ethanol, hydrocarbons, natural gas, hydrogen, plastics, polymers, and proteins.

[0005] Although existing patents disclose methods for biohydrogen production that can generate hydrogen, these methods often require the purchase of one or more fermentation-producing bacteria and fail to consider that beet pulp, an industrial by-product of agricultural products, can also be used as a fermentation substrate. Therefore, using bacteria enriched in underground in-situ coal seams or surface coalbed methane drainage water as hydrogen-producing bacteria and beet pulp as a substrate for hydrogen production is more economical and conducive to commercial application. Summary of the Invention

[0006] The present invention addresses the shortcomings of the prior art by providing a method for biohydrogen production by coupling coalbed microbial communities with sugar beet pulp waste. This method utilizes microorganisms native to underground coalbeds or surface coalbed methane well drainage water, and uses sugar beet pulp, a waste product from the sugar industry, as a hydrogen production substrate. This method addresses the bottlenecks of existing biohydrogen production technologies, such as high strain costs and limited substrate applicability, by constructing a low-cost hydrogen production system. This system offers advantages such as ease of operation, cost-effectiveness, and environmental friendliness, and has promising prospects for industrial application.

[0007] To solve the above technical problems, the present invention adopts a technical solution: a method for producing hydrogen by coupling coal seam microbial communities with sugar beet pulp waste, characterized in that the method comprises the following steps:

[0008] Step 1: Coal seam microorganism collection:

[0009] Collect samples from underground in-situ coal seams and surface coalbed methane well drainage water, and then seal and store them at low temperatures to obtain underground in-situ coal seam samples and surface coalbed methane well drainage water containing microorganisms;

[0010] Step 2: Sample pretreatment:

[0011] The underground in-situ coal seam coal sample with microorganisms obtained in step 1 is aseptically crushed, and then sealed and stored at low temperature to obtain a crushed underground in-situ coal seam coal sample; part of the crushed underground in-situ coal seam coal sample and the crushed beet pulp are dried and sterilized to obtain a sterile coal sample and sterile beet pulp;

[0012] Step 3: Preparation of culture medium:

[0013] The culture medium is prepared by using an inorganic culture medium, a reducing solution, a trace element solution and a vitamin solution;

[0014] Step 4: Bacteria enrichment culture:

[0015] Add the culture solution prepared in step 3 to the crushed underground in-situ coal seam sample obtained in step 2, then replace it with nitrogen, seal it and culture it, and obtain an anaerobic fermentation bacterial liquid of the underground in-situ coal seam sample; add the surface coalbed methane well drainage water obtained in step 1 and the culture solution prepared in step 3 to the sterile coal sample obtained in step 2, then replace it with nitrogen, seal it and culture it, and obtain an anaerobic fermentation bacterial liquid of the surface coalbed methane well drainage water; the volume ratio of the mass of the crushed underground in-situ coal seam sample to the culture solution is 10 to 30:300, and the ratio of the mass of the sterile coal sample to the total volume of the surface coalbed methane well drainage water and the culture solution is 10 to 30:300, wherein the unit of mass is g, the unit of volume is mL, and the volume ratio of the surface coalbed methane well drainage water to the culture solution is 1:1 to 5;

[0016] Step 5: Construction of biohydrogen production system:

[0017] The culture solution and methane inhibitor prepared in step 3 and the underground in-situ coal seam coal sample anaerobic fermentation bacterial liquid or the surface coalbed methane well drainage water anaerobic fermentation bacterial liquid obtained in step 4 are added to the sterile beet pulp obtained in step 2, and then nitrogen is replaced, and the mixture is sealed and cultured to obtain hydrogen-rich gas; the ratio of the mass of the sterile beet pulp, the volume of the culture solution and the mass of the methane inhibitor is 1-10:150-300:0.15-0.3, wherein the unit of mass is g and the unit of volume is mL; and the volume ratio of the culture solution to the underground in-situ coal seam coal sample anaerobic fermentation bacterial liquid or the surface coalbed methane well drainage water anaerobic fermentation bacterial liquid is 10-100:1.

[0018] The present invention uses underground in-situ coal seam samples containing microorganisms and surface coalbed methane well drainage water as microbial sources. The underground in-situ coal seam samples contain microorganisms directly from the coal seam, making them more pristine and representing the true in-situ microbial ecosystem within the coal seam. In contrast, the microorganisms in surface coalbed methane well drainage water are affected by the mining process, causing changes in the microbial flora, but are easier to obtain and culture. The sugar beet pulp is dried and sterilized to prevent the introduction of other bacteria.

[0019] When the present invention uses underground in-situ coal seam coal samples with microorganisms as the source of microorganisms, they are aseptically crushed and then added with culture medium for cultivation to obtain anaerobic fermentation bacterial liquid of the underground in-situ coal seam coal samples, and then sterile beet pulp, culture medium and methane inhibitor are added to prepare hydrogen.

[0020] When the present invention uses surface coalbed methane well drainage water containing microorganisms as the source of microorganisms, partially crushed underground in-situ coal seam coal samples are dried and sterilized to enrich the microorganisms in the surface coalbed methane well drainage water. At this time, the sterile coal sample acts as a substrate. The sterile coal sample and culture medium are added to the surface coalbed methane well drainage water for cultivation to obtain the surface coalbed methane well drainage water anaerobic fermentation bacterial liquid, and then sterile beet pulp, culture medium and methane inhibitor are added to prepare hydrogen.

[0021] The methane inhibitors in the present invention can significantly inhibit the activity of methanogens in anaerobic fermentation broth. Anaerobic fermentation broth enriched with coalbed methane well drainage water and underground in-situ coal seam coal samples contains methanogens that consume hydrogen. Adding a methane inhibitor inhibits the conversion of hydrogen into methane, thereby improving hydrogen production efficiency. Vitamins and trace elements act as enzyme cofactors and participate in and catalyze various metabolic reactions within microorganisms, helping them complete various reactions in their metabolic pathways and improving the efficiency of the fermentation process and product quality. Microbial growth requires a suitable environment. The addition of a reducing solution provides an ideal redox environment for the microorganisms, thereby ensuring smooth microbial life activities and hydrogen production.

[0022] The beet pulp in the present invention is a by-product of the beet sugar production process and has a high yield. The production of hydrogen from beet pulp has multiple advantages such as low cost, abundant raw materials, environmental friendliness, great technical potential, and improved energy security. At the same time, it can also reduce the environmental burden of agricultural waste and promote sustainable development.

[0023] The aforementioned method for biohydrogen production by coupling coal seam microbial communities with sugar beet pulp waste is characterized in that the aseptic operations in steps 1 and 2 are performed within a clean bench, and the low temperature in steps 1 and 2 is 2°C to 5°C. The present invention controls aseptic operations to prevent the introduction of foreign bacteria, and by controlling the low temperature and storage temperature, slows the metabolism of microorganisms, preventing their activity from decreasing or even dying.

[0024] The above-mentioned method for coupling coal seam microbial communities with sugar beet pulp waste for biohydrogen production is characterized in that the particle size of the crushed underground in-situ coal seam coal sample in step 2 is 0.5 cm to 1 cm; the particle size of the sterilized sugar beet pulp is 0.15 mm to 0.60 mm; the drying temperature is 100°C to 110°C and the time is 4 hours to 6 hours; and the sterilization is irradiation sterilization under a UV lamp for 30 minutes to 60 minutes. The present invention facilitates the enrichment of microorganisms by controlling the particle size of the crushed underground in-situ coal seam coal sample, while ensuring the feasibility and efficiency of the experimental operation. By controlling the particle size of the sterilized sugar beet pulp, the surface area, mass transfer efficiency and fermentation system stability during the microbial degradation process are better balanced. By controlling the drying parameters, not only can the free water in the coal and sugar beet pulp be effectively removed and some microorganisms be killed, but the loss of organic matter by pyrolysis can also be avoided, ensuring the efficiency and stability of subsequent microbial degradation. By controlling the sterilization process and parameters, the miscellaneous bacteria present in the coal and sugar beet pulp are effectively killed, preventing them from competing for nutrients with the target degradation strain.

[0025] The above-mentioned method for producing biohydrogen by coupling coal seam microbial communities with beet pulp waste is characterized in that the process of preparing the culture medium in step 3 is: adding a trace element solution to an inorganic culture medium, then sterilizing at 121°C under closed conditions and cooling to obtain an inorganic culture medium containing trace elements, then adding a reducing solution and a vitamin solution to the obtained inorganic culture medium containing trace elements, stirring evenly, and sterilizing again under ultraviolet light for 30 minutes to 60 minutes, and finally adjusting the pH to 6.8 to 7.2 to obtain a culture medium. The present invention controls the process of preparing the culture medium so that the reducing solution and vitamin solution are added after high-temperature sterilization, thereby preventing them from deteriorating during the high-temperature sterilization process.

[0026] The aforementioned method for biohydrogen production by coupling coalbed microbial communities with sugar beet pulp waste is characterized in that the inorganic culture medium in step 3 is prepared from KCl, NaCl, MgCl2, NH4Cl, KH2PO4, K2HPO4, yeast extract, and distilled water, and the mass ratio of KCl, NaCl, MgCl2, NH4Cl, KH2PO4, K2HPO4, yeast extract, and distilled water in the culture medium is 0.2:2.0:2.0:1.0:0.4:0.4:1.0:1000. This method promotes hydrogen production from sugar beet pulp degradation by microorganisms by controlling the composition and content of the inorganic culture medium, optimizing the ion ratio, and adding yeast extract to provide growth factors, thereby offering the advantages of low cost and high stability.

[0027] The aforementioned method for biohydrogen production by coupling coalbed microbial communities with sugar beet pulp waste is characterized in that the reducing solution in step three is prepared from Na2S, NaHCO3, L-cysteine, and distilled water, and the mass ratio of Na2S, NaHCO3, L-cysteine, and distilled water in the culture medium is 0.2:1:0.5:1000. By controlling the composition and content of the reducing solution, providing a strong reducing environment with NaS and L-cysteine ​​to maintain a low redox potential in the system, and buffering acid production metabolism with NaHCO3, the present invention can both protect the activity of strictly anaerobic hydrogen-producing bacteria and promote the hydrolysis and fermentation of sugar beet pulp, offering the dual advantages of rapidly establishing anaerobic conditions and stabilizing hydrogen production efficiency.

[0028] The above-mentioned method for producing hydrogen by coupling coal seam microbial communities with beet pulp waste is characterized in that the trace element solution in step 3 consists of MgCl2·6H2O, FeCl2·4H2O, CoCl·6H2O, H3BO3, ZnSO4·7H2O, CuSO4·5H2O, NiSO4·6H2O, MnCl2·4H2O, (NH4)6Mo7O 24 ·4H2O and distilled water, wherein the culture solution contains MgCl2·6H2O, FeCl2·4H2O, CoCl·6H2O, H3BO3, ZnSO4·7H2O, CuSO4·5H2O, NiSO4·6H2O, MnCl2·4H2O, (NH4)6Mo7O 24 The mass ratio of 4H2O to distilled water is 0.05:0.06:0.0015:0.001:0.001:0.0005:0.0045:0.05:0.0064:1000. By controlling the composition and content of the trace element solution and providing a variety of essential trace elements, the present invention supports the metabolic activity of coal seam microorganisms, promotes the degradation of beet pulp, and thus promotes the efficient production of hydrogen.

[0029] The above-mentioned method for biological hydrogen production by coupling coal seam microbial communities with beet pulp waste is characterized in that the vitamin solution in step three is made of vitamin B6, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B12, lipoic acid, para-aminobenzoic acid, vitamin B7, vitamin B9 and distilled water, and the mass ratio of vitamin B6, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B12, lipoic acid, para-aminobenzoic acid, vitamin B7, vitamin B9 and distilled water in the culture solution is 0.0001:0.00005:0.00005:0.00005:0.00005:0.00005:0.00005:0.00005:0.00002:0.00002:1000. The present invention provides necessary nutritional support for microorganisms by controlling the composition and content of the vitamin solution, promotes their metabolic activity, and thus improves the efficiency of biohydrogen production. At the same time, the optimization of the component ratio helps maintain the stability and function of the microbial community.

[0030] The aforementioned method for biohydrogen production by coupling coalbed microbial communities with sugar beet pulp waste is characterized in that the culture temperature in step 4 is maintained at 25°C to 35°C and the culture duration is maintained at 14 to 28 days. This method controls the culture temperature to the optimal temperature for the growth and metabolism of most coalbed microorganisms. At this temperature, the microorganisms' metabolic rate and enzyme activity are optimized, significantly improving degradation efficiency. Controlling the culture time window provides the microorganisms with a sufficient growth period, allowing them to adapt to the environment and achieve optimal activity.

[0031] The aforementioned method for biohydrogen production by coupling coalbed microbial communities with sugar beet pulp waste is characterized in that the methane inhibitor in step five is prepared from sodium 2-bromoethylsulfonate and distilled water in a mass ratio of 1:1000; the incubation temperature in step five is 25°C to 35°C, and the incubation time is 56 to 63 days. By controlling the composition and content of the methane inhibitor and binding to the active site of methyl-CoA reductase, the present invention specifically inhibits the metabolic activity of methanogens. This method exhibits strong selectivity and minimal impact on other metabolic pathways, thereby helping to increase hydrogen yield. By controlling the incubation temperature and time, optimal hydrogenase activity is maintained while ensuring sufficient degradation of sugar beet pulp, achieving efficient and long-lasting biohydrogen production.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] 1. The microorganisms used in the present invention are original microorganisms in underground in-situ coal seams or surface coalbed methane well drainage water, thereby reducing the economic cost of the microbial hydrogen production process and facilitating commercial application. In addition, beet pulp, a waste product of the sugar industry, is used as a hydrogen production substrate. It is a by-product of beet sugar production and has the advantages of being safe, easily available, low cost, and having a high gas yield. In response to the bottlenecks of existing biological hydrogen production technologies such as high strain cost and limited substrate applicability, a low-cost hydrogen production system is constructed, which has the advantages of simple operation, economic efficiency, and green environmental protection, and has good prospects for industrial application.

[0034] 2. In the present invention, a methane inhibitor is added during the hydrogen production process to significantly inhibit the activity of methanogens in the anaerobic fermentation broth, thereby improving the hydrogen production efficiency. In addition, a culture solution composed of an inorganic culture medium, a reducing solution, a trace element solution, and a vitamin solution is added. The vitamins and trace elements promote the growth of microorganisms, help microorganisms complete various reactions in the metabolic pathway, and promote the improvement of the efficiency of the fermentation process and the quality of the product. The reducing solution provides an ideal redox environment for the microorganisms, ensuring the normal growth of the microorganisms and the smooth progress of the hydrogen production process.

[0035] 3. The present invention facilitates the enrichment of microorganisms by controlling the particle size of the crushed underground in-situ coal seam coal sample, while ensuring the feasibility and efficiency of the experimental operation. By controlling the particle size of the sterile beet pulp, the surface area, mass transfer efficiency and fermentation system stability during the microbial degradation process are better balanced.

[0036] 4. The present invention can maintain the optimal hydrogen production enzyme activity and ensure the full degradation of sugar beet pulp by controlling the culture temperature and time, thereby achieving efficient and long-lasting biological hydrogen production.

[0037] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a curve diagram of hydrogen production from microbial degradation of sugar beet pulp in an underground in-situ coal seam coal sample in Example 1 of the present invention.

[0039] Figure 2 This is a curve diagram of hydrogen production from microbial degradation of sugar beet pulp in coalbed methane well drainage water in Example 2 of the present invention.

[0040] Figure 3 This is a curve diagram of hydrogen production from microbial degradation of sugar beet pulp in an underground in-situ coal seam coal sample in Example 3 of the present invention.

[0041] Figure 4 This is a curve diagram of hydrogen production from microbial degradation of sugar beet pulp in coalbed methane well drainage water in Example 4 of the present invention. DETAILED DESCRIPTION

[0042] Example 1

[0043] This embodiment includes the following steps:

[0044] Step 1: Coal seam microorganism collection:

[0045] Collect samples from underground in-situ coal seams, put them into sealed bags on site, fill them with nitrogen to replace the air, store them in an insulated box at 2°C to 5°C, and bring them back to the laboratory to obtain underground in-situ coal seam samples containing microorganisms;

[0046] Step 2: Sample pretreatment:

[0047] The underground in-situ coal seam coal sample with microorganisms obtained in step 1 is aseptically crushed in a clean workbench, placed in a sealed bag, filled with nitrogen to replace the air, and stored in a refrigerator at 4°C to obtain a crushed underground in-situ coal seam coal sample with a particle size of 0.5 cm to 1 cm. The crushed beet pulp is dried at 110°C for 4 hours and sterilized under ultraviolet light for 40 minutes to obtain sterilized beet pulp with a particle size of 0.15 mm to 0.60 mm.

[0048] Step 3: Preparation of culture medium:

[0049] Place the inorganic culture medium in a conical flask and add the trace element solution. Seal the conical flask with medical absorbent cotton wrapped in cloth. Sterilize the flask in an autoclave at 121°C for 20 minutes. After sterilization, cool to room temperature and place it in a clean bench to obtain the inorganic culture medium containing trace elements. Add the reducing solution and vitamin solution to the obtained inorganic culture medium containing trace elements, stir evenly, and sterilize it again under ultraviolet light for 40 minutes. Finally, adjust the pH to 6.8-7. 2. Obtain a culture solution; the culture solution comprises KCl, NaCl, MgCl2, NH4Cl, KH2PO4, K2HPO4, yeast extract, Na2S, NaHCO3, L-cysteine, MgCl2·6H2O, FeCl2·4H2O, CoCl·6H2O, H3BO3, ZnSO4·7H2O, CuSO4·5H2O, NiSO4·6H2O, MnCl2·4H2O, (NH4)6Mo7O 244H2O, vitamin B6, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B12, lipoic acid, p-aminobenzoic acid, vitamin B7, vitamin B9 and distilled water are prepared in the following mass ratio: 0.2: 2.0: 2.0: 1.0: 0.4: 0.4: 1.0: 0.2: 1: 0.5: 0.05: 0.06: 0.0015: 0.001: 0.001: 0.0005: 0.0045: 0.05: 0.0064: 0.0001: 0.00005: 0.00005: 0.00005: 0.00005: 0.00005: 0.00005: 0.00002: 0.00002: 1000;

[0050] Step 4: Bacteria enrichment culture:

[0051] 30 g of the crushed underground in-situ coal seam sample obtained in step 2 was placed in an anaerobic bottle, and 300 mL of the culture solution obtained in step 3 was added. The bottle was then placed in an anaerobic glove box, and nitrogen was used as the carrier gas. The gas was replaced three times continuously to ensure that the oxygen in the glove box was completely removed. The bottle was sealed with an isobutyl stopper and a sealing film, and placed in a constant temperature incubator at 35°C in the dark for continuous cultivation for 28 days to obtain an anaerobic fermentation bacterial solution of the underground in-situ coal seam coal sample.

[0052] Step 5: Construction of biohydrogen production system:

[0053] 2 g of the sterile beet pulp obtained in step 2 is placed in an anaerobic bottle, and 150 mL of the culture medium obtained in step 3, 0.15 g of a methane inhibitor prepared by sodium 2-bromoethylsulfonate and distilled water in a mass ratio of 1:1000, and 1.5 mL of the underground in-situ coal seam coal sample anaerobic fermentation bacterial liquid obtained in step 4 are added. Then, the bottle is placed in an anaerobic glove box, and nitrogen is used as a carrier gas. The gas is replaced three times continuously to ensure that the oxygen in the glove box is completely removed. After that, an isobutyl stopper and a sealing film are used to seal the bottle, and the bottle is placed in a constant temperature incubator at 25°C to 35°C and cultured in the dark for 56 days to obtain hydrogen-rich gas.

[0054] In step 5 of this embodiment, the cells were cultured in a constant temperature incubator in the dark. The gas components were tested by gas chromatography on days 0, 7, 14, 21, 28, 35, 49, and 56, and the hydrogen content was calculated. The test results are shown in Tables 1 and Figure 1 , Table 1 and Figure 1 Parallel 1 and Parallel 2 in the figure indicate that two experiments were performed under the same conditions.

[0055] Table 1

[0056]

[0057] Example 2

[0058] This embodiment includes the following steps:

[0059] Step 1: Coal seam microorganism collection:

[0060] Collect samples from the drainage water of the surface coalbed methane well, fill the sample bottle with drainage water to prevent air from mixing, store it in a 2℃ to 5℃ incubator, and then store it in a 4℃ refrigerator in the laboratory to obtain the surface coalbed methane well drainage water with microorganisms;

[0061] Step 2: Sample pretreatment:

[0062] The underground in-situ coal seam sample with microorganisms obtained in step 1 is aseptically crushed in a clean workbench to obtain a crushed underground in-situ coal seam sample; the partially crushed underground in-situ coal seam sample and the crushed beet pulp are dried at 110° C. for 4 hours and sterilized under a UV lamp for 50 minutes to obtain a sterile coal sample with a particle size of 0.15 mm to 0.60 mm and a sterile beet pulp with a particle size of 0.15 mm to 0.60 mm;

[0063] Step 3: Preparation of culture medium:

[0064] Place the inorganic culture medium in a conical flask and add the trace element solution. Seal the conical flask with medical absorbent cotton wrapped in cloth. Sterilize the flask in an autoclave at 121°C for 20 minutes. After sterilization, cool to room temperature and place it in a clean workbench to obtain the inorganic culture medium containing trace elements. Add the reducing solution and vitamin solution to the obtained inorganic culture medium containing trace elements, stir evenly, and sterilize it again under ultraviolet light for 50 minutes. Finally, adjust the pH to 6.8-7. 2. Obtain a culture solution; the culture solution comprises KCl, NaCl, MgCl2, NH4Cl, KH2PO4, K2HPO4, yeast extract, Na2S, NaHCO3, L-cysteine, MgCl2·6H2O, FeCl2·4H2O, CoCl·6H2O, H3BO3, ZnSO4·7H2O, CuSO4·5H2O, NiSO4·6H2O, MnCl2·4H2O, (NH4)6Mo7O 244H2O, vitamin B6, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B12, lipoic acid, p-aminobenzoic acid, vitamin B7, vitamin B9 and distilled water are prepared in the following mass ratio: 0.2: 2.0: 2.0: 1.0: 0.4: 0.4: 1.0: 0.2: 1: 0.5: 0.05: 0.06: 0.0015: 0.001: 0.001: 0.0005: 0.0045: 0.05: 0.0064: 0.0001: 0.00005: 0.00005: 0.00005: 0.00005: 0.00005: 0.00005: 0.00002: 0.00002: 1000;

[0065] Step 4: Bacteria enrichment culture:

[0066] 30 g of the sterile coal sample obtained in step 2 was placed in an anaerobic bottle, and 100 mL of the ground coalbed methane well drainage water containing microorganisms obtained in step 1 and 200 mL of the culture medium obtained in step 3 were added. The bottle was then placed in an anaerobic glove box, and nitrogen was used as the carrier gas. The gas was replaced three times in a row to ensure that the oxygen in the glove box was completely removed. The bottle was sealed with an isobutyl stopper and a sealing film, and placed in a constant temperature incubator at 35° C. in the dark for continuous cultivation for 28 days to obtain an anaerobic fermentation bacterial solution of ground coalbed methane well drainage water.

[0067] Step 5: Construction of biohydrogen production system:

[0068] 2 g of the sterile beet pulp obtained in step 2 was placed in an anaerobic bottle, and 150 mL of the culture solution obtained in step 3, 0.15 g of a methane inhibitor prepared by sodium 2-bromoethylsulfonate and distilled water in a mass ratio of 1:1000, and 1.5 mL of the anaerobic fermentation bacterial liquid of the ground coalbed methane well drainage water obtained in step 4 were added. Then, the bottle was placed in an anaerobic glove box, and nitrogen was used as a carrier gas. The gas was replaced three times in a row to ensure that the oxygen in the glove box was completely removed. After that, an isobutyl stopper and a sealing film were used to seal the bottle. The bottle was placed in a constant temperature incubator at 35°C and protected from light for 56 days to obtain hydrogen-rich gas.

[0069] In step 5 of this embodiment, the cells were cultured in a constant temperature incubator in the dark. The gas components were tested by gas chromatography on days 0, 7, 14, 21, 28, 35, 49, and 56, and the hydrogen content was calculated. The test results are shown in Tables 2 and Figure 2 , Table 2 and Figure 2 Parallel 1 and Parallel 2 in the figure indicate that two experiments were performed under the same conditions.

[0070] Table 2

[0071]

[0072] Example 3

[0073] This embodiment includes the following steps:

[0074] Step 1: Coal seam microorganism collection:

[0075] Collect samples from underground in-situ coal seams, put them into sealed bags on site, fill them with nitrogen to replace the air, store them in an insulated box at 2°C to 5°C, and bring them back to the laboratory to obtain underground in-situ coal seam samples containing microorganisms;

[0076] Step 2: Sample pretreatment:

[0077] The underground in-situ coal seam coal sample with microorganisms obtained in step 1 is aseptically crushed in a clean workbench, placed in a sealed bag, filled with nitrogen to replace the air, and stored in a refrigerator at 4°C to obtain a crushed underground in-situ coal seam coal sample with a particle size of 0.5 cm to 1 cm. The crushed beet pulp is dried at 100°C for 6 hours and sterilized under ultraviolet light for 30 minutes to obtain sterile beet pulp with a particle size of 0.15 mm to 0.60 mm;

[0078] Step 3: Preparation of culture medium:

[0079] Place the inorganic culture medium in a conical flask and add the trace element solution. Seal the conical flask with medical absorbent cotton wrapped in cloth. Sterilize the flask in an autoclave at 121°C for 20 minutes. After sterilization, cool to room temperature and place it in a clean workbench to obtain the inorganic culture medium containing trace elements. Add the reducing solution and vitamin solution to the obtained inorganic culture medium containing trace elements, stir evenly, and sterilize it again under ultraviolet light for 30 minutes. Finally, adjust the pH to 6.8-7. 2. Obtain a culture solution; the culture solution comprises KCl, NaCl, MgCl2, NH4Cl, KH2PO4, K2HPO4, yeast extract, Na2S, NaHCO3, L-cysteine, MgCl2·6H2O, FeCl2·4H2O, CoCl·6H2O, H3BO3, ZnSO4·7H2O, CuSO4·5H2O, NiSO4·6H2O, MnCl2·4H2O, (NH4)6Mo7O 244H2O, vitamin B6, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B12, lipoic acid, p-aminobenzoic acid, vitamin B7, vitamin B9 and distilled water are prepared in the following mass ratio: 0.2: 2.0: 2.0: 1.0: 0.4: 0.4: 1.0: 0.2: 1: 0.5: 0.05: 0.06: 0.0015: 0.001: 0.001: 0.0005: 0.0045: 0.05: 0.0064: 0.0001: 0.00005: 0.00005: 0.00005: 0.00005: 0.00005: 0.00005: 0.00002: 0.00002: 1000;

[0080] Step 4: Bacteria enrichment culture:

[0081] 10 g of the crushed underground in-situ coal seam sample obtained in step 2 was placed in an anaerobic bottle, and 300 mL of the culture solution obtained in step 3 was added. The bottle was then placed in an anaerobic glove box, and nitrogen was used as the carrier gas. The gas was replaced three times continuously to ensure that the oxygen in the glove box was completely removed. The bottle was sealed with an isobutyl stopper and a sealing film, and placed in a constant temperature incubator at 30°C in the dark for continuous cultivation for 20 days to obtain an anaerobic fermentation bacterial solution of the underground in-situ coal seam coal sample.

[0082] Step 5: Construction of biohydrogen production system:

[0083] 1 g of the sterile beet pulp obtained in step 2 was placed in an anaerobic bottle, and 200 mL of the culture medium obtained in step 3, 0.2 g of a methane inhibitor prepared by sodium 2-bromoethylsulfonate and distilled water in a mass ratio of 1:1000, and 4.0 mL of the underground in-situ coal seam coal sample anaerobic fermentation bacterial liquid obtained in step 4 were added. Then, the bottle was placed in an anaerobic glove box, and nitrogen was used as a carrier gas. The gas was replaced three times in a row to ensure that the oxygen in the glove box was completely removed. After that, an isobutyl stopper and a sealing film were used to seal the bottle. The bottle was placed in a constant temperature incubator at 30°C and protected from light for continuous culture for 63 days to obtain hydrogen-rich gas.

[0084] In step 5 of this embodiment, the cells were cultured in a constant temperature incubator in the dark. The gas components were tested by gas chromatography on days 0, 7, 14, 21, 28, 35, 49, 56, and 63, and the hydrogen content was calculated. The test results are shown in Tables 3 and Figure 3 , Table 3 and Figure 3 Parallel 1 and Parallel 2 in the figure indicate that two experiments were performed under the same conditions.

[0085] Table 3

[0086]

[0087] Example 4

[0088] This embodiment includes the following steps:

[0089] Step 1: Coal seam microorganism collection:

[0090] Collect samples from the drainage water of the surface coalbed methane well, fill the sample bottle with drainage water to prevent air from mixing, store it in a 2℃ to 5℃ incubator, and then store it in a 4℃ refrigerator in the laboratory to obtain the surface coalbed methane well drainage water with microorganisms;

[0091] Step 2: Sample pretreatment:

[0092] The underground in-situ coal seam sample with microorganisms obtained in step 1 is aseptically crushed in a clean workbench to obtain a crushed underground in-situ coal seam sample; the partially crushed underground in-situ coal seam sample and the crushed beet pulp are dried at 105° C. for 5 h and sterilized under a UV lamp for 60 min to obtain a sterile coal sample with a particle size of 0.15 mm to 0.60 mm and a sterile beet pulp with a particle size of 0.15 mm to 0.60 mm;

[0093] Step 3: Preparation of culture medium:

[0094] Place the inorganic culture medium in a conical flask and add the trace element solution. Seal the conical flask with medical absorbent cotton wrapped in cloth. Sterilize the flask in an autoclave at 121°C for 20 minutes. After sterilization, cool to room temperature and place it in a clean bench to obtain the inorganic culture medium containing trace elements. Add the reducing solution and vitamin solution to the obtained inorganic culture medium containing trace elements, stir evenly, and sterilize it again under ultraviolet light for 60 minutes. Finally, adjust the pH to 6.8-7. 2. Obtain a culture solution; the culture solution comprises KCl, NaCl, MgCl2, NH4Cl, KH2PO4, K2HPO4, yeast extract, Na2S, NaHCO3, L-cysteine, MgCl2·6H2O, FeCl2·4H2O, CoCl·6H2O, H3BO3, ZnSO4·7H2O, CuSO4·5H2O, NiSO4·6H2O, MnCl2·4H2O, (NH4)6Mo7O 244H2O, vitamin B6, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B12, lipoic acid, p-aminobenzoic acid, vitamin B7, vitamin B9 and distilled water are prepared in the following mass ratio: 0.2: 2.0: 2.0: 1.0: 0.4: 0.4: 1.0: 0.2: 1: 0.5: 0.05: 0.06: 0.0015: 0.001: 0.001: 0.0005: 0.0045: 0.05: 0.0064: 0.0001: 0.00005: 0.00005: 0.00005: 0.00005: 0.00005: 0.00005: 0.00002: 0.00002: 1000;

[0095] Step 4: Bacteria enrichment culture:

[0096] 20 g of the sterile coal sample obtained in step 2 was placed in an anaerobic bottle, and 150 mL of the ground coalbed methane well drainage water containing microorganisms obtained in step 1 and 150 mL of the culture medium obtained in step 3 were added. The bottle was then placed in an anaerobic glove box, and nitrogen was used as the carrier gas. The gas was replaced three times in a row to ensure that the oxygen in the glove box was completely removed. The bottle was sealed with an isobutyl stopper and a sealing film, and placed in a constant temperature incubator at 25° C. in the dark for 14 days to obtain an anaerobic fermentation bacterial solution of ground coalbed methane well drainage water.

[0097] Step 5: Construction of biohydrogen production system:

[0098] 10 g of the sterile beet pulp obtained in step 2 was placed in an anaerobic bottle, and 300 mL of the culture solution obtained in step 3, 0.3 g of a methane inhibitor prepared by sodium 2-bromoethylsulfonate and distilled water in a mass ratio of 1:1000, and 30 mL of the anaerobic fermentation bacterial liquid of the ground coalbed methane well drainage water obtained in step 4 were added. Then, the bottle was placed in an anaerobic glove box, and nitrogen was used as a carrier gas. The gas was replaced three times in a row to ensure that the oxygen in the glove box was completely removed. After that, an isobutyl stopper and a sealing film were used to seal the bottle. The bottle was placed in a constant temperature incubator at 25° C. and cultured in the dark for 63 days to obtain hydrogen-rich gas.

[0099] In step 4 of this embodiment, the mass of the sterile coal sample can also be 10 g, and the volume of the culture solution obtained in step 3 can also be 500 mL.

[0100] In step 5 of this embodiment, the cells were cultured in a constant temperature incubator in the dark. The gas components were tested by gas chromatography on days 0, 7, 14, 21, 28, 35, 49, 56, and 63, and the hydrogen content was calculated. The test results are shown in Tables 4 and Figure 4 , Table 4 and Figure 4 Parallel 1 and Parallel 2 in the figure indicate that two experiments were performed under the same conditions.

[0101] Table 4

[0102]

[0103] In Examples 1 to 4, the underground in-situ coal seams were taken from gas coal in Baode, Shanxi, the surface coalbed methane well drainage water was taken from the coalbed methane wells in Baode, Shanxi, and the beet pulp was the residue left over from squeezing beet roots and tubers in a sugar factory in Gansu.

[0104] In Examples 1 to 4, the anaerobic bottles (500 mL), glass rods, beakers and other instruments used were sterilized in a high-temperature autoclave at 121° C. for 20 minutes before use.

[0105] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the technical essence of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for producing biohydrogen by coupling coal seam microbial communities with sugar beet pulp waste, characterized in that: The method comprises the following steps: Step 1: Coal seam microorganism collection: Collect samples from underground in-situ coal seams and surface coalbed methane well drainage water, and then seal and store them at low temperatures to obtain underground in-situ coal seam samples and surface coalbed methane well drainage water containing microorganisms; Step 2: Sample pretreatment: The underground in-situ coal seam coal sample with microorganisms obtained in step 1 is aseptically crushed, and then sealed and stored at low temperature to obtain a crushed underground in-situ coal seam coal sample; part of the crushed underground in-situ coal seam coal sample and the crushed beet pulp are dried and sterilized to obtain a sterile coal sample and sterile beet pulp; Step 3: Preparation of culture medium: The culture medium is prepared by using an inorganic culture medium, a reducing solution, a trace element solution and a vitamin solution; Step 4: Bacteria enrichment culture: Add the culture solution prepared in step 3 to the crushed underground in-situ coal seam sample obtained in step 2, then replace it with nitrogen, seal it and culture it, and obtain an anaerobic fermentation bacterial liquid of the underground in-situ coal seam sample; add the surface coalbed methane well drainage water obtained in step 1 and the culture solution prepared in step 3 to the sterile coal sample obtained in step 2, then replace it with nitrogen, seal it and culture it, and obtain an anaerobic fermentation bacterial liquid of the surface coalbed methane well drainage water; the volume ratio of the mass of the crushed underground in-situ coal seam sample to the culture solution is 10 to 30:300, and the ratio of the mass of the sterile coal sample to the total volume of the surface coalbed methane well drainage water and the culture solution is 10 to 30:300, wherein the unit of mass is g, the unit of volume is mL, and the volume ratio of the surface coalbed methane well drainage water to the culture solution is 1:1 to 5; Step 5: Construction of biohydrogen production system: The culture solution and methane inhibitor prepared in step 3 and the underground in-situ coal seam coal sample anaerobic fermentation bacterial liquid or the surface coalbed methane well drainage water anaerobic fermentation bacterial liquid obtained in step 4 are added to the sterile beet pulp obtained in step 2, and then nitrogen is replaced, and the mixture is sealed and cultured to obtain hydrogen-rich gas; the ratio of the mass of the sterile beet pulp, the volume of the culture solution and the mass of the methane inhibitor is 1-10:150-300:0.15-0.3, wherein the unit of mass is g and the unit of volume is mL; and the volume ratio of the culture solution to the underground in-situ coal seam coal sample anaerobic fermentation bacterial liquid or the surface coalbed methane well drainage water anaerobic fermentation bacterial liquid is 10-100:

1.

2. The method for producing hydrogen by coupling coal seam microbial communities with sugar beet pulp waste according to claim 1, characterized in that: The aseptic operation in step 1 and step 2 is completed in a clean workbench, and the low temperature in step 1 and step 2 is 2° C. to 5° C.

3. The method for producing hydrogen by coupling coal seam microbial communities with sugar beet pulp waste according to claim 1, characterized in that: The particle size of the crushed underground in-situ coal seam coal sample in step 2 is 0.5cm~1cm; the particle size of the sterilized beet pulp is 0.15mm~0.60mm; the drying temperature is 100℃~110℃, and the time is 4h~6h; the sterilization is irradiation sterilization under ultraviolet light for 30min~60min.

4. The method for producing hydrogen by coupling coal seam microbial communities with sugar beet pulp waste according to claim 1, characterized in that: The process of preparing the culture medium described in step three is: adding the trace element solution to the inorganic culture medium, then sterilizing and cooling at a high temperature of 121°C under closed conditions to obtain an inorganic culture medium with trace elements, and then adding the reducing solution and the vitamin solution to the obtained inorganic culture medium with trace elements, respectively, stirring evenly, and sterilizing again under ultraviolet light for 30 minutes to 60 minutes, and finally adjusting the pH to 6.8 to 7.2 to obtain the culture medium.

5. The method for producing hydrogen by coupling coal seam microbial communities with sugar beet pulp waste according to claim 1, characterized in that: The inorganic culture medium in step 3 is prepared from KCl, NaCl, MgCl2, NH4Cl, KH2PO4, K2HPO4, yeast extract and distilled water. The mass ratio of KCl, NaCl, MgCl2, NH4Cl, KH2PO4, K2HPO4, yeast extract and distilled water in the culture solution is 0.2:2.0:2.0:1.0:0.4:0.4:1.0:1000.

6. The method for producing hydrogen by coupling coal seam microbial communities with sugar beet pulp waste according to claim 1, characterized in that: The reducing solution in step 3 is made of Na2S, NaHCO3, L-cysteine ​​and distilled water, and the mass ratio of Na2S, NaHCO3, L-cysteine ​​and distilled water in the culture solution is 0.2:1:0.5:1000.

7. The method for producing hydrogen by coupling coal seam microbial communities with sugar beet pulp waste according to claim 1, characterized in that: The trace element solution in step 3 is composed of MgCl2·6H2O, FeCl2·4H2O, CoCl·6H2O, H3BO3, ZnSO4·7H2O, CuSO4·5H2O, NiSO4·6H2O, MnCl2·4H2O, (NH4)6Mo7O 24 ·4H2O and distilled water, wherein the culture solution contains MgCl2·6H2O, FeCl2·4H2O, CoCl·6H2O, H3BO3, ZnSO4·7H2O, CuSO4·5H2O, NiSO4·6H2O, MnCl2·4H2O, (NH4)6Mo7O 24 The mass ratio of 4H2O to distilled water is 0.05:0.06:0.0015:0.001:0.001:0.0005:0.0045:0.05:0.0064:1000.

8. The method for producing hydrogen by coupling coal seam microbial communities with sugar beet pulp waste according to claim 1, characterized in that: The vitamin solution in step 3 is prepared from vitamin B6, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B12, lipoic acid, p-aminobenzoic acid, vitamin B7, vitamin B9 and distilled water, and the mass ratio of vitamin B6, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B12, lipoic acid, p-aminobenzoic acid, vitamin B7, vitamin B9 and distilled water in the culture solution is 0.0001:0.00005:0.00005:0.00005:0.00005:0.00005:0.00005:0.00005:0.00002:0.00002:1000.

9. The method for producing hydrogen by coupling coal seam microbial communities with sugar beet pulp waste according to claim 1, characterized in that: The culture temperature in step 4 is 25° C. to 35° C., and the culture time is 14 to 28 days.

10. The method for producing hydrogen by coupling coal seam microbial communities with sugar beet pulp waste according to claim 1, characterized in that: The methane inhibitor in step 5 is prepared from sodium 2-bromoethylsulfonate and distilled water in a mass ratio of 1:1000; the culture temperature in step 5 is 25°C to 35°C, and the culture time is 56 days to 63 days.

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