CN105755049B - Method for preparing hydrogen by fermenting with xylose as substrate - Google Patents
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Abstract
The invention relates to a method for preparing hydrogen by fermenting xylose serving as a substrate. Specifically, a xylose solution is used as a substrate, the xylose solution is a xylose water solution or cellulose hydrolysate, anaerobic digested sludge, clostridium butyricum INET1 or enterococcus faecium INET2 is used as a hydrogen-producing strain, and the hydrogen is prepared by fermentation under an anaerobic condition, wherein the maximum hydrogen production amount of the xylose is 105-330 mL/100mL, and the hydrogen production efficiency is 66-228 mL/g of the xylose. The implementation of the invention effectively solves the problem that the cellulose hydrolysate can not be fully utilized due to the difficulty of utilizing xylose by microorganisms. Xylose is a main hydrolysate of plant cellulose, plant fiber raw materials are rich in resources, cheap and easy to obtain, and the important hydrolysate of the plant cellulose, namely xylose, is used as a substrate for biological hydrogen production, so that the cost of raw materials for biological fermentation hydrogen production can be reduced, and the cellulose raw materials can be fully utilized.
Description
Technical Field
The invention belongs to the technical field of hydrogen production by biological fermentation, and particularly relates to a method for preparing hydrogen by fermentation by taking xylose as a substrate.
Background
Hydrogen energy is a secondary energy source, and the existing hydrogen production processes mainly comprise hydrogen production by water electrolysis, hydrogen production by pyrolysis, photochemical hydrogen production, nuclear hydrogen production, ore fuel hydrogen production, biological hydrogen production and the like. The hydrogen production by water electrolysis is widely applied and mature at present. The efficiency of hydrogen production by water electrolysis is generally 75-85%, the process is simple, pollution-free and high in power consumption, so that the application of the hydrogen production is limited to a certain extent. Photochemical hydrogen production is a method for preparing hydrogen by taking water as a raw material and carrying out photocatalytic decomposition. The hydrogen production by mineral fuel is the most important method for producing hydrogen at present, and coal, petroleum and natural gas are used as raw materials. The use of nuclear and solar energy to decompose water to produce hydrogen is a subject that is being sought.
Compared with the hydrogen production process, the biological fermentation hydrogen production method has the characteristics of mild reaction conditions, low energy consumption, high environmental protection and the like, and the hydrogen is prepared by the fermentation method, so that the environmental benefit of the hydrogen can be exerted to the greatest extent.
The substrate is a key factor for limiting the development of biological hydrogen production, and the hydrogen production cost can be reduced only by adopting the cheap substrate, so that the application of the technology is promoted. As a big agricultural country, China has over fifteen billions of acres of cultivated land and generates a large amount of plant fiber wastes every year. According to incomplete statistics, the yield of the plant straws and the hulls in China can reach more than 7 hundred million tons every year. Most of the organic matters are not effectively utilized and are mainly treated in a local burning mode, so that not only is the resource wasted, but also the environment is polluted. If the cellulose can be used for producing hydrogen by fermentation, the cellulose can be more fully utilized.
Lignocellulose is used as a main component of crop straws, 40% of crude fiber (lignin and cellulose) and 20% of hemicellulose can be obtained after physical and chemical pretreatment or enzymolysis treatment, the hemicellulose and the cellulose are hydrolyzed into monosaccharide molecules under the action of enzyme or acid, the monosaccharide molecules mainly comprise glucose and xylose, the glucose can be utilized by almost all microorganisms, about 30-40% of pentose, namely the xylose, is difficult to be utilized by the microorganisms, and a large amount of compounds which can inhibit the activity of the microorganisms exist in cellulose hydrolysate. The utilization of xylose in cellulose hydrolysate is the focus and hot spot of the current research. The essence of the research papers and patents reported at present for producing hydrogen by using cellulose hydrolysate is to use glucose in the hydrolysate as a fermentation substrate. For example, some thermophilic hydrogen-producing strains can grow by utilizing xylose in cellulose hydrolysate, but the hydrogen production efficiency of xylose in the thermophilic hydrogen-producing strains is not reported (Zhuyuhong, high-temperature fermentation hydrogen-producing strain W16 and the fermentation hydrogen production characteristics thereof [ D ]. Harbin industry university, 2008; Liuyan, thermophilic anaerobic clostridium for degrading cellulose and hydrogen production characteristics research [ D ]. Shandong university, 2008); research on cow and motherland shows that xylose in cellulose hydrolysate can inhibit the hydrogen production effect of microorganisms by influencing the metabolic pathways of the microorganisms (cow and motherland. Klebsiella pneumoniae ECU-15 strain dark fermentation hydrogen production process analysis and experimental research on utilization of lignocellulose hydrolysate [ D ]. China east university of science, 2010 ]. Therefore, the key to the efficient utilization of the cellulose hydrolysate is to utilize xylose in the cellulose hydrolysate to produce hydrogen energy, so that the recycling efficiency of cellulose substances can be improved undoubtedly.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for preparing hydrogen by fermenting with xylose as a substrate.
A method for preparing hydrogen by fermentation by taking xylose as a substrate comprises the following steps:
1) pre-culturing hydrogen-producing strains;
2) using xylose solution as a substrate, adding nutrient solution to prepare a fermentation hydrogen production culture medium, inoculating hydrogen production strains pre-cultured in the step 1) into the fermentation hydrogen production culture medium, blowing off nitrogen for more than 3min to provide an oxygen-free environment, placing a reactor in a constant-temperature water bath shaking table, controlling the temperature at 25-40 ℃, the rotating speed of the shaking table at 80-120 r/min, adjusting the initial pH of fermentation liquor to be 6.0-9.0, performing fermentation hydrogen production, collecting fermentation gas, removing CO (carbon monoxide), and removing CO (carbon monoxide) from the fermentation gas2To obtain H2。
The hydrogen-producing strain in the step 1) is anaerobic digested sludge subjected to irradiation pretreatment, or a mixed strain of Clostridium butyricum (Clostridium butyricum) INET1 and Enterococcus Faecium (Enterococcus faecalis) INET2, or a single strain of Clostridium butyricum INET1 or a single strain of Enterococcus Faecium INET 2; the preservation number of the clostridium butyricum INET1 in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC1.5199, and the preservation date is 2015, 6 months and 3 days; the preservation number of the enterococcus faecium INET2 in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms is CGMCC1.15321, and the preservation date is 2015, 6 months and 3 days. The general microbiological center of China Committee for culture Collection of microorganisms is No. 3 Xilu No.1 on North Chen of the Chaozhou city.
The irradiation pretreatment is gamma ray irradiation treatment with 5kGy dosage, and adopts60The Co source acts as a radioactive source.
The clostridium butyricum INET1 and the enterococcus faecium INET2 are obtained by separating anaerobic digested sludge subjected to gamma-ray irradiation treatment with 5kGy dosage, and the radioactive source is60Co。
The pre-culture of the anaerobic digestion sludge after irradiation pretreatment comprises the following components: 20g/L of xylose, 10g/L of peptone, 0.5g/L of yeast powder and 10mL/100mL of nutrient solution;
the pre-culture of clostridium butyricum INET1 and enterococcus faecium INET2 comprises the following components in percentage by weight: 50g/L of glucose, 10g/L of peptone, 0.5g/L of yeast powder and 10mL/100mL of nutrient solution.
The xylose solution in the step 2) is a xylose aqueous solution or a cellulose hydrolysate, and the xylose solution is the cellulose hydrolysate which is prepared by directly using the cellulose hydrolysate to prepare a fermentation hydrogen production culture medium.
The fermentation hydrogen production culture medium comprises the following components: the xylose solution is 90mL/100mL, and the nutrient solution is 10mL/100 mL.
The inoculation proportion of the hydrogen-producing strain in the fermented hydrogen-producing culture medium is 10mL/100 mL.
The nutrient solution comprises the following components: NaHCO 2340g/L，NH4Cl 5g/L，NaH2PO4·2H2O 5g/L，K2HPO4·3H2O 5g/L，FeSO4·7H2O 0.25g/L，MgCl2·6H2O 0.085g/L，NiCl2·6H2O 0.004g/L。
The xylose concentration in the fermentation hydrogen production culture medium is 5-50 g/L, the maximum hydrogen production amount of xylose is 105-330 mL/100mL, and the hydrogen production efficiency is 66-228 mL/g xylose.
The invention has the beneficial effects that: the lignocellulose hydrolysate is used as a substrate for hydrogen production, and the hydrogen production strain disclosed by the invention not only can fully utilize glucose in the lignocellulose hydrolysate, but also can effectively utilize xylose in the lignocellulose hydrolysate for hydrogen production. Xylose is used as an important hydrolysate of lignocellulose, and the xylose is used as a substrate to prepare hydrogen through fermentation, so that the substrate range of biological hydrogen production is expanded, the utilization rate of plant fiber raw materials is improved, the cost of hydrogen production substrate is reduced, the reduction and the resource utilization of plant fiber waste are facilitated, and the aim of changing waste into valuable is really fulfilled. The successful application of the invention has important significance for realizing the industrialization of biological hydrogen production, directly promotes the further full utilization of cellulose substances, and provides a new method for the disposal of plant wastes.
Detailed Description
The present invention will be further described with reference to the following embodiments. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
Example 1
Inoculating anaerobic digested sludge subjected to irradiation pretreatment by taking xylose as a substrate to ferment and produce hydrogen:
the anaerobic digestion sludge is irradiated and pretreated by adopting gamma rays with 5kGy dosage so as to60Co acts as a radioactive source. Carrying out enrichment pre-culture on the anaerobic digestion sludge subjected to irradiation pretreatment, wherein the pre-culture medium comprises the following components: 20g/L of xylose, 10g/L of peptone, 0.5g/L of yeast powder and 10mL/100mL of nutrient solution. After pre-culture, inoculating 10mL of the culture medium into 100mL of the fermentation hydrogen-producing culture medium, wherein the fermentation hydrogen-producing culture medium comprises the following components: 50g/L of xylose and 10mL/100mL of nutrient solution, adjusting the initial pH to 7.0 by using 5mol/L HCl solution or 5mol/L NaOH solution, testing the air tightness by blowing off nitrogen and removing oxygen in the bottle, placing the reaction bottle in a constant-temperature water bath shaking table, fermenting to produce hydrogen at the temperature of 35 ℃ and under the reaction of 100r/min, and recording the gas production rate at intervals of 2 h. After 48 hours, the hydrogen production reaction is finished, and the gas chromatographic analysis shows that no methane is generated in the reaction process, so that methane bacteria are effectively killed by visible radiation treatment. The maximum accumulated hydrogen yield obtained by the experiment is 330mL/100mL, the hydrogen production efficiency is 66mL/g xylose, and the maximum hydrogen production rate is 20.3 mL-100 mL-1·h-1The lag time is 2.4 h.
Example 2
Inoculating anaerobic digested sludge subjected to irradiation pretreatment by taking xylose as a substrate to ferment and produce hydrogen:
the anaerobic digestion sludge is irradiated and pretreated by adopting gamma rays with 5kGy dosage so as to60Co acts as a radioactive source. Carrying out enrichment pre-culture on the anaerobic digestion sludge subjected to irradiation pretreatment, wherein the pre-culture medium comprises the following components: 20g/L of xylose, 10g/L of peptone, 0.5g/L of yeast powder and 10mL/100mL of nutrient solution. After pre-culture, inoculating 10mL of the culture medium into 100mL of the fermentation hydrogen-producing culture medium, wherein the fermentation hydrogen-producing culture medium comprises the following components: 5g/L xylose and 10mL/100mL nutrient solution, adjusting the initial pH to 7.0 by using 5mol/L HCl solution or 5mol/L NaOH solution, testing the air tightness by blowing off nitrogen and removing oxygen in the bottle, placing the reaction bottle in a constant-temperature water bath shaking table, fermenting at 35 ℃, reacting at 100r/min to produce hydrogen, and recording at intervals of 2hRecording the gas production rate. After 48 hours, the hydrogen production reaction is finished, and the gas chromatographic analysis shows that no methane is generated in the reaction process, so that methane bacteria are effectively killed by visible radiation treatment. The maximum accumulated hydrogen yield obtained by the experiment is 114mL/100mL, the hydrogen production efficiency is 228mL/g xylose, and the maximum hydrogen production rate is 7.8 mL-100 mL-1·h-1The lag time is 1.8 h.
Example 3
Inoculating clostridium butyricum INET1 to ferment and produce hydrogen by taking xylose as a substrate:
the bacterial colony of clostridium butyricum INET1 is picked from a plate culture medium and inoculated into 200mL of enrichment culture solution for enrichment pre-culture, and the enrichment culture solution comprises the following components: 50g/L of glucose, 10g/L of peptone, 0.5g/L of yeast powder and 10mL/100mL of nutrient solution. The strain grows into a logarithmic phase after 8-10h, at the moment, 10mL of bacterial liquid in the logarithmic phase is inoculated into 100mL of hydrogen-producing fermentation medium, and the hydrogen-producing fermentation medium comprises the following components: 10g/L of xylose and 10mL/100mL of nutrient solution, adjusting the initial pH to 7.0 by using 5mol/L HCl solution or 5mol/L NaOH solution, testing the air tightness by using nitrogen stripping and removing oxygen in the bottle, placing the reaction bottle in a constant-temperature water bath shaking table, fermenting at 35 ℃ and 100r/min to produce hydrogen, and recording the gas production rate at intervals of 2 h. After 56h, the hydrogen production reaction is finished, the maximum accumulated hydrogen production obtained by the experiment is 105mL/100mL, the hydrogen production efficiency is 105mL/g xylose, and the maximum gas production rate is 9.3 mL-100 mL-1·h-1The lag time is 36.4 h.
Example 4
Inoculating enterococcus faecium INET2 to ferment and produce hydrogen by taking xylose as a substrate:
the bacterial colony of clostridium faecium INET2 is picked from a plate culture medium and inoculated into 200mL of enrichment culture solution for enrichment pre-culture, and the enrichment culture solution comprises the following components: 50g/L of glucose, 10g/L of peptone, 0.5g/L of yeast powder and 10mL/100mL of nutrient solution. The strain grows into a logarithmic phase after 8-10h, at the moment, 10mL of bacterial liquid in the logarithmic phase is inoculated into 100mL of hydrogen-producing fermentation medium, and the hydrogen-producing fermentation medium comprises the following components: 10g/L of xylose and 10mL/100mL of nutrient solution, adjusting the initial pH to 7.0 by using 5mol/L HCl solution or 5mol/L NaOH solution, testing airtightness by using nitrogen stripping and removing oxygen in a bottle, placing a reaction bottle in a constant-temperature water bath shaking table, reacting at 35 ℃ under the condition of 100r/min, and recording the gas production rate at intervals of 2 h. After 70h, the hydrogen production reaction is finished, and the maximum accumulated hydrogen production obtained by the experiment is 3mL/100mL, which indicates that the single strain enterococcus faecium INET2 has weak capability of producing hydrogen by xylose fermentation.
Example 5
Taking xylose as a substrate, inoculating clostridium butyricum INET1 and enterococcus faecium INET2 at the same time for fermentation and hydrogen production:
colonies of clostridium butyricum INET1 and enterococcus faecium INET2 are picked from a plate culture medium and are respectively inoculated into 200mL of enrichment culture solution for enrichment pre-culture, and the enrichment culture solution comprises the following components: 50g/L of glucose, 10g/L of peptone, 0.5g/L of yeast powder and 10mL/100mL of nutrient solution. The two strains grow in the logarithmic phase after 8-10h, at the moment, 5mL of two kinds of bacteria liquid in the logarithmic phase are respectively inoculated in 100mL of xylose fermentation medium, and the fermentation hydrogen production medium comprises the following components: 10g/L of xylose and 10mL/100mL of nutrient solution, adjusting the initial pH to 7.0 by using 5mol/L HCl solution or 5mol/L NaOH solution, testing the air tightness by using nitrogen stripping, removing oxygen in the reaction bottle, placing the reaction bottle in a constant-temperature water bath shaking table, reacting at the temperature of 35 ℃ at 100r/min, and recording the gas production rate at intervals of 2 h. After 64h, the hydrogen production reaction is finished, the maximum accumulated hydrogen production obtained by the experiment is 114mL/100mL, the hydrogen production efficiency is 114mL/g xylose, and the maximum hydrogen production rate is 10.8 mL-100 mL-1·h-1The lag time was 49.7 h.
Example 6
Taking lignocellulose hydrolysate as a substrate, inoculating anaerobic digestion sludge subjected to irradiation pretreatment for fermentation to produce hydrogen:
the anaerobic digestion sludge is irradiated and pretreated by adopting gamma rays with 5kGy dosage so as to60Co acts as a radioactive source. Carrying out enrichment pre-culture on the anaerobic digestion sludge subjected to irradiation pretreatment, wherein the pre-culture medium comprises the following components: 20g/L of xylose, 10g/L of peptone, 0.5g/L of yeast powder and 10mL/100mL of nutrient solution. After pre-culture, inoculating 10mL of the culture medium into 100mL of the fermentation hydrogen-producing culture medium, wherein the fermentation hydrogen-producing culture medium comprises the following components: 90mL/100mL of lignocellulose hydrolysate, 10mL/100mL of nutrient solution and xylose concentration in the lignocellulose hydrolysateThe yield is 18g/L, xylose in the xylose is used as a substrate, the xylose is used as the substrate to produce hydrogen through fermentation, and glucose and the like in the xylose are used to produce hydrogen through fermentation. Adjusting the initial pH to 7.0 by using 5mol/L HCl solution or 5mol/L NaOH solution, testing the air tightness by using nitrogen stripping and expelling oxygen in the bottle, placing the reaction bottle in a constant-temperature water bath shaking table, fermenting to produce hydrogen at the temperature of 35 ℃ and the speed of 100r/min, and recording the gas production rate at intervals of 2 h. After 60h, the hydrogen production reaction is finished, the utilization rate of glucose reaches 90%, hydrogen produced by glucose and the like is deducted, the maximum accumulated hydrogen production of xylose in the experiment is 152mL/100mL, and the hydrogen production efficiency is 94mL/g xylose.
Claims (4)
1. A method for preparing hydrogen by fermentation by taking xylose as a substrate is characterized by comprising the following steps:
1) pre-culturing hydrogen-producing strains; the hydrogen-producing strain is a mixed flora of Clostridium butyricum (Clostridium butyricum) INET1 and Enterococcus Faecium (Enterococcus faecalis) INET 2; the preservation number of the clostridium butyricum (clostridium butyricum) INET1 in the China general microbiological culture collection management center is CGMCC NO.1.5199, and the preservation number of the Enterococcus Faecium (Enterococcus faecalis) INET2 in the China general microbiological culture collection management center is CGMCC NO. 1.15321;
2) using xylose solution as a substrate, adding nutrient solution to prepare a fermentation hydrogen production culture medium, inoculating hydrogen production strains pre-cultured in the step 1) into the fermentation hydrogen production culture medium, blowing off nitrogen for more than 3min to provide an oxygen-free environment, placing a reactor in a constant-temperature water bath shaking table, controlling the temperature at 25-40 ℃, the rotating speed of the shaking table at 80-120 r/min, adjusting the initial pH of fermentation liquor to be 6.0-9.0, performing fermentation hydrogen production, collecting fermentation gas, removing CO (carbon monoxide), and removing CO (carbon monoxide) from the fermentation gas2To obtain H2；
The xylose solution is xylose water solution or cellulose hydrolysate;
the xylose concentration in the fermentation hydrogen production culture medium is 5-50 g/L;
the nutrient solution comprises the following components: NaHCO 2340g/L，NH4Cl 5g/L，NaH2PO4·2H2O 5g/L，K2HPO4·3H2O5g/L，FeSO4·7H2O 0.25g/L，MgCl2·6H2O 0.085g/L，NiCl2·6H2O 0.004g/L。
2. The method according to claim 1, wherein the pre-culture of Clostridium butyricum (Clostridium butyricum) INET1, Enterococcus Faecium (Enterococcus faecalis) INET2, the medium composition is: 50g/L of glucose, 10g/L of peptone, 0.5g/L of yeast powder and 10mL/100mL of nutrient solution;
the nutrient solution comprises the following components: NaHCO 2340g/L，NH4Cl 5g/L，NaH2PO4·2H2O 5g/L，K2HPO4·3H2O5g/L，FeSO4·7H2O 0.25g/L，MgCl2·6H2O 0.085g/L，NiCl2·6H2O 0.004g/L。
3. The method according to claim 1, wherein the fermentation hydrogen production medium comprises the following components: the xylose solution is 90mL/100mL, and the nutrient solution is 10mL/100 mL.
4. The method as claimed in claim 1, wherein the inoculation ratio of the hydrogen-producing bacterial species in the hydrogen-producing fermentation medium is 10mL/100 mL.
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| CN106554974B (en) * | 2016-11-28 | 2021-02-19 | 东北大学 | Method for producing hydrogen by fermentation by using modified peanut shells as supplementary substrate |
| CN107012175B (en) * | 2017-03-22 | 2020-12-11 | 东北大学 | Method for pretreating peanut shells as biological hydrogen production supplement substrate by combining ultralow temperature and crushing |
| CN109825459B (en) * | 2019-03-29 | 2021-09-14 | 天津科技大学 | Dissimilatory iron reduction bacterium for coupling hydrogen production |
| CN113583921A (en) * | 2021-09-18 | 2021-11-02 | 北京北方华科科技有限公司 | Hydrogen producing bacterium and method for producing hydrogen by using same |
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