Method for synergistically promoting cotton stalk aerobic composting through cotton stalk aerobic composting biological promoter and bacterial enzyme

Abstract

The invention belongs to the technical field of resources and environments, and particularly relates to a method for synergistically promoting cotton stalk aerobic composting by a cotton stalk aerobic composting biological promoter and a bacterial enzyme. The cotton stalk aerobic composting biological accelerant provided by the invention comprises an independent compound microbial agent and an independent compound enzyme. Aspergillus niger has strong capability of decomposing phenol structures of gossypol, candida tropicalis has strong capability of decomposing side chain structures of gossypol, and trichoderma viride has strong capability of decomposing fatty acid chains of gossypol. The compound microbial agent is inoculated in the initial period of aerobic composting organisms of cotton stalks, so that the microbial decomposition and degradation capacity of aerobic composting can reach the highest efficiency, the high-temperature period of composting is prolonged, the phytotoxicity of gossypol is reduced, the humification process is promoted, the microbial community structure is optimized, and the composting quality and efficiency are improved. The compound enzyme is inoculated in the decomposition period of cotton stalk aerobic composting organisms, so that the humification process can be promoted, the content of effective components such as humic acid is increased, and the utilization efficiency and value of cotton stalks are improved.

Description

Technical Field

[0001] This invention belongs to the field of resource and environmental technology, and specifically relates to a method for promoting aerobic composting of cotton stalks in synergistic effect between a biological promoter for aerobic composting of cotton stalks and microbial enzymes. Background Technology

[0002] After cotton harvesting, a large amount of byproducts remain—cotton stalks. The main component of cotton stalks is natural lignocellulose (composed of cellulose, hemicellulose, and lignin, etc.), which is a lignocellulose biomass resource with development potential. Through biodegradation and transformation, it can be developed into biofuels (such as ethanol, hydrogen, and biogas), animal feed, organic fertilizer, and used as a cultivation substrate for edible fungi.

[0003] Among crop straws, cotton stalks are harder and more difficult to decompose than rice straw, wheat straw, and corn straw. Currently, due to the lack of microorganisms (or enzymes) with efficient decomposition capabilities for cotton stalks, their bioconversion (including energy conversion and feed conversion) efficiency is very low, and the biological utilization of cotton stalks is mostly limited to direct return to the field. However, direct return of cotton stalks to the field also has problems. Because cotton stalks have a high lignocellulose content, they are extremely difficult to decompose and have low natural decomposition levels, making it difficult to become fertilizer in the same year. Returning them to the field not only has little effect on soil fertilization but also affects the planting of crops the following year due to the introduction of pests and diseases. Most of the unused cotton stalks are disposed of by farmers through direct open-air burning, which releases smoke and dust that seriously affects the environment and public transportation.

[0004] Aerobic composting is an ideal method for utilizing cotton stalks as a resource. However, the presence of gossypol, a toxic substance in cotton stalks, can have various adverse effects on the composting process and the quality of the final product. These effects include inhibiting the growth and metabolic activities of microorganisms that decompose cellulose, hemicellulose, and lignin, reducing the decomposition efficiency of organic matter, and prolonging the composting cycle. Degrading gossypol in cotton stalks is currently a challenge in cotton stalk composting. Exploring a method that can efficiently degrade gossypol in cotton stalks and improve the degree of humification in cotton stalk compost is of great practical significance for the cleaning and secondary utilization of cotton stalks. Summary of the Invention

[0005] The purpose of this invention is to address the problems of difficult degradation of gossypol in cotton stalks and low degree of composting humification by providing a method for promoting aerobic composting of cotton stalks through the synergistic effect of a biological promoter and microbial enzymes. This method efficiently degrades gossypol, increases the degradation and conversion rate of cotton stalks, promotes the humification process, and increases the content of effective components such as humic acid, thereby realizing the resource utilization and harmlessness of cotton stalks and improving the utilization efficiency and value of cotton stalks.

[0006] To achieve the above objectives, the present invention provides a cotton stalk aerobic composting biological promoter, which includes two independently used components: component one is a compound microbial agent, and component two is a compound enzyme. The compound microbial agent includes Aspergillus niger, Candida tropicalis, and Trichoderma viride; the total effective viable count of the compound microbial agent is ≥2×10⁻⁶. 8 cfu / g; The complex enzyme includes polyphenol oxidase, catalase and β-glucosidase; the total enzyme activity of the complex enzyme is 800~1800 U / L.

[0007] Preferably, in the compound microbial agent, the effective viable count ratio of Aspergillus niger and Candida tropicalis is (1~1.5):(1~3). In the compound microbial agent, the effective viable count ratio of Aspergillus niger and Trichoderma viride is (1~1.5):(1~4.5).

[0008] Preferably, in the composite enzyme, the enzyme activity ratio of polyphenol oxidase to catalase is (1~2):(1~2); In the complex enzyme, the enzyme activity ratio of polyphenol oxidase to β-glucosidase is (1~2):(2~10).

[0009] This invention provides the application of the cotton stalk aerobic composting biological promoter described in the above technical solution in promoting aerobic composting of cotton stalks.

[0010] Preferably, promoting aerobic composting of cotton stalks includes promoting the degradation of gossypol in cotton stalk compost and / or increasing the humification of cotton stalk compost.

[0011] This invention provides a method for promoting aerobic composting of cotton stalks through synergistic effects of bacteria and enzymes, comprising the following steps: After mixing cotton stalks and livestock manure, the carbon-to-nitrogen ratio of the mixture is adjusted to 25-35 and the moisture content is 60%-70% to obtain compost material; The composting material and compound microbial agent are mixed for aerobic composting. During the high-temperature period, the compost is turned over once a week. When the temperature drops below 40°C during the maturation period, compound enzymes are inoculated. The compound microbial agent is the compound microbial agent in the cotton stalk aerobic composting biological promoter described in the above technical solution; the compound enzyme is the compound enzyme in the cotton stalk aerobic composting biological promoter described in the above technical solution.

[0012] Preferably, the dry matter mass ratio of the cotton stalks to the livestock and poultry manure is (2~4):(1~2); the livestock and poultry manure includes one or more of pig manure, cow manure, horse manure and chicken manure.

[0013] Preferably, the mass ratio of the compost material to the compound microbial agent is 100:(0.2~0.5).

[0014] Preferably, the mass ratio of the compost material to the compound enzyme is 100:(0.1~0.3).

[0015] Preferably, the aerobic composting time is 25-35 days; the ventilation rate of the aerobic composting is 0.3-0.6 L / (kg DM·min), each ventilation lasts 10-30 minutes, and the interval between two adjacent ventilations is 50-80 minutes.

[0016] Beneficial effects: The aerobic composting bio-promoter for cotton stalks provided by this invention comprises two independently usable components: component one is a compound microbial inoculant, and component two is a compound enzyme; the compound microbial inoculant includes Aspergillus niger, Candida tropicalis, and Trichoderma viride; the total effective viable count of the compound microbial inoculant is ≥2×10⁻⁶. 8 The compound enzyme comprises polyphenol oxidase, catalase, and β-glucosidase; the total enzyme activity of the compound enzyme is 800-1800 U / L. Inoculating the compound microbial agent during the initial stage of aerobic composting of cotton stalks maximizes the efficiency of microbial decomposition and degradation in aerobic composting. All three agents have a certain decomposition ability on the main, difficult-to-decompose component—cellulose. Furthermore, all three agents exhibit significant degradation effects on specific gossypol compounds, demonstrating strong targeting: *Aspergillus niger* has a strong ability to decompose the phenolic structure of gossypol, *Candida tropicalis* has a strong ability to decompose the side-chain structure of gossypol, and *Trichoderma viride* has a strong ability to decompose the fatty acid chains of gossypol. Compared with conventional microbial agents, adding the compound agent during aerobic composting can prolong the high-temperature period of composting, reduce the phytotoxicity of gossypol, promote the humification process, optimize the microbial community structure, and improve compost quality and efficiency. The interaction of the mixed agents rapidly decomposes cellulose and proteases, increasing the decomposition rate, and accelerates the degradation rate by altering the structure of gossypol. Inoculating the compound enzyme during the aerobic composting stage of cotton stalks can promote the humification process, increase the content of effective components such as humic acid, realize the resource utilization and harmlessness of cotton stalks, and improve the utilization efficiency and value of cotton stalks.

[0017] Furthermore, this invention involves turning the cotton stalks during the high-temperature period of aerobic composting. The synergistic effect of turning and microbial enzymes enhances the humification process and removes gossypol from the compost. Turning, by periodically loosening the pile and replenishing oxygen, converts local anaerobic zones to aerobic zones, promoting rapid mineralization of easily degradable components and providing more readily accessible substrates for subsequent compound microbial agents. The inoculated compound microbial agents precisely replenish functional microbial communities during the high-activity phase, maintaining the continuous secretion of gossypol-degrading enzymes. Inoculating the aerobic compost with compound enzymes directly cleaves lignocellulose and releases humic precursors, accelerating aromatic condensation and polymerization reactions, leading to rapid accumulation of humic acid and earlier compost maturation, thereby improving the degree of humification and product stability. The coupling of turning and the synergistic effect of microbial enzymes not only improves the conversion efficiency of humic precursors but also significantly increases the total removal rate of gossypol, ultimately achieving simultaneous compliance with compost maturity and safety standards. Detailed Implementation

[0018] This invention provides a cotton stalk aerobic composting biological promoter, which includes two independently used components: component one is a compound microbial agent, and component two is a compound enzyme. The compound microbial agent includes Aspergillus niger, Candida tropicalis, and Trichoderma viride; the total effective viable count of the compound microbial agent is ≥2×10⁻⁶. 8 cfu / g; The complex enzyme includes polyphenol oxidase, catalase and β-glucosidase; the total enzyme activity of the complex enzyme is 800~1800 U / L.

[0019] In one embodiment, the *Aspergillus niger* of this invention is *Aspergillus niger* with the serial number CGMCC 3.15663. In another embodiment, the *Candida tropicalis* of this invention is *Candida tropicalis* with the serial number CGMCC 2.3967. In another embodiment, the *Trichoderma viride* of this invention is *Trichoderma viride* with the serial number CGMCC 3.15484. In one embodiment, the effective viable count ratio of *Aspergillus niger* to *Candida tropicalis* in the composite microbial agent of this invention is (1~1.5):(1~3); in another embodiment, the effective viable count ratio of *Aspergillus niger* to *Candida tropicalis* in the composite microbial agent of this invention is 1:2. In one embodiment, the effective viable count ratio of *Aspergillus niger* to *Trichoderma viride* in the composite microbial agent of this invention is (1~1.5):(1~4.5); in another embodiment, the effective viable count ratio of *Aspergillus niger* to *Trichoderma viride* in the composite microbial agent of this invention is 1:(2~3). In one implementation scheme, the effective viable count ratio of *Aspergillus niger*, *Candida tropicalis*, and *Trichoderma viride* in the compound microbial agent of this invention is 1:(2~3):(2~3). The addition of these three agents maximizes the microbial decomposition and degradation efficiency of aerobic composting. All three agents have a certain decomposition ability on cellulose, a major component that is difficult to decompose. Furthermore, *Aspergillus niger* has a strong ability to decompose the phenolic structure of gossypol, *Candida tropicalis* has a strong ability to decompose the side-chain structure of gossypol, and *Trichoderma viride* has a strong ability to decompose the fatty acid chain of gossypol. The synergistic effect of the three agents significantly reduces the degradation effect on gossypol compared to single strains, demonstrating greater specificity. Compared to conventional microbial agents, adding the compound agent during aerobic composting can prolong the high-temperature period of composting, reduce the phytotoxicity of gossypol, promote the humification process, optimize the microbial community structure, and improve compost quality and efficiency. The interaction of the mixed agents rapidly decomposes cellulose and proteases, increasing the decomposition rate, and accelerates the degradation rate by altering the structure of gossypol. The compound microbial agent provided by this invention can promote the decomposition and transformation of organic matter in cotton stalks.

[0020] In one embodiment, the activity ratio of polyphenol oxidase to catalase in the composite enzyme of the present invention is (1~2):(1~2). In another embodiment, the activity ratio of polyphenol oxidase to catalase in the composite enzyme of the present invention is 2:1.5. In one embodiment, the activity ratio of polyphenol oxidase to β-glucosidase in the composite enzyme of the present invention is (1~2):(2~10); in another embodiment, the activity ratio of polyphenol oxidase to β-glucosidase in the composite enzyme of the present invention is 1.5:(2.5~3). In one embodiment, the activity ratio of polyphenol oxidase, catalase, and β-glucosidase in the composite enzyme of the present invention is (1.5~2):(1.5~2):(2.5~3). In one embodiment, the solvent of the composite enzyme of the present invention is phosphate buffer.

[0021] This invention provides the application of the cotton stalk aerobic composting biological promoter described in the above technical solution in promoting aerobic composting of cotton stalks.

[0022] As one implementation scheme, the method for promoting aerobic composting of cotton stalks according to the present invention includes promoting the degradation of gossypol in cotton stalk compost and / or increasing the humification of cotton stalk compost.

[0023] This invention provides a method for promoting aerobic composting of cotton stalks through synergistic effects of bacteria and enzymes, comprising the following steps: After mixing cotton stalks and livestock manure, the carbon-to-nitrogen ratio of the mixture is adjusted to 25-35 and the moisture content is 60%-70% to obtain compost material; The composting material and compound microbial agent are mixed for aerobic composting. During the high-temperature period, the compost is turned over once a week. When the temperature drops below 40°C during the maturation period, compound enzymes are inoculated. The compound microbial agent is the compound microbial agent in the cotton stalk aerobic composting biological promoter described in the above technical solution; the compound enzyme is the compound enzyme in the cotton stalk aerobic composting biological promoter described in the above technical solution.

[0024] This invention involves mixing cotton stalks and livestock manure, adjusting the carbon-to-nitrogen ratio of the mixture to 25-35 and the moisture content to 60-70%, to obtain compost. In one embodiment, the cotton stalks have a particle size of 4-5 cm. In another embodiment, the dry matter mass ratio of the cotton stalks to the livestock manure is (2-4):(1-2); the livestock manure includes one or more of pig manure, cow manure, horse manure, and chicken manure. In yet another embodiment, the carbon-to-nitrogen ratio of the mixture is adjusted to 30 and the moisture content to 65%.

[0025] After obtaining the compost material, the present invention mixes the compost material with the compound microbial agent for aerobic composting, turns the compost once a week during the high-temperature period, and inoculates the compost with compound enzymes when the temperature drops below 40°C during the maturation period.

[0026] The composite microbial agent is the same as the composite microbial agent in the aerobic composting bio-promoter for cotton stalks described in the above technical solution; the composite enzyme is the same as the composite enzyme in the aerobic composting bio-promoter for cotton stalks described in the above technical solution. In this invention, the compost is turned once a week during the high-temperature period. Without adding external equipment, the outer layer of low-temperature, low-oxygen, and slightly dry material is periodically and thoroughly mixed with the inner layer of high-temperature, high-humidity, and oxygen-deficient material. This allows oxygen to refill the pores, activating thermophilic bacteria to continuously and efficiently decompose organic matter, avoiding the generation of odors and methane in the anaerobic zone. Simultaneously, uniform heat dissipation prevents localized overheating that inhibits microbial activity, promotes moisture evaporation, reduces water content, shortens the composting cycle by approximately 7-10 days, and improves the humic content and nutrient stability of the finished product, achieving low-cost, high-efficiency, and environmentally friendly organic waste resource utilization. In this invention, the compost is not turned during the composting period. As one implementation scheme, during the aerobic composting process of this invention, the compost is turned once a week when the temperature is ≥55℃, stops turning when the temperature drops below 55℃, and is inoculated with the composite enzyme when the temperature drops below 40℃.

[0027] In one embodiment, the mass ratio of compost material to compound microbial agent in this invention is 100:(0.2~0.5); in another embodiment, the mass ratio is 100:(0.3~0.4). In the initiation stage of aerobic composting of cotton stalks, the above-mentioned compound microbial agent is inoculated, allowing *Aspergillus niger*, *Candida tropicalis*, and *Trichoderma viride* to rapidly colonize. First, these microorganisms rapidly proliferate using soluble sugars, galacturonic acid, and free amino acids in the cotton stalks, secreting pectinase, amylase, and protease to quickly convert easily degradable components into microbial biomass and heat energy, rapidly raising the compost temperature to around 40°C, creating a suitable temperature environment for humification. Second, β-glucosidase and endoglucanase secreted by *Aspergillus niger* can cleave long cellulose chains in the cell walls of cotton stalks, and the laccase-peroxidase complex of *Aspergillus* interacts with the quinone reductase of yeast to condense polyphenol-quinone intermediates with amino acids, constructing humic acid precursors. Xylanase and laccase secreted by *Trichoderma viride* effectively cleave the xylan and lignin network, exposing more humic precursors and promoting their transformation. The secreted extracellular polysaccharides act as cementing agents, promoting the stable granulation of humic colloids, thus achieving simultaneous detoxification of cotton stalks and high-quality humic material. Furthermore, the synergistic effect of these three bacteria optimizes the microbial community structure in compost, enhances the microbial capacity to decompose complex organic matter, and accelerates the transformation of organic matter and the formation of humic material. The polysaccharides and organic acids produced by microbial metabolism not only serve as humic precursors but also act as cementing agents, promoting the stable granulation of humic colloids and further enhancing the degree of humification. Therefore, the three bacteria in the initial stage, through multi-pathway synergistic action, not only efficiently degrade gossypol but also lay a solid foundation for the subsequent humification process, significantly improving the overall efficiency and quality of compost. With the rapid consumption of easily degradable components, the compost temperature exceeds 45℃ within 48 hours, entering the high-temperature period. *Trichoderma viride* continuously decomposes hemicellulose and lignin fragments, maintaining a high temperature of 50-65℃ for 5-7 days. During this stage, the thermostable enzyme systems of *Aspergillus niger* and *Trichoderma viride* remain active, continuing to oxidize the aromatic rings of gossypol and cleave the branches, resulting in an exponential decrease in gossypol content. The synergistic effect of *Aspergillus niger*, *Candida tropicalis*, and *Trichoderma viride* has a significant positive impact on the composting process and quality improvement. These microorganisms maintain high activity under high temperature conditions, and their secreted thermostable enzyme systems, such as cellulase, hemicellulase, and ligninase, continuously and efficiently decompose complex organic matter in cotton stalks, accelerating the degradation of cellulose, hemicellulose, and lignin, and promoting the transformation of organic matter. In addition, these microorganisms form a synergistic warming community with thermophilic microorganisms in the compost pile, maintaining a high-temperature environment for composting, ensuring the effective inactivation of pathogens and weed seeds, and improving the safety of compost. Polysaccharides and organic acids produced by microbial metabolism serve as precursors to humus, promoting the formation and accumulation of humus, while the cementing effect of extracellular polysaccharides further stabilizes the humic colloid, increasing the degree of humification.

[0028] In one embodiment, the mass ratio of compost material to compound enzyme in this invention is 100:(0.1~0.3); in another embodiment, the mass ratio is 100:0.2. This invention inoculates the compound enzyme during the maturation stage, when the pile temperature has gradually decreased from a high-temperature stage to a range suitable for enzyme activity, avoiding damage to the enzyme from high temperatures. Simultaneously, most of the easily degradable organic matter in the compost has been decomposed, leaving mostly recalcitrant substances. Adding exogenous enzymes at this stage helps to further decompose these substances, promotes humus formation, and improves the stability and fertilizer efficiency of the compost. Furthermore, the microbial community tends to be stable at this time, making the effect of the compound enzyme less susceptible to strong competition or inhibition. This invention inoculates a mixture of polyphenol oxidase, catalase, and β-glucosidase, which, on the one hand, allows for limited hydrolysis of the remaining recalcitrant matrix, and on the other hand, promotes the condensation of intermediate products and the formation of humus molecular structures through polyphenol oxidation and controlled oxidation environment, thereby accelerating the maturation and humification process and more effectively improving compost quality.

[0029] In one embodiment, the aerobic composting time of the present invention is 25-35 days; in another embodiment, the aerobic composting time of the present invention is 30 days. In one embodiment, the ventilation rate of the aerobic composting of the present invention is 0.3-0.6 L / (kg DM·min); in another embodiment, the ventilation rate of the aerobic composting of the present invention is 0.4 L / (kg DM·min). In one embodiment, the ventilation time of each ventilation of the aerobic composting of the present invention is 10-30 minutes; in another embodiment, the ventilation time of each ventilation of the aerobic composting of the present invention is 10 minutes. In one embodiment, the interval between two adjacent ventilations of the aerobic composting of the present invention is 50-80 minutes; in another embodiment, the interval between two adjacent ventilations of the aerobic composting of the present invention is 60 minutes.

[0030] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a method for synergistic promotion of aerobic composting of cotton stalks by a biological promoter and microbial enzymes, is provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 1. Composting raw materials: Crush cotton stalks to a particle size of 4-5cm, mix cow dung with crushed cotton stalks at a mass ratio of 1:4, add urea to adjust the carbon-nitrogen ratio of the mixture to 25, add water to adjust the moisture content to 65%, and obtain compost material.

[0032] 2. Preparation of compound biological agent: Aspergillus niger, Candida tropicalis, and Trichoderma viride are mixed to obtain a compound biological agent; wherein, the total viable count of the compound biological agent is ≥2×10⁻⁶. 8The cfu / g ratio of viable Aspergillus niger, Candida tropicalis, and Trichoderma viride in the compound biological agent is 1:2:3.

[0033] 3. Preparation of the complex enzyme: Prepare 1 L of phosphate buffer, then weigh out 300 U of polyphenol oxidase, 300 U of catalase and 500 U of β-glucosidase, and add them to the buffer solution in sequence. Stir gently with a magnetic stirrer until completely dissolved. To avoid contamination, filter the enzyme solution for sterilization or sterilize it at low temperature by bathing in a 60℃ water bath for 30 min. Finally, dispense the prepared enzyme solution into 50 mL or 100 mL portions, seal them and store them at 4℃ for a short period or freeze them at -20℃.

[0034] 4. Composting Unit: 60L fermentation tank. It adopts bottom ventilation, with a ventilation device connected to the bottom of the fermentation tank. Air inlets and outlets are located at the bottom and top of the reactor, respectively. A sieve plate is installed inside to facilitate uniform gas entry. The reactor has a built-in temperature sensor and ventilation system. An automated control system (PLC) controls the ventilation volume and frequency during composting, and can monitor and record temperature data, uploading it to the cloud and a mobile app.

[0035] 5. Inoculate the compound biological agent obtained in step 2 into the compost material at a ratio of 0.2 wt.% (the mass percentage of the compound biological agent in the compost material). After mixing evenly, place the composting device for aerobic composting for 30 days. Set the aeration rate of the composting to 0.4 L kg / min (DM), with each aeration lasting 10 minutes and an interval of 60 minutes. During the composting process, measure the temperature of the pile daily. During the high-temperature period (temperature ≥55℃), turn the pile once a week. Stop turning the pile when the temperature drops below 55℃. When the temperature drops below 40℃, inoculate the compound enzyme obtained in step 3 at a ratio of 0.3 wt.% (the mass percentage of the compound enzyme in the compost material).

[0036] Example 2 1. Composting raw materials: Crush cotton stalks to a particle size of 4-5cm, mix cow dung with crushed cotton stalks at a mass ratio of 1:4, add urea to adjust the carbon-nitrogen ratio of the mixture to 25, add water to adjust the moisture content to 65%, and obtain compost material.

[0037] 2. Preparation of compound biological agent: Aspergillus niger, Candida tropicalis, and Trichoderma viride are mixed to obtain a compound biological agent; wherein, the total viable count of the compound biological agent is ≥2×10⁻⁶. 8 The cfu / g ratio of viable Aspergillus niger, Candida tropicalis, and Trichoderma viride in the compound biological agent is 1:3:2.

[0038] 3. Preparation of the complex enzyme: Prepare 1 L of phosphate buffer, then weigh out 400 U of polyphenol oxidase, 400 U of catalase and 600 U of β-glucosidase, and add them to the buffer solution in sequence. Stir gently with a magnetic stirrer until completely dissolved. To avoid contamination, filter the enzyme solution for sterilization or sterilize it at low temperature by bathing in a 60°C water bath for 30 min. Finally, dispense the prepared enzyme solution into 50 mL or 100 mL portions, seal them and store them at 4°C for a short period or freeze them at -20°C.

[0039] 4. Composting Unit: 60L fermentation tank. It adopts bottom ventilation, with a ventilation device connected to the bottom of the fermentation tank. Air inlets and outlets are located at the bottom and top of the reactor, respectively. A sieve plate is installed inside to facilitate uniform gas entry. The reactor has a built-in temperature sensor and ventilation system. An automated control system (PLC) controls the ventilation volume and frequency during composting, and can monitor and record temperature data, uploading it to the cloud and a mobile app.

[0040] 5. Inoculate the compound biological agent obtained in step 2 into the composting raw materials at a ratio of 0.3 wt.% (the mass percentage of the compound biological agent in the composting material), mix thoroughly, and then place in a composting device for aerobic composting for 30 days. Set the composting ventilation rate to 0.4 L kg / min (DM), with each ventilation lasting 10 minutes and an interval of 60 minutes. Measure the temperature of the compost pile daily during the composting process. Once the temperature drops below 40℃, perform a turning operation and inoculate with the compound enzyme obtained in step 3 at a ratio of 0.3 wt.% (the mass percentage of the compound enzyme in the composting material).

[0041] Example 3 1. Composting raw materials: Cotton stalks are crushed and pretreated. Cow manure and cotton stalks are mixed at a mass ratio of 1:4. Urea is added to adjust the carbon-nitrogen ratio of the mixture to 25, and water is added to adjust the moisture content to 65%.

[0042] 2. Preparation of compound biological agent: Aspergillus niger, Candida tropicalis, and Trichoderma viride are mixed to obtain a compound biological agent; wherein, the total viable count of the compound biological agent is ≥2×10⁻⁶. 8 The cfu / g ratio of viable Aspergillus niger, Candida tropicalis, and Trichoderma viride in the compound biological agent is 1:2:2.

[0043] 3. Preparation of the complex enzyme: Prepare 1 L of phosphate buffer, then weigh out 400 U of polyphenol oxidase, 300 U of catalase and 600 U of β-glucosidase, and add them to the buffer solution in sequence. Stir gently with a magnetic stirrer until completely dissolved. To avoid contamination, filter the enzyme solution for sterilization or sterilize it at low temperature by bathing in a 60°C water bath for 30 min. Finally, dispense the prepared enzyme solution into 50 mL or 100 mL portions, seal them and store them at 4°C for a short period or freeze them at -20°C.

[0044] 4. Composting Unit: 60L fermentation tank. It adopts bottom ventilation, with a ventilation device connected to the bottom of the fermentation tank. Air inlets and outlets are located at the bottom and top of the reactor, respectively. A sieve plate is installed inside to facilitate uniform gas entry. The reactor has a built-in temperature sensor and ventilation system. An automated control system (PLC) controls the ventilation volume and frequency during composting, and can monitor and record temperature data, uploading it to the cloud and a mobile app.

[0045] 5. Inoculate the compound biological agent obtained in step 2 into the composting raw materials at a ratio of 0.3 wt.% (the mass percentage of the compound biological agent in the composting material), mix thoroughly, and then place in a composting device for aerobic composting for 30 days. Set the aeration rate of the composting to 0.4 L kg / min (DM), with each aeration lasting 10 minutes and an interval of 60 minutes. During the composting process, measure the temperature of the pile daily. Once the temperature drops below 40℃, perform a turning operation and inoculate with the compound enzyme obtained in step 3 at a ratio of 0.3 wt.% (the mass percentage of the compound enzyme in the composting material).

[0046] Comparative Example 1 1. Composting raw materials: Cotton stalks are crushed and pretreated. Cow manure and cotton stalks are mixed at a mass ratio of 1:4. Urea is added to adjust the carbon-nitrogen ratio of the mixture to 25, and water is added to adjust the moisture content to 65%.

[0047] 2. Composting Unit: 60L fermentation tank. It employs bottom ventilation, with a ventilation system connected to the bottom of the fermentation tank. Air inlets and outlets are located at the bottom and top of the reactor, respectively. An internal sieve plate facilitates even gas flow. The reactor incorporates a temperature sensor and ventilation system. An automated control system (PLC) controls the ventilation volume and frequency during composting, monitoring and recording temperature data and uploading it to the cloud and a mobile app.

[0048] 3. Place the composting materials directly into the composting device for aerobic composting for 30 days. Set the aeration rate to 0.4 L kg / min (DM), with each aeration lasting 10 minutes and an interval of 60 minutes. Monitor the temperature of the compost pile daily during the composting process. Once the temperature drops below 40℃, turn the pile over. Do not inoculate with compound enzymes.

[0049] Comparative Example 2 1. Composting raw materials: Cotton stalks are crushed and pretreated. Cow manure and cotton stalks are mixed at a mass ratio of 1:4. Urea is added to adjust the carbon-nitrogen ratio of the mixture to 25, and water is added to adjust the moisture content to 65%.

[0050] 2. Preparation of compound biological agent: Aspergillus niger, Candida tropicalis, and Trichoderma viride are mixed to obtain a compound biological agent; wherein, the total viable count of the compound biological agent is ≥2×10⁻⁶. 8 The cfu / g ratio of viable Aspergillus niger, Candida tropicalis, and Trichoderma viride in the compound biological agent is 1:2:2.

[0051] 3. Composting Unit: 60L fermentation tank. It employs bottom ventilation, with a ventilation system connected to the bottom of the fermentation tank. Air inlets and outlets are located at the bottom and top of the reactor, respectively. An internal sieve plate facilitates even gas flow. The reactor incorporates a temperature sensor and ventilation system. An automated control system (PLC) controls the ventilation volume and frequency during composting, monitoring and recording temperature data and uploading it to the cloud and a mobile app.

[0052] 4. Inoculate the compound biological agent from step 2 into the composting raw materials at a ratio of 0.3 wt.%, mix thoroughly, and place in a composting device for aerobic composting for 30 days. Set the composting ventilation rate to 0.4 L kg / min (DM), with each ventilation lasting 10 minutes and an interval of 60 minutes. Monitor the temperature of the compost pile daily during the composting process. Once the temperature drops below 40℃, perform a turning operation, without inoculating with the compound enzyme.

[0053] Comparative Example 3 1. Composting raw materials: Cotton stalks are crushed and pretreated. Cow manure and cotton stalks are mixed at a mass ratio of 1:4. Urea is added to adjust the carbon-nitrogen ratio of the mixture to 25, and water is added to adjust the moisture content to 65%.

[0054] 2. Composting Unit: 60L fermentation tank. It employs bottom ventilation, with a ventilation system connected to the bottom of the fermentation tank. Air inlets and outlets are located at the bottom and top of the reactor, respectively. An internal sieve plate facilitates even gas flow. The reactor incorporates a temperature sensor and ventilation system. An automated control system (PLC) controls the ventilation volume and frequency during composting, monitoring and recording temperature data and uploading it to the cloud and a mobile app.

[0055] 3. The composting materials were directly placed in the composting device for aerobic composting for 30 days. The aeration rate was set at 0.4 L kg / min (DM), with each aeration lasting 10 minutes and an interval of 60 minutes. The temperature of the compost pile was measured daily during the composting process. After the temperature dropped below 40°C, the pile was turned over, and the compound enzyme obtained in step 3 of Example 3 was inoculated at a ratio of 0.3 wt.% (the mass percentage of the compound enzyme in the compost material).

[0056] Comparative Example 4 1. Composting raw materials: Cotton stalks are crushed and pretreated. Cow manure and cotton stalks are mixed at a mass ratio of 1:4. Urea is added to adjust the carbon-nitrogen ratio of the mixture to 25, and water is added to adjust the moisture content to 65%.

[0057] 2. Preparation of compound biological agent: Aspergillus niger and Trichoderma viride are mixed to obtain a compound biological agent; wherein the total viable count of the compound biological agent is ≥2×10⁻⁶. 8 The cfu / g ratio of viable Aspergillus niger to Trichoderma viride in the compound biological agent is 1:2.

[0058] 3. Composting Unit: 60L fermentation tank. It employs bottom ventilation, with a ventilation system connected to the bottom of the fermentation tank. Air inlets and outlets are located at the bottom and top of the reactor, respectively. An internal sieve plate facilitates even gas flow. The reactor incorporates a temperature sensor and ventilation system. An automated control system (PLC) controls the ventilation volume and frequency during composting, monitoring and recording temperature data and uploading it to the cloud and a mobile app.

[0059] 4. Inoculate the compound biological agent from step 2 into the composting raw materials at a ratio of 0.3 wt.%, mix thoroughly, and place in a composting device for aerobic composting for 30 days. Set the aeration rate of the composting to 0.4 L kg / min (DM), with each aeration lasting 10 minutes and an interval of 60 minutes. Measure the temperature of the compost pile daily during the composting process. Once the temperature drops below 40°C, turn the pile and inoculate with the compound enzyme obtained in step 3 of Example 3 at a ratio of 0.3 wt.% (the percentage of the compound enzyme by mass in the composting material).

[0060] Comparative Example 5 1. Composting raw materials: Cotton stalks are crushed and pretreated. Cow manure and cotton stalks are mixed at a mass ratio of 1:4. Urea is added to adjust the carbon-nitrogen ratio of the mixture to 25, and water is added to adjust the moisture content to 65%.

[0061] 2. Preparation of compound biological agent: Aspergillus niger and Candida albicans are mixed to obtain a compound biological agent; wherein, the total viable count of the compound biological agent is ≥2×10⁻⁶. 8The cfu / g ratio of Aspergillus niger to Candida albicans in the compound biological agent is 1:2.

[0062] 3. Composting Unit: 60L fermentation tank. It employs bottom ventilation, with a ventilation system connected to the bottom of the fermentation tank. Air inlets and outlets are located at the bottom and top of the reactor, respectively. An internal sieve plate facilitates even gas flow. The reactor incorporates a temperature sensor and ventilation system. An automated control system (PLC) controls the ventilation volume and frequency during composting, monitoring and recording temperature data and uploading it to the cloud and a mobile app.

[0063] 4. Inoculate the compound biological agent from step 2 into the composting raw materials at a ratio of 0.3 wt.%, mix thoroughly, and place in a composting device for aerobic composting for 30 days. Set the aeration rate of the composting to 0.4 L kg / min (DM), with each aeration lasting 10 minutes and an interval of 60 minutes. Measure the temperature of the compost pile daily during the composting process. Once the temperature drops below 40°C, turn the pile and inoculate with the compound enzyme obtained in step 3 of Example 3 at a ratio of 0.3 wt.% (the percentage of the compound enzyme by mass in the composting material).

[0064] Comparative Example 6 1. Composting raw materials: Cotton stalks are crushed and pretreated. Cow manure and cotton stalks are mixed at a mass ratio of 1:4. Urea is added to adjust the carbon-nitrogen ratio of the mixture to 25, and water is added to adjust the moisture content to 65%.

[0065] 2. Preparation of compound biological agent: Trichoderma viride and Candida albicans are mixed to obtain a compound biological agent; wherein, the total viable count of the compound biological agent is ≥2×10⁻⁶. 8 The cfu / g ratio of viable Trichoderma viride to Candida albicans in the compound biological agent is 1:2.

[0066] 3. Composting Unit: 60L fermentation tank. It employs bottom ventilation, with a ventilation system connected to the bottom of the fermentation tank. Air inlets and outlets are located at the bottom and top of the reactor, respectively. An internal sieve plate facilitates even gas flow. The reactor incorporates a temperature sensor and ventilation system. An automated control system (PLC) controls the ventilation volume and frequency during composting, monitoring and recording temperature data and uploading it to the cloud and a mobile app.

[0067] 4. Inoculate the compound biological agent from step 2 into the composting raw materials at a ratio of 0.3 wt.%, mix thoroughly, and place in a composting device for aerobic composting for 30 days. Set the aeration rate of the composting to 0.4 L kg / min (DM), with each aeration lasting 10 minutes and an interval of 60 minutes. Measure the temperature of the compost pile daily during the composting process. Once the temperature drops below 40°C, turn the pile and inoculate with the compound enzyme obtained in step 3 of Example 3 at a ratio of 0.3 wt.% (the percentage of the compound enzyme by mass in the composting material).

[0068] Test Example 1 Samples were taken from the compost bins on days 0, 6, 14, 22, and 30 of the composting in Examples 1-3 and Comparative Examples 1-6, using a five-point sampling method for uniform sampling. The gossypol content in the samples was determined according to GB / T13086-2020, the method for determining free gossypol in feed; the contents of humic acid, fulvic acid, and humic acid in the samples were determined according to LY / T 1238-1999, the method for determining the composition of humic substances in forest soils. The results are shown in Tables 1-4.

[0069] Table 1. Gossypol content (mg / kg) in compost products under different composting methods

[0070] Table 2 Humic acid content (g / kg) in compost products under different composting methods

[0071] Table 3. Fulvic acid content (mg / kg) in compost products under different composting methods

[0072] Table 4. Humus content (mg / kg) in compost products under different composting methods

[0073] As can be seen from Tables 1-4, the example group inoculated with humifying bacteria and humifying enzymes showed better phenol degradation and higher humic content and its components. Inoculation with enzymes or bacteria alone also promoted humification of the pile, but the effect was worse than that of the example group. Overall, synergistic inoculation with bacteria and exogenous enzymes can achieve effective degradation of phenol in cotton stalks and promote the maturation of the pile to a great extent.

[0074] As can be seen from the above, the present invention can promote the aerobic composting of cotton stalks, degrade gossypol, and improve humification.

[0075] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bio-promoter for aerobic composting of cotton stalks, characterized in that, The cotton stalk aerobic composting bio-promoter includes two independently used components: component one is a compound microbial agent, and component two is a compound enzyme. The compound microbial agent includes Aspergillus niger (… Aspergillus niger ), Tropical Candida ( Candida tropicalis ) and green Trichoderma ( Trichoderma viride The total effective viable count of the compound microbial agent is ≥2×10⁻⁶. 8 cfu / g; The complex enzyme includes polyphenol oxidase, catalase and β-glucosidase; the total enzyme activity of the complex enzyme is 800~1800 U / L.

2. The cotton stalk aerobic composting bio-promoter according to claim 1, characterized in that, In the compound microbial agent, the effective viable count ratio of Aspergillus niger and Candida tropicalis is (1~1.5):(1~3). In the compound microbial agent, the effective viable count ratio of Aspergillus niger and Trichoderma viride is (1~1.5):(1~4.5).

3. The cotton stalk aerobic composting bio-promoter according to claim 1, characterized in that, In the complex enzyme, the enzyme activity ratio of polyphenol oxidase to catalase is (1~2):(1~2); In the complex enzyme, the enzyme activity ratio of polyphenol oxidase to β-glucosidase is (1~2):(2~10).

4. The application of the cotton stalk aerobic composting bio-promoter according to any one of claims 1 to 3 in promoting aerobic composting of cotton stalks.

5. The application according to claim 4, characterized in that, The promotion of aerobic composting of cotton stalks includes promoting the degradation of gossypol in cotton stalk compost and / or increasing the humification of cotton stalk compost.

6. A method for promoting aerobic composting of cotton stalks through synergistic effects of bacteria and enzymes, characterized in that, Includes the following steps: After mixing cotton stalks and livestock manure, the carbon-to-nitrogen ratio of the mixture is adjusted to 25-35 and the moisture content is 60%-70% to obtain compost material; The composting material and compound microbial agent are mixed for aerobic composting. During the high-temperature period, the compost is turned over once a week. When the temperature drops below 40°C during the maturation period, compound enzymes are inoculated. The compound microbial agent is the compound microbial agent in the aerobic composting bio-promoter for cotton stalks according to any one of claims 1 to 3; the compound enzyme is the compound enzyme in the aerobic composting bio-promoter for cotton stalks according to any one of claims 1 to 3.

7. The method according to claim 6, characterized in that, The dry matter mass ratio of the cotton stalks to the livestock and poultry manure is (2~4):(1~2); the livestock and poultry manure includes one or more of pig manure, cow manure, horse manure and chicken manure.

8. The method according to claim 6, characterized in that, The mass ratio of the compost material to the compound microbial agent is 100:(0.2~0.5).

9. The method according to claim 6, characterized in that, The mass ratio of the compost material to the compound enzyme is 100:(0.1~0.3).

10. The method according to any one of claims 6 to 9, characterized in that, The aerobic composting time is 25-35 days; the ventilation rate of the aerobic composting is 0.3-0.6 L / (kg DM·min), each ventilation lasts 10-30 minutes, and the interval between two adjacent ventilations is 50-80 minutes.

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