A method for improving methanogenesis from lignocellulose based on mixed-culture fermentation strategy

Abstract

This invention discloses a method for improving methanogenesis from lignocellulose based on a mixed-culture fermentation strategy, belonging to the field of anaerobic fermentation technology. The method includes the following steps: (1) inoculating activated *Trichoderma echinosporum* into a culture medium containing lignocellulose and loofah sponge as a biological carrier material for fermentation to obtain a fermentation broth; (2) activating activated sludge using a revitalizing solution; (3) inoculating the activated sludge from step (2) into the fermentation broth of *Trichoderma echinosporum* from step (1) for anaerobic fermentation to produce methanogens. This method achieves a methane yield of 262 mL / g VS, which is more than 30% higher than that of traditional anaerobic fermentation for methanogenesis, and is of great significance for improving the bioconversion of lignocellulose resources.

Description

Technical Field

[0001] This invention belongs to the field of anaerobic fermentation technology, specifically involving a method for efficiently decomposing lignocellulose with Trichoderma echinosporum and using biological carrier materials to fix the growth of Trichoderma echinosporum, thereby improving the anaerobic fermentation of methane in activated sludge. Background Technology

[0002] As human society continues to progress, conventional fossil fuels are increasingly unable to meet the ever-growing energy demand, and the large amounts of greenhouse gases released from their combustion exacerbate global warming. Biomass energy is a renewable energy source that can reduce the need for fossil fuels. Lignocellulose, derived from agricultural and forestry waste, is a renewable, low-carbon biomass resource that demonstrates enormous potential to replace traditional fossil fuels.

[0003] Globally, lignocellulose biomass production amounts to 181.5 million tons. Lignocellulose is primarily composed of cellulose (35%–50%), hemicellulose (23%–32%), and lignin (15%–30%), making it the most abundant renewable biomass resource in nature. It includes agricultural waste such as rice straw, rice husks, corn stalks, cottonseed, lotus root stalks, corn cobs, as well as garden waste, pine wood, and maple wood. However, only 3% of lignocellulose is effectively utilized in a circular bioeconomy. Most is disposed of through simple methods such as landfill and incineration. In contrast, converting it into methane using anaerobic fermentation technology can significantly enhance its utilization value.

[0004] Methanogenesis through anaerobic fermentation of lignocellulose is a complex cascade process involving the synergistic effects of multiple functional microbial communities, including hydrolytic acidifiers, hydrogen-producing and acetic acid-producing bacteria, and methanogens. The metabolic coupling and mass transfer efficiency among these communities influence the final gas production efficiency. However, the microbial communities inherent in anaerobic digestion are not fully exposed to lignocellulose, often exhibiting low lignocellulose degradation efficiency. Therefore, the strategy of adding exogenous lignocellulose-degrading microorganisms based on mixed microbial approaches has significant application potential. However, it is worth noting that not all microorganisms with degradation capabilities can effectively enhance the overall efficiency of anaerobic digestion. This may be because while enhanced hydrolysis increases substrate availability, it shifts the limiting factors to subsequent stages (such as methanogenesis). Therefore, when selecting exogenous functional bacteria, their adaptability and survival ability to the existing anaerobic digestion community must be comprehensively considered, and their metabolism should be as conducive to methanogenesis as possible. Furthermore, increasing the inoculum size is also crucial for enhancing the effect, often employing a multiple inoculation strategy. A single inoculation often requires a higher dose. Otherwise, exogenous bacteria may be gradually replaced by other microorganisms in the microbial community due to insufficient competitiveness, or even disrupt the original microbial community balance and inhibit methane production. Simultaneously, the pH value for anaerobic fermentation to produce methane should be above 6.6, ideally between 6.8 and 7.2. The ideal state for efficient methane production is when the production and consumption of volatile fatty acids (VFA) reach equilibrium. Under this state, hydrolytic and acidifying bacteria efficiently convert organic matter into appropriate amounts of VFA (mainly acetic acid), which is then promptly utilized by methanogenic bacteria to produce methane. However, if hydrolysis is too rapid, it can lead to system instability, often resulting in "acid collapse" during methane fermentation.

[0005] To address the difficulties in colonizing exogenous functional bacteria and the poor stability of the system, introducing biological carriers has become an important means to overcome this bottleneck. Traditional artificial carriers often suffer from drawbacks such as high cost and poor degradation. However, loofah sponge is a widely available and inexpensive natural agricultural and forestry byproduct in my country. Its internal structure is a three-dimensional porous network formed by multiple layers of interwoven filamentous fibers, possessing advantages such as high porosity and large specific surface area. It has extremely high application value in the fields of anaerobic digestion and microbial immobilization. This study's strategy involves targeted supplementation of key lignocellulose-hydrolyzing microorganisms and the timely addition of loofah sponge as a biological carrier to construct a synergistic reinforcement alliance, promoting the stable operation of the anaerobic digestion system. Summary of the Invention

[0006] To address the problems existing in current technologies, the present invention aims to provide a method for improving methane production from lignocellulose based on a mixed-culture fermentation strategy. The method involves fermenting *Trichoderma echinococcus* in a culture medium containing lignocellulose and a biological carrier, loofah sponge, to obtain a fermentation broth. This broth is then mixed with activated sludge for fermentation, enhancing the activated sludge's ability to degrade lignocellulose and thus increasing methane production. This provides an effective way to treat agricultural waste and "turn waste into treasure," and is also a highly efficient path to achieving carbon neutrality and a circular economy.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: A method for enhancing methanogenesis from lignocellulose using a mixed-culture fermentation strategy involves using lignocellulose as the fermentation substrate, leveraging the high lignocellulose-producing capacity of *Trichoderma echinosporum*, and growing it via a loofah sponge biocarrier to improve the methanogenesis capacity of activated sludge through anaerobic fermentation. The specific steps include: (1) The activated Trichoderma hydatids was inoculated into a fermentation medium containing lignocellulose and loofah sponge for fermentation to obtain fermentation broth; (2) Add a revival solution to the activated sludge to activate the microbial community. Once the sludge stops producing gas, it can be used for subsequent fermentation. (3) The activated sludge obtained in step (2) is inoculated into the fermentation broth of Trichoderma hygroscopicum in step (1) for anaerobic fermentation to produce methane.

[0008] The inoculum size of the activated *Trichoderma hydathodes* in step (1) is 1-20% of the fermentation medium volume; the fermentation medium containing lignocellulose is formulated as follows: 0.2-0.4 g / L urea, 1-2 g / L (NH4)2SO4, 1-3 g / L KH2PO4, 0.1-0.4 g / L CaCl2, 0.1-0.4 g / L MgSO4·7H2O, 0.001-0.01 g / L FeSO4·7H2O, 0.001-0.002 g / L MnSO4·H2O, 0.001-0.002 g / L ZnSO4·7H2O, 0.001-0.003 g / L CoCl2, 20-80 g / L lignocellulose, 3-10 g / L corn steep liquor powder, with water as the solvent, pH adjusted to 4.5-7.5, sterilized at 121℃ for 20 minutes. The fermentation conditions are: fermentation temperature 25-37 ℃, fermentation time 24-120 h, fermentation pH 4.5-7.5, and rotation speed 0-180 rpm.

[0009] The lignocellulose is derived from corn stalks, wheat stalks, rice stalks, corn cobs, etc.

[0010] Preferably, the concentration of lignocellulose in the fermentation medium containing lignocellulose is 40 g / L.

[0011] Preferably, the fermentation time of *Trichoderma echinococcus* in step (1) is 24-120 hours, and more preferably 72 hours. The activated sludge is inoculated into the fermentation broth obtained in (1), and fermentation broth from *Trichoderma echinococcus* aged 3 days is selected. At this time, the filter paper enzyme activity is high, which can effectively alleviate the metabolic burden on the hydrolytic bacteria. If the inoculation time is too early (before 24 hours), the cellulase activity is low, which is not conducive to the subsequent efficient degradation of lignocellulose; if the inoculation time is too late (after 120 hours of *Trichoderma echinococcus* fermentation), the culture time of *Trichoderma echinococcus* will be too long, and some cellulase will lose its activity.

[0012] The Trichoderma mentioned in step (1) is Trichoderma acicularis ( Trichoderma asperellum LYS1.

[0013] This invention incorporates loofah sponge as a biological carrier material into the fermentation medium. The loose internal structure and large specific surface area of ​​the loofah sponge provide attachment sites for *Trichoderma echinosporum* growth, forming a stable biofilm. This reduces hyphal loss and maintains biomass; the hyphae penetrate deep into the pores, increasing the contact area with the substrate and alleviating the "hydrolysis rate limit" bottleneck. *Trichoderma* degradation reduces the crystallinity of the substrate cellulose and the lignin barrier, improving the substrate utilization efficiency of subsequent activated sludge. Its maximum outer diameter is 2-10 cm, its overall height is 1-3 cm, and its weight is approximately 8-20 g / L.

[0014] Adding loofah sponge can efficiently stabilize functional microbial communities, enhance the hydrolysis and acidification process, increase methane production, and realize the resource utilization of natural biomaterials. The loofah sponge can be added at the beginning of fermentation or during fermentation; preferably, it is added during the 0-24 h fermentation of Trichoderma echinosporum, and more preferably, it is added to the fermentation medium at 12 h fermentation.

[0015] Preferably, the fermentation conditions in step (1) are: fermentation temperature 30℃, fermentation time 72 h, fermentation pH 5.5, and rotation speed 180 rpm.

[0016] The activation medium for *Trichoderma echinococcus* is formulated as follows: 0.3 g / L urea, 1.4 g / L (NH4)2SO4, 2.0 g / L KH2PO4, 0.3 g / L CaCl2, 0.3 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O, 0.00156 g / L LmnSO4·H2O, 0.0014 g / L ZnSO4·7H2O, 0.002 g / L CoCl2, 10 g / L glucose, with water as the solvent, and the pH adjusted to 5.0-6.0. The activation conditions are as follows: 0.1-0.8 mL of *Trichoderma echinococcus* mycelium solution is coated onto PDA medium and incubated at 25-37℃ for 48-120 h. The colonies in the PDA solid medium are rinsed with 0.5-3.0 mL of sterile water and then inoculated onto the activation medium, and activated at 25-37℃ for 48-96 h.

[0017] The activated sludge in step (2) is the residual activated sludge in the secondary sedimentation tank of the sewage treatment plant after resuspension, which is used as the fermentation substrate.

[0018] The formula for the awakening solution in step (2) is: glucose 300-600 g / L, KH2PO4 7.00 g / L, MgSO4·7H2O 2.25 g / L, NH4HCO3 45.15 g / L, and water as the solvent.

[0019] Preferably, the activation conditions for the microbial community in step (2) are: a temperature of 37°C, adding 0.1 mL of revival solution to 50 mL of activated sludge every 24 hours for 3 consecutive days, a fermentation pH of 7.0, and anaerobic static culture. Adding too much revival solution can easily lead to acid collapse during anaerobic fermentation. Adding too little revival solution will result in slower microbial community activation and prolong the fermentation cycle. Once it stops producing gas, it can be used for subsequent anaerobic fermentation.

[0020] The activated sludge in step (3) is inoculated as follows: the activated sludge is inoculated into the fermentation broth obtained in step (1) at an inoculation rate of 20-60% (v / v). The fermentation conditions are: fermentation temperature 35-40℃, fermentation time 20-30 days, fermentation pH 6.8-7.4, and anaerobic static culture.

[0021] Preferably, the fermentation temperature is 37 ℃, the fermentation time is 25 days, and the fermentation pH is 7.

[0022] Among them, Trichoderma acicularis ( Trichoderma asperellumThe strain, LYS1, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20211179 and a deposit date of September 15, 2021. This strain can efficiently secrete cellulase to degrade corn stalks; it has extremely high application value in the production of energy from lignocellulose.

[0023] This study selected Trichoderma hyacinth ( Trichoderma asperellum This strain is used as a fortifying strain. Its advantage lies in the fact that its fortifying effect does not depend on its long-term survival in an anaerobic environment. Under aerobic conditions, this strain can secrete a highly efficient and synergistic complex of cellulase and hemicellulase, effectively degrading the lignocellulose structure and producing intermediate products that are easily metabolized later. However, when used alone to enhance methanogenesis in anaerobic fermentation, this strain can easily cause microenvironmental imbalance, which is detrimental to the stable operation of the anaerobic digestion process. When using loofah sponge as a biological carrier, the hyphae may better promote electron transfer between bacteria and methanogens, while fully preserving the strain's original highly efficient lignocellulose degradation ability, achieving a synergistic fortifying effect between *Trichoderma echinosporum* and the loofah sponge biological carrier.

[0024] Beneficial effects: The exogenous addition of Trichoderma hygroscopicum grown on loofah sponge can effectively increase the hydrolysis efficiency of lignocellulose, stabilize the entire anaerobic digestion process, reduce pH fluctuations, create a suitable microenvironment for strains with different oxygen requirements, enhance the electron transfer efficiency between methanogens and symbiotic bacteria, and ultimately significantly increase methane production. Attached Figure Description

[0025] Figure 1 The effect of exogenous addition of Trichoderma hygroscopicum fermentation broth on methanogenesis by anaerobic fermentation of activated sludge; Figure 2 The effect of exogenous addition of Trichoderma hygroscopicum on anaerobic fermentation methanogenesis at different temperatures; Figure 3 The effect of exogenous Trichoderma hyacinth addition on the methanogenic effect of anaerobic fermentation under different activated sludge inoculum amounts; Figure 4 Image of Trichoderma hyacinthiae growing on a loofah sponge biocarrier; Figure 5 Comparison of the effects of loofah sponge-based biological carrier-immobilized Trichoderma hydathodes and free Trichoderma hydathodes on promoting methanogenesis from anaerobic digestion of activated sludge; Figure 6 The effect of loofah sponge addition time on methanogenesis in Trichoderma hygroscopic culture on methanogenesis from activated sludge. Detailed Implementation

[0026] Trichoderma is classified and named Trichoderma acicularis ( Trichoderma asperellum The strain number is LYS1, and the preservation number is CCTCC NO: M 20211179.

[0027] The activated sludge is a biofloc formed by microbial communities, organic and inorganic suspended solids, and is a conventional biological treatment material in the field of wastewater treatment. In this field, conventional sources are used, such as anaerobic aeration tank mixed liquor or secondary sedimentation tank return sludge from municipal wastewater treatment plants or industrial wastewater treatment stations, which can be concentrated, acclimated, or used directly. In the following examples, the activated sludge was collected from the residual activated sludge of the anaerobic bioreactor at Nanjing University of Technology after resuspension, as the fermentation substrate.

[0028] Example 1: Effect of exogenous addition of Trichoderma echinosporum fermentation broth on methanogenesis from anaerobic fermentation of activated sludge Activated sludge was activated with a revitalizing solution at 37℃. Every 24 hours, 0.1 mL of the revitalizing solution was added to 50 mL of activated sludge, and this was repeated for 3 days. The fermentation pH was maintained at 7.0, and the sludge was incubated anaerobicly. The revitalizing solution consisted of: 500 g / L glucose, 7.00 g / L KH₂PO₄, 2.25 g / L MgSO₄·7H₂O, and 45.15 g / L NH₄HCO₃, with water as the solvent. Once the solution ceased producing gas, it was used for subsequent anaerobic fermentation to produce methane.

[0029] 0.2 mL of Trichoderma LYS1 bacterial suspension was coated onto PDA solid medium (46 g / L potato dextrose, 20 g / L agar) and incubated at 30℃ for 72 h. The colonies in the PDA solid medium were rinsed with 1 mL of sterile water and inoculated onto activation medium, which was then activated at 30℃ for 24 h. The activation medium consisted of: 0.3 g / L urea, 1.4 g / L (NH4)2SO4, 2.0 g / L KH2PO4, 0.3 g / L CaCl2, 0.3 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O, 0.00156 g / L LmnSO4·H2O, 0.0014 g / L ZnSO4·7H2O, 0.002 g / L CoCl2, and 10 g / L glucose, with water as the solvent, and the pH adjusted to 5. The activated Trichoderma hydathodes were inoculated into the fermentation medium at 10% (v / v). The fermentation medium consisted of: 0.3 g / L urea, 1.4 g / L (NH4)2SO4, 2.0 g / L KH2PO4, 0.3 g / L CaCl2, 0.3 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O, 0.00156 g / L MnSO4·H2O, 0.0014 g / L ZnSO4·7H2O, 0.002 g / L CoCl2, 40 g / L corn straw, and 7.5 g / L corn steep liquor powder. The solvent was water, and the pH was adjusted to 5. Fermentation was carried out for 72 h. The activated sludge was anaerobic fermented with Trichoderma hygroscopicum fermentation broth at 25% (v / v) to produce methanogens, and the pH was adjusted to 7.

[0030] The group with exogenous Trichoderma echinococcus fermentation broth: Activated sludge was used as inoculum (inoculum amount of 25% v / v) and inoculated into 50 mL of Trichoderma echinococcus fermentation broth. The control group was tested under the following conditions: 12.5 mL of activated sludge was added to corn straw containing 40 g / L, and the mixture was directly subjected to static anaerobic fermentation.

[0031] Fermentation conditions were as follows: temperature 37℃, fermentation pH 7, anaerobic static culture. Cultured for 25 days, with three replicates per group. Methane content was measured every 2 days during the culture process.

[0032] The methane yield in the experimental group with exogenous addition of Trichoderma echinosporum was approximately 200 mL / g VS, which was about 15% higher than that in the control group without Trichoderma echinosporum. This experiment demonstrates that exogenous addition of Trichoderma echinosporum can effectively promote the degradation of straw and increase methane yield.

[0033] Example 2: Effect of exogenous addition of Trichoderma hygroscopicum at different temperatures on anaerobic fermentation for methanogenesis The method was the same as in Example 1, except that the fermentation temperatures for the experimental groups were set at 25, 30, 37, and 45°C, respectively, and the fermentation time was 25 days, with three replicates per group. The methane content was measured every two days.

[0034] During the experiment, methane content was measured every two days. The methane content reached its maximum at 37℃ (204 mL / g VS), approximately 50% higher than that of anaerobic fermentation at 25℃. The experiment verified that low temperatures inhibit methane production, while excessively high temperatures initially promote methane production. This is likely because high temperatures significantly accelerate the enzyme activity and metabolic rate of hydrolytic and acid-producing bacteria. Complex substrates such as lignocellulose are rapidly decomposed into large amounts of monosaccharides and amino acids, which further ferment to produce high concentrations of volatile fatty acids (VFAs), hydrogen, and carbon dioxide. However, slow-growing and environmentally sensitive methanogenic bacteria cannot maintain their high methane production metabolic state, leading to a decrease in methane yield in the later stages.

[0035] Example 3: Effect of exogenous Trichoderma echinosporum addition on methanogenesis in anaerobic fermentation under different activated sludge inoculum amounts. The method is the same as in Example 1, except that the fermentation temperature is selected as 37°C, and the activated sludge inoculum amounts of 5, 15, 25, and 50% (v / v) are selected and inoculated into the fermentation broth of Trichoderma echinosporum.

[0036] During the experiment, the methane content was measured every 2 days. When 25% (v / v) activated sludge was inoculated into the Trichoderma effusus fermentation broth, the methane production reached approximately 206 mL / g VS. A lower initial inoculum size resulted in insufficient numbers of hydrolytic bacteria, acid-producing bacteria, and the crucial methanogenic archaea, hindering efficient methanogenesis. Conversely, an excessively high inoculum size may have caused a decrease in methane production, similar to the effect of excessively high temperatures in Example 1.

[0037] Example 4: Growth images of Trichoderma echinosporum on loofah biocarrier 0.2 mL of *Trichoderma hygroscopica* suspension was coated onto PDA solid medium (46 g / L potato dextrose, 20 g / L agar) and incubated at 30°C for 72 h. Colonies in the PDA solid medium were rinsed with 1 mL of sterile water and inoculated onto activation medium, which was then activated at 30°C for 24 h. The activation medium consisted of: 0.3 g / L urea, 1.4 g / L (NH4)2SO4, 2.0 g / L KH2PO4, 0.3 g / L CaCl2, 0.3 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O, 0.00156 g / L LmnSO4·H2O, 0.0014 g / L ZnSO4·7H2O, 0.002 g / L CoCl2, and 10 g / L glucose, with water as the solvent, and the pH adjusted to 5. The activated *Trichoderma hygroscopicum* was inoculated into the fermentation medium at a rate of 10% (v / v). The fermentation medium consisted of: 0.3 g / L urea, 1.4 g / L (NH4)2SO4, 2.0 g / L KH2PO4, 0.3 g / L CaCl2, 0.3 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O, 0.00156 g / L MnSO4·H2O, 0.0014 g / L ZnSO4·7H2O, 0.002 g / L CoCl2, 40 g / L corn stalks, and 7.5 g / L corn steep liquor powder. The solvent was water, and the pH was adjusted to 5. Loofah sponges with a maximum outer diameter of 2-10 cm, an overall height of 1-3 cm, and a weight of approximately 8-20 g / L were then added.

[0038] Fermentation conditions were as follows: temperature 30 ℃, fermentation pH: 5, rotation speed: 180 rpm, fermentation culture for 3 days; The growth of *Trichoderma echinosporum* on the loofah biocarrier material was as follows... Figure 4 As shown, when using loofah sponge as a biological carrier, the hyphae may better promote electron transfer in bacteria and methanogens, while fully preserving the strain's original high-efficiency lignocellulose degradation ability.

[0039] Example 5: Comparison of the effects of immobilized and free Trichoderma echinosporum on promoting methanogenesis in activated sludge using loofah sponge as a biological carrier. The method for culturing Trichoderma echinosporum is the same as in Example 4. Trichoderma echinosporum is grown by adding exogenous biological carriers to fix and non-fix Trichoderma echinosporum, and then inoculating with 25% (v / v) activated sludge for anaerobic static fermentation.

[0040] Methane content was measured every 2 days after 25 days of cultivation. The methane yield reached 242 mL / g VS by exogenously adding activated sludge immobilized with *Trichoderma echinococcus* using loofah sponge as a biological carrier, approximately 20% higher than the control group without loofah sponge immobilization. This experiment demonstrates that immobilizing *Trichoderma echinococcus* using a biological carrier can effectively increase methane production. It may provide a suitable growth environment for microorganisms with different oxygen requirements, and the immobilized hyphae may also better promote electron transfer between bacteria and methanogens. This provides favorable conditions for anaerobic digestion.

[0041] Example 6: Effect of adding loofah sponge at different fermentation times of Trichoderma echinosporum on methanogenesis in activated sludge. The method for activating Trichoderma echinosporum is the same as in Example 4, except that the time for adding exogenous loofah sponge (maximum outer diameter of 2-10 cm, overall height of 1-3 cm, and weight of approximately 8-20 g / L) during Trichoderma echinosporum fermentation is different, and is set at 0h, 12h, 24h, 36h, 48h, and 60h respectively.

[0042] By controlling the duration of exogenous addition of loofah sponge to immobilize *Trichoderma echinococcus*, and after culturing for 3 days, anaerobic static fermentation was carried out with 25% (v / v) activated sludge, and cultured for 25 days. Methane content was measured every 2 days during the culture process. It was found that the highest methane yield was achieved with exogenous addition of loofah sponge during 12 hours of fermentation, reaching a maximum of 262 mL / g VS. This may be because *Trichoderma echinococcus* requires higher oxygen levels in the early stages of fermentation; otherwise, its cellulase production is lower. The 12-hour activation process allowed *Trichoderma echinococcus* to grow fully and efficiently degrade lignocellulose.

Claims

1. A method for improving the methanogenic potential of lignocellulosic biomass using a mixed culture fermentation strategy, characterized in that, Includes the following steps: (1) The activated Trichoderma hygroscopicum was inoculated into a fermentation medium containing lignocellulose and loofah sponge biological carrier material for fermentation to obtain fermentation broth; (2) Add a revival solution to the activated sludge to activate the microbial community. Once the microbial community stops producing gas, it can be used for subsequent fermentation. (3) Inoculate the activated sludge into the fermentation broth of Trichoderma hygroscopicum and anaerobic ferment to produce methane.

2. The method according to claim 1, characterized in that, Step (1) the Trichoderma asperellum is Trichoderma asperellum (ATCC 96517) Trichoderma asperellum ) LYS1.

3. The method of claim 1, wherein, After fermenting Trichoderma hygroscopica for 24-120 hours in step (1), the activated sludge obtained in step (2) is inoculated into the fermentation broth obtained in step (1) for anaerobic fermentation to produce methane.

4. The method of claim 1, wherein, The amount of activated Trichoderma hydatids in step (1) is 5-20% of the volume of the fermentation medium.

5. The method of claim 1, wherein, The maximum outer diameter of the loofah sponge mentioned in step (1) is 2-10 cm, the overall height is 1-3 cm, and the amount added is 8-20 g / L.

6. The method of claim 1, wherein, The fermentation conditions described in step (1) are: fermentation temperature 25-37℃, fermentation pH 4.5-7.5, and rotation speed 0-180 rpm.

7. The method according to claim 1, characterized in that, The fermentation medium containing lignocellulose in step (1) is formulated as follows: 0.2-0.4 g / L urea, 1-2 g / L (NH4)2SO4, 1-3 g / L KH2PO4, 0.1-0.4 g / L CaCl2, 0.1-0.4 g / L MgSO4·7H2O, 0.001-0.01 g / L FeSO4·7H2O, 0.001-0.002 g / L MnSO4·H2O, 0.001-0.002 g / L ZnSO4·7H2O, 0.001-0.003 g / L CoCl2, 20-80 g / L lignocellulose, 3-10 g / L corn steep liquor powder, with water as the solvent, and the pH adjusted to 4.5-7.

5.

8. The method according to claim 1, characterized in that, The activation method of the activated sludge in step (2) is as follows: every 24-48 hours, add 0.05-0.3 mL of revival solution to 25-100 mL of activated sludge, and continue for 3-5 days.

9. The method according to claim 1, characterized in that, The fermentation conditions described in step (3) are: fermentation temperature 35-40℃, fermentation time 20-30 days, fermentation pH 6.8-7.4, and static culture.

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