Method for producing methane through trichoderma reesei assisted artificial mixed bacteria liquid fermentation after genetic engineering modification

Abstract

The invention relates to the technical field of biology, in particular to a method for producing methane through trichoderma reesei assisted artificial mixed bacteria liquid fermentation after genetic engineering modification. According to the method, an endogenous beta-glucosidase overexpression strain-Trichoderma reesei SEU-7 is utilized to assist artificial bacteria mixing-biogas residue-alkali treatment of corn straw-pig urine to carry out a liquid fermentation methane production test, so that biomass energy conversion of an artificial bacteria mixing-biogas residue-alkali treatment of corn straw-pig urine liquid fermentation system is improved; in addition, methane synthesis bacteria, namely methanosarcina barkeri, are also added into the system, so that the methane synthesis potential is further enhanced.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for producing methane by artificial mixed bacteria liquid fermentation assisted by genetically engineered Trichoderma reesei. Background Art

[0002] At present, lignocellulosic biomass has been applied to important livelihood fields such as the preparation of new energy and the development of medical carriers. Lignocellulose is composed of cellulose, hemicellulose and lignin, and cellulose and hemicellulose have been used to prepare new energy materials such as cellulosic ethanol and methane (McKendry P, Energy production from biomass (part 1): Overview of biomass, Bioresource Technology, 2002, Vol. 83, No. 1, pp. 37-46).

[0003] Currently, lignocellulosic biomass has been used in the methane fermentation process, but its own "stubbornness" hinders the bioavailability of polysaccharides such as cellulose and hemicellulose. Existing studies have found that microorganisms such as bacteria and fungi in nature can secrete laccases, peroxidases, etc. to degrade and depolymerize lignocellulose, thereby enhancing the potential for biomass conversion. Among them, T. reesei has been verified to have a strong ability to secrete cellulases and hemicellulases. To date, Methanosarcinales, Methanobacteriales, Methanomicrobiales, Methanococcales, Methanopyrales, Methanocellales and Methanoplasmatales have been isolated and applied in the methane production process (Ruan D et al., Enhancing methane production of anaerobic sludge digestion by microaeration: Enzyme activity stimulation, semi-continuous reactor validation and microbial community analysis, Bioresour Technol, 2019, Vol. 289, 121643; Dhaked RK et al., Biomethanation under psychrophilic conditions, Waste Manag, 2010, Vol. 30, No. 12, pp. 2490-2496).

[0004] The fermentable sugars in lignocellulose are tightly surrounded by lignin, which makes it difficult to release polysaccharides such as glucans. At the same time, the low relative abundance of methanogens during biogas fermentation is also a major factor causing the unsatisfactory biomass conversion efficiency. Summary of the invention

[0005] In view of this, the present invention provides a method for producing methane by liquid fermentation of artificial mixed bacteria assisted by genetically engineered Trichoderma reesei, wherein an endogenous β-glucosidase overexpressing strain, Trichoderma reesei SEU-7, is used to assist artificial mixed bacteria-biogas residue-alkali-treated corn straw-pig urine in a liquid fermentation methane production experiment, thereby improving the biomass conversion capacity of the artificial mixed bacteria-biogas residue-alkali-treated corn straw-pig urine liquid fermentation system, and then methane-synthesizing bacteria, Methanosarcina barkeri, are added to the system to enhance the methane synthesis potential.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a bacterial strain combination, comprising:

[0008] (i) a strain of the genus Cronobacter sp. with a deposit number of CGMCC NO. 31759; and

[0009] (ii) a strain of the genus Bacteroides sp. with a deposit number of CGMCC NO. 31760; and

[0010] (iii) Enterobacter sp strain with a deposit number of CGMCC NO. 31757; and

[0011] (iv) Enterococcus sp strain with the deposit number of CGMCC NO. 31758.

[0012] In some specific embodiments of the present invention, the above bacterial strain combination also includes:

[0013] (v) Trichoderma reesei SEU-7 with a deposit number of CCTCC M 2016492.

[0014] In some specific embodiments of the present invention, the above bacterial strain combination also includes:

[0015] (vi) Methanosarcina barkeri with the deposit number of ATCC 51582.

[0016] The present invention also provides a methane fermentation system, which comprises biogas residue, alkali-pretreated straw, pig urine and the above bacterial species combination.

[0017] In some specific embodiments of the present invention, the relationship between the amounts of (i) the strain, (ii) the strain, (iii) the strain and (iv) the strain in the above fermentation system is: OD 600 When it is 1.5, the volume ratio is 1:1:1:1.

[0018] In some specific embodiments of the present invention, the relationship between the amount of (v) Trichoderma reesei SEU-7 and (vi) Methanosarcina barkeri in the fermentation system is: OD 600 When it is 1.0, the volume ratio is 1:1.

[0019] In some specific embodiments of the present invention, the relationship between the amounts of (i) the strain, (ii) the strain, (iii) the strain, (iv) the strain, (v) the Trichoderma reesei SEU-7 and (vi) the Methanosarcina barkeri in the fermentation system is: OD 600 The strain (i) has an OD of 1.5 600 The strain (ii) has an OD of 1.5 600 (iii) The strain and OD of 1.5 600 (iv) strain, OD of 1.5 600 The Trichoderma reesei SEU-7 (V) was 1.0 with an OD of 600 The volume ratio of (vi) Methanosarcina barkeri to 1.0 is 1:1:1:1:2:2.

[0020] In some specific embodiments of the present invention, the method for preparing the alkali-pretreated straw in the fermentation system comprises: mixing 10 g of straw and 9 mL of sodium hydroxide with a concentration of 11 g / L, and reacting at 120° C. for 30 min.

[0021] In some specific embodiments of the present invention, the volume of the fermentation system is 300 mL, and the inoculation ratio of the biogas residue is 2%, and the inoculation ratio of the straw is 20%, calculated by volume.

[0022] In some specific embodiments of the present invention, the fermentation system further comprises activated carbon, Tween-80 and manganese ions.

[0023] In some specific embodiments of the present invention, the activated carbon in the above fermentation system is 5 g.

[0024] In some specific embodiments of the present invention, the Tween-80 in the above fermentation system is 50 mg / L.

[0025] In some specific embodiments of the present invention, the manganese ion in the above fermentation system is 15 mg / L.

[0026] In some specific embodiments of the present invention, the straw in the above fermentation system is corn straw.

[0027] In some specific embodiments of the present invention, the fermentation time of the above fermentation system is 17 days.

[0028] The present invention also provides a method for producing methane, which is based on the above-mentioned bacterial strain combination production.

[0029] In some specific embodiments of the present invention, the above production method comprises: mixing any combination of the above bacterial species with a methane fermentation system, and fermenting to obtain methane.

[0030] The present invention also provides a method for producing methane, which is produced based on the above fermentation system.

[0031] In some specific embodiments of the present invention, the production method comprises: mixing the biogas residue, the alkali-pretreated straw, the pig urine and the bacterial combination, and fermenting them to obtain methane.

[0032] The method of the present invention has the following effects:

[0033] On the one hand, in order to address the problem of polysaccharide conversion barriers caused by the structural complexity of lignocellulosic biomass itself, which in turn leads to low product synthesis efficiency during methane fermentation, the present invention first optimizes the lignocellulosic biomass liquid fermentation methane production system, and utilizes different types of pretreatment to depolymerize the lignocellulosic biomass, thereby improving the ability of renewable resources to synthesize methane by liquid fermentation.

[0034] On the other hand, the present invention uses the response surface method to obtain a model. The test results show that the model is extremely significant (p<0.01) and the lack of fit term is not significant (p>0.05), indicating that the model has a good degree of fit and little interference from unknown factors. The model determination coefficient R 2 =0.9858, Corrected determination coefficient R 2Adj=0.9675, indicating that 96.75% of the response value changes can be explained by the model, and the methane content can be effectively analyzed and predicted. The present invention uses the response surface methodology to optimize the artificial mixed bacteria-assisted biogas residue-alkali pretreatment corn straw-pig urine system. The results show that when the activated carbon is 5 g, Tween-80 is 50 mg / L and the manganese ion content is 15 mg / L, the gas accumulation and methane synthesis content of the fermentation system are increased to 550 mL and 5.37%, respectively.

[0035] In addition, the experiment showed that based on the T. reesei SEU-7-artificial mixed bacteria-biogas residue-alkali pretreated corn straw-pig urine fermentation system, the gas volume and methane amount increased by 970 mL and 8.95% respectively after adding M. barkeri ATCC 51582, which were 76.36% and 66.67% higher than those of the group without additional strain addition. However, the gas volume of the group with only M. barkeri ATCC 51582 decreased, and the methane content only increased by 8.94%; the gas volume of the group with only T. reesei SEU-7 increased by only 1.82%, and the methane content only increased by 35.01%. It can be considered that T. reesei SEU-7 and M. barkeri ATCC 51582 synergistically achieved a significant yield increase effect.

[0036] Biological Deposit Description

[0037] Biological material: HK-EPT-7, classification name: Enterobacter sp., deposited on August 26, 2024 at the General Microbiology Center of China Culture Collection Administration. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the collection number is CGMCC No. 31757.

[0038] Biological material: HK-EPT-8, classification name: Enterococcus sp., deposited on August 26, 2024 in the General Microbiology Center of China Culture Collection Administration. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the collection number is CGMCC No. 31758.

[0039] Biological material: HK-EPT-9, classification name: Cronobacter sp., deposited on August 26, 2024 in the General Microbiology Center of China Culture Collection Administration. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the collection number is CGMCC No. 31759.

[0040] Biological material: HK-EPT-10, classification name: Bacteroides sp., deposited on August 26, 2024 in the General Microbiology Center of China Culture Collection Administration. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the collection number is CGMCC No. 31760. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0042] Figure 1 Shown are (a) biogas volume and (b) methane content under different treatments. DETAILED DESCRIPTION

[0043] The present invention discloses a method for producing methane by artificial mixed-bacteria liquid fermentation assisted by genetically engineered Trichoderma reesei. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0044] The present invention utilizes genetic engineering technology to enhance the ability of T. reesei to depolymerize biomass and release polysaccharides to improve the ability of an artificial mixed bacteria-biogas residue-alkali-treated corn straw-pig urine liquid fermentation system to convert biomass, and adds methanogens to the above system to further enhance the potential for liquid fermentation to produce methane.

[0045] The present invention is based on an artificial mixed bacteria combination - Cronobacter. sp strains, Bacteroides. sp strains, Enterobacter. sp strains, Enterococcus. sp strains assisted digestate-alkali treated corn straw-pig urine liquid fermentation system, the liquid fermentation system is 300 mL, the fermentation cycle is 17 days, the inoculum volume ratio is 2%, the solid inoculation ratio is 20% (volume ratio), and pig urine is used to make up the remaining liquid volume. At the same time, the amount of activated carbon added is 5 g of activated carbon, Tween-80 is 50 mg / L, and the manganese ion content is 15 mg / L. In addition, the inoculation amount of the above-mentioned strains is 1 mL each (OD 600 is 1.5).

[0046] In some specific embodiments, the Cronobacter sp strain is a strain with a deposit number of CGMCC NO. 31759.

[0047] In some specific embodiments, the Bacteroides sp strain is a strain with a deposit number of CGMCC NO. 31760.

[0048] In some specific embodiments, the Enterobacter sp strain is a strain with a deposit number of CGMCC NO. 31757.

[0049] In some specific embodiments, the Enterococcus sp strain is a strain with a deposit number of CGMCC NO. 31758.

[0050] In the specific embodiment, Agrobacterium-mediated and T-DNA mutagenesis techniques were used to construct T. reesei SEU-7 and T. reesei TRB1 to assist in the liquid fermentation and methane production test of the artificial mixed bacteria-biogas residue-alkali pretreated corn straw-pig urine system, and the inoculation amount of T. reesei SEU-7 was 2 mL (OD 600 2.0), with the deposit number: CCTCC M2016492, which has been disclosed in other patent applications (e.g., CN201710024825.0).

[0051] In the specific embodiment, the liquid fermentation methane production test was carried out by using Methanosarcina barkeri (M. barkeri) with the deposit number of ATCC 51582 to assist T. reesei SEU-7-artificial mixed bacteria-biogas residue-alkali pretreated corn straw-pig urine system, and the inoculation volume was 2 mL (OD 600 is 1.0).

[0052] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0053] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0054] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0055] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0056] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0057] The biogas residue involved in this application comes from the biogas digesters of farmers' homes and is made through long-term fermentation of livestock manure and straw.

[0058] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0059] The present invention will be further described below in conjunction with the embodiments.

[0060] Example 1

[0061] First, the amount of biomass added in the process of preparing methane by feces-corn straw-liquid fermentation was optimized. Among them, the amount of corn straw added was set to 4%, 8%, 15%, 20% and 30% (volume ratio) respectively. At the same time, the liquid fermentation system of the present invention is 300mL, the liquid fermentation is 17 days, the volume of the inoculum (pig feces) is 2% (volume ratio), and the rest of the reaction system is supplemented by pig urine. The results are shown in Table 1.

[0062] Table 1: Liquid fermentation capacity at different corn straw addition ratios

[0063]

[0064] The results showed that when liquid fermentation was carried out using different corn straw addition ratios, the gas accumulation showed a trend of first increasing and then decreasing with the increase of corn straw addition ratio (Table 1). Among them, when the corn straw addition ratio was 15%, the gas volume was higher than that of other experimental groups. As for the methane synthesis efficiency, when the corn straw addition ratio was 15%, its methane content was the highest and reached 1.57% (Table 1).

[0065] Then, corn straw was depolymerized by different types of pretreatments - alkali pretreatment and ethylenediamine pretreatment. The alkali pretreatment conditions were: 10 g corn straw, 9 mL sodium hydroxide (11 g / L), reaction temperature 120°C, and reaction time 30 min; the ethylenediamine pretreatment conditions were: 10 g corn straw, 9 mL ethylenediamine, reaction temperature 130°C, and reaction time 60 min. The corn straw after the above pretreatment was dried, and the methane production test was carried out with different types of pretreated corn straw inoculation ratios (4%, 8%, 15%, 20%, and 30%). The results are shown in Table 2.

[0066] Table 2: Liquid fermentation capacity of corn straw with different types of pretreatment

[0067]

[0068] From the methane production test of manure-ethylenediamine pretreated corn straw-liquid fermentation in Table 2, it can be seen that with the increase in the proportion of pretreated biomass added in the above system, the gas volume gradually increases and the methane content is low. Compared with the above pretreated corn straw, when 20% pretreated biomass is added to the manure-sodium hydroxide pretreated corn straw-liquid fermentation system, the gas accumulation volume reaches 325 mL. At the same time, the methane content in the above system has increased to 2.35%.

[0069] Example 2

[0070] Liquid fermentation and methane production experiments were carried out with artificial mixed bacteria, biogas residue, alkali-treated corn stalks and pig urine as substrates. The liquid fermentation system was 300 mL, the fermentation cycle was 17 d, the inoculum (biogas residue) inoculation ratio was 2%, the solid inoculation ratio was 20% (i.e., the biomass inoculation ratio, in this embodiment, alkali-pretreated corn stalks), and pig urine was used to make up the remaining liquid volume. The artificial mixed bacteria were Cronobacter. sp strains with a preservation number of CGMCC NO. 31759, Bacteroides. sp strains with a preservation number of CGMCC NO. 31760, Enterobacter. sp strains with a preservation number of CGMCC NO. 31757, and Enterococcus. sp strains with a preservation number of CGMCC NO. 31758, and the inoculation amount was 1 mL (OD 600 is 1.5).

[0071] The conditions for the alkaline pretreatment are: 10 g corn stalks and 9 mL of sodium hydroxide with a concentration of 11 g / L are mixed, the reaction temperature is 120°C, and the reaction time is 30 min. The specific steps are: 10 g corn stalks (dry weight) and 9 mL of sodium hydroxide solution are added to the reactor and stirred evenly. After tightening the reactor, the reactor is placed in a 120°C constant temperature oil bath. After reacting for 30 min, the reactor is taken out and cooled to room temperature with tap water. The reacted corn stalks are washed 3 times with 100 mL of distilled water each time and filtered with gauze. The washed stalks are dried in an oven at 60°C overnight to a moisture content of less than 10%, and stored in a ziplock bag at room temperature.

[0072] The response surface analysis software Design-Expert8.0 was used for experimental design. The amount of activated carbon added, Tween-80 content, and trace heavy metal (manganese) content were used as response variables, and the methane content was used as the response value. The experiment was carried out according to the response surface design scheme and the response values ​​were filled in for data analysis (Table 3). The quadratic polynomial regression equation obtained was:

[0073] Y=5.19 - 0.021 A + 0.016 B + 0.032 C - 0.070 AB +0.096 AC - 0.13 BC -0.75 A 2 – 0.52 B 2 -1.59 C 2 .

[0074] Table 3: Box-Behnken test plan and results

[0075]

[0076] Table 4: Regression model and ANOVA

[0077]

[0078] As shown in Table 4, the model is extremely significant (p < 0.01) and the lack of fit term is not significant (p > 0.05), indicating that the model has a good fit and little interference from unknown factors. 2 =0.9858, Corrected determination coefficient R 2 Adj=0.9675. The above results show that 96.75% of the response value changes can be explained by this model, and it can effectively analyze and predict the methane content. 2 , B 2 and C 2The influence on methane content was significant (p<0.01). According to the p value of each factor, the order of influence of each parameter on methane content can be obtained: trace heavy metal (manganese) content > activated carbon addition > Tween-80 content.

[0079] The response surface methodology was used to optimize the artificial mixed bacteria-assisted biogas residue-alkali pretreatment corn straw-pig urine system. The results showed that when the activated carbon was 5 g, Tween-80 was 50 mg / L and the manganese ion content was 15 mg / L, the gas accumulation and methane synthesis content increased to 550 mL and 5.37%, respectively.

[0080] Example 3

[0081] Based on the artificial mixed bacteria-biogas residue-alkali pretreatment corn straw-pig urine system, a total of 2 mL OD 600 2.0 strains, and the compositions of the strains in different experimental groups are:

[0082] M. barkeri ATCC 51582;

[0083] T. reesei TRB1;

[0084] T. reesei SEU-7;

[0085] T. reesei SEU-7+M. barkeri ATCC 51582.

[0086] The liquid fermentation system is 300 mL, the fermentation cycle is 17 days, the inoculum (sludge) inoculation ratio is 2%, the solid inoculation ratio is 20% (i.e. the biomass inoculation ratio, in this embodiment, alkali pretreated corn stalks), and the remaining liquid volume is made up with pig urine. At the same time, the amount of activated carbon added is 5 g, the Tween-80 content is 50 mg / L, and the manganese ion content is 15 mg / L.

[0087] The artificial mixed bacteria are: Cronobacter.sp strain with a preservation number of CGMCC NO. 31759, Bacteroides.sp strain with a preservation number of CGMCC NO. 31760, Enterobacter.sp strain with a preservation number of CGMCC NO. 31757, and Enterococcus.sp strain with a preservation number of CGMCC NO. 31758. The inoculation volume of each strain is 1 mL (OD 600 is 1.5).

[0088] The conditions of the alkaline pretreatment are: 10 g corn stalks and 9 mL of sodium hydroxide with a concentration of 11 g / L are mixed, the reaction temperature is 120°C, and the reaction time is 30 min. The specific steps are: 10 g corn stalks (dry weight) and 9 mL of sodium hydroxide solution are added to the reactor and stirred evenly. After tightening the reactor, put the reactor into a 120°C constant temperature oil bath. After reacting for 30 min, take out the reactor and cool it to room temperature with tap water. The reacted corn stalks are washed 3 times with 100 mL of distilled water each time and filtered with gauze. The washed stalks are dried in a 60°C oven overnight to a moisture content of less than 10%, and stored in a ziplock bag at room temperature.

[0089] The experimental results are as follows Figure 1 , as shown in Table 5.

[0090] Table 5: Gas production capacity of different types of liquid fermentation

[0091]

[0092] See also Figure 1 , Table 5, from the gas accumulation and methane synthesis rate, it can be seen that after T. reesei SEU-7 was added to the above liquid fermentation system, its gas volume did not change much, but the methane content further increased to 7.25%. Compared with T. reesei SEU-7, after T. reesei TRB1 was added to the artificial mixed group-biogas residue-alkali pretreated corn straw-pig urine system, its methane synthesis ability was inhibited.

[0093] Surprisingly, based on the fermentation system of T. reesei SEU-7-artificial mixed bacteria-biogas residue-alkali pretreated corn straw-pig urine, after M. barkeri ATCC 51582 was mixed into it, its gas volume and methane amount increased by 970 mL and 8.95%, respectively, which were 76.36% and 66.67% higher than those without additional strains. In contrast, the gas volume of the group with only M. barkeriATCC 51582 was reduced, and the methane content was only increased by 8.94%. The gas volume of the group with only T. reesei SEU-7 increased by only 1.82%, and the methane content was only increased by 35.01%. It is obvious that T. reesei SEU-7 and M. barkeri ATCC 51582 synergistically achieved a significant increase in production. In summary, the present invention provides a strategy with reference significance for the preparation of methane by biomass liquid fermentation.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bacterial strain combination, characterized in that: include: (i) Cronobacter sp strain with a deposit number of CGMCC NO. 31759; and (ii) Bacteroides sp strain with a deposit number of CGMCC NO. 31760; and (iii) Enterobacter sp strain with a deposit number of CGMCC NO. 31757; and (iv) Enterococcus sp strain with the deposit number of CGMCC NO. 31758.

2. The bacterial strain combination according to claim 1, characterized in that: Also includes: (v) Trichoderma reesei SEU-7 with a deposit number of CCTCC M 2016492.

3. The bacterial strain combination according to claim 1 or 2, characterized in that: Also includes: (vi) Methanosarcina barkeri with the deposit number of ATCC 51582.

4. A methane fermentation system, characterized in that: The method comprises biogas residue, alkali-pretreated straw, pig urine and the bacterial strain combination according to any one of claims 1 to 3.

5. The fermentation system according to claim 4, characterized in that The preparation method of the alkali-pretreated straw comprises: mixing 10 g of straw and 9 mL of sodium hydroxide with a concentration of 11 g / L, and reacting at 120° C. for 30 min.

6. The fermentation system according to claim 4 or 5, characterized in that: The volume of the fermentation system is 300 mL. In terms of volume ratio, the inoculation ratio of the biogas residue is 2%, and the inoculation ratio of the straw is 20%.

7. The fermentation system according to any one of claims 4 to 6, characterized in that: Also included are activated carbon, Tween-80 and manganese ions.

8. The fermentation system according to claim 7, characterized in that The activated carbon is 5 g, the Tween-80 is 50 mg / L, and the manganese ion is 15 mg / L.

9. The fermentation system according to any one of claims 4 to 6, characterized in that: The straw is corn straw.

10. A method for producing methane, characterized in that: Produced based on any of the following: (a) The bacterial strain combination according to any one of claims 1 to 3; (b) The fermentation system according to any one of claims 4 to 9.

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