Strain combination, methane fermentation system and preparation method

Abstract

The invention discloses a strain combination, a methane fermentation system and a preparation method, and belongs to the technical field of microorganisms. In the methane fermentation system provided by the invention, lignocellulose in crop straws is firstly and preliminarily hydrolyzed into soluble saccharides by natural hydrolytic flora in an inoculum, and then the soluble saccharides are fermented by lactobacillus plantarum to generate intermediate products such as lactic acid, acetic acid, ethanol, hydrogen, carbon dioxide and the like; further, intermediate products such as lactic acid and ethanol are converted into methane-producing precursors such as hydrogen and acetic acid through natural microbial flora in the inoculum; subsequently, in the methane production stage, methanosarcina utilizes hydrogen and carbon dioxide to produce methane in a hydrogen nutrition mode, utilizes acetic acid to produce methane in an acetic acid nutrition mode and / or utilizes methyl substances to produce methane in a methyl nutrition mode, accumulation of intermediate products is avoided, and the conversion efficiency of a substrate and the methane yield are improved.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, specifically involving strain combinations, methane fermentation systems, and preparation methods. Background Technology

[0002] Methane is a clean and renewable energy source with high utilization value.

[0003] In related technologies, the methane fermentation system includes inoculum and crop straw. The natural microbial community contained in the inoculum decomposes and transforms the lignocellulose in the crop straw to produce methane.

[0004] However, the number of functional bacteria in natural microbial communities fluctuates greatly and their activity is unstable, leading to unstable fermentation efficiency and thus affecting methane yield. Summary of the Invention

[0005] This invention discloses a combination of microbial strains, a methane fermentation system, and a preparation method to address the problem in existing technologies where the number of functional microbial communities in natural microbial communities fluctuates greatly and their activity is unstable, leading to unstable fermentation efficiency and thus affecting methane yield.

[0006] To solve the above-mentioned technical problems, the present invention is implemented as follows: In a first aspect, the present invention provides a bacterial strain combination, including *Lactobacillus plantarum* with accession number NRRL NO.B-14768. Lactiplantibacillus plantarum ) and Methanococcus ostreatus with accession number ATCC NO. 51582 ( Methanosarcina barkeri ).

[0007] Secondly, this application provides a methane fermentation system comprising crop straw, inoculum, water, and the aforementioned combination of microorganisms.

[0008] In some embodiments, the volume percentage of the crop straw in the fermentation system is 8% to 12%, the volume percentage of the inoculum is 8% to 12%, the volume percentage of the microbial combination is 1% to 3%, and the volume percentage of the water is 73% to 83%.

[0009] In some embodiments, in the bacterial strain combination, *Lactobacillus plantarum* (… Lactiplantibacillus plants ) and methanogenic octopus ( Methanosarcina barkeri The volume ratio of ) is 1:(0.8~1.2).

[0010] In some embodiments, the *Lactobacillus plantarum* ( Lactiplantibacillus plantarum ) of OD 600 The value is 0.8~1.2; And / or, the methanogenic octopus ( Methanosarcina barkeri ) of OD 600 The value is 0.8 to 1.2.

[0011] In some embodiments, the crop straw includes at least one of corn straw, rice straw, and wheat straw.

[0012] In some embodiments, the inoculum includes at least one of livestock manure and activated sludge; And / or, the water includes sterile water.

[0013] In some embodiments, when the inoculum comprises livestock manure and activated sludge, the volume ratio of the livestock manure and activated sludge is 1:(0.8~1.2).

[0014] Thirdly, this application provides a method for preparing methane, including: Crop straw, inoculum, and water are mixed to obtain a liquid fermentation system; The combination of microorganisms as described in the first aspect is added to the liquid fermentation system to obtain the methane fermentation system as described in the second aspect, and methane is obtained by anaerobic fermentation.

[0015] In some embodiments, the anaerobic fermentation time is 15 to 20 days, and the fermentation temperature is 32 to 38 ℃.

[0016] This invention discloses a microbial combination, a methane fermentation system, and a preparation method. In this invention, during the acid production stage, the lignocellulose in crop straw is initially hydrolyzed into soluble sugars by natural hydrolytic bacteria in the inoculum. Subsequently, *Lactobacillus plantarum* ferments these soluble sugars, generating intermediate products such as lactic acid, acetic acid, ethanol, hydrogen, and carbon dioxide. Further, syntrophic oxidizing bacteria and other microorganisms in the inoculum convert the intermediate products such as lactic acid and ethanol into methanogenic precursors such as hydrogen and acetic acid. Then, during the methanogenesis stage, *Methanococcus methanogens* utilizes hydrogen and carbon dioxide for hydrogen-based methanogenesis, utilizes acetic acid for acetic-based methanogenesis, and / or utilizes methyl compounds (such as methanol and methylamine) for methyl-based methanogenesis. This avoids the accumulation of intermediate products and improves the substrate conversion efficiency and methane yield. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the fixed scope of the present invention.

[0018] It should be understood that the phrase "one embodiment" or "an embodiment" throughout the specification means that a specific feature, structure, or characteristic related to the embodiment is included in at least one embodiment of the invention. Therefore, "in one embodiment" or "in an embodiment" appearing throughout the specification do not necessarily refer to the same embodiment. Furthermore, these specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0019] Methane fermentation is a process that uses microorganisms to decompose organic matter into methane and carbon dioxide under anaerobic conditions. It can reduce environmental pollution by processing organic matter and generate renewable energy at the same time. It is suitable for decomposing various organic materials such as agricultural and forestry waste, human and animal excrement, and urban domestic waste.

[0020] This application provides a microbial strain combination for use in a methane fermentation system. This microbial strain combination includes *Lactobacillus plantarum* with accession number NRRL NO.B-14768. Lactiplantibacillus plantarum ) and Methanococcus ostreatus with accession number ATCC NO. 51582 ( Methanosarcina barkeri ).

[0021] In this embodiment, Lactobacillus plantarum ( Lactiplantibacillus plantarum As acidifying bacteria in anaerobic fermentation systems, *Methanococcus faecium* can convert organic matter into small-molecule acids and hydrogen precursors through metabolic activities. For example, it can utilize soluble sugars in the anaerobic fermentation system to produce large amounts of lactic acid through homolactic fermentation, lowering the environmental pH and creating a suitable acidifying environment. Simultaneously, its metabolic process also produces key intermediate products such as acetic acid, ethanol, hydrogen, and carbon dioxide. *Methanococcus faecium* (…) Methanosarcina barkeri As a broad substrate-utilizing methanogen, it can synthesize methane using hydrogen and carbon dioxide via a hydrogenotrophic pathway, directly produce methane by cleaving acetic acid molecules via an acetic acidotrophic pathway, and also produce methane via a methylotrophic pathway using methyl compounds such as methanol and methylamine.

[0022] Lactobacillus plantarum ( Lactiplantibacillus plantarum Purchased from the Agricultural Research Service Culture Collection (NRRL), accession number NRRLNO.B-14768; *Methanococcus faecium* (… Methanosarcina barkeri Purchased from the American Type Culture Collection (ATCC), accession number ATCC NO.51582.

[0023] In the following embodiments, Lactiplantibacillus plantarum Abbreviated as L. plantarum ,Will Methanosarcina barkeri Abbreviated as M. barkeri .

[0024] This application also provides a methane fermentation system, including crop straw, inoculum, microbial culture combination and sterile water.

[0025] In the methane fermentation system of this embodiment, the lignocellulose in crop straw is first initially hydrolyzed into soluble sugars by the natural hydrolytic bacteria in the inoculum, and then... L. plantarum These soluble sugars are fermented to produce intermediate products such as lactic acid, acetic acid, ethanol, hydrogen, and carbon dioxide. Further, symbiotic oxidizing bacteria and other microorganisms in the inoculum convert these intermediate products into methanogenic precursors such as hydrogen and acetic acid. Subsequently, in the methanogenesis stage... M. barkeri Hydrogen and carbon dioxide are used for hydrogenotrophic methanogenesis, acetic acid is used for acetic acidotrophic methanogenesis, and / or methyl compounds (such as methanol and methylamine) are used for methylotrophic methanogenesis. This avoids the accumulation of intermediate products and improves the substrate conversion efficiency and methane yield.

[0026] Methyl compounds such as methanol mainly originate from the microbial degradation of pectin components in crop straw, while substances such as methylamine mainly originate from the anaerobic decomposition of proteins and amino acids in crop straw.

[0027] In some embodiments, crop straw includes at least one of corn straw, rice straw, and wheat straw. In the methane fermentation system, at least one of corn straw, rice straw, and wheat straw serves as a fermentation raw material, rich in organic matter such as cellulose and hemicellulose, and can provide carbon source and energy for the entire anaerobic fermentation process.

[0028] In some embodiments, the inoculum includes at least one of livestock manure and activated sludge, which are rich in nitrogen sources and diverse natural microbial communities. For example, livestock manure contains livestock-derived microbial communities, while activated sludge includes a high concentration of hydrolytic bacteria and acid-producing bacteria. These natural microbial communities can promote the initial degradation of cellulose, hemicellulose, etc., thereby enhancing the metabolic activity of the methane fermentation system.

[0029] In some embodiments, when livestock manure and activated sludge are combined as inoculum, a dominant microbial community can be established, the fermentation environment optimized, and [the process can be carried out]... L. plantarum and M. barkeri Anaerobic fermentation provides energy and enhances the metabolic activity of the fermentation system, thereby improving the degradation efficiency and methane yield of crop straw.

[0030] In some embodiments, the inoculum includes livestock manure and activated sludge, with a volume ratio of livestock manure to activated sludge of 1:(0.8~1.2).

[0031] In this embodiment, the volume ratio of livestock manure to activated sludge is controlled within the range of 1:(0.8~1.2), so that the livestock and poultry-derived microbial community rich in livestock manure complements the hydrolytic bacteria and acidifying bacteria in activated sludge. This is beneficial for converting intermediate products such as alcohols generated in the acid production stage into substrates such as hydrogen and acetic acid. In this way, the risk of ammonia nitrogen inhibition caused by a slightly higher proportion of livestock manure can be avoided, and the acidification of the system caused by an excessive proportion of activated sludge can be prevented. This maintains the diversity of the microbial community in the fermentation system and improves the methane yield.

[0032] In some embodiments, in the methane fermentation system, the volume percentage of crop straw is 8% to 12%, the volume percentage of inoculum is 8% to 12%, the volume percentage of microbial combination is 1% to 3%, and the volume percentage of water is 73% to 83%.

[0033] In this embodiment, when the volume ratio of crop straw is controlled at 8% to 12%, it can ensure that there is a sufficient carbon source for the metabolic activities of microbial communities, while avoiding the problems of uneven mass transfer or system acidification caused by excessively high proportion of crop straw.

[0034] When the volume percentage of the inoculum is controlled at 8% to 12%, it can ensure that the methane fermentation system has a relatively rich and diverse microbial community and a relatively sufficient nitrogen source, which is conducive to accelerating the start-up of the methane fermentation system and improving the stability of the anaerobic fermentation process.

[0035] When the volume ratio of the bacterial strain combination is controlled at 1%~3%, it can enhance... L. plantarum and M. barkeri The leading role, while avoiding the influence of L. plantarum and M. barkeri The excessive addition of these two exogenous microbial agents disrupted the original ecological balance of the methane fermentation system.

[0036] When the volume ratio of water is controlled at 73% to 83%, the fluidity and mass transfer conditions of the methane fermentation system can be improved, which is conducive to full contact between crop straw, inoculum and microbial combination, providing a suitable liquid phase environment for fermentation reaction and increasing methane production.

[0037] For example, the proportions are: crop straw 10%, inoculum 10%, microbial culture combination 2%, and water 78%; or: crop straw 12%, inoculum 12%, microbial culture combination 3%, and water 73%; or: crop straw 9%, inoculum 12%, microbial culture combination 3%, and water 76%. Furthermore, to avoid introducing other contaminating microorganisms, the water in the methane fermentation system is sterile water.

[0038] In some embodiments, in the strain combination, L. plantarum and M.barkeri The volume ratio is 1:(0.8~1.2).

[0039] In this embodiment, when L. plantarum and M.barkeri When the volume ratio is controlled at 1:(0.8~1.2), a sufficient quantity can be ensured during the acid production stage. L. plantarum This promotes the decomposition of crop straw, thereby promoting substrate generation and providing a sufficient amount of substrate for the subsequent methanogenesis stage; at the same time, it can also provide sufficient substrate for the methanogenesis stage. M. barkeri Improve the understanding L. plantarum The utilization rate of substrates produced by decomposition increases methane production, while also preventing acid accumulation that could acidify the methane fermentation system and thus inhibit the fermentation rate.

[0040] In some embodiments, L. plantarum OD 600 The value is 0.8 to 1.2.

[0041] It should be noted that OD 600 Optical Density at 600 nm refers to the optical density value obtained by measuring a bacterial sample at a wavelength of 600 nm using a spectrophotometer. It is used to characterize the concentration or density of bacteria in the bacterial sample. The higher the optical density value, the more bacteria there are and the higher the concentration in the bacterial sample.

[0042] In this embodiment, when L. plantarum OD 600 A value between 0.8 and 1.2 can ensure vaccination. L. plants During the late logarithmic growth phase to the early stationary phase, this stage... L. plantarum Not only does it have abundant biomass, but it also has vigorous metabolic activity, enabling it to quickly initiate and perform hydrolysis and acidification, providing sufficient substrate for the methanogenesis stage.

[0043] In some embodiments, M.barkeri OD 600 The value is also controlled between 0.8 and 1.2 to ensure vaccination. M. barkers During the late logarithmic growth phase to the early stationary phase, it possesses high activity and biomass, enabling it to utilize resources relatively promptly and efficiently. L. plantarum The resulting substrates prevent acid accumulation from inhibiting the fermentation rate, thereby increasing the fermentation rate and methane yield.

[0044] This application also provides a method for preparing methane, comprising the following steps: mixing crop straw, inoculum, and water to establish a liquid fermentation system; continuing to add the above-mentioned bacterial strain combination to the liquid fermentation system to obtain a methane fermentation system, and performing anaerobic fermentation to obtain methane, wherein the anaerobic fermentation time is 15-20 days and the fermentation temperature is 32-38 ℃.

[0045] Among these measures, controlling the fermentation temperature within the range of 32~38 ℃ can provide for, among other things, fermentation. L. plantarum and M. barkers The microbial community provides suitable metabolic conditions, ensuring that various bacteria maintain high activity, thereby increasing the degradation rate of crop straw and methane production. At the same time, setting the fermentation time to 15-20 days provides sufficient time for the anaerobic fermentation process, which is conducive to the decomposition and transformation of crop straw by the microbial community, allowing the acid production and methanogenesis stages to be fully completed, thus increasing the methane yield.

[0046] The technical solution of the present invention will be further described below with reference to the embodiments.

[0047] Example 1 Using a 300 mL methane fermentation system as a baseline, corn stalks were weighed at volume ratios of 5%, 10%, 15%, 20%, and 30%, respectively. Activated sludge was measured at a volume ratio of 1:1 between corn stalks and activated sludge. The remaining portion was supplemented with sterile water to bring the total volume to 300 mL. After mixing all the above components evenly, the mixture was placed at a constant temperature of 35 °C for anaerobic fermentation for 28 days.

[0048] By comparing the effects of different corn stalk volume ratios on biogas volume and methane production, the amount of corn stalk added was optimized. The results are shown in Table 1.

[0049] Table 1

[0050] As shown in Table 1, in the methane fermentation system without added functional bacteria, when the amount of corn stalks added increased from 10% to 15%, a phenomenon occurred where the volume of biogas was high while the volume ratio of methane decreased. This is because excessive corn stalks provided an excessive amount of substrate for the natural hydrolytic and acid-producing bacteria in the methane fermentation system, causing their metabolic activity to become too vigorous. This resulted in the rapid production of large amounts of organic acids (such as acetic acid and propionic acid) and hydrogen / carbon dioxide in a short period. The rapid accumulation of these acidic products exceeded the metabolic capacity of the natural methanogens in the methane fermentation system, leading to a decrease in the pH value of the fermentation system. This, in turn, inhibited the activity of the natural methanogens, reducing their conversion efficiency for intermediate products such as acetic acid and hydrogen. Ultimately, this resulted in an increase in the proportion of carbon dioxide in the biogas and a relative decrease in the proportion of methane. Therefore, in this embodiment, the preferred volume ratio of corn stalks is 10%, achieving a balance between the amount of corn stalks and the amount of inoculum added.

[0051] Example 2 Using a 300 mL methane fermentation system as a baseline, corn stalks were weighed at a volume ratio of 10%, and the volume ratios of corn stalks to activated sludge were set at 9:1, 7:1, 3:1, 1:1, 1:3, 1:7, and 1:9 to measure the activated sludge. The remaining portion was supplemented with sterile water to bring the total volume to 300 mL. After thoroughly mixing all the components, the mixture was placed in a constant temperature environment of 35°C for anaerobic fermentation for 28 days.

[0052] By comparing the effects of different corn stalk to inoculum volume ratios on biogas volume and methane production, the optimal ratio of corn stalk to inoculum volume was achieved, and the results are shown in Table 2.

[0053] Table 2

[0054] Table 2 shows that in the methane fermentation system without added functional bacteria, the volume ratio of corn straw to activated sludge affects the balance between the substrate and the natural microbial community, thus determining the gas production efficiency. When the proportion of corn straw is too high (e.g., 9:1, 7:1), there is an excess of carbon source and insufficient natural hydrolytic and acid-producing bacteria, resulting in a large amount of corn straw that cannot be effectively hydrolyzed, leading to insufficient substrate utilization and low gas production and methane content. As the proportion of activated sludge increases (e.g., 3:1), the hydrolytic bacteria gradually become sufficient, deepening the degradation of corn straw and steadily improving gas production performance. The optimal balance is reached at a 1:1 ratio, where the hydrolytic and acid-producing bacteria in the activated sludge can fully degrade the straw and maintain a stable acid-base environment, increasing the methane production efficiency of the natural methanogenic bacteria. When the proportion of activated sludge is further increased (e.g., from 1:3 to 1:9), although the hydrolytic bacteria and acid-producing bacteria communities are sufficient, the corn stalks available for degradation are relatively insufficient. The hydrolytic bacteria and acid-producing bacteria communities have reduced metabolic activity due to the lack of sufficient carbon sources, which in turn leads to a decrease in gas production efficiency and methane synthesis capacity.

[0055] Example 3 Based on a methane fermentation system with a total volume of 300 mL, corn stalks were weighed at a volume ratio of 10%, and activated sludge was measured at a volume ratio of 1:1 (corn stalks to activated sludge). Three groups of microbial cultures were then established: the first group consisted of 1% (corn stalks by volume). L. plantarum The second group has a volume percentage of 1%. M.barkeri The third group has a volume percentage of 0.5%. L. plantarum With a volume percentage of 0.5% M.barkeri The resulting microbial culture combination also included a control group, with the remaining components supplemented with sterile water to a total volume of 300 mL. After thoroughly mixing all the components, the mixture was placed in a constant temperature environment of 35°C for anaerobic fermentation for 28 days.

[0056] By comparing the effects of different bacterial strains on biogas volume and methane production, the bacterial strains were optimized, and the results are shown in Table 3.

[0057] Table 3

[0058] Table 3 shows that adding corn stalks and activated sludge... L. plantarum and M.barkeri At that time, the volume of biogas and the proportion of methane in the biogas volume increased to 1750 mL and 13.4%, respectively. However, only adding... Fifty plants The proportion of biogas volume to methane volume in the control group was lower than that in the control group, mainly because: L. plantarum Metabolic activity disrupted the microecological balance of the methane fermentation system. L. plantarum As a front-end acidifying bacterium, it converts the sugars produced by the hydrolysis of crop straw into lactic acid, causing the pH value of the fermentation system to drop rapidly in a short period of time. Under this acidic environment, the activity of the original natural microbial community in the inoculum (such as activated sludge or livestock manure) is inhibited, which in turn leads to a reduction in the volume of biogas and methane.

[0059] Example 4 Based on a methane fermentation system with a total volume of 300 mL, corn stalks were weighed out at a volume ratio of 10%, activated sludge was measured out at a volume ratio of 1:1 (corn stalks to activated sludge), and microbial culture combinations were measured out at a volume ratio of 1%. These combinations were then set up in the microbial culture system. L. plantarum and M. barkeri The volume ratios of the components were 1:4, 1:3, 1:1, 3:1, and 4:1, with the remainder supplemented with sterile water to a total volume of 300 mL. After thoroughly mixing all the components, the mixture was placed in a constant temperature environment of 35°C for anaerobic fermentation for 28 days.

[0060] By comparing different L. plantarum and M. barkeri The effect of volume ratio on biogas volume and methane production was investigated to optimize the proportion of each strain in the strain combination. The results are shown in Table 4.

[0061] Table 4

[0062] As shown in Table 4, when L. plantarum When relatively too little, L. plantarum If too little substrate is produced, the amount of substrate provided will be relatively insufficient. M. barkeri The inability to fully utilize the biogas and methane volumes results in relatively small proportions of both; however, an optimal balance is achieved when the ratio reaches 1:1. L. plantarum The resulting substrate can just be M. barkers When fully converted to methane, both the total volume of biogas and the proportion of methane volume reach their peak, i.e., when... L. plantarum and M. barkeri When the volume ratio of biogas to methane is 1:1, the methane fermentation system exhibits superior gas production performance, with a biogas volume reaching 1750 mL and a relatively high methane volume ratio of 13.7% in the biogas volume; however, as... L. plants Continued increase led to L. plantarum The generated substrate cannot be fully utilized, which increases the acidity of the system, and in turn inhibits... M. barkeri The metabolic activity of the biogas and the proportion of methane volume decreased.

[0063] Example 5 Based on a methane fermentation system with a total volume of 300 mL, corn stalks were weighed at a volume ratio of 10%, and activated sludge was measured at a volume ratio of 1:1 (corn stalks to activated sludge). Inoculum combinations were then measured at volume percentages of 0.5%, 1%, 2%, and 3%, respectively. Each inoculum combination included a 1:1 volume ratio of... L. plantarum and M. barkers The remaining portion was supplemented with sterile water to bring the total volume to 300 mL. After thoroughly mixing all the above components, the mixture was placed in a constant temperature environment of 35°C for anaerobic fermentation for 28 days.

[0064] By comparing the effects of different strain combinations on biogas volume and methane yield, the volume ratio of strain combinations in the methane fermentation system was optimized. The results are shown in Table 5.

[0065] Table 5

[0066] Table 5 shows that when the volume percentage of the microbial combination increased from 0.5% to 2%, the acid production and methanogenesis stages transitioned smoothly, and substrate conversion was sufficient. Therefore, biogas production and methane content continued to increase to their peak values. However, when the amount of microbial combination added further increased to 3%, excessive exogenous microorganisms caused the acid production or methanogenesis stages to proceed too rapidly, leading to the accumulation of metabolic byproducts and disrupting the microbial ecological balance of the system. This resulted in a slight decrease in biogas production efficiency and methane yield. When the volume percentage of the microbial combination was 2%, the methane fermentation system exhibited optimal biogas production performance, with a biogas volume of 1875 mL and the highest methane volume percentage (14.7%).

[0067] Example 6 Based on a 300 mL methane fermentation system, corn stalks were weighed at a volume ratio of 10% to prepare three inoculum groups: the first group consisted of 10% livestock manure; the second group consisted of 10% activated sludge; and the third group consisted of both 5% livestock manure and 5% activated sludge. Additionally, a microbial culture combination was measured at a volume ratio of 1%, comprising a 1:1 ratio of... L. plantarum and M. barkeri The remaining portion was supplemented with sterile water to bring the total volume to 300 mL. After thoroughly mixing all the above components, the mixture was placed in a constant temperature environment of 35°C for anaerobic fermentation for 28 days.

[0068] By comparing the effects of different inoculums on biogas volume and methane production, the selection of inoculum types was optimized, and the results are shown in Table 6.

[0069] Table 6

[0070] Table 6 shows that when livestock manure and activated sludge are mixed as inoculum, the methane fermentation system exhibits superior gas production performance, with a biogas volume of 1870 mL and a methane volume ratio of 14.7%. This is because activated sludge is rich in hydrolytic and acid-producing bacteria, which can promote the degradation of lignocellulose in crop straw, while livestock manure provides a rich nitrogen source and livestock-derived microbial community. When the two are mixed in an appropriate ratio, the microbial community in the activated sludge is responsible for decomposing the straw into fermentable substrate, while the livestock manure supplements the fermentation system with nitrogen to maintain the optimal carbon-nitrogen ratio. Furthermore, its inherent livestock-derived microbial community can further promote the decomposition of intermediate products generated during the acid-producing stage.

[0071] Example 7 Based on a 300 mL methane fermentation system, corn stalks were weighed at a 10% volume ratio, and a mixed inoculum consisting of livestock manure and activated sludge was measured at a 10% volume ratio. The volume ratios of livestock manure to activated sludge were set at 9:1, 7:3, 1:1, 3:7, and 1:9, respectively. Simultaneously, a microbial culture combination was measured at a 1% volume ratio, consisting of [missing information - likely a specific microbial composition]. L. plantarum and M. barkeri The remaining portion was supplemented with sterile water to bring the total volume to 300 mL. After thoroughly mixing all the above components, the mixture was placed in a constant temperature environment of 35°C for anaerobic fermentation for 28 days.

[0072] By comparing the effects of different livestock manure to activated sludge volume ratios on biogas volume and methane production, the optimal livestock manure to activated sludge volume ratio was achieved. The results are shown in Table 7.

[0073] Table 7

[0074] As shown in Table 7, the effect of the mixing ratio of livestock manure and activated sludge on the fermentation effect first increases and then decreases, reaching a peak at 1:1. When the ratio is 9:1, the fermentation system is close to pure livestock manure, with sufficient nitrogen source but fewer hydrolyzing and acidifying bacteria, resulting in insufficient degradation of crop straw. When adjusted to 7:3, the hydrolyzing and acidifying bacteria of activated sludge begin to appear, improving gas production performance. At 1:1, the two achieve the best balance, with livestock manure providing nitrogen source and livestock-derived microbial flora, and activated sludge playing a hydrolysis role. When the ratio further leans towards activated sludge (3:7 to 1:9), although the hydrolysis capacity continues to be enhanced, the nutritional supplementation function of livestock manure gradually weakens, causing the methane fermentation system to gradually return to the characteristics of pure activated sludge. The synergistic effect of livestock manure and activated sludge gradually diminishes, causing the proportion of biogas volume and methane volume to slowly decline from the peak.

[0075] It should be noted that the various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0076] Although alternative embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make further changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the alternative embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0077] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used merely to distinguish one entity from another, and do not necessarily require or imply any such actual relationship or order between these entities. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that an article or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such an article or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the article or terminal device that includes that element.

[0078] The technical solution provided by the present invention has been described in detail above. Specific examples have been used to illustrate the principle and implementation of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation and application scope based on the principle and implementation of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A combination of microbial strains, characterized in that, Including Lactobacillus plantarum with accession number NRRL NO.B-14768 ( Lactiplantibacillus plantarum ) and Methanococcus ostreatus with accession number ATCC NO. 51582 ( Methanosarcina barkeri ).

2. A methane fermentation system, characterized in that, It includes crop straw, inoculum, water, and the microbial combination as described in claim 1.

3. The methane fermentation system according to claim 2, characterized in that, In the fermentation system, the volume percentage of crop straw is 8% to 12%, the volume percentage of inoculum is 8% to 12%, the volume percentage of the microbial combination is 1% to 3%, and the volume percentage of water is 73% to 83%.

4. The methane fermentation system according to claim 3, characterized in that, In the strain combination, Lactobacillus plantarum ( Lactiplantibacillus plantarum ) and methanogenic octopus ( Methanosarcina barkeri The volume ratio of ) is 1:(0.8~1.2).

5. The methane fermentation system according to claim 4, characterized in that, The plant lactobacillus ( Lactiplantibacillus plantarum OD 600 The value is 0.8~1.2; And / or, the methanogenic octopus ( Methanosarcina barkeri OD 600 The value is 0.8~1.

2.

6. The methane fermentation system according to claim 2, characterized in that, The crop straw includes at least one of corn straw, rice straw, and wheat straw.

7. The methane fermentation system according to claim 2, characterized in that, The inoculum includes at least one of livestock manure and activated sludge; And / or, the water includes sterile water.

8. The methane fermentation system according to claim 7, characterized in that, When the inoculum includes livestock manure and activated sludge, the volume ratio of the livestock manure and activated sludge is 1:(0.8~1.2).

9. A method for preparing methane, characterized in that, include: Crop straw, inoculum, and water are mixed to obtain a liquid fermentation system; Add the strain combination as described in claim 1 to the liquid fermentation system to obtain the methane fermentation system as described in any one of claims 2 to 8, and carry out anaerobic fermentation to obtain methane.

10. The method for preparing methane according to claim 9, characterized in that, The anaerobic fermentation process takes 15 to 20 days and is carried out at a temperature of 32 to 38 ℃.

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