A compound microbial agent for sugarcane filter mud and sugarcane leaf mixed compost and an application method thereof

Abstract

The application relates to a composite microbial inoculum for mixed composting of sugarcane filter mud and sugarcane leaves and an application method thereof, and belongs to the technical field of microorganisms. The composite microbial inoculum comprises a high-temperature microbial inoculum, the high-temperature microbial inoculum is prepared from thermophilic carbon monoxide Streptomyces G-4-7, soil Brevibacillus G-5-2 and thermophilic Chaetomium; the composite microbial inoculum further comprises a medium-temperature microbial inoculum, the medium-temperature microbial inoculum is prepared from Trichoderma longibrachiatum, Staphylococcus warneri LH-403 and Bacillus thuringiensis LMU-81; and the composite microbial inoculum adopts a two-stage inoculation method. The composite microbial inoculum can effectively enhance the degradation of lignocellulose and further promote composting maturity, further improves the humification degree of the compost through a biological strengthening effect, is beneficial to the nitrogen fixation of the compost and the improvement of the germination index of the compost, can promote the resource utilization of organic matters rich in lignocellulose, and provides a technical basis for a green treatment method of agricultural wastes.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, specifically a compound inoculant for composting sugarcane filter mud and sugarcane leaves, and its application method. Background Technology

[0002] Economic development and population growth have driven the expansion of agricultural production, resulting in a large amount of agricultural waste. Sugarcane is the world's most widely cultivated sugar crop and also the most important energy and feed crop, possessing high ecological and economic value.

[0003] Step-by-step harvesting is a mode of mechanized sugarcane harvesting that breaks down the complete sugarcane harvesting process (such as cutting, stripping leaves, and transporting the sugarcane) into multiple independent stages. First, the sugarcane is cut; then, a transporter transfers the sugarcane to transport vehicles; and finally, specialized equipment separates the leaves and tops. Step-by-step harvesting is more suitable for scenarios with scattered plots, special crop characteristics, or insufficient adaptability of agricultural machinery in the planting area. However, the promotion of step-by-step harvesting currently faces a serious challenge: the timely disposal of stripped leaves. Due to the lack of mature and feasible high-value processing technologies, most of the leaves removed during step-by-step harvesting can only be piled up at the work site, occupying a large amount of land and posing a high risk of fire. With the further promotion of step-by-step harvesting, the amount of leaves produced will continue to increase. Therefore, exploring and realizing the on-site resource utilization of leaves has become crucial for the comprehensive advancement of sugarcane mechanized harvesting.

[0004] Sugarcane filter mud is a byproduct of sugarcane sugar production, an organic mixture that precipitates out after sugarcane juice has been filtered and clarified by an clarifier. Filter mud is rich in organic matter, nitrogen, phosphorus, potassium, and other fertilizer nutrients, as well as trace elements such as calcium, magnesium, and sulfur, making it a high-quality raw material for organic fertilizer. However, current utilization of filter mud mainly relies on simple composting or direct return to the field. Due to a lack of efficient composting technology, utilization efficiency is very low, and it easily leads to problems such as pests and diseases, soil-borne diseases, and root and seedling burn. In recent years, the industry has explored and practiced the preparation of organic fertilizer from filter mud, but existing technologies still generally suffer from long fermentation cycles, insufficient microbial activity, and unstable fertilizer effects, limiting the increase in product added value and making it difficult to support the efficient utilization of sugarcane filter mud.

[0005] Sugarcane leaves and sugarcane filter mud are both byproducts of sugarcane production and are rich in lignocellulose, making them suitable as raw materials for organic fertilizer. Mixing them results in a complementary carbon-to-nitrogen ratio and balanced nutrient elements. Adding fermenting microorganisms to produce organic fertilizer allows for the comprehensive utilization of waste. However, during fermentation, the recalcitrant nature of lignocellulose, especially its lignin component, often leads to low degradation efficiency in aerobic composting of lignocellulose-rich solid waste. To promote lignocellulose degradation and shorten the process, high-temperature composting is commonly used. The high-temperature period in composting is crucial for the biological decomposition of organic matter and ensuring the harmlessness of the compost. However, microorganisms are often affected by the high temperature during this stage, resulting in a general decrease in population size and activity, and a short residence time at high temperatures, thus limiting the rapid decomposition of organic matter.

[0006] Although existing technologies document numerous compound microbial agents for promoting composting and techniques for composting sugarcane filter mud and sugarcane leaves together, there is still room for improvement in the degradation capacity of these agents for lignocellulose in sugarcane filter mud-leaf compost. During the lignocellulose degradation process, the decomposition of lignin and cellulose is not sustained over a long period, resulting in a lower-than-expected degradation rate of the final substrate. Secondly, after being added to the compost, the compound microbial agents undergo different heating and cooling stages, and the individual microorganisms react differently to these temperatures, leading to reduced activity, inactivation, or even death of some microorganisms, thus affecting the efficacy of the compound microbial agents. Summary of the Invention

[0007] This invention addresses the problems of low lignocellulose degradation efficiency, long composting process, and low compost quality and humification degree in sugarcane filter mud and sugarcane leaf mixed compost. It provides a compound microbial agent for use in sugarcane filter mud and sugarcane leaf mixed compost, which can effectively enhance the degradation rate of lignocellulose and the compost maturity. The technical solution of this invention is as follows:

[0008] A compound microbial agent for composting sugarcane filter mud and sugarcane leaves, comprising a high-temperature microbial agent and a mesophilic microbial agent, which are packaged separately. The high-temperature microbial agent is suitable for composting at a temperature range of ≥50℃, while the mesophilic microbial agent is suitable for composting at a temperature range of <50℃.

[0009] Thermophilic bacterial agent is composed of thermophilic carbon monoxide-loving Streptomyces ( Streptomyces thermocarboxydus G-4-7, Soil-borne Bacillus ( Brevibacillus agri G-5-2 and thermophilic Chaetomium ( Chaetomium thermophilum )preparation;

[0010] The thermophilic carbon monoxide streptomyces ( Streptomyces thermocarboxydusG-4-7 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 30, 2025, with accession number CGMCC No. 36417.

[0011] The soil short-spore bacteria ( Brevibacillus agri G-5-2 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 30, 2025, with accession number CGMCC No. 36418.

[0012] The thermophilic Chaetomium ( Chaetomium thermophilum The sample is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC 3.17990.

[0013] Mesophilic bacterial agent composed of Staphylococcus warwick (Staphylococcus warneri) LH-403, Trichoderma longifolia ( Trichoderma longibrachiatum Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 preparation;

[0014] The Staphylococcus wartii (Staphylococcus warneri) LH-403 was deposited at the China General Microbiological Culture Collection Center on October 9, 2025, with accession number CGMCC No. 36214;

[0015] The long-branched Trichoderma ( Trichoderma longibrachiatum It is deposited at the China Industrial Microbial Culture Collection Center, accession number CICC 41185;

[0016] The Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36215.

[0017] The method for preparing the thermophilic bacterial agent includes the following steps: mixing *Streptomyces thermophilus* (a type of bacteria) with carbon monoxide. Streptomyces thermocarboxydus G-4-7, Soil-borne Bacillus ( Brevibacillus agri G-5-2 and thermophilic Chaetomium ( Chaetomium thermophilum Inoculate separately into liquid culture medium and incubate at 50°C, adjusting the colony count to 1×10⁻⁶. 8 CFU / mL; mix equal volumes of each strain to obtain the high-temperature bacterial agent.

[0018] The preparation method of the mesophilic bacterial agent includes the following steps: Staphylococcus warwick... (Staphylococcus warneri) LH-403, Trichoderma longifolia ( Trichoderma longibrachiatum Bacillus thuringiensis (Bacillus thuringiensis LMU-81 was inoculated into liquid culture medium and cultured at 28°C, with the colony count adjusted to 1×10⁻⁶. 8 CFU / mL; mix equal volumes of each strain to obtain a mesophilic bacterial agent.

[0019] The composting method of the compound microbial agent adopts a two-stage inoculation: a high-temperature microbial agent is inoculated in the initial stage of composting; after the compost enters the cooling period and the temperature drops below 50°C, a medium-temperature microbial agent is inoculated.

[0020] The total inoculum amount of the compound microbial agent in compost is 6% (v / w), of which the inoculum amount of thermophilic microbial agent and mesophilic microbial agent each accounts for 3%.

[0021] The application of the compound microbial agent in the degradation of lignocellulose-based agricultural waste.

[0022] The beneficial effects of this invention are:

[0023] 1. Aside from some purchased strains, the microorganisms used in this invention were selected and extracted from a composting system consisting of sugarcane leaves obtained from a sugarcane distributed harvesting site in Long'an County, Nanning City, Guangxi Province, and sugarcane filter mud obtained from a sugar factory in Long'an County. The mesophilic strains were collected from mangrove wetland soil in Fangchenggang City, Guangxi Province. The microbial advantage stems from the unique, harsh, and variable growth environment of mangroves. The abundance of high-lignin, high-cellulose fallen leaves and branches in mangroves, coupled with high salinity and oxygen deficiency, provides a unique "training ground" for microorganisms, resulting in higher degradation activity and efficiency compared to terrestrial microorganisms. Furthermore, mangrove soil is frequently oxygen-deficient due to periodic tidal inundation. Therefore, the strains must be capable of both aerobic respiration and anaerobic fermentation, which is particularly important for the traditionally believed requirement of strictly aerobic degradation of lignocellulose. In real-world industrialization projects, the strain's sensitivity to oxygen levels is crucial to success, highlighting the significant research and application value of mangrove wetland microbial strains.

[0024] 2. The present invention prepares a composite microbial agent containing a large number of lignocellulose-degrading microorganisms. These microorganisms exhibit no antagonism and significant synergistic effects during degradation, demonstrating highly efficient lignocellulose degradation capabilities, including that of natural lignocellulose such as sugarcane leaves. Firstly, in the high-temperature stage, the high-temperature composite microbial agent rapidly decomposes lignin, breaking down the "defense system" constructed by lignocellulose fibers. The high temperature itself softens lignin, while the mechanical destruction of the lignocellulose structure makes it more susceptible to attack by ligninases. Then, in the second stage, the mesophilic microbial agent further attacks the cellulose exposed from the lignin, causing a large-scale decomposition of cellulose. Simultaneously, lignin continues to degrade until the final stage of composting, achieving relative degradation rates of cellulose, hemicellulose, and lignin of up to 67.59%, 55.54%, and 52.37%, respectively.

[0025] 3. The two-stage inoculation method of this invention can extend the number of days in the high-temperature period of composting and increase the temperature during this period, thereby enhancing compost quality and promoting nitrogen fixation. Through biofortification, it increases the degradation rate of lignocellulose in the compost, further improving the degree of humification. This invention is simple and easy to operate, enabling efficient aerobic composting of sugarcane filter mud and sugarcane leaves. It helps solve the problems of resource waste and environmental pollution caused by sugarcane filter mud waste, and also addresses the difficulty in degrading and reusing sugarcane leaves accumulated at multi-stage harvesting sites, possessing profound social and economic value. Attached Figure Description

[0026] Figure 1 This is a graph showing the temperature changes during the composting process.

[0027] Figure 2 This is a graph showing the pH changes during the composting process.

[0028] Figure 3 This is a graph showing the changes in EC during the composting process;

[0029] Figure 4 This is a graph showing the changes in organic matter during the composting process;

[0030] Figure 5 NO3 during composting - -N variation curve;

[0031] Figure 6 NH4 during composting + -N variation curve;

[0032] Figure 7 This is a graph showing the change in total Kjeldahl nitrogen during the composting process;

[0033] Figure 8 This is a graph showing the change in the carbon-to-nitrogen ratio during composting.

[0034] Figure 9 This is a graph showing the change in the germination index during the composting process.

[0035] Figure 10 This is a graph showing the changes in humus content during composting.

[0036] Figure 11 This is a graph showing the change in fulvic acid content during composting.

[0037] Figure 12 This is a graph showing the changes in humic acid content during composting.

[0038] As shown in the uploaded microbial preservation certificate and microbial survival certificate, the strain preservation information is as follows:

[0039] thermophilic carbon monoxide streptomyces ( Streptomyces thermocarboxydus G-4-7 was deposited on October 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 36417; and is referred to as strain G-4-7 in the following examples.

[0040] Soil brevicorbacterium ( Brevibacillus agri G-5-2 was deposited on October 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 36418; and is referred to as strain G-5-2 in the following examples.

[0041] thermophilic chamomile ( Chaetomium thermophilum The sample was deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC 3.17990 and was purchased by the applicant from the CGMCC. In the following examples, it is referred to as *Chaetomium thermophilum*.

[0042] Staphylococcus wartii (Staphylococcus warneri) LH-403 was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 36214; in the following examples, it is abbreviated as strain LH-403.

[0043] Trichoderma longifolia ( Trichoderma longibrachiatum The sample, deposited at the China Industrial Microbial Culture Collection Center (CICC 41185), was purchased by the applicant from the CICC. In the following examples, it is abbreviated as Trichoderma longibranchii.

[0044] Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 36215; and is referred to as strain LMU-81 in the following examples. Detailed Implementation

[0045] Example 1

[0046] The preparation method of high-temperature compound microbial agent includes the following steps:

[0047] (1) Strains G-4-7 and G-5-2 were cultured in LB liquid medium for 2 days at 50℃ and 180 rpm. The bacterial count in the liquid medium was determined by viable plate counting method. The bacterial count was adjusted with sterile water to reach 1×10⁻⁶. 8 CFU / mL;

[0048] (2) The activated thermophilic Chaetomium was inoculated onto a PDA solid plate, and the fungal spores on the surface of the PDA plate were collected. The plate was then placed in PDB liquid medium and cultured at 50°C and 180 rpm for 7 days. The spore concentration was measured using a hemocytometer and adjusted to 1×10⁻⁶ with sterile water. 8 CFU / mL concentration;

[0049] (3) Mix strain G-4-7, strain G-5-2 and thermophilic Chaetomium in equal proportions to obtain a high-temperature bacterial agent.

[0050] The preparation method of mesophilic bacterial agents includes the following steps:

[0051] (1) Strains LH-403 and LMU-81 were cultured in LB liquid medium for 2 days at 28℃ and 180 rpm. The bacterial count in the liquid medium was determined by the serial dilution plate count method. The bacterial count was adjusted with sterile water to reach 1×10⁻⁶. 8 CFU / mL;

[0052] (2) The activated Trichoderma longifolia was inoculated onto a PDA solid plate, and the fungal spores on the surface of the PDA plate were collected. The plate was then placed in PDB liquid medium and cultured at 28°C and 180 rpm for 7 days. The spore concentration was measured using a hemocytometer and adjusted to 1×10⁻⁶ with sterile water. 8 CFU / mL concentration;

[0053] (3) Mix strain LH-403, strain LMU-81 and Trichoderma longifolia in equal proportions to obtain a mesophilic inoculum.

[0054] Example 2

[0055] Cellulase or ligninase activity was determined for strains G-4-7, G-5-2, thermophilic Chaetomium, thermophilic fungal agent, strain LMU-81, strain LH-403, Trichoderma longifolia, and mesophilic fungal agent.

[0056] (1) Preparation of crude enzyme solution

[0057] Each bacterial agent was cultured into a bacterial suspension and inoculated into the enzyme-producing fermentation medium at a volume ratio of 5%. Strains G-4-7, G-5-2, thermophilic Chaetomium, and thermophilic agents were cultured at 50℃, while strains LMU-81, LH-403, Trichoderma longifolia, and mesophilic agents were cultured at 28℃ and 180 rpm with shaking for 5 days. 5 mL of fermentation broth was centrifuged at 8000 rpm, and the supernatant after centrifugation for 10 min was the crude enzyme solution.

[0058] (2) Plot the glucose standard curve

[0059] Using a pipette, pipette 0.50 mL of glucose standard series solutions into 25 mL test tubes. Then, add 1.50 mL of 0.05 M pH 5.00 citrate buffer to each tube. For the control group, add 2 mL of the same concentration of citrate buffer. Finally, add 3 mL of DNS reagent to all tubes and mix thoroughly. Perform triplicate for each sample and control tube. Place the tubes in a boiling water bath for 10 minutes to allow the reaction to proceed, then immediately remove and cool. Adjust the volume to 25 mL with distilled water and mix thoroughly again. Using a blank tube as a control, measure the absorbance at 540 nm. Plot a standard curve with glucose concentration as the X-axis and absorbance as the Y-axis, and calculate the linear regression equation.

[0060] (3) Cellulase activity assay

[0061] Eng Activity Assay: The amount of reducing sugar released during hydrolysis was determined by the DNS colorimetric method, and the cellulase activity was calculated. 1 mL of 1% carboxymethyl cellulose solution was used as the substrate, and 0.50 mL of enzyme solution was added. The mixture was vortexed and incubated at 50°C for 30 min to complete the enzyme reaction. After the reaction was complete, 3 mL of DNS colorimetric reagent was immediately added to the mixture to stop the enzyme reaction. The treated sample was heated in a boiling water bath for 10 min and then cooled in water to maintain color stability. Next, an inactivated enzyme solution was used as a blank control, and the absorbance was measured at 540 nm using a UV spectrophotometer. Each experimental sample was tested in triplicate. Cellulase activity is defined as the amount of enzyme required to release 1 µmol of glucose from the corresponding substrate in 1 min at 50°C, expressed as U / mL. Enzyme activity is calculated using Formula 1: Where G is the glucose content (mg) determined by the standard curve; V is the total reaction volume (mL); T is the reaction time (min); and v is the enzyme volume (mL).

[0062] Exg activity assay: Using 1 mL of 1% microcrystalline cellulose as substrate, add 0.50 mL of enzyme solution, vortex to mix thoroughly, incubate the mixture at 50℃ for 30 min, remove the test tube, and immediately add 3 mL of DNS chromogenic reagent to stop the enzyme reaction. Heat in a boiling water bath for 10 min, cool in water to maintain color stability, use inactivated enzyme solution as a blank, and measure the absorbance at 540 nm using a UV spectrophotometer. Each experimental sample is performed in triplicate, and enzyme activity is calculated as shown in Formula 1.

[0063] β-Glu activity assay: Using 1 mL of 1% salicin as substrate, add 0.50 mL of enzyme solution, vortex to mix thoroughly, incubate the mixture at 50℃ for 30 min, remove the test tube, and immediately add 3 mL of DNS chromogenic reagent to stop the enzyme reaction. Heat in a boiling water bath for 10 min, cool in water to maintain color stability, use inactivated enzyme solution as a blank, and measure the absorbance at 540 nm using a UV spectrophotometer. Each experimental sample was performed in triplicate, and enzyme activity was calculated as shown in Formula 1.

[0064] Filter paper activity (FPA) assay: Using 50 mg of starch-free filter paper as substrate, add 0.50 mL of enzyme solution and 1 mL of 0.05 M pH 5.00 citrate buffer, vortex to mix thoroughly, and incubate the mixture at 50 °C for 30 min. Remove the test tube and immediately add 3 mL of DNS chromogenic reagent to stop the enzyme reaction. Heat in a boiling water bath for 10 min, then cool in water to maintain color stability. Use inactivated enzyme solution as a blank. Measure the absorbance at 540 nm using a UV spectrophotometer. Perform three replicates for each sample. Calculate enzyme activity as shown in Formula 1.

[0065] LiP activity assay: 1.50 mL of 0.10 M tartaric acid buffer, 1 mL of 10 mM resveratrol, 0.40 mL of crude enzyme solution were added sequentially, followed by 0.10 mL of 10 mM H₂O₂. The reaction was initiated at 30 °C. The absorbance change of the reaction solution at 310 nm wavelength was measured within the first 3 minutes. One unit of enzyme activity is defined as the amount of enzyme required to oxidize resveratrol to produce 1 μmol of veratraldehyde per minute.

[0066] MnP activity assay: MnP can convert Mn 2+ Oxidized to Mn 3+Add 2 mL of 0.05 M succinate buffer, 0.50 mL of 15 mM MnSO4, and 0.4 mL of crude enzyme solution sequentially, and finally add 0.10 mL of 10 mM H2O2 to start the reaction at 30 °C. Detect the absorbance change of the reaction solution at 240 nm wavelength within the first 3 minutes. One unit of enzyme activity is defined as the oxidation of 1 μmol Mn per minute. 2+ The required amount of enzyme.

[0067] Lac activity assay: 0.50 mL of 0.60 mM ABTS, 2 mL of 0.05 M citrate buffer, and finally 1 mL of crude enzyme solution were added sequentially. The reaction was started at 25°C, and the absorbance change of the reaction solution at 420 nm wavelength was detected in the first 3 minutes. One unit of enzyme activity is defined as the amount of enzyme required to catalyze 1 μmol of ABTS per minute.

[0068] The formulas for calculating LiP, MnP, and Lac enzyme activities are shown in Formula 2: Where ΔA is the absorbance change; ε is the molar absorptivity (mol) -1 ·L·cm -1 ); d is the optical path length of the cuvette (cm); V is the total reaction volume (mL); v is the crude enzyme solution volume (mL); T is the reaction time (min).

[0069] The following enzyme activity data were obtained:

[0070] The Lac, LiP, and MnP enzyme activities of strain G-4-7 were 3.48 U / mL, 19.54 U / mL, and 17.25 U / mL, respectively.

[0071] The Lac, LiP, and MnP enzyme activities of strain G-5-2 were 0.33 U / mL, 15.77 U / mL, and 8.85 U / mL, respectively.

[0072] The activities of Lac, LiP, and MnP enzymes in *Chaetomium thermophilum* were 8.85 U / mL, 3.52 U / mL, and 2.44 U / mL, respectively.

[0073] The enzyme activities of the high-temperature bacterial agents Lac, LiP, and MnP were 9.42 U / mL, 22.70 U / mL, and 19.50 U / mL, respectively.

[0074] The strain LMU-81 exhibited the following activity levels: exoglucanase (Exg) 12.42 U / mL, endoglucanase (Eng) 3.52 U / mL, β-glucosidase (β-glu) 1.41 U / mL, filter paper enzyme (FPA) 8.07 U / mL, laccase (Lac) 11.59 U / mL, lignin peroxidase (LiP) 22.65 U / mL, and manganese peroxidase (MnP) 6.62 U / mL.

[0075] The strain LH-403 exhibited the following activity levels: exoglucanase (Exg) 8.71 U / mL, endoglucanase (Eng) 13.91 U / mL, β-glucosidase (β-glu) 4.87 U / mL, filter paper enzyme (FPA) 7.80 U / mL, laccase (Lac) 2.05 U / mL, lignin peroxidase (LiP) 3.85 U / mL, and manganese peroxidase (MnP) 5.41 U / mL.

[0076] Trichoderma longifolia exhibited the following activity levels: exoglucase (Exg) 1.06 U / mL, endoglucase (Eng) 6.09 U / mL, β-glucosidase (β-glu) 12.01 U / mL, filter paper enzyme (FPA) 9.07 U / mL; laccase (Lac) 3.46 U / mL, lignin peroxidase (LiP) 12.67 U / mL, and manganese peroxidase (MnP) 14.30 U / mL.

[0077] The mesophilic bacterial inoculum exhibited the following activity levels: exoglucase (Exg) 15.67 U / mL, endoglucase (Eng) 15.23 U / mL, β-glucosidase (β-glu) 13.27 U / mL, filter paper enzyme (FPA) 13.71 U / mL; laccase (Lac) 13.22 U / mL, lignin peroxidase (LiP) 25.20 U / mL, and manganese peroxidase (MnP) 16.45 U / mL.

[0078] composting experiment

[0079] Sugarcane leaves and sugarcane filter mud samples were collected. The sugarcane leaves came from a sugarcane distributed harvesting point in Long'an County, Nanning City, Guangxi Province, and the sugarcane filter mud came from a sugar factory in Long'an County, Guangxi Province.

[0080] The sugarcane leaves used need to be dried to constant weight and then crushed using a crushing device to a length of less than 2cm to ensure uniform particle size of the sample.

[0081] Sugarcane leaves and sugarcane filter mud (wet weight) were thoroughly mixed at a weight ratio of approximately 1:1.5, adjusting the initial carbon-to-nitrogen ratio to approximately 25 and the initial moisture content to approximately 55%. Three composting experiments were conducted. The first group was the initial inoculation group (T1), where the two groups of compound bacteria were simultaneously inoculated into the compost at the initial stage (day 0). The second group was the two-stage inoculation group (T2), where thermotolerant compound bacteria were inoculated at the initial stage (day 0) and mesophilic compound bacteria were inoculated during the cooling stage (day 12). The control group (CK) received no microbial inoculation throughout the composting process. Equal volumes of sterile culture medium were added to the CK group on days 0 and 12. The total inoculation amount for both T1 and T2 groups was 6% (v / w), with 3% each of thermotolerant and mesophilic bacteria. The initial characteristics of the compost materials are shown in Table 1.

[0082]

[0083] The composting experiment was conducted by placing the experimental materials in a 60L plastic container with a breathable bottom in a well-ventilated room. To ensure adequate ventilation during composting, each compost pile was manually turned and sampled on days 0, 3, 7, 12, 18, 25, 35, and 45. Three samples were taken from each of the three different depths of the pile. All samples were thoroughly mixed and stored at -20°C for the determination of the compost's physicochemical parameters.

[0084] Example 3

[0085] Composting temperature measurement:

[0086] Throughout the composting process, probe-type electronic thermometers are inserted from the top of the compost reactor daily to measure the temperature at three specific locations in the central composting area, and the room temperature is recorded.

[0087] Temperature is considered the most critical parameter for controlling the composting process. Temperature fluctuations during composting reflect changes in the microbial population and the degradation process of organic matter.

[0088] As attached Figure 1 As shown, the temperature in group T2 exceeded 50℃ on the first day of composting, entering the high-temperature period earlier than groups CK and T1. The peak temperature of group T2 (75.1℃) was higher than that of groups CK (65℃) and T1 (69.2℃), and the duration of the high-temperature period was 4 days and 2 days longer than that of groups CK and T1, respectively. These results indicate that two-stage inoculation is more conducive to improving the metabolic activity of thermophilic microorganisms during the high-temperature period, thereby prolonging the duration of the high-temperature period and accelerating the composting fermentation process.

[0089] Example 4

[0090] pH determination of compost: Weigh 5.00g of fresh sample, add deionized water, shake at room temperature for 30 min, filter the supernatant and measure its pH value with a pH meter.

[0091] pH plays an important role in microbial growth and metabolism and is closely related to changes in compost composition, making it one of the important indicators reflecting the composting process.

[0092] As attached Figure 2 As shown, the pH value of the compost ranged from 7.4 to 8.5 throughout the composting process. During the high-temperature period of composting, the pH value of all composting groups increased significantly. This is likely because thermophilic microorganisms are more active at high temperatures, effectively enhancing the decomposition and ammonification of organic matter such as organic acids, proteins, and amino acids in the compost, leading to the accumulation of NH3 in the compost pile. As the temperature decreased, the decomposition of carbon sources by microorganisms, the accumulation of small-molecule organic acids, and the ammonia loss during the high-temperature period reduced the buffering capacity of the composting system, causing the pH value to begin to decrease after day 18 and gradually stabilize over time. At the end of composting, the pH values ​​of the CK and T1 groups were higher than those of the T2 group, which may have stimulated ammonia volatilization, leading to greater nitrogen loss. The results indicate that the pH value of the T2 group was less than 8, closest to neutral, and more suitable for seedling growth.

[0093] Example 5

[0094] EC value determination of compost: The EC value of the filtrate of the compost sample can be determined by using a conductivity meter.

[0095] EC indicates soluble salt content and compost maturity, and is used to evaluate the toxic effects of compost on plants.

[0096] As attached Figure 3 As shown, the EC values ​​of the three composting groups exhibited similar trends, all increasing during the high-temperature phase. This increase was attributed to the degradation of organic matter during this period, which produced a large amount of mineral salts, thus increasing the content of soluble salts. The EC values ​​of groups T1 and T2 decreased after the high-temperature phase, while those of the CK group began to decrease on day 7. This decrease was attributed to the presence of inorganic ions (such as NH4+). + It is released as gaseous NH3. During the cooling and maturation stages of composting, the EC value gradually increases due to the reduction in the mass of compost material and the formation of soluble salts from the degradation of organic matter. At the end of composting, the EC values ​​of all groups are below 4 mS / cm, meeting the safety requirements for agronomic production.

[0097] Example 6

[0098] Determination of organic matter content in compost: After drying the compost sample to constant weight at 105℃, weigh 0.50g of the dry sample, ignite it in a muffle furnace for 5h, and then calculate the organic matter content of the sample.

[0099] The degradation of organic matter involves the process of converting organic matter into H₂S and mineralizing it into inorganic matter in compost.

[0100] As attached Figure 4As shown, the organic matter content of each compost group gradually decreased during the composting process. Thermophilic microorganisms preferentially utilize soluble organic matter to obtain energy to meet their own proliferation and metabolic needs, and efficiently decompose macromolecules such as lignin, hemicellulose, and cellulose. Therefore, the organic matter degradation rate is highest during the high-temperature period. From day 0 to 18, the organic matter content of group T1 was the lowest. From day 18 to 45, the organic matter content of group T2 was lower than that of groups CK and T1, indicating that group T2 was beneficial for the degradation of organic matter in the later stages of composting. At the end of composting, the organic matter degradation rates of groups CK, T1, and T2 were 10.76%, 12.18%, and 12.43%, respectively, indicating that the inoculation of the compound bacteria improved the degradation rate of organic matter in the compost. Although the organic matter degradation rates of groups T1 and T2 were similar, further comparative analysis of the degradation rates and humification parameters of various components of lignocellulose in the compost revealed that the lignocellulose degradation rate of group T2 was higher than that of group T1. This may be because microorganisms in group T2 can more effectively convert organic matter such as lignocellulose into more stable humic macromolecular organic matter, and the mineralization of organic matter in group T2 is weaker than that in group T1.

[0101] Example 7

[0102] Compost NO3 - -N content determination: Weigh 5.00 g of compost sample, add 50 mL of 2M potassium chloride solution (2M potassium chloride solution preparation method: weigh 149.00 g KCl, dissolve in distilled water, and make up to 1 L). Shake at room temperature for 1 h, centrifuge and collect the supernatant. Add deionized water and activated carbon, and measure the absorbance of the supernatant at 220 nm and 275 nm.

[0103] The conversion of nitrogen in composting is a complex process, with ammoniation and nitrification being the most important nitrogen conversion steps.

[0104] As attached Figure 5 As shown, NO3 in all composting groups - -N concentrations showed a similar trend. In the initial stages of composting, NO3 was almost undetectable. - The formation of -N is mainly due to the significant inhibitory effect of the higher temperature and pH in composting on the activity and growth of nitrifying bacteria. As composting progresses, the pH and temperature decrease, leading to the accumulation of large amounts of NH4+ in the compost pile during the high-temperature phase. + -N promotes nitration. NH4 + -N concentration decreased significantly by day 12 and reached a stable state by day 25. NO3... - The -N concentration only increased rapidly after day 25, and its accumulation rate was significantly delayed. This is because the NH4+ concentration increased rapidly in the early stages of composting. + -N will also be released as NH3, rather than being completely converted into nitrate.

[0105] The results showed that at the end of composting, NO3 levels in the CK, T1, and T2 groups were significantly lower. - The NO3- concentrations were 52.71 mg / kg, 57.13 mg / kg, and 64.92 mg / kg, respectively. Compared with the initial material, NO3- concentrations decreased. - The concentrations of NO3- increased by 686.25%, 853.56%, and 955.92%, respectively. For many plants, the NO3- in organic fertilizers... - -N is more than NH4 + -N is a better form of nitrogen, indicating that two-stage inoculation helps improve the quality of mature compost.

[0106] Example 8

[0107] Compost NH4 + -N content determination: Take the NO3 content determined above. - Add 1 mL of the supernatant without activated carbon to the -N content sample, then add 9 mL of potassium chloride. After mixing and allowing to stand, add 40 mL of colorimetric reagent (sodium nitroprusside-phenol colorimetric reagent preparation method: weigh 0.80 g of sodium nitroprusside dihydrate, dissolve in distilled water, and bring the volume to 1 L; weigh 70.00 g of phenol, dissolve in distilled water, and bring the volume to 1 L; measure 15 mL of sodium nitroprusside dihydrate solution, 15 mL of phenol solution, and 750 mL of distilled water, and mix well). After 6 hours, measure the absorbance at 630 nm.

[0108] NH4 + -N inhibits aerobic fermentation in compost, and its concentration can be used as one of the important indicators for assessing the degree of compost maturity.

[0109] As attached Figure 6 As shown, the NH4 in each treatment group + -N concentrations all showed a trend of first increasing and then decreasing. NH4 + The -N concentration increased rapidly from day 0 to 3, with NH4+ concentrations in the CK, T1, and T2 groups increasing significantly. + The peak concentrations of -N were 483.60 mg / kg, 839.37 mg / kg, and 788.93 mg / kg, respectively. At high temperatures, ammonifying bacteria in the compost promote the rapid degradation of large amounts of nitrogen-containing organic matter, while nitrification is relatively weak, thus stimulating the production of NH4+. + The large-scale generation of -N. After the high-temperature period of composting, the activity of nitrifying microorganisms increases, leading to a surge in NH4+. + -N is converted to NO3 - -N. Simultaneously, the pile body releases NH3, causing NH4... + The concentration of -N decreased rapidly and then remained stable.

[0110] Studies have shown that at the end of composting, the NH4+ levels in the CK, T1, and T2 groups were significantly lower.+ -N concentrations decreased to 3.43 mg / kg, 3.55 mg / kg, and 3.43 mg / kg, respectively. In mature compost, NH4+... + The concentration of -N should generally be below 400 mg / kg. (NH4+ in three groups of compost) + -N concentrations all meet the standards for composting.

[0111] Example 9

[0112] Total Kjeldahl nitrogen and carbon-nitrogen ratio

[0113] (1) Total Kjeldahl Nitrogen (TKN) content: Weigh 0.5 g of dry compost sample, add copper sulfate, potassium sulfate and concentrated sulfuric acid, digest for 4 hours and cool, then transfer the digestion liquid into a fully automatic Kjeldahl nitrogen analyzer to determine the TKN content.

[0114] TKN is the sum of organic nitrogen and ammonia nitrogen, often used as a substitute indicator for total nitrogen content in compost, and is an important parameter for measuring compost quality.

[0115] As attached Figure 7 As shown, the TKN content decreased in all composting groups from day 0 to day 3, and then increased until the end of composting. The decrease in TKN content was mainly attributed to the increase in pH and temperature during composting, which led to an increase in NH4+. + The chemical balance between -N and NH3 is disrupted, leading to a large release of NH3. After the third day, the TKN content gradually increases. On the one hand, during composting fermentation, organic nitrogen is consumed by microbial metabolism, and ammonification and mineralization reactions also promote a large loss of nitrogen; on the other hand, the nitrogen-fixing effect of nitrogen-fixing bacteria in the compost reduces nitrogen loss, while the lignocellulose-degrading bacteria decompose a large amount of organic matter, resulting in a decrease in the dry weight of the compost. The reduction in organic carbon is greater than the reduction in organic nitrogen, leading to a gradual increase in the relative TKN content.

[0116] Studies have shown that at the end of composting, the TKN contents of the CK group, T1 group, and T2 group were 3.31%, 3.34%, and 3.46%, respectively, with the TKN content of the T2 group being the highest. This indicates that the two-stage inoculation has a better nitrogen retention effect.

[0117] (2) Carbon-nitrogen ratio: The organic matter content is converted into total organic carbon content, and the total Kjeldahl nitrogen content is calculated as total nitrogen content. The ratio of the two is the C / N of the compost sample.

[0118] The C / N ratio is one of the key indicators for measuring the nutrient balance and maturity of compost during the composting process. When the C / N ratio of compost drops below 20, it can be used as an important sign of compost maturity. Generally, the suitable C / N ratio for composting starting materials is 25 to 30. This ratio helps microorganisms efficiently absorb carbon and nitrogen, promoting their rapid reproduction and metabolism.

[0119] As attached Figure 8 As shown, during days 0-3, the TKN content was low, leading to an increase in the C / N ratio. After day 3, the degradation of organic carbon exceeded that of nitrogen-containing organic matter, and the C / N ratio gradually decreased. The C / N ratio decreased at the fastest rate during the high-temperature period because the metabolism of organic carbon is more rapid under high-temperature conditions.

[0120] Studies showed that on day 12, the C / N ratios of the CK, T1, and T2 groups were 20.36, 19.24, and 18.59, respectively, with the T1 and T2 groups having reached the maturity standard. After 45 days of composting, the C / N ratios of each composting group decreased to 13.56 (CK), 13.24 (T1), and 12.78 (T2) (all below 20), with the T2 group showing relatively better maturity.

[0121] Example 10

[0122] Seed germination index: Measure 5 mL of the filtrate used for pH and EC determination, add it to a petri dish containing filter paper, add an equal volume of deionized water to the blank group, select 10 cabbage seeds of uniform size, and evenly place them on the filter paper containing the filtrate using tweezers. Seal the dish with plastic wrap and incubate at 25 ℃ for 48 h. Calculate the seed germination index using the following formula (Formula 3): GI, as an important biological indicator, is widely used to assess the phytotoxicity and maturity of compost. This indicator was initially proposed by Zucconi et al. Compost is considered mature when its GI is ≥70%.

[0123] As attached Figure 9 As shown, on day 0 of composting, the GI values ​​for the CK, T1, and T2 groups were 51.90%, 52.53%, and 52.98%, respectively. Immature compost contains a large amount of small-molecule organic acids, salts, and ammonium nitrogen produced by the decomposition of organic matter. These substances can have varying degrees of toxicity to crops and inhibit crop growth. With the degradation of phytotoxic substances and the increase in the degree of humification in the compost, the GI values ​​of all treatment groups showed an upward trend.

[0124] Studies have shown that at the end of composting, the GI of groups T1 (117.92%) and T2 (128.77%) were both higher than that of the CK group (110.37%), indicating that the addition of the compound microbial agent of this invention can improve the maturity and safety of compost through biofortification, thereby promoting plant growth. The effect was particularly significant with the two-stage inoculation method. Ultimately, all three compost groups showed no phytotoxicity and can be safely applied to the soil.

[0125] Example 11

[0126] Determination of Lignocellulose Content in Compost

[0127] Compostable materials contain abundant organic macromolecules such as lignocellulose, proteins, and lipids. Lignocellulose is the most stable component, and its degradation products, such as oligosaccharides, monosaccharides, phenols, and quinones, are important precursors and frameworks for hygroscopic hemicellulose (HS). The complex structure of lignocellulose, with hemicellulose and cellulose encapsulated by lignin, significantly limits its biodegradation efficiency, thus hindering the humification process of composting. Therefore, accelerating the degradation of lignocellulose during composting is crucial.

[0128] The degradation rates of cellulose, hemicellulose, and lignin in compost samples were determined using the Van der Waals washing method.

[0129] The cellulose content in all composting groups showed a decreasing trend. The initial cellulose contents in the compost materials of the CK, T1, and T2 groups were 92.13 g / kg, 91.93 g / kg, and 91.30 g / kg, respectively. Throughout the composting process, the cellulose content in the CK group was consistently higher than that in the T1 and T2 groups. During the high-temperature period of composting, the cellulose degradation rate was highest in all groups, at which point thermophilic microorganisms dominated and preferentially decomposed readily available cellulose and hemicellulose. On day 12, the high-temperature period ended, and the cellulose degradation rates in the CK, T1, and T2 groups reached 23.65%, 32.03%, and 36.15%, respectively. As the composting temperature decreased, the cellulose degradation rate gradually declined. At the end of composting, the cellulose degradation rate in the T2 group (67.59%) was 1.53 times that of the CK group (44.32%) and 1.11 times that of the T1 group (61.00%), indicating that two-stage inoculation was beneficial to the degradation of cellulose in the compost.

[0130] Hemicellulose, the lowest molecular weight and most easily degraded component of lignocellulose, can be directly used as a carbon and energy source for microorganisms. The initial hemicellulose content in the compost materials of the CK, T1, and T2 groups was 58.90 g / kg, 59.23 g / kg, and 58.13 g / kg, respectively. Similar to cellulose, the degradation rate of hemicellulose increased significantly during the high-temperature period, with degradation rates of 17.56%, 23.72%, and 25.61% in the CK, T1, and T2 groups, respectively. This can be attributed to the activity of thermophilic microorganisms that promote hemicellulose degradation. At the end of composting, the hemicellulose degradation rate in the T2 group reached 55.54%, significantly higher than that in the CK group (38.82%) and the T1 group (50.55%). This indicates that the two-stage inoculation stimulated the activity of microorganisms in the compost, thereby accelerating the decomposition of hemicellulose.

[0131] Lignin is insoluble in water and possesses a highly branched and irregular aromatic complex three-dimensional organic structure, which gives it strong resistance to degradation. Initial lignin content: T1 group (40.77 g / kg), T2 group (40.60 g / kg), and CK group (39.83 g / kg). Due to its recalcitrant nature, lignin exhibited the lowest degradation rate among the three groups. At the end of composting, the lignin degradation rate of T2 group (52.37%) was 1.64 and 1.16 times that of CK group (32.02%) and T1 group (44.90%), respectively. The results indicate that inoculation with a lignocellulose-degrading complex can improve the lignin degradation rate in compost, and two-stage inoculation is more effective. The lignocellulose degradation rate in compost is shown in Table 2.

[0132]

[0133] Example 12

[0134] Determination of humus and its components in compost

[0135] Extraction and determination of the contents of humic acid (HS), fulvic acid (HA), and fulvic acid (FA) in compost:

[0136] (1) Extraction of HS: Weigh 1.00 g of dry compost sample and place it in a 100 mL Erlenmeyer flask. Add 40 mL of alkaline sodium pyrophosphate solution (pH 13.00) (Preparation method of alkaline sodium pyrophosphate solution (pH 13.00): Weigh 4.00 g NaOH, 26.607 g Na4P2O7⋅10H2O, dissolve in ultrapure water, and make up to 1 L). Extract at 25℃ and shake at 180 rpm for 24 h. Pour out the mixture and centrifuge at 10000 rpm for 15 min. Dilute the supernatant and filter it through a 0.45 μm filter membrane to obtain the HS solution.

[0137] (2) Extraction of HA and FA: The pH of the undiluted HS solution was adjusted to 1 with 6 M HCl (6 M HCl preparation method: measure 50 mL of concentrated hydrochloric acid, add it to ultrapure water for dilution, and finally make up to 100 mL with ultrapure water). After standing for 12 h, the solution was centrifuged at 10000 rpm for 15 min at 4℃. The supernatant was diluted and filtered through a 0.45 μm filter membrane to obtain FA. The precipitate was repeatedly washed with 0.05 M HCl (0.05 M HCl preparation method: measure 4.167 mL of concentrated hydrochloric acid, add it to ultrapure water for dilution, and finally make up to 1 L with ultrapure water). Then it was dissolved with 0.05 M NaHCO3 (0.05 M NaHCO3 preparation method: weigh 4.20 g of NaHCO3, add it to ultrapure water for dissolution, and make up to 1 L). After dilution, the solution was filtered through a 0.45 μm filter membrane to obtain the HA solution.

[0138] (3) Use a total organic carbon analyzer to detect the total organic carbon content of the above three solutions.

[0139] During aerobic composting, organic matter such as lignocellulose degrades to produce precursors such as amino acids, reducing sugars, and polyphenols, forming HS (humic acid). As a key product of composting, HS content is an important indicator for evaluating compost quality and humification.

[0140] As attached Figure 10 As shown, the HS content changes in the three composting groups showed a consistent trend. During the high-temperature period (days 0-3), thermophilic microorganisms exhibited high activity and vigorous metabolism, utilizing unstable HS as a carbon source. Inoculation with microorganisms may have enhanced the intensity of the metabolic reaction, leading to a more significant decrease in HS content. From days 3-7, the HS content increased, mainly due to the rapid decomposition of easily degradable organic matter in the raw materials, resulting in an increase in humic precursors. Simultaneously, the high temperature reduced microbial activity, with HS synthesis dominating the microbial consumption and synthesis process. As the composting temperature gradually decreased, microbial reproduction intensified, again competing for substrate, leading to a further decrease in HS content. At the end of composting, the HS content of group T2 (86.59 mg / g) was significantly higher than that of group T1 (80.13 mg / g). P <0.05) and CK group (76.33 mg / g) ( P <0.01).

[0141] HA and FA are the main components of HS (humic acid compost), and their changes can reflect the humification process of aerobic composting. HA is a structurally stable high-molecular-weight substance with multiple functional groups. As the final product of humification, its content changes can characterize the maturity and stability of compost products. In addition, HA is an important beneficial nutrient in soil and plays a vital role in increasing crop yield.

[0142] In this study, as shown in the appendix Figure 11 As shown, except for a slight fluctuation on day 35 (consistent with the changes in HS during this stage), the HA content showed an upward trend throughout the composting process. The T2 group showed the fastest increase in HA content between days 35 and 45. This may be due to two factors: firstly, the second inoculation may have promoted the degradation of lignocellulose, leading to an increase in HA precursors (such as phenols, quinones, and reducing sugars); secondly, the decrease in temperature may have facilitated the conversion of unstable humic components (FA) into stable HA through aromatization and polymerization. The final HA content of the T2 group (25.80 mg / g) was significantly higher than that of the CK group (16.92 mg / g). P <0.001) and 20.00 mg / g in group T1 ( P <0.01). The results show that the inoculation with the compound microbial agent of the present invention significantly improves the maturity and stability of compost.

[0143] Compared to HA, FA has a simpler structure and lower molecular weight, making it an active and unstable component of the HS (Hydrogen-Water) system. It is easily absorbed and utilized by microorganisms as a carbon source during composting. Under the action of microorganisms, FA and HA can interconvert, with the conversion of FA to stable HA being the dominant reaction. Therefore, unlike the trend of HA content changes, FA typically decreases during composting. (See attached image) Figure 12 As shown, the trend of FA changes from day 0 to 18 was consistent with that of HS. From day 0 to 3, the rate at which FA was utilized by microorganisms was higher than the rate of mineralization and synthesis of organic matter, leading to a decrease in FA content. Due to the more active microbial metabolism in group T2 during the high-temperature composting period, the decrease in FA content was most significant. As composting progressed, the available organic matter gradually decreased, and FA was used by microorganisms for energy consumption, transforming into the more structurally stable HA through condensation reactions during compost maturation. Ultimately, the FA content in groups CK, T1, and T2 decreased by 15.78%, 20.52%, and 16.93%, respectively. The results indicate that inoculation with the compound microbial agent of this invention contributes to a more stable formation of HS.

Claims

1. A compound microbial agent for composting sugarcane filter mud and sugarcane leaves, characterized in that, The compound microbial agent includes a high-temperature microbial agent and a medium-temperature microbial agent. The high-temperature microbial agent and the medium-temperature microbial agent are packaged separately. The high-temperature microbial agent is suitable for composting at a temperature range of ≥50℃, while the medium-temperature microbial agent is suitable for composting at a temperature range of <50℃. Thermophilic bacterial agent is composed of thermophilic carbon monoxide-loving Streptomyces ( Streptomyces thermocarboxydus G-4-7, Soil-borne Bacillus ( Brevibacillus agri G-5-2 and thermophilic Chaetomium ( Chaetomium thermophilum )preparation; The thermophilic carbon monoxide streptomyces ( Streptomyces thermocarboxydus G-4-7 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 30, 2025, with accession number CGMCC No. 36417. The soil short-spore bacteria ( Brevibacillus agri G-5-2 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 30, 2025, with accession number CGMCC No. 36418. The thermophilic Chaetomium ( Chaetomium thermophilum The sample is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC 3.17990. Mesophilic bacterial agent composed of Staphylococcus warwick (Staphylococcus warneri) LH-403, Trichoderma longifolia ( Trichoderma longibrachiatum Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 preparation; The Staphylococcus wartii (Staphylococcus warneri) LH-403 was deposited at the China General Microbiological Culture Collection Center on October 9, 2025, with accession number CGMCC No. 36214; The long-branched Trichoderma ( Trichoderma longibrachiatum It is deposited at the China Industrial Microbial Culture Collection Center, accession number CICC 41185; The Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36215.

2. The compound microbial agent for composting sugarcane filter mud and sugarcane leaves according to claim 1, characterized in that, The method for preparing the thermophilic bacterial agent includes the following steps: mixing *Streptomyces thermophilus* (a type of bacteria) with carbon monoxide. Streptomyces thermocarboxydus G-4-7, Soil-borne Bacillus ( Brevibacillus agri G-5-2 and thermophilic Chaetomium ( Chaetomium thermophilum Inoculate separately into liquid culture medium and incubate at 50°C, adjusting the colony count to 1×10⁻⁶. 8 CFU / mL; mix equal volumes of each strain to obtain the high-temperature bacterial agent.

3. The compound microbial agent for composting sugarcane filter mud and sugarcane leaves according to claim 1, characterized in that, The preparation method of the mesophilic bacterial agent includes the following steps: Staphylococcus warwick... (Staphylococcus warneri) LH-403, Trichoderma longifolia ( Trichoderma longibrachiatum Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 was inoculated into liquid culture medium and cultured at 28°C, with the colony count adjusted to 1×10⁻⁶. 8 CFU / mL; mix equal volumes of each strain to obtain a mesophilic bacterial agent.

4. A composting method using the compound microbial agent according to claim 1, characterized in that, A two-stage inoculation method was adopted: a high-temperature microbial agent was inoculated at the initial stage of composting; and a medium-temperature microbial agent was inoculated after the compost entered the cooling period and the temperature dropped below 50°C.

5. The composting method of the compound microbial agent according to claim 4, characterized in that, The total inoculum amount of the compound microbial agent in compost is 6% (v / w), of which the inoculum amount of thermophilic microbial agent and mesophilic microbial agent each accounts for 3%.

6. The application of the compound microbial agent as described in claim 1 in the degradation of lignocellulosic agricultural waste.

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