Method for domesticating rumen microorganisms to adapt to in-vitro environment
By controlling temperature, solids content, and carbon-nitrogen ratio, a stable rumen microbial interaction network was constructed in stages, solving the problem of low adaptation and degradation efficiency of rumen microorganisms in in vitro applications and realizing efficient utilization of biomass resources.
Patent Information
- Application Number
- CN202610047706.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-14
- Publication Date
- 2026-03-03
AI Technical Summary
Rumen microorganisms have difficulty adapting to new environments in in vitro applications, their interaction networks are unstable, and their degradation efficiency and VFA production are low, which limits their large-scale application in the utilization of biomass resources.
By controlling the temperature, solids content, and substrate carbon-nitrogen ratio during in vitro fermentation, a multi-microbial community with strong interactions was constructed in stages to form a stable in vitro rumen microbial interaction network, with the priority being temperature > solids content > carbon-nitrogen ratio.
It enabled the efficient application of rumen microorganisms in the in vitro environment, increasing the yield of reducing sugars by 118.75%, VFAs by 280.98%, and improving the degradation efficiency of cellulosic biomass to 45%~65%, thus solving the problems of difficult in vitro adaptation and low degradation efficiency.
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Figure CN121592572A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of resources and environmental technology, and specifically to a method for domesticating rumen microorganisms to adapt to the in vitro environment. Background Technology
[0002] The rumen microbiome is a complex ecosystem composed of various microorganisms, including bacteria, fungi, and archaea. It plays a crucial role in the transformation of matter within the rumen of ruminants, gradually hydrolyzing and acidifying cellulose biomass into volatile fatty acids (VFAs). These metabolites are then absorbed and utilized by ruminants, becoming an important source of energy and nutrition. Within this complex community, various microorganisms perform their specific functions and work synergistically: the number of fungi in the rumen is approximately 10... 3 -10 5 The number of bacteria per mL is close to one hundred genera, and their core function is to colonize and penetrate plant cell walls, providing a wider range of attachment sites for subsequent rumen bacteria. The number of bacteria in the rumen is even greater, approximately 102. 9 -10 11 With a density of over 600 identifiable genera, bacteria and fungi form stable interactions, jointly promoting the efficient degradation of recalcitrant substances such as cellulose and hemicellulose. Furthermore, archaea play a crucial regulatory role in the community, mitigating rancidity caused by excessive VFA production and maintaining rumen stability. Among the numerous rumen microorganisms, cellulose-degrading bacteria (such as *Ruminococcus albus*, *Ruminococcus flamefaciens*, and *Fibrobacter uccinogenes*) have become the core functional flora dominating plant tissue degradation processes due to their unique functional advantages and rich metabolic diversity. However, despite their excellent degradation performance in vivo, their in vitro pure culture and application have not yet been achieved.
[0003] In recent years, with the increasing demand for biomass resource utilization, researchers have extensively conducted research on the in vitro application of rumen microorganisms, achieving some progress in several fields such as co-methanation of cellulosic biomass and the hydrolysis of food waste to produce VFAs. However, it is undeniable that the interaction mechanisms of rumen microorganisms are extremely complex, and approximately 80% of bacteria and fungi cannot be cultured in vitro using existing technologies, posing a significant challenge to their in vitro application. To date, no researchers have systematically investigated the interaction patterns between rumen microorganisms and environmental factors such as temperature, solids content, and carbon-nitrogen ratio when they are transferred from the stable in vivo environment to artificial in vitro conditions. Furthermore, effective solutions are still lacking for key technologies that rapidly promote the adaptation of bacteria and fungi to new environmental conditions and simultaneously improve their degradation efficiency and metabolic production performance during the initial stage of in vitro culture, severely limiting the large-scale application of rumen microorganisms in the in vitro biomass resource utilization. Summary of the Invention
[0004] The purpose of this invention is to provide a method for domesticating rumen microorganisms to adapt to the in vitro environment, thereby solving the problems of difficulty in in vitro adaptation of rumen microorganisms, unstable interaction networks, low degradation efficiency and low VFA production, and realizing the efficient application of rumen microorganisms in the in vitro environment.
[0005] To achieve the above objectives, the technical solution of this application is as follows: a method for domesticating rumen microorganisms to adapt to the in vitro environment, comprising: regulating the abundance of functional microbial communities, expression of related genes and proportion of metabolites by controlling three environmental factors of in vitro fermentation: temperature, solid content and substrate carbon-nitrogen ratio, constructing strong multi-microbial community interaction relationships in stages, and forming a stable in vitro rumen microbial interaction network, wherein the control priority of the environmental factors is temperature > solid content > carbon-nitrogen ratio.
[0006] In another implementation of the present invention, the temperature control range is 35℃~42℃ or 50℃~60℃, and the relative abundance of the acidophilic bacteria Prevotella is 23.5%~25.5% and the relative abundance of the thermotolerant bacteria Clostridium is 12.5%~14.5% through temperature control.
[0007] In another implementation of the present invention, the acetic acid production ratio is controlled to 50%~65% by temperature control and bacterial abundance adjustment.
[0008] In another implementation of the present invention, the solid content is controlled within the range of 5 mg / L to 12.5 mg / L, and the abundance of the acidifying functional bacteria genus Sphaerochaeta is maintained at 4.5% to 7.0% through this solid content control.
[0009] In another implementation of the present invention, the yield of volatile fatty acids (VFAs) is increased to 1000 mg / L to 3500 mg / L by adjusting the solid content and enhancing the activity of acidifying functional bacteria.
[0010] In another implementation of the present invention, the substrate carbon-nitrogen ratio is regulated within the range of 18.5 to 24.5, and this carbon-nitrogen ratio regulation enables the expression level of the cellulase gene Glu to reach 1.5 × 10⁻⁶. 6 ~4.5×10 6 .
[0011] In another implementation of the present invention, the degradation efficiency of cellulosic biomass is increased to 45%~65% by regulating the carbon-nitrogen ratio and enhancing cellulase gene expression. degradation .
[0012] In another implementation of the present invention, the specific process of constructing the interaction relationship in stages is as follows: firstly, by controlling the temperature, a strong interaction relationship is formed between the fungi Wallemia and Caecomyces and the bacteria NK4A214_group (P < 0.001); secondly, by controlling the solid content, a strong interaction relationship is formed between Candida and Wallemia, Caecomyces, and NK4A214_group (P < 0.05); finally, by controlling the carbon-nitrogen ratio, a strong interaction relationship is formed between Psychrobacter and Candida, Wallemia, Caecomyces, and NK4A214_group (P < 0.05).
[0013] In another implementation of the present invention, the proportion of acetic acid is controlled within the range of 50% to 65% and the concentration of reducing sugar is controlled within the range of 150 mg / L to 200 mg / L, so as to achieve the dominant hydrolysis stage of Wallemia, Caecomyces and NK4A214_group.
[0014] In another implementation of the present invention, the concentration of VFAs is simultaneously controlled within the range of 1000 mg / L to 3500 mg / L and the degradation efficiency is controlled within the range of 45% to 65% VFAs. degradation Within this range, the acidification phase is achieved, dominated by Candida and Psychrobacter.
[0015] By adopting the above technical solution, the present invention can achieve the following technical effects: 1. By regulating environmental factors in stages, a new stable interaction network was formed, dominated by fungi such as Wallemia and Caecomyces in the hydrolysis stage and bacteria such as Candida and Psychrobacter in the acidification stage, thus solving the problem of unstable in vitro interactions of rumen microorganisms.
[0016] 2. Compared with existing technologies, the yield of reducing sugars is increased by 118.75%, the yield of VFAs is increased by 280.98%, and the acetic acid ratio is optimized to 50%~65%, which enhances the utilization value of the product.
[0017] 3. By regulating environmental factors, rumen microorganisms can rapidly adapt to the external environment, increasing the degradation efficiency of cellulosic biomass to 45%~65% VS degradation This solves the problems of difficult in vitro adaptation and low degradation efficiency.
[0018] 4. This method is applicable to the resource utilization of various cellulosic biomass such as corn stalks and kitchen waste. It can be used in fields such as biohydrogen production and VFAs production, reducing production costs and having significant economic and environmental benefits. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0020] Figure 1 This is a graph showing the change of reducing sugar concentration over time under different operating conditions in this embodiment of the invention. Figure 2 This is a schematic diagram illustrating the succession characteristics of the rumen microbial community during the hydrolysis and acidification stages in an embodiment of the present invention. Figure 3 This is a collinear network diagram of rumen microbes during the acidification phase. Detailed Implementation
[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to specific embodiments. All experimental methods used are conventional methods, and all experimental equipment, materials, and reagents used are commercially available.
[0022] Example 1 Fermentation substrate preparation: Naturally air-dried corn stalks were selected as cellulose biomass raw materials. They were first pre-crushed using a conventional pulverizer to obtain stalk particles with a particle size ≤2cm. Then, the pre-crushed particles were sent to an ultra-micro pulverizer for secondary crushing. After crushing, the particles were screened using a standard inspection sieve to collect stalk powder with a particle size ≤0.15mm, which was used as the fermentation substrate in this embodiment. The powder was sealed and stored for later use to avoid moisture absorption and deterioration.
[0023] Inoculum preparation: Select healthy adult ruminants (such as dairy cows) and collect fresh rumen fluid under aseptic conditions. Maintain an oxygen-free environment throughout the collection process to prevent rumen microorganisms from being inactivated by air contact. After collection, quickly transfer the rumen fluid to a pre-sterilized anaerobic container, seal it, and store it temporarily at a constant temperature of 37°C for later inoculation.
[0024] Buffer solution preparation: A buffer solution simulating the composition of ruminant saliva was used as the conditioning water for the fermentation system to simulate the physiological environment within the rumen. The specific formula is shown in Table 1 below. Weigh each chemical reagent component according to the formula and add them sequentially to deionized water while stirring to ensure complete dissolution. After preparation, allow to stand for later use.
[0025] Table 1 Buffer Solutions Fermentation system setup and parameter control: The prepared fermentation substrate, spare rumen fluid, and buffer solution were added to the anaerobic fermenter in a mass ratio of 1:5:10. After adding the materials, the fermenter was quickly sealed to ensure an anaerobic environment. The sealed fermenter was placed in a constant-temperature fermentation apparatus, and the fermentation program was started. The core environmental parameters were controlled as follows: fermentation temperature was set at 39℃, fermentation solids content was controlled at 12.5 mg / L, and the substrate carbon-to-nitrogen ratio was adjusted to 24.5. Throughout the fermentation process, the environmental factor control priority was strictly followed: temperature > solids content > carbon-to-nitrogen ratio, ensuring that each parameter was stably maintained within the set range.
[0026] Fermentation process monitoring and result analysis: Microbial community succession characteristics: During fermentation, the composition of the microbial community at different stages was dynamically monitored using high-throughput sequencing technology. The results are as follows: Figure 2As shown. During the hydrolysis stage, the bacterial community is mainly composed of Bacteroidetes and Proteobacteria, while the fungal community is dominated by Basidiomycota and Ascomycota. Among them, fungi such as Wallemia and Caecomyces, as well as bacteria such as NK4A214_group and Mrakia, form a core hydrolysis network. Under the joint regulation of temperature, solids content, and carbon-nitrogen ratio, they efficiently promote the decomposition of lignocellulose. After entering the acidification stage, the microbial community gradually evolves to be dominated by acid-producing fungi and bacteria. Prevotella, Sphaerochaeta, Treponema, Christensenellaceae_R-7_group, as well as fungi such as Candida, Debaryomyces, Psychrobacter, and Leucosporidium, form a new interaction network to achieve further hydrolysis of recalcitrant polysaccharides and convert intermediate products into volatile fatty acids (VFAs). Community succession analysis revealed that temperature has the greatest impact on microbial community structure, followed by solids content, while the carbon-nitrogen ratio has the least impact.
[0027] Changes in reducing sugar concentration: The DNS method was used to monitor the reducing sugar concentration in real time during fermentation. The results are as follows: Figure 1 As shown, the reducing sugar concentration rapidly increased during the first exponential growth phase (0–8 h), indicating that the hydrolytic bacteria quickly initiated the hydrolysis reaction. During the 8–72 h range, the reducing sugar concentration remained at a high level, indicating that the hydrolytic bacteria were in a highly efficient hydrolysis state. After 72 h, the second exponential growth phase began, and the reducing sugar was further utilized by the acidifying bacteria, causing the concentration to gradually decrease. In this embodiment, under the optimal operating conditions represented by the MNS reactor, the reducing sugar concentration was significantly higher than that of the control system LML. During the 8–72 h increase range, the maximum difference between the two systems reached 18.60%, and the overall maximum difference reached 116.66%. This indicates that under conditions of 39°C, high solids content, and a suitable carbon-to-nitrogen ratio, the synergistic effect of Caecomyces and NK4A214_group significantly improved the cellulose hydrolysis efficiency, enabling the reducing sugar yield during the hydrolysis stage to reach a maximum of 118.75% of the control system.
[0028] VFAs concentration changes: The concentration of VFAs during fermentation was monitored using gas chromatography, and the results are as follows: Figure 1As shown, VFA concentrations continuously increased over time during the first exponential growth phase, reaching a stable increase around 48 hours after the transition from hydrolysis to acidification. This indicates that the acidifying bacteria gradually became dominant and fully utilized intermediate products such as reducing sugars and pyruvate for acid production metabolism. At the end of the second exponential growth phase, VFA concentrations decreased under some conditions, indicating further substrate transformation. Significant differences in VFA concentrations were observed under different operating conditions. The highest VFA concentration in the reactor under optimal conditions reached 280.98% of the lowest concentration, further verifying that temperature had the greatest impact on VFA formation, followed by solids content, while the carbon-to-nitrogen ratio (C / N ratio) had a relatively small impact on VFA formation within the range of 18–25.
[0029] Example 2 Fermentation substrate preparation: Naturally air-dried wheat straw was selected as raw material. After initial crushing to a particle size ≤2cm, it was further crushed using an ultra-micro pulverizer. The resulting wheat straw powder with a particle size ≤0.15mm was screened and used as the fermentation substrate. It was then sealed and stored in a dry environment for later use.
[0030] Inoculum preparation: Collect fresh rumen fluid from healthy goats, ensuring strict oxygen isolation during the collection process. Immediately after collection, seal the fluid in an anaerobic bottle and store it in a 38°C incubator for a short period to ensure that the activity of rumen microorganisms is not affected, for use in inoculation.
[0031] Buffer solution preparation: The simulated ruminant saliva buffer solution with the exact same formula as in Example 1 was prepared accurately according to the same preparation method, and allowed to stand until completely dissolved before use.
[0032] Fermentation system setup and parameter control: The components were added to the anaerobic fermenter at a mass ratio of substrate:rumen fluid:buffer solution = 1:6:12, sealed, and placed in a constant-temperature fermentation system. Environmental parameters were controlled as follows: fermentation temperature 55℃, fermentation solids content 5 mg / L, and substrate C / N ratio 18.5. The priority for environmental factor control was temperature > solids content > C / N ratio, maintaining parameter stability during fermentation.
[0033] Fermentation process monitoring and result analysis: Microbial community succession: High-throughput sequencing results showed that during the hydrolysis stage, the bacterial community was dominated by Bacteroidetes and Proteobacteria, while the fungal community included Basidiomycota and Ascomycota. Wallemia, Caecomyces, NK4A214_group, and other species constituted the core hydrolytic bacteria, which efficiently decomposed lignocellulose in wheat straw. During the acidification stage, the community structure was dominated by acid-producing bacteria. Prevotella, Sphaerochaeta, Candida, Psychrobacter, and other species formed a stable interaction network, promoting the conversion of intermediate products into VFAs. Temperature had the most significant driving effect on community succession.
[0034] Reducing sugar and VFAs yield: The reducing sugar concentration increased rapidly from 0 to 10 h and remained at a high level from 10 to 70 h, with the highest yield being 102% higher than the control system without the control method of this invention; the VFAs concentration entered a stable increasing range around 50 h, with the highest concentration reaching 3100 mg / L, which is 275% higher than the lowest concentration condition, fully demonstrating the effect of the technical solution of this invention on improving acid production performance.
[0035] Example 3 Fermentation substrate preparation: Rice straw is used as raw material. After being naturally air-dried, it is crushed to ≤2cm. After ultra-fine grinding, the powder with a particle size ≤0.15mm is screened as the fermentation substrate and sealed for later use.
[0036] Inoculum preparation: Collect fresh rumen fluid from sheep, store it in an anaerobic container in an oxygen-free environment, and temporarily refrigerate it at 4°C. One hour before inoculation, restore it to 37°C to ensure microbial activity.
[0037] Buffer solution preparation: Using the buffer solution formulation described in Example 1, accurately weigh each component and dissolve it in deionized water. Once prepared, set aside for later use.
[0038] Fermentation system setup and parameter control: The substrate, rumen fluid, and buffer solution were mixed at a mass ratio of 1:4:9 and added to the fermentation apparatus. After sealing, fermentation was started. The controlled environmental factors were: fermentation temperature 38℃, fermentation solids content 8 mg / L, and substrate C / N ratio 20.5. The control priority was temperature > solids content > C / N ratio.
[0039] Fermentation process monitoring and result analysis: Community and metabolic performance: During the hydrolysis stage, a hydrolysis network with Wallemia, Caecomyces, and NK4A214_group as its core is formed, and during the acidification stage, it evolves into an interaction system dominated by acid-producing bacteria. The highest yield of reducing sugar is 95% higher than that of the control system, and the highest concentration of VFAs reaches 2700 mg / L, which is 260% higher than that of the lowest concentration condition, verifying the applicability and stability of the technical solution of this invention in different cellulosic biomass raw materials.
[0040] The above embodiments, through different raw materials and parameter settings, systematically verified the effectiveness of the technical solution of the present invention. By precisely controlling three environmental factors—temperature, solids content, and carbon-nitrogen ratio—and following specific priorities, the orderly succession of the rumen microbial community can be guided, a stable hydrolysis-acidification functional bacterial community interaction network can be constructed, and the degradation efficiency of cellulosic biomass and the yield of reducing sugars and VFAs can be significantly improved, providing reliable technical support for the resource utilization of cellulosic biomass.
[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.
Claims
1. A method for acclimating rumen microorganisms to an in vitro environment, characterized in that, include: By regulating three environmental factors—temperature, solids content, and substrate carbon-nitrogen ratio—in vitro fermentation, the abundance of functional microbial communities, the expression of related genes, and the proportion of metabolites are adjusted. Multi-microbial communities with strong interactions are constructed in stages to form a stable in vitro rumen microbial interaction network. The priority of the environmental factors is temperature > solids content > carbon-nitrogen ratio.
2. The method for domesticating rumen microorganisms to adapt to the in vitro environment according to claim 1, characterized in that, The temperature is controlled within a range of 35℃~42℃ or 50℃~60℃, thereby controlling the relative abundance of the acidophilic bacteria Prevotella to 23.5%~25.5% and the relative abundance of the thermotolerant bacteria Clostridium to 12.5%~14.5%.
3. The method for domesticating rumen microorganisms to adapt to the in vitro environment according to claim 2, characterized in that, The acetic acid production ratio was adjusted to 50%~65% by controlling temperature and adjusting bacterial abundance.
4. The method for domesticating rumen microorganisms to adapt to the in vitro environment according to claim 1, characterized in that, The solid content is controlled within the range of 5 mg / L to 12.5 mg / L, and the abundance of the acidifying functional bacteria genus Sphaerochaeta is maintained at 4.5% to 7.0% through this solid content control.
5. The method for domesticating rumen microorganisms to adapt to the in vitro environment according to claim 4, characterized in that, By regulating the solid content and enhancing the activity of acidifying functional bacteria, the yield of volatile fatty acids (VFAs) can be increased to 1000 mg / L~3500 mg / L.
6. The method for domesticating rumen microorganisms to adapt to the in vitro environment according to claim 1, characterized in that, The substrate carbon-nitrogen ratio is regulated within the range of 18.5–24.5, and this carbon-nitrogen ratio regulation enables the expression level of the cellulase gene Glu to reach 1.5 × 10⁻⁶. 6 ~4.5×10 6 .
7. The method for domesticating rumen microorganisms to adapt to the in vitro environment according to claim 6, characterized in that, By regulating the carbon-to-nitrogen ratio and enhancing cellulase gene expression, the degradation efficiency of cellulosic biomass was increased to 45%–65% VS. degradation .
8. The method for domesticating rumen microorganisms to adapt to the in vitro environment according to claim 1, characterized in that, The specific process of establishing interaction relationships in stages is as follows: First, by regulating temperature, a strong interaction relationship is formed between the fungi Wallemia and Caecomyces and the bacteria NK4A214_group (P < 0.001); second, by regulating the solids content, a strong interaction relationship is formed between Candida and Wallemia, Caecomyces, and NK4A214_group (P < 0.05); finally, by regulating the carbon-nitrogen ratio, a strong interaction relationship is formed between Psychrobacter and Candida, Wallemia, Caecomyces, and NK4A214_group (P < 0.05).
9. The method for domesticating rumen microorganisms to adapt to the in vitro environment according to claim 3, characterized in that, Simultaneously, the acetic acid ratio is controlled within the range of 50% to 65% and the reducing sugar concentration is controlled within the range of 150 mg / L to 200 mg / L to achieve the dominant hydrolysis stage dominated by Wallemia, Caecomyces and NK4A214_group.
10. The method for domesticating rumen microorganisms to adapt to the in vitro environment according to claim 5, characterized in that, Simultaneously, the concentration of VFAs was controlled within the range of 1000 mg / L to 3500 mg / L, and the degradation efficiency was controlled within the range of 45% to 65% VS. degradation Within this range, the acidification phase is achieved, dominated by Candida and Psychrobacter.