Method for producing methane through dry anaerobic fermentation

Abstract

The invention provides a method for producing methane through dry anaerobic fermentation, and relates to the technical field of anaerobic ferment.The method is characterized in that a charcoal immobilized compound enzyme is added in the dry anaerobic fermentation process, and the charcoal immobilized compound enzyme is formed by immobilizing enzyme on charcoal, so that the stability and protein degradation resistance of the enzyme can be enhanced; the service life of the enzyme is prolonged, and the inactivation speed of the enzyme is reduced, so that the yield of methane is increased. In addition, the biochar has the characteristics of relatively high specific surface area and capability of promoting interspecific electron transfer, so that the yield of methane can be increased. Moreover, in the biochar immobilized compound enzyme, the biochar immobilizes the enzyme on the surface or in the biochar, more reaction active sites can be provided, and the enrichment of methanogens is facilitated, so that the fermentation reaction period is shortened, and the anaerobic fermentation reaction rate is improved. In conclusion, the method for producing methane through dry anaerobic fermentation, provided by the invention, is relatively high in reaction rate and methane yield.

Description

Technical Field

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

[0002] Dry anaerobic fermentation is one of the commonly used methods for treating solid organic waste. It can convert organic matter into methane and carbon dioxide, which is of great significance for achieving energy transformation and environmental protection. However, the increase in organic load during dry anaerobic fermentation is prone 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 inhibiting 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 invention is: how to further improve the reaction rate and methane yield of dry anaerobic fermentation.

[0004] In order 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 composite enzyme;

[0006] Step S2, mixing cow dung and crop straw as a substrate, adding inoculants and biochar-immobilized complex enzymes 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] The mixed enzyme, biochar and citric acid-sodium citrate buffer solution are mixed, followed by stirring and centrifugation. 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 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, and a precipitate obtained by centrifugation is washed and then dried to obtain glutaraldehyde-biochar;

[0012] After the glutaraldehyde-biochar, mixed enzyme and citric acid-sodium citrate buffer solution are mixed, 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 composite 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 the mixed enzyme, 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 the 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.

[0015] Optionally, in the mixed enzyme, 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; the mass ratio of the volatile solids in the inoculant 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 days 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, namely, adsorption, covalent bonding, and adsorption-crosslinking. The enzyme can enhance the stability and resistance to protein degradation of the enzyme, prolong 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 facilitating the increase of methane production. 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 facilitating the increase of methane production. 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 facilitating the increase of the reaction rate of anaerobic fermentation. In summary, the reaction rate and methane yield of the dry anaerobic fermentation methanogenesis 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] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with 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 interpreted as being limited to the embodiments described herein. On the contrary, 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 only for exemplary purposes 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 of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0025] The term "including" and its variations used in this article are open inclusions, that is, "including but not limited to"; the term "based on" is "based at least in part on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one other embodiment"; the term "some embodiments" means "at least some embodiments"; the term "optionally" means "optional embodiments". The relevant definitions of other terms will be given in the following description. It should be noted that the concepts of "first" and "second" mentioned in the present invention are used to distinguish different objects, rather than to describe a specific order or a primary and secondary relationship. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, the meaning of "multiple" is two or more. It should be noted that in the present invention, the dry weight is the weight of the object after drying and removing water.

[0026] In the related art, the dry anaerobic fermentation process is improved by adding biochar and enzymes (such as cellulase and laccase) respectively, thereby improving the reaction efficiency and methane production. However, this method has some disadvantages, such as poor enzyme stability, easy inactivation, poor reusability, and weak interaction between biochar and enzyme, which leads to enzyme shedding and reduced activity. Therefore, 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 composite enzyme;

[0029] Step S2, mixing cow dung and crop straw as a substrate, adding inoculants and biochar-immobilized complex enzymes 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 fixes the enzyme to the biochar by three methods, namely, adsorption, covalent bonding, and adsorption-crosslinking. This can enhance the stability and resistance to protein degradation of the enzyme, prolong 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 facilitating the increase in methane production. 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 facilitating the increase in methane production. 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 facilitating the increase in the reaction rate of anaerobic fermentation.

[0032] The following are three methods for preparing biochar-immobilized composite enzymes. The specific method for immobilizing the enzyme on biochar by adsorption to obtain the biochar-immobilized composite enzyme is as follows:

[0033] After mixing the mixed enzyme, biochar and citric acid-sodium citrate buffer solution, stirring treatment and centrifugation treatment are carried out in sequence, and the precipitate obtained by centrifugation is washed with 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 to biochar by physical adsorption, which is simple to operate and therefore has a relatively low preparation cost.

[0035] The specific method of immobilizing the enzyme on biochar by covalent bonding to obtain the biochar-immobilized composite 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 performed in sequence. 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 the glutaraldehyde-biochar, the mixed enzyme and the citric acid-sodium citrate buffer solution are mixed, 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 composite 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 glutaraldehyde-biochar, the mixed enzyme and the citric acid-sodium citrate buffer solution, the mass ratio of glutaraldehyde-biochar to the volume of the citric acid-sodium citrate buffer solution is (1:20) g / ml.

[0038] This method is to fix the enzyme on biochar through chemical bonds, making the combination of enzyme and biochar more firmly and improving the stability and durability of the enzyme.

[0039] The specific method of fixing the enzyme on biochar by adsorption-crosslinking method to obtain biochar-immobilized composite 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 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, 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] The method is to initially fix the enzyme on the biochar by adsorption, and then further enhance the binding force between the enzyme and the biochar by 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 inoculant 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 days 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, 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 made by high-temperature pyrolysis of organic matter from plants or animals under anaerobic conditions. In some embodiments of the present invention, illustratively, the method for preparing biochar includes: crushing corn cobs and sieving them, washing them with deionized water, and then soaking them in anhydrous ethanol for 24 hours to remove soluble organic impurities, drying them at 105°C to constant weight, and obtaining dried corn cob particles; heating the corn cob particles to 500°C in a nitrogen atmosphere, keeping them warm 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 in conjunction with specific examples. It should be noted that the manufacturer of the citric acid-sodium citrate buffer solution used in the following examples is Isejiu (Jiangsu Lianyungang) Biotechnology Co., Ltd., the product model is 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 is the biochar prepared 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 in 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] The cellulase and laccase were mixed in a mass ratio of 1:1 to obtain a mixed enzyme.

[0052] Add 60 mg of the mixed enzyme into 20 ml of citric acid-sodium citrate buffer solution and mix well 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 composite enzyme; wherein the stirring speed was 120 rpm and the time was 3 h; the centrifugal speed was 10000 rpm and the time was 10 min.

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

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

[0056] 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; wherein the volume fraction of glutaraldehyde in the glutaraldehyde aqueous solution is 4%; the first stirring treatment speed is 120 rpm, and the time is 3 hours; the first centrifugation treatment speed is 10000 rpm, and the time is 10 minutes.

[0057] Add 60 mg of the mixed enzyme into 20 ml of citric acid-sodium citrate buffer solution and mix well to obtain an enzyme solution.

[0058] 1 g of biochar was added to the enzyme solution and mixed evenly. The solution was stirred for the second time at room temperature to allow the enzyme to adsorb on the biochar. The solution was then centrifuged for the second time. 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 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. The second centrifugation speed was 10,000 rpm for 10 min.

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

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

[0061] Add 60 mg of the mixed enzyme into 20 ml of citric acid-sodium citrate buffer solution and mix well 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 the enzyme activity could not be detected in the washing solution, and then freeze-dried at -20°C to obtain a biochar-immobilized composite enzyme; wherein the stirring speed was 120 rpm and the time was 3 h; the centrifugal speed was 10000 rpm and the time was 10 min.

[0064] Example 5

[0065] A1. Mix cow dung and corn stalks as a substrate, add inoculants and biochar-immobilized complex enzymes 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 inoculant 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 preparation method of the inoculant comprises: using cow dung as a raw material, anaerobically fermenting at 35° C. for more than 30 days in an anaerobic fermentation methanogenesis reactor, and the output of the anaerobic fermentation methanogenesis reactor is the inoculant.

[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 be 18%, the temperature of the dry anaerobic fermentation is 38° C., and the 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] Embodiment 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. Mix cow dung and corn stalks as a substrate, add inoculant, biochar and mixed enzyme 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 inoculant to volatile solids in the substrate is 0.75:1, and the combination of biochar and mixed enzyme is used as the first composition, the mass ratio of biochar to mixed enzyme in the first composition is 3:50, the mixed enzyme is composed 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 preparation method of the inoculant 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 output of the anaerobic fermentation methanogenesis reactor is the inoculant.

[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 be 18%, the temperature of the dry anaerobic fermentation is 38° C., and the 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] The methane production and maximum methane production rate of dry anaerobic fermentation in Examples 5 to 13 and Comparative Examples 1 to 3 were statistically analyzed, and the results are shown in Table 1. It can be seen from Table 1 that compared with Comparative Examples 1 to 3, the methane production and maximum methane production rate of dry anaerobic fermentation in Examples 5 to 13 are both higher. Compared with Examples 5 and 8, the methane production and maximum methane production rate of dry anaerobic fermentation in Example 11 are both higher, compared with Examples 6 and 9, the methane production and maximum methane production rate of dry anaerobic fermentation in Example 12 are both higher, and compared with Examples 7 and 10, the methane production and maximum methane production rate of dry anaerobic fermentation in Example 13 are both higher, indicating that under the same addition amount, compared with the biochar immobilized composite enzyme prepared by the adsorption method and the covalent binding method for dry anaerobic fermentation, the biochar immobilized composite enzyme prepared by the adsorption-crosslinking method has a higher methane production and a higher maximum methane production rate when dry anaerobic fermentation is performed. In addition, in Examples 5 to 7, the methane production and maximum methane production rate of the dry anaerobic fermentation in Example 7 are both high, indicating that when the biochar immobilized complex enzyme prepared in Example 2 (adsorption method) is added during the dry anaerobic fermentation, when the mass ratio of volatile solids in the biochar immobilized complex enzyme to volatile solids in the substrate is 8%, the methane production and maximum methane production rate of the dry anaerobic fermentation are both high. In Examples 8 to 10, the methane production and maximum methane production rate of the dry anaerobic fermentation in Example 9 are both high, indicating that when the biochar immobilized complex enzyme prepared in Example 3 (covalent binding method) is added during the dry anaerobic fermentation, when the mass ratio of volatile solids in the biochar immobilized complex enzyme to volatile solids in the substrate is 6%, the methane production and maximum methane production rate of the dry anaerobic fermentation are both high. Among Examples 11 to 13, the methane production and the maximum methane production rate of the dry anaerobic fermentation in Example 12 are both relatively high, indicating that when the biochar immobilized complex enzyme prepared in Example 4 (adsorption-crosslinking method) is 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 is 6%, the methane production and the maximum methane production rate of the dry anaerobic fermentation are both relatively high.

[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 composite enzyme; Step S2, mixing cow dung and crop straw as a substrate, adding an inoculant 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.

2. The method for producing methane by dry anaerobic fermentation according to claim 1, characterized in that: In the step S1, the preparation of the biochar-immobilized complex enzyme comprises: The mixed enzyme, biochar and citric acid-sodium citrate buffer solution are mixed, followed by stirring and centrifugation. 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 composite enzyme; wherein the mixed enzyme includes cellulase and laccase.

3. The method for producing methane by dry anaerobic fermentation according to claim 1, characterized in that: In the step S1, the preparation of the biochar-immobilized complex enzyme comprises: The biochar is dispersed in a glutaraldehyde aqueous solution, and a first stirring treatment and a first centrifugation treatment are performed in sequence, and a precipitate obtained by centrifugation is washed and then dried to obtain glutaraldehyde-biochar; After the glutaraldehyde-biochar, mixed enzyme and citric acid-sodium citrate buffer solution are mixed, 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 composite enzyme; wherein the mixed enzyme includes cellulase and laccase.

4. The method for producing methane by dry anaerobic fermentation according to claim 1, characterized in that: In the step S1, the preparation of the biochar-immobilized complex enzyme comprises: After mixing the mixed enzyme, 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 the 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.

5. The method for producing methane by dry anaerobic fermentation according to any one of claims 2 to 4, characterized in that: In the mixed enzyme, the mass ratio of the cellulase to the laccase is 1:

1.

6. The method for producing methane by dry anaerobic fermentation according to claim 2 or 4, characterized in that: The mass ratio of the mixed enzyme to the biochar is 3:

50.

7. The method for producing methane by dry anaerobic fermentation according to claim 3, characterized in that: The mass ratio of the mixed enzyme to the glutaraldehyde-biochar is 3:

50.

8. The method for producing methane by dry anaerobic fermentation according to any one of claims 1 to 4, characterized in that: In the 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%.

9. The method for producing methane by dry anaerobic fermentation according to any one of claims 1 to 4, characterized in that: In the 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 inoculant to the volatile solids in the substrate is (0.7 to 0.8):

1.

10. The method for producing methane by dry anaerobic fermentation according to any one of claims 1 to 4, characterized in that: In step S3, during the dry anaerobic fermentation, 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 days to 36 days.

Images