Microbial agent and method for producing methane by anaerobic fermentation of straw and application thereof

Abstract

The present application provides a kind of microbial inoculum for methane production by straw anaerobic fermentation, belongs to the technical field of microbial fermentation.The microbial inoculum is fermented Lactobacillus mucus and Lactobacillus plantarum.The methane production of the microbial inoculum is 17.64 times compared with the group without microbial inoculum, indicating that the microbial inoculum composed of fermented Lactobacillus mucus and Lactobacillus plantarum can effectively utilize straw anaerobic fermentation to produce a large amount of methane, and the specific synergistic effect between microbial inoculums.The method significantly improves the effect of methane production by promoting the hydrolysis of fermentation substrate, and has the advantages of low cost, sustainability, simple operation and high organic matter utilization rate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and particularly relates to a microbial agent and method for producing methane by anaerobic fermentation of straw and application thereof. BACKGROUND

[0002] Anaerobic fermentation technology, as an important way of biomass resource utilization, can convert organic waste such as straw into clean energy such as methane. However, in crop straw, lignin forms a close cross-linked structure with cellulose and hemicellulose, making it difficult for microorganisms to effectively contact and degrade the straw itself, resulting in problems such as long start-up time and low fermentation efficiency in the fermentation process of straw.

[0003] The research status of bioaugmentation anaerobic fermentation Bioaugmentation technology mainly optimizes and improves the efficiency of anaerobic fermentation through the addition of microbial strains and flora, enzyme preparations and genetically engineered bacteria. Bioaugmentation technology can strengthen the degradation of complex substances in the system by screening and culturing microbial strains with specific functions, and can also create conditions conducive to the growth and reproduction of microorganisms by adjusting key parameters in the fermentation system, thereby efficiently enriching or inhibiting certain microorganisms. However, when crop straw is used as a single substrate for anaerobic fermentation, the gas production period is long, and due to the high carbon content of the substrate, high concentrations of volatile fatty acids are easily produced, causing a sharp drop in pH, resulting in acid inhibition of the fermentation system and even failure of gas production. Ultimately, the anaerobic fermentation process fails. In order to solve this problem, crop straw and livestock manure are usually mixed for anaerobic fermentation, thereby improving the buffering capacity of the fermentation system. However, due to the fact that manure can only be obtained in livestock farms, and the supply of manure raw materials is insufficient in some areas, only crop straw can be used for single-substrate anaerobic fermentation. Therefore, it is necessary to develop a method for maintaining stable anaerobic fermentation using straw as a single substrate in practical production and application. At present, there is an urgent need for a microbial agent to enhance the hydrolysis efficiency of straw, degrade macromolecular substances in straw, and increase methane production. SUMMARY

[0004] In view of this, the present application aims to provide a microbial agent and method for producing methane by anaerobic fermentation of straw and application thereof.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a microbial agent for producing methane by anaerobic fermentation of straw, wherein the microbial agent is a fermentation of Lactobacillus muci den and Lactobacillus plantarum.

[0007] Preferably, the mass ratio of the fermentation of Lactobacillus muci den to the fermentation of Lactobacillus muci den is 1:0.8-1.2.

[0008] Preferably, the viable cell count of the L. fermentum is 7*10 5 9*10 5 CFU / mL; the viable cell count of the L. plantarum is 6.5*10 7 9.5*10 7 CFU / mL.

[0009] Preferably, the mass ratio of the L. fermentum to the L. fermentum is 1:1.

[0010] Preferably, the accession number of the L. fermentum is CICC6233; the accession number of the L. plantarum is CICC22696.

[0011] The application provides an application of the microbial agent in preparation of methane.

[0012] The application provides a method for producing methane by anaerobic fermentation of straw, which comprises the following steps:

[0013] The microbial agent is mixed with a fermentation substrate for fermentation.

[0014] The fermentation substrate is straw and sludge.

[0015] Preferably, the straw is wheat straw, the sludge is sludge from a secondary sedimentation tank of a sewage treatment plant, and the total solid content of the fermentation substrate is greater than 15%.

[0016] Preferably, the fermentation is anaerobic fermentation at 32-38 DEG C for 28-35 days.

[0017] Preferably, 0.6 g of the L. fermentum is added per gram of volatile solid of the fermentation substrate, and 0.6 g of the L. plantarum is added per gram of volatile solid of the fermentation substrate.

[0018] The mass-volume ratio of the straw to the sludge is (9.15-9.30):100.

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

[0020] The microbial agent for producing methane by anaerobic fermentation of straw has a methane production yield which is 17.64 times that of a group without the microbial agent, indicating that the microbial agent composed of the L. fermentum and the L. plantarum can effectively produce a large amount of methane by anaerobic fermentation of straw, and the microbial agent has a specific synergistic effect. The method can promote hydrolysis of the fermentation substrate, significantly improve the methane production effect, and has the advantages of low cost, sustainability, simple operation and high organic matter utilization rate. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 6 is a diagram showing the change of methane production of wheat straw dry anaerobic fermentation under the action of Lactobacillus fermentum CICC6233, (a) is the daily methane production change of wheat straw dry anaerobic fermentation under the action of Lactobacillus fermentum CICC6233, (b) is the total methane production change of wheat straw dry anaerobic fermentation under the action of Lactobacillus fermentum CICC6233;

[0022] Figure 2 Figure 7 is a diagram showing the change of methane production of wheat straw dry anaerobic fermentation under the action of Lactobacillus helveticus CICC20977, (a) is the daily methane production change of wheat straw dry anaerobic fermentation under the action of Lactobacillus helveticus CICC20977, (b) is the total methane production change of wheat straw dry anaerobic fermentation under the action of Lactobacillus helveticus CICC20977;

[0023] Figure 3 Figure 8 is a diagram showing the change of methane production of wheat straw dry anaerobic fermentation under the action of Lactobacillus plantarum CICC22696, (a) is the daily methane production change of wheat straw dry anaerobic fermentation under the action of Lactobacillus plantarum CICC22696, (b) is the total methane production change of wheat straw dry anaerobic fermentation under the action of Lactobacillus plantarum CICC22696. DETAILED DESCRIPTION

[0024] The present application analyzes the methane production kinetics characteristics of the anaerobic fermentation process by the Modified Gompertz model, and studies the characteristics of the methane production activity of each Lactobacillus. It is found that the methane production of Lactobacillus fermentum increases rapidly at the initial stage of fermentation and gradually stabilizes, indicating that Lactobacillus fermentum can start methane production faster, thereby accelerating the degradation of the substrate and increasing the methane generation rate at the initial stage of anaerobic fermentation. The methane production of Lactobacillus helveticus increases slowly at the initial stage of fermentation, and the methane production of Lactobacillus plantarum increases slowly throughout the fermentation period and stabilizes at the late stage of fermentation, indicating that the addition of Lactobacillus helveticus and Lactobacillus plantarum requires more time to adapt to the environmental changes and start their own metabolic activities during the anaerobic fermentation process.

[0025] Based on the methane production kinetics characteristics of different Lactobacillus, the present application studies the influence of the combined application of different microorganisms on the methane production activity, and the results show that the selection of the strains in the Lactobacillus combination is crucial for methane production, and not all combinations of two or more Lactobacillus can produce a large amount of methane through anaerobic fermentation and have a synergistic effect. Compared with the addition of a single Lactobacillus, the two-by-two combination or the combination of two or more Lactobacillus may also reduce the methane production content.

[0026] Therefore, the present application provides a microbial inoculant for producing methane by straw anaerobic fermentation, and the microbial inoculant is Lactobacillus fermentum and Lactobacillus plantarum.

[0027] The present application researches and finds that compared with no addition of Lactobacillus group or Lactobacillus alone, the use of fermented Lactobacillus muciadis and Lactobacillus plantarum can greatly improve the ability of producing methane, and the total methane production rate is 17.64 times, 12.47 times and 12.08 times compared with no addition of Lactobacillus group, fermented Lactobacillus muciadis or Lactobacillus plantarum group. Therefore, the present application utilizes fermented Lactobacillus muciadis and Lactobacillus plantarum to produce methane, which has a synergistic effect. As a preferred embodiment, the mass ratio of the fermented Lactobacillus muciadis to the fermented Lactobacillus muciadis is 1:0.8-1.2, further preferably 1:0.9-1.1, and more preferably 1:1. The viable bacterial count of the fermented Lactobacillus muciadis is 7×10 5 ~9×10 5 CFU / mL; the viable bacterial count of the Lactobacillus plantarum is 6.5×10 7 ~9.5×10 7 CFU / mL. The preservation number of the fermented Lactobacillus muciadis is CICC6233; the preservation number of the Lactobacillus plantarum is CICC22696. In the present application, the culture method of the fermented Lactobacillus muciadis and the fermented Lactobacillus muciadis is not particularly limited, and known methods in the art can be used for culture preparation.

[0028] The present application provides an application of the above-mentioned microbial inoculant in preparing methane.

[0029] The present application provides a method for producing methane by anaerobic fermentation of straw, comprising the following steps:

[0030] Mixing the above-mentioned microbial inoculant with fermentation substrate for fermentation;

[0031] The fermentation substrate is straw and sludge.

[0032] In the present application, the straw is wheat straw, and the basic physicochemical parameters of the wheat straw are TS of 90.94%, VS of 73.72%, TC of 46.52%, and TN of 0.75%. The wheat straw is obtained by natural air drying and then crushing to obtain wheat straw particles, and the particle size of the crushed particles is 1.0-3.0 mm. The sludge is the sludge from the secondary sedimentation tank of a sewage treatment plant, such as the sludge from the secondary sedimentation tank of the sewage treatment plant in Yangling Demonstration Zone, Shaanxi Province, and the basic physicochemical parameters are pH of 8.32, TS of 8.03%, VS of 4.59%, TC of 27.44%, and TN of 3.91%. According to the volatile solids of the fermentation substrate, 0.6 g of Lactobacillus mucosus is added per gram of volatile solids of the fermentation substrate, and 0.6 g of Lactobacillus plantarum is added per gram of volatile solids of the fermentation substrate. The mass-volume ratio of the straw and sludge is (9.15-9.30):100. The total solids of the fermentation substrate are more than 15%, such as 15%, to achieve the purpose of dry anaerobic fermentation. The fermentation is anaerobic fermentation at 32-38℃ for 28-35 days, further preferably anaerobic fermentation at 33-36℃ for 30-33 days, and more preferably anaerobic fermentation at 35℃ for 32 days.

[0033] In the present application, the above-mentioned microbial agent is directly used for dry anaerobic fermentation, and the method is simple to operate without complex fermentation mode, and can significantly improve the content of methane production.

[0034] In the present application, all raw material components are commercially available products well known to those skilled in the art, unless otherwise specified.

[0035] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0036] In the following examples, the statistical methods are:

[0037] The obtained data were statistically analyzed using Excel 2019 and SPSS 26.0, all physicochemical parameters and functional gene abundance were represented as "mean ± standard deviation", and the graphs were drawn by OriginPro 2022b.

[0038] Example 1

[0039] 1. Test materials:

[0040] Wheat straw was collected from the experimental field of Northwest A&F University in Yangling, Shaanxi Province. After air-drying, the straw was ground into particles with a size of 1.0-3.0 mm using a grinder (FW-100, TAISITE, Tianjin, China) and stored at room temperature for later use. Inoculum was collected from the sludge of the secondary sedimentation tank of a wastewater treatment plant in Yangling, Shaanxi Province. Tap water was added to adjust the total solid (TS) and volatile solid (VS) concentrations. The basic physicochemical properties of the experimental materials were determined in triplicate and averaged. The basic physicochemical properties of the experimental materials are shown in Table 1.

[0041] Table 1 Basic physicochemical properties of anaerobic fermentation raw materials and inoculum

[0042]

[0043] The selected lactic acid bacteria species included Limosilactobacillus fermentum (heterofermentative), Lactobacillus helveticus (homofermentative), and Lactiplantibacillus plantarum (facultative fermentative), all purchased from the CICC China Industrial Microbial Culture Collection Center (Beijing, China) with CICC numbers CICC6233, CICC20977, and CICC22696, respectively.

[0044] 2. Test method

[0045] Anaerobic fermentation bottles of 500 mL were used, with a total working volume of 350 mL and an inoculum ratio of 30%. According to the results of the pre-experiment, 9.18 g of wheat straw and 100 g of sludge from the secondary sedimentation tank of the sewage treatment plant in Yangling, Shaanxi Province were mixed to make the final total TS of the fermentation substrate 15%. The amount of bacterial liquid added was set to 0.2 g / g VS~0.8 g / g VS according to the volatile solid (VS) ratio. For subsequent analysis, the fermentation L. mucosus CICC6233, L. helveticus CICC20977 and L. plantarum CICC22696 were labeled as R1, R2 and R3, respectively. The addition amounts of 0 g / g VS, 0.2 g / g VS, 0.4 g / g VS, 0.6 g / g VS and 0.8 g / g VS of each strain were labeled as CK, A, B, C and D, respectively. Nitrogen gas was blown into the 500 mL anaerobic fermentation bottle at a stable flow rate for 1 min, and the fermentation bottle was quickly sealed with a sealing device to ensure an anaerobic environment in the anaerobic fermentation reactor. The fermentation bottles were placed in a 35°C constant temperature incubator (SPM-1008, Ningbo, China) for batch anaerobic fermentation experiments for 32 days. The gas volume was collected every two days by a 2L aluminum foil gas collection bag, and the specific gas composition was determined. Every 2 days, 1 mL of mixed solution sample was collected from the fermentation bottle and diluted, and after centrifugation at 10000 r / min for 10 min, the solid sample was reserved for measurement. The liquid sample was subjected to SCOD, total carbohydrate and total organic acid content determination, while the system pH change was monitored. After sampling, nitrogen gas was introduced to maintain the anaerobic conditions inside the reactor. Each treatment was repeated 4 times.

[0046] Bacterial liquid preparation: According to the physicochemical properties of the bacterial liquid and the addition concentration set by the experiment, the corresponding amount of lactic acid bacterial liquid was inoculated in an 800 mL centrifuge bottle at a 1% inoculation amount, and shaken at 220 rpm until the logarithmic growth phase. When collecting the bacterial cells, centrifuge at 4000 r / min for 15 min at room temperature, and wash 3 times with sterile phosphate buffered saline solution (PBS, pH=7.4) to remove residual culture medium and metabolic byproducts. After each washing, the supernatant was discarded and the precipitated bacterial cells were retained. Finally, the bacterial cells were resuspended in an appropriate amount of PBS and stored at 4°C for later use (storage time not exceeding 24 h), or immediately used for anaerobic fermentation inoculation. At this time, the OD 600 of the fermentation L. mucosus CICC6233, L. helveticus CICC20977 and L. plantarum CICC22696 bacterial liquid were 1.259, 1.081 and 1.870, respectively, and the CFU were 7.18×10 5 ~8.63×10 5 CFU / mL, 3.60×10 5 ~9.14×10 5 CFU / mL and 6.84×10 7 ~9.12×10 7CFU / mL.

[0047] Determination of biogas composition and volume: Biogas composition was determined using a gas chromatograph (GC-2014, Shimadzu, Japan). The temperature program was set as follows: column temperature 80℃, DTCD1 temperature 150℃, and DINJ temperature 100℃. Argon was used as the carrier gas, the flow rate was 30 mL / min, and the injection volume was 1 μL. Biogas volume was determined using the water displacement method.

[0048] (1) Methanogenic characteristics of dry anaerobic fermentation of wheat straw under the action of Lactobacillus myxoidis CICC6233

[0049] Figure 1 The results in step a showed that, throughout the 32-day fermentation cycle, the daily methane production of the fermented *Lactobacillus mucinus* CICC6233 treatment groups exhibited the same trend, showing an initial increase followed by a decrease. The CK group reached its peak methane production on day 14 (4.32 mL / g VS). The R1A, R1B, R1C, and R1D groups reached their peak methane production on days 12, 14, 10, and 12, respectively, at 9.32 mL / g VS, 5.97 mL / g VS, 9.37 mL / g VS, and 7.84 mL / g VS, indicating that the addition of *Lactobacillus mucinus* advanced the peak methane production by 2–4 days.

[0050] like Figure 1 As shown in (b), after fermentation, the total methane yield of group CK was 55.50 mL / g VS. The total methane yields of groups R1 were 78.25 mL / g VS, 58.64 mL / g VS, 79.12 mL / g VS, and 77.95 mL / g VS, respectively. The addition of *Lactobacillus mucilaginosus* increased the total methane yield of the anaerobic fermentation system by 5.67%–42.56%, with group R1C achieving the highest total methane yield.

[0051] (2) Methanogenic characteristics of dry anaerobic fermentation of wheat straw under the action of Lactobacillus helveticus CICC20977

[0052] Figure 2 The results in (a) show that the daily methane production in group R2 also exhibited a trend of first increasing and then decreasing. Peak methane production occurred in group R2 on days 8, 14, 14, and 16, at 4.22 mL / g VS, 8.01 mL / g VS, 5.27 mL / g VS, and 6.75 mL / g VS, respectively. Except for group R2A, all values ​​were higher than those in group CK (4.32 mL / g VS). This indicates that the addition of *Lactobacillus helveticus* CICC20977 can effectively improve the system's methane production performance, with group R2B showing the best effect.

[0053] like Figure 2As shown in (b), after fermentation, the total methane production of groups R2A, R2B, R2C, and R2D were 58.13 mL / g VS, 72.81 mL / g VS, 71.05 mL / g VS, and 68.85 mL / g VS, respectively. The addition of *Lactobacillus helveticus* CICC20977 increased the total methane production of the anaerobic fermentation system by 4.73%–31.18%, with group R2B achieving the highest total methane production.

[0054] (3) Methanogenic characteristics of dry anaerobic fermentation of wheat straw under the action of Lactobacillus plantarum CICC22696

[0055] like Figure 3 As shown in (a), the daily methane production in group R3 also showed a trend of first increasing and then decreasing. The peak methane production in group R3 occurred on days 16, 16, 18 and 16, respectively, at 4.83 mL / g VS, 7.18 mL / g VS, 6.30 mL / g VS and 5.73 mL / g VS. The daily methane production and the amount of *Lactobacillus plantarum* added were in the order R3B>R3C>R3D>R3A.

[0056] like Figure 3 As shown in (b), after fermentation, the total methane production of groups R3 was 58.22 mL / g VS, 79.88 mL / g VS, 81.89 mL / g VS, and 56.37 mL / g VS, respectively. The total methane production followed the order of R3C > R3B > R3D > R2A. The addition of Lactobacillus plantarum CICC22696 increased the cumulative methane production of the anaerobic fermentation system by 1.56% to 47.54%, with group R3C achieving the highest total methane production.

[0057] In summary, *Lactobacillus plantarum* CICC22696 exhibited the best methanogenic performance. The R3C group showed the most significant improvement (47.54%) in total methane production at the end of fermentation, with a total yield of 81.89 mL / g VS compared to the CK group.

[0058] (4) Analysis of methanogenesis characteristics based on the Modified Gompertz model

[0059] Kinetic analysis: The kinetics of methanogenesis (P) from wheat straw was analyzed using the Modified Gompertz model (Equation 1).

[0060]

[0061] In the formula, P is the methane production at time t; Pmax is the maximum methane production; Rmax is the maximum methane production rate; λ is the lag time; and e is the natural constant.

[0062] The system Modified Gompertz model fitting curve under the action of lactic acid bacteria can be seen that the fitting curve effect is good. The methane production of R1 group increases rapidly in the early fermentation period (4-12 days), and gradually tends to be stable at the 28th day, and the final yield is close to 80 mL / g VS. The methane production of R2 group increases slowly in the early fermentation period (4-12 days), and the final yield is slightly lower than that of R1 group. The methane production of R3 group presents a relatively gentle growth during the whole fermentation period, and reaches stability in the late fermentation period, and the final yield is 81.89 mL / g VS.

[0063] The Modified Gompertz model is used to analyze the methane production kinetics characteristics of the anaerobic fermentation process. The kinetic parameter results are shown in Table 2. From the fitting results, the model R 2 between 0.995 and 0.999, which indicates that the Modified Gompertz model has good simulation effect on the experiment. The lag time (λ) can represent the methane production activity, the absorption of microorganisms to the specific substrate and the adaptation to the environmental conditions during the anaerobic fermentation process. The lag time of R1 group is shorter, which indicates that the fermentation of L. mucosus can start methane production faster, thereby accelerating the degradation of the substrate and increasing the methane generation rate in the early anaerobic fermentation period. The lag time of R2 group and R3 group is relatively long, which means that the addition of L. helveticus and L. plantarum needs to consume more time to adapt to the environmental changes and start their own metabolic activities during the anaerobic fermentation process. The maximum methane production rate (Rm, mL / g VS d) of R1, R2 and R3 groups is greater than that of CK group. Compared with the other two groups, R1 group shows a higher Rm, in which the Rm value of R1C is the largest, which is 7.93 mL / g VS d, which indicates that the fermentation of L. mucosus can effectively promote the hydrolysis of the substrate, increase the nutrients available to the microorganisms, thereby providing sufficient substrate for the methanogens and accelerating the anaerobic fermentation process. In addition, the high Rm value of R1 group can also improve the microbial community structure and function, and enhance the metabolic activity and efficiency of the whole anaerobic fermentation system. R3 group has the largest methane production (Pm, mL / g VS), especially the Pm value of R3C reaches 83.49 mL / g VS.

[0064] Table 2 Kinetic parameters of anaerobic fermentation system under the action of different lactic acid bacteria

[0065]

[0066] Example 2

[0067] Different lactic acid bacteria combined for methane production in dry anaerobic fermentation of wheat straw

[0068] Anaerobic fermentation bottles of 500 mL were used, with a total working volume of 350 mL and an inoculum ratio of 30%. According to the results of the pre-experiment, 9.18 kg of wheat straw described in Example 1 and 100 kg of sludge from the secondary sedimentation tank of the Yangling demonstration area sewage treatment plant in Shaanxi described in Example 1 were mixed to make the final total TS of the fermentation substrate 15%. Then 0.6 g / gVS of Lactobacillus fermentum CICC6233 broth (denoted as R1C group), 0.4 g / gVS of Lactobacillus helveticus CICC20977 broth (denoted as R2B group), 0.6 g / gVS of Lactobacillus plantarum CICC22696 broth (denoted as R3C group), a mixture of 0.6 g / gVS of Lactobacillus fermentum CICC6233 broth and 0.4 g / gVS of Lactobacillus helveticus CICC20977 broth (denoted as R1C+R2B group), a mixture of 0.6 g / gVS of Lactobacillus fermentum CICC6233 broth and 0.6 g / gVS of Lactobacillus plantarum CICC22696 broth (denoted as R1C+R3C group), a mixture of 0.4 g / gVS of Lactobacillus helveticus CICC20977 broth and 0.6 g / gVS of Lactobacillus plantarum CICC22696 broth (denoted as R2B+R3C group), and a mixture of 0.6 g / gVS of Lactobacillus fermentum CICC6233 broth, 0.4 g / gVS of Lactobacillus helveticus CICC20977 broth and 0.6 g / gVS of Lactobacillus plantarum CICC22696 broth (denoted as R1C+R2B+R3C group) were added. At the same time, no lactic acid bacteria strains were added as the CK group. Nitrogen gas was blown into the 500 mL anaerobic fermentation bottle at a stable flow rate for 1 min, and the fermentation bottle was quickly sealed with a sealing device to ensure an anaerobic environment in the anaerobic fermentation reactor. The fermentation bottles were placed in a 35°C constant temperature incubator (SPM-1008, Ningbo, China) for batch anaerobic fermentation experiments for 32 days. The gas volume was collected every two days by a 2L aluminum foil gas collection bag, and the specific gas composition was determined. Every 2 days, 1 mL of mixed solution sample was collected from the fermentation bottle, diluted, and centrifuged at 10000 r / min for 10 min. The solid sample was stored for testing. The liquid sample was subjected to SCOD, total carbohydrate and total organic acid content determination, and the system pH change was monitored. Nitrogen gas was introduced after sampling to maintain the anaerobic conditions inside the reactor. Each treatment was repeated 4 times in this experiment.

[0069] wherein the broth preparation was the same as in Example 1.

[0070] Biogas composition and volume determination: Gas chromatography (GC-2014, Shimadzu, Japan) was used to determine the composition of biogas, with the temperature program set as follows: column temperature 80°C, DTC D1 temperature 150°C and DINJ temperature 100°C, carrier gas argon, flow rate 30 mL / min, injection volume 1 μL. The biogas volume was determined by the drainage method. The mean values of each group were calculated.

[0071] The results of Table 3 show that compared with the CK group, the use of fermented L. muciadis CICC6233, L. helveticus CICC20977 and L. plantarum CICC22696 alone can improve the total methane production, but the efficiency of improvement is not high. Among them, L. helveticus CICC20977 and L. plantarum CICC22696 (R2B+R3C) or the combination of the three strains does not improve the total methane production compared with the total methane production of the single lactobacillus, but rather reduces the total methane production. When fermented L. muciadis CICC6233 is combined with L. helveticus CICC20977 (R1C+R2B) or fermented L. muciadis CICC6233 is combined with L. plantarum CICC22696 (R1C+R3C) for dry anaerobic fermentation of wheat straw methane, the total methane production enhancement rate is 5.12 and 17.64 times, respectively, compared with the CK group, especially when fermented L. muciadis CICC6233 is combined with L. plantarum CICC22696 (R1C+R3C) for dry anaerobic fermentation of wheat straw methane, the total methane production is higher.

[0072] Table 3 Results of methane production of different combinations of lactic acid bacteria

[0073]

[0074] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of a microbial inoculum for methanogenesis by anaerobic fermentation of straw in the preparation of methane, characterized in that, The microbial agent is fermented Lactobacillus muci denii and Lactobacillus plantarum; The preservation number of the fermented Lactobacillus muci denii is CICC6233; and the preservation number of the Lactobacillus plantarum is CICC22696.

2. Use according to claim 1, characterized in that, The viable cell count of the fermented L. muci- de is 7 x 10 5 9 x 10 5 CFU / mL; the viable cell count of the L. plantarum is 6.5 x 10 7 9.5 x 10 7 CFU / mL.

3. Use according to claim 1, characterized in that, The mass ratio of the fermented Lactobacillus muci denii to the Lactobacillus plantarum is 1:

1.

4. A method for producing methane by anaerobic fermentation of straw, characterized by, The method comprises the following steps: The microbial agent and the fermentation substrate are mixed for fermentation. The fermentation substrate is straw and sludge.

5. The method of claim 4, wherein, The straw is wheat straw, and the sludge is sludge from a secondary sedimentation tank of a sewage treatment plant; and the total solid of the fermentation substrate is more than 15%.

6. The method of claim 4, wherein, The fermentation is anaerobic fermentation at 32-38℃ for 28-35 days.

7. The method of claim 4, wherein, 0.6g of the fermented Lactobacillus muci denii is added per gram of volatile solid of the fermentation substrate, and 0.6g of the Lactobacillus plantarum is added per gram of volatile solid of the fermentation substrate; The mass-volume ratio of the straw and the sludge is (9.15-9.30):100.

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