Multistage anaerobic fermentation microbial acclimation method
By employing a multi-stage anaerobic fermentation method, hydrolytic, acid-producing, and methanogenic microorganisms were specifically domesticated, solving the problem of the inability to optimize microorganisms separately in single-stage anaerobic fermentation and improving fermentation efficiency and stability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-16
- Publication Date
- 2026-03-27
AI Technical Summary
In existing single-stage anaerobic fermentation technology, the microorganisms in each fermentation stage cannot be in optimal growth conditions, resulting in poor stability of the fermentation system and easy failure due to imbalance in a certain stage.
A multi-stage anaerobic fermentation method was adopted, and hydrolytic, acid-producing and methanogenic microorganisms were optimized at different stages through successive generations of domestication. Different carbon sources and inoculums were used for microbial domestication under specific pH, temperature and anaerobic conditions until the microbial community structure at each stage was stable.
The optimization of microorganisms at each stage has been achieved, improving anaerobic fermentation efficiency and system stability, and ensuring the efficient operation of the multi-stage fermentation process.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial domestication technology and relates to a method for domesticating multi-stage anaerobic fermentation microorganisms. Background Technology
[0002] Anaerobic fermentation refers to the process by which organic matter such as human and animal manure, straw, and weeds are decomposed and metabolized by a series of microorganisms with different functions under certain moisture, temperature and anaerobic conditions to form a combustible mixture of gases such as methane and carbon dioxide. It is the core technology of biogas engineering.
[0003] Traditional anaerobic fermentation technology typically employs a single-stage fermentation process. The entire biotransformation process, from feed to discharge, occurs within a single closed reactor. Each fermentation stage occurs simultaneously, with microorganisms from each stage coexisting in a relatively disordered, "chaotic" state. The key functional microorganisms at each stage cannot operate under their optimal growth conditions, thus hindering the full realization of their potential activity. Furthermore, the fermentation system exists in a delicate balance; disruption of this balance at any stage can lead to the failure of the entire system.
[0004] Currently, the development trend of anaerobic fermentation technology, both domestically and internationally, is from single-stage to multi-stage. For example, European countries have already achieved industrial applications of two-phase fermentation, including a hydrogen-producing phase and a methanogenic phase, as well as two-stage fermentation with a first-stage dry fermentation and a second-stage wet fermentation. The most classic biotransformation process theory of anaerobic fermentation, both domestically and internationally, is the "three-stage" theory. (See attached...) Figure 1 As shown, the hydrolysis-fermentation stage is mainly driven by microorganisms that secrete various hydrolytic enzymes, breaking down various biomolecules into smaller biomolecules such as monosaccharides. Microorganisms at this stage typically possess a certain degree of acid and oxygen tolerance. The hydrogen- and acetic acid-producing stage primarily involves acid-producing bacteria gradually converting soluble small molecules such as glucose into acetic acid. Microorganisms at this stage generally have good acid tolerance. The methanogenesis stage mainly involves methanogens converting the acetic acid produced in the acidogenesis stage into methane and carbon dioxide. Microorganisms at this stage typically require a neutral pH and are strictly anaerobic. Microorganisms at different stages progressively convert organic matter into biogas. Multi-stage fermentation is currently a relatively new technology, and how to ensure that each stage of multi-stage fermentation has optimized microorganisms suitable for that stage remains to be solved. Summary of the Invention
[0005] The purpose of this invention is to provide a method for the domestication of microorganisms in multi-stage anaerobic fermentation, so as to solve the problem of microorganisms not being optimized in stages during multi-stage anaerobic fermentation.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: Based on the multi-stage anaerobic fermentation process, which includes hydrolysis, acid production, and methanogenesis stages, this application provides a method for the domestication of multi-stage anaerobic fermentation microorganisms. This domestication method includes: Hydrolysis stage: Using macromolecular organic matter as the hydrolysis carbon source and anaerobic sludge as the hydrolysis inoculum, the microorganisms are domesticated generation by generation at a pH of 7-9; when the hydrolysis rate of the hydrolysis carbon source is the highest, the microorganisms are subcultured. Acid production stage: Using small molecule organic matter as the carbon source for acid production and anaerobic sludge as the inoculum, the microorganisms are domesticated generation by generation under strict anaerobic conditions with a pH of 5-7; when the acetic acid production rate is the highest, the microorganisms are subcultured. Methanogenesis stage: Using acetic acid or acetate as the carbon source for methanogenesis and anaerobic sludge from methane production as the inoculum, the microorganisms are domesticated generation by generation under strict anaerobic conditions at pH 7-8; when the acetic acid consumption rate or methanogenesis rate is the highest, the microorganisms are subcultured. The domestication process ends when the microbial community structure of the hydrolysis stage, the acid production stage, and the methanogenesis stage becomes stable.
[0007] The present invention has the following beneficial effects: This application involves targeted microbial domestication for each stage of multi-stage anaerobic fermentation, which can yield fermentation microorganisms with optimized microbial community structures at each stage, thereby improving anaerobic fermentation efficiency and overall stability. Attached Figure Description
[0008] Figure 1 This is a theoretical diagram of the three stages of the biotransformation process in anaerobic fermentation at the present stage. Figure 2 This is a diagram illustrating the domestication process during the hydrolysis stage. Figure 3 This is a diagram illustrating the domestication process during the acid-producing stage. Figure 4 This is a diagram illustrating the domestication process during the methanogenesis stage. Detailed Implementation
[0009] The multi-stage anaerobic fermentation in this application refers to fermentation that includes attached... Figure 1 The anaerobic fermentation process includes the hydrolysis stage, acid production stage, and methanogenesis stage. Based on this multi-stage anaerobic fermentation, this application provides a method for the domestication of multi-stage anaerobic fermentation microorganisms, which includes: Hydrolysis stage: Using macromolecular organic matter as the hydrolysis carbon source and anaerobic sludge as the hydrolysis inoculum, the hydrolysis carbon source and hydrolysis inoculum are mixed at a mass-to-volume ratio of 1g:20mL, and water is added to a final volume of 500mL; wherein the mass ratio of solids in the hydrolysis carbon source and hydrolysis inoculum is 1:1. Microorganisms are acclimatized generation by generation under conditions of pH 7-9 and acclimatization temperatures of 35-38℃ or 55-60℃. During acclimatization, the hydrolysis rate of the hydrolysis carbon source or the glucose production rate is analyzed. When the hydrolysis rate of the hydrolysis carbon source or the glucose production rate reaches its maximum, the culture is used as inoculum for subculturing. In this application, macromolecular organic matter includes at least one or more of cellulose, starch, protein, and lipids.
[0010] Acid production stage: Using small-molecule organic matter as the carbon source for acid production and anaerobic sludge as the inoculum, the carbon source and inoculum are mixed at a mass-to-volume ratio of 1g:20mL, and water is added to bring the volume to 1000mL; the mass ratio of solids in the carbon source and inoculum is 1:1. After adding nitrogen and trace elements necessary for the growth of hydrolytic microorganisms, the microorganisms are domesticated generation by generation under the following conditions: pH 5-7, strict anaerobic conditions, acclimatization temperature of 35-38℃ or 55-60℃, and redox potential of -150 to -400mV. During the acclimatization process, the acetic acid production rate is analyzed, and subculturing is performed when the acetic acid production rate reaches its maximum. In this application, the small-molecule organic matter includes at least one or more of glucose, amino acids, and fatty acids.
[0011] Methanogenesis stage: Using acetic acid or acetate as the carbon source for methanogenesis and anaerobic sludge from methane-producing sludge as the inoculum, the carbon source and inoculum are mixed at a mass-to-volume ratio of 1 g: 20 mL, and water is added to a final volume of 1000 mL; the mass ratio of solids in the carbon source and inoculum is 1:1. After adding the necessary nitrogen source and trace elements for methanogenic microorganisms, and then adding the necessary nitrogen source and trace elements for acidogenic microorganisms, the microorganisms are acclimatized generation by generation under conditions of pH 7-8, strict anaerobic conditions, acclimatization temperatures of 35-38℃ or 55-60℃, and redox potential below -300 mV. During acclimatization, the rate of acetic acid consumption or methanogenesis is analyzed. When the rate of acetic acid consumption or methanogenesis reaches its maximum, the microorganisms are subcultured.
[0012] After successive generations of domestication, when the microbial community structure of the hydrolysis, acid production, and methanogenesis stages tends to stabilize, the corresponding dominant microorganisms become dominant and can be used as inoculum for each stage of multi-stage anaerobic fermentation. At this point, domestication ends. Typically, after 6-10 generations of domestication, the microbial community structure of each stage of multi-stage anaerobic fermentation tends to stabilize, and the corresponding bioconversion rate remains high.
[0013] The technical solution of the present invention will be further explained and described below through specific embodiments.
[0014] Example 1 This application provides a method for the domestication of multi-stage anaerobic fermentation microorganisms, the method comprising: Hydrolysis stage: Using cellulose as the carbon source and anaerobic sludge as the inoculum, the carbon source and inoculum were mixed at a mass-to-volume ratio of 1g:20mL, and water was added to bring the volume to 500mL; the mass ratio of solids in the carbon source and inoculum was 1:1. After adding the nitrogen source and trace elements necessary for the growth of hydrolytic microorganisms, the microorganisms were acclimatized generation by generation at pH 8 and acclimatization temperature of 37℃. During the acclimatization process, the content of cellulose or glucose was measured at different time points, and the cellulose hydrolysis rate or glucose production rate at each time point was calculated. When the cellulose hydrolysis rate or glucose production rate reached its maximum, the culture was used as inoculum for subculturing, as shown in the attached figure. Figure 2 The diagram shows the domestication process. After eight generations of domestication, stable cellulose-hydrolyzing bacteria were obtained during the hydrolysis stage.
[0015] Acid production stage: Using glucose as the carbon source for acid production and anaerobic sludge as the inoculum, the carbon source and inoculum were mixed at a mass-to-volume ratio of 1g:20mL, and water was added to bring the volume to 1000mL; the mass ratio of solids in the carbon source and inoculum was 1:1. After adding the nitrogen source and trace elements necessary for the growth of acid-producing microorganisms, the microorganisms were acclimatized generation by generation under the conditions of pH 6, strict anaerobic conditions, acclimatization temperature of 37℃, and redox potential of -300mV. During the acclimatization process, the acetic acid content was measured at different time points, and the acetic acid production rate at each time point was calculated. When the acetic acid production rate reached its maximum, the culture was used as inoculum for subculturing, as shown in the attached figure. Figure 3 The diagram shows the domestication process. After six generations of domestication, stable acid-producing bacteria were obtained during the acid-producing stage.
[0016] Methanogenesis stage: Acetic acid was used as the carbon source for methanogenesis, and anaerobic sludge from methane production was used as the inoculum. The carbon source and inoculum were mixed at a mass-to-volume ratio of 1 g:20 mL, and water was added to a final volume of 1000 mL. The mass ratio of solids in the carbon source to the inoculum was 1:1. After adding the necessary nitrogen source and trace elements for methanogenic microbial growth, the microorganisms were acclimatized generation by generation under the conditions of pH 8, strict anaerobic conditions, acclimatization temperature of 37℃, and redox potential of -250 mV. During the acclimatization process, the content of acetic acid or methane was measured at different time points, and the consumption rate of acetic acid or the methanogenesis rate was calculated. When the acetic acid consumption rate or the methanogenesis rate reached its maximum, the culture was used as inoculum for subculturing, as shown in the attached figure. Figure 4The diagram shows the domestication process. After six generations of domestication, stable methanogenic bacteria were obtained during the methanogenic stage.
[0017] Example 2 This application provides a method for the domestication of multi-stage anaerobic fermentation microorganisms, the method comprising: Hydrolysis stage: Using protein as the carbon source and anaerobic sludge as the inoculum, the carbon source and inoculum were mixed at a mass-to-volume ratio of 1 g:20 mL, and water was added to bring the volume to 500 mL; the mass ratio of solids in the carbon source and inoculum was 1:1. After adding nitrogen and trace elements necessary for the growth of hydrolytic microorganisms, the microorganisms were acclimatized generation by generation at pH 7 and an acclimatization temperature of 55℃. During the acclimatization process, the protein or amino acid content was measured at different time points, and the protein hydrolysis rate or amino acid production rate was calculated at each time point. When the protein hydrolysis rate or amino acid production rate reached its maximum, the culture was used as inoculum for subculturing. After 7 generations of acclimatization, stable protein-hydrolyzing bacteria were obtained.
[0018] Acid-producing stage: Using amino acids as the carbon source and anaerobic sludge as the inoculum, the carbon source and inoculum were mixed at a mass-to-volume ratio of 1g:20mL, and water was added to bring the volume to 1000mL; the mass ratio of solids in the carbon source and inoculum was 1:1. After adding nitrogen and trace elements necessary for the growth of hydrolytic microorganisms, the microorganisms were acclimatized generation by generation under conditions of pH 5, strict anaerobic conditions, acclimatization temperature of 60℃, and redox potential of -150mV. During the acclimatization process, the acetic acid content was measured at different time points, and the acetic acid production rate at each time point was calculated. When the acetic acid production rate reached its maximum, the culture was used as inoculum for subculturing. After 6 generations of acclimatization, stable acid-producing bacteria were obtained.
[0019] Methanogenesis stage: Sodium acetate was used as the carbon source for methanogenesis, and anaerobic sludge from methane production was used as the inoculum. The carbon source and inoculum were mixed at a mass-to-volume ratio of 1 g:20 mL, and water was added to bring the volume to 1000 mL; the mass ratio of solids in the carbon source to the inoculum was 1:1. After adding the nitrogen source and trace elements necessary for the growth of methanogenic microorganisms, the microorganisms were acclimatized generation by generation under the conditions of pH 7, strict anaerobic conditions, acclimatization temperature of 35℃, and redox potential of -200 mV. During the acclimatization process, the content of sodium acetate or methane was measured at different time points, and the consumption rate of sodium acetate or the methanogenesis rate was calculated. When the sodium acetate consumption rate or the methanogenesis rate reached its maximum, the culture was used as inoculum for subculturing. After 6 generations of acclimatization, stable methanogenic bacteria were obtained.
[0020] Example 3 This application provides a method for the domestication of multi-stage anaerobic fermentation microorganisms, the method comprising: Hydrolysis stage: Using oil as the carbon source and anaerobic sludge as the inoculum, the carbon source and inoculum were mixed at a mass-to-volume ratio of 1 g:20 mL, and water was added to bring the volume to 500 mL; the mass ratio of solids in the carbon source and inoculum was 1:1. After adding nitrogen and trace elements necessary for the growth of hydrolytic microorganisms, the microorganisms were acclimatized generation by generation at pH 9 and acclimatization temperature of 38℃. During the acclimatization process, the content of oil or fatty acids was measured at different time points, and the oil hydrolysis rate or fatty acid production rate at each time point was calculated. When the oil hydrolysis rate or fatty acid production rate reached its maximum, the culture was used as inoculum for subculturing. After 7 generations of acclimatization, stable oil-hydrolyzing bacteria were obtained.
[0021] Acid production stage: Using fatty acids as the carbon source and anaerobic sludge as the inoculum, the carbon source and inoculum were mixed at a mass-to-volume ratio of 1g:20mL, and water was added to bring the volume to 1000mL; the mass ratio of solids in the carbon source and inoculum was 1:1. After adding nitrogen and trace elements necessary for the growth of hydrolytic microorganisms, the microorganisms were acclimatized generation by generation under conditions of pH 7, strict anaerobic conditions, acclimatization temperature of 55℃, and redox potential of -400mV. During the acclimatization process, the acetic acid content was measured at different time points, and the acetic acid production rate at each time point was calculated. When the acetic acid production rate reached its maximum, the culture was used as inoculum for subculturing. After 6 generations of acclimatization, stable acid-producing bacteria were obtained.
[0022] Methanogenesis stage: Acetic acid was used as the carbon source for methanogenesis, and anaerobic sludge from methane production was used as the inoculum. The carbon source and inoculum were mixed at a mass-to-volume ratio of 1 g:20 mL, and water was added to a final volume of 1000 mL; the mass ratio of solids in the carbon source to the inoculum was 1:1. After adding the necessary nitrogen source and trace elements for methanogenic microbial growth, the microorganisms were acclimatized generation by generation under the conditions of pH 8, strict anaerobic conditions, acclimatization temperature of 38℃, and redox potential of -200 mV. During the acclimatization process, the content of acetic acid or methane was measured at different time points, and the acetic acid consumption rate or methanogenesis rate was calculated. When the acetic acid consumption rate or methanogenesis rate reached its maximum, the culture was used as inoculum for subculturing. After 6 generations of acclimatization, stable methanogenic bacteria were obtained.
[0023] Example 4 This application provides a method for the domestication of multi-stage anaerobic fermentation microorganisms, the method comprising: Hydrolysis stage: Starch was used as the carbon source for hydrolysis, and anaerobic sludge was used as the inoculum. The carbon source and inoculum were mixed at a mass-to-volume ratio of 1 g: 20 mL, and water was added to bring the volume to 500 mL; the mass ratio of solids in the carbon source and inoculum was 1:1. After adding nitrogen and trace elements necessary for the growth of hydrolytic microorganisms, the microorganisms were acclimatized generation by generation at pH 7.5 and an acclimatization temperature of 55℃. During the acclimatization process, the starch or glucose content was measured at different time points, and the starch hydrolysis rate or glucose production rate at each time point was calculated. When the starch hydrolysis rate or glucose production rate reached its maximum, the culture was used as inoculum for subculturing. After 8 generations of acclimatization, stable starch-hydrolyzing bacteria were obtained.
[0024] Acid production stage: Using glucose as the acid-producing carbon source and anaerobic sludge as the acid-producing inoculum, the carbon source and inoculum were mixed at a mass-to-volume ratio of 1g:20mL, and water was added to bring the volume to 1000mL; the mass ratio of solids in the carbon source and inoculum was 1:1. After adding nitrogen and trace elements necessary for the growth of hydrolytic microorganisms, the microorganisms were domesticated generation by generation under conditions of pH 6, strict anaerobic conditions, acclimatization temperature of 55℃, and redox potential of -200mV. During the acclimatization process, the acetic acid content was measured at different time points, and the acetic acid production rate at each time point was calculated. When the acetic acid production rate reached its maximum, the culture was used as inoculum for subculturing. After 6 generations of acclimatization, stable acid-producing bacteria were obtained.
[0025] Methanogenesis stage: Acetic acid was used as the carbon source for methanogenesis, and anaerobic sludge from methane production was used as the inoculum. The carbon source and inoculum were mixed at a mass-to-volume ratio of 1 g:20 mL, and water was added to bring the volume to 1000 mL; the mass ratio of solids in the carbon source to the inoculum was 1:1. After adding the nitrogen source and trace elements necessary for the growth of methanogenic microorganisms, the microorganisms were acclimatized generation by generation under the conditions of pH 8, strict anaerobic conditions, acclimatization temperature of 38℃, and redox potential of -100 mV. During the acclimatization process, the content of acetic acid or methane was measured at different time points, and the consumption rate of acetic acid or the methanogenesis rate was calculated. When the acetic acid consumption rate or the methanogenesis rate reached its maximum, the culture was used as inoculum for subculturing. After 7 generations of acclimatization, stable methanogenic bacteria were obtained.
[0026] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. A method for domesticating multi-stage anaerobic fermentation microorganisms, characterized in that, The multi-stage anaerobic fermentation includes a hydrolysis stage, an acid production stage, and a methanogenesis stage, and the acclimation method includes: Hydrolysis stage: Using macromolecular organic matter as the hydrolysis carbon source and anaerobic sludge as the hydrolysis inoculum, the microorganisms are domesticated generation by generation at a pH of 7-9; when the hydrolysis rate of the hydrolysis carbon source is the highest, the microorganisms are passaged for cultivation. Acid production stage: Using small molecule organic matter as the carbon source for acid production and anaerobic sludge as the inoculum for acid production, the microorganisms are domesticated generation by generation under strict anaerobic conditions with a pH of 5-7; when the acetic acid production rate is the highest, the microorganisms are subcultured. Methanogenesis stage: Using acetic acid or acetate as the carbon source for methanogenesis and anaerobic sludge from methane production as the inoculum, the microorganisms are domesticated generation by generation under strict anaerobic conditions at pH 7-8; when the acetic acid consumption rate or methanogenesis rate is the highest, the microorganisms are subcultured. The domestication process ends when the microbial community structure of the hydrolysis stage, the acid production stage, and the methanogenesis stage becomes stable.
2. The method for domesticating multi-stage anaerobic fermentation microorganisms according to claim 1, characterized in that, The macromolecular organic compounds include at least one or more of cellulose, starch, protein, and fats.
3. The method for domesticating multi-stage anaerobic fermentation microorganisms according to claim 1, characterized in that, The small molecule organic compounds include at least one or more of glucose, amino acids, and fatty acids.
4. The method for domesticating multi-stage anaerobic fermentation microorganisms according to claim 1, characterized in that, The acclimatization temperature is 35-38℃ or 55-60℃.
5. The method for domesticating multi-stage anaerobic fermentation microorganisms according to claim 1, characterized in that, During the acid production stage, the redox potential of the acclimatization environment is -150 to -400 mV.
6. The method for domesticating multi-stage anaerobic fermentation microorganisms according to claim 1, characterized in that, During the methanogenesis stage, the redox potential of the acclimatization environment is below -300mV.
7. The method for domesticating multi-stage anaerobic fermentation microorganisms according to claim 1, characterized in that, The domestication process takes 6-10 generations.
8. The method for domesticating multi-stage anaerobic fermentation microorganisms according to claim 1, characterized in that, The mass-to-volume ratio of the hydrolyzed carbon source to the hydrolyzed inoculum is 1 g: 20 mL, and the mass ratio of the solids in the hydrolyzed carbon source to the hydrolyzed inoculum is 1:
1.
9. The method for domesticating multi-stage anaerobic fermentation microorganisms according to claim 1, characterized in that, The mass-to-volume ratio of the acid-producing carbon source to the acid-producing inoculum is 1 g: 20 mL, and the mass ratio of the solids in the acid-producing carbon source to the solids in the acid-producing inoculum is 1:
1.
10. The method for domesticating multi-stage anaerobic fermentation microorganisms according to claim 1, characterized in that, The mass-to-volume ratio of the methanogenic carbon source to the methanogenic inoculum is 1 g: 20 mL, and the mass ratio of the solids in the methanogenic carbon source to the solids in the methanogenic inoculum is 1:1.