CN114958924A - Coal-based biomass dark fermentation hydrogen production method with function of coal bed flora remodeling - Google Patents
Coal-based biomass dark fermentation hydrogen production method with function of coal bed flora remodeling Download PDFInfo
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Abstract
A coal-based biomass dark fermentation hydrogen production method with a function of coal bed flora remodeling belongs to the technical field of biomass hydrogen production. Taking methanogenic flora in the coal bed as a basic bacteria source, and performing coal-biomass combined induction fermentation to make the coal bed flora adapt to a biomass fermentation environment and obtain methanogenic flora; the method comprises the steps of utilizing metabolic substrate domestication and sensitive ion and antibiotics combined regulation and control, further utilizing a regulation and control factor induction method to cut off key hydrogen-producing metabolic biological metabolism points of non-hydrogen-producing acid production, hydrogen-producing methane and hydrogen-producing organic acid in a targeted mode, remodeling the metabolism function of a coal bed flora, realizing the conversion of the metabolism function of the coal-based methane-producing flora from methane production to hydrogen production, and realizing efficient and stable hydrogen production by virtue of regulating and controlling the dominant strain structure balance, substrate metabolism selectivity and utilization rate and key metabolite inhibition factors of a new hydrogen-producing flora, and hydrogen production industrial application. The advantages are that: reduces the cost of hydrogen production by fermentation, enriches the source structure of the substrate and ensures sustainable biological hydrogen production.
Description
Technical Field
The invention relates to the technical field of biomass hydrogen production, in particular to a coal-based biomass dark fermentation hydrogen production method with a function of coal bed flora remodeling.
Background
The explosion of hydrogen production technology is motivated by the development of clean energy industry and the requirement of carbon emission control. Wherein, the biological anaerobic dark fermentation hydrogen production technology is an important technical method in various hydrogen production technologies. Currently, the hydrogen production is mainly obtained by separating from soil, various animal excreta and extreme environments.
The soil source flora survives in an aerobic environment for a long time, wherein the content of anaerobic bacteria is relatively low, anaerobic hydrogen-producing bacteria and aerobic bacteria, facultative anaerobic bacteria and various fungi are symbiotic, the flora with the anaerobic hydrogen-producing function, which is obtained by taking the soil bacteria source as the only bacteria source, has relatively weak decomposition capacity on cellulose and hemicellulose, thereby limiting the hydrogen-producing capacity, the adoption of the exogenous bacteria introduction method has great difficulty in the strain fusion technology, and the stability of the artificial mixed bacteria constructed by the multisource flora is poor.
Animal excrement strains exist in a chyme medium formed by a pre-digestion system, the decomposition capacity of hydrogen-producing bacteria directly separated on cellulose and hemicellulose is relatively weak, the suitable temperature of the bacteria is higher than 35 ℃ under the selection of intestinal tracts, and the bacteria even need an environment of more than 42 ℃ to reach the optimal metabolic rate, so the energy consumption of industrial application is high, and the degradation capacity of biomass is poor.
The flora in the extreme environment is suitable for various special environments such as high temperature, ultralow temperature, strong acid, strong alkali and the like, and the flora is difficult to stably survive in the normal temperature environment.
In the prior art of hydrogen production by biological anaerobic dark fermentation, a single bacterial source is difficult to exist, and the hydrogen production method has excellent gas production at the environmental temperature below 30 ℃, is suitable for anaerobic environment and has strong substrate degradation capability.
Disclosure of Invention
The technical problem is as follows: the invention aims to provide a coal-based biomass dark fermentation hydrogen production method for remodeling functions of coal bed flora, and solves the problem that multi-source bacteria compounding is needed to realize high-efficiency hydrogen production, so that the construction of a bacteria source and the implementation of a technology are complex.
The technical scheme is as follows: the aim of the invention is realized by taking methanogenic floras in a coal bed as a basic bacteria source, and by coal-biomass combined induction fermentation, the coal bed floras are adapted to a biomass fermentation environment and obtain methanogenic floras; the method comprises the steps of utilizing metabolic substrate domestication and sensitive ion and antibiotics combined regulation and control, further utilizing a regulation and control factor induction method to cut off key hydrogen-producing metabolic biological metabolism points of non-hydrogen-producing acid production, hydrogen-producing methane and hydrogen-producing organic acid in a targeted mode, remodeling the metabolism function of a coal bed flora, realizing the conversion of the metabolism function of the coal-based methane-producing flora from methane production to hydrogen production, and realizing efficient and stable hydrogen production by virtue of regulating and controlling the dominant strain structure balance, substrate metabolism selectivity and utilization rate and key metabolite inhibition factors of a new hydrogen-producing flora, and hydrogen production industrial application.
The method comprises the following specific steps:
step 1, obtaining coal-based methanogenic flora
The method comprises the following steps of searching a coal bed which simultaneously meets the following conditions for obtaining the coal bed by a bacterial sample, wherein the selection of the bacterial sample for obtaining the coal bed needs to simultaneously meet the following requirements:
(1) the coal seam burial depth is more than 600 m;
(2) the temperature of the coal bed is 24-30 ℃;
(3) hydraulic measures such as fracturing, water injection and the like are not carried out on the coal seam;
(4) the coal bed water is not communicated with the surface water system;
(5) the coal bed contains coal bed gas, and the coal bed gas contains biogas components;
selecting new denuded coal from the coal bed obtained by the bacteria sample, taking 10kg of blocky coal sample with the diameter of 5-10cm by adopting a channeling method, and immediately sealing the coal sample in an anaerobic aseptic tank;
the initial 100% CO concentration in the tank 2 Filling, placing coal sample, replacing with nitrogen and detecting CO 2 Concentration until CO in the tank 2 The concentration is less than 1%; the coal sample is kept sealed and sent to a pre-incubation laboratory under the condition that the preservation temperature is less than 30 ℃.
Step 2, inducing fermentation strain for producing methane by combining coal-based coal and biomass
Firstly, culturing a methanogenic flora in a coal sample by taking coal as a substrate; then, gradually increasing the content of the biomass according to a gradient of 10% every 7-14 days, wherein the biomass adopted in the step needs to be sterilized before use; along with the specific gravity increase of biomass in the substrate, enabling methanogens in the coal seam to gradually adapt to the biomass substrate; until the coal: the mass ratio of the biomass is 1:9, and the concentration of methane in the biogas is stable and is more than 50%, so that the induction of the coal-based coal-biomass combined methane-producing fermentation strain is completed; the coal-based coal-biomass combined methane-producing fermentation strain is referred to as a coal-based methane-producing fermentation strain group for short.
Step 3, separating key strains from the coal-based methane-producing fermentation flora and identifying the types of the strains
Separating and enriching cellulose and hemicellulose hydrolytic bacteria, hydrogen producing bacteria, non-hydrogen producing acid producing bacteria, hydrogen phagosting acid producing bacteria and methanogen strains in a coal-based methane producing fermentation flora, wherein the relative abundance of each enriched strain is more than 97.00 percent; the separation and enrichment do not need strain purification; the microbial diversity analysis method is utilized to establish the coal-based methane-producing fermentation flora and the strain type of each key strain.
Step 4, classifying the H metabolic pathway and hydrogen production functional bacteria in biomass degradation
The metabolic pathway analysis method is utilized to analyze the methanogenesis metabolic pathway of the coal-based methanogenesis zymocyte, so that the H production in the methanogenesis metabolic pathway of the biomass is cleared 2 And macrophage H 2 And (4) metabolic distribution, namely determining point position distribution and distribution weight of a typical anaerobic hydrogen production path in an H metabolic pathway, and screening the level types of the phyla and the genera of the hydrogen producing bacteria from the birth substances.
Step 5, establishing key biological point positions and functional strain categories of hydrogen phagocytosis
Combining the metabolic pathway of H and the key hydrogen-producing bacteria type to determine H 2 Produce CH 4 And H 2 Key biological point sites for producing acetic acid or butyric acid, and the main hydrogen-feeding point sites in the metabolism of methanogen flora are determined to form the strain category.
Step 6, biological characteristic analysis and community cooperation relationship of strains
The biological characteristics of cellulose and hemicellulose hydrolytic bacteria, hydrogen producing bacteria, non-hydrogen producing acid producing bacteria, hydrogen producing acid producing bacteria and methanogen are clarified, and H production surrounding a target biological point is revealed 2 Acetogenic-phagocytic H 2 An inter-fungal symbiosis-cooperation topological relation;
wherein the biological characteristic analysis of each strain comprises the following indexes:
temperature adaptability;
pH adaptability;
key nutritional requirement and nutrient ion tolerance range;
growth curve;
antibiotic sensitivity characteristics;
trace ion demand and sensitivity;
·H 2 and CO 2 Partial pressure sensitivity;
acetic, butyric and salt partial pressure sensitivity;
wherein the nutritive ions comprise ions composed of N, P, K, Na, S, Mg, Ca, Fe, Cl and Se; the antibiotics mainly comprise:
(1) penicillin antibiotics;
(2) cephalosporin antibiotics;
(3) cephamycins antibiotics;
(4) monocyclic beta-lactam antibiotics;
(5) oxacephem antibiotics;
(6) beta-lactamase inhibitor and a combination preparation of beta-lactam antibiotics and beta-lactamase inhibitor;
(7) carbapenem antibiotics;
(8) aminoglycoside antibiotics;
(9) fluoroquinolones antibacterial agents;
(10) macrolide antibacterial agents;
(11) vancomycin and other glycopeptide antibiotics;
(12) tetracycline and chloramphenicol antibiotics;
(13) lincomycin and clindamycin;
(14) a polypeptide antibiotic;
(15) rifamycin antibiotics;
(16) sulfonamides, nitroimidazoles, furans antibacterial agents;
(17) an anti-mycobacterial drug, single antibiotic;
and a composite antibiotic formulated from two or more antibiotics at different gradient doses;
the trace ions include: NO 3 - ，NO 2 - Cu, Hg, Co, As, and other coal bed water at 1000-100 ppm.
Step 7, preparation and induction method of regulation and control culture medium for blocking hydrogen-phagocytosis target point position and weakening non-hydrogen-production acid-producing bacteria
Analyzing response rules of cellulose and hemicellulose hydrolytic bacteria, hydrogen producing bacteria, non-hydrogen producing acid producing bacteria, hydrogen producing acid producing bacteria and methanogen to changes of environmental factors; by combining the biological characteristics of the target strains analyzed in the step 6, sensitive factors which have excellent tolerance to cellulose and hemicellulose hydrolytic bacteria and hydrogen producing bacteria and can obviously inhibit non-hydrogen producing acid producing bacteria, hydrogen producing acid producing bacteria and methanogenic bacteria are searched by a sensitive source analysis method, and a method for configuring a regulation and control culture medium is further established by a step experiment and a response surface experiment method based on the sensitive source analysis result; combining the temperature adaptability, pH value adaptability and growth rate difference of the main strains determined in the step 6 and the step, further adjusting and controlling the composition, content level and supply method of sensitive factors in the culture medium by using a cascade experiment and a response surface experiment method, and establishing an induction method for blocking the hydrogen-phagocytosis target point position and weakening non-hydrogen-production acid-producing bacteria; the establishing of hydrogen phagemid target point position blocking is directional methanogen inhibition and target H blocking 2 Produce CH 4 And H 2 And (4) producing an organic acid metabolic node.
Wherein, the environmental factors mainly include:
the temperature;
·pH；
key nutrient ion concentration;
trace ion concentration.
Wherein the sensitivity factors include:
key trophic ions;
trace ions;
antibiotics;
a water-soluble factor.
Step 8, construction of coal-based hydrogen-producing flora and clear and stable hydrogen-producing regulation and control method based on biological diversity
The method is clear in the process from methanogenesis to hydrogen production regulation and control of the flora function, the association of the formation of the hydrogen-producing flora and the environmental factors and sensitive factors in the step 7 is further determined by the following response rule of the cooperative relationship between dominant bacteria and bacteria to the regulation and control of the metabolic site, the method optimizes the 'induction method for blocking the hydrogen-producing targeting site and weakening the non-hydrogen-producing acid-producing bacteria' completed in the step 7 by taking the construction of the hydrogen-producing flora as a target comprehensive band interaction orthogonal experiment and response surface experiment method, and the stable biomass hydrolysis and the enhanced H production are constructed 2 Inhibit non-hydrogen production of acid and H 2 Produce CH 4 And H 2 Preparation method and accurate regulation and control method of hydrogen-producing bacteria induction culture medium for producing organic acid, clear formation internal cause of dominant bacteria in new flora, flora balance condition and self-stability capability, optimize and definitely dredge coal-produced H 2 Metabolic pathway, blocking phagocytosis H 2 The compound factor of the metabolic point regulates and controls the core metabolic pathway conversion mode and the community structure evolution rule in the reconstructed flora function; and (3) inducing and constructing the coal-based hydrogen-producing flora based on the coal-based methane-producing fermentation flora obtained in the second step.
Step 9, degrading selectivity of biomass hydrogen production substrate and optimal substrate H 2 Analysis of conversion
Aiming at the coal-based hydrogen-producing flora with new structure, H is compared by theoretical derivation, calculation simulation and biomass substrate group occurrence analysis means 2 Produce CH 4 The selective difference of the flora on the substrate groups before and after the targeted blocking is clear, the nutrition obtaining tendency of the hydrogen-producing flora is clear, and the optimal substrate H is deduced by combining the metabolic mode of the hydrogen-producing flora 2 Conversion rate;
wherein, the biomass hydrogen production substrate can be single or mixture of various plants; or a mixture of various single or mixed plants and coal.
Step 10, metabolite partial pressure vs. H 2 Analysis of influence of conversion
Exploring the influence rule of the partial pressure of the hydrogen-producing fermentation fingerprint metabolites on the hydrogen production of the biomass, clarifying the tolerance of the coal-based hydrogen-producing flora to the change of the biomass category, and determining the key metabolite influence factors of stabilizing the hydrogen production rate and improving the effective conversion rate of the substratePrime and control conditions; the hydrogen-producing fermentation fingerprint metabolite partial pressure comprises the following components: h 2 Partial pressure, CO 2 Partial pressure, acetic acid or acetate partial pressure, butyric acid or butyrate partial pressure.
The hydrogen-producing fermentation fingerprint metabolite partial pressure comprises the following components:
·H 2 partial pressure;
·CO 2 partial pressure;
acetic acid or acetate partial pressure;
butyric acid or butyrate partial pressure.
Step 11, optimizing and establishing biomass hydrogen production technical scheme of coal-based hydrogen producing bacteria
On the basis of the experimental method and the experimental result in the step 8, the analysis results in the step 9 and the step 10 are superposed to serve as analysis factors, an ideal hydrogen-producing flora structure model and a diversity driving factor model of the biomass category to be implemented are constructed, a regulation and control method for realizing substrate hydrogen production maximization is optimized, the structural stability and the functional reliability of the hydrogen-producing flora under regulation and control are optimized through multi-cycle analog simulation analysis, and a biomass hydrogen production technical scheme of the coal-based hydrogen-producing bacteria is established.
The biomass hydrogen production technology of the coal-based hydrogen producing bacteria is based on the ground temperature and pH condition of the coal bed obtained by the coal sample, and the biomass dark fermentation hydrogen production can stably run at the temperature of 24-30 ℃.
Step 12, implementation of biomass hydrogen production technology constructed by coal-based hydrogen producing bacteria
The technical proposal of biomass hydrogen production constructed according to the coal-based hydrogen producing bacteria is as follows:
step 12-1, performing propagation culture on the coal-based hydrogen-producing flora constructed in the step 8, wherein the quantity of the propagation culture liquid is more than 10% of the total volume of the tank body according to the volume of the fermentation tank body;
step 12-2, randomly extracting 10 expanding culture bacteria samples to perform a biomass hydrogen production stability test, and when the H of each sample is 2 Content (wt.)>55 percent of maximum fluctuation of substrate hydrogen conversion rate and conversion amount<When 10%, the strain propagation meets the requirement of industrialized strain introduction;
12-3, inoculating the coal-based hydrogen-producing bacteria in the fermentation tank according to the strain introduction rate of 10%, paving a bacteria sample with the thickness of 1 meter and the particle size of 10-20cm at the bottom of the tank to obtain coal serving as a coal-based hydrogen-producing bacteria bed and a buffer medium, and performing hydrogen production by coal-based biomass;
the biomass hydrogen production technology of the coal-based hydrogen producing bacteria is based on the ground temperature and pH condition of a coal layer obtained by a coal sample, and the biomass dark fermentation hydrogen production can stably run at the temperature of 24-30 ℃;
step 12-4, pumping and discharging gas metabolites in real time to keep the gas pressure in the tank body at 1.05-1.10atm standard atmospheric pressure;
and 12-5, monitoring the partial pressure of the water-soluble metabolites of the liquid in the tank body in real time, and controlling the concentration of the water-soluble metabolites in the tank by using a dilution method, a neutralization method or an acetic acid and butyric acid separation method to realize the implementation of the high-efficiency stable biomass hydrogen production technology.
The method has the beneficial effects that the method is adopted, and based on the coal-bed coal-based methanogenic flora, the coal-based hydrogen-producing flora is constructed by domesticating the adaptive capacity of the biomass substrate, performing targeted regulation and control on a metabolic pathway and blocking the hydrogen-phagocytosis metabolic point, and is further applied to the hydrogen production by biomass dark fermentation. The constructed flora is naturally selected for a long time to adapt to strict anaerobic environment, the growth temperature of 25-30 ℃ and the survival condition taking difficultly degraded organic matters as substrates, thereby solving the various defects of soil strains, excrement strains and extreme environment strains. Because the degradation difficulty of the biomass is far lower than that of coal, the domesticated strain can realize the high-efficiency hydrogen production target by taking the biomass as a substrate,
the device is suitable for the temperature environment of 25-35 ℃, biological high-efficiency hydrogen production at a lower temperature is realized, and energy consumption required by tank body heating is greatly saved; the microbial community can actively adapt to strict anaerobic environment, does not need exogenous bacteria supplementation or matched fermentation of aerobic bacteria and fungi, and forms a natural symbiotic relationship through natural selection of flora, so that the community structure stability is strong; the bacterial source is converted from coal which is difficult to degrade as a substrate into biomass which is easy to degrade, so that the bacterial source can quickly adapt to the substrate condition and realize high-efficiency biological hydrogen production;
the invention blocks the metabolism sites of hydrogen-producing methane and hydrogen-producing acid-producing organisms, synchronously inhibits the non-hydrogen-producing acid-producing fermentation process, enhances the effective conversion rate of substrate hydrogen production, improves the utilization rate of the substrate, and further reduces the hydrogen production cost; the fermentation system utilizes the bacteria source to obtain the massive coal of the coal bed to form a natural bacteria bed, and can further stabilize the stability of the constructed hydrogen-producing bacteria, thereby improving the reliability of the biological hydrogen-producing fermentation system.
By taking methanogenic flora in the coal bed as a basic bacterial source and adopting the energy of the coal bed flora, the biomass hydrogen production efficiency is improved, and the following beneficial effects are achieved: (1) the adaptability of anaerobic environment is strong: coal bed bacteria exist in a strict anaerobic environment of an underground coal bed for a long time, a bacteria source which is mainly composed of anaerobic bacteria can form a symbiotic relation more easily and stably, and can better adapt to the strict anaerobic condition and the anaerobic environment; (2) the temperature adaptability is strong: the temperature of a 600-1000 m coal seam is more between 25 and 30 ℃, the environment of a target bacteria source can be kept at the environment temperature of between 25 and 35 ℃ for a long time, and the bacteria source can be stably activated under the temperature condition through natural selection, so that the requirement on the temperature of a cultivation system is lower; (3) stronger substrate degradation capacity: coal is an organic matter formed by giant molecules with complex structures, the biodegradation difficulty is more difficult than cellulose and hemicellulose, coalbed bacteria can only survive in a coalbed for a long time and only take the organic matter in the coal as a carbon source, the bacteria source nutrient source is the cellulose and the hemicellulose, and even the organic matter is more difficult to degrade, so that the constructed hydrogen-producing flora has strong biomass degradation capability, and has stronger biomass degradation capability compared with soil and excrement flora. The method integrates the characteristics of the coal bed anaerobic environment, the coal bed temperature and the nutrient source, obtains hydrogen-producing bacteria based on coal bed microorganisms and is used for biomass hydrogen production, and can meet the technical targets that other bacteria sources are difficult to realize.
The problems that multi-source bacteria compounding is needed for realizing high-efficiency hydrogen production, and the construction of bacteria sources and the implementation of technology are complex are solved, and the aim of the invention is achieved.
The advantages are that: the hydrogen-producing flora constructed on the basis of the coal bed microbial community has the advantages of good low-temperature adaptability, strong biomass hydrolysis capability, stable flora structure and the like, reduces the fermentation hydrogen production cost, enriches the substrate source structure and ensures sustainable biological hydrogen production.
Description of the drawings:
FIG. 1 is a technical roadmap for the present invention.
Detailed Description
Example 1: the invention relates to a coal-based biomass dark fermentation hydrogen production method with function remodeling of coal bed flora, which takes methanogenic flora in a coal bed as a basic bacteria source, and leads the coal bed flora to adapt to a biomass fermentation environment and obtain the methanogenic flora through coal-biomass combined induced fermentation; the method comprises the steps of utilizing metabolic substrate domestication and sensitive ion and antibiotics combined regulation and control, further utilizing a regulation and control factor induction method to cut off key hydrogen-producing metabolic biological metabolism points of non-hydrogen-producing acid production, hydrogen-producing methane and hydrogen-producing organic acid in a targeted mode, remodeling the metabolism function of a coal bed flora, realizing the conversion of the metabolism function of the coal-based methane-producing flora from methane production to hydrogen production, and realizing efficient and stable hydrogen production by virtue of regulating and controlling the dominant strain structure balance, substrate metabolism selectivity and utilization rate and key metabolite inhibition factors of a new hydrogen-producing flora, and hydrogen production industrial application.
The method comprises the following specific steps:
step 1, obtaining coal-based methanogenic flora
The method comprises the following steps of searching a coal bed which simultaneously meets the following conditions for obtaining the coal bed by a bacterial sample, wherein the selection of the bacterial sample for obtaining the coal bed needs to simultaneously meet the following requirements:
(1) the coal seam burial depth is more than 600 m;
(2) the temperature of the coal bed is 24-30 ℃;
(3) hydraulic measures such as fracturing, water injection and the like are not carried out on the coal seam;
(4) the coal bed water is not communicated with the surface water system;
(5) the coal seam contains coal seam gas, and the coal seam gas contains biogas components.
In the technical implementation, the specific coal type is not limited except for the above conditions.
Selecting new denuded coal in the coal bed obtained by the bacterial sample, wherein the denuded mode can be selected from a tunneling machine, an excavator or blast mining, and the tunneling or coal mining process which possibly causes bacterial pollution such as hydraulic punching cannot be used; 10kg of blocky coal samples with the diameter of 5-10cm are taken by adopting a slotting method, and the coal samples are immediately sealed in an anaerobic aseptic tank.
Initially using 100% concentration in the tankCO 2 Filling, placing coal sample, replacing with nitrogen and detecting CO 2 Concentration until CO in the tank 2 The concentration is less than 1%. The coal sample is kept sealed and sent to a pre-incubation laboratory under the condition that the preservation temperature is less than 30 ℃.
Step 2, inducing fermentation strains for jointly producing methane by coal-based coal and biomass
Firstly, taking coal as a substrate, selecting any inorganic salt methanogenic bacteria culture medium to culture a methanogenic flora in a coal sample, then gradually increasing the biomass content according to a gradient of 10% every 7-14 days, and sterilizing the biomass adopted in the step before use; along with the increase of the specific gravity of the biomass in the substrate, the methanogens of the coal bed are gradually adapted to the biomass substrate; along with the increase of the specific gravity of the biomass, the degradable components per unit mass are increased, and the amount of the biological gas is increased; in the adaptive domestication of the coal bed methane-producing bacteria to the biomass substrate, the strict geometric proportion corresponding relation between the increase rate of the organic matter content and the change of the gas production rate is not required, and only the proportional relation is required; until the coal: the mass ratio of biomass is 1:9, and the concentration of methane in the biogas is stable and is more than 50%, so that the induction of the coal-based coal-biomass combined methanogenic fermentation strain (hereinafter referred to as coal-based methanogenic fermentation flora) is completed.
Step 3, separating key strains from the coal-based methane-producing fermentation flora and identifying the types of the strains
Separating and enriching cellulose and hemicellulose hydrolytic bacteria, hydrogen producing bacteria, non-hydrogen producing acid producing bacteria, hydrogen phagosting acid producing bacteria and methanogen strains in a coal-based methane producing fermentation flora, wherein the relative abundance of each enriched strain is more than 97.00 percent; the culture medium of various characteristic bacteria can be any conventional special culture medium which can meet the strain enrichment requirement; the separation and enrichment do not need strain purification; the microbial diversity analysis method is utilized to establish the coal-based methane-producing fermentation flora and the strain type of each key strain.
Step 4, differentiating the H metabolic pathway and the main hydrogen production functional bacteria in biomass degradation
The metabolic pathway analysis method is utilized to analyze the methanogenesis metabolic pathway of the coal-based methanogenesis zymocyte, so that the H production in the methanogenesis metabolic pathway of the biomass is cleared 2 And macrophage H 2 Metabolic distribution, namely determining point position distribution and distribution weight of a typical anaerobic hydrogen-producing pathway in an H metabolic pathway, and screening the level types of a biont hydrogen-producing bacteria phylum and genus.
Step 5, establishing key biological point positions and functional strain categories of hydrogen phagocytosis
Combining the metabolic pathway of H and the key hydrogen-producing bacteria type to determine H 2 Produce CH 4 And H 2 Key biological point sites for producing acetic acid or butyric acid, and the main hydrogen-feeding point sites in the metabolism of methanogen flora are determined to form the strain category.
Step 6, biological characteristic analysis and community cooperation relationship of strains
The biological characteristics of cellulose and hemicellulose hydrolytic bacteria, hydrogen producing bacteria, non-hydrogen producing acid producing bacteria, hydrogen producing acid producing bacteria and methanogen are clarified; wherein the biological characteristics of the cellulose and hemicellulose hydrolytic bacteria and the hydrogenogen bacteria are mainly analyzed for the tolerance limit; the biological characteristics of non-hydrogen-producing acid-producing bacteria, hydrogen-producing acid-producing bacteria and methanogenic bacteria are mainly analyzed to inhibit conditions; revealing the product H around a target biological site 2 Acetogenic-phagocytic H 2 Symbiosis between the bacteria-cooperative topological relation.
Wherein the biological characteristic analysis of each strain comprises the following indexes:
temperature adaptability;
pH adaptability;
critical nutritional requirements and nutrient ion tolerance range;
growth curve;
antibiotic sensitivity characteristics;
trace ion demand and sensitivity;
·H 2 and CO 2 Partial pressure sensitivity;
acetic, butyric and salt partial pressure sensitivity.
Wherein the nutritive ions comprise ions composed of N, P, K, Na, S, Mg, Ca, Fe, Cl and Se; the antibiotics mainly comprise:
(1) penicillin antibiotics;
(2) cephalosporin antibiotics;
(3) cephamycins antibiotics;
(4) monocyclic beta-lactam antibiotics;
(5) oxacephem antibiotics;
(6) beta-lactamase inhibitor and a combination preparation of beta-lactam antibiotics and beta-lactamase inhibitor;
(7) carbapenem antibiotics;
(8) aminoglycoside antibiotics;
(9) fluoroquinolones antibacterial agents;
(10) macrolide antibacterial agents;
(11) vancomycin and other glycopeptide antibiotics;
(12) tetracycline and chloramphenicol antibiotics;
(13) lincomycin and clindamycin;
(14) a polypeptide antibiotic;
(15) rifamycin antibiotics;
(16) sulfonamides, nitroimidazoles, furans antibacterial agents;
(17) single antibiotic of antimycobacterial medicine.
And a composite antibiotic formulated from two or more antibiotics at different gradient doses.
The trace ions include: NO 3 - ，NO 2 - Cu, Hg, Co, As, and other coal bed water at 1000-100 ppm.
Step 7, preparation and induction method of regulation and control culture medium for blocking hydrogen-phagocytosis target point position and weakening non-hydrogen-production acid-producing bacteria
Analyzing response rules of cellulose and hemicellulose hydrolytic bacteria, hydrogen producing bacteria, non-hydrogen producing acid producing bacteria, hydrogen producing acid producing bacteria and methanogen to changes of environmental factors; by combining the biological characteristics of the target strains analyzed in the step 6, sensitive factors which have excellent tolerance to cellulose and hemicellulose hydrolytic bacteria and hydrogen producing bacteria and can obviously inhibit non-hydrogen producing acid producing bacteria, hydrogen producing acid producing bacteria and methanogenic bacteria are searched by a sensitive source analysis method, and a method for configuring a regulation and control culture medium is further established by a step experiment and a response surface experiment method based on the sensitive source analysis result;
combining the temperature adaptability, pH value adaptability and growth rate difference of the main strains determined in the step 6 and the step, further adjusting and controlling the composition, content level and supply method of sensitive factors in the culture medium by using a cascade experiment and a response surface experiment method, and establishing an induction method for blocking the hydrogen-phagocytosis target point position and weakening non-hydrogen-production acid-producing bacteria; the establishing of hydrogen phagemid target point position blocking is directional methanogen inhibition and target H blocking 2 Produce CH 4 And H 2 And producing an organic acid metabolic node.
Wherein, the environmental factors mainly include:
the temperature;
·pH；
key nutrient ion concentration;
trace ion concentration.
Wherein the sensitivity factors include:
key trophic ions;
trace ions;
antibiotics
A water-soluble factor.
Step 8, construction of coal-based hydrogen-producing flora and clear and stable hydrogen-producing regulation and control method based on biological diversity
The method is clear in the process from methanogenesis to hydrogen production regulation and control of the flora function, the association of the formation of the hydrogen-producing flora and the environmental factors and sensitive factors in the step 7 is further determined by the following response rule of the cooperative relationship between dominant bacteria and bacteria to the regulation and control of the metabolic site, the method optimizes the 'induction method for blocking the hydrogen-producing targeting site and weakening the non-hydrogen-producing acid-producing bacteria' completed in the step 7 by taking the construction of the hydrogen-producing flora as a target comprehensive band interaction orthogonal experiment and response surface experiment method, and the stable biomass hydrolysis and the enhanced H production are constructed 2 Inhibiting non-hydrogen production of acid and H 2 Produce CH 4 And H 2 Preparation method and accurate regulation and control method of hydrogen-producing bacteria induction culture medium for producing organic acid, clear formation internal cause of dominant bacteria in new flora, flora balance condition and self-stability capability, optimize and definitely dredge coal-produced H 2 Metabolic pathway, blockadePhage H 2 The compound factor at the metabolic point regulates and controls a core metabolic pathway conversion mode and a community structure evolution rule in the function of the reconstructed flora; and (3) inducing and constructing the coal-based hydrogen-producing flora based on the coal-based methane-producing fermentation flora obtained in the second step.
Step 9, degrading selectivity of biomass hydrogen production substrate and optimal substrate H 2 Analysis of conversion
Aiming at the coal-based hydrogen-producing flora with new structure, H is compared by theoretical derivation, calculation simulation and biomass substrate group occurrence analysis means 2 Produce CH 4 The selective difference of the flora on the substrate groups before and after the targeted blocking is clear, the nutrition obtaining tendency of the hydrogen-producing flora is clear, and the optimal substrate H is deduced by combining the metabolic mode of the hydrogen-producing flora 2 And (4) conversion rate.
Wherein, the biomass hydrogen production substrate can be single or mixture of various plants; or a mixture of various single or mixed plants and coal.
Step 10, metabolite partial pressure vs. H 2 Analysis of influence of conversion
Exploring the influence rule of the partial pressure of the hydrogen-producing fermentation fingerprint metabolites on the hydrogen production of the biomass, clarifying the tolerance of the coal-based hydrogen-producing flora to the change of the biomass category, and determining key metabolite influence factors and control conditions for stabilizing the hydrogen production rate and improving the effective conversion rate of the substrate; the hydrogen-producing fermentation fingerprint metabolite partial pressure comprises the following components:
·H 2 partial pressure;
·CO 2 partial pressure;
acetic acid or acetate partial pressure;
butyric acid or butyrate partial pressure.
Step 11, optimizing and establishing biomass hydrogen production technical scheme of coal-based hydrogen producing bacteria
And 8, on the basis of the experimental method and the experimental result, overlapping the analysis results of the steps 9 and 10 as analysis factors, constructing an ideal hydrogen-producing flora structural model and a diversity driving factor model of the biomass category to be implemented, optimizing a regulation and control method for realizing substrate hydrogen production maximization, optimizing the structural stability and the functional reliability of the hydrogen-producing flora under regulation and control through multi-cycle analog simulation analysis, and determining a biomass hydrogen production technical scheme of the coal-based hydrogen-producing bacteria.
The biomass hydrogen production technology of the coal-based hydrogen producing bacteria is based on the ground temperature and pH condition of the coal bed obtained by the coal sample, and the biomass dark fermentation hydrogen production can stably run at the temperature of 24-30 ℃.
Step 12, implementation of biomass hydrogen production technology constructed by coal-based hydrogen producing bacteria
The technical proposal of biomass hydrogen production constructed according to the coal-based hydrogen producing bacteria is as follows:
step 12-1, performing propagation culture on the coal-based hydrogen-producing flora constructed in the step 8, wherein the quantity of the propagation culture liquid is more than 10% of the total volume of the tank body according to the volume of the fermentation tank body;
step 12-2, randomly extracting 10 expanding culture bacteria samples to perform a biomass hydrogen production stability test, and when the H of each sample is 2 Content (wt.)>55 percent, maximum fluctuation of substrate hydrogen conversion rate and conversion amount<When 10%, the strain propagation meets the requirement of industrialized strain introduction;
12-13, inoculating coal-based hydrogen-producing bacteria in the fermentation tank according to the strain introduction rate of 10%, paving a bacteria sample with the thickness of 1 meter and the particle size of 10-20cm at the bottom of the tank to obtain coal serving as a coal-based hydrogen-producing bacteria bed and a buffer medium, and performing hydrogen production by coal-based biomass; during inoculation of the strains, the liquid injection amount of the fermentation tank can be gradually increased by adopting four steps of 30%, 50%, 70% and 100%, so that on one hand, the preparation amount of inoculated strains is reduced, and on the other hand, the coal-based hydrogen-producing bacteria are gradually adapted to the internal environment of the fermentation tank; thirdly, the quick stable implantation in the fungus bed is facilitated.
The biomass hydrogen production technology of the coal-based hydrogen producing bacteria is based on the ground temperature and pH condition of the coal bed obtained by a coal sample, and the biomass dark fermentation hydrogen production can stably run at the temperature of 24-30 ℃.
Step 12-4, pumping gas metabolites in real time to keep the gas pressure in the tank body at 1.05-1.10atm standard atmospheric pressure;
and step 12-5, monitoring the partial pressure of the water-soluble metabolites of the liquid in the tank body in real time, and controlling the concentration of the water-soluble metabolites in the tank by using a dilution method, a neutralization method or an acetic acid and butyric acid separation method, so as to realize the implementation of the high-efficiency stable biomass hydrogen production technology.
Claims (8)
1. A coal-based biomass dark fermentation hydrogen production method with a function of coal bed flora remodeling is characterized by comprising the following steps: taking methanogenic flora in the coal bed as a basic bacteria source, and performing coal-biomass combined induction fermentation to make the coal bed flora adapt to a biomass fermentation environment and obtain methanogenic flora; the method comprises the steps of utilizing metabolic substrate domestication and sensitive ion and antibiotics combined regulation and control method, further utilizing a regulation and control factor induction method to cut off key hydrogen-producing metabolism biological metabolism sites of non-hydrogen-producing acid production, hydrogen-producing methane and hydrogen-producing organic acid in a targeted mode, remodeling the metabolism function of a coal bed flora, realizing that the metabolism function of the coal-based methane-producing flora is converted from methane production to hydrogen production, and realizing efficient and stable coal-based biomass dark fermentation hydrogen production and hydrogen production industrial application by regulating and controlling the dominant strain structure balance, substrate metabolism selectivity and utilization rate and key metabolite inhibition factors of the new-structure hydrogen-producing flora.
2. The method for producing hydrogen by dark fermentation of coal-based biomass with function of coalbed flora remodeled as claimed in claim 1, which is characterized in that:
the method comprises the following specific steps:
step 1, obtaining coal-based methanogenic flora
Selecting new denuded coal from the coal bed obtained by the bacteria sample, taking 10kg of block coal sample with the diameter of 5-10cm by adopting a channeling method, and immediately sealing the coal sample in an anaerobic aseptic tank; the initial use of 100% CO in the canister 2 Filling, placing coal sample, replacing with nitrogen and detecting CO 2 Concentration until CO in the tank 2 The concentration is less than 1%; the coal sample is kept sealed and is sent to a pre-culture laboratory under the condition that the preservation temperature is lower than 30 ℃;
step 2, inducing fermentation strain for producing methane by combining coal-based coal and biomass
Firstly, culturing a methanogenic flora in a coal sample by taking coal as a substrate; then, gradually increasing the content of the sterile biomass according to a gradient of 10 percent every 7 to 14 days to ensure that methanogens in the coal seam gradually adapt to the biomass substrate; when the coal: the mass ratio of the biomass is 1:9, and the concentration stability of methane in the biogas is more than 50%, so that the induction of the coal-based coal-biomass combined methane-producing fermentation strain is completed; the coal-based coal-biomass combined methane-producing fermentation strain is referred to as a coal-based methane-producing fermentation flora for short;
step 3, separating key strains from the coal-based methane-producing fermentation flora and identifying the types of the strains
Separating and enriching cellulose and hemicellulose hydrolytic bacteria, hydrogen producing bacteria, non-hydrogen producing acid producing bacteria, hydrogen phagosting acid producing bacteria and methanogen strains in a coal-based methane producing fermentation flora, wherein the relative abundance of each enriched strain is more than 97.00 percent; the separation and enrichment do not need strain purification; determining the coal-based methane-producing fermentation flora and the strain type of each key strain by using a microbial diversity analysis method;
step 4, classifying key H metabolic pathway and main hydrogen production functional bacteria in biomass degradation
The metabolic pathway analysis method is utilized to analyze the methanogenesis metabolic pathway of the coal-based methanogenesis zymocyte, so that the H production in the methanogenesis metabolic pathway of the biomass is cleared 2 And macrophage H 2 Metabolic distribution, namely determining point position distribution and distribution weight of a typical anaerobic hydrogen production path in an H metabolic pathway, and screening the level types of main phyla and genera of the biomass hydrogen producing bacteria;
step 5, establishing key biological point positions and functional strain categories of hydrogen phagocytosis
Combining the metabolic pathway of H and the key hydrogen-producing bacteria type to determine H 2 Produce CH 4 And H 2 Key biological point sites for producing acetic acid or butyric acid, and defining main hydrogen-feeding point sites in metabolism of methanogen flora to form strain categories;
step 6, biological characteristic analysis and community cooperation relation of main strains
The biological characteristics of cellulose and hemicellulose hydrolytic bacteria, hydrogen producing bacteria, non-hydrogen producing acid producing bacteria, hydrogen producing acid producing bacteria and methanogen are clarified, and H production surrounding a target biological point is revealed 2 Acetogenic-phagocytic H 2 An inter-fungal symbiosis-cooperation topological relation;
step 7, preparation and induction method of hydrogen phagocytosis targeting point blocking regulation and control culture medium
Analysis of cellulose and hemicellulose hydrolytic bacteria, hydrogen producing bacteria and non-hydrogen producing bacteriaResponse rules of acid bacteria, hydrogen-phagostimulant acid-producing bacteria and methanogen to environmental factor changes; by combining the biological characteristics of the target strains analyzed in the step 6, sensitive factors which have excellent tolerance to cellulose and hemicellulose hydrolytic bacteria and hydrogen producing bacteria and can obviously inhibit non-hydrogen producing acid producing bacteria, hydrogen producing acid producing bacteria and methanogenic bacteria are searched by a sensitive source analysis method, and a method for regulating and controlling the configuration of the culture medium is established; combining the growth rate difference of the main strains determined in the step 6 and the step, establishing the oriented methanogen inhibition and the targeted H blocking 2 Produce CH 4 And H 2 An induction method for producing organic acid metabolic nodes and weakening non-hydrogen-producing acid-producing bacteria;
step 8, construction of coal-based hydrogen-producing flora and clear and stable hydrogen-producing regulation and control method based on biological diversity
The method has the advantages that the following response rule of dominant bacteria formation and cooperative relationship among bacteria to metabolic point regulation is established in the regulation and control process from methane production to hydrogen production, the hydrolysis of stable biomass and the enhanced H production are established 2 Inhibit non-hydrogen production of acid and H 2 Produce CH 4 And H 2 Preparation method and accurate regulation and control method of hydrogen-producing bacteria induction culture medium for producing organic acid, clear formation internal cause of dominant bacteria in new flora, flora balance condition and self-stability capability, optimize and definitely dredge coal-produced H 2 Metabolic pathway, blocking phagocytosis H 2 The compound factor of the metabolic point regulates and controls the core metabolic pathway conversion mode and the community structure evolution rule in the reconstructed flora function; inducing and constructing a coal-based hydrogen-producing flora based on the coal-based methane-producing fermentation flora obtained in the second step;
step 9, degrading selectivity of biomass hydrogen production substrate and optimal substrate H 2 Analysis of conversion
Aiming at the coal-based hydrogen-producing flora with new structure, H is compared by theoretical derivation, calculation simulation and biomass substrate group occurrence analysis means 2 Produce CH 4 The selective difference of the flora on the substrate groups before and after the targeted blocking is clear, the nutrition obtaining tendency of the hydrogen-producing flora is clear, and the optimal substrate H is deduced by combining the metabolic mode of the hydrogen-producing flora 2 Conversion rate;
step 10, metabolite partial pressure vs. H 2 Analysis of influence of conversion
Exploring the influence rule of the partial pressure of the hydrogen-producing fermentation fingerprint metabolites on the hydrogen production of the biomass, clarifying the tolerance of the coal-based hydrogen-producing flora to the change of the biomass category, and determining key metabolite influence factors and control conditions for stabilizing the hydrogen production rate and improving the effective conversion rate of the substrate; the hydrogen-producing fermentation fingerprint metabolite partial pressure comprises the following components: h 2 Partial pressure, CO 2 Partial pressure of acetic acid or acetate, partial pressure of butyric acid or butyrate;
step 11, optimizing and establishing biomass hydrogen production technical scheme of coal-based hydrogen producing bacteria
Constructing an ideal hydrogen-producing flora structural model and a diversity driving factor model of biomass categories to be implemented, optimizing a regulation and control method for realizing substrate hydrogen production maximization, performing multi-cycle analog simulation analysis to optimize the structural stability and functional reliability of hydrogen-producing flora under regulation and control, and establishing a biomass hydrogen production technical scheme of coal-based hydrogen-producing bacteria;
step 12, implementation of biomass hydrogen production technology constructed by coal-based hydrogen producing bacteria
The technical proposal of biomass hydrogen production constructed according to the coal-based hydrogen producing bacteria is as follows:
step 12-1, performing expanded culture on the coal-based hydrogen-producing bacterial flora constructed in the step 8, wherein the expanded culture liquid amount is more than 10% of the total volume of the tank body according to the volume of the fermentation tank body;
step 12-2, randomly extracting 10 expanding culture bacteria samples to perform a biomass hydrogen production stability test, and when the H of each sample is 2 Content (wt.)>55 percent, maximum fluctuation of substrate hydrogen conversion rate and conversion amount<When 10%, the strain propagation meets the requirement of industrialized strain introduction;
12-3, inoculating a coal-based hydrogen-producing bacteria group in the fermentation tank according to the strain introduction rate of 10%, paving a bacteria sample with the thickness of 1 meter and the particle size of 10-20cm at the bottom of the tank to obtain coal serving as a coal-based hydrogen-producing bacteria bed and a buffer medium, and performing hydrogen production by using coal-based biomass;
the biomass hydrogen production technology of the coal-based hydrogen producing bacteria is based on the ground temperature and pH condition of a coal layer obtained by a coal sample, and the biomass dark fermentation hydrogen production can stably run at the temperature of 24-30 ℃;
step 12-4, pumping and discharging the gas metabolites in real time to keep the gas pressure in the tank body at 1.05-1.10atm standard atmospheric pressure;
and 12-5, monitoring the partial pressure of the water-soluble metabolites of the liquid in the tank body in real time, and controlling the concentration of the water-soluble metabolites in the tank by using a dilution method, a neutralization method or an acetic acid and butyric acid separation method to realize the implementation of the high-efficiency stable biomass hydrogen production technology.
3. The method for producing hydrogen by dark fermentation of coal-based biomass with function of coalbed flora remodeled as claimed in claim 2, which is characterized in that: in the step 1, the selection of the coal bed obtained by the bacterial sample simultaneously meets the following requirements: (1) the coal seam burial depth is more than 600 m; (2) the temperature of the coal bed is 24-30 ℃; (3) hydraulic measures such as fracturing, water injection and the like are not carried out on the coal seam; (4) the coal bed water is not communicated with the surface water system; (5) the coal seam contains coal seam gas, and the coal seam gas contains biogas components.
4. The method for producing hydrogen by dark fermentation of coal-based biomass with function of coalbed flora remodeled as claimed in claim 2, which is characterized in that: in step 6, the biological characteristic analysis of cellulose and hemicellulose hydrolytic bacteria, hydrogen producing bacteria, non-hydrogen producing acid producing bacteria, hydrogen producing acid producing bacteria and methanogen comprises the following indexes: environmental suitability for temperature and pH factors, critical nutrient requirement and nutrient ion tolerance range, growth curve, antibiotic sensitivity characteristics, trace ion requirement and sensitivity, H 2 And CO 2 Partial pressure sensitivity and acetic acid, butyric acid and salt partial pressure sensitivity; wherein the nutritive ions comprise ions composed of N, P, K, Na, S, Mg, Ca, Fe, Cl and Se; the antibiotics mainly comprise: (1) penicillin antibiotics, (2) cephalosporin antibiotics, (3) cephamycin antibiotics, (4) monocyclic beta-lactam antibiotics, (5) oxycephalosporane antibiotics, (6) beta-lactamase inhibitors and combined preparations of the beta-lactam antibiotics and the beta-lactamase inhibitors, (7) carbapenem antibiotics, (8) aminoglycoside antibiotics, (9) fluoroquinolone antibiotics, (10) macrolide antibiotics, (11) vancomycin and other glycopeptide antibiotics, (12) tetracyclines and chloramphenicol antibiotics, (13) lincomycin and clindamycin, (14) polypeptide antibiotics, (15) rifamycin antibiotics, antibiotics for treating chronic infectious diseases, and the like,(16) Sulfonamides, nitroimidazoles and furans, an antimycobacterial drug (17) an antimycobacterial single antibiotic, and a compound antibiotic which is prepared by two or more antibiotics according to different gradient doses; the trace ions include: NO 3 - ，NO 2 - Cu, Hg, Co, As, and other coal seam water at 1000-100 ppm.
5. The method for producing hydrogen by dark fermentation of coal-based biomass with function of coalbed flora remodeled as claimed in claim 2, which is characterized in that: in step 7, the environmental factors include: temperature, pH, critical nutrient ion concentration, and trace ion concentration.
6. The method for producing hydrogen by dark fermentation of coal-based biomass with function of coalbed flora remodeled as claimed in claim 2, which is characterized in that: in step 7, the sensitive factors of cellulose, hemicellulose hydrolytic bacteria and hydrogen producing bacteria which have excellent tolerance and can obviously inhibit non-hydrogen producing acid-producing bacteria, hydrogen-producing acid-producing bacteria and methanogen comprise: key water-soluble factors of nutrient ions, trace ions and antibiotics.
7. The method for producing hydrogen by dark fermentation of coal-based biomass with function of coalbed flora remodeled as claimed in claim 2, which is characterized in that: in step 9, the biomass hydrogen production substrate can be a single plant or a mixture of various plants; or a mixture of various single or mixed plants and coal.
8. The method for producing hydrogen by dark fermentation of coal-based biomass with function of coalbed flora remodeled as claimed in claim 2, which is characterized in that: in step 11, the biomass hydrogen production technology of the coal-based hydrogen producing bacteria is based on the ground temperature and pH condition of the coal bed obtained by the coal sample, and the biomass hydrogen production by dark fermentation can stably operate at the temperature of 24-30 ℃.
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