Method for utilizing agricultural ferment yellow silage straw and anaerobic fermentation of yellow silage straw

Abstract

The invention belongs to the technical field of yellow corn silage of straw, and particularly relates to a yellow corn silage straw and yellow corn silage straw anaerobic fermentation method using agricultural enzymes, and the method comprises the following steps: S1, mixing disinfected waste fruits and vegetables and withered straw according to a mass ratio of 1: 0.5-2, then inoculating 0.5-3% of an acid-resistant cellulose degradation bacterial system to obtain mixed waste, and carrying out anaerobic fermentation at 22-28 DEG C for 60 d or more to obtain mixed waste; filtering and taking a liquid part as an agricultural enzyme; and S2, diluting the agricultural enzyme until the mass ratio is 25-70%, then adding the withered straws into the diluted agricultural enzyme, controlling the water content of the system to be 55-65%, supplementing 0.5-2g / L lysine and 0.5-2g / L methionine, uniformly mixing to obtain the yellow corn silage, compacting the yellow corn silage, and sealing and storing. According to the method, the withered straw can be stored for a long time, the storage stability is improved, lignocellulose degradation is promoted, and the methane production potential of the yellow corn silage straw is improved.

Description

Technical Field

[0001] The invention relates to the technical field of straw yellowing, and in particular to a method for yellowing straw using agricultural enzymes and anaerobic fermentation of the yellowed straw. Background Art

[0002] my country has abundant crop straw resources. Except for a small amount used as feed and directly returned to the field, most of them are used as fuel or burned directly in the fields, which not only pollutes the environment, but also causes a huge waste of resources. Anaerobic digestion is an effective means to turn crop straw into treasure. Under anaerobic conditions, organic waste such as straw can be decomposed and utilized by anaerobic microorganisms to produce biogas. Returning biogas residue to the field can also help maintain soil fertility, improve humus and the nutrient cycle between soil and crops, and has key environmental and economic benefits. However, there is a contradiction between the continuous operation of biogas projects and the seasonal supply of crop straw. Crop straw is usually harvested in autumn and must be properly stored to provide high-quality raw materials for biogas production throughout the year. In addition, crop straw is usually harvested after wilting or drying. The semi-dry straw loses a lot of water and sugar, is highly fibrous and lignified, and has a high polymer content and dense structure. Reduce the biodegradability and conversion efficiency of the yellow straw, which easily leads to problems such as low biogas production efficiency. Therefore, how to achieve long-term storage of yellow straw has become an important issue facing straw biogas projects.

[0003] Silage and yellowing technology was first applied to the field of feed storage. Among them, fresh straw with a moisture content of 60%-75% is used for silage. The harvested fresh straw is crushed and directly compacted and sealed. Under suitable humidity conditions, the microorganisms contained in the fresh straw are used for anaerobic fermentation to produce organic acids, which reduces the pH value of the silage raw material to 4.0-5.0, thereby inhibiting the growth of microorganisms, so as to achieve the purpose of long-term preservation of straw and reduce nutrient loss. In recent years, silage technology has been applied to the pretreatment of straw. Silage fermentation can loosen the lignocellulose structure in the straw, improve the dry matter retention rate and the biodegradability of lignocellulose, and play a positive role in increasing biogas production. However, for air-dried and yellowed straw, the moisture content of the raw material is too low, and the water-soluble carbohydrates (WSCs) and microorganisms are lost, which will significantly affect its biological accessibility, making it unsuitable for direct silage operations. Yellow storage is a straw fermentation method relative to silage. It mainly uses dry straw as raw material. After crushing, it is added with appropriate amount of water and biological agents, and then baled and bagged to achieve good preservation of nutrients in the raw material. Yellow storage has lower requirements for raw materials and is not limited by straw moisture content and straw harvesting time. It has a broader application prospect in straw biogas projects. However, this storage method will cause a large loss of nutrient components in the straw raw material, enhance the degradation of lignocellulose, and face many problems such as long anaerobic gas production cycle, low conversion rate, and low gas production. In addition, additives such as inoculation of lactic acid bacteria (LAB) or supplementation of WSCs-rich materials (such as fresh straw) are required to promote acid production and enhance the yellow storage effect. The application of these additives is often limited by high cost or regional factors. Therefore, finding a low-cost yellow storage method suitable for subsequent straw fermentation is a key issue that needs to be urgently solved for efficient straw fermentation to produce biogas. Summary of the invention

[0004] In view of the above technical problems in the prior art, the present invention provides a method for yellowing straw using agricultural enzymes. The method of the present invention can achieve long-term stable storage of withered straw, while helping to improve the biodegradability of the straw and enhance the methane production potential of the yellowing straw.

[0005] To achieve the above object, the present invention is specifically implemented through the following technical solutions:

[0006] The first aspect of the present invention provides a method for yellowing straw using agricultural enzymes, comprising the following steps:

[0007] S1. Mix the sterilized discarded fruits and vegetables and withered straw in a mass ratio of 1:0.5-2, then inoculate 0.5-3% of acid-resistant cellulose-degrading bacteria to obtain mixed waste, perform anaerobically fermentation at 22-28°C for 60 days or more, and filter the liquid portion as agricultural enzyme;

[0008] S2. Dilute the agricultural enzyme to a mass ratio of 25-70%, then add the withered straw into the diluted agricultural enzyme, control the water content of the system to 55-65%, supplement 0.5-2g / L lysine and 0.5-2g / L methionine, mix well to obtain yellow storage material, compact the yellow storage material and seal it for storage.

[0009] Furthermore, in step S1, the acid-resistant cellulose degrading bacteria are obtained by restricted subculture, comprising the following steps:

[0010] S11, inoculating the microorganisms isolated from nature into a seed culture medium, and culturing anaerobically at 22-28° C. for 48 h, wherein the seed culture medium comprises the following components: 10 g / L glucose, 5 g / L microcrystalline cellulose, 5 g / L peptone, 3 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and 2 g / L sodium phosphate, with a pH of 7.0;

[0011] S12, performing gradient restricted subculture on the culture of step S11, wherein:

[0012] During the first generation of subculture, the glucose in the seed culture medium was adjusted to 5 g / L, the microcrystalline cellulose to 5 g / L and the pH to 6.5, and the culture was performed for 48 hours;

[0013] During the second generation subculture, the glucose in the seed culture medium was adjusted to 3 g / L, the microcrystalline cellulose to 7 g / L and the pH to 6.0, and the culture was performed for 48 hours;

[0014] During the third generation subculture, the glucose in the seed culture medium was adjusted to 2 g / L, the microcrystalline cellulose to 8 g / L and the pH to 5.5, and the culture was carried out for 48 hours;

[0015] During the fourth generation of subculture, the glucose in the seed culture medium was adjusted to 1 g / L, the microcrystalline cellulose to 9 g / L and the pH to 5.5, and the culture was carried out for 48 hours;

[0016] During the fifth generation of subculture, the glucose in the seed culture medium was adjusted to 0.5 g / L, the microcrystalline cellulose to 10 g / L and the pH to 5.5, and the culture was carried out for 48 hours;

[0017] The same conditions as those for the fifth generation were used to continue subculture for 10 generations, and the microorganisms in the culture solution were separated to obtain the acid-resistant cellulose degrading bacterial strain.

[0018] Furthermore, in step S11, the microorganisms isolated from nature are derived from microorganisms in soil and compost; in step S12, the pH value is adjusted by L-lactic acid.

[0019] Furthermore, in step S1, the inoculation amount of the acid-resistant cellulose degrading bacteria is 1%.

[0020] Furthermore, in step S1, the mass ratio of the discarded fruits and vegetables to the withered straw is 1:1.

[0021] Furthermore, in step S1, the mass ratio of the mixed waste, the brown sugar and the water is 3:1:10; the mixture is anaerobically fermented at 25°C for 60 days, and the liquid portion is filtered and taken as the agricultural enzyme.

[0022] Furthermore, in step S1, the discarded fruits and vegetables are selected from at least one of oranges, apples, pears, cabbages, Chinese cabbages and tomatoes; and the withered straw is selected from at least one of corn straws, sorghum straws, sugarcane straws, rice straws and wheat straws.

[0023] The second aspect of the present invention provides a yellow silage straw, which is prepared by the method of using agricultural enzyme yellow silage straw as described above.

[0024] The third aspect of the present invention provides the use of the above-mentioned silage straw in anaerobic fermentation to produce biogas.

[0025] A fourth aspect of the present invention provides a method for anaerobic fermentation of silage straw, comprising the following steps:

[0026] The yellow silage straw prepared above is mixed with anaerobic digestion sludge in a total solid ratio of 1:3-5, and medium-temperature anaerobic fermentation is carried out.

[0027] The advantages and positive effects of the present invention are:

[0028] 1. The present invention uses discarded withered straw and fruits and vegetables as raw materials, and adds acid-resistant cellulose-degrading bacteria that can adapt to acidic environments and use cellulose as a substrate, and sugar carbon sources, and performs co-fermentation under anaerobic conditions. The prepared agricultural enzyme has a high content of water-soluble carbohydrates and a high content of VFAs such as lactic acid, formic acid, acetic acid, and acetic acid, and has efficient cellulose degradation ability. It can recruit beneficial bacteria and supplement VFA during the yellow storage process to inhibit the growth of undesirable microorganisms, reduce the dry matter loss of straw, and achieve long-term stable quality storage of withered straw. At the same time, the addition of the agricultural enzyme of the present invention helps to increase the cellulose degradation rate of the straw, enhance biodegradability, and thereby enhance the methane production potential of the yellow storage straw and improve the efficiency of the biogas project.

[0029] 2. The agricultural enzyme of the present invention is an acidic liquid obtained by anaerobic co-fermentation of rotten fruits and vegetables and dry withered straw, which has the advantages of low cost and easy acquisition of raw materials; it can reduce the cost of yellow storage straw and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 The physicochemical properties of the yellow silage straw of the embodiment of the present invention are shown in Figure (a), wherein the dry matter content, organic dry matter content and dry matter loss rate of the yellow silage straw, Figure (b) is the volatile fatty acid content and pH value of the yellow silage straw, Figure (c) is the water-soluble carbohydrate content of the yellow silage straw, and Figure (d) is the lignocellulose content of the yellow silage straw;

[0032] Figure 2 This is an analysis of the microbial composition of yellow silage straw in an embodiment of the present invention, wherein Figure (a) shows the abundance of species communities at the phylum level, and Figure (b) shows the abundance of species communities at the genus level;

[0033] Figure 3 The present invention is an analysis of key microbial groups in yellow silage straw, wherein Figure (a) is a cladogram of LEfSe analysis, and Figure (b) is a network interaction diagram of Pearson correlation analysis;

[0034] Figure 4 It is a Pearson correlation heat map between the microbial community and environmental factors of the embodiment of the present invention;

[0035] Figure 5 A network interaction diagram between microbial communities and environmental factors according to an embodiment of the present invention;

[0036] Figure 6 This is a curve diagram of the cumulative methane production of yellow silage straw according to an embodiment of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0039] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, etc. should be understood as modified by the word "approximately" in all cases. The term "approximately" has its usual meaning, which is used to indicate that a value includes the inherent variation of the error of the device or method used to determine the value, or contains a value close to the value, such as within 10% of the value (or range of values). Therefore, unless otherwise specified, the numerical parameters listed in the specification and the attached claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained.

[0040] In addition, it should be noted that the terms "include", "comprises", "contains", "has" and similar words are non-restrictive, that is, other steps and other components that do not affect the results can be added.

[0041] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0042] The embodiment of the present invention provides a method for yellowing straw using agricultural enzymes, comprising the following steps:

[0043] S1. Preparing agricultural enzymes: Mixing sterilized discarded fruits and vegetables and withered straw in a mass ratio of 1:0.5-2, and then inoculating 0.5-3% (wt) of an acid-resistant cellulose-degrading bacterial system to obtain mixed waste; adding brown sugar and water to the mixed waste in a mass ratio of 3:0.5-2:5-15, that is, the mass ratio of the mixed waste, the brown sugar and the water is 3:0.5-2:5-15, and after mixing evenly, anaerobically fermenting at 22-28°C for 60 days or more, and filtering the liquid portion as the agricultural enzyme;

[0044] S2. Straw yellowing storage: dilute the agricultural enzyme to a mass percentage of 25-70%, that is, the mass percentage of the agricultural enzyme in the diluted solution is 25-70%, add the aforementioned diluted solution into the withered straw, control the water content of the system to 55-65%, add 0.5-2g / L lysine and 0.5-2g / L methionine solution, mix well to obtain yellowing storage material, compact the yellowing storage material and seal it for storage.

[0045] The invention uses abandoned wilted stover (WS) and fruits and vegetables as raw materials, and simultaneously adds acid-resistant cellulose-degrading bacteria that can adapt to acidic environment and use cellulose as substrate and sugar carbon source, and performs co-fermentation under anaerobic conditions. The prepared agricultural enzyme (Agriculturaljiaoossu, AJ) has a high content of water-soluble carbohydrates and a high content of lactic acid, formic acid, acetic acid, and acetic acid substances, a pH value between 4 and 5, and has a high efficiency in degrading cellulose substances. The biologically active ingredients contained in the enzyme are not only beneficial to improving the nutritional value of the yellow storage straw, but also introduce beneficial exogenous microorganisms, VFA and amino acids such as lysine and methionine, which can recruit beneficial bacteria in the yellow storage process, accelerate the reproduction of acid-producing bacteria and cellulose-degrading bacteria and maintain their activity to inhibit the growth of undesirable microorganisms, which is beneficial to carbohydrates and nutrients. The invention can preserve the material, reduce the dry matter loss of straw, and promote the degradation of lignin, cellulose, hemicellulose, etc. in the straw, so that the biodegradability of the straw is rapidly improved, and then it is conducive to improving the conversion and utilization degree of each component by anaerobic digestion microorganisms such as methanogenic bacteria, and improving the methane production potential of the straw; and it is conducive to changing the microbial community structure in yellow storage, enhancing the proliferation rate of microorganisms with antibacterial effects such as Bacillus and Paenibacillus, inhibiting the growth of spoilage bacteria such as Clostridium and Enterococcus, and then it is conducive to improving the antibacterial effect of yellow storage and then the quality of yellow storage straw, realizing the long-term stable quality storage of withered straw, and can provide high-quality straw raw materials for large-scale biogas projects throughout the year. In addition, the agricultural enzyme of the present invention is an acidic liquid obtained by anaerobic co-fermentation of rotten fruits and vegetables and dry withered straw, with the advantages of low cost and easy acquisition of raw materials.

[0046] After testing, compared with conventional clear water yellow storage, the present invention uses agricultural enzyme yellow storage to make the dry matter loss of yellow storage straw less than 1%, and the concentration of organic acids mainly lactic acid is increased by more than 2 times, which greatly reduces the risk of straw corruption and deterioration during yellow storage; the abundance of Bacillus and Paenibacillus is increased by more than 300 times, and these genera help to inhibit miscellaneous bacteria and improve the methane production potential of yellow storage; the total abundance of Clostridia and Enterococcus is reduced by more than 20 times, and these genera will cause the loss of organic dry matter and methane production potential of yellow storage. The present invention reduces the loss of dry matter during yellow storage, promotes the degradation of lignin and cellulose and the ability to generate organic acids, and supplements soluble carbohydrates, etc., and finally significantly improves the stability and methane production potential of yellow storage, so that the methane production potential of straw is greatly improved compared with conventional yellow storage.

[0047] Optionally, in step S1, the waste fruits and vegetables include but are not limited to: oranges, apples, pears, cabbages, Chinese cabbages and tomatoes; the withered straws include but are not limited to: withered and dry yellow corn straws, sorghum straws, sugarcane straws, rice straws and wheat straws. The waste withered straws are usually chopped into 1-2 cm small segments and then mixed with the waste fruits and vegetables, preferably with a mass ratio of 1:1.

[0048] The purpose of disinfecting discarded fruits and vegetables and withered straw is to inhibit the growth of bacteria or harmful microorganisms. The disinfection method does not need to be particularly strict. During the first production, pasteurization can be used, that is, the discarded fruits and vegetables and withered straw are heated to 65°C-70°C and kept for about 15 seconds. In continuous production, the agricultural enzyme prepared in the previous batch can be used, which has a lower pH value (pH4-5). By sealing and soaking the discarded fruits and vegetables and withered straw in the aforementioned agricultural enzyme liquid for a period of time, such as 12-36 hours, the growth and reproduction of bacteria can be effectively inhibited. It is understandable that an acidic liquid with a pH of 4-5 prepared by an inorganic acid (such as hydrochloric acid, sulfuric acid) or an organic acid (such as lactic acid, acetic acid, propionic acid) can also be used for sealing and soaking to inhibit the growth of bacteria.

[0049] Optionally, in step S1, the acid-resistant cellulose-degrading bacteria are obtained by restricted subculture, which specifically includes the following steps:

[0050] S11, inoculating microorganisms isolated from nature into a seed culture medium, and anaerobically culturing at 22-28° C. for 48 hours, wherein the seed culture medium comprises the following components: 10 g / L glucose, 5 g / L microcrystalline cellulose, 5 g / L peptone, 3 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and 2 g / L sodium phosphate, with a pH of 7.0; this step is intended to enrich microorganisms;

[0051] S12, performing gradient restricted subculture on the culture of step S11, wherein:

[0052] In the first subculture, the glucose in the seed culture medium was adjusted to 5 g / L, the microcrystalline cellulose to 5 g / L, and the pH to 6.5, and the other conditions remained unchanged for 48 h.

[0053] In the second generation subculture, the glucose in the seed culture medium was adjusted to 3 g / L, the microcrystalline cellulose to 7 g / L and the pH to 6.0, and the other conditions remained unchanged for 48 h.

[0054] In the third generation of subculture, the glucose in the seed culture medium was adjusted to 2 g / L, the microcrystalline cellulose to 8 g / L and the pH to 5.5, and the other conditions remained unchanged for 48 h.

[0055] In the fourth generation of subculture, the glucose in the seed culture medium was adjusted to 1 g / L, the microcrystalline cellulose to 9 g / L, and the pH to 5.5, and the other conditions remained unchanged for 48 h.

[0056] In the fifth generation of subculture, the glucose in the seed culture medium was adjusted to 0.5 g / L, the microcrystalline cellulose to 10 g / L, and the pH to 5.5, and the other conditions remained unchanged for 48 h.

[0057] The same conditions as those for the fifth generation were used to continue subculture for 10 generations, and the microorganisms in the culture solution were separated to obtain the acid-resistant cellulose degrading bacterial strain.

[0058] An environment of low sugar, high cellulose and low pH is the actual condition in agricultural enzymes and yellow storage fermentation. The present invention adopts a gradient restricted subculture method to screen acid-resistant cellulose-degrading bacterial strains, by gradually reducing the glucose concentration in the culture medium and increasing the microcrystalline cellulose concentration, while adjusting the pH value, to gradually adapt and screen out acid-resistant and efficient lignocellulose-degrading strains. Specifically, glucose, as a soluble carbon source, is easily utilized by microorganisms. Gradually reducing the glucose concentration during the screening process can force the microorganisms to gradually adapt to microcrystalline cellulose as the main carbon source, thereby screening out strains that can effectively utilize cellulose; microcrystalline cellulose is a carbon source that is difficult to degrade, and increasing its concentration can increase the selection pressure on cellulose degradation ability, and screen out strains with stronger cellulosic material degradation ability; the pH value is gradually reduced to 5.5, which can effectively screen out and increase the concentration of the target strains that have a high ability to degrade cellulose and are acid-resistant. The resulting bacterial strain can adapt to the low pH, low sugar, and high cellulose environment of agricultural enzymes and yellow storage fermentation and proliferate rapidly, thereby effectively inhibiting harmful microorganisms and improving the degradation efficiency of cellulose and lignin, which has a positive effect on improving the yellow storage effect of straw and subsequent biogas production capacity.

[0059] Optionally, in step S11, the microorganisms isolated from nature can specifically come from microorganisms in soil and compost. The present invention does not specifically limit its source. The inoculation amount of the microorganism in the culture medium has little effect on the screening of the bacterial strain. In steps S11-S12, the conventional inoculation amount in the art can be used.

[0060] Optionally, in step S12, the pH value is adjusted by L-lactic acid. L-lactic acid is the main organic acid produced during agricultural enzymes and yellow storage fermentation. By simulating the acidic environment, strains that can tolerate the actual harsh environment in production can be screened out, thereby maintaining the strains' efficient cellulose degradation ability under acidic conditions.

[0061] In step S1, the inoculation amount of the acid-resistant cellulose degrading bacteria system is based on the total weight of the discarded fruits and vegetables and the withered straw, that is, inoculating 0.5-3% (wt) of the acid-resistant cellulose degrading bacteria system means that the acid-resistant cellulose degrading bacteria system accounts for 0.5-3% of the total weight of the discarded fruits and vegetables and the withered straw. In a specific embodiment, it can be 0.5%, 1%, 1.5%, 2%, 3% or any value between 0.5-3%. Preferably, the inoculation amount of the acid-resistant cellulose degrading bacteria system is 1%.

[0062] Preferably, in step S1, the mass ratio of the mixed waste, the brown sugar and the water is 3:1:10; the mixture is anaerobically fermented at 25°C for 60 days, and the liquid portion is filtered and taken as the agricultural enzyme.

[0063] During the yellow storage process, too high acid concentration will lead to excessive dissolution of lignin and cellulose, resulting in increased dry matter loss. Appropriate acid concentration is beneficial to improving the quality of yellow storage straw and the biogas production of yellow storage straw. Preferably, in step S2, the solvent for diluting the agricultural enzyme is water, and the agricultural enzyme is diluted to a mass ratio of 50%, and the water content of the control system is 60%. Thus, the mass ratio of the agricultural enzyme to the withered straw is 3:4, and then 1g / L lysine and 1g / L methionine solution are added and mixed to obtain yellow storage material. Under this condition, the organic acid concentration of yellow storage can be increased by 297.6%, which significantly reduces the risk of corruption and deterioration of straw during the yellow storage process, and is beneficial to improving the methane production potential, which makes an important contribution to promoting the resource utilization of straw and improving the efficiency of bioenergy.

[0064] The yellow storage straw prepared by the yellow storage method of the present invention is rich in acid-producing bacteria and organic acids, especially lactic acid and soluble carbohydrates that are beneficial to biogas production. The straw has good biodegradability and a dry matter loss of <1%. It is used for anaerobic fermentation to produce biogas, which can significantly increase the biogas yield and gas production rate of anaerobic fermentation. Therefore, another embodiment of the present invention provides the use of yellow storage straw prepared by the method of using agricultural enzyme yellow storage straw as described above in anaerobic fermentation to produce biogas.

[0065] Based on the same inventive concept as above, an embodiment of the present invention further provides a method for anaerobic fermentation of silage straw, comprising the following steps:

[0066] The yellow silage straw prepared above is mixed with anaerobic digestion sludge at a total solids (TS) ratio of 1:3-5, and medium-temperature anaerobic fermentation is performed.

[0067] The straw yellow storage obtained by the invention is used for straw anaerobic fermentation to produce biogas, and the cumulative methane production can reach 110mL / gVS. Compared with conventional clear water yellow storage to produce biogas, the methane production potential is increased by more than 10%, and the optimal conditions can be increased by 45.0%.

[0068] Optionally, the temperature of the mesophilic anaerobic fermentation is generally 35-38°C.

[0069] Optionally, the total solid ratio of the silage straw to the anaerobic digestion sludge is 1:4.

[0070] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0071] Example

[0072] 1. Screening of acid-resistant cellulose-degrading bacteria

[0073] The acid-resistant cellulose-degrading bacterial system is obtained by restricted subculture, comprising the following steps: (1) inoculating microorganisms isolated from nature into a seed culture medium, and anaerobic culturing at 25° C. for 48 hours to enrich the microorganisms, wherein the seed culture medium comprises the following components: 10 g / L glucose, 5 g / L microcrystalline cellulose, 5 g / L peptone, 3 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and 2 g / L sodium phosphate, and the pH value is 7.0; (2) subjecting the culture of step (1) to gradient restricted subculture, and culturing for a total of 15 generations, wherein the gradient restricted subculture comprises the following operations:

[0074] In the first subculture, the glucose in the seed culture medium was adjusted to 5 g / L, the microcrystalline cellulose to 5 g / L, and the pH to 6.5, and the other conditions were kept the same as the seed culture medium, and the culture was carried out for 48 h;

[0075] In the second generation subculture, the glucose in the seed culture medium was adjusted to 3 g / L, the microcrystalline cellulose to 7 g / L and the pH to 6.0, and the other conditions were kept the same as the seed culture medium, and the culture was carried out for 48 h;

[0076] In the third generation of subculture, the glucose in the seed culture medium was adjusted to 2 g / L, the microcrystalline cellulose to 8 g / L and the pH to 5.5, and the other conditions were kept the same as the seed culture medium, and the culture was carried out for 48 h;

[0077] In the fourth generation of subculture, the glucose in the seed culture medium was adjusted to 1 g / L, the microcrystalline cellulose to 9 g / L and the pH to 5.5, and the other conditions were kept the same as the seed culture medium, and the culture was carried out for 48 h.

[0078] In the fifth generation of subculture, the glucose in the seed culture medium was adjusted to 0.5 g / L, the microcrystalline cellulose to 10 g / L, and the pH to 5.5. The other conditions were kept the same as the seed culture medium and cultured for 48 h.

[0079] The same conditions as the fifth generation were used to continue subculture for 10 generations, and solid-liquid separation was performed to obtain the microorganisms in the culture solution, which were the acid-resistant cellulose-degrading bacterial strains.

[0080] In the preliminary experiments of the present invention, it was found that by adopting the same concentration screening scheme, that is, keeping the concentration of glucose and microcrystalline cellulose unchanged during the screening process (for example, always keeping the concentration of glucose 10g / L, microcrystalline cellulose 5g / L, pH7.0), the growth ability of the screened strains under low concentration glucose and high concentration cellulose conditions was weakened, and the acid resistance and cellulose degradation ability of the strains were also weakened, and it was difficult to occupy a dominant position in the enzyme solution after inoculation. By adopting a positive concentration increase scheme, that is, the concentration of glucose and microcrystalline cellulose is positively increased (for example, glucose is gradually increased to 10g / L, microcrystalline cellulose is gradually increased to 10g / L, and pH is gradually increased to 7.0), strains that are insensitive to the type of carbon source will be screened out. These strains perform poorly in low sugar and high acid environments, cannot effectively degrade cellulose, and the acid production effect is not ideal.

[0081] The acid cellulose degradation bacterial system screened by gradient restricted subculture of the present invention has the following advantages: (1) improving the concentration and acid resistance of the target bacteria: by gradually adapting to the low-sugar, high-cellulose and low pH environment, simulating the acidic conditions in enzyme fermentation and silage, screening out strains that tolerate lactic acid and grow well, thereby improving the adaptability and stability of the target bacteria in practical applications; (2) improving the cellulose degradation ability: gradually increasing the concentration of microcrystalline cellulose, forcing the strain to adapt to the high-cellulose environment, and still being able to effectively degrade cellulose in a low-glucose, high-cellulose environment, which is beneficial to improving the ability of yellow storage fermentation to degrade cellulose and significantly improving the degradation efficiency of cellulose; (3) improving the acid production ability: gradually reducing the pH value, the strain screened out can still grow and metabolize efficiently in an acidic environment, can significantly increase the yield and acid production ability of organic acids, reduce the risk of growth of miscellaneous bacteria, and is suitable for acidic conditions in practical applications. Therefore, the bacterial system of the present invention is more suitable as an inoculated bacterial agent for enzyme fermentation, which is beneficial to improving the yellow storage effect and quality of straw and enhancing the methane production potential.

[0082] 2. Preparation of agricultural enzymes

[0083] Corn stover was collected from Nanbin Farm, Sanya, Hainan Province, China (18.36°N, 109.18°E), and consisted of stems and leaves, excluding cobs and husks. After corn was harvested at maturity, the stover was withered in the field for 1 month, and then the withered stover (WS) was collected and cut into small pieces of 1-2 cm for later use. The physical and chemical properties of WS are shown in Table 1.

[0084] Table 1 Physicochemical properties of withered straw in this example

[0085]

[0086]

[0087] Note: FW means fresh weight, %FW means the percentage of fresh weight, %DM means the percentage of dry matter, g / kgDM means the weight of target compound per kilogram of dry matter, ND means not detected, all data were determined three times and expressed in mean ± SD mode.

[0088] Preparation of agricultural enzyme (AJ): (1) Mix the discarded fruits and vegetables and 1-2 cm withered straw in a mass ratio of 1:1 and add them to a 15L fermentation tank, add the fermented agricultural enzyme and soak them anaerobically for 1 day to inhibit the growth of bacteria in the above raw materials. When there is a lack of agricultural enzyme, pasteurization can be used as an alternative disinfection. Then filter out the agricultural enzyme, and inoculate the acid-resistant cellulose degrading bacteria obtained by the above screening at an inoculation amount of 1% (wt) of the total weight of the discarded fruits and vegetables and withered straw to form a mixed waste; (2) Add brown sugar and water to the mixed waste, and calculate the mass ratio of mixed waste: brown sugar: water = 3:1:10. After mixing evenly, anaerobically ferment at 25°C for 60 days, and filter the liquid part to obtain the agricultural enzyme (AJ) of the present invention. The physical and chemical properties of AJ are shown in Table 2.

[0089] Table 2 Physicochemical properties of the agricultural enzymes of this example

[0090]

[0091] Note: FW means fresh weight, %FW means the percentage of fresh weight, and %TS means the percentage of total solids.

[0092] 3. Yellow storage of withered straw

[0093] Agricultural enzyme is used as a yellowing additive for straw yellowing. The method includes: diluting the agricultural enzyme and water in different mass ratios, and then adding the diluted agricultural enzyme to the withered straw. The withered straw itself has a certain moisture content, and its solid accounts for 90.28%. By mass ratio, the diluted agricultural enzyme (solid): withered straw = 6:4, so as to maintain the moisture content of the system at 60%, and the withered straw accounts for 40%, which is suitable for yellowing fermentation. Then, 1g / L lysine and 1g / L methionine solution are added, mixed to obtain yellowing material, and the yellowing material is compacted and sealed for storage. The amount of agricultural enzyme added in the yellow silage was adjusted by adjusting the dilution ratio of the agricultural enzyme. The specific configuration ratios were 0% (agricultural enzyme: water = 0:1), 25% (agricultural enzyme: water = 1:3), 50% (agricultural enzyme: water = 1:1) and 100% (agricultural enzyme: water = 1:0) of agricultural enzyme dilutions, which were named CK, QA, HA and A experimental groups respectively. The raw material ratios of each experimental group are shown in Table 3.

[0094] Table 3 Proportion of agricultural enzymes and withered straw in yellow silage of this embodiment

[0095]

[0096] 300 g of yellow silage from each treatment was packed into polyethylene bags and vacuumed using a vacuum packaging machine (FW-3150, FreshWorld, China). Each treatment was repeated three times. The polyethylene bags were stored at 25°C ± 3°C for 60 days. The yellow silage samples were stored at -20°C for further analysis.

[0097] 3.1 Analysis of physical and chemical properties of yellow silage straw

[0098] Using Porter and Murray [1] The dry matter (DM), organic dry matter (ODM) content and DM loss of silage straw were determined by the method reported by Cai et al. [2] The contents of total solids (TS), volatile solids (VS), and volatile fatty acids (VFA) were determined using the methods reported by Thomas et al. pH was measured using a pH meter (SX610; Sanxin, Shanghai, China). [3] Soluble carbohydrates (WSC) were determined by anthrone colorimetry according to the method reported by Vansoest et al. Lignocellulose content was measured by ANKOM 220 fiber analyzer according to Vansoest et al. [4] The total carbon (TC) and total nitrogen (TN) contents were measured using a Flasharc2000 elemental analyzer.

[0099] Figure 1 The figure shows the changes in the physical and chemical properties of withered straw after yellow storage, where (a) shows the DM content, ODM content and DM loss rate of yellow storage straw, (b) shows the VFA content and pH value of yellow storage straw, (c) shows the water-soluble carbohydrate (WSC) content of yellow storage straw, and (d) shows the lignocellulose content of yellow storage straw.

[0100] from Figure 1 (a) As can be seen in the figure, the DM and ODM contents of the CK group that was not treated with agricultural enzymes (AJ) were close to those of the yellow silage straw treated with AJ, but the DM loss of the CK group increased significantly (p<0.05), with the DM loss of the CK group being 2.87%, while the DM loss of all AJ treatments was <1%. This is due to the rapid decrease in pH caused by the added agricultural enzymes rich in VFA and acid-producing bacteria, which can inhibit the excessive decomposition of the straw to a certain extent and achieve good preservation of chemical nutrients such as sugars and cellulose. There are slight differences in the yellow silage straw group treated with AJ because 25% of the agricultural enzymes have already shown strong antibacterial ability, and when the concentration is >25%, it has little effect on the antibacterial ability. [5]The DM loss of experimental group A was greater than that of experimental group HA, which may be due to the excessive dissolution of lignocellulose caused by the increased acid concentration, resulting in increased DM loss.

[0101] Figure 1 Figure (b) shows the VFA concentration and pH value of each treatment. During long-term storage of straw, the growth of microorganisms and the loss of DM are mainly driven by high concentrations of protons (H + ) and VFA. In the complex scenarios of actual application, the risk of yellow silage straw failure is higher, and accelerating acidification and lowering pH to obtain a low pH and high VFA environment is beneficial to improving the quality of yellow silage. The pH value of the CK group was 4.7, while the pH of all AJ-treated silage liquids was about 4.1, similar to the added AJ raw material (see Table 2), indicating that the use of AJ with a lower pH helps to recruit more acid-resistant microbial communities and produce high concentrations of VFA, thereby further reducing the yellow silage pH, which is not only beneficial to increase the inhibition rate of undesirable microorganisms, but also to significantly extend the inhibition time and achieve long-term stable preservation of yellow silage straw. The total VFA (TVFA) content in all AJ treatment groups was significantly higher than that in CK (p<0.05), but there was no significant difference between different AJ treatment groups; this indicates that after the addition of AJ, acid-producing bacteria may continue to metabolize until the VFA concentration reaches its tolerance limit. It is noteworthy that the main VFA component of the CK group was acetic acid, while the main VFA component of all AJ treatments was lactic acid, with significantly lower acetic acid levels; this change in acid composition may be attributed to changes in the microbial community, leading to different metabolic pathways. The composite acid dominated by lactic acid can produce synergistic advantages and improve the subsequent biogas production potential of straw.

[0102] from Figure 1 (c) As can be seen from the figure, with the increase of AJ addition, the level of WSC in silage straw also showed an upward trend, among which the increased WSC mainly came from AJ and partly from lignocellulose degradation.

[0103] The improvement of lignocellulose biodigestibility is the main reason for the increase in methane production from silage straw. Figure 1 (d) As can be seen in the figure, the cellulose content in the fermentation liquid of the yellow storage in all AJ treatment groups was significantly lower than that in the CK group. There was no significant difference in the cellulose, hemicellulose and lignin content in CK compared with WS, while the cellulose, hemicellulose and lignin content in all AJ treatment groups were significantly reduced compared with CK. This shows that under control conditions, the ability of clear water yellow storage to degrade lignin and cellulose is limited, and the addition of AJ improves the ability to utilize these components through acid and microbial communities, promoting the digestion of lignocellulose more effectively. The degradation of cellulose and lignin helps to break down the structure of lignocellulose and create areas that are more susceptible to microbial activity and enzymatic hydrolysis. Therefore, this will increase the biodegradability of lignocellulose during anaerobic digestion of biogas.

[0104] 3.2. Illumina MiSeq sequencing analysis of microbial diversity

[0105] 10 g of yellow husk straw was mixed with 40 mL of sterile water and incubated on a shaker at 120 rpm for 2 h. The main plant tissues were removed by filtering with a double cotton mesh, and the filtrate was centrifuged at 1200 g and 4 °C for 30 min. The supernatant was discarded, and the precipitate was used to extract microbial genomic DNA using a DNA kit (purchased from OmegaBio-tek, catalog number D3350-01). Zheng et al. [6] The reported primers and PCR procedure were used to amplify the V3-V4 hypervariable region of the bacterial 16S rRNA gene, and the purified amplification products were sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing using the Illumina MiSeq platform.

[0106] 3.2.1 Species composition analysis

[0107] Operational taxonomic units (OTUs) with a similarity of more than 97% were integrated. The OTU representative sequences were compared with the database to obtain the species annotation information of all OTUs in each sample, and the relative abundance was calculated based on the OTU abundance table and species annotation information. Figure 2 The microbial composition of silage straw in different treatment groups is shown. Figure (a) shows the species community abundance at the phylum level, and Figure (b) shows the species community abundance at the genus level.

[0108] Some groups in the Firmicutes phylum showed stronger tolerance to extreme environments due to their thicker cell walls. The relative abundance of Firmicutes was over 0.7 in all treatments, but its abundance advantage became more obvious after the addition of AJ, and increasing the abundance of Firmicutes could improve the quality of silage straw.

[0109] At the genus level, compared with all the yellow silage straws treated with AJ, the CK group had more significant abundance differences among different genera, with Lactobacillus, Clostridium sensu stricto_1 and Enterobacter being the dominant genera. Lactobacillus has good adaptability to acidic environments and can secrete various organic acids, such as lactic acid and acetic acid, making it ubiquitous in most yellow silage fermentation products. Clostridium sensustricto_1 belongs to a subgenus of the genus Clostridium in the phylum Actinobacteria, and studies have shown that its abundance is negatively correlated with methane production during anaerobic digestion. Enterobacter, which belongs to the phylum Proteobacteria, is a common pathogenic species. Compared with other major biochemical pathways, the biochemical pathways of Clostridium sensu stricto_1 and Enterobacter will lead to greater ODM and biomethane potential (BMP) losses, which is consistent with the above results. Figure 1 The DM loss presented in the middle (a) figure is consistent.

[0110] In the AJ treatment group, Bacillus, Pediococcus and Paenibacillus replaced Clostridium and Enterobacter and became the new dominant genera. Bacillus, Pediococcus and Paenibacillus can metabolize antimicrobial compounds or degrade lignocellulose, thereby improving the stability and BMP of straw storage. Although these three genera have been reported in some silage or yellow storage studies, they usually do not show advantages, and the relative abundance does not exceed 1%. Therefore, the AJ prepared by the present invention can produce a temporary inhibitory effect on most microorganisms through its acid-based antimicrobial components, creating a good environment for beneficial bacteria such as Bacillus present in the straw, so that they show advantages. Once they gain an advantage, they can further inhibit the growth of other microorganisms, which may help prevent yellow storage failure and reduce DM loss after aerobic exposure.

[0111] In the A and HA experimental groups, the abundance of lactic acid bacteria was similar to that in the CK group. However, the relative abundance of lactic acid bacteria in the QA treatment was higher than that in the A and HA experimental groups. This may be due to the fact that when the AJ concentration is high, lactic acid bacteria are inhibited by lactic acid feedback, which hinders their proliferation. In contrast, when the AJ concentration is low, light supplementation of WSC and VFA is beneficial to the growth of certain genera, which leads to further utilization of WSC and lignocellulose. Therefore, the straw VFA concentration was increased until the pH reached a level similar to that of the A and HA treatments. This phenomenon explains why the VFA of the A, HA, and QA treatments did not have the same trend with the addition of AJ (see Figure 1(middle (b) figure).

[0112] 3.2.2 Identification of key species

[0113] The sequencing data were analyzed by Linear discriminant analysis Effect Size (LEfSe) to compare the microbial groups among multiple groups and find species with significant abundance differences among different groups as potential microbial markers. The nonparametric Kruskal-Wallis test and Wilcoxon rank test were used to determine the significant differences in microbial abundance (p<0.05). LEfSe was also used to analyze the effects of different classification levels on the abundance of microbiota and determine the significant differences in the abundance of microbiota among different treatments (p<0.05). The top 100 genera in relative abundance were selected for Pearson correlation analysis to construct an association network. All nodes (i.e., species) with an absolute value of R (Pearson correlation value) > 0.8 and a p value < 0.05 (significant differences between groups) in the LEfSe analysis were described. Red nodes indicate positive correlations between species (R>0), and green nodes indicate negative correlations between species (R<0). Based on the correlation, the nodes were divided into two modules (M1 and M2) using Cytoscape v3.8.0 software. M1 contained 25 nodes and M2 contained 63 nodes. In each module, red lines indicated synergy between populations, while green lines between the two modules indicated antagonism between specific groups. Due to the small microbial differences between the A, HA, and QA experimental groups, these samples were merged into a group labeled "AJ" and compared with CK using LEfSe analysis to screen marker species. The marker species of CK and AJ were distributed in M1 and M2, respectively, indicating that a new symbiotic community was established after the addition of AJ, competing for ecological niches with the native community in CK. Therefore, among the six genera discussed above, Lactobacillus and Pediococcus did not appear in the network because they did not meet the filtering criteria for correlation with other genera.

[0114] Related results can be found in Figure 3 , where (a) is the evolutionary branch diagram of LEfSe analysis. The circles radiating from the inside to the outside represent the classification level from phylum to genus. Each circle at a different classification level represents a species classification at that level. The diameter of the circle is proportional to the relative abundance. (b) is the network interaction diagram of Pearson correlation analysis. Different nodes represent different dominant genera. The connection between nodes indicates that there is a correlation between two genera. The red line indicates a positive correlation, and the green line indicates a negative correlation. The number of node connections can be used to find species that are more associated with other members of the bacterial community. In addition, the node color is defined by the phylum level. The same node color indicates that these genera belong to the same phylum.

[0115] According to the association network and LEfSe analysis, key groups were determined based on the following principles: 1) Abundance stability: key groups must persist in most (at least 75%) samples during the fermentation process, indicating that they have strong environmental adaptability and stability; 2) Centrality: by analyzing the microbial co-occurrence network, the centrality parameters (closeness centrality, betweenness centrality and degree centrality) of each group were calculated. Key groups usually score high in these parameters, indicating that they are central to the microbial community and can significantly affect the structure and function of the microbial community; 3) LEfSe analysis: through the linear discriminant analysis effect size (LEfSe), the microbial groups that are significantly present under different treatment conditions are identified. Key groups show statistically significant differences in this analysis, proving their important role in specific treatments. Based on the above principles, two key groups, Brevibacillus and Thermoactinomyces, were screened out. They play an important role in promoting lignocellulose degradation, maintaining fermentation stability and increasing biogas production, and can effectively improve fermentation efficiency and system sustainability, thereby providing new microbial regulation strategies for the improvement of biogas production technology.

[0116] In addition, Clostridium sensu stricto_1(1-13) and Enterobacter(1-7) were found in M1, and Bacillus(2–29) and Paenibacillus(2–19) were found in M2 with higher abundance (ranging from 0.432-0.52), indicating that they had relatively greater impact on the microbial community. Dominant and key taxa play unique and important roles in microbial communities. This study found that Bacillus, Paenibacillus, Brevibacillus, and Thermoactinomyces were key drivers of changes in microbial community structure and function. To further explore potentially influential taxa, nodes connected to these genera were filtered, and a new network was constructed, consisting of five nodes from M1 and M2, respectively, and almost all nodes were interconnected, indicating that these books play an indispensable role in the interaction network.

[0117] 3.2.3 Relationship between yellow storage fermentation characteristics and microbial communities

[0118] To further investigate the relationship between environmental factors (lactic acid, acetic acid, dry matter, organic dry matter, water-soluble carbohydrates, pH, cellulose, hemicellulose, lignin, AJ addition, etc.) and microbial community evolution during WS yellow storage, a Pearson correlation heat map was constructed (see Figure 4), among which. In the cluster, Firmicutes is widely distributed, while Proteobacteria and Actinobacteria do not have any obvious pattern, indicating that the clustering results are less related to phylogeny (the phylum to which the microorganisms belong). Figure 3 The modular divisions shown in are basically the same, named M1S and M2S.

[0119] The abundance of Clostridium sensu stricto_1 and Enterobacter was negatively correlated with WSC, indicating their potential resistance to low-nutrient environments. In addition, their abundance was positively correlated with pH, ​​indicating that the decrease in pH during yellow storage inhibited their growth. Cellulose content was significantly negatively correlated with the abundance of 17 genera, including dominant genera (Bacillus and Paenibacillus) and key species (Brevibacillus and Thermoactinomyces), all of which have the ability to degrade cellulose.

[0120] In addition, a two-factor association network of microorganisms and fermentation characteristics was constructed (see Figure 5 ), it was found that the genera negatively correlated with cellulose had a large overlap with the microbiome of M2S, indicating that these widely connected genera are likely to be closely related to cellulose degradation. In addition to reducing pH, the correlation analysis results showed that reducing acetic acid concentration or increasing WSC content helped expand the cellulose-degrading bacterial community.

[0121] In general, WSC, cellulose, ODM, AJ concentration and total VFA (TVFA) have the most microbial connection nodes, 32, 31, 30, 30 and 26 edges, respectively. This indicates that the microorganisms are most sensitive to these factors, or that changes in the microbial community have the greatest impact on these factors. It can be inferred that the addition of AJ in the present invention mainly regulates the microbial community and lignocellulose degradation by supplementing WSC, ODM and VFA, and the improved microbial community and cellulose degradation further improve the stability and biogas production capacity of yellow silage straw.

[0122] 4. Anaerobic fermentation

[0123] The anaerobic digested sludge used for biogas production came from a mesophilic (37 °C) anaerobic digester (Sanhe, Hebei, China) with continuous dosing of corn straw and cow dung. The total solids (TS) content, volatile solids (VS) content and pH value of the anaerobic digested sludge were 49.0 g / L, 26.7 g / L and 7.86, respectively.

[0124] After yellow storage, untreated withered straw (WS) and four groups of treated yellow storage straw were used for anaerobic digestion. The yellow storage straw was mixed with anaerobic digestion sludge at a total solid (TS) ratio of 1:4. The working volume of all reactors was 200 mL, and the total solid (TS) was controlled to account for 5% of the weight of the anaerobic fermentation digestate. Medium temperature (37 ° C) anaerobic fermentation was carried out, and the reactor was purged with nitrogen for 3-5 minutes to remove oxygen. The biogas and methane content were measured every day. In addition, AJ was used instead of yellow storage straw for anaerobic fermentation alone to measure its direct contribution to biogas (methane) production, which was then subtracted from the cumulative methane production of other treatment groups, that is, AJ-treated yellow storage gas production-AJ gas production alone = AJ-treated straw gas production after excluding AJ. Distilled water was used instead of yellow storage straw as a blank control.

[0125] 4.1 Measurement of biogas production and composition

[0126] The pressure test was performed using an AZ82100 pressure gauge (AZ Instruments, Taiwan, China) with an accuracy of 0.1 kPa. Based on the pressure difference between the inside and outside of the reactor, the biogas yield was calculated using formula (3). The biogas composition was detected using Shimadzu gas chromatography-2014 (Kyoto, Japan). The specific operating details of the gas chromatograph were referred to Cai et al. [2] The methane (CH4) content was determined by Yuan et al. [7] The cumulative methane production was calculated using the modified Gompertz model.

[0127] V biogas =(ΔP×V headspace ×C) / (R×T) (1);

[0128] P=P max ×exp{-exp[P max ×e×(λ-t) / P max +1]} (2).

[0129] In the above formula, V biogas : Daily biogas production (mL); ΔP: Absolute pressure difference (kPa); V headspace : headspace volume (mL); C: molar volume (22.41 L / mol at 273.15 K and 101.325 kPa); R: universal gas constant (=8.314 Lmbar / K / mol), T: absolute temperature (Kelvin, K); P: cumulative methane production (mL / g total solids, mL / g TS); P max : Maximum potential methane production (mL / g TS); R max: maximum methanogenesis rate (mL / g total solids·day, mL / g TS-d); λ: lag period (day, d); t: experimental duration (day, d); e is a constant (=2.7183).

[0130] The cumulative methane production of all treatment groups gradually increased over time and stabilized at 30 days (see Figure 6 TS of AJ and methane produced by AJ were excluded from the calculation. Cumulative methane production was higher in the AJ treatment compared with that in CK (p < 0.05). The correlation coefficient (R 2 The range of the model parameters is 0.980-0.998, and the model parameters are shown in Table 4). On the 30th day, the cumulative methane production of each treatment (mL / gVS) was: CK=100.61, QA=113.88, HA=145.85, A=121.11 and WS=114.78. It can be seen that the straw BMP can be enhanced by the straw storage method of the present invention because it can improve the biodegradability of lignocellulose when consuming WSC.

[0131] Table 4 Gompertz model parameters

[0132]

[0133] BMP in CK was significantly lower than that in other treatments (p<0.05), which may be due to greater loss of ODM and BMP caused by biochemical pathways of Clostridium sensu stricto_1 and Enterobacter, while the addition of AJ significantly suppressed the abundance of these two genera (p<0.05). The best methane production was observed in the HA treatment, which is due to the increased accessibility of the remaining lignocellulose due to the partial dissolution of lignocellulose during ensilage. After excluding the methane produced by AJ, there was no significant difference among the QA, A and WS treatments, indicating that the addition of AJ during ensilage helped to suppress the reduction of BMP in WS.

[0134] It should be noted that the present invention uses Origin 2021 for data processing and statistical analysis. Variance analysis uses Duncan multiple range test, and the significance threshold is set to p < 0.05. SPSS25.0 is used for variance calculation and Pearson correlation analysis. Subsequently, Cytoscape v3.8.0 (NRNB, San Diego, CA, USA) is used to visualize the association network. Photoshop CC 2019 (Adobe, Mountain View, CA, USA) is used to process the images.

[0135] In addition, the references cited in the present invention are listed as follows:

[0136] [1]PORTER M G,MURRAY R S.The volatility of components of grass silageon oven drying and the inter-relationship between dry-matter contentestimated by different analytical methods[J].Grass and Forage Science,2001,56(4):405-11.

[0137] [2]CAI Y,HU K,ZHENG Z,et al.Effects ofadding EDTA and Fe2+on theperformance of reactor and microbial community structure in two simulatedphases of anaerobic digestion[J].Bioresour Technol,2019,275:183-91.

[0138] [3]THOMAS T A.AUTOMATED PROCEDURE FOR DETERMINATION OF SOLUBLECARBOHYDRATES IN HERBAGE[J].Journal of the Science of Food and Agriculture,1977,28(7):639-42.

[0139] [4]VANSOEST P J,ROBERTSON J B,LEWIS B A.METHODS FOR DIETARY FIBER,NEUTRAL DETERGENT FIBER,AND NONSTARCH POLYSACCHARIDES IN RELATIONTOANIMALNUTRITION[J].Journal ofDairy Science,1991,74(10):3583-97.

[0140] [5]ZHANG Y,GAO Y,ZHENG Z,et al.A microbial ecosystem:agriculturalJiaosu achieves effective and lasting antifungal activity against Botrytiscinerea[J].Amb Express,2020,10(1).

[0141] [6] ZHENG Z, CAI Y, ZHANG Y, et al. The effects of C / N(10-25) on relationship of substrates, metabolites, and microorganisms in "inhibitedsteady-state" of anaerobic digestion[J]. Water Res, 2021,188:116466-.

[0142] [7]YUAN

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for yellowing straw using agricultural enzymes, characterized in that: The following steps are involved: S1. Mix the sterilized discarded fruits and vegetables and withered straw in a mass ratio of 1:0.5-2, then inoculate 0.5-3% of acid-resistant cellulose-degrading bacteria to obtain mixed waste, perform anaerobically fermentation at 22-28°C for 60 days or more, and filter the liquid portion as agricultural enzyme; S2. Dilute the agricultural enzyme to a mass ratio of 25-70%, then add the withered straw into the diluted agricultural enzyme, control the water content of the system to 55-65%, supplement 0.5-2g / L lysine and 0.5-2g / L methionine, mix well to obtain yellow storage material, compact the yellow storage material and seal it for storage.

2. The method for storing straw using agricultural enzymes according to claim 1, characterized in that: In step S1, the acid-resistant cellulose-degrading bacteria are obtained by restricted subculture, which includes the following steps: S11, inoculating the microorganisms isolated from nature into a seed culture medium, and culturing anaerobically at 22-28° C. for 48 h, wherein the seed culture medium comprises the following components: 10 g / L glucose, 5 g / L microcrystalline cellulose, 5 g / L peptone, 3 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate and 2 g / L sodium phosphate, with a pH of 7.0; S12, performing gradient restricted subculture on the culture of step S11, wherein: During the first generation of subculture, the glucose in the seed culture medium was adjusted to 5 g / L, the microcrystalline cellulose to 5 g / L and the pH to 6.5, and the culture was performed for 48 hours; During the second generation subculture, the glucose in the seed culture medium was adjusted to 3 g / L, the microcrystalline cellulose to 7 g / L and the pH to 6.0, and the culture was performed for 48 hours; During the third generation subculture, the glucose in the seed culture medium was adjusted to 2 g / L, the microcrystalline cellulose to 8 g / L and the pH to 5.5, and the culture was carried out for 48 hours; During the fourth generation of subculture, the glucose in the seed culture medium was adjusted to 1 g / L, the microcrystalline cellulose to 9 g / L and the pH to 5.5, and the culture was carried out for 48 hours; During the fifth generation of subculture, the glucose in the seed culture medium was adjusted to 0.5 g / L, the microcrystalline cellulose to 10 g / L and the pH to 5.5, and the culture was carried out for 48 hours; The same conditions as those for the fifth generation were used to continue subculture for 10 generations, and the microorganisms in the culture solution were separated to obtain the acid-resistant cellulose degrading bacterial strain.

3. The method for storing straw using agricultural enzymes according to claim 2, characterized in that: In step S11, the microorganisms separated from nature are derived from microorganisms in soil and compost; in step S12, the pH value is adjusted by L-lactic acid.

4. The method for storing straw using agricultural enzymes according to claim 1, characterized in that: In step S1, the inoculation amount of the acid-resistant cellulose degrading bacteria is 1%.

5. The method for storing straw using agricultural enzymes according to claim 1, characterized in that: In step S1, the mass ratio of the discarded fruits and vegetables to the withered straw is 1:

1.

6. The method for storing straw using agricultural enzymes according to claim 1, characterized in that: In step S1, the mass ratio of the mixed waste, the brown sugar and the water is 3:1:

10.

7. The method for storing straw using agricultural enzymes according to claim 1, characterized in that: In step S1, the waste fruits and vegetables are selected from at least one of oranges, apples, pears, cabbages, Chinese cabbages and tomatoes; the withered straw is selected from at least one of corn straws, sorghum straws, sugarcane straws, rice straws and wheat straws.

8. A yellow silage straw, characterized in that: The product is prepared by the method of utilizing agricultural enzyme to yellow straw as claimed in claim 1.

9. Use of the yellow silage straw as claimed in claim 8 in producing biogas by anaerobic fermentation.

10. A method for anaerobic fermentation of silage straw, characterized in that: The following steps are involved: The yellow silage straw as claimed in claim 8 is mixed with anaerobic digestion sludge in a total solid ratio of 1:3-5, and subjected to medium-temperature anaerobic fermentation.

Images