Kitchen garbage composite microbial inoculant, preparation method and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a kitchen garbage composite microbial inoculant, a preparation method and application thereof. The kitchen garbage composite microbial inoculant comprises bacteria and fungi; the bacteria comprise Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pumi, Bacillus, Pristinamycetium koreensis and Pristinamycetium magnum; and the fungi comprise Mycogone peridium and Fusarium verticillioides. The present application utilizes the synergistic effect of bacteria and fungi to specifically solve the problem of hydrophobic film formed by oil and grease and the problem of difficult degradation of cellulose, thereby significantly improving the biodegradation ability of organic dry matter in kitchen garbage to realize real source reduction. Meanwhile, a solid microbial inoculant optimized by fermentation process and carrier adsorption process is provided, so that the solid microbial inoculant can maintain high activity and high stability for a long time in the actual engineering environment of continuous feeding and salt accumulation, and effectively meet the requirement of long-term continuous operation of distributed kitchen garbage treatment equipment.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a compound microbial agent for kitchen waste, its preparation method, and its application. Background Technology

[0002] Food waste, due to its high water content, rich organic matter, and susceptibility to decay, can cause hygiene and environmental pollution problems if improperly disposed of. Currently, centralized treatment is the mainstream model, but it often results in high costs, high carbon emissions, and secondary pollution due to long-distance transportation, especially in areas with dispersed waste sources. Therefore, distributed (in-situ) treatment models have emerged, which avoid transportation difficulties by installing small-scale treatment equipment at the source; however, their treatment effectiveness heavily depends on the degradation performance of the microbial agents used.

[0003] Currently, distributed processing commonly employs small-scale biological treatment equipment equipped with heating, insulation, and stirring functions, along with the addition of commercially available general-purpose compound microbial agents (such as general-purpose EM agents). While this approach achieves some volume reduction, several key issues remain in practical applications, hindering its engineering adoption and long-term stable operation:

[0004] (1) The actual degradation rate is low, mainly relying on physical dehydration: general-purpose microbial agents are not optimized for the complex composition of kitchen waste, and their extracellular enzyme activity is limited. Although the treatment process can achieve a certain reduction in wet weight, it mainly comes from the evaporation of water caused by equipment heating and stirring. The actual organic matter biodegradation rate is not high (usually less than 70%), and a large amount of solid organic matter cannot be effectively decomposed and still needs further treatment.

[0005] (2) Problem of insufficient degradation capacity for high oil and cellulose components: Kitchen waste is rich in oil and cellulose. Oil easily forms a hydrophobic film on the surface of materials, which hinders the contact and degradation of microorganisms; general-purpose microbial agents lack highly efficient lipase-producing strains, making it difficult to break through the oil film barrier. At the same time, the cellulose structure in vegetables and fruit peels is dense, and general-purpose microbial agents lack highly efficient cellulose-degrading fungi, making it difficult for these components to be effectively decomposed, thus limiting the overall degradation rate.

[0006] (3) Poor long-term operational stability and easy degradation and failure of microbial agents: During continuous operation, salt gradually accumulates in the system and osmotic pressure increases. At the same time, the auxiliary carrier is gradually broken down due to mechanical stirring and the air permeability decreases. Commercially available general-purpose microbial agents have poor tolerance to environmental stress and are prone to problems such as decreased activity and population imbalance in the middle and late stages of operation, resulting in a significant reduction in treatment efficiency and making it difficult to ensure long-term stable operation. Summary of the Invention

[0007] The purpose of this invention is to provide a composite microbial agent for kitchen waste, its preparation method, and its application, thereby overcoming the shortcomings of existing technologies. By screening and compounding specific strains with high extracellular enzyme activity to construct a highly efficient composite microbial community, the synergistic effect of bacteria and fungi specifically addresses the problems of hydrophobic film obstruction caused by grease formation and the difficulty in cellulose degradation, thus significantly improving the biodegradation capacity of organic dry matter in kitchen waste to achieve true source reduction. Simultaneously, a solid microbial agent optimized through both fermentation and carrier adsorption processes is provided, enabling it to maintain high activity and stability for a longer period even in actual engineering environments with continuous feeding and salt accumulation, effectively meeting the requirements for long-term continuous operation of distributed kitchen waste treatment equipment.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides a compound microbial agent for kitchen waste, comprising bacteria and fungi;

[0010] Bacteria include peanut bacteria. Bacillus arachidis Priestella megaterium Priestia megaterium Bacillus thuringiensis Bacillus thuringiensis, Bacillus Bacillus sp. Bacillus Widmansii Bacillus wiedmannii Korean Priestella Priestia koreensis;

[0011] Fungi include *Smuts*. Filobasidium magnum and Fusarium Fusarium proliferatum;

[0012] Bacillus arachidii, Priestella megaterium, Bacillus thuringiensis 、 Bacillus, Bacillus Widmansii, *Priscilla koreanum*, *Saccharomyces cerevisiae* and All Fusarium species have been deposited at the China General Microbiological Culture Collection Center, with a deposit date of January 19, 2026. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0013] Among them, the preservation numbers for *Bacillus arachidica* are CGMCC No. 36635, *Priscilla megaterium* is CGMCC No. 36636, *Bacillus thuringiensis* is CGMCC No. 36631, *Bacillus* is CGMCC No. 36632, *Bacillus Widmansii* is CGMCC No. 36633, *Priscilla koreanum* is CGMCC No. 36634, *Ustilago maydis* is CGMCC No. 39052, and *Fusarium solani* is CGMCC No. 42321.

[0014] The composite microbial agent constructed in this invention was obtained by targeted screening of four substrates—starch, protein, oil, and cellulose—from kitchen waste residue and soil, using the ratio of the diameter of the transparent zone to the diameter of the colony (D / d value) as a quantitative indicator. Its coexistence stability was confirmed through plate confrontation experiments. The composite microbial agent possesses the ability to synergistically degrade starch, protein, oil, and cellulose. Specifically, the highly active lipase secreted by bacteria effectively destroys the hydrophobic oil film covering the surface of organic matter, relieving the inhibition of microbial attachment by oil. Simultaneously, through the cellulase produced by fungi and the physical penetration of mycelium, it disintegrates the dense cell wall structure in vegetable and fruit peels, thereby achieving efficient synergistic degradation of the complex components of kitchen waste.

[0015] More specifically, *Bacillus arachidica* possesses a multi-enzyme system composed of α-amylase, neutral protease, and lipase, which can efficiently and synergistically degrade the three core substrates in kitchen waste residue. During the degradation process, this strain can also rapidly convert ammonia nitrogen produced from the decomposition of nitrogenous organic matter into bacterial protein through efficient nitrogen assimilation, thereby significantly reducing ammonia emissions and possessing both environmental purification and resource recovery value.

[0016] Priestella megaterium secretes highly active alkaline protease and glycerol ester hydrolase, which have a specific ability to degrade kitchen waste oil and animal fats. Its enzyme system can target and cleave the ester bonds in oil molecules, effectively breaking the physical barrier formed by "oil encapsulation" and solving the problem of anaerobic spoilage caused by oil blockage.

[0017] Bacillus thuringiensis can secrete proteases, chitinases, and Cryotoxins, possessing both biodegradation and biocontrol functions. Its proteases and chitinases can synergistically degrade proteins, cellulose, and insect remains in kitchen waste residues, effectively cutting off the pathogen transmission chain; while Cryotoxins can specifically inhibit the reproduction of harmful organisms such as fly larvae, significantly improving hygiene and safety.

[0018] Bacillus, as the core "basic microbial community" of kitchen waste composting, possesses excellent environmental adaptability (tolerant to pH fluctuations of 4-9 and temperatures of 40-60℃) and a multi-enzyme degradation system (amylase, protease, and lipase). This microbial community can broadly and efficiently degrade starch, protein, and oil in kitchen waste residues, and can maintain high metabolic activity even during the high-temperature period of composting, ensuring the stable decomposition of organic matter.

[0019] Bacillus Wedemannii possesses excellent low-temperature tolerance and extracellular enzyme secretion capabilities, allowing it to remain active during the composting initiation stage or under low-temperature conditions. It also competitively inhibits the growth of other microorganisms by secreting antibacterial substances, creating a favorable microecological environment for subsequent fermentation.

[0020] Korean Priestella can secrete metalloproteinases and phospholipases, forming an enzyme system complementary to that of Priestella megaterium. The synergistic effect of these two bacteria can efficiently degrade proteins and oils in kitchen waste residue, significantly reducing ammonia emissions from the decomposition of nitrogenous organic matter and alleviating odor pollution during composting.

[0021] *Ustilago maydis* can secrete exocellulase, β-glucosidase, and amylase, exhibiting strong ability to degrade starch and cellulose, as well as acid and ethanol resistance. This strain can efficiently decompose cellulose-based kitchen waste such as straw and vegetable leaves, promoting subsequent fermentation through sugar production, and has important application value in bioenergy conversion.

[0022] Fusarium moniliformes secretes endonuclease, xylanase, and laccase, which have a strong ability to degrade cellulose and starch in kitchen waste and can partially decompose lignin. Its enzyme system can accelerate the structural depolymerization of lignocellulosic waste, significantly shorten the composting maturity cycle, promote humus formation, and improve the quality of compost products.

[0023] In some other embodiments, the mixed volume ratio of bacteria and fungi is (2-4):1.

[0024] Bacillus arachidii, Priestella megaterium, Bacillus thuringiensis 、 The mixed volume ratios of Bacillus, Bacillus Widmansii, and Priestella Korea were the same.

[0025] Black yeast and The mixed volume ratio of Fusarium moniliformes is the same.

[0026] Preferably, the mixed volume ratio of bacteria and fungi is 3:1; Bacillus arachidica, Priestella megaterium, and Bacillus thuringiensis. 、 The mixed volume ratio of Bacillus, Bacillus Widmansii, and Priscilla koreanum was 1:1:1:1:1:1; *Ustilago maydis* and The volume ratio of Fusarium sp. was 1:1.

[0027] In some other embodiments, the viable bacteria count of the kitchen waste compound microbial agent is greater than 8.0 × 10⁻⁶. 7 CFU / mL.

[0028] In some other embodiments, the kitchen waste compound microbial agent is a solid or liquid preparation.

[0029] Solid formulations also include carriers, which can be agricultural waste or inorganic porous materials. In addition, any other organic or inorganic porous materials with a certain porous structure, non-toxicity, and the ability to provide attachment sites for microorganisms can be used as carriers. Specifically, agricultural waste includes one or more of rice husks, wheat bran, sawdust, corn cob powder, peanut shell powder, and straw powder. Inorganic porous materials include one or more of biochar, zeolite, and porous ceramic particles. Rice husks are preferred as a carrier, and are sterilized by high-temperature and high-pressure steam sterilization at 121°C for 20 minutes to eliminate interference from other microorganisms.

[0030] In some other embodiments, the loading rate of the kitchen waste compound microbial agent in the solid preparation is 72-76%. Specifically, the effective loading rate of the rice husk-based solid microbial agent reaches 74.45%, proving that microorganisms can firmly attach to the carrier by secreting extracellular polymers, and the microbial agent is more resistant to mechanical stirring and less likely to fall off in practical applications.

[0031] Secondly, the present invention provides a method for preparing a compound microbial agent for kitchen waste, comprising the following steps:

[0032] After inoculating each single strain into LB liquid medium, the liquid bacterial agent was prepared by intermittent shaking and aeration at 30-40℃ for 72-120 hours. Intermittent shaking and aeration was performed by shaking and aeration at 100-150 rpm / min for 10-12 hours, followed by stopping the aeration for 10-12 hours, and this cycle was repeated continuously.

[0033] Specifically, the bacteria were cultured at 30, 35, or 40°C using intermittent shaking with oxygen supply for 72, 96, or 120 hours. This culture condition balanced dissolved oxygen supply with fluid shear force, satisfying the aerobic growth of Bacillus while protecting the fungal hyphal structure from damage, thus obtaining a high-concentration composite bacterial solution. Intermittent shaking with oxygen supply involved using an air-bath constant-temperature shaker at 130 rpm for 12 hours, followed by a 12-hour oxygen supply interruption, and this cycle was repeated.

[0034] In some other embodiments, the liquid bacterial agent is mixed with a carrier for adsorption loading, and then vacuum dried and frozen to obtain a solid formulation.

[0035] In some other embodiments, the temperature of the adsorption loading is 25-35°C, the time is 6-12 hours, and 0.5-1.5 g of carrier is added per milliliter of liquid bacterial agent.

[0036] Specifically, the adsorption loading temperature was 25, 30, or 35 °C, the time was 6 h, 8 h, 10 h, or 12 h, and the mass ratio of liquid inoculum to carrier was 2:1 mL / g, 1:1 mL / g, or 2:3 mL / g. Under these specific conditions, the secretion of extracellular polymeric substances (EPS) by microorganisms was most suitable, achieving firm adhesion of the bacteria to the pores of rice husks and solving the problems of easy detachment and inactivation of the inoculum.

[0037] Thirdly, the present invention provides the application of the kitchen waste compound microbial agent of the first aspect in kitchen waste treatment, composting and decomposition of agricultural organic waste and treatment of livestock and poultry manure.

[0038] Specifically, the microbial agent of this invention is rich in cellulase and protease, and can be directly used for the rapid composting of agricultural waste such as crop straw, discarded vegetable leaves, and orchard fallen leaves, promoting their decomposition and conversion into organic fertilizer. Utilizing the high-temperature tolerance and deodorizing ability of this compound microbial agent (through the rapid degradation of odor-causing organic precursors), it can be used for the aerobic fermentation treatment of livestock and poultry manure in farms, accelerating the deodorization and drying process. For municipal solid waste with high moisture content, short-term biological drying treatment using the microbial agent of this invention before incineration utilizes microbial fermentation to generate heat, reducing moisture content and increasing the calorific value of the waste, thereby improving incineration efficiency.

[0039] In some other embodiments, the method for treating kitchen waste is as follows: mixing kitchen waste with a kitchen waste compound microbial agent at a mass ratio of 20:(2-5), and intermittently stirring during continuous treatment.

[0040] During continuous processing, add compound microbial agent for kitchen waste every 7-20 days, with a mass ratio of (10-5):1.

[0041] Specifically, during initial addition, the ratio of kitchen waste mass to solid microbial agent mass is 20:2, 20:3, or 20:5. The temperature is 30 or 35℃, with intermittent stirring, and the operation cycle is 24 hours. During continuous operation, supplementary addition is performed every 12-14 days. This addition ratio and supplementary cycle are the optimal points optimized based on engineering experiments, achieving the highest dry matter reduction rate and long-term operational stability at the lowest cost.

[0042] The beneficial effects of this invention are:

[0043] (1) This invention selectively screened six bacterial strains (mainly including Bacillus thuringiensis, Bacillus Wedmansii, and Priestella) and two fungal strains (Ustilago maydis and Fusarium moniliforme) from kitchen waste residue and surrounding soil samples. Based on the verification of no antagonistic effect between the strains using the plate confrontation method, a composite microbial agent with the ability to synergistically degrade starch, protein, oil, and cellulose was constructed. This microbial system utilizes the highly active lipase secreted by bacteria to effectively destroy the hydrophobic oil film covering the surface of organic matter, thereby relieving the inhibition of microbial attachment by oil. At the same time, through the cellulase produced by fungi and the physical penetration of mycelium, the dense cell wall structure in vegetable and fruit peels is broken down, thus achieving efficient synergistic degradation of complex components of kitchen waste.

[0044] (2) This invention innovatively employs a bacterial-fungal synergistic compounding scheme: on the one hand, it utilizes highly efficient lipases secreted by screened Bacillus Wedmansii to rapidly break down the hydrophobic oil film encapsulating organic matter, thereby relieving the inhibition of degradation by oils; on the other hand, it introduces fungi such as Ustilago maydis, whose secreted cellulase and mycelial physical penetration can effectively disintegrate the dense cell wall structure of plant waste. This synergistic effect significantly improves the overall degradation rate of complex waste components.

[0045] (3) To address the problem of easy cell detachment in the preparation and application of existing solid bacterial agents, this invention optimizes the preparation process using response surface methodology: During the liquid fermentation stage, an intermittent oscillation process is employed to obtain a high-concentration bacterial solution under conditions of balanced dissolved oxygen and shear force; during the carrier adsorption stage, constant-temperature adsorption conditions are controlled to induce the secretion of extracellular polymers by microorganisms, utilizing their bioadhesive properties to firmly load the cells onto the rice husk carrier, achieving an effective loading rate of 74.45%. This significantly enhances the stability of the bacterial agent under mechanical stirring conditions.

[0046] (4) In view of the problem that the activity of the microbial agent is easily degraded due to salt accumulation and carrier caking during long-term operation of the existing technology, the composite microbial agent system constructed in this invention has stronger environmental tolerance. In the continuous 15-day full-load operation test, the microbial agent always maintains high degradation activity; it can effectively overcome the efficiency decay in the later stage of operation and ensure the long-term stable and low-maintenance operation of the distributed treatment system. Attached Figure Description

[0047] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0048] Figure 1 This is a technical roadmap illustrating the entire process from strain screening to final engineering application in Example 3 of the present invention.

[0049] Figure 2The diagram shows the results of optimizing the mixed fermentation conditions of the strains in Example 2 of the present invention; where a is a contour plot of the interaction between culture time and culture temperature on the number of viable cells, b is the response surface plot corresponding to a, c is a contour plot of the interaction between culture time and oxygen supply conditions on the number of viable cells, and d is the response surface plot corresponding to c.

[0050] Figure 3 The graph shows the results of optimizing the effective loading rate of the compound microbial agent in Example 2 of the present invention; where a is a contour plot showing the interaction between temperature and the mass ratio of bacterial solution to carrier on the effective loading rate, b is the response surface plot corresponding to a, c is a contour plot showing the interaction between temperature and time on the effective loading rate, and d is the response surface plot corresponding to c. Detailed Implementation

[0051] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] The specific bacteria used include Bacillus angiotensinii. Bacillus arachidis Priestella megaterium Priestia megaterium Bacillus thuringiensis Bacillus thuringiensis, Bacillus Bacillus sp Bacillus Widmansii Bacillus wiedmannii Korean Priestella Priestia koreensis; Black yeast Filobasidium magnum and Fusarium Fusarium proliferatum; All samples have been deposited with the China General Microbiological Culture Collection Center (CGMCC) on January 19, 2026, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession numbers for the following samples are: *Bacillus arachidica* (CGMCC No. 36635), *Priscilla megaterium* (CGMCC No. 36636), *Bacillus thuringiensis* (CGMCC No. 36631), *Bacillus* (CGMCC No. 36632), *Bacillus Widmansii* (CGMCC No. 36633), *Priscilla koreanum* (CGMCC No. 36634), *Ustilago maydis* (CGMCC No. 39052), and *Fusarium solani* (CGMCC No. 42321).

[0053] The current centralized collection and transportation of kitchen waste from remote towns and scattered units is costly and prone to secondary pollution. The biological agents used in the existing distributed in-situ treatment technology have technical problems such as poor targeting of specific components of kitchen waste (such as oil and cellulose), low dry matter reduction rate, and poor stability under continuous operation.

[0054] This invention constructs a specific microbial community system with synergistic metabolic functions, and solidifies it through specific fermentation and adsorption process parameters, ultimately achieving efficient application in conjunction with biological treatment equipment. The following detailed embodiments further illustrate the solution of this invention:

[0055] Example 1

[0056] This embodiment provides a method for constructing a composite microbial inoculant system:

[0057] This compound microbial agent (ZL) consists of eight non-antagonistic strains selected through specific screening. Specifically, it includes: Bacillus thuringiensis (Bt). Bacillus thuringiensis ZLX1, accession number CGMCC No. 36631), Bacillus Widmansii ( Bacillus cereus ZLX9, accession number CGMCC No. 36633), Bacillus arachidica ( Bacillus wiedmannii ZLF2, accession number CGMCC No. 36635), Bacillus ( Bacillus sp. ZLX5, accession number CGMCC No. 36632), *Priscilla koreanum* ( Priestia koreensis ZLX10, accession number CGMCC No. 36634) and Priestella megaterium ( Priestia megaterium ZLF12, accession number CGMCC No. 36636). The main technical function of these bacterial components is the secretion of highly active amylases, proteases, and lipases. *Ustilago maydis* (ZLF12, accession number CGMCC No. 36636). Filobasidium magnum ZLZ5, accession number CGMCC No. 39052) and Fusarium moniliforme ( Fusarium proliferatum ZLZ6, accession number CGMCC No.42321).

[0058] The technical function of these fungal components is to disrupt the cell wall structure of vegetable and fruit peels by secreting cellulase and utilizing the physical penetration ability of mycelium.

[0059] The above-mentioned strains were obtained from kitchen waste residue and soil through targeted screening targeting four substrates: starch, protein, oil, and cellulose, using the ratio of the diameter of the transparent zone to the colony diameter (D / d value) as a quantitative indicator. Their coexistence stability was confirmed through plate confrontation experiments. Specifically, the experiments demonstrating no antagonistic interaction between the strains are as follows:

[0060] Two test strains were seeded in pairs on opposite sides of an LB agar plate and incubated at 30 °C for approximately 48 h. The presence of an inhibition zone or a clear inhibition line at the growth interface between the two strains was observed. A "+" indicates antagonistic activity. “” indicates no antagonistic effect, and “ / ” indicates consistency. The experimental results are shown in Table 1.

[0061] Table 1 shows the results of the plate confrontation test.

[0062]

[0063] As shown in Table 1, ZLX1, ZLX5, ZLX9, ZLX10, ZLF2, ZLF12, ZLZ5 and ZLZ6 grew well at the plate interface and no obvious inhibition zone was observed, which indicates that these strains have good compatibility and can be cultured together.

[0064] Example 2

[0065] This embodiment optimizes the preparation process of the compound microbial agent in Example 1, as follows:

[0066] (1) Optimize the fermentation conditions of mixed strains:

[0067] Using Design Expert 10.0 software, a Box-Behnken design (BBD) response surface methodology was employed. A three-factor, three-level experiment was designed, selecting culture time, oxygen supply conditions, and culture temperature as significant factors. LB liquid medium was used. After inoculation, each strain was placed in a constant-temperature air bath shaking incubator. Each group had three parallel experiments. The average viable cell count (CFU / mL) of the fermentation broth, measured by the concentration gradient dilution plate counting method, was used as the response value for secondary response surface analysis. The experimental factors and levels for secondary response surface analysis are shown in Table 2, and the experimental groups and results are shown in Table 3. In Table 3, 0 corresponds to intermittent oscillation (oscillation with oxygen supply for 12 h at a rotation speed of 100-150 rpm / min, followed by a 12 h oxygen supply interruption, and this cycle was repeated continuously) in the oxygen supply conditions. -1 corresponds to static conditions in Table 2, and +1 corresponds to continuous oscillation in Table 2.

[0068] Table 2 shows the levels of experimental factors for optimizing mixed fermentation conditions.

[0069]

[0070] Table 3 shows the results of the experiment on optimizing the mixed fermentation conditions.

[0071]

[0072] The results of optimizing the liquid fermentation conditions of the compound microbial agent using the Box-Behnken Design method are as follows: Figure 2 As shown, the contour plot illustrates the interaction between culture time and culture temperature on the number of viable bacteria. Figure 2 a) and response surface plot ( Figure 2 (b) Contour plot showing the interaction between culture time and oxygen supply conditions on viable cell count. Figure 2 c) and response surface plot ( Figure 2 As shown in d), under optimized fermentation conditions (culture temperature 35 ℃, culture time 96 h, intermittent shaking oxygen supply), the average viable count in the fermentation broth reached 8.08 × 10⁻⁶. 7 The CFU / mL value had an error of only 3.81% compared to the model prediction, demonstrating the high efficiency and controllability of the fermentation process.

[0073] (2) Optimize and improve the effective loading rate of compound microbial agents:

[0074] To prepare a standardized bacterial suspension for subsequent experiments, the optimal fermentation broth was centrifuged, washed and resuspended with sterile physiological saline, and the turbidity was adjusted to [value missing]. OD 600 =1.0. Based on this, sterilized rice husks were selected as the carrier (sterilized by high-temperature and high-pressure steam sterilization at 121℃ for 20 minutes to eliminate interference from other microorganisms). A three-factor, three-level experiment was conducted using the BBD method to examine the effects of three significant factors: time, the ratio of bacterial volume to carrier mass, and temperature. Specific factors and levels are shown in Table 4. Each group had three parallel experiments, and the effective loading rate of the solid bacterial agent was set as the response value. The experimental groups and results are shown in Table 5. Specifically, -1 in the bacterial volume to carrier mass ratio in Table 5 corresponds to a bacterial volume to carrier mass ratio of 2:1 mL / g in Table 4; 0 in the bacterial volume to carrier mass ratio in Table 5 corresponds to a bacterial volume to carrier mass ratio of 1:1 mL / g in Table 4; and 1 in the bacterial volume to carrier mass ratio in Table 5 corresponds to a bacterial volume to carrier mass ratio of 2:3 mL / g in Table 4.

[0075] After the experimental treatment, the rice husks were freeze-dried under vacuum to constant weight. Samples were taken and added to sterile water for elution by shaking. The number of viable bacteria in the eluent was determined by plate counting method, and then the effective loading rate of the carrier adsorbed with viable bacteria per unit mass relative to the initial number of viable bacteria was calculated.

[0076] Table 4 shows the levels of experimental factors for optimizing the preparation conditions of microbial agents.

[0077]

[0078] Table 5 shows the levels of experimental factors for optimizing the preparation conditions of microbial agents.

[0079]

[0080] The results of optimizing the effective loading rate of the compound microbial agent using the Box-Behnken Design method are as follows: Figure 3 As shown, the contour plot illustrates the interaction between temperature and the ratio of bacterial culture to carrier mass on the effective loading rate. Figure 3 a) and response surface plot ( Figure 3 (b) Contour plot showing the interaction between temperature and time on effective load factor ( Figure 3 c) and response surface plot ( Figure 3 As shown in d), the optimal preparation conditions for the compound bacterial agent are: temperature 30 ℃, time 8 h, and bacterial volume to carrier mass ratio (mL / g) of 10:10. Under these conditions, the effective loading rate reaches 75.25%. Three parallel verification experiments were conducted under these conditions, and the average actual effective loading rate was measured to be 74.45%. The relative error between the actual value and the predicted value was only 1.06%, verifying the accuracy and reliability of the response surface model.

[0081] Example 3

[0082] This embodiment provides an application of the in-situ processing system:

[0083] This application solution uses existing small-scale biological treatment equipment (such as Sanyi Quanfang, Sf-2015-2KG), which includes a reaction unit (containing a reaction chamber with a temperature-controlled heating jacket and a mechanical agitator) and a ventilation and deodorization unit.

[0084] Dosing and Operational Technical Parameters: A specific mass ratio of 20:3 is adopted, that is, 3 parts by weight of compound solid microbial agent ZL are added for every 20 parts by weight of kitchen waste. This ratio ensures that the initial biomass at system startup is sufficient to complete the main degradation of organic matter within 24 hours. The temperature of the reaction chamber is constantly controlled at 30-35℃, and the agitator operates on an intermittent stirring program. Under these conditions, combined with the characteristics of the microbial agent, a wet weight reduction rate of 88.21% and a dry matter reduction rate of 80.89% can be achieved within a 24-hour treatment cycle. To address the problem of activity decay caused by salt accumulation during continuous operation, the technical solution specifies a maintenance method of "replenishing the microbial agent every 12-14 days," that is, adding new microbial agent to the system approximately every two weeks to maintain the abundance of dominant bacterial communities in the reaction chamber and ensure long-term operational stability.

[0085] In a small-scale biological treatment machine for kitchen waste, the optimal dosing ratio (20:3) was set, and the composite microbial agent ZL prepared in this invention was used to treat 1000g of standardized kitchen waste for 24 hours.

[0086] The experimental results show that the wet weight loss rate of the group with 150 g of the compound microbial agent ZL added reaches 88.21%, and the dry matter weight loss rate reaches 80.89%. The experimental data directly prove that the microbial agent of the present invention has a relatively high dry matter biodegradation rate after removing the interference of moisture, significantly improving the substantial degradation ability of organic matter.

[0087] Stability verification under continuous operation conditions. Simulating the actual use scenario, a continuous feeding operation experiment was carried out for 15 days. 1000 g of fresh kitchen waste was put in every day, and the change of the degradation performance of the system was monitored. Experimental results: In the first 5 days of operation, the dry matter weight loss rate of the system increased rapidly and remained at a high level of about 80%, reaching a peak of 81.20% on the 3rd day. As the operation time extended, affected by the salt accumulation and the change of the physical properties of the carrier, the efficiency began to decline slowly after the 7th day, but still remained at the level of 72.50% on the 15th day. The experiment proves that the microbial agent of the present invention has good environmental tolerance and anti-shock load ability, and accordingly an engineering maintenance strategy of supplementing the microbial agent once every 12 - 14 days was established, verifying its engineering feasibility in the distributed long-term operation scenario.

[0088] Figure 1 It shows the whole-process technical roadmap of the present invention from strain screening to final engineering application. This flow chart details the process of screening 8 target strains based on the ratio of the transparent circle to the colony diameter and constructing the compound microbial agent ZL with cross-border compounding of bacteria and fungi. Subsequently, liquid fermentation was carried out under the conditions of 35 °C and 96 h with intermittent oscillation to prepare a high-concentration microbial liquid, and then adsorption was carried out using rice husk at a liquid-solid ratio of 1:1 under the conditions of 30 °C and 8 h. Finally, it was added to the in-situ treatment equipment at a mass ratio of 20:3 and periodic microbial agent supplementation maintenance was carried out.

[0089] Comparative Example 1

[0090] Different from Example 3, only 150 g of sterilized rice husk carrier was added, and other steps were the same as those in Example 3.

[0091] Comparative Example 2

[0092] Different from Example 3, only 150 g of a commercially available general EM microbial agent was added. It was purchased from a specific welfare biotechnology company, with the model being EM Bokashi (Obstacle Welfare Service Office Company, model EM Bokashi), and other steps were the same as those in Example 3.

[0093] Comparative Example 3

[0094] Different from Example 3, only 100 mL of the liquid compound microbial agent ZL in Example 2 of the present invention was added to ensure that the total viable bacteria count of the initial addition was the same as that of adding 150 g of the sterilized rice husk carrier, and other steps were the same as those in Example 3.

[0095] Comparative Example 4

[0096] Unlike Example 3, only 150 g of the solid compound bacterial agent ZL (Bacillus arachidica, Priestella megaterium, Bacillus thuringiensis) containing bacteria from Example 2 of this invention was added. 、 (Bacillus, Bacillus Widmansii, and Priestella Korea), other steps are the same as in Example 3.

[0097] Studies have found that when using only bacterial solid-state compound inoculants to treat kitchen waste, the system lacks the ability to deeply decompose stubborn lignocellulosic components. Although bacterial communities can rapidly consume easily degradable substrates such as starch, protein, and oil, the lack of highly active lignin-degrading enzyme systems (such as laccase) and hyphal structures capable of physically penetrating plant cell walls prevents them from effectively breaking down the dense cross-linked structures of cellulose and lignin in straw and fruit peels. This lack of physical and biochemical attack capabilities prevents the release of internal nutrients encased in cell walls, causing bacteria to prematurely enter a dormant state in the later stages of degradation due to the inability to access locked carbon sources. Ultimately, this treatment group not only resulted in a large amount of cellulose residue retention, significantly reducing volume reduction efficiency, but also easily induced secondary fermentation and foul odor problems due to the loose structure and insufficient maturity of the products, confirming that the lack of the synergistic mechanism of "fungal physical cell wall disruption - rapid bacterial assimilation" leads to the breakage of the degradation chain.

[0098] Comparative Example 5

[0099] Unlike Example 3, only 150 g of the solid compound microbial agent ZL (Saccharomyces cerevisiae and Fusarium moniliforme) containing fungi from Example 2 of this invention was added, and the other steps were the same as in Example 3.

[0100] Studies have found that using only fungal solid composite microbial agents to treat kitchen waste is insufficient to meet the engineering requirements for rapid degradation. On one hand, fungi have long growth cycles and slow initiation, making it difficult to quickly establish a dominant population to cope with the highly perishable kitchen waste substrate in the early stages of treatment. This results in a large amount of starch and protein not being consumed in time, leading to acidification and ammonia volatilization. On the other hand, fungi lack the heat resistance and efficient extracellular lipid-degrading enzyme system unique to bacteria. When the temperature of the pile increases (>50℃) due to hydrolysis heat generated during equipment operation or external heating, fungal activity is significantly inhibited or even inactivated. Furthermore, given the high-oil environment commonly found in kitchen waste, the lack of bacterial lipases for demulsification and hydrolysis makes fungal hyphae easily encased in oil films and suffocated, preventing them from contacting the substrate. Therefore, single-fungal systems exhibit significant degradation lag and incompleteness, failing to achieve rapid and harmless treatment of kitchen waste.

[0101] Table 6 shows the treatment effects of Example 3 and Comparative Examples 1-3. The formula for calculating the wet matter weight loss rate is: ;

[0102] in, The wet weight loss rate (%) The initial wet weight (g) of the mixture of kitchen waste and microbial agent at the time of addition. The wet weight (g) of the mixture inside the processor at the end of operation.

[0103] The formula for calculating the dry matter weight loss rate is: ;

[0104] in, Dry matter weight loss rate (%) The dry matter mass (g) of the mixture inside the processor at the end of the operation. The dry matter mass (g) inside the processing unit at the end of the operation.

[0105] Table 6 shows the treatment effects of Example 3 and Comparative Examples 1-3.

[0106]

[0107] Table 6 clearly shows that comparing the weight loss effects of the commercially available EM bacterial agent groups (Comparative Examples 1 and 2) with the ZL bacterial agent group in Example 3 of this invention after 24 hours of treatment, the data trend indicates that the dry matter weight loss rate of the ZL bacterial agent group of this invention is as high as 80.89%, significantly higher than the 67.72% of the commercially available EM bacterial agent group (Comparative Example 2) and 34.35% of Comparative Example 1. This is because the rice husk carrier in Example 3 provides crucial physical support and a bioadsorption interface for the composite bacterial agent, and its unique porous structure and rough surface become an ideal microenvironment for microbial colonization. The microorganisms in the bacterial agent adhere firmly to the inside and surface of the rice husk pores by secreting extracellular polymers, forming a resilient biofilm structure. This tight physical bond not only effectively prevents the bacterial strains from detaching and being lost due to severe shear force during mechanical stirring, but also provides a relatively stable sheltered space for the microorganisms, enabling them to resist drastic fluctuations in the external environment, thereby ensuring the high-density retention and long-lasting biological activity of the core functional bacterial community in the reaction system.

[0108] The synergistic effect of rice husks and compound microbial agents is mainly manifested in a dual-effect mechanism of physical structure improvement and enhanced biochemical degradation. On the one hand, rice husks, with their rigid structure, physically grind and penetrate kitchen waste during mechanical stirring, increasing the specific surface area of ​​the substrate. Simultaneously, their loose texture significantly improves the porosity and aeration of the compost pile, providing ample oxygen transport channels for aerobic Bacillus bacteria and regulating moisture balance. On the other hand, the microbial agents attached to the carrier can utilize this spatial advantage to directionally secrete highly active amylases, proteases, and other hydrolytic enzyme systems, deeply chemically degrading the physically broken organic substrate. This mechanism of physical breaking assisted chemical hydrolysis greatly improves mass transfer efficiency, making the degradation rate and volume reduction effect of the immobilized microbial agent system significantly better than that of a single free microbial agent.

[0109] In summary, this invention not only theoretically constructs a reasonable bacterial-fungal synergistic system, but also demonstrates its operability in preparation process and its efficient degradation ability of kitchen waste in practical application through rigorous experimental data, thus possessing full feasibility for industrial application.

[0110] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound microbial agent for kitchen waste, characterized in that, Including bacteria and fungi; The bacteria include Bacillus arachidii. Bacillus arachidis Priestella megaterium Priestia megaterium Bacillus thuringiensis Bacillus thuringiensis, Bacillus sp., Bacillus Widmansii Bacillus wiedmannii Korean Priestella Priestia koreensis; The fungus includes *Ustilago maydis*. Filobasidium magnum and Fusarium Fusarium proliferatum; The aforementioned Bacillus arachidii, Priestella megaterium, and Bacillus thuringiensis 、 Bacillus, Bacillus Widmansii, Priestella Korea, Ustilago maydis, and Fusarium moniliforme have all been sent to the China General Microbiological Culture Collection Center, with a deposit date of January 19, 2026. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Among them, the preservation numbers for *Bacillus arachidica* are CGMCC No. 36635, *Priscilla megaterium* is CGMCC No. 36636, *Bacillus thuringiensis* is CGMCC No. 36631, *Bacillus* is CGMCC No. 36632, *Bacillus Widmansii* is CGMCC No. 36633, *Priscilla koreanum* is CGMCC No. 36634, *Ustilago maydis* is CGMCC No. 39052, and *Fusarium solani* is CGMCC No. 42321.

2. The compound microbial agent for kitchen waste according to claim 1, characterized in that, The mixed volume ratio of the bacteria and fungi is (2-4):1; The aforementioned Bacillus arachidii, Priestella megaterium, and Bacillus thuringiensis 、 The mixed volume ratios of Bacillus, Bacillus Widmansii, and Priestella Korea were the same; The volume ratio of *Saccharomyces macrocarpa* and *Fusarium solani* is the same.

3. The kitchen waste compound microbial agent according to claim 1, characterized in that, The viable bacteria count of the kitchen waste compound microbial agent is greater than 8.0 × 10⁻⁶. 7 CFU / mL.

4. The compound microbial agent for kitchen waste according to claim 1, characterized in that, The formulation of the kitchen waste compound microbial agent is either a solid formulation or a liquid formulation; The solid formulation also includes a carrier, which is agricultural waste and inorganic porous material; The agricultural waste includes one or more of the following: rice husks, wheat bran, sawdust, corn cob powder, peanut shell powder, and straw powder; The inorganic porous material includes one or more of biochar, zeolite, and porous ceramic particles.

5. The kitchen waste compound microbial agent according to claim 4, characterized in that, The loading rate of the compound microbial agent for kitchen waste in solid preparations is 72-76%.

6. A method for preparing a compound microbial agent for kitchen waste according to any one of claims 1-5, characterized in that, Includes the following steps: After each single strain was inoculated into LB liquid medium, it was cultured at 30-40℃ for 72-120 h using intermittent shaking oxygen supply to obtain liquid bacterial agent; the intermittent shaking oxygen supply was performed by shaking oxygen supply for 10-12 h at a speed of 100-150 rpm / min, followed by stopping oxygen supply for 10-12 h, and this cycle was repeated continuously.

7. The method for preparing the compound microbial agent for kitchen waste according to claim 6, characterized in that, It also includes mixing liquid bacterial agents with a carrier for adsorption loading, followed by vacuum drying and freezing to obtain a solid formulation.

8. The method for preparing the compound microbial agent for kitchen waste according to claim 7, characterized in that, The temperature for adsorption loading is 25-35℃, the time is 6h-12h, and 0.5-1.5 g of carrier is added per milliliter of liquid bacterial agent.

9. The application of the kitchen waste compound microbial agent according to any one of claims 1-5 in kitchen waste treatment, composting and maturation of agricultural organic waste and treatment of livestock and poultry manure.

10. The application according to claim 9, characterized in that, The method for treating kitchen waste is as follows: Mix kitchen waste with kitchen waste compound microbial agent at a mass ratio of 20:(2-5) and stir intermittently during continuous treatment; during continuous treatment, add kitchen waste compound microbial agent every 7-20 days, with a mass ratio of (10-5):1.

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