Composite microbial inoculant for efficient degradation of tobacco stalk lignin, preparation method and application thereof

Abstract

This invention discloses a compound microbial agent for the efficient degradation of lignin in tobacco straw, its preparation method, and its application. The compound microbial agent is composed of *Bacillus velezensis* L13 and *Bacillus cereus* L15. The compound agent contains at least 1 × 10⁻⁶ viable bacteria in each component. 7 The above two strains, each containing CFU / mL, were mixed in a 1:1 volume ratio. This invention also provides a method for preparing this compound microbial agent and its application in degrading lignin in tobacco straw and producing organic fertilizer. Furthermore, this invention provides a method for producing organic fertilizer, which involves mixing crushed tobacco straw and sheep manure at a mass ratio of 7:3, inoculating with the compound microbial agent, adjusting the carbon-to-nitrogen ratio to 25:1 and the moisture content to 60%-65%, and then aerobically fermenting at 40-60℃ for 50-60 days. This compound microbial agent can efficiently and synergistically degrade lignocellulose in tobacco straw, significantly accelerate the composting process, improve the quality of compost products, and realize the resource utilization of agricultural waste.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a compound microbial agent for the efficient degradation of lignin in tobacco straw, its preparation method, and its application. More specifically, this invention relates to a specific microbial composition composed of Bacillus velezensis L13 and Bacillus cereus L15, and its application in the preparation of a compound microbial agent for the efficient degradation of tobacco straw and in the production of organic fertilizer. Background Technology

[0002] my country is the world's largest tobacco producer, generating a large amount of tobacco straw waste annually. Statistics show that my country produces as much as 2.25-3 million tons of tobacco straw resources each year. Tobacco straw is rich in cellulose, hemicellulose, and lignin, offering potential for resource utilization. However, it is precisely the complex and dense structure formed by lignin, cellulose, and hemicellulose that makes tobacco straw difficult to degrade effectively in the natural environment. Currently, traditional methods of tobacco straw disposal mainly involve on-site dumping or direct burning, which not only wastes resources but also causes serious environmental pollution problems, such as air pollution and soil structure damage, and poses fire hazards.

[0003] Microbial degradation is considered an environmentally friendly and efficient core biotechnology approach for the resource utilization of agricultural waste. Its principle mainly involves inoculating straw with microbial agents containing specific functional microorganisms (such as lignocellulose-degrading bacteria). The enzymes secreted by these microorganisms decompose the large organic molecules in straw into smaller molecules that are easily absorbed and utilized by plants, or form stable humus, thereby enabling straw return to the field or the production of organic fertilizer. However, in practical applications, especially in the variable natural environment of the field, efficient straw degradation still faces many challenges, such as:

[0004] 1. Degradation efficiency problem: Single microbial strains usually have difficulty secreting the complete enzyme system required to degrade lignocellulose, resulting in limited degradation efficiency of the complex tobacco straw, and the degradation process is slow and incomplete.

[0005] 2. Environmental adaptability issues: The natural environment in the field (such as temperature, moisture, and competition among soil microbial communities) is complex and variable. Many microbial agents that perform well in the laboratory may not be able to fully exert their degradation capabilities under field conditions due to inhibited activity. For example, low temperatures can significantly slow down microbial metabolism, and antagonistic interactions between microorganisms can also affect the colonization and functional expression of target strains.

[0006] 3. The Challenge of Synergistic Effects in Compound Microbial Agents: While using compound microbial agents is a common approach to address the limitations of single-strain functions, a simple mixture of multiple strains does not necessarily produce a synergistic effect. If antagonistic interactions exist between strains, they can inhibit each other, leading to poor degradation. Therefore, screening for harmonious symbiotic and complementary strain combinations is crucial. Existing technologies have disclosed some compound microbial agents for degrading tobacco straw. For example, some solutions use a combination of three bacteria: *Bacillus zinniae*, *Acinetobacter ininus*, and *Bacillus*, while others use a combination of three bacteria: *Bacillus zinniae*, *Bacillus subtilis*, and another type of *Bacillus*. While these solutions have achieved some success, the search continues for strain combinations and solutions with higher degradation efficiency, better synergistic effects, and stronger environmental adaptability.

[0007] Furthermore, using degraded tobacco straw to produce high-quality organic fertilizer and verifying its actual effects on promoting crop growth (such as vegetable cultivation) is of great significance for achieving closed-loop recycling of tobacco straw and promoting sustainable agricultural development. However, how to use specific compound microbial agents not only to accelerate the decomposition of tobacco straw but also to improve the maturity and fertilizer efficiency of the final compost product is a direction that current technology needs to be further optimized.

[0008] Therefore, there is an urgent need in this field for a compound microbial agent that can efficiently and rapidly degrade tobacco straw, especially one with strong lignin degradation ability, good inter-strain synergy, and strong environmental adaptability, as well as a complete technical solution for its application in the production of high-quality organic fertilizer. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a compound microbial agent specifically for the efficient degradation of lignin in tobacco straw, its preparation method, and its application. This compound microbial agent, through the synergistic effect of two specifically screened strains, can significantly accelerate the degradation and composting process of tobacco straw, improve the quality of compost products, and ultimately promote tobacco growth.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] Firstly, a microbial composition.

[0012] The present invention provides a microbial composition, the key of which is composed of Bacillus velezensis L13 and Bacillus cereus L15.

[0013] Bacillus velezensis L13 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35382 on July 25, 2025.

[0014] Bacillus cereus L15 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37864 and deposit date of March 6, 2026.

[0015] Preferably, the ratio of viable bacteria of Bacillus belyssus L13 and Bacillus cereus L15 is in the range of 1:0.5 to 1:2.

[0016] Preferably, the viable counts of both *Bacillus belyi* L13 and *Bacillus cereus* L15 are not less than 1 × 10⁻⁶. 7 The concentration was CFU / mL and mixed at a volume ratio of 1:1. This specific ratio and concentration has been shown to produce the best synergistic degradation effect.

[0017] Secondly, a compound microbial agent and its preparation method.

[0018] This invention provides a multifunctional, highly efficient compound microbial agent for degrading lignin in tobacco stalks, composed of the above-mentioned microbial composition. Its preparation method includes the following steps:

[0019] Strain activation and seed culture preparation: The preserved Bacillus belye L13 and Bacillus cereus L15 were activated on LB solid medium, and then single colonies were picked and inoculated into LB liquid medium. The culture was shaken at 28-30℃ and 180-200rpm for 18-24 hours to obtain their respective seed cultures.

[0020] Fermentation culture: The above seed culture is inoculated into LB liquid medium at a certain inoculation amount (e.g., volume fraction 3%-5%) and cultured with shaking at 28-30℃ and 180-200rpm for 24-48 hours.

[0021] Cell collection and preparation: Centrifuge the fermentation broth (e.g., 8000 r / min, 10 min), collect the cell precipitate, wash with sterile water, centrifuge again, and resuspend in sterile water. Adjust the bacterial concentration to achieve a final concentration of 1×10⁻⁶ for both *Bacillus belye* L13 and *Bacillus cereus* L15. 7 CFU / mL.

[0022] Compound preparation: The two bacterial solutions prepared above are mixed evenly at a volume ratio of 1:1 to obtain the compound bacterial agent.

[0023] Thirdly, the application of the compound microbial agent.

[0024] This invention claims protection for the use of the above-mentioned compound microbial agent in the following aspects:

[0025] Application in the preparation of a multifunctional compound microbial agent for the efficient degradation of lignin in tobacco straw.

[0026] Its application in the production of organic fertilizer, especially in the process of producing organic fertilizer using tobacco straw as the main raw material.

[0027] Fourthly, a method for producing organic fertilizer.

[0028] This invention further provides a method for producing organic fertilizer using the above-mentioned compound microbial agent, characterized by comprising the following steps:

[0029] Raw material preparation: Crush tobacco stalks into small pieces of 2-5cm. Mix sheep manure with the crushed tobacco stalks at a dry matter mass ratio of 3:7.

[0030] Inoculation agent and parameter adjustment: Add the compound microbial agent described in this invention to the mixed raw materials and mix evenly to ensure that the number of effective viable bacteria in the pile is ≥1×10⁻⁶. 8 CFU / g. Meanwhile, urea is added to adjust the carbon-to-nitrogen ratio (C / N) of the mixture to 25:1, and water is added to adjust the moisture content to 60%-65%.

[0031] Aerobic fermentation: The prepared raw materials are piled in a composting facility equipped with ventilation and drainage systems, and aerobic fermentation is carried out at 40-60℃ for 50-60 days. During fermentation, ventilation and oxygen supply are provided by turning the pile regularly, and moisture is added as needed.

[0032] Beneficial effects of this invention

[0033] Compared with the prior art, the present invention has the following significant advantages:

[0034] 1. Outstanding Synergistic Effect of Strains: The combination of Bacillus belyssus L13 and Bacillus cereus L15 screened in this invention exhibits a significant synergistic effect in degrading tobacco straw. Experimental data show that the degradation rate of the mixed inoculant (19.82%) is significantly higher than that of any single strain treatment group (L13: 15.74%; L15: 13.38%), effectively solving the problem of limited degradation capacity of single strains.

[0035] 2. High degradation efficiency and strong targeting: This compound microbial agent has a high degradation capacity for lignin, cellulose and hemicellulose in tobacco straw, which can effectively destroy the complex physical structure of tobacco straw, accelerate its decomposition and shorten the composting cycle.

[0036] 3. Improve the quality of compost products: The seed germination index (GI) of the final product of composting treated with this compound microbial agent is significantly improved (up to 124%), and the degree of humification is higher (E4 / E6 value is lower), indicating that the compost is more thoroughly decomposed, the product quality is better, and it is safer for plant growth.

[0037] 4. Promote crop growth and realize resource utilization: Potted bok choy has proven that the application of organic fertilizer produced by this technology can significantly improve the agronomic traits of bok choy, increase its yield, realize the high-value resource utilization of tobacco straw, and conform to the direction of green agricultural development.

[0038] 5. Mature technology and easy to promote: The composting method provided by this invention has clear parameters, is easy to operate, has low cost, and is easy to promote and apply in actual production. Attached Figure Description

[0039] Figure 1 This is a graph showing the change in lignin content in the compost pile during the composting process in Example 3 of the present invention;

[0040] Figure 2 This is a graph showing the change in cellulose content in the compost pile during the composting process in Example 3 of the present invention;

[0041] Figure 3 This is a graph showing the change in hemicellulose content in the compost pile during the composting process of Example 3 of the present invention;

[0042] Figure 4 This is a diagram showing the pH changes in the compost pile during the composting process in Example 3 of the present invention.

[0043] Figure 5 This is a graph showing the change in electrical conductivity of the compost pile during the composting process in Embodiment 3 of the present invention;

[0044] Figure 6 This is a graph showing the change in humus content in the compost pile during the composting process in Example 3 of the present invention.

[0045] Figure 7 This is a graph showing the change in seed germination index in the compost pile during the composting process of Embodiment 3 of the present invention;

[0046] Figure 8A This is a graph showing the change in the fresh weight of the whole plant during the cultivation of *Vaccaria segetalis* in Example 4 of the present invention.

[0047] Figure 8B This is a graph showing the change in the fresh weight of the above-ground parts during the cultivation of *Vaccaria segetalis* in Example 4 of the present invention.

[0048] Figure 9A This is a graph showing the change in the dry weight of the whole plant during the cultivation of *Vaccaria segetalis* in Example 4 of the present invention.

[0049] Figure 9B This is a graph showing the change in aboveground dry weight during the cultivation of *Vaccaria segetalis* in Example 4 of the present invention. Detailed Implementation

[0050] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the invention is further described below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention.

[0051] Example 1: Preparation of Compound Microbial Agent

[0052] 1. Activation of microbial strains and preparation of seed culture

[0053] Bacillus velezensis L13 and Bacillus cereus L15 strains, preserved at low temperature (-80℃), were streaked onto LB solid medium (formula: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15-20 g / L agar, pH 7.0-7.2).

[0054] Place in a 28℃ constant temperature incubator for 24-48 hours to activate and culture until plump, single colonies appear.

[0055] Select single, typical colonies of L13 and L15 respectively and inoculate them into 250mL Erlenmeyer flasks containing 100mL LB liquid medium.

[0056] The Erlenmeyer flasks were placed in a constant temperature shaking incubator and shaken at 28℃ and 180 rpm for 18-24 hours to obtain turbid, vigorously growing L13 and L15 seed solutions.

[0057] 2. Fermentation culture

[0058] Prepare LB liquid culture medium (i.e., 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0-7.2), dispense 250 mL into 500 mL Erlenmeyer flasks, and autoclave at 121 °C for 20 minutes for later use.

[0059] The L13 seed solution and L15 seed solution prepared in step 1 were aseptically inoculated into the sterilized culture medium at an inoculation rate of 5% by volume.

[0060] After inoculation, the Erlenmeyer flasks were placed in a constant temperature shaking incubator and cultured for 24 hours at 28°C and 180 rpm.

[0061] 3. Cell collection and concentration adjustment

[0062] The fermented L13 and L15 bacterial cultures were poured into 50 mL centrifuge tubes and centrifuged at 4 °C and 8000 r / min for 10 minutes. The supernatant was discarded and the bacterial precipitate was collected.

[0063] Add an appropriate amount of sterile physiological saline (0.85% NaCl solution) to the centrifuge tube containing bacterial precipitate, vortex to fully resuspend the bacterial cells, and then centrifuge and wash again under the same conditions. Repeat this process three times to obtain pure bacterial cells.

[0064] The washed bacterial cells were resuspended in sterile physiological saline, and the absorbance (OD) of the bacterial suspension at a wavelength of 600 nm was measured using a UV spectrophotometer. 600 value).

[0065] The concentrations of both L13 and L15 bacterial suspensions were adjusted to 1×10⁻⁶ by adding sterile saline. 7 CFU / mL. This concentration is determined by a pre-established OD. 600 The values ​​were calibrated and validated using a standard curve of viable cell counts.

[0066] 4. Compound formulation of compound microbial agents

[0067] The concentration of the sample prepared in step 3 is 1×10 7 A CFU / mL suspension of Bacillus belye L13 and a suspension of Bacillus cereus L15 were aseptically mixed in a sterile container at a 1:1 volume ratio (e.g., 50 mL of each).

[0068] The multifunctional tobacco stalk lignin-efficient degrading compound microbial agent is obtained by thoroughly mixing the mixed bacterial solution using a shaker or magnetic stirrer.

[0069] The compound microbial agent prepared in this embodiment has two strains in a highly active state and in a synergistic ratio. It can be directly used for subsequent composting applications or low-temperature storage (4℃ storage is recommended, with a shelf life of 30 days).

[0070] This embodiment provides two multifunctional lignocellulose-degrading bacteria, identified as Bacillus velezensis (L13) and Bacillus cereus (L15), respectively. They are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 35382 and CGMCC No. 37864, respectively.

[0071] In this embodiment, Bacillus berleis (L13) and Bacillus cereus (L15) were screened from tobacco straw compost products from the Luoning Tobacco Base in Luoyang City, Henan Province. The colony morphology of Bacillus berleis is as follows: the colony surface is smooth and slightly convex, the edges are irregular, and it is milky white. The colony morphology of Bacillus cereus is as follows: opaque, milky white, round, the surface is not smooth, and it is not raised.

[0072] Example 2: Verification of the degradation effect of compound microbial agents on tobacco straw

[0073] This embodiment aims to quantitatively verify the degradation efficiency of the compound microbial agent of the present invention on tobacco straw through laboratory simulated degradation experiments, and in particular to demonstrate the synergistic effect of the combined use of Bacillus belyss L13 and Bacillus cereus L15.

[0074] 1. Preparation of experimental materials

[0075] Tobacco straw processing: Collect tobacco straw from the field, wash it, and dry it in an 80℃ oven until constant weight. Crush it using a pulverizer and pass it through a 20-mesh sieve to obtain uniform tobacco straw powder for later use.

[0076] Culture medium: LB liquid medium (formulation same as in Example 1).

[0077] Test inoculum:

[0078] Treatment group L13: The concentration of Bacillus belyss L13 single-strain inoculum prepared in Example 1 was adjusted to 1×10⁻⁶. 8 CFU / mL.

[0079] Treatment group L15: The concentration of Bacillus cereus L15 single-strain inoculum prepared in Example 1 was adjusted to 1×10⁻⁶. 8 CFU / mL.

[0080] Treatment group L13+L15 (this invention): The above-mentioned L13 and L15 bacterial agents are mixed at a volume ratio of 1:1 to obtain a compound bacterial agent. After mixing, the effective viable count of each bacteria remains at 1×10⁻⁶. 8 CFU / mL.

[0081] Control group (CK): An equal volume of sterile water was used instead of the bacterial agent.

[0082] Basic culture medium: The culture medium used for degradation experiments. Its composition is: sodium nitrate (NaNO3) added to deionized water to make its concentration meet the nitrogen source required for microbial growth.

[0083] 2. Experimental Methods

[0084] Degradation experiment setup: Weigh 5.00 g (accurate to 0.01 g) of the pretreated tobacco straw powder and place it in a 250 mL Erlenmeyer flask. Add 30 mL of the above-mentioned basal culture medium to each Erlenmeyer flask and supplement with 2.0 g of sodium nitrate.

[0085] Inoculation with bacterial agent: Add 10 mL of the corresponding bacterial agent (treatment groups L13, L15, L13+L15) or 10 mL of sterile water (control group CK) to each conical flask. Each treatment was set up in 3 parallel replicates.

[0086] Culture conditions: Place the inoculated conical flask in a constant temperature shaking incubator and shake it at 28℃ and 180 rpm to provide sufficient oxygen and promote full contact between the inoculant and the straw.

[0087] Sample collection and analysis: On days 10 and 20 of culture, conical flasks from each treatment were randomly selected (each sampling included parallel samples from all treatments). The contents of the flasks were filtered through three layers of gauze to separate the solid residue from the filtrate. The solid residue was collected, gently rinsed with deionized water to remove attached bacteria and water-soluble substances, and then dried in an 80°C oven to constant weight. The dry weight was weighed and recorded.

[0088] 3. Result Calculation and Data Analysis

[0089] The degradation rate (weight loss rate) of tobacco straw is calculated using the following formula:

[0090] Degradation rate (%) = (W0 - W1) / W0 × 100% where:

[0091] W0 represents the initial dry weight (g) of tobacco straw in the conical flask before inoculation.

[0092] W1 represents the dry weight (g) of tobacco straw residue collected after the cultivation period.

[0093] Experimental data are expressed as mean ± standard deviation, and significance analysis was performed using appropriate statistical methods (such as Duncan's new multiple range method). The results are shown in the table below.

[0094] Table 1. Degradation effect of compound microbial agent on tobacco straw (degradation rate %)

[0095] Processing group 10 days of cultivation 20 days of cultivation Control group (CK) 5.12±0.35c 8.07±0.28d L13 single-strain agent 10.59±0.21b 15.74±0.14b L15 single-strain agent 7.23±0.45c 13.38±0.35c L13+L15 compound microbial agent (this invention) 13.32±0.61a 19.82±0.11a

[0096] Note: Different lowercase letters after the data in the same column indicate that the difference is significant at the P<0.05 level.

[0097] 4. Experimental Conclusions

[0098] The results of this embodiment clearly demonstrate that:

[0099] Significant synergistic effect: After 20 days of cultivation, the L13+L15 compound microbial agent of this invention achieved a degradation rate of 19.82% for tobacco straw, which was significantly higher than that of any single microbial agent treatment group (L13: 15.74%; L15: 13.38%) and the control group. This fully demonstrates that the combination of Bacillus belyssus L13 and Bacillus cereus L15 produced a synergistic degradation effect of "1+1>2".

[0100] Degradation efficiency increases over time: As the culture time is extended, the degradation rate of all inoculated bacterial agents in the treatment group increases, but the composite bacterial agent of the present invention shows the greatest increase, demonstrating its continuous and efficient degradation ability.

[0101] Example 3: Application of compound microbial agents in indoor aerobic composting of tobacco straw (e.g.) Figure 1-7 )

[0102] This embodiment aims to simulate actual production conditions and verify the application effect of the compound microbial agent described in this invention in the aerobic composting process of tobacco straw and sheep manure, focusing on evaluating its effect on accelerating the composting process and improving the quality of the final compost product.

[0103] 1. Preparation and pretreatment of compost raw materials

[0104] Tobacco straw: sourced from a tobacco planting base in Henan Province, with an initial moisture content of 43.56%, total carbon content of 42.15%, and total nitrogen content of 1.54% (initial C / N ratio of tobacco straw to sheep manure at a mass ratio of 7:3 is 27.33:1). Before use, the tobacco straw is sun-dried for 3-5 days to further reduce moisture, and then shredded into small pieces of 2-5cm in length using a straw shredder for later use.

[0105] Livestock and poultry manure: Sheep manure provided by local large-scale farms is used, with an initial moisture content of 55.94%, a total carbon content of 32.10%, and a total nitrogen content of 2.10%.

[0106] Compound bacterial agent: A compound bacterial agent of Bacillus belye L13 and Bacillus cereus L15 prepared by the method described in Example 1, with an effective viable count of 1×10⁻⁶ for each. 7 CFU / mL, and mixed at a 1:1 volume ratio.

[0107] Other auxiliary materials: Urea (46% nitrogen content) is used to adjust the initial C / N ratio of compost.

[0108] 2. Experimental Design and Composting Initiation

[0109] Experimental setup: This experiment consists of 3 treatments:

[0110] CK (control group): tobacco straw + sheep manure + equal volume of sterile water.

[0111] T1 (Control Group): Tobacco straw + sheep manure + another commercially available compound microbial agent M1 (as a process comparison).

[0112] T2 (Invention Group): Tobacco straw + sheep manure + the compound microbial agent of the present invention (L13+L15).

[0113] Composting Start-up:

[0114] Raw material mixing: In each compost bin (60L capacity, with ventilation and drainage holes at the bottom), weigh 7.0kg of crushed tobacco straw and 3.0kg of sheep manure at a dry matter mass ratio of 7:3, and mix them evenly.

[0115] Inoculation agent and adjustment parameters: The compound microbial agent of this invention was introduced into the mixed raw materials of the T2 treatment group, and the inoculation amount ensured that the effective viable bacteria count in the pile reached ≥1×10⁻⁶. 8 CFU / g. The CK group was given an equal volume of sterile water, and the T1 group was inoculated with an equal volume of commercially available bacterial agent M1.

[0116] Adjust key parameters: Add urea to all composting materials to precisely adjust the carbon-to-nitrogen ratio (C / N) of the mixture to 25:1. At the same time, spray sterile water to adjust the initial moisture content of the compost pile to 60%-65%.

[0117] Initial index determination: On the day of composting start-up (day 0), a representative sample of 300g was taken from each treatment to determine its initial pH, electrical conductivity (EC), lignin, cellulose and hemicellulose content.

[0118] 3. Composting process management

[0119] Evenly pile the inoculated and properly calibrated compost into the compost bin, to a height of about 50cm.

[0120] Fermentation conditions: Place the compost bin in a constant temperature incubation room and control the ambient temperature to maintain the internal temperature of the compost pile between 40-60℃. The entire fermentation cycle is 50-60 days.

[0121] Process Management:

[0122] Oxygen supply and ventilation: Oxygen is supplied by regularly turning the pile. In the initial stage (days 1-15), turn the pile every 2 days; in the middle stage (days 16-40), turn the pile every 3 days; and in the later stage (day 41 to the end), turn the pile once a week. Ensure that the materials on both sides are mixed evenly when turning the pile.

[0123] Moisture control: Weigh the total weight of the compost bin every 3-5 days and replenish the moisture lost through evaporation by spraying sterile water, so that the moisture content of the compost pile is always maintained within the suitable range of 50%-65% throughout the fermentation period.

[0124] 4. Sample collection and indicator monitoring

[0125] Sampling points: Samples were taken on days 0, 7, 21, 42, and 56 of composting.

[0126] Sampling method: A five-point sampling method was used to collect about 500g of fresh sample from different positions at the top, middle and bottom of the pile. After being mixed evenly, the sample was divided into two portions. One portion was used to determine the physicochemical properties, and the other portion was frozen at -20℃ for enzyme activity determination.

[0127] Monitoring indicators and methods:

[0128] Basic physicochemical properties: pH value (potential method), EC value (conductivity method).

[0129] Lignocellulose degradation rate: Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were determined using the Pantheon detergent fiber analysis method, and the contents of cellulose, hemicellulose, and lignin were calculated.

[0130] Maturity index:

[0131] Humic index (E4 / E6): The ratio of absorbance of compost water extract at 465 nm and 665 nm.

[0132] Seed germination index (GI): determined using a radish seed germination test.

[0133] 5. Results and Analysis

[0134] The changes in key indicators during composting clearly demonstrate the effectiveness of the compound microbial agent of this invention:

[0135] Lignocellulose degradation: such as Figure 1-3 As shown, in all treatments, the contents of lignin, cellulose, and hemicellulose all decreased with composting time. At the end of composting, the T2 treatment group (the present invention) exhibited the highest degradation rates of lignin, cellulose, and hemicellulose, reaching 19.48%, 64.69%, and 64.75%, respectively, significantly higher than the CK group (9.23%, 58.96%, 62.33%) and the T1 group (12.59%, 50.51%, 57.83%). This indicates that the composite microbial agent of the present invention can effectively and synergistically degrade the complex lignocellulose structure in tobacco straw.

[0136] The ripening process is accelerated: such as Figure 4 and Figure 5 As shown, the pH of all treatments eventually stabilized at around 8.0 (meeting organic fertilizer standards), and the EC values ​​were all less than 4 mS / cm (non-toxic to plants). The T2 treatment group showed a significant advantage in key indicators characterizing decomposition maturity.

[0137] Seed germination index (GI): The GI value of the T2 treatment group rose rapidly in the middle of composting and was the first to exceed the 80% maturity standard. It reached 124% at the end of composting, which was significantly higher than that of the CK group (104%) and the T1 group (111%), indicating that it was the most thoroughly matured and had the lowest plant toxicity.

[0138] Humification index (E4 / E6): At the end of composting, the T2 treatment group had the lowest E4 / E6 value (2.4), indicating that it had the highest degree of humification and that the compost product was more stable and of better quality.

[0139] 6. Experimental Conclusions

[0140] This embodiment fully demonstrates that applying the compound microbial agent described in this invention to aerobic composting of tobacco straw and sheep manure can:

[0141] It significantly accelerates the degradation of lignocellulose, especially exhibiting highly efficient decomposition ability for recalcitrant lignin.

[0142] It significantly shortens the composting cycle and improves the degree of decomposition, as evidenced by the seed germination index (GI) reaching the decomposition standard faster and having a higher final value.

[0143] Improving the quality of the final compost product results in a more stable and safer product, which is beneficial to crop growth.

[0144] In summary, this embodiment discloses in detail the complete process and verification data of composting using the compound microbial agent of the present invention, fully demonstrating the feasibility and superiority of this technology in achieving efficient resource utilization of tobacco straw.

[0145] Example 4: Verification of the effect of organic fertilizer produced by compound microbial agent on the growth of spinach, as shown in Figures 8(AB)-9(AB).

[0146] This embodiment aims to verify the effect of tobacco straw organic fertilizer and compound microbial agent produced using the compound microbial agent of the present invention on promoting the growth of spinach under actual planting conditions, and to demonstrate its end-use application value.

[0147] 1. Overview and Design of the Experimental Site

[0148] Experimental location and time: The experiment was conducted in 2025 in a greenhouse at the Institute of Horticulture, Anhui Academy of Agricultural Sciences.

[0149] Test materials:

[0150] Vegetable seedlings used in the experiment: Chinese spinach, provided by the Institute of Horticulture.

[0151] Test fertilizer: Tobacco straw organic fertilizer produced by the method described in Example 3 was applied.

[0152] Experimental Design:

[0153] Organic fertilizer should be applied at 1% of the dry matter weight of the potting soil, and compound microbial agent should be applied at a rate of 1×10⁻⁶. 7 CFU / g.

[0154] OF1: Apply organic fertilizer T1

[0155] OF2: Apply organic fertilizer T2

[0156] 2. Methods

[0157] Routine management: The potted Chinese cabbage plants in each treatment were managed in accordance with local vegetable management methods.

[0158] Measurement indicators and methods:

[0159] Fresh weight: First, use absorbent paper to remove the residual moisture from the corresponding part of the black cabbage, and then weigh it using an electronic balance with an accuracy of 0.01g.

[0160] Dry weight: First, use a 105℃ oven to blanch for half an hour, then set the oven temperature to 75℃ and dry until constant weight, then weigh using an electronic balance with an accuracy of 0.001g.

[0161] 3. Results Analysis

[0162] Fresh weight: such as Figure 8A , 8B As shown, the OF2 treatment with the organic fertilizer of this invention increased the whole plant fresh weight by 32.47% at 45 days compared with the OF1 treatment; the aboveground fresh weight at 45 days also increased by 32.47% compared with the OF1 treatment. The whole plant, aboveground and underground fresh weights of the OF2 treatment at 45 days were all significantly higher than those of the OF1 treatment.

[0163] Dry weight: such as Figure 9A , 9B As shown, the OF2 treatment, which applied the organic fertilizer of this invention, increased the whole plant dry weight by 34.25% at 45 days compared to the OF1 treatment; and increased the aboveground dry weight by 36.93% at 45 days compared to the OF1 treatment. The OF2 treatment showed a significant increase in both the whole plant and aboveground dry weight at 45 days compared to the OF1 treatment.

[0164] 4. Experimental Conclusions

[0165] This embodiment demonstrates through a pot experiment of *Brucea javanica*:

[0166] Applying tobacco straw organic fertilizer produced by the compound microbial agent of this invention can significantly promote the growth and development of spinach, laying a good foundation for the production of high-quality vegetables.

[0167] This technology enables the high-value recycling of tobacco straw, transforming agricultural waste into high-quality resources that promote crop growth, thus forming a sound ecological cycle model, which is of great significance for promoting the sustainable development of vegetables.

[0168] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A microbial composition, characterized in that, It contains Bacillus velezensis L13, accession number CGMCC No. 35382 and Bacillus cereus L15, accession number CGMCC No. 37864.

2. The microbial composition according to claim 1, characterized in that, The ratio of viable bacteria of Bacillus belyss L13 and Bacillus cereus L15 ranges from 1:0.5 to 1:

2.

3. A compound microbial agent, characterized in that, It comprises the microbial composition according to claim 1 or 2.

4. The compound microbial agent according to claim 3, characterized in that, The concentrations of both *Bacillus belyi* L13 and *Bacillus cereus* L15 are not less than 1 × 10⁻⁶. 7 CFU / mL.

5. The method for preparing the compound microbial agent according to claim 3 or 4, characterized in that, Includes the following steps: Bacillus belye L13 and Bacillus cereus L15 were cultured by liquid fermentation to obtain high-concentration bacterial solutions, and then the two bacterial solutions were mixed in a certain proportion.

6. The preparation method according to claim 5, characterized in that, The liquid fermentation culture uses LB medium, with a culture temperature of 28-30℃ and a culture time of 24-48 hours.

7. The application of the compound microbial agent according to claim 3 or 4 in the tobacco straw composting process.

8. The application of the compound microbial agent according to claim 3 or 4 in the preparation of organic fertilizer.

9. A method for producing organic fertilizer, characterized in that, Includes the following steps: Tobacco straw, sheep manure and the compound microbial agent described in claim 3 or 4 are mixed, and the carbon-nitrogen ratio and moisture content of the mixture are adjusted to carry out aerobic fermentation.

10. The method according to claim 9, characterized in that, The mass ratio of tobacco straw to sheep manure is 7:3; the carbon-nitrogen ratio is adjusted to 25:1; the moisture content is adjusted to 60%-65%; the temperature for aerobic fermentation is 40-60℃, and the time is 50-60 days.

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