Anaerobic inoculant and method for improving methane conversion rate of medium-high temperature anaerobic digestion cellulose

Abstract

The invention discloses an anaerobic bacterial agent and a method for improving the methane conversion rate of medium-high temperature anaerobic digestion cellulose. The compound anaerobic microorganism liquid bacterial agent is prepared from the following raw materials: clostridium cellobiose producing bacteria, clostridium alpinum bacteria and clostridium amylolyticum, and the compound anaerobic microorganism liquid bacterial agent is prepared from the following raw materials: clostridium cellobiose producing bacteria, clostridium alpinum bacteria, clostridium amylolyticum bacteria, and the compound anaerobic microorganism liquid bacterial agent is prepared from the following raw materials: the clostridium cellobiose producing bacteria, the clostridium alpinum bacteria and the clostridium amylolyticum bacteria, the clostridium alpinum bacteria and the clostridium amylolyticum bacteria. The microbial inoculum is formed by compounding three commercially available synergistic bacteria, the strains are convenient to obtain, the culture process is simple and easy to implement, and when the microbial inoculum is applied to medium-high temperature (40-50 DEG C) anaerobic digestion, the conversion rate of cellulose to methane (greater than 15%) can be remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental microorganisms, and in particular relates to an anaerobic bacterial agent and a method for improving the methane conversion rate of cellulose by medium- and high-temperature anaerobic digestion. Background Art

[0002] Converting biomass waste into biogas energy is a key path for resource recycling. The current mainstream anaerobic fermentation raw materials (crop straw, livestock and poultry manure, food waste, etc.) are all based on cellulose / hemicellulose as the main components. However, because cellulose and hemicellulose are usually wrapped with a layer of complex, dense and extremely difficult to degrade lignin components, it hinders the direct contact of extracellular hydrolases with them, ultimately leading to defects such as low hydrolysis rate and methane yield. Therefore, how to improve the conversion of cellulosic substances is a key technical bottleneck problem that the anaerobic biogas production industry of biomass waste urgently needs to break through.

[0003] At present, mesophilic fermentation and thermophilic fermentation are commonly used in anaerobic biogas production projects. Among them, the temperature range of mesophilic fermentation is 30-40°C, and the temperature range of thermophilic fermentation is above 50°C. Within their respective temperature ranges, the most suitable temperature range for mesophilic fermentation is 35-40°C, and the most suitable temperature range for thermophilic fermentation is 50-55°C. However, it is worth noting that between these two temperature zones, there is also a transition temperature range of 40-50°C for mesophilic and thermophilic fermentation. Research and engineering applications have shown that in the mesophilic and thermophilic transition range, mesophilic and thermophilic bacteria can coexist and cooperate, and the reaction efficiency is superimposed, which can significantly improve the efficiency and stability of anaerobic biogas production from biomass.

[0004] However, existing technologies lack research on medium- and high-temperature anaerobic biogas-producing microorganisms suitable for the medium- and high-temperature transition temperature range, and there is also a lack of anaerobic agent products suitable for this temperature range. Therefore, the current urgent task is to build a complete technical solution: on the one hand, it is urgent to systematically discover and identify specialized microbial resources that can efficiently degrade cellulose and simultaneously drive methanogenesis under medium- and high-temperature conditions; on the other hand, based on these core bacterial resources, develop standardized, highly active medium- and high-temperature anaerobic microbial functional agents and establish a corresponding innovative application technology system. Summary of the Invention

[0005] The present invention aims to provide an anaerobic bacterial agent and method specifically for improving the methane conversion rate of cellulose in medium- and high-temperature anaerobic digestion, so as to solve the problems of difficult cellulose degradation and low volumetric gas production rate in existing biomass waste, and at the same time partially establish a medium- and high-temperature biomass high-efficiency anaerobic biogas production agent and its supporting innovative technology system.

[0006] The technical solutions specifically adopted in the present invention are as follows:

[0007] In a first aspect, the present invention provides an anaerobic bacterial agent for improving the methane conversion rate of cellulose in medium- and high-temperature anaerobic digestion, wherein the medium- and high-temperature temperature range is 40 to 50°C, and the anaerobic bacterial agent is a neutral composite anaerobic microbial liquid agent prepared from a cellobiose-producing Clostridium (Ruminiclostridium cellulolyticum) bacterial liquid, a Clostridium lituseburense bacterial liquid, and a Clostridium amylolyticum bacterial liquid.

[0008] As a preferred embodiment of the first aspect, the Ruminiclostridium cellulolyticum, Clostridium lituseburense and Clostridium amylolyticum are all purchased from the China Anaerobic Culture Collection Center, with the accession numbers being CCAM 52, CCAM 1085 and CCAM 1591, respectively.

[0009] As a preferred embodiment of the first aspect, the volume ratio of the cellobiose-producing Clostridium (Ruminiclostridium cellulolyticum) bacterial solution, the Ivory Coast Clostridium (Clostridium lituseburense) bacterial solution and the amylolyticum (Clostridium amylolyticum) bacterial solution in the anaerobic bacterial agent is (2-3): (3-4): (3-4), and the anaerobic bacteria content in each bacterial solution is not less than 200 million / ml.

[0010] As a preference for the first aspect above, the volume ratio of the bacterial solution of Ruminiclostridium cellulolyticum, the bacterial solution of Clostridium lituseburense and the bacterial solution of Clostridium amylolyticum in the bacterial agent is 2:3:3.

[0011] As a preference for the first aspect above, the pH of the anaerobic agent is 6.8-7.2.

[0012] In a second aspect, the present invention provides a method for preparing an anaerobic agent for improving the methane conversion rate of cellulose during medium- and high-temperature anaerobic digestion, comprising:

[0013] S1: Inoculate Ruminiclostridium cellulolyticum, Clostridium lituseburense, and Clostridium amylolyticum into their respective liquid culture media, expand the culture in an anaerobic environment until the anaerobic bacteria count is not less than 200 million cells / mL, and obtain three bacterial cultures;

[0014] S2: The three bacterial solutions were mixed in an anaerobic atmosphere at a volume ratio of (2-3):(3-4):(3-4), and the pH was adjusted to 6.8-7.2 with a buffer solution to obtain a composite anaerobic microbial liquid agent.

[0015] As a preferred embodiment of the second aspect, the buffer solution is potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer solution.

[0016] As a preferred embodiment of the second aspect, the expanded culture needs to be cultured at a temperature of 44-46° C. and with shaking at 160-200 rpm for 48-72 hours.

[0017] In a third aspect, the present invention provides a method for improving the methane conversion rate of cellulose in medium- and high-temperature anaerobic digestion, which is performed by adding the anaerobic bacterial agent for improving the methane conversion rate of cellulose in medium- and high-temperature anaerobic digestion as described in any of the schemes in the first aspect above to an anaerobic sludge system containing methanogens and cellulose, mixing them evenly, and conducting anaerobic fermentation in a medium- and high-temperature temperature range of 40 to 50°C. The cellulose is gradually degraded into primary volatile fatty acids through the complementary cellulase clusters of the three anaerobic bacteria, thereby improving the methane conversion rate of cellulose.

[0018] As a preferred embodiment of the third aspect, the dosage of the anaerobic bacteria agent is 1-10% of the volume of the anaerobic sludge, and the temperature range of the anaerobic fermentation is 43-47°C.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The strains selected in the present invention can grow and metabolize rapidly in a medium-high temperature (40-50°C, preferably 43-47°C) environment, and the content of a single bacterial colony is not less than 200 million per milliliter;

[0021] (2) The present invention can achieve step-by-step degradation of cellulose through the complementary cellulase clusters among the three strains, significantly improving the cellulose-to-methane conversion rate of anaerobic digestion (greater than 15%);

[0022] (3) The bacterial agent of the present invention is constructed by combining three commercially available synergistic bacteria. The bacterial strains are easy to obtain, the culture process is simple and easy, and the preparation is convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Cumulative methane production in the cellulose anaerobic digestion test of the composite microbial agent of the present application at medium-high temperature (45℃).

[0024] Figure 2 Cumulative methane production in the cellulose anaerobic digestion test of the composite microbial agent of the present application at medium-high temperature (45℃). DETAILED DESCRIPTION

[0025] The present application will be further described and illustrated in conjunction with the accompanying drawings and specific embodiments. The technical features of each embodiment of the present application can be combined accordingly without conflict, provided that there is no conflict. The test materials used in the following examples are commercially available products, unless otherwise specified.

[0026] In order to avoid ambiguity, the temperature range corresponding to medium-high temperature in the present application is defined as 40-50℃.

[0027] The present application provides a composite anaerobic microbial liquid agent specially used for improving the methane conversion rate of cellulose in medium-high temperature anaerobic digestion (43-47℃), which is prepared from Ruminiclostridium cellulolyticum bacterial liquid, Clostridium lituseburense bacterial liquid and Clostridium amylolyticum bacterial liquid, and is a neutral pH composite anaerobic microbial liquid agent.

[0028] In an embodiment of the present invention, the three bacterial agents constituting the anaerobic bacterial agent are all commercially available bacterial agents, among which Ruminiclostridium cellulolyticum is purchased from the China Anaerobic Microorganism Culture Collection Administration Center with a preservation number of CCAM No. 52; Clostridium lituseburense is purchased from the China Collection of Anaerobic Microorganisms (CCAM) with a preservation number of CCAM No. 1085; and Clostridium amylolyticum is purchased from the China Collection of Anaerobic Microorganisms (CCAM) with a preservation number of CCAM No. 1591. These three selected bacterial species can grow and metabolize rapidly in a medium to high temperature environment (40-50° C., preferably 43-47° C.). Of course, the bacterial agent purchased by CCAM is only one implementation method. In theory, other commercially available products, as long as they belong to the same strain and have the same characteristics, can replace CCAM's bacterial agent products and be used to prepare the above-mentioned composite anaerobic microbial liquid bacterial agent of the present invention.

[0029] In order to ensure the effect after the addition of the inoculum, in this embodiment, the bacterial content in the composite anaerobic microbial liquid inoculum cannot be too low. Each bacterial solution that makes up the anaerobic inoculum can be expanded to reach a certain bacterial content, and then used to prepare the composite anaerobic microbial liquid inoculum. Preferably, the anaerobic bacteria content in each bacterial solution that makes up the composite anaerobic microbial liquid inoculum should be no less than 200 million cells / ml. In addition, the ratio of the three bacterial solutions in the composite anaerobic microbial liquid inoculum can be optimized and adjusted according to the actual effect. After experimental optimization, the mixing volume ratio of the cellulolytic bacteria solution of Ruminiclostridium cellulolyticum, the Ivory Coast Clostridium (Clostridium lituseburense) and the amylolytic bacteria solution during preparation is 2-3:3-4:3-4, and more preferably 2:3:3.

[0030] In addition, the pH of the composite anaerobic microbial liquid inoculum should be adjusted to neutral, which can be achieved by using potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffers. The final pH is preferably 6.8 to 7.2, and optimally 7.0.

[0031] In addition, the present invention also provides a method for preparing the above-mentioned composite anaerobic microbial liquid inoculant, the specific process of which is as follows:

[0032] S1: respectively inoculate Ruminiclostridium cellulolyticum, Clostridium lituseburense and Clostridium amylolyticum into respective liquid culture medium, and expand culture in an anaerobic environment to an anaerobic bacteria content of not less than 200 million per milliliter, to obtain three kinds of bacterial liquids, respectively, Ruminiclostridium cellulolyticum bacterial liquid, Clostridium lituseburense bacterial liquid and Clostridium amylolyticum bacterial liquid.

[0033] The above expansion culture can be carried out in an anaerobic bottle, that is, the liquid culture medium is placed in the anaerobic bottle for culture after inoculation of the corresponding bacterial agent. The expansion culture can be carried out under the following conditions: 44-46℃ temperature, 160-200rpm oscillation for 48-72h, preferably 45℃ temperature, 180rpm oscillation for 48h.

[0034] S2: The three kinds of bacterial liquids are mixed in an anaerobic atmosphere at a volume ratio of (2-3):(3-4):(3-4), and the pH is adjusted to 6.8-7.2 with a buffer solution to obtain a composite anaerobic microbial liquid bacterial agent.

[0035] Preferably, the anaerobic atmosphere is 100% N2 atmosphere or 80% N2 / 20% CO2 atmosphere, the buffer solution is preferably a buffer solution composed of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and the final pH is preferably 7.0.

[0036] It should be noted that each of the three different bacterial agents needs to have a corresponding liquid culture medium, and the specific culture medium formula can be adjusted and optimized according to the growth needs of the bacterial species.

[0037] In addition, the present application also provides a method for improving the cellulose conversion methane rate of mesophilic and thermophilic anaerobic digestion by using the above-mentioned anaerobic bacterial agent, which is as follows:

[0038] The bacterial agent is taken in a proportion of 1-10% (preferably 5%) of the total volume of anaerobic sludge, and the above-mentioned anaerobic bacterial agent is directly added to an anaerobic sludge system containing methanogenic bacteria and cellulose, and stirred uniformly, and anaerobic fermentation is carried out in a mesophilic and thermophilic temperature range of 40-50℃ (preferably 43-47℃), and cellulose is gradually degraded to primary volatile fatty acids by complementary cellulase clusters of the three kinds of anaerobic bacteria, so as to improve the cellulose conversion methane rate.

[0039] The anaerobic fermentation process can be carried out in an anaerobic digestion tank. In the anaerobic fermentation process, the cellulose is gradually degraded into primary volatile fatty acids by the complementary cellulase clusters of the three bacteria which still have high specific activity under medium-high temperature conditions, so as to improve the cellulose conversion rate to methane. The specific complementary cellulase clusters are as follows: Ruminiclostridium cellulolyticum provides endo-cellulase, Clostridium lituseburense provides cellobiohydrolase, and Clostridium amylolyticum provides starch side chain debunching enzyme.

[0040] It should be noted that the cellulose in the anaerobic sludge system in the present application can be externally added or self-provided by the sludge system. The methanogens are widely present in the anaerobic sludge system, especially in the sludge of anaerobic biogas production projects and the sludge of kitchen waste treatment projects, and the content is higher, and generally no additional addition is required. Therefore, the composite anaerobic microbial liquid inoculant of the present application is especially suitable for use in anaerobic biogas production projects and kitchen waste treatment projects.

[0041] The preparation of the above-mentioned anaerobic bacterial agent and the application effect of the present application will be shown through multiple examples.

[0042] Example 1

[0043] In this embodiment, a composite anaerobic microbial liquid inoculant for improving the cellulose conversion rate to methane in medium-high temperature (40-50℃) anaerobic digestion is prepared, which is as follows:

[0044] S1: Ruminiclostridium cellulolyticum, Clostridium lituseburense and Clostridium amylolyticum are respectively inoculated into anaerobic bottles containing corresponding liquid medium at 2.5% v / v, and cultured at a temperature of 45℃ and 180rpm oscillation for 48h to realize the expansion culture, so as to obtain Ruminiclostridium cellulolyticum bacterial liquid, Clostridium lituseburense bacterial liquid and Clostridium amylolyticum bacterial liquid.

[0045] In this embodiment, the corresponding liquid medium used for the expansion culture of the three bacteria is as follows:

[0046] The medium formula of Ruminiclostridium cellulolyticum is as follows: ammonium sulfate 1.30 g, potassium dihydrogen phosphate 1.50 g, potassium phosphate dibasic trihydrate 2.90 g, yeast extract 2.00 g, magnesium chloride hexahydrate 0.20 g, calcium chloride dihydrate 75.00 mg, cellobiose 6.00 g, sodium carbonate 1.50 g, L-cysteine hydrochloride 0.50 g, 0.1% resazurin sodium solution 0.50 mL, trace element solution 1.00 mL, and sterile water 1.00 L;

[0047] The medium formula of Clostridium lituseburense is as follows: beef extract powder 3.00 g, peptone 10.00 g, yeast extract 5.00 g, sodium phosphate dibasic 4.00 g, glucose 1.50 g, soluble starch 0.5 g, L-cysteine hydrochloride 0.50 g, 0.1% resazurin sodium solution 0.50 mL, anticoagulant lysed horse blood (sterile) 50.00 mL, and sterile water 1.00 L;

[0048] The medium formula of Clostridium amylolyticum is as follows: peptone 10.00 g, yeast extract 5.00 g, glucose 1.00 g, L-cysteine hydrochloride 0.50 g, 0.1% resazurin sodium solution 0.50 mL, and sterile water 1.00 L;

[0049] The formula of the trace element solution used in each medium is as follows: ferrous sulfate heptahydrate 1.00 g, zinc sulfate heptahydrate 1.00 g, boric acid 6.00 mg, manganese sulfate monohydrate 150.00 mg, copper sulfate pentahydrate 3.00 mg, cobalt chloride hexahydrate 190.00 mg, nickel chloride hexahydrate 24.00 mg, sodium molybdate dihydrate 36.00 mg, sodium tungstate dihydrate 15.00 mg, potassium aluminum sulfate dodecahydrate 10.00 mg, nitrilotriacetic acid 1.50 g, and sterile water 1.00 L.

[0050] The total number of bacteria in the three kinds of bacterial liquid obtained after expansion culture was detected under a microscope by a hemocytometer method, and the anaerobic bacterial content of Ruminiclostridium cellulolyticum liquid, Clostridium lituseburense liquid, and Clostridium amylolyticum liquid was measured to be 3.46×10 11 / mL, 3.01×10 11 / mL, and 2.91×10 11 / ml, meeting the requirement that the anaerobic bacteria content in each anaerobic bacterial solution is not less than 200 million / ml.

[0051] The total bacterial count of each bacterial solution prepared in S1 was detected by a hemocytometer under a microscope, and the anaerobic bacteria content in each anaerobic bacterial solution was found to be no less than 200 million per milliliter.

[0052] S2: The three bacterial liquids prepared in S1 were mixed under anaerobic conditions (80% N2 / 20% CO2 atmosphere) at a volume ratio of 2:3:3 for Ruminiclostridium cellulolyticum, Clostridium lituseburense, and Clostridium amylolyticum, and the pH was adjusted to 7.0 with potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer to obtain a composite anaerobic microbial liquid agent.

[0053] Example 2

[0054] In this example, the composite anaerobic microbial liquid inoculum prepared in Example 1 was used in anaerobic fermentation of cellulose to study methane production and cellulose-to-methane conversion rate, as follows:

[0055] (1) Control experiment of microbial agent application

[0056] XW group (microbial agent addition group): The composite anaerobic microbial liquid microbial agent prepared in Example 1 was directly added to a 500 mL anaerobic digestion bottle containing 380 mL of mesophilic anaerobic sludge (from the Tianziling restaurant waste treatment project in Hangzhou, which is rich in methanogens) at a concentration of 20 mL based on the total volume of the anaerobic sludge liquid (400 mL). 5 g of microcrystalline cellulose was added and the mixture was stirred continuously. The cells were cultured in a water bath at 45°C (representing medium-high temperature, marked as XW45 group) and 35°C (representing medium temperature, marked as XW35 group), respectively. Each treatment group was replicated three times.

[0057] CK group (control group): Sterile water was added directly at 5% (20 mL) of the total liquid volume (400 mL) into a 500 mL anaerobic digestion bottle containing 380 mL of the same mesophilic anaerobic sludge as the XW group, and 5 g of microcrystalline cellulose was added. The mixture was stirred continuously and cultured in a water bath at 45°C (representing medium-high temperature, marked as CK45 group) and 35°C (representing medium temperature, marked as CK35 group), respectively. Each treatment group was replicated three times.

[0058] The daily methane production of the XW45, XW35, CK45, and CK35 groups was recorded every day using the drainage gas collection method using a dedicated Bichen anaerobic fermentation device. The experiment was terminated three days after all treatment groups stopped producing gas, and the cumulative methane production was calculated.

[0059] (2) Sample testing method

[0060] Sample pretreatment: The sample after the reaction is terminated is passed through a freeze dryer to remove moisture and obtain a freeze-dried sample.

[0061] Determination of cellulose content in materials (Fan's method): Weigh X0 g of a freeze-dried sample (passed through a 40-mesh sieve) and place it in a beaker. Add an acidic detergent (2% hexadecyltrimethylammonium bromide), anhydrous sodium sulfate, and decahydronaphthalene. Boil and maintain a slight boil for 60 min. Filter the hot sample through a glass crucible (X1 g). Rinse the crucible and filter residue repeatedly with boiling water until the filtrate is neutral. Rinse the filter residue with a small amount of acetone until the filtrate is colorless, and then remove all the acetone. Dry the crucible in a 105°C oven, cool it in a desiccator, and weigh it to a constant weight (X2 g). The residue is acid detergent fiber (X2 - X1 g). Add 72% sulfuric acid to the acid detergent fiber and digest it at 20°C for 3 h. Rinse it with deionized water and filter it until the filtrate is neutral. Dry the filter residue, cool it, and weigh it (X3 g). The cellulose content is (X2 - X1 - X3) / X0 × 100%.

[0062] (3) Test results

[0063] The experiment lasted for 21 days. The CK35 and CK45 groups stopped producing gas on the 10th day, and the other experimental groups stopped producing gas on the 18th to 20th day. The cumulative methane production of each experimental group is shown in Figure 1 .Depend on Figure 1 As can be seen, the experimental group with the highest cumulative methane production was the XW45 group, with an average of 802.6 mL, significantly higher than the control groups of CK35 (424.0 mL) and CK45 (498.0 mL), indicating that the addition of the inoculum can effectively increase methane production during medium- and high-temperature anaerobic digestion. However, the XW35 group produced zero methane, indicating that the inoculum is only suitable for medium- and high-temperature anaerobic digestion.

[0064] The cellulose conversion to methane rate of each experimental group is shown in Figure 2 .Depend on Figure 2 It can be seen that the XW45 group had the highest cellulose to methane conversion rate, reaching 53.69%, which was significantly higher than the 28.52% of the control group CK35 and 34.62% of CK45, indicating that the addition of bacterial agents can effectively improve the cellulose to methane conversion rate of medium and high temperature anaerobic digestion.

[0065] The mechanism of the improvement effect shown in this example was explored, and it was found that this was because during use of the bacterial agent of the present invention, the complementary cellulase clusters of three bacteria still had high specific activity under medium and high temperature conditions, thereby achieving the gradual degradation of cellulose into primary volatile fatty acids, thereby improving the cellulose to methane conversion rate. The complementary cellulase clusters are as follows: Ruminiclostridium cellulolyticum provides cellulase endonuclease, Clostridium lituseburense provides cellobiohydrolase, and Clostridium amylolyticum provides starch side chain debranching enzyme.

[0066] The embodiment described above is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

Claims

1. An anaerobic agent for improving the methane conversion rate of cellulose during medium-high temperature anaerobic digestion, wherein the medium-high temperature range is 40-50°C, characterized in that: The anaerobic bacterial agent is a neutral composite anaerobic microbial liquid bacterial agent prepared from a bacterial solution of Ruminiclostridium cellulolyticum, a bacterial solution of Clostridium lituseburense and a bacterial solution of Clostridium amylolyticum.

2. The anaerobic bacterial agent for improving the methane conversion rate of cellulose during medium- and high-temperature anaerobic digestion according to claim 1, characterized in that: The Ruminiclostridium cellulolyticum, Clostridium lituseburense and Clostridium amylolyticum were all purchased from the China Anaerobic Culture Collection Center, with the collection numbers being CCAM 52, CCAM 1085 and CCAM 1591, respectively.

3. The anaerobic bacterial agent for improving the methane conversion rate of cellulose during medium- and high-temperature anaerobic digestion according to claim 1, characterized in that: The volume ratio of the cellobiose-producing Clostridium (Ruminiclostridium cellulolyticum) bacterial solution, the Ivory Coast Clostridium (Clostridium lituseburense) bacterial solution and the amylolyticum Clostridium (Clostridium amylolyticum) bacterial solution in the anaerobic bacterial agent is (2-3): (3-4): (3-4), and the anaerobic bacteria content in each bacterial solution is not less than 200 million per milliliter.

4. The anaerobic bacterial agent for improving the methane conversion rate of cellulose during medium- and high-temperature anaerobic digestion according to claim 3, characterized in that: The volume ratio of the bacterial solution of Ruminiclostridium cellulolyticum, the bacterial solution of Clostridium lituseburense and the bacterial solution of Clostridium amylolyticum in the bacterial agent is 2:3:

3.

5. The anaerobic bacterial agent for improving the methane conversion rate of cellulose during medium- and high-temperature anaerobic digestion according to claim 1, characterized in that: The pH of the anaerobic bacterial agent is 6.8-7.

2.

6. A method for preparing an anaerobic agent for improving the methane conversion rate of cellulose during medium- and high-temperature anaerobic digestion, characterized in that: include: S1: Inoculate Ruminiclostridium cellulolyticum, Clostridium lituseburense, and Clostridium amylolyticum into their respective liquid culture media, expand the culture in an anaerobic environment until the anaerobic bacteria count is not less than 200 million cells / mL, and obtain three bacterial cultures; S2: The three bacterial solutions were mixed in an anaerobic atmosphere at a volume ratio of (2-3):(3-4):(3-4), and the pH was adjusted to 6.8-7.2 with a buffer solution to obtain a composite anaerobic microbial liquid agent.

7. The method for preparing an anaerobic agent for improving the methane conversion rate of cellulose during medium- and high-temperature anaerobic digestion according to claim 6, characterized in that: The buffer solution is potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer solution.

8. The method for preparing an anaerobic bacterial agent for improving the methane conversion rate of cellulose during medium- and high-temperature anaerobic digestion according to claim 6, characterized in that: The expanded culture needs to be cultured at a temperature of 44-46° C. and with shaking at 160-200 rpm for 48-72 hours.

9. A method for improving the methane conversion rate of cellulose during medium- and high-temperature anaerobic digestion, characterized in that: The anaerobic bacterial agent for improving the methane conversion rate of cellulose in medium- and high-temperature anaerobic digestion according to any one of claims 1 to 5 is added to an anaerobic sludge system containing methanogens and cellulose and mixed evenly. Anaerobic fermentation is carried out in a medium- and high-temperature temperature range of 40 to 50°C. The complementary cellulase clusters of the three anaerobic bacteria gradually degrade the cellulose into primary volatile fatty acids, thereby improving the methane conversion rate of cellulose.

10. The method for improving the methane conversion rate of cellulose during medium- and high-temperature anaerobic digestion according to claim 9, characterized in that: The dosage of the anaerobic bacteria agent is 1-10% of the volume of the anaerobic sludge, and the temperature range of the anaerobic fermentation is 43-47°C.

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