A kind of bacteria agent for improving humification efficiency of biological drying and rotting process and method thereof

Abstract

The application discloses a kind of microbial inoculum for improving humification efficiency of biological drying and rotting process and method thereof.The effective component of the microbial inoculum is composed of high-temperature-resistant strains, such as Saccharomonospora viridis, Pseudomonas taiwanensis, Microtus luteus, Bacillus licheniformis and Bacillus subtilis.The microbial inoculum provided by the application can significantly increase bacterial abundance when applied to the biological drying and rotting system of organic waste, accelerate the temperature rise and dehydration of the substrate in the biological drying stage, and make the system enter the high-temperature stage earlier.The lignin degradation rate is increased by more than 20%, the demethoxylation rate of lignin and the breaking rate of β-O-4 bond are increased, and more aromatic skeletons rich in phenolic hydroxyl groups are produced as key humic acid precursors for HA synthesis.The lignin-protein and polyphenol pathways are enhanced, and the seed germination index and humification ratio of compost products are significantly increased by more than 20%.

Description

Technical Field

[0001] This invention belongs to the field of organic waste treatment and environmental microbiology technology, specifically involving a microbial agent and method for improving the humification efficiency of biological drying and composting processes. It can be used to achieve targeted and rapid composting of lignin-rich organic wastes such as kitchen waste, biogas residue, and large-scale dairy farming waste. Background Technology

[0002] Aerobic composting is a key technology for the full resource utilization of organic waste such as kitchen waste, biogas residue, and waste from large-scale dairy farming. Its core product, humic acid (HA), has significant ecological value. However, organic waste generally has a high moisture content (>70%), and traditional single-phase aerobic composting processes often face problems such as slow start-up and heating (3-7 days), limited microbial activity, long fermentation cycle (15-60 days), and low product maturity and humification, making it difficult to meet the engineering requirements for efficiently and rapidly obtaining compost products with high HA content.

[0003] The integrated two-phase process of biological drying and decomposition replaces the long heating period of traditional single-phase processes with a 1-2 day biological drying pretreatment, enabling lignin-rich organic waste such as kitchen waste, biogas residue, and waste from large-scale dairy farming to mature within 10 days. During the biological drying stage, high-frequency ventilation and stirring improve the pile's pore structure, enhance oxygen transport, and promote the rapid proliferation of aerobic microorganisms, which degrade organic matter and generate heat, thus driving rapid heating and dehydration of the pile. Subsequently, under high-temperature, oxygen-rich, and suitable moisture conditions, thermophilic microorganisms rapidly accumulate, efficiently degrading organic matter and generating humic acid precursors, thereby accelerating HA synthesis. However, with increasing constraints on land resources, growing demand for soil improvement, and continuously rising pressure for emission reduction and carbon sequestration, further improving the humification efficiency of this process to obtain high-quality products with higher HA content is of great significance for promoting its engineering application in the field of organic waste fertilizer.

[0004] Previous studies have found that during the bio-drying and composting process of organic waste, the rapid temperature rise caused by bio-drying can significantly enhance the demethoxylation and β-O-4 bond breaking reaction of lignin in organic waste, promoting lignin depolymerization and generating an aromatic skeleton rich in phenolic hydroxyl groups. This aromatic skeleton, as a key humic acid precursor in the lignin-protein and polyphenol humification pathways that dominate the bio-drying and composting process, provides numerous highly reactive sites for the condensation of other precursors with the skeleton, thereby accelerating HA molecular chain elongation. Therefore, enhancing the demethoxylation and β-O-4 bond breaking of lignin during the bio-drying and composting process is an effective strategy to increase the supply of key humic acid precursors and further improve the humification efficiency of organic waste.

[0005] Compared to common strategies for improving humification efficiency, such as the addition of metal catalysts, auxiliary materials, and exogenous humic acid precursors, exogenous microbial agents are lower in cost, have less secondary pollution and environmental risk, and have better potential for engineering application. Therefore, there is an urgent need to develop microbial agents that can specifically enhance the directed and rapid humification pathway of the biological drying and composting process of organic waste such as kitchen waste, biogas residue, and waste from large-scale dairy farming, in order to overcome existing technological bottlenecks and improve process stability and product quality. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems in the prior art and to provide a microbial agent and method for improving the humification efficiency of the biological drying and composting process, so as to increase the supply of key humic acid precursors in the biological drying and composting process of organic waste such as kitchen waste, biogas residue, and waste from large-scale dairy farming, and improve HA synthesis efficiency.

[0007] The specific technical solution adopted in this invention is as follows:

[0008] In a first aspect, the present invention provides a microbial agent for improving the humification efficiency of a biological drying and composting process. The effective components of the microbial agent are composed of a compound of non-antagonistic Saccharomonospora viridis, Halopseudomonas formosensis, Oceanobacillus luteolus, Bacillus licheniformis, and Bacillus subtilis.

[0009] The Bacillus licheniformis strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 8, 2022, with accession number GDMCC NO. 62362.

[0010] The Bacillus subtilis strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 8, 2022, with accession number GDMCC NO. 62361.

[0011] As a preferred embodiment of the first aspect above, the Saccharomonospora viridis is a commercially available strain purchased from the American Type Culture Collection Center (ATCC) with accession number ATCC 33517.

[0012] As a preferred strain in the first aspect mentioned above, *Halopseudomonas formosensis* is a commercially available strain purchased from the China Industrial Microbial Culture Collection Center, with accession number CICC 23006.

[0013] As a preferred strain in the first aspect mentioned above, Oceanobacillus luteolus is a commercially available strain purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC 1.12636.

[0014] As a preferred embodiment of the first aspect above, the microbial agent is a solid composite microbial agent prepared by mixing bacterial solutions of Saccharomonos poraviridis, Halopseudomonas formosensis, Oceanobacillus luteolus, Bacillus licheniformis, and Bacillus subtilis in a certain proportion with a solid matrix and then aerobic fermentation.

[0015] As a preferred embodiment of the first aspect above, the mass ratio of Saccharomonospora viridis, Halopseudomonas formosensis, Oceanobacillus luteolus, Bacillus licheniformis, and Bacillus subtilis in the solid matrix is ​​(1.8~2.2): (1.8~2.2): (1.8~2.2): (0.8~1.2):1.

[0016] As a preferred embodiment of the first aspect above, the solid matrix is ​​composed of corn cobs, bamboo shavings, and rice straw mixed in a mass ratio of (3.5~4.5):(0.5~1.5):1, and by percentage of the total mass of the mixture, the following are added: 4~6 wt% corn starch, 4~6 wt% soybean meal, 0.4~0.6 wt% (NH4)2SO4, 0.4~0.6 wt% KH2PO4, 0.08~0.12 wt% NaCl, 0.08~0.12 wt% MgSO4·7H2O, and 0.008~0.012 wt% MnSO4.

[0017] As a preferred embodiment of the first aspect above, the total effective viable count of the microbial agent is (5.3~8.5) × 10⁻⁶. 9cfu·g -1 The viable count of each strain was no less than 2.1 × 10⁻⁶. 8 cfu·g -1 .

[0018] In a second aspect, the present invention provides a method for preparing a microbial agent for improving the humification efficiency of a biological drying and composting process as described in any of the embodiments of the first aspect above, comprising:

[0019] The *Saccharomonospora viridis* and *Oceanobacillus luteolus* were inoculated into TSB medium, respectively; *Halopseudomonas formosensis* was inoculated into NB medium; and *Bacillus licheniformis* and *Bacillus subtilis* were inoculated into LB liquid medium, respectively. The inoculated medium was then placed at 54–56 °C with shaking at 140–160 rpm for at least 48 h. The inoculated medium was then further cultured with *Halopseudomonas formosensis* and *Oceanobacillus luteolus*. The culture medium containing luteolus was placed at 44-46℃ and shaken at 140-160 rpm for at least 48 h. The five bacterial solutions obtained from the shaking culture were sprayed onto the solid substrate, the moisture content was adjusted to 58-62%, and the mixture was mixed evenly before aerobic fermentation. The pile was turned 1-2 times a day. After the pile temperature rose to 45-55℃, it was spread out and placed at 44-46℃ for constant temperature drying until the moisture content was less than 20%, thus obtaining the bacterial agent.

[0020] Thirdly, this invention provides a process for biological drying and composting of organic waste. Specifically, the process involves inoculating the organic waste in a biological drying chamber with the microbial agent described in the first aspect, which enhances the humification efficiency of the biological drying and composting process. Sufficient oxygen is provided to the pile through forced ventilation and stirring, and the pile is biologically dried for 22-26 hours, raising the pile temperature to above 50°C and reducing the moisture content to below 60%. Then, 1 / 3 to 1 / 2 of the biologically dried pile material is retained as inoculum for the next batch of fresh organic waste, and biological drying continues for the next batch of organic waste entering the biological drying chamber. The remaining biologically dried pile material is then transferred to a high-temperature composting chamber for further composting for 7-9 days, after which it is discharged from the high-temperature composting chamber as a dried composting product.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) The five bacterial strains in the bacterial agent of the present invention have complementary functions and can synergistically improve the humification efficiency of the bio-drying and composting process of organic waste rich in lignin, such as kitchen waste, biogas residue, and waste from large-scale dairy farming. Among them, S. viridis and H. formosensis are thermosensitive key functional bacteria that can mediate the demethoxylation and β-O-4 bond cleavage of lignin to generate an aromatic skeleton rich in phenolic hydroxyl groups and HA synthesis; O. luteolus can provide H. formosensis with one-way feeding, providing the energy, cofactors and reaction substrates required to maintain cell homeostasis and efficiently degrade lignin at high temperature; B. licheniformis and B. subtilis have high protein, cellulose, fat and starch degrading enzyme activity, which can promote the heat generation of organic matter degradation, drive the rapid temperature rise of the organic waste bio-drying and composting system, so as to form a high temperature and oxygen-rich environment suitable for the growth and metabolism of the other three strains of bacteria, weaken interspecies competition, strengthen metabolic cooperation, and concentrate the resources in the system on completing the complex tasks of lignin degradation and HA synthesis.

[0023] (2) When the microbial agent of the present invention is applied to the biological drying and composting system of organic waste rich in lignin, such as kitchen waste, biogas residue, and waste from large-scale dairy farming, it can significantly increase the abundance of bacteria, increase the total organic matter degradation rate by more than 30%, and significantly increase the temperature of the biological drying bin, allowing it to enter the high-temperature stage earlier; the lignin degradation rate is increased by more than 20%, the demethoxylation and β-O-4 bond breaking is enhanced, and more phenolic hydroxyl groups are exposed to increase the supply of key humic acid precursors; the lignin-protein and polyphenol pathways are significantly enhanced, and the seed germination index and Hu-Fu ratio of compost products are increased by more than 20%.

[0024] (3) The total effective viable bacteria count of the bacterial agent of the present invention reaches (5.3~8.5) × 10 9 cfu·g -1The viable count of each strain was no less than 2.1 × 10⁻⁶. 8 cfu·g -1 It meets the technical indicators for the effective live bacteria count of the product in "Organic Material Composting Agent" (NY 609-2002) and "Agricultural Microbial Agents" (GB20287-2006). Attached Figure Description

[0025] Figure 1 The changes in the relative abundance of bacterial communities during the bio-drying-promoted composting (BEC) and bio-enhanced composting (BMC) processes in the embodiments of the present invention are shown. (a) and (b) show the top 10 bacteria with the highest average relative abundance at the phylum level, and (c) and (d) show the bacteria with the highest average relative abundance at the genus level.

[0026] Figure 2 This is a metabolic schematic diagram of MAG_48 (S. viridis) and MAG_387 (H. formosensis) participating in the lignin β-O-4 bond cleavage and demethoxylase cascade reaction in an embodiment of the present invention;

[0027] Figure 3 This refers to the OD levels during the individual and co-culture processes of H. formosensis and O. luteolus in the embodiments of the present invention. 600 And the results of changes in the corresponding gene copy number;

[0028] Figure 4 This invention relates to the effect of the microbial agent on the change in bacterial 16S rRNA copy number during the bio-drying and composting process of organic waste.

[0029] Figure 5 This invention relates to the effect of microbial agents on temperature changes during the biological drying and composting process of organic waste in the embodiments of the present invention.

[0030] Figure 6 This invention relates to the effect of microbial agents on the change in moisture content during the biological drying and composting process of organic waste.

[0031] Figure 7 This invention relates to the effect of microbial agents on the degradation of organic matter during the biological drying and composting process of organic waste.

[0032] Figure 8 This invention relates to the effect of microbial agents on the seed germination index during the biological drying and decomposition process of organic waste in the embodiments of the present invention.

[0033] Figure 9 This invention relates to the effect of microbial agents on the Hufb ratio changes during the biological drying and composting process of organic waste in the embodiments of the present invention.

[0034] Biological Preservation

[0035] Of the five microbial strains in this invention, three—*Saccharomonos poraviridis*, *Halopseudomonas formosensis*, and *Oceanobacillus luteolus*—are commercially available strains, while *Bacillus licheniformis* and *Bacillus subtilis* are separately preserved strains. The preservation information for *Bacillus licheniformis* and *Bacillus subtilis* is as follows:

[0036] Bacillus licheniformis was deposited on April 8, 2022, at the Guangdong Provincial Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO. 62362.

[0037] Bacillus subtilis was deposited on April 8, 2022, at the Guangdong Provincial Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO. 62361. Detailed Implementation

[0038] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The technical features of each embodiment of the present invention can be combined accordingly without mutual conflict. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0039] This invention provides a microbial agent to improve the humification efficiency of biological drying and decomposition processes. The effective components of the microbial agent are composed of a compound of non-antagonistic Saccharomonospora viridis, Halopseudomonas formosensis, Oceanobacillus luteolus, Bacillus licheniformis, and Bacillus subtilis.

[0040] For ease of description, Saccharomonospora viridis, Halopseudomonas formosensis, Oceanobacillus luteolus, Bacillus licheniformis, and Bacillus subtilis are abbreviated as S. viridis, H. formosensis, O. luteolus, B. licheniformis, and B. subtilis, respectively.

[0041] The aforementioned B. licheniformis and B. subtilis strains were previously screened from other high-temperature composting systems and were deposited at the Guangdong Provincial Microbial Culture Collection Center on April 8, 2022, with accession numbers GDMCC NO. 62362 and GDMCC NO. 62361, respectively.

[0042] The other three strains, *S. viridis*, *H. formosensis*, and *O. luteolus*, can all be commercially available strains. In the embodiments of this invention, the commercially available strain of *S. viridis* can be purchased from the American Type Culture Collection (ATCC), accession number ATCC 33517. The commercially available strain of *H. formosensis* can be purchased from the China Industrial Microbiological Culture Collection Center, accession number CICC23006. The commercially available strain of *O. luteolus* can be purchased from the China General Microbiological Culture Collection Center, accession number CGMCC 1.12636. However, it should be noted that since *S. viridis*, *H. formosensis*, and *O. luteolus* are all commercially available strains, the above-mentioned purchase methods are actually only one possible method, but the same strains can also be obtained through other commercially available channels, and there is no limitation on this.

[0043] Among the aforementioned microbial agents, *S. viridis*, *H. formosensis*, *O. luteolus*, *B. licheniformis*, and *B. subtilis* form a synergistic combination. These five bacteria have complementary functions and can synergistically enhance the humification efficiency of the bio-drying and composting process of lignin-rich organic waste. To facilitate understanding of this synergistic effect, the selection criteria for these five bacteria and their respective roles in the microbial agent are detailed below.

[0044] In the bacterial agent of this invention, *S. viridis* and *H. formosensis* are strains selected through metagenomic sequencing analysis that highly contribute to the demethoxylation and β-O-4 bond cleavage of lignin and the synthesis of humic acid (HA) during the bio-drying and composting process of organic waste. *S. viridis* carries the genes ligL, vanAB, ligM, and metF, and their abundance is significantly correlated with humification indicators such as Humophilia during the bio-drying and composting process of organic waste. *H. formosensis* carries the genes ligD, ligF, vanA, and metF. Pure culture experiments using lignin model compounds as reaction substrates verified that *S. viridis* can remove the methoxy groups of vanillic acid and 3-O-methylgallic acid, and *H. formosensis* can cleave the β-O-4 bond of guaiacol-glycerol-β-guaiacol ether. Both can promote the exposure of free phenolic hydroxyl groups in lignin, generating an aromatic skeleton rich in phenolic hydroxyl groups as a key humic acid precursor for HA synthesis.

[0045] In the bacterial agent of this invention, *O. luteolus* is a metabolic cooperative bacterium screened through metagenomic sequencing analysis that can unidirectionally feed *H. formosensis*. It can reduce the metabolic costs of *H. formosensis* in obtaining the energy, cofactors, and reaction substrates required to maintain cell homeostasis and efficiently degrade lignin at high temperatures by providing intermediate metabolites. Pure culture experiments have verified that *H. formosensis* can utilize amino acids / peptides and analogues, small molecule organic acids, lipids, and lipid molecules produced by the metabolism of *O. luteolus* to promote its own proliferation.

[0046] In subsequent embodiments of the present invention, the above-mentioned pure culture experiment is performed in two steps:

[0047] Step 1: Inoculate 1 mL of *H. formosensis* culture, 1 mL of *O. luteolus* culture, and a mixture of 1 mL of each into 200 mL of TSB liquid medium, and incubate at 45°C. o C. Incubate at 150 rpm for 48 h, and determine the OD value of the culture medium. 600 The effect of mixed culture on the proliferation of H. formosensis was determined by the changes in the 16S rRNA gene copy number concentration of the two bacteria in the culture medium.

[0048] Step 2: The bacterial cells collected from the culture medium obtained after culturing H. formosensis for 48 h were washed with pH 7.2 phosphate-buffered saline (PBS) to prepare a bacterial suspension. This suspension was then inoculated at 1% (v / v) into the supernatant of O. luteolus culture medium after sterilization via filtration through a 0.22 μm filter membrane. The mixture was incubated at 45°C. o C. Continue culturing at 150 rpm for 48 h, and determine the OD value of the culture medium. 600 The changes in the types and amounts of metabolites in the culture medium were used to assess the potential of H. formosensis to utilize O. luteolus metabolites.

[0049] In the bacterial agent of this invention, *B. licheniformis* and *B. subtilis* are thermophilic bacteria with high activity of protein, cellulose, fat, and starch-degrading enzymes. They can efficiently degrade organic matter to generate heat, promote the heating and dehydration of the pile, and thus accelerate the formation of a high-temperature, oxygen-rich environment suitable for the growth and metabolism of *S. viridis*, *H. formosensis*, and *O. luteolus* in the bio-drying and composting system of organic waste. This high-temperature environment can also drive niche differentiation, weaken bacterial competition in the bio-drying and composting system, selectively eliminate metabolic competitors of *S. viridis* and *H. formosensis*, and strengthen metabolic cooperation and division of labor among bacteria to collaboratively complete complex tasks such as lignin degradation and HA synthesis. At the same time, the enhanced protein, cellulose, fat, and starch degradation by *B. licheniformis* and *B. subtilis* can produce humic acid precursors such as amino acids, reducing sugars, and fatty acids, which condense with the phenolic hydroxyl groups on the aromatic skeleton generated by the depolymerization of lignin by *S. viridis* and *H. formosensis*, promoting HA synthesis.

[0050] It should be noted that *S. viridis*, *H. formosensis*, *O. luteolus*, *B. licheniformis*, and *B. subtilis* are the active ingredients of the microbial agent. However, in practical applications, other culture medium components or excipients may be added depending on the type of microbial agent. Theoretically, the type of microbial agent can be either a liquid or a solid microbial agent. In the embodiments of this invention, the type of microbial agent is preferably a solid microbial agent. This type of solid microbial agent is a solid composite microbial agent prepared by mixing the bacterial solutions of *S. viridis*, *H. formosensis*, *O. luteolus*, *B. licheniformis*, and *B. subtilis* in a certain mass ratio into a solid matrix and then carrying out aerobic fermentation. The specific mixing ratio of the bacterial solution can be optimized and adjusted according to actual conditions. In the embodiments of the present invention, the preferred mixing mass ratio of *S. viridis*, *H. formosensis*, *O. luteolus*, *B. licheniformis*, and *B. subtilis* in the solid matrix is ​​(1.8~2.2):(1.8~2.2):(1.8~2.2):(0.8~1.2):1, and more preferably 2:2:2:1:1. Considering the effect of use, the total effective viable bacteria count in the final bacterial agent should be (5.3~8.5) × 10⁻⁶. 9 cfu·g -1 The viable count of each strain must be no less than 2.1 × 10⁻⁶. 8 cfu·g -1 .

[0051] Furthermore, the solid substrate used to prepare the solid composite microbial agent can be adjusted according to the actual growth requirements of the microbial agent. In the embodiments of the present invention, the solid substrate is preferably a mixture of corn cobs, bamboo shavings and rice straw in a mass ratio of (3.5~4.5):(0.5~1.5):1, and by percentage of the total mass of the mixture, the following are added: 4~6 wt% corn starch, 4~6 wt% soybean meal, 0.4~0.6 wt% (NH4)2SO4, 0.4~0.6 wt% KH2PO4, 0.08~0.12 wt% NaCl, 0.08~0.12 wt% MgSO4·7H2O and 0.008~0.012 wt% MnSO4. More preferably, the solid matrix is ​​a mixture of corn cobs, bamboo shavings and rice straw in a mass ratio of 4:1:1, and contains 5 wt% corn starch, 5 wt% soybean meal, 0.5 wt% (NH4)2SO4, 0.5 wt% KH2PO4, 0.1 wt% NaCl, 0.1 wt% MgSO4·7H2O and 0.01 wt% MnSO4, by percentage of the total mass of the mixture.

[0052] In an embodiment of the present invention, a specific preparation method for the above-mentioned solid composite microbial agent is further provided, the steps of which are as follows:

[0053] Step 1: Inoculate S. viridis and O. luteolus into TSB liquid medium (preferred formulation: 17.0 g / L tryptone). -1 3.0 g·L soybean peptone -1 NaCl 5.0 g·L -1 K2HPO4 2.5 g·L -1 2.5 g·L glucose -1 The *H. formosensis* was inoculated into NB liquid medium (preferred formulation: 10.0 g / L tryptone, pH 7.1–7.5). -1 3.0 g·L beef extract -1 NaCl 5.0 g·L -1 (pH 7.0–7.4), B. licheniformis and B. subtilis were inoculated into LB liquid medium (formulation: 10.0 g / L tryptone) respectively. -1 Yeast extract 5.0 g·L -1 NaCl 5.0 g·L -1 The culture medium inoculated with *S. viridis*, *B. licheniformis*, and *B. subtilis* was kept at 54–56°C (preferably 55°C) and shaken at 140–160 rpm (preferably 150 rpm) for 48 h. Or more, and at the same time, the culture medium after inoculating H. formosensis and O. luteolus is placed at 44~46℃ (preferably 45℃) and shaken at 140~160 rpm (preferably 150 rpm) for 48 h or more, and five bacterial solutions are obtained after each shaking culture is completed.

[0054] Step 2: Based on the obtained five bacterial suspensions, antagonistic tests were conducted on each of the five strains (S. viridis, H. formosensis, O. luteolus, B. licheniformis, and B. subtilis) in pairs. The test method was as follows: the bacterial suspension of one strain was evenly spread on a heat-resistant NB solid medium (preferred preparation method: adding 20.0 g / L gellan gum and 4 mL / L 20 mM CaCl2 solution to NB liquid medium, sterilizing at 121℃ for 30 min, and then cooling to solidify). Filter paper soaked in the bacterial suspension of the other strain was then placed on the spread plate, and incubated upside down at 45℃ for 24-48 h. The presence or absence of an inhibition zone around the filter paper was used to determine whether the two strains were antagonistic. The test results showed that the five strains were not antagonistic to each other and could be used to formulate bacterial agents.

[0055] Step 3: Spray the five bacterial suspensions (S. viridis, H. formosensis, O. luteolus, B. licheniformis, and B. subtilis) obtained from the shaking culture onto a solid substrate at a mass ratio of 2:2:2:1:1. The substrate is preferably prepared by mixing corn cobs, bamboo shavings, and rice straw at a mass ratio of 4:1:1, and adding 5 wt% corn starch, 5 wt% soybean meal, 0.5 wt% (NH4)2SO4, 0.5 wt% KH2PO4, 0.1 wt% NaCl, 0.1 wt% MgSO4·7H2O, and 0.01 wt% MnSO4, followed by autoclaving at 121℃ for 30 minutes. In a foam box, adjust the moisture content to 58-62% (preferably about 60%) and mix evenly. Place the mixture in a foam box for aerobic fermentation, turning the pile 1-2 times a day (preferably twice). After the pile temperature rises to 45-55℃, spread it out and place it in an oven at 44-46℃ (preferably 45℃) for constant temperature drying until the moisture content is below 20%, and the microbial agent can be obtained.

[0056] It should be noted that the antagonism test in the above preparation method is only carried out by this invention to prove that the five strains do not antagonize each other. However, in actual industrial applications, if the five strains have already been verified to be non-antagonistic, it is not necessary to repeat the antagonism test.

[0057] In addition, the present invention also provides a method for improving the humification efficiency of the biological drying and composting process of organic waste using the above-mentioned microbial agent, as follows:

[0058] Organic waste rich in lignin, such as kitchen waste, biogas residue, and waste from large-scale dairy farming, is fed into the biological drying chamber of an organic waste biological drying and composting system. The microbial agent is inoculated into the biological drying chamber and mixed with the organic waste (since the moisture content of the microbial agent and organic waste differs, the inoculation ratio can be controlled by the final moisture content of the material; preferably, the microbial agent is continuously added and thoroughly mixed with the organic waste until the moisture content of the mixture is adjusted to 66%~70%, preferably about 68%). Sufficient oxygen is provided to the pile through forced ventilation (preferably intermittent ventilation, with a 37-minute cycle, ventilation duration / stop duration of 1 min / 36 min, and ventilation volume of 120 L / min) and high-frequency stirring (preferably intermittent stirring, with a 74-minute cycle, stirring duration / stop duration of 2 min / 72 min, and stirring speed of 12 rpm) to promote the growth of *B. licheniformis* and *B.*. The proliferation of aerobic microorganisms, represented by *S. viridis*, which have high organic matter degradation and heat generation capabilities, drives the rapid heating and dehydration of the compost pile, and accumulates various humic acid precursors. After bio-drying for 22-26 hours (preferably 24 hours), the temperature of the compost pile rises above 50°C, and the moisture content drops below 60%, forming a suitable warm, humid, and oxygen-rich environment for thermotolerant key functional bacteria such as *S. viridis*, *H. formosensis*, and *O. luteolus*, which have high lignin demethoxylation and β-O-4 bond cleavage to generate aromatic skeletons rich in phenolic hydroxyl groups, or participate in HA synthesis, as well as their metabolic collaborators. Then, 1 / 3 to 1 / 2 (preferably 1 / 2) of the bio-dried compost pile material is retained as inoculum for the next batch of fresh organic waste and mixed with the next batch of organic waste entering the bio-drying chamber for further bio-drying. The remaining bio-dried compost pile material is then placed in a high-temperature composting chamber for 7-9 days (preferably 8 days). During this process, *S. viridis*, *H. formosensis*, and *O. luteolus*... luteolus, B. licheniformis, and B. subtilis can rapidly proliferate in the high-temperature, oxygen-rich environment of the high-temperature composting chamber and synergistically and efficiently perform functions such as degradation heat generation, lignin demethoxylation and β-O-4 bond breaking, and HA synthesis. The material after completing the high-temperature composting stage can be discharged from the high-temperature composting chamber as a dried composting product.

[0059] It should be noted that the bio-drying chamber and high-temperature composting chamber in this invention are commonly used devices in organic waste bio-drying and composting systems, and can be implemented using commercially available equipment in this field. The entire equipment operates in a batch processing mode. Organic waste is periodically fed into the bio-drying chamber, and after bio-drying, a portion is retained as inoculum for subsequent fresh materials, while the remainder is fed into the high-temperature composting chamber and discharged after composting. Therefore, the microbial agent is only added during the equipment startup phase. Once the equipment is running normally, it is sufficient to periodically retain 1 / 3 to 1 / 2 of the bio-dried stockpile material and mix it with fresh organic waste; no additional microbial agent is required.

[0060] The following examples further illustrate the strain screening, preparation process, and specific technical effects of the above-mentioned microbial agents of the present invention when applied to the organic waste biological drying and composting system.

[0061] Example 1

[0062] In this embodiment, amplicon sequencing, metagenomic sequencing analysis, and pure culture experiments were used to identify strains that significantly contribute to the demethoxylation and β-O-4 bond breaking of lignin and HA synthesis during the bio-drying and composting process of lignin-rich organic waste. The synergistic relationships among the strains were also elucidated, as follows:

[0063] S1: Amplicon sequencing analysis of samples from organic waste bio-drying and bio-enhanced composting (BEC) processes and bio-enhanced composting (BMC) processes without bio-drying pretreatment.

[0064] Amplicon sequencing analysis results of bio-drying and bio-enhanced composting processes of kitchen waste without pre-treatment are as follows: Figure 1 As shown, the results revealed changes in the bacterial community. It was found that the bio-drying and ripening process began to enrich Bacillus within 0–1 day, with an abundance of 0.17–3.11%, slightly higher than the 0.05–1.93% in the bio-enhanced ripening process. By day 3, when the pile temperature reached 61.4 ± 4.7℃, aerobic and heat-resistant Bacillus dominated in the bio-drying and ripening process (53.83 ± 1.83%), while in the bio-enhanced ripening process, Bacillus abundance only began to increase significantly and reach a peak (34.70 ± 2.84%) within 5–9 days, and the peak value was significantly lower (p < 0.001). Pearson correlation analysis showed that during the 0–3 days of bio-drying and decomposition, the abundance of Bacillus was significantly positively correlated with the pile temperature (p < 0.01). Bacillus is a key thermophilic functional bacterium enriched under the oxygen supply conditions optimized by bio-drying forced ventilation and high-frequency stirring, and drives the rapid temperature rise of the pile through rapid degradation of organic matter.

[0065] S2: Metagenomic sequencing analysis of organic waste samples after bio-drying and decomposition.

[0066] S2-1: Assemble and bin the data obtained from the DNA metagenomic sequencing of samples extracted during the bio-drying and composting process of organic waste, and align the protein sequences of the obtained metagenomic assembled genomes (MAGs) to the sequences of genes encoding lignin O-demethylase (vanA, vanB, and ligM) obtained from the KEGG database and genes related to lignin β-O-4 bond breakage (ligD, ligL, ligE, ligF, and ligG) obtained from GenBank for functional prediction and annotation. Figure 2 A metabolic schematic diagram of the involvement of MAG_48 (S. viridis) and MAG_387 (H. formosensis) in the lignin β-O-4 bond cleavage and demethoxylation cascade reaction is presented. The results show that among the MAGs significantly enriched during the high-temperature composting stage (≥ 55℃) of organic waste bio-drying and ripening, 22 carry at least one gene related to lignin demethoxylation and β-O-4 bond cleavage, excluding metF. Specifically, MAG_48 (S. viridis) carries ligL, vanAB, ligM, and metF genes; MAG_387 (H. formosensis) carries ligD, ligF, vanA, and metF genes. These MAGs carry the most types of related functional genes and contribute significantly to the abundance of these genes, making them key thermophilic functional bacteria mediating lignin demethoxylation and β-O-4 bond cleavage during the organic waste bio-drying and ripening process.

[0067] S2-2: Pearson correlation analysis of the abundance of MAGs significantly enriched during the high-temperature composting stage (≥ 55℃) of organic waste bio-drying and composting and the humification indicators such as seed germination index and Hufb ratio revealed that the abundance of MAG_48 (S. viridis) showed a significant positive correlation with these humification indicators (p < 0.05), and its abundance was the highest among all MAGs that showed a strong correlation with humification indicators. It is a key thermophilic functional bacterium involved in HA synthesis in the organic waste bio-drying and composting system.

[0068] S2-3: A metabolic interaction network of MAGs in an organic waste bio-drying and composting system was constructed using the iNAP2 platform. Results showed that the rapid and sustained high temperatures caused by the enrichment of thermotolerant Bacillus during 24-hour bio-drying pretreatment and its efficient degradation of organic matter promoted niche differentiation, selectively eliminating potential competitors with poorer heat tolerance, such as Lactobacillaceae and Enterococcus, weakening interbacterial competition, and reducing nutrient competition pressure on S. viridis and H. formosensis, allowing them to concentrate more metabolic resources on lignin degradation and HA synthesis. Meanwhile, high temperature also enhances the metabolic cooperation among bacteria, which is mainly based on unidirectional feeding. This allows H. formosensis to obtain intermediate metabolites such as coenzymes and their derivatives, carbohydrates and their derivatives, short-chain organic acids and their derivatives, and amino acids / peptides and their analogues from MAG_115 (O. luteolus), which is also enriched during the high-temperature composting stage (≥ 55℃). This allows them to maintain cell homeostasis at high temperatures with lower metabolic costs and obtain the energy, cofactors, and reaction substrates required for lignin degradation, thereby improving degradation efficiency.

[0069] S3: Functional verification of lignin demethoxylation and β-O-4 bond breaking in S. viridis and H. formosensis.

[0070] Commercial strains *S. viridis* (ATCC 33517) and *H. formosensis* (CICC 23006), which are phylogenetically close to MAG_48 and MAG_387 obtained in the metagenomic sequencing analysis, were purchased from the China General Microbiological Culture Collection Center and the China Industrial Microbiological Culture Collection Center, respectively, for verification of lignin demethoxylation and β-O-4 bond breaking functions.

[0071] S3-1: Based on the gene carried by *S. viridis*, *S. viridis* possesses the potential for demethoxylation, but cannot independently complete the full β-O-4 bond cleavage process. Therefore, vanillic acid and 3-O-methylgallic acid were used as model compounds of lignin carrying methoxy groups to verify its lignin demethoxylation ability. The specific liquid culture medium formulation is as follows: tryptone 2.83 g·L⁻¹ -1 0.50 g·L soybean peptone -1 0.42 g·L glucose -1 K2HPO4 2.50 g·L -1 NaCl 5.00 g·L -1 Tetrahydrofolate 22.5 mg·L -1 Reduced glutathione 154 mg·L -1Vanillic acid 42 mg·L -1 3-O-methylgallic acid 46 mg·L -1 The pH was 6.8–7.2. *S. viridis* was inoculated into the above liquid culture medium and cultured at 55°C with shaking at 150 rpm for 48 h. The changes in phenolic acid content in the culture medium were determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS / MS). The results showed that *S. viridis* (ATCC 33517) could remove the methoxy groups from vanillic acid and 3-O-methylgallic acid, generating products richer in phenolic hydroxyl groups, such as protocatechuic acid and gallic acid.

[0072] S3-2: Based on the gene carried by *H. formosensis*, *H. formosensis* can catalyze the cleavage of β-O-4 bonds to generate phenolic acid monomers, but the demethoxylation pathway is incomplete. Therefore, guaiacol-glycerol-β-guaiacol ether was used as a lignin model compound containing β-O-4 bonds to verify its lignin β-O-4 bond cleavage ability. The specific liquid culture medium formulation is as follows: tryptone 5.0 g·L⁻¹ -1 1.50 g·L beef extract -1 NaCl 1.50 g·L -1 Tetrahydrofolate 22.5 mg·L -1 Reduced glutathione 154 mg·L -1 Guaiacinol-β-guaiacyl ether 40 mg·L -1 The pH was 6.8–7.2. *H. formosensis* was inoculated into the above liquid medium and cultured at 45°C with shaking at 150 rpm for 48 h. The changes in phenolic acid content in the culture medium were determined by HPLC-MS / MS. The results showed that *H. formosensis* could cleave the β-O-4 bond in guaiacol-β-guaiacol ether, generating vanillic acid and vanillin, thus promoting the exposure of phenolic hydroxyl groups.

[0073] S4: Validation of the potential of H. formosensis to obtain O. luteolus metabolites to promote its own growth and reproduction.

[0074] A commercial strain of *O. luteolus* (CGMCC 1.12636), which has a similar phylogenetic distance to MAG_115 obtained in the metagenomic sequencing analysis, was purchased from the China General Microbiological Culture Collection Center to verify its unidirectional feeding ability to *H. formosensis*.

[0075] S4-1: Inoculate 1 mL of *H. formosensis* culture, 1 mL of *O. luteolus* culture, and a mixture of both into 200 mL of TSB liquid medium, respectively. Incubate at 45℃ and 150 rpm for 48 h. Analyze the OD values ​​of the culture medium. 600 The effect of co-culture on the proliferation of *H. formosensis* was determined by the changes in the 16S rRNA gene copy number concentration of the two bacteria in the culture medium. *H. formosensis* was annealed at 51.8℃ using primers γ-proteobacteriaF(5′-TCGTCAGCTCGTGTYGTGA-3′) / R(5′-CGTAAGGGCCATGATG-3′) as shown in SEQ ID No. 1 and SEQ ID No. 2, respectively; *O. luteolus* was annealed at 50.7℃ using primers FirmicutesF(5′-GAAACTYAAAGGAATTGACG-3′) / R(5′-ACCATGCACCACCTGTC-3′) as shown in SEQ ID No. 3 and SEQ ID No. 4, respectively. Results are as follows: Figure 3 As shown, the OD of the two-bacterial mixed culture system can be observed. 600 The growth rate was faster than that of any single-strain culture, and the 16S rRNA gene copy number of *H. formosensis* after 12 h of co-culture with *O. luteolus* was significantly higher than that of single-strain culture by about an order of magnitude (p < 0.05), indicating that co-culture with *O. luteolus* promotes the rapid growth and reproduction of *H. formosensis*. Endogenous death due to nutrient depletion may have occurred between 24 and 48 h, leading to a decrease in OD in the co-culture system. 600 And the number of bacteria decreases even faster.

[0076] S4-2: Bacterial cells collected from the culture medium obtained after culturing *H. formosensis* for 48 h were washed with pH 7.2 phosphate-buffered saline (PBS) to prepare a bacterial suspension. This suspension was then inoculated at 1% (v / v) into the supernatant of *O. luteolus* culture medium after sterilization via filtration through a 0.22 μm filter. The culture was further incubated at 45°C and 150 rpm for 48 h. The bacterial suspension was then analyzed based on the OD of the culture medium. 600 The study assessed the potential of *H. formosensis* to utilize *O. luteolus* metabolites by examining changes in the types and concentrations of metabolites in the culture medium. Results showed that *H. formosensis* can utilize metabolites produced by *O. luteolus*, including amino acids / peptides and analogues, small organic acids, and lipids and lipid molecules, leading to an increase in the OD value of the culture medium after inoculation with *H. formosensis*. 600The OD value rose rapidly, peaking at 24 h, indicating that *H. formosensis* can promote its own growth and reproduction by utilizing metabolites produced by *O. luteolus*. Endogenous death due to nutrient depletion likely occurred between 24 and 48 h, leading to a decrease in the OD value of the *H. formosensis* culture medium. 600 decline.

[0077] In summary, based on the molecular biological analysis and pure culture functional verification results of S1~S4, this embodiment uses S. viridis (ATCC 33517), H. formosensis (CICC 23006), and O. luteolus (CGMCC1.12636), as well as B. licheniformis (GDMCC NO. 62362) with high cellulose, fat, and starch degrading enzyme activity obtained in the previous screening, and B. subtilis (GDMCC NO. 62361) with high protein and starch degrading enzyme activity, to construct a microbial agent that improves the humification efficiency of the bio-drying and composting process of organic waste. Among them, *S. viridis* and *H. formosensis* can synergistically complete the enzymatic cascade reaction of lignin demethoxylation and β-O-4 bond cleavage at high temperatures, causing lignin depolymerization and exposing the aromatic skeleton rich in phenolic hydroxyl groups as a key humic acid precursor. *O. luteolus* can provide *H. formosensis* with energy, cofactors, and substrates to alleviate high-temperature stress and support lignin degradation, reducing the metabolic burden of *H. formosensis* and further improving lignin degradation efficiency. *B. licheniformis* and *B. subtilis* can release bioheat through rapid degradation of organic matter, driving rapid heating and dehydration of the pile, accelerating the formation of a high-temperature and oxygen-rich environment suitable for the growth and metabolism of heat-resistant *S. viridis*, *H. formosensis*, and *O. luteolus*, and weakening the metabolic competition between these three key functional bacteria and other microorganisms by enhancing high-temperature stress, strengthening metabolic cooperation mainly based on unidirectional feeding (including unidirectional feeding of *O. luteolus* to *H. formosensis*), and further improving the resource utilization and metabolic efficiency of the three key functional bacteria. Meanwhile, *B. licheniformis* and *B. subtilis* efficiently degrade proteins, cellulose, fats, and starches, and also promote the formation of humic acid precursors such as amino acids, reducing sugars, and fatty acids. These precursors can condense with phenolic hydroxyl groups on the aromatic backbone formed by the depolymerization of lignin by *S. viridis* and *H. formosensis*, forming highly aromatic macromolecules (HA). *S. viridis* can mediate this HA synthesis process. In summary, *S. viridis*, *H. formosensis*, *O. luteolus*, *B. licheniformis*, and *B. subtilis* each play different roles in the humification process, and all are indispensable for improving the humification efficiency of the biological drying and composting process for organic waste.

[0078] Example 2

[0079] In this embodiment, a microbial agent was prepared to improve the humification efficiency of the biological drying and composting process of organic waste, as detailed below:

[0080] S1: Large-scale culture of bacterial strain.

[0081] S. viridis and O. luteolus were inoculated into TSB liquid medium (17.0 g·L⁻¹ tryptone). -1 3.0 g·L soybean peptone -1 NaCl 5.0 g·L -1 K2HPO4 2.5 g·L -1 2.5 g·L glucose -1 H. formosensis was inoculated into NB liquid medium (pH 7.1–7.5) and cultured at 10.0 g / L tryptone. -1 3.0 g·L beef extract -1 NaCl 5.0 g·L -1 (pH 7.0–7.4), B. licheniformis and B. subtilis were inoculated into LB liquid medium (10.0 g / L tryptone) respectively. -1 Yeast extract 5.0 g·L -1 NaCl 5.0 g·L -1 Five bacterial cultures were obtained by incubating at pH 7.0-7.2, with S. viridis, B. licheniformis and B. subtilis at 55℃ and H. formosensis and O. luteolus at 45℃ and shaking at 150 rpm for 48 h.

[0082] S2: Antagonism test.

[0083] Antagonism tests were conducted on *S. viridis*, *H. formosensis*, *O. luteolus*, *B. licheniformis*, and *B. subtilis*. One strain was evenly spread on a thermostable NB solid medium (NB liquid medium was sterilized at 121°C for 30 min with 20.0 g / L gellan gum and 4 mL / L 20 mM CaCl2 solution added, then cooled and solidified). Filter paper soaked in the bacterial solution of another strain was placed on the spread plate, and the plates were incubated upside down at 45°C for 24–48 h. The presence or absence of an inhibition zone around the filter paper was used to determine antagonism between the two strains. The results showed that the five strains did not antagonize each other and could be used to formulate bacterial agents.

[0084] S3: Solid compound microbial agent fermentation.

[0085] Five bacterial solutions of *S. viridis*, *H. formosensis*, *O. luteolus*, *B. licheniformis*, and *B. subtilis* were sprayed at a mass ratio of 2:2:2:1:1 onto a solid substrate composed of corn cobs, bamboo shavings, and rice straw in a mass ratio of 4:1:1, with the addition of 5 wt% corn starch, 5 wt% soybean meal, 0.5 wt% (NH4)2SO4, 0.5 wt% KH2PO4, 0.1 wt% NaCl, 0.1 wt% MgSO4·7H2O, and 0.01 wt% MnSO4, and sterilized by high-pressure steam at 121℃ for 30 min. The moisture content was adjusted to approximately 60%, and the mixture was thoroughly mixed. The substrate was then placed in a foam box for aerobic fermentation, turned twice daily. Once the temperature of the pile reached 45-55℃, it was spread out and dried in a 45℃ oven until the moisture content was below 20%, thus obtaining the bacterial agent.

[0086] S4: Determination of the effective viable count of the microbial agent.

[0087] The viable count of the prepared microbial agent was performed according to the serial dilution method in "Agricultural Microbial Inoculants" (GB 20287-2006). The total viable count of the agent was determined to be (5.3~8.5) × 10⁻⁶. 9 cfu·g -1 The viable count of each strain was no less than 2.1 × 10⁻⁶. 8 cfu·g -1 It exceeds the technical indicators for the effective live bacteria count of products in "Organic Material Composting Agent" (NY 609-2002) and "Agricultural Microbial Agents" (GB20287-2006).

[0088] Example 3

[0089] In this embodiment, the bacterial agent prepared in Example 2 was added to the organic waste biological drying and composting system to verify the effect of improving the humification efficiency, as detailed below:

[0090] The operating procedure of the organic waste biological drying and composting system is as follows: The initial material in the biological drying and composting system is a mixture of fresh organic waste raw materials with added microbial agents or auxiliary materials and substrate after 24 hours of biological drying. After biological drying, half of the substrate after 24 hours of biological drying is retained as inoculum for the next batch of fresh organic waste, and the remaining substrate is put into the high-temperature composting chamber for further composting for 8 days. The operating parameters of the biological drying chamber are as follows: operating time 48 hours, residence time 2 days, stirring frequency 2 min / 72 min, speed 12 rpm, positive pressure ventilation frequency 1 min / 36 min, air volume 120 L / min, negative pressure exhaust frequency 10 min / 36 min, air volume 57 L / min. The operating parameters of the high-temperature composting chamber are as follows: operating time 8 days, residence time 8 days, turning once / day, positive pressure ventilation frequency 1 min / 36 min, air volume 120 L / min, negative pressure exhaust frequency 5 min / 36 min, air volume 57 L / min. When the temperature of the pile is detected to be higher than 65°C, start stirring / turning, positive pressure ventilation, and negative pressure exhaust until the temperature drops to 55°C to avoid excessive temperature causing a large number of microorganisms to become inactive.

[0091] (1) Control experiment setup

[0092] Two control groups were set up: a TG group (with added microbial agent) and a CK group (with added control group). The TG group was given a compound microbial agent without any additional excipients; the CK group was given excipients with the same composition and proportion as the solid matrix of the microbial agent. The initial moisture content of both groups was adjusted to approximately 68%.

[0093] (2) Sample testing methods

[0094] 16S rRNA gene copy number: The abundance of 16S rRNA gene in compost samples was determined by qPCR. Primers used were 341F (5′-CCTAYGGGRBGCASCAG'-3′) / 806R (5′-GGACTACNNGGGTATCTAAT-3′) as shown in SEQ ID No. 5 and SEQ ID No. 6, respectively. The amplification program was 95℃ pre-denaturation for 5 min, followed by 40 cycles (95℃ for 15 s, 60℃ for 30 s).

[0095] Temperature: The temperature of the upper, middle and lower layers of the stack was measured using an electronic thermometer, and the average value was taken.

[0096] Moisture content and volatile solids percentage: Referring to the "Sampling and Analysis Methods for Municipal Solid Waste" (CJ / T 313-2009), fresh compost samples were dried to constant weight at 105℃, and the moisture content (%) and total solids content (TS) were calculated using the difference method. The dried samples were then calcined in a muffle furnace at 550℃ for 2 h, and the volatile solids content (VS) and ash content were calculated using the difference method. VS / TS (%) and ash percentage (%) are the ratios of VS and ash content to TS, respectively.

[0097] Organic matter content: Starch content was determined using the anthrone colorimetric method, protein content using the Kjeldahl nitrogen determination method, fat content using the Soxhlet extraction method, and lignin, cellulose, and hemicellulose content using the Panthen washing method. The organic matter content was corrected using the following formula to eliminate the composting concentration effect:

[0098]

[0099] Among them, OM loss OM represents the percentage (%) of organic matter loss in a sample at a certain stage compared to the initial sample. initial and OM current Organic matter content (g·100 g) of the initial sample and a sample from a certain stage, respectively. -1 (dry basis), Ash initial and Ash current These represent the ash content percentage (%) of the corresponding samples.

[0100] Lignin methoxyl content and β-O-4 bond ratio: Lignin in compost samples was separated using a γ-valerol binary solvent system with a solid-liquid ratio of 1:10. The ratio of γ-valerol to water in the mixed solvent was 4:1 (w / w), and 0.075 mol·L⁻¹ was added. -1 The reaction was carried out in H2SO4 solution (10 wt% of solvent) at 160 °C for 24 h. 2D-HSQC NMR spectra of lignin were collected using DMSO-d6 as solvent, with 64 scans and a relaxation time of 2.0 s. The δ-ray diffraction (δ) in the spectrum... C / δ H 56.3 / 3.7 ppm represents the C–H (–OCH3) in the methoxy group, δ C / δ H 71.4~72.6 / 4.7~5.0 ppm represents C in the β-O-4 structure (A) and the α-acetylated β-O-4 structure (A'''). α –H α ((A, A''') α ), δ C / δ H 84.5 / 4.3 ppm represents C in the β–β resin alcohol structure (B). α–H α (B) α ), δ C / δ H 86.8 / 5.5 ppm represents C in the β–5-phenylcoumarin structure (C). α –H α (C) α ), δ C / δ H 104.4 / 6.7 ppm represents C in the syringyl unit (S). 2,6 –H 2,6 (S) 2,6 ), δ C / δ H 110.7~111.6 / 6.9~7.1 ppm represents C2–H2 (G2) in the guaiac wood matrix unit (G), δ C / δ H 111.8 / 7.3 ppm represents oxidized guaiac-based units (G', C). α =O) in C2–H2(G'2), δ C / δ H 128.9 / 7.2 ppm represents the C in the p-hydroxyphenyl unit (H). 2,6 –H 2,6 (H) 2,6 The methoxy content is calculated using the aromatic unit (Ar) as an internal standard, according to the following formula:

[0101]

[0102]

[0103] Where IC9 is the aromatic unit integral, I –OCH3 For the methoxy integral, IS 2,6 IG2, IH 2,6 The integrals are for the S, G, and H unit signals, respectively. The relative content of each type of bonding bond is expressed as the proportion of its integral to the total integral of the bonding bond signal.

[0104] Total phenolic hydroxyl content of lignin: 0.4 mmol of chromium acetylacetonate (III) and 0.5 mmol of N-hydroxysuccinimide were dissolved in 10 mL of pyridine-d5 to obtain 50 mmol·L⁻¹. -1 Internal standard solution. Dissolve 10 mg of lignin sample in 400 μL of LCDCl3-d6 and 200 μL of internal standard solution, add 80 μL of 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxophosphazenecyclopentane (TMDP) as phosphating agent, and shake vigorously for 5 min before proceeding. 31 P NMR analysis was performed, with 32 scans and a relaxation time of 4.0 s. The δp spectra in the spectrum are shown.P 151.40 ppm represents the –OH group in the internal standard reagent N-hydroxysuccinimide, δ P 144.0–140.5 ppm represents the –OH group and δ group in guaiacyl (S) phenols. P 140.5–138.0 ppm represents the –OH group in coniferyl (G) phenols, δ P 138.0–135.5 ppm represents the –OH group in p-hydroxyphenyl (H)phenols. Hydroxyl group content (C) –OH (mmol·g) -1 Calculate using the following formula:

[0105]

[0106] Where A is the integral area of ​​the hydroxyl peak, A' is the integral area of ​​the internal standard peak, w is the mass of the lignin sample (g), and 0.01 is the number of moles of the internal standard reagent (mmol). The total phenolic hydroxyl content is the sum of the –OH contents of S, G, and H type phenols.

[0107] Seed germination index: The GI of fresh compost samples was determined according to Appendix F of "Organic Fertilizers" (NY / T 525-2021). Specifically, a filter paper was placed in a petri dish, 5.0 mL of distilled water (blank control) or leachate (treatment group) was added, then 10 radish seeds were added, the lid was closed, and the dish was placed in the dark at 25℃ for 3 days. The number of germinated seeds and root length were measured. Each sample required 4 replicates, and the seed germination index (GI) was calculated using the following formula:

[0108]

[0109] Hu Fubi: The freeze-dried and ground compost sample was mixed with 0.1 mol·L⁻¹ -1 Na4P2O7·10H2O and 0.1 mol·L -1 The NaOH mixture was prepared at a ratio of 1:20 (w / v) and extracted with shaking at 25°C and 180 rpm for 6 h. The mixture was then centrifuged at 12000 rpm for 10 min, and the supernatant was collected as the humic acid extract (HS). The HS was filtered through a 0.45 μm filter membrane and then purified with 6 mol·L⁻¹ water. -1 Adjust the pH to 1-2 with HCl solution, incubate overnight at 4°C, then centrifuge at 12000 rpm for 10 min to separate the crude HA precipitate. Use 0.05 mol·L⁻¹ water to extract the crude HA precipitate. -1 Dissolve the HA in NaOH solution and adjust to neutral. Centrifuge at 12000 rpm for 10 min, and freeze-dry the supernatant to obtain HA. The contents of HA, fulvic acid (FA), and HS are all expressed as carbon content, denoted as C. HA C FA and C HSC HS and C HA C was measured using a TOC analyzer. FA The difference between the two is defined by Hoogewerf as C. HA / C FA The humic acid content was corrected using the same method as the organic matter content determination to eliminate the compost concentration effect.

[0110] (3) Test results

[0111] Changes in bacterial 16S rRNA copy number in the TG and CK groups during the experiment are as follows: Figure 4 As shown, the 16S rRNA copy number in the TG group was consistently significantly higher than that in the CK group (p < 0.05).

[0112] Temperature changes in the TG and CK groups during the experiment are as follows: Figure 5 As shown, the temperature in the TG group was consistently significantly higher than that in the CK group during the 24-hour bio-drying period (p < 0.05), and it entered the high-temperature stage earlier.

[0113] The changes in moisture content of the TG and CK groups during the experiment are as follows: Figure 6 As shown, rapid heating further promoted efficient dehydration. At the end of bio-drying, the moisture content of the TG group material decreased to 58.17 ± 0.98%, which was significantly lower than that of the CK group (59.99 ± 0.43%) (p < 0.05).

[0114] The organic matter degradation rates of the TG and CK groups during the experiment were as follows: Figure 7 As shown, the total degradation rate of macromolecular components in the TG group was significantly higher than that in the CK group by 32.84 ± 0.36% (p < 0.01), with the lignin degradation rate showing the largest increase, which was 23.75 ± 2.23% higher than that in the CK group (p < 0.01).

[0115] The methoxy group content, β-O-4 bond ratio, and total phenolic hydroxyl content of lignin in the TG and CK groups during the experiment are shown in Table 1. The lowest values ​​of methoxy group content (3.99 / Ar) and β-O-4 bond content (50.89%) in the TG group during lignin depolymerization were 12.69% and 16.30% lower than those in the CK group, respectively. The intense demethoxylation and β-O-4 bond depolymerization resulted in a peak total phenolic hydroxyl content of 3.88 mmol·g in the TG group. -1 It was 1.11 times the peak value of the CK group, indicating an increased supply of aromatic skeletons rich in phenolic hydroxyl groups.

[0116] Table 1. Changes in lignin methoxy, β-O-4 bond and total phenolic hydroxyl content in the TG and CK groups.

[0117]

[0118] Seed germination index of TG group and CK group during the experiment is as follows: Figure 8 As shown, the seed germination index of the TG group increased more rapidly during the high-temperature composting stage (3-9 days), reaching 74.10 ± 3.43% on day 5, exceeding the 70% composting standard stipulated in "Organic Fertilizer" (NY / T 525-2021), and rising to 90.35 ± 3.34% at the end of composting on day 9, which was significantly higher than the 74.05 ± 4.40% of the CK group by 22.51 ± 11.43% (p < 0.01).

[0119] Hu Furu, from the TG group and CK group, during the experiment Figure 9 As shown, the glutathione ratio of the product in the TG group was 2.97 ± 0.15, which was significantly higher than that in the CK group by 23.97 ± 11.79% (p < 0.01).

[0120] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A microbial agent for improving the humification efficiency of biological drying and decomposition processes, characterized in that: The active ingredient of the bacterial agent is composed of non-antagonistic green sucralose (Saccharomyces cerevisiae). Saccharomonospora viridis ), Taiwan Pseudomonas ( Halopseudomonas formosensis ), Bacillus microepiploicus ( Oceanobacillus luteolus ), Bacillus licheniformis ( Bacillus licheniformis ) and Bacillus subtilis ( Bacillus subtilis It is composed of composite components; The Bacillus licheniformis ( Bacillus licheniformis It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 8, 2022, with accession number GDMCC NO. 62362; The Bacillus subtilis ( Bacillus subtilis It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 8, 2022, with accession number GDMCC NO. 62361; The green sucrose monosporum ( Saccharomonospora viridis The strain was a commercially available strain, purchased from the American Type Culture Collection Center (ATCC), with accession number ATCC 33517. Taiwan Pseudomonas ( Halopseudomonas formosensis The strain was a commercially available strain purchased from the China Industrial Microbial Culture Collection Center, with accession number CICC 23006. Bacillus microepiploicus ( Oceanobacillus luteolus The strain was a commercially available strain purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC 1.12636.

2. The microbial agent for improving the humification efficiency of the biological drying and composting process as described in claim 1, characterized in that: The inoculum is *Sucralose* (a type of fungus). Saccharomonospora viridis ), Taiwan Pseudomonas ( Halopseudomonas formosensis ), Bacillus microepiploicus ( Oceanobacillus luteolus ), Bacillus licheniformis ( Bacillus licheniformis ) and Bacillus subtilis ( Bacillus subtilis Solid composite microbial agent is prepared by mixing bacterial solutions of 1,0 ...

3. The microbial agent for improving the humification efficiency of the biological drying and composting process as described in claim 2, characterized in that, The green sucrose monosporum ( Saccharomonospora viridis ), Taiwan Pseudomonas ( Halopseudomonas formosensis ), Bacillus microepiploicus ( Oceanobacillus luteolus ), Bacillus licheniformis ( Bacillus licheniformis ) and Bacillus subtilis ( Bacillus subtilis The mixing mass ratio of the components in the solid matrix is ​​(1.8~2.2): (1.8~2.2): (1.8~2.2): (0.8~1.2):

1.

4. The microbial agent for improving the humification efficiency of the biological drying and composting process as described in claim 2, characterized in that, The solid matrix is ​​made by mixing corn cobs, bamboo shavings and rice straw in a mass ratio of (3.5~4.5):(0.5~1.5):1, and by adding 4~6 wt% corn starch, 4~6 wt% soybean meal, 0.4~0.6 wt% (NH4)2SO4, 0.4~0.6 wt% KH2PO4, 0.08~0.12 wt% NaCl, 0.08~0.12 wt% MgSO4·7H2O and 0.008~0.012 wt% MnSO4, based on the percentage of the total mass of the mixture.

5. The microbial agent for improving the humification efficiency of the biological drying and composting process as described in claim 1, characterized in that: The total effective viable count of the microbial agent is (5.3~8.5) × 10⁻¹⁰. 9 cfu·g -1 The viable count of each strain was no less than 2.1 × 10⁻⁶. 8 cfu·g -1 .

6. A method for preparing a microbial agent for improving the humification efficiency of a biological drying and composting process as described in any one of claims 1 to 5, characterized in that, include: The green sucrose monoporum ( Saccharomonospora viridis ) and Bacillus microepiploicus ( Oceanobacillus luteolus The *Pseudomonas taiwanensis* (Taiwan Pseudomonas) were inoculated separately into TSB medium. Halopseudomonas formosensis ) was inoculated into NB medium, and the Bacillus licheniformis ( Bacillus licheniformis ) and Bacillus subtilis ( Bacillus subtilis ) were inoculated separately into LB liquid medium; then the inoculated *Saccharomyces cerevisiae* ( Saccharomonospora viridis ), Bacillus licheniformis ( Bacillus licheniformis ) and Bacillus subtilis ( Bacillus subtilis After incubation, the culture medium was placed at 54-56℃ with shaking at 140-160 rpm for at least 48 h, and then inoculated with *Pseudomonas taiwanensis* (…). Halopseudomonas formosensis ) and Bacillus microepiploicus ( Oceanobacillus luteolus The culture medium was placed at 44-46℃ and shaken at 140-160 rpm for at least 48 h. The five bacterial solutions obtained from the shaking culture were sprayed onto the solid substrate, the moisture content was adjusted to 58-62%, and the mixture was mixed evenly before aerobic fermentation. The pile was turned 1-2 times a day. After the pile temperature rose to 45-55℃, it was spread out and placed at 44-46℃ for constant temperature drying until the moisture content was less than 20%, thus obtaining the bacterial agent.

7. A biological drying and composting process for organic waste, characterized in that: The microbial agent for improving the humification efficiency of the bio-drying and composting process as described in any one of claims 1 to 5 is inoculated into the organic waste in the bio-drying chamber. Sufficient oxygen is provided to the pile through forced ventilation and stirring, and the pile is bio-dried for 22 to 26 hours, so that the pile temperature rises to above 50°C and the moisture content drops to below 60%. Then, 1 / 3 to 1 / 2 of the bio-dried pile material is retained as inoculum for the next batch of fresh organic waste, and the next batch of organic waste entering the bio-drying chamber is bio-dried. The remaining pile material that has completed bio-drying is all transferred to the high-temperature composting chamber for composting for 7 to 9 days, and then discharged from the high-temperature composting chamber as a dried composting product.

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