A method for producing methane by dry anaerobic fermentation

Abstract

The present invention provides a method for producing methane by dry anaerobic fermentation, which relates to the technical field of anaerobic fermentation. In the present invention, a biochar-immobilized complex enzyme is added during the dry anaerobic fermentation process. The biochar-immobilized complex enzyme is achieved by fixing the enzyme to biochar, which can enhance the stability and resistance to protein degradation of the enzyme, extend the service life of the enzyme, and reduce the inactivation rate of the enzyme, thereby facilitating an increase in methane production. In addition, biochar has a high specific surface area and the characteristics of promoting interspecies electron transfer, which is conducive to increasing methane production. Moreover, in the biochar-immobilized complex enzyme, biochar fixes the enzyme on its surface or interior, which can provide more reactive sites, which is conducive to the enrichment of methanogens, thereby shortening the fermentation reaction cycle and facilitating an increase in the reaction rate of anaerobic fermentation. In summary, the reaction rate and methane yield of the method for producing methane by dry anaerobic fermentation provided by the present invention are both high.

Description

Technical Field

[0001] The present invention relates to the technical field of anaerobic fermentation, in particular to a method for producing methane through dry anaerobic fermentation. Background Art

[0002] Dry anaerobic fermentation is a commonly used method for treating solid organic waste. It can convert organic matter into methane and carbon dioxide, which is of great significance for achieving energy transition and environmental protection. However, the increase in organic load during dry anaerobic fermentation can easily lead to acidification instability, affecting the progress of anaerobic fermentation. In addition, problems such as poor mass transfer, slow startup, easy accumulation of inhibitory substances, and high solid content that inhibits microbial activity also restrict the efficiency of dry anaerobic fermentation. Therefore, the reaction rate and methane yield of dry anaerobic fermentation need to be further improved. Summary of the Invention

[0003] The problem solved by the present invention is how to further improve the reaction rate and methane yield of dry anaerobic fermentation.

[0004] To solve the above problems, the present invention provides a method for producing methane by dry anaerobic fermentation, comprising:

[0005] Step S1, preparing biochar-immobilized complex enzyme;

[0006] Step S2: mixing cow dung and crop straw as a substrate, adding an inoculum and a biochar-immobilized complex enzyme to obtain a mixture to be fermented;

[0007] Step S3: subjecting the mixed material to be fermented to dry anaerobic fermentation.

[0008] Optionally, in step S1, the preparation of the biochar-immobilized complex enzyme includes:

[0009] After mixing the mixed enzyme, biochar and citric acid-sodium citrate buffer solution, stirring and centrifuging are carried out in sequence. The precipitate obtained by centrifugation is washed with citric acid-sodium citrate buffer solution until enzyme activity can no longer be detected in the washing liquid, and then freeze-dried to obtain the biochar-immobilized composite enzyme; wherein the mixed enzyme includes cellulase and laccase.

[0010] Optionally, in step S1, the preparation of the biochar-immobilized complex enzyme includes:

[0011] The biochar is dispersed in a glutaraldehyde aqueous solution, and a first stirring treatment and a first centrifugation treatment are performed in sequence. The precipitate obtained by centrifugation is washed and then dried to obtain glutaraldehyde-biochar;

[0012] After mixing the glutaraldehyde-biochar, mixed enzyme and citric acid-sodium citrate buffer solution, a second stirring treatment and a second centrifugation treatment are performed, and the precipitate obtained by centrifugation is washed with a citric acid-sodium citrate buffer solution until the enzyme activity can no longer be detected in the washing liquid, and then freeze-dried to obtain the biochar-immobilized complex enzyme; wherein the mixed enzyme includes cellulase and laccase.

[0013] Optionally, in step S1, the preparation of the biochar-immobilized complex enzyme includes:

[0014] After mixing a mixed enzyme, a citric acid-sodium citrate buffer solution, and glutaraldehyde, biochar is added, stirred, and centrifuged. The precipitate obtained by centrifugation is washed with a citric acid-sodium citrate buffer solution until no enzyme activity can be detected in the washing liquid, and then freeze-dried to obtain the biochar-immobilized complex enzyme; wherein the mixed enzyme includes cellulase and laccase.

[0015] Optionally, in the enzyme mixture, the mass ratio of the cellulase to the laccase is 1:1.

[0016] Optionally, the mass ratio of the mixed enzyme to the biochar is 3:50.

[0017] Optionally, the mass ratio of the mixed enzyme to the glutaraldehyde-biochar is 3:50.

[0018] Optionally, in step S2, the mass ratio of the volatile solids in the biochar-immobilized complex enzyme to the volatile solids in the substrate is 4% to 8%.

[0019] Optionally, in step S2, the dry weight ratio of the cow dung to the crop straw is 1:3; and the mass ratio of the volatile solids in the inoculum to the volatile solids in the substrate is (0.7 to 0.8):1.

[0020] Optionally, in step S3, during the dry anaerobic fermentation process, the solid content of the reaction system is controlled to be 17.5% to 18.5%, the temperature of the dry anaerobic fermentation is 37.5° C. to 38.5° C., and the time is 34 to 36 days.

[0021] Compared with the related art, in the present invention, a biochar-immobilized complex enzyme is added during the dry anaerobic fermentation process. The biochar-immobilized complex enzyme is fixed to the biochar by three methods: adsorption, covalent bonding, and adsorption-crosslinking. This can enhance the stability and resistance of the enzyme to protein degradation, extend the service life of the enzyme, and reduce the inactivation rate of the enzyme, which is beneficial to further improve the stability of the anaerobic fermentation process, thereby helping to increase the production of methane. In addition, biochar has a high specific surface area and the characteristics of promoting interspecies electron transfer, which is also beneficial to further improve the stability of the dry anaerobic fermentation process, thereby helping to increase the production of methane. Moreover, in the biochar-immobilized complex enzyme, the biochar fixes the enzyme on its surface or inside, which can provide more reactive sites, which is beneficial to the enrichment of methanogens, thereby shortening the fermentation reaction cycle and helping to increase the reaction rate of anaerobic fermentation. In summary, the reaction rate and methane yield of the dry anaerobic fermentation methane production method provided by the present invention are both high. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the process of dry anaerobic fermentation to produce methane in an embodiment of the present invention. DETAILED DESCRIPTION

[0023] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. Although certain embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as being limited to the embodiments described herein. Instead, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0025] As used herein, the term "including" and its variations are open-ended, meaning "including but not limited to"; the term "based on" means "based, at least in part, on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments." Definitions of other terms are provided in the following description. It should be noted that the concepts of "first" and "second" mentioned in this disclosure are used to distinguish different objects, not to describe a specific order or hierarchy. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of technical features indicated. Therefore, features specified as "first" or "second" may explicitly or implicitly include one or more of such features. In the description of this disclosure, unless otherwise specified, "plurality" means two or more. It should be noted that, in this disclosure, dry weight refers to the weight of an object after it has been dried and its moisture content has been removed.

[0026] Related technologies have been used to improve dry anaerobic fermentation by adding biochar and enzymes (such as cellulase and laccase), thereby increasing reaction efficiency and methane production. However, this approach has drawbacks, such as poor enzyme stability, easy inactivation, and poor reusability. Furthermore, weak interaction between biochar and enzymes can lead to enzyme shedding and reduced activity. Consequently, the reaction rate and methane yield of dry anaerobic fermentation need to be further improved.

[0027] Based on the above considerations, if Figure 1 As shown, an embodiment of the present invention provides a method for producing methane by dry anaerobic fermentation, comprising:

[0028] Step S1, preparing biochar-immobilized complex enzyme;

[0029] Step S2: mixing cow dung and crop straw as a substrate, adding an inoculum and a biochar-immobilized complex enzyme to obtain a mixture to be fermented;

[0030] Step S3: subjecting the mixed material to be fermented to dry anaerobic fermentation.

[0031] In an embodiment of the present invention, a biochar-immobilized complex enzyme is added during the dry anaerobic fermentation process. The biochar-immobilized complex enzyme is fixed to the biochar by three methods: adsorption, covalent bonding, and adsorption-crosslinking. This can enhance the stability and resistance of the enzyme to protein degradation, extend the service life of the enzyme, and reduce the inactivation rate of the enzyme, thereby helping to improve the stability of the anaerobic fermentation process and thus help to increase the production of methane. In addition, biochar has a high specific surface area and the characteristics of promoting interspecies electron transfer, which is also conducive to improving the stability of the dry anaerobic fermentation process and thus helps to increase the production of methane. Moreover, in the biochar-immobilized complex enzyme, the biochar fixes the enzyme on its surface or inside, which can provide more reactive sites, which is conducive to the enrichment of methanogens, thereby shortening the fermentation reaction cycle and helping to increase the reaction rate of anaerobic fermentation.

[0032] The following are three methods for preparing biochar-immobilized complex enzymes. The specific methods for immobilizing the enzymes on biochar by adsorption are as follows:

[0033] After mixing the mixed enzyme, biochar and citric acid-sodium citrate buffer solution, stirring and centrifuging are carried out in sequence, and the precipitate obtained by centrifugation is washed with a citric acid-sodium citrate buffer solution until the enzyme activity can no longer be detected in the washing liquid, and then freeze-dried to obtain a biochar-immobilized composite enzyme; wherein the mixed enzyme includes cellulase and laccase; in the mixed enzyme, the mass ratio of the cellulase to the laccase is 1:1, the mass ratio of the mixed enzyme to the biochar is 3:50, the pH of the citric acid-sodium citrate buffer solution is 4.8, the citrate concentration in the citric acid-sodium citrate buffer solution is 0.1 mol / L, the ratio of the mass of biochar to the volume of the citric acid-sodium citrate buffer solution is (1:20) g / ml, the stirring speed is 110 rpm to 130 rpm, and the time is 2.5 h to 3.5 h.

[0034] This method is to fix the enzyme on biochar by physical adsorption, which is simple to operate and therefore has a relatively low preparation cost.

[0035] The specific method for immobilizing the enzyme on biochar by covalent bonding to obtain biochar-immobilized complex enzyme is as follows:

[0036] The biochar is dispersed in a glutaraldehyde aqueous solution, and a first stirring treatment and a first centrifugation treatment are sequentially performed. The precipitate obtained by centrifugation is washed and then dried to obtain glutaraldehyde-biochar. In this process, the volume fraction of glutaraldehyde in the glutaraldehyde aqueous solution is 4%.

[0037] After mixing the glutaraldehyde-biochar, the mixed enzyme and the citric acid-sodium citrate buffer solution, a second stirring treatment and a second centrifugation treatment are performed, and the precipitate obtained by centrifugation is washed with a citric acid-sodium citrate buffer solution until the enzyme activity can no longer be detected in the washing liquid, and then freeze-dried to obtain a biochar-immobilized complex enzyme; wherein the mixed enzyme includes cellulase and laccase, and in the mixed enzyme, the mass ratio of the cellulase to the laccase is 1:1, the mass ratio of the mixed enzyme to the glutaraldehyde-biochar is 3:50, and the citrate concentration in the citric acid-sodium citrate buffer solution is 0.1 mol / L; in the solution obtained by mixing the glutaraldehyde-biochar, the mixed enzyme and the citric acid-sodium citrate buffer solution, the ratio of the mass of glutaraldehyde-biochar to the volume of the citric acid-sodium citrate buffer solution is (1:20) g / ml.

[0038] This method fixes the enzyme on biochar through chemical bonds, making the combination of enzyme and biochar stronger and improving the stability and durability of the enzyme.

[0039] The specific method for immobilizing the enzyme on biochar by adsorption-crosslinking method to prepare biochar-immobilized complex enzyme is as follows:

[0040] After mixing a mixed enzyme, a citric acid-sodium citrate buffer solution, and glutaraldehyde, biochar is added, stirred and centrifuged, and the precipitate obtained by centrifugation is washed with a citric acid-sodium citrate buffer solution until no enzyme activity can be detected in the washing liquid, and then freeze-dried to obtain a biochar-immobilized complex enzyme; wherein the mixed enzyme includes cellulase and laccase; in the mixed enzyme, the mass ratio of the cellulase to the laccase is 1:1, the mass ratio of the mixed enzyme to the biochar is 3:50, the pH of the citric acid-sodium citrate buffer solution is 4.8, and the citrate concentration in the citric acid-sodium citrate buffer solution is 0.1 mol / L; in the solution obtained by mixing the mixed enzyme, the citric acid-sodium citrate buffer solution, and glutaraldehyde, the ratio of the mass of biochar to the volume of the citric acid-sodium citrate buffer solution is (1:20) g / ml.

[0041] This method is to initially fix the enzyme on biochar by adsorption, and then further enhance the binding force between the enzyme and biochar through a cross-linking agent.

[0042] In some embodiments of the present invention, in step S2, the mass ratio of the volatile solids in the biochar-immobilized complex enzyme to the volatile solids in the substrate is 4% to 8%.

[0043] In some embodiments of the present invention, in step S2, the dry weight ratio of the cow dung to the crop straw is 1:3; the mass ratio of the volatile solids in the inoculum to the volatile solids in the substrate is (0.7 to 0.8):1.

[0044] In some embodiments of the present invention, in step S3, during the dry anaerobic fermentation process, the solid content of the reaction system is controlled to be 17.5% to 18.5%, the temperature of the dry anaerobic fermentation is 37.5° C. to 38.5° C., and the time is 34 to 36 days.

[0045] In some embodiments of the present invention, in step S2, the method for preparing the inoculum includes: using cow dung as raw material, and anaerobically fermenting it at 34 to 36° C. in an anaerobic fermentation methanogenesis reactor for more than 30 days, and the output of the anaerobic fermentation methanogenesis reactor is the inoculum.

[0046] It should be noted that biochar in the present invention refers to a black solid produced by high-temperature pyrolysis of plant or animal organic matter under anoxic conditions. In some embodiments of the present invention, the biochar preparation method illustratively includes: crushing corn cobs and sieving them, washing them with deionized water, then soaking them in anhydrous ethanol for 24 hours to remove soluble organic impurities, and drying them at 105°C to constant weight to obtain dried corn cob particles; heating the corn cob particles to 500°C under a nitrogen atmosphere, holding the temperature for 2 hours, cooling them to room temperature, crushing them, and passing them through a 100-mesh sieve to obtain biochar.

[0047] The present invention is further described below with reference to specific examples. It should be noted that the citric acid-sodium citrate buffer solution used in the following examples is manufactured by Isejiu (Jiangsu Lianyungang) Biotechnology Co., Ltd., product model BO163. The pH of the citric acid-sodium citrate buffer solution is 4.8, and the citrate concentration in the citric acid-sodium citrate buffer solution is 0.1 mol / L. The biochar used in Examples 5 to 13 and Comparative Examples 1 to 3 was the biochar produced in Example 1.

[0048] Example 1: Preparation of biochar

[0049] The corn cob was crushed and sieved, washed with deionized water, and then soaked in anhydrous ethanol for 24 hours to remove soluble organic impurities, and dried at 105°C to constant weight to obtain dry corn cob particles; the corn cob particles were heated to 500°C under a nitrogen atmosphere, kept warm for 2 hours, cooled to room temperature, crushed, and passed through a 100-mesh sieve to obtain biochar.

[0050] Example 2: Preparation of biochar-immobilized complex enzyme by adsorption method

[0051] Cellulase and laccase were mixed in a mass ratio of 1:1 to obtain a mixed enzyme.

[0052] 60 mg of the mixed enzyme was added to 20 ml of citric acid-sodium citrate buffer solution and mixed evenly to obtain an enzyme solution.

[0053] 1 g of biochar was added to the enzyme solution, mixed evenly, stirred at room temperature to allow the enzyme to adsorb on the biochar, and then centrifuged. The precipitate obtained by centrifugation was washed with a citric acid-sodium citrate buffer solution until the enzyme activity could not be detected in the washing liquid, and then freeze-dried at -20°C to obtain a biochar-immobilized complex enzyme; wherein, the stirring speed was 120 rpm and the time was 3 h; the centrifugal speed was 10,000 rpm and the time was 10 min.

[0054] Example 3: Preparation of biochar-immobilized complex enzyme by covalent binding

[0055] Cellulase and laccase were mixed in a mass ratio of 1:1 to obtain a mixed enzyme.

[0056] The biochar was dispersed in a glutaraldehyde aqueous solution, and a first stirring treatment and a first centrifugation treatment were performed in sequence. The precipitate obtained by centrifugation was washed and then dried to obtain glutaraldehyde-biochar; wherein the volume fraction of glutaraldehyde in the glutaraldehyde aqueous solution was 4%; the first stirring treatment was performed at a speed of 120 rpm for 3 hours; and the first centrifugation treatment was performed at a speed of 10,000 rpm for 10 minutes.

[0057] 60 mg of the mixed enzyme was added to 20 ml of citric acid-sodium citrate buffer solution and mixed evenly to obtain an enzyme solution.

[0058] 1 g of biochar was added to the enzyme solution and mixed evenly. The solution was stirred for a second time at room temperature to allow the enzyme to adsorb on the biochar. The solution was then centrifuged for a second time. The precipitate obtained by centrifugation was washed with a citric acid-sodium citrate buffer solution until no enzyme activity was detected in the washing solution. The solution was then freeze-dried at -20°C to obtain a biochar-immobilized composite enzyme. The second stirring speed was 120 rpm for 3 h, and the second centrifugation speed was 10,000 rpm for 10 min.

[0059] Example 4: Preparation of biochar-immobilized complex enzyme by adsorption-crosslinking method

[0060] Cellulase and laccase were mixed in a mass ratio of 1:1 to obtain a mixed enzyme.

[0061] 60 mg of the mixed enzyme was added to 20 ml of citric acid-sodium citrate buffer solution and mixed evenly to obtain an enzyme solution.

[0062] Glutaraldehyde is added to the enzyme solution to obtain a mixed solution; the volume fraction of glutaraldehyde in the mixed solution is 4%.

[0063] 1 g of biochar was added to the mixed solution, mixed evenly, stirred at room temperature, and then centrifuged. The precipitate obtained by centrifugation was washed with a citric acid-sodium citrate buffer solution until no enzyme activity was detected in the washing solution, and then freeze-dried at -20°C to obtain a biochar-immobilized complex enzyme; wherein the stirring speed was 120 rpm and the time was 3 h; the centrifugal speed was 10,000 rpm and the time was 10 min.

[0064] Example 5

[0065] A1. Cow dung and corn stalks are mixed as a substrate, and an inoculum and a biochar-immobilized complex enzyme are added to obtain a mixture to be fermented; wherein the dry weight ratio of cow dung to corn stalks is 1:3, the mass ratio of volatile solids in the inoculum to volatile solids in the substrate is 0.75:1, the biochar-immobilized complex enzyme is a biochar-immobilized complex enzyme prepared by the method in Example 2, and the mass ratio of volatile solids in the biochar-immobilized complex enzyme to volatile solids in the substrate is 4%; the method for preparing the inoculum comprises: using cow dung as a raw material, anaerobically fermenting at 35° C. in an anaerobic fermentation methanogenesis reactor for more than 30 days, and the discharge of the anaerobic fermentation methanogenesis reactor is the inoculum.

[0066] A2. The mixed material to be fermented is subjected to dry anaerobic fermentation; during the dry anaerobic fermentation, the solid content of the reaction system is controlled to 18%, the temperature of the dry anaerobic fermentation is 38° C., and the fermentation time is 35 days.

[0067] Example 6

[0068] The difference from Example 5 is that in step A1, the mass ratio of the volatile solids in the biochar-immobilized complex enzyme to the volatile solids in the substrate is 6%.

[0069] Example 7

[0070] The difference from Example 5 is that in step A1, the mass ratio of the volatile solids in the biochar-immobilized complex enzyme to the volatile solids in the substrate is 8%.

[0071] Example 8

[0072] The difference from Example 5 is that the biochar-immobilized complex enzyme used in step A1 is the biochar-immobilized complex enzyme prepared by the method in Example 3.

[0073] Example 9

[0074] The difference from Example 8 is that in step A1, the mass ratio of the volatile solids in the biochar-immobilized complex enzyme to the volatile solids in the substrate is 6%.

[0075] Example 10

[0076] The difference from Example 8 is that in step A1, the mass ratio of the volatile solids in the biochar-immobilized complex enzyme to the volatile solids in the substrate is 8%.

[0077] Example 11

[0078] The difference from Example 5 is that the biochar-immobilized complex enzyme used in step A1 is the biochar-immobilized complex enzyme prepared by the method in Example 4.

[0079] Example 12

[0080] The difference from Example 11 is that in step A1, the mass ratio of the volatile solids in the biochar-immobilized complex enzyme to the volatile solids in the substrate is 6%.

[0081] Example 13

[0082] The difference from Example 11 is that in step A1, the mass ratio of the volatile solids in the biochar-immobilized complex enzyme to the volatile solids in the substrate is 8%.

[0083] Comparative Example 1

[0084] A1. Cow dung and corn stalks are mixed as a substrate, and an inoculum, biochar and mixed enzyme are added respectively to obtain a mixture to be fermented; wherein, the dry weight ratio of cow dung to corn stalks is 1:3, the mass ratio of volatile solids in the inoculum to volatile solids in the substrate is 0.75:1, and the combination of biochar and mixed enzyme is used as a first composition, the mass ratio of biochar to mixed enzyme in the first composition is 3:50, the mixed enzyme consists of cellulase and laccase in a mass ratio of 1:1, and the mass ratio of volatile solids in the first composition to volatile solids in the substrate is 4%; the method for preparing the inoculum comprises: using cow dung as raw material, anaerobically fermenting at 35°C in an anaerobic fermentation methanogenesis reactor for more than 30 days, and the output of the anaerobic fermentation methanogenesis reactor is the inoculum.

[0085] A2. The mixed material to be fermented is subjected to dry anaerobic fermentation; during the dry anaerobic fermentation, the solid content of the reaction system is controlled to 18%, the temperature of the dry anaerobic fermentation is 38° C., and the fermentation time is 35 days.

[0086] Comparative Example 2

[0087] The difference from Comparative Example 1 is that in step A1, the mass ratio of the volatile solids in the first composition to the volatile solids in the substrate is 6%.

[0088] Comparative Example 3

[0089] The difference from Comparative Example 1 is that in step A1, the mass ratio of the volatile solids in the first composition to the volatile solids in the substrate is 8%.

[0090] Experimental example

[0091] Table 1 shows the methane production and maximum methane production rate of dry anaerobic fermentation in Examples 5 to 13 and Comparative Examples 1 to 3. Table 1 shows that the methane production and maximum methane production rate of dry anaerobic fermentation in Examples 5 to 13 were higher than those in Comparative Examples 1 to 3. The methane production and maximum methane production rate of dry anaerobic fermentation in Example 11 were higher than those in Examples 5 and 8. The methane production and maximum methane production rate of dry anaerobic fermentation in Example 12 were higher than those in Examples 6 and 9. The methane production and maximum methane production rate of dry anaerobic fermentation in Example 13 were higher than those in Examples 7 and 10. This indicates that, at the same addition amount, dry anaerobic fermentation using the biochar-immobilized complex enzyme prepared by the adsorption-crosslinking method resulted in higher methane production and a higher maximum methane production rate than dry anaerobic fermentation using the biochar-immobilized complex enzyme prepared by the adsorption method and the covalent binding method. In addition, among Examples 5 to 7, the dry anaerobic fermentation in Example 7 exhibited higher methane production and maximum methane production rates, indicating that when the biochar-immobilized complex enzyme prepared in Example 2 (adsorption method) was added to the dry anaerobic fermentation process, the methane production and maximum methane production rates were both higher when the mass ratio of volatile solids in the biochar-immobilized complex enzyme to volatile solids in the substrate was 8%. Among Examples 8 to 10, the dry anaerobic fermentation in Example 9 exhibited higher methane production and maximum methane production rates, indicating that when the biochar-immobilized complex enzyme prepared in Example 3 (covalent binding method) was added to the dry anaerobic fermentation process, the methane production and maximum methane production rates were both higher when the mass ratio of volatile solids in the biochar-immobilized complex enzyme to volatile solids in the substrate was 6%. Among Examples 11 to 13, the methane production and maximum methane production rate of the dry anaerobic fermentation in Example 12 were both higher, indicating that when the biochar-immobilized complex enzyme prepared in Example 4 (adsorption-crosslinking method) was added during the dry anaerobic fermentation process, when the mass ratio of volatile solids in the biochar-immobilized complex enzyme to volatile solids in the substrate was 6%, the methane production and maximum methane production rate of the dry anaerobic fermentation were both higher.

[0092] Table 1

[0093]

[0094] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for producing methane by dry anaerobic fermentation, characterized in that: include: Step S1, preparing biochar-immobilized complex enzyme; Step S2, mixing cow dung and crop straw as a substrate, adding an inoculum and the biochar-immobilized complex enzyme to obtain a mixture to be fermented; Step S3, subjecting the mixed material to be fermented to dry anaerobic fermentation; The preparation of the biochar-immobilized complex enzyme is carried out by any one of the following three methods: Method 1: After mixing the mixed enzyme, biochar and citric acid-sodium citrate buffer solution, stirring and centrifuging are performed in sequence, washing the precipitate obtained by centrifugation with citric acid-sodium citrate buffer solution until no enzyme activity can be detected in the washing solution, and then freeze-drying to obtain the biochar-immobilized composite enzyme; wherein the mixed enzyme includes cellulase and laccase; Method 2: The biochar is dispersed in a glutaraldehyde aqueous solution, and a first stirring treatment and a first centrifugation treatment are performed in sequence. The precipitate obtained by centrifugation is washed and then dried to obtain glutaraldehyde-biochar; After mixing the glutaraldehyde-biochar, the mixed enzyme and the citric acid-sodium citrate buffer solution, performing a second stirring treatment and a second centrifugation treatment, washing the precipitate obtained by centrifugation with the citric acid-sodium citrate buffer solution until no enzyme activity can be detected in the washing solution, and then freeze-drying to obtain the biochar-immobilized complex enzyme; wherein the mixed enzyme includes cellulase and laccase; Method 3: After mixing a mixed enzyme, a citric acid-sodium citrate buffer solution, and glutaraldehyde, biochar is added, stirred, and centrifuged. The precipitate obtained by centrifugation is washed with a citric acid-sodium citrate buffer solution until no enzyme activity can be detected in the washing liquid, and then freeze-dried to obtain the biochar-immobilized complex enzyme; wherein the mixed enzyme includes cellulase and laccase.

2. The method for producing methane by dry anaerobic fermentation according to claim 1, characterized in that: In the enzyme mixture, the mass ratio of the cellulase to the laccase is 1:

1.

3. The method for producing methane by dry anaerobic fermentation according to claim 1, characterized in that: In the method one and the method three, the mass ratio of the mixed enzyme to the biochar is 3:

50.

4. The method for producing methane by dry anaerobic fermentation according to claim 1, wherein: In the second method, the mass ratio of the mixed enzyme to the glutaraldehyde-biochar is 3:

50.

5. The method for producing methane by dry anaerobic fermentation according to claim 1, characterized in that: In step S2, the mass ratio of the volatile solids in the biochar-immobilized complex enzyme to the volatile solids in the substrate is 4% to 8%.

6. The method for producing methane by dry anaerobic fermentation according to claim 1, characterized in that: In step S2, the dry weight ratio of the cow dung to the crop straw is 1:3; the mass ratio of the volatile solids in the inoculum to the volatile solids in the substrate is (0.7 to 0.8):

1.

7. The method for producing methanogen by dry anaerobic fermentation according to claim 1, characterized in that: In step S3, during the dry anaerobic fermentation process, the solid content of the reaction system is controlled to be 17.5% to 18.5%, the temperature of the dry anaerobic fermentation is 37.5° C. to 38.5° C., and the time is 34 to 36 days.

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