Staphylococcus warneri as well as complex microbial inoculant, preparation method and application thereof

Abstract

The invention discloses staphylococcus warneri as well as a complex microbial inoculant, a preparation method and application thereof, and belongs to the technical field of microorganisms, the staphylococcus warneri has the strain name of LH-403 and the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.36214, and the complex microbial inoculant is prepared from the staphylococcus warneri, Trichoderma longibrachiatum and Bacillus thuringiensis LMU-81 together. According to the invention, the three strains are compounded to generate a synergistic effect, so that the lignocellulase can be massively produced at normal temperature, and the method is suitable for degradation of bagasse lignocellulose.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Staphylococcus wartii and its compound inoculum, preparation method and application. Background Technology

[0002] Natural lignocellulose undergoes a long degradation cycle and has low utilization under natural conditions. Therefore, the selection of low-cost, environmentally friendly, and easy-to-operate microbial methods for degrading natural lignocellulose has attracted widespread attention. However, using single microorganisms for lignocellulose degradation has limitations, such as long cultivation times and low efficiency. To achieve effective biodegradation, composite microbial communities composed of different strains can be used. Utilizing the synergistic effect between different functional microorganisms is key to efficient degradation; compared to any single strain, composite microbial systems exhibit stronger decomposition capabilities and more stable lignocellulase activity. Due to their higher substrate utilization, composite microorganisms have gradually replaced single strains as the preferred method for biodegrading lignocellulose. Exploring the role of composite microbial communities in lignocellulose degradation is of great significance. Currently, composite microorganisms are mainly constructed through targeted domestication and artificial creation. Targeted domestication of composite microorganisms can significantly improve their function, but the domestication time is long. Artificially constructed composite microbial communities have greater advantages, promoting complementary advantages among microorganisms and improving the overall performance of the composite microorganism.

[0003] Artificial construction is not simply a matter of mechanically mixing microorganisms together; it requires regulating and promoting their synergistic effects to further advance the biochemical reaction process. Therefore, balancing the production needs of different microorganisms is key to promoting their cooperation. In the selection of strains, cross-species combinations of fungi and bacteria often yield more unexpected results than concentric combinations of fungi and fungi. This is because bacteria and fungi avoid competition, preventing efficiency reduction and minimizing competition for resources and space; the stability of the entire degradation system depends on the survival status of the fungi, and the degradation process will quickly halt if the environment becomes unsuitable; fungi provide the physical scaffold for bacterial survival and the nutrient source after degradation; and bacteria, by rapidly consuming small molecule products, create favorable conditions for the continuous enzymatic hydrolysis activity of fungi. Therefore, exploring the different effects of various bacterial + fungal composite inoculants on the degradation of lignocellulose is a direction that those skilled in the art are continuously exploring.

[0004] Staphylococcus wartii ( Staphylococcus warneri*Staphylococcus warwick*, belonging to the genus *Staphylococcus*, is widely found in the normal flora of human skin. This strain has a diameter of 0.5–1.5 μm and can form single, diploid, or grape-like arrangements, tolerating high-salt environments. It is mainly used in the development of pathogen detection chips, classification research, and the development of fermented foods. Previous studies have shown that most lignocellulases originate from fungi, particularly *Trichoderma* and *Aspergillus*. There are few existing reports on the use of *Staphylococcus warwick* for decomposing lignocellulose. For example, Chinese patent: *Staphylococcus warwick* AACE8 that can promote the growth of *Armillaria mellea* and its application; publication number: CN119639609A; the *Staphylococcus warwick* AACE8 described has the ability to degrade cellulose, but the specific degradation ability is unknown.

[0005] In the existing technology, although many compound microbial agents for the degradation of lignocellulose have emerged, many compound microbial agents can only achieve the effect of efficiently degrading one or two components of cellulose, hemicellulose and lignin, and the degradation ability is not balanced; secondly, when applied to substrates of various sources and different component ratios, the degradation ability of existing compound microbial agents is not satisfactory. Summary of the Invention

[0006] This invention discloses a strain of *Staphylococcus wartii*, its compound inoculant, preparation method, and application. This *Staphylococcus wartii* strain is combined with other lignocellulose-degrading strains to obtain a compound inoculant with higher degradation efficiency. This addresses the problem of insufficient lignocellulose degradation efficiency in existing compound inoculants, resulting in a more comprehensive range of lignocellulose enzymes with higher enzyme activity. It exhibits particularly high efficiency in degrading sugarcane bagasse lignocellulose.

[0007] A strain of Staphylococcus warwick, classified and named as: Staphylococcus warwick ( Staphylococcus warneri The strain name is LH-403, accession number: CGMCC No.36214, depositary institution: China General Microbiological Culture Collection Center; deposit date: October 9, 2025.

[0008] The application of *Staphylococcus wartii* in the preparation of a compound microbial agent that promotes the degradation of lignocellulose.

[0009] The application of *Staphylococcus wartii* in the preparation of cellulase and ligninase biological agents; the cellulase includes one or more of the following: exoglucosidase Exg, endoglucosidase Eng, β-glucosidase β-glucoside and filter paper enzyme FPA; the ligninase includes one or more of the following: laccase Lac, lignin peroxidase LiP and manganese peroxidase MnP.

[0010] The compound microbial agent is composed of Staphylococcus warwick. (Staphylococcus warneri) LH-403, Trichoderma longifolia ( Trichoderma longibrachiatum Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 preparation; The Staphylococcus wartii (Staphylococcus warneri) LH-403 was deposited at the China General Microbiological Culture Collection Center on October 9, 2025, with accession number CGMCC No. 36214; The long-branched Trichoderma ( Trichoderma longibrachiatum It is deposited at the China Industrial Microbial Culture Collection Center, accession number CICC 41185; The Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36215.

[0011] The preparation method of the compound microbial agent is as follows: Staphylococcus warwick... (Staphylococcus warneri) LH-403, Trichoderma longifolia ( Trichoderma longibrachiatum Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 was inoculated into liquid culture medium and cultured at 28 ℃, with the colony count adjusted to 1×10⁻⁶. 8 CFU / mL; mix equal volumes of each strain to obtain the compound bacterial agent.

[0012] The application of the aforementioned compound microbial agent in the degradation of sugarcane sugar production waste, such as bagasse, sugarcane leaves, and sugarcane filter mud.

[0013] The beneficial effects of this invention are:

[0014] 1. The strain LH-403 described in this invention not only possesses cellulose decomposition ability but also lignin decomposition ability, exhibiting high cellulase and ligninase activity, thus demonstrating highly efficient lignocellulose degradation characteristics. Based on these characteristics, it can be applied to lignocellulose degradation and can be combined with other microorganisms, especially fungi, to form compound inoculants, expanding the combination methods and selection range of lignocellulose-degrading strains. Because bacteria have a greater advantage in reproduction speed, they can rapidly multiply in large quantities during composting, accelerating the degradation process. Furthermore, a complex synergistic relationship exists between bacteria and fungi, utilizing the intermediate products of initial fungal degradation, thus forming a highly efficient degradation alliance, highlighting the advantages of bacterial-fungal compound inoculants.

[0015] 2. The Bacillus thuringiensis described in this invention (Bacillus thuringiensis) LMU-81 and Staphylococcus wartii (Staphylococcus warneri)LH-403 was collected from mangrove wetland soil in Fangchenggang City, Guangxi Province. Its advantages stem from the unique, harsh, and variable growth environment of mangroves. Mangroves contain an abundance of high-lignin, high-cellulose dead branches and leaves, and the environment is high-salt and oxygen-deficient. This ecosystem provides a special training environment for microorganisms, resulting in degradation activity and efficiency often exceeding that of microorganisms in terrestrial environments. Secondly, mangrove soil is frequently in an oxygen-deficient state due to periodic tidal inundation. Therefore, the microorganisms must be able to perform both aerobic respiration and anaerobic fermentation. This is particularly important for the traditionally believed strict aerobic degradation of lignocellulose, as the strain's sensitivity to oxygen levels is crucial to success in real-world industrialization projects. This is where the immense scientific and applied value of mangrove wetland microbial strains lies.

[0016] 3. This invention achieves a good synergistic effect by combining specifically screened microorganisms, resulting in a compound microbial agent exhibiting a strong ability to degrade lignocellulose. In this synergistic process, the microorganisms exhibit varying degrees of secretion of cellulase and ligninase; after compounding, the enzyme activity indicators of both ligninase and cellulase in the microbial agent reach high and balanced levels, making it particularly suitable for the degradation of lignocellulose in sugarcane bagasse.

[0017] 4. The composite microbial agent of this invention can adapt to a variety of natural lignocellulose materials, exhibiting broad substrate adaptability and stable degradation performance. This confirms its practical application capability in degrading natural lignocellulose and further demonstrates its potential application value in the field of biodegradation. Attached Figure Description

[0018] Figure 1 These are photographs of the colony morphology and phylogenetic tree of strain LH-403; among them Figure 1 (a) is a photograph of the colony morphology; Figure 1 (b) is a phylogenetic tree diagram; Figure 2 These are photographs of the colony morphology and phylogenetic tree of strain LMU-81; among them Figure 2 (a) Photograph of colony morphology; Figure 2 (b) is a phylogenetic tree diagram; Figure 3 Electrophoresis images of PCR products from strains LH-403 and LMU-81; Figure 4 Bar chart showing cellulase activity of single strains and compound inoculants; Figure 5 Bar graphs showing the ligninase activity of single strains and compound inoculants; Figure 6 Bar chart showing filter paper weight loss for single strains and compound bacterial agents; Figure 7Bar chart showing the degradation rate of lignocellulose in sugarcane bagasse for single strains and compound inoculants; Figure 8 A line graph showing the time trend of the rate of decomposition of sugarcane bagasse lignocellulose by compound microbial agents; Figure 9 Bar chart showing the degradation rate of lignocellulose biomass on different substrates by compound microbial agents; Figure 10 SEM image of the process of compound microbial agent degrading sugarcane bagasse; Figure 10 (a) Surface morphology of sugarcane bagasse that has not been degraded by compound microbial agents; Figure 10 (b) Internal morphology of sugarcane bagasse that has not been degraded by compound microbial agents; Figure 10 (c) Surface morphology of sugarcane bagasse after 48 hours of degradation; Figure 10 (d) Internal morphology of sugarcane bagasse after 48 hours of degradation; Figure 10 (e) Surface morphology of bagasse after 72 hours of degradation; Figure 10 (f) Internal morphology of sugarcane bagasse after 72 hours of degradation; Figure 10 (g) Surface morphology of bagasse after 168 h of degradation; Figure 10 (h) Internal morphology of sugarcane bagasse after 168 hours of degradation.

[0019] As shown in the uploaded microbial preservation certificate and microbial survival certificate, the strain preservation information is as follows:

[0020] Staphylococcus wartii (Staphylococcus warneri) LH-403 was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 36214; in the following examples, it is abbreviated as strain LH-403. Trichoderma longifolia ( Trichoderma longibrachiatum The sample, deposited at the China Industrial Microbial Culture Collection Center (CICC 41185), was purchased by the applicant from the China Industrial Microbial Culture Collection Center. In the following examples, it is abbreviated as Trichoderma longibranchii. Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 36215; and is referred to as strain LMU-81 in the following examples. Detailed Implementation

[0021] Preparation of culture media and solutions used in the examples Culture medium: LB liquid culture medium (g·L) -1): NaCl 10.00 g, tryptone 10.00 g, yeast extract 5.00 g, adjust pH to approximately 7.0.

[0022] LB solid medium (g·L) -1 ): LB liquid medium with 20.00 g agar.

[0023] PDB medium (g·L) -1 ): 200.00 g of potatoes, cut into chunks and boil for about 30 minutes, collect the filtrate and add 20.00 g of glucose.

[0024] PDA medium (g·L) -1 ): PDB medium with 20.00 g agar.

[0025] CMC-Na liquid medium: Same as CMC-Na solid medium, but without agar powder.

[0026] Alkali lignin solid culture medium: 5 g alkali lignin, 2 g (NH4)2SO4, 0.50 g MgSO4·7H2O, 1 g K2HPO4, 0.50 g NaCl, 20 g agar powder, add water to 1000 mL, sterilize at 121℃ for 20 min and set aside.

[0027] Lignin enzyme production medium (g·L) -1 ): Alkali lignin 2.00 g, K2HPO4 1.00 g, MgSO4·7H2O 0.50 g, NaCl 0.50 g, (NH4)2SO4 2.00 g, CaCl2 0.10 g, MnSO4 0.02 g, FeSO4 0.05 g.

[0028] Reagent solution: 50% Glycerin: 50 g glycerin, 50 g H2O, sterilize by moist heat at 121 ℃ for 20 min.

[0029] 0.10 M tartaric acid buffer (pH 3.00): Mix 0.10 M tartaric acid solution and 0.10 M sodium tartrate solution until the pH is 3.00.

[0030] 10 mM resveratrol: Weigh 1.682 g of resveratrol, dissolve it in distilled water, and bring the volume to 1 L.

[0031] 10 mM H2O2: Measure 1.02 mL of 30% H2O2 and add distilled water to bring the volume to 1 L.

[0032] 0.05 M Succinate Buffer (pH 4.50): Mix 0.05 M succinic acid solution and 0.05 M sodium succinate solution until the pH reaches 4.50. (0.05 M succinic acid solution: Weigh 0.59 g of succinic acid, dissolve in distilled water, and bring the volume to 100 mL; 0.05 M sodium succinate solution: Weigh 1.35 g of sodium succinate, dissolve in distilled water, and bring the volume to 100 mL).

[0033] 15 mM MnSO4: Weigh 2.535 g MnSO4, dissolve in distilled water, and bring the volume to 1 L.

[0034] 0.60 mM ABTS: Weigh 0.033 g ABTS, dissolve in distilled water, and bring the volume to 100 mL.

[0035] 0.05 M citric acid buffer solution (pH 5.00): Measure 20.50 mL of 0.10 M citric acid solution and 29.50 mL of 0.10 M sodium citrate solution, and add distilled water to a final volume of 100 mL.

[0036] Example 1

[0037] Screening of strains LH-403 and LMU-81 (1) Sample collection: Soil samples were collected from the mangrove wetland in Fangchenggang City.

[0038] (2) Enrichment: Take 10g of the sample and place it in a sterile conical flask containing sterile water and glass beads. Incubate in a shaker at 28 ℃ and 180 rpm for 1 h. Take 5 mL of the suspension and inoculate it into 100 mL of CMC-Na liquid medium and alkali lignin liquid medium, respectively. Incubate at 28 ℃ and 130 rpm for 3-5 days to enrich the sample.

[0039] (3) Separation and purification: Transfer 1 mL of the sample suspension to a test tube containing 9 mL of sterile water, then transfer 1 mL to another identical new test tube. Repeat this process stepwise to prepare 10 mL of the solution. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Sample solutions at different dilutions. Use a pipette to pipette 200 μL of each solution. -4 10 -5 and 10 -6Diluted solutions were spread onto CMC-Na and alkali lignin solid plates using a sterile spreader. The plates were allowed to stand at room temperature for 5-10 minutes to allow the bacterial culture to penetrate the medium. The solid plates were then inverted and incubated at 28 °C for 5 days. The culture was then isolated and purified on PDA solid medium.

[0040] Example 2

[0041] Molecular biological identification of strains LH-403 and LMU-81 Morphological and molecular identification of strain LH-403: as shown in the appendix. Figure 1 As shown in (a), the colonies of strain LH-403 grown on LB agar plates are round or oval, white and opaque, and relatively small. In Gram staining, the cells of LH-403 stain purple, indicating it is a Gram-positive bacterium. Microscopic observation reveals that the colonies are spherical and arranged in a grape-like pattern, a typical morphological characteristic of the Staphylococcus family.

[0042] After strain LH-403 was cultured in liquid medium, its genomic DNA was extracted and used as a template. The 16S rRNA gene of the strain was amplified using universal bacterial primers. The results of agarose gel electrophoresis of the PCR products are shown in the attached figure. Figure 3 As shown, specific bands of the expected size are displayed.

[0043] As attached Figure 1 As shown in (b), strain LH-403 is compared with Staphylococcus warwick in the phylogenetic tree. Staphylococcus warneri Because they are most closely related, LH-403 is classified as Staphylococcus warwick. Staphylococcus warneri It belongs to the Firmicutes phylum. (Firmicutes), Bacillus (Bacilli), Order Bacillales, Family Staphylococcaceae, Genus Staphylococcus ( Staphylococcus ).

[0044] Morphological and molecular identification of strain LMU-81: as shown in the appendix Figure 2 As shown in (a), the colonies formed by strain LMU-81 on LB agar plates are round or oval with irregular edges, a characteristic consistent with the typical colony features of Bacillus thuringiensis. Under a microscope, the cells of LMU-81 are stained purple and rod-shaped, arranged in short chains, indicating that it is a Gram-positive bacterium.

[0045] After culturing strain LMU-81 in liquid medium, its genomic DNA was extracted and used as a template to amplify the strain's 16S rRNA gene using universal bacterial primers. The results of agarose gel electrophoresis of the PCR products are shown in the attached figure. Figure 3 As shown, specific bands of the expected size are displayed.

[0046] As attached Figure 2 As shown in (b), strain LMU-81 is compared with Bacillus thuringiensis (Bt) in the phylogenetic tree. Bacillus thuringiensis The most closely related species, LMU-81, is classified as Bacillus thuringiensis. Bacillus thuringiensis It belongs to the phylum Firmicutes, class Bacilli, order Bacillales, family Bacillaceae, and genus Bacillus. Bacillus ).

[0047] Example 3

[0048] Antagonism experiment: The culture medium used in this embodiment was prepared as follows: PDA culture medium (g·L) -1 Weigh out 200.00g of peeled potatoes, 20.00g of glucose, and 20.00g of agar. Cut the potatoes into chunks and boil them in water for about 30 minutes. Then filter the solution through gauze, collect the filtrate, and add glucose and agar to the filtrate. Stir with a glass rod until the glucose and agar are completely dissolved, then add ultrapure water to 1000mL and sterilize at 121℃ for 20 minutes.

[0049] A plate antagonism experiment was conducted on strains LH-403, LMU-81, and *Trichoderma longicornis*. The antagonism between strains LH-403 and LMU-81 was verified using a cross-streaking assay, observing the growth status of the strains at the cross-streaked areas. If the two strains did not contact each other or showed weak growth, it indicated antagonism between the strains. If they could contact each other and both grew well, it indicated no antagonism between the two strains.

[0050] To verify the antagonistic relationship between strain LH-403 and Trichoderma longifolia, and between strain LMU-81 and Trichoderma longifolia, strains LH-403 and LMU-81 can be mixed with solid culture medium and poured into plates. Then, a plug made of Trichoderma longifolia using a sterile punch can be inoculated into the bacterial plate. After culturing for 3-5 days, observe whether an inhibition zone is formed. If no inhibition zone is formed, it indicates that there is no antagonistic effect.

[0051] Analysis of the plate antagonism experiment results, as shown in Table 1, revealed that there was no antagonistic effect among the three strains during the culture process, and they can be used to construct a compound bacterial agent.

[0052]

[0053] In the table, "-" indicates no antagonism, "+" indicates slight antagonism, and "++" indicates antagonism.

[0054] Example 4

[0055] Preparation method of compound microbial agent: (1) Strains LH-403 and LMU-81 were cultured in LB liquid medium for 2 days at 28 ℃ and 180 rpm. The bacterial count in the liquid medium was determined by the serial dilution plate count method. The bacterial count was adjusted with sterile water to reach 1×10⁻⁶. 8 CFU / mL; (2) The activated Trichoderma longifolia was inoculated onto a PDA solid plate, and the fungal spores on the surface of the PDA plate were collected. The plate was then placed in PDB liquid medium and cultured at 28 ℃ and 180 rpm for 7 days. The spore concentration was measured using a hemocytometer and adjusted to 1×10⁻⁶ with sterile water. 8 CFU / mL concentration; (3) Mix strain LH-403, strain LMU-81 and Trichoderma longifolia in equal proportions to obtain a compound microbial agent.

[0056] Example 5

[0057] Lignocellulase Activity Assay (1) Preparation of crude enzyme solution Strains LH-403, LMU-81, and Trichoderma longifolia were cultured into bacterial suspensions and inoculated into enzyme-producing fermentation medium at a volume ratio of 5%. The suspensions were cultured at 28 ℃ and 180 rpm for 5 days with shaking. 5 mL of the fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant obtained was the crude enzyme solution.

[0058] (2) Plot the glucose standard curve Using a pipette, pipette 0.50 mL of the glucose standard series solution into 25 mL test tubes. Then, add 1.50 mL of 0.05 M pH 5.00 citrate buffer to each tube. For the control group, add 2 mL of the same concentration of citrate buffer. Finally, add 3 mL of DNS reagent to all tubes and mix thoroughly. Perform triplicate for each sample and control tube. Place the tubes in a boiling water bath for 10 minutes, then immediately remove and cool. Adjust the volume to 25 mL with distilled water and mix again. Using a blank tube as a control, measure the absorbance at 540 nm. Plot a standard curve with glucose concentration as the X-axis and absorbance as the Y-axis, and calculate the linear regression equation.

[0059] (3) Cellulase activity assay Eng Activity Assay: The amount of reducing sugar released during hydrolysis was determined by the DNS colorimetric method, and the cellulase activity was calculated. 1 mL of 1% carboxymethyl cellulose solution was used as the substrate, and 0.50 mL of enzyme solution was added. The mixture was vortexed and incubated at 50°C for 30 min to complete the enzyme reaction. After the reaction was complete, 3 mL of DNS colorimetric reagent was immediately added to the mixture to stop the enzyme reaction. The treated sample was heated in a boiling water bath for 10 min and then cooled in water to maintain color stability. Next, an inactivated enzyme solution was used as a blank control, and the absorbance was measured at 540 nm using a UV spectrophotometer. Each experimental sample was tested in triplicate. Cellulase activity is defined as the amount of enzyme required to release 1 µmol of glucose by hydrolyzing the corresponding substrate in 1 min at 50°C, expressed as U / mL. Enzyme activity is calculated as shown in Formula 1:

[0060] Where G is the glucose content (mg) determined by the standard curve; V is the total reaction volume (mL); T is the reaction time (min); and v is the enzyme volume (mL).

[0061] Exg activity assay: Using 1 mL of 1% microcrystalline cellulose as substrate, add 0.50 mL of enzyme solution, vortex to mix thoroughly, incubate the mixture at 50 ℃ for 30 min, remove the test tube, and immediately add 3 mL of DNS chromogenic reagent to stop the enzyme reaction. Heat in a boiling water bath for 10 min, cool in water to maintain color stability, use inactivated enzyme solution as a blank, and measure the absorbance at 540 nm using a UV spectrophotometer. Each experimental sample is performed in triplicate, and enzyme activity is calculated as shown in Formula 1.

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

[0063] Filter paper activity (FPA) assay: Using 50 mg of starch-free filter paper as substrate, add 0.50 mL of enzyme solution and 1 mL of 0.05 M pH 5.00 citrate buffer, vortex to mix thoroughly, and incubate the mixture at 50℃ for 30 min. Remove the test tube and immediately add 3 mL of DNS chromogenic reagent to stop the enzyme reaction. Heat in a boiling water bath for 10 min, then cool in water to maintain color stability. Use inactivated enzyme solution as a blank. Measure the absorbance at 540 nm using a UV spectrophotometer. Perform three replicates for each sample. Calculate enzyme activity as shown in Formula 1.

[0064] (4) Ligninase activity assay LiP activity assay: 1.50 mL of 0.10 M tartaric acid buffer, 1 mL of 10 mM resveratrol, 0.40 mL of crude enzyme solution were added sequentially, followed by 0.10 mL of 10 mM H₂O₂. The reaction was initiated at 30 °C. The absorbance change of the reaction solution at 310 nm was measured within the first 3 minutes. One unit of enzyme activity is defined as the amount of enzyme required to produce 1 μmol of veratraldehyde per minute by oxidizing resveratrol.

[0065] MnP activity assay: MnP can convert Mn 2+ Oxidized to Mn 3+ 2 mL of 0.05 M succinate buffer, 0.50 mL of 15 mM MnSO4, and 0.4 mL of crude enzyme solution were added sequentially, and finally 0.10 mL of 10 mM H2O2 was added to start the reaction at 30 °C. The absorbance change of the reaction solution at 240 nm wavelength was detected within the first 3 minutes. One unit of enzyme activity is defined as the oxidation of 1 μmol Mn per minute. 2+ The required amount of enzyme.

[0066] Lac activity assay: 0.50 mL of 0.60 mM ABTS, 2 mL of 0.05 M citrate buffer, and finally 1 mL of crude enzyme solution were added sequentially. The reaction was started at 25 °C, and the absorbance change of the reaction solution at 420 nm wavelength was detected within the first 3 minutes. One unit of enzyme activity is defined as the amount of enzyme required to catalyze 1 μmol of ABTS per minute.

[0067] The above calculations of LiP, MnP, and Lac enzyme activities are given by Formula 2: Where ΔA is the absorbance change; ε is the molar absorptivity (mol) -1 ·L·cm -1 ); d is the optical path length of the cuvette (cm); V is the total reaction volume (mL); v is the crude enzyme solution volume (mL); T is the reaction time (min).

[0068] Conclusion: As attached Figure 4-5 As shown, the activity of the compound microbial agent was determined to be: exoglucase (Exg) 15.67 U / mL, endoglucase (Eng) 15.23 U / mL, β-glucosidase (β-glu) 13.27 U / mL, filter paper enzyme (FPA) 13.71 U / mL; laccase (Lac) 13.22 U / mL, lignin peroxidase (LiP) 25.20 U / mL, and manganese peroxidase (MnP) 16.45 U / mL.

[0069] The levels of exoglucanase Exg in strain LH-403 were determined to be 8.71 U / mL, endoglucanase Eng 13.91 U / mL, β-glucosidase β-glu 4.87 U / mL, filter paper enzyme FPA 7.80 U / mL, laccase Lac 2.05 U / mL, lignin peroxidase LiP 3.85 U / mL, and manganese peroxidase MnP 5.41 U / mL.

[0070] The exonuclease Exg of strain LMU-81 was determined to be 12.42 U / mL, the endonuclease Eng was 3.52 U / mL, the β-glucosidase β-glu was 1.41 U / mL, the filter paper enzyme FPA was 8.07 U / mL, the laccase Lac was 11.59 U / mL, the lignin peroxidase LiP was 22.65 U / mL, and the manganese peroxidase MnP was 6.62 U / mL.

[0071] The levels of exoglucanase (Exg) in *Trichoderma longicornis* were determined to be 1.06 U / mL, endoglucanase (Eng) 6.09 U / mL, β-glucosidase (β-glu) 12.01 U / mL, filter paper enzyme (FPA) 9.07 U / mL; laccase (Lac) 3.46 U / mL, lignin peroxidase (LiP) 12.67 U / mL, and manganese peroxidase (MnP) 14.30 U / mL.

[0072] Example 6

[0073] Determination of filter paper weight loss rate for single bacterial strains and compound bacterial agents: Strains LH-403, LMU-81, *Trichoderma longifolia*, and a compound microbial agent were inoculated into filter paper strip culture medium, and three parallel experiments were performed. Three quantitative filter paper strips (50 mg each, 6 cm × 1 cm in size) were placed in each culture bottle and incubated at 28℃ for 10-15 days, gently shaken every 6 hours. A filter paper strip culture medium without the strains was used as a control. The disintegration of the filter paper strips was observed periodically. The degree of disintegration was used to determine the strains' ability to degrade cellulose. The relative weight loss rate of the filter paper was determined using the weight loss method, calculated as follows: Filter paper weight loss rate (%) = [(Filter paper weight - Residual filter paper weight) / Filter paper weight] × 100%. The compound microbial agent was then incubated in the filter paper strip culture medium for 96 hours. (See attached...) Figure 6 As shown, the filter paper weight loss rate of the compound microbial agent was 60.3%; the filter paper weight loss rate of strain LH-403 was 42.6%; the filter paper weight loss rate of strain LMU-81 was 47.7%; and the filter paper weight loss rate of Trichoderma longifolia was 49.0%.

[0074] Example 7

[0075] Determination of sugarcane bagasse lignocellulose degradation rate by single strains and compound inoculants: The sugarcane bagasse used for the determination of lignocellulose degradation rate was taken from a sugar factory in Long'an County, Guangxi. It was dried to constant weight, and the dried sample was crushed using a pulverizing device. Finally, it was passed through a 100-mesh sieve to ensure uniform particle size.

[0076] Strains LH-403, LMU-81, *Trichoderma longifolia*, and a compound inoculum were inoculated at 10% each into 100 mL of lignocellulose degradation liquid medium and cultured in a constant-temperature shaker at 180 rpm and 28°C. After culture, a mixture of dilute hydrochloric acid and dilute nitric acid was added to the fermentation broth to remove residual cells. The broth was then repeatedly rinsed with distilled water. The culture was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the precipitate was washed several times with a small amount of distilled water to remove degradation residues until the effluent was clear. The effluent was then dried in a drying oven at 105°C to constant weight. The changes in cellulose, hemicellulose, and lignin content were calculated by measuring the contents of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), and ash in the degradation residues during the washing process. Cellulose content was calculated as the difference between ADF and ADL, hemicellulose content as the difference between NDF and ADF, and lignin content as the difference between ADL and Ash. A lignocellulose degradation liquid culture medium without added microorganisms was used as a control. The relative degradation rate of lignocellulose was determined, and the lignocellulose degradation rate was calculated using the following formula: Lignocellulose degradation rate (%) = [(bagasse weight - residual bagasse weight) / bagasse weight] × 100% Fermentation was conducted using sugarcane bagasse as the sole carbon source to evaluate the ability of a compound microbial agent to degrade lignocellulose. After 7 days of cultivation, the lignocellulose degradation rates of single-strain and compound microbial agents are shown in the attached figure. Figure 7 As shown, the degradation rate of the compound microbial agent reached 49.3%; the degradation rate of strain LMU-81 was 44.1%; the degradation rate of strain LH-403 was 36.2%; and the degradation rate of Trichoderma longicornis was 38.6%. The degradation rate of lignocellulose by the compound microbial agent is shown in the attached figure. Figure 8 As shown, the development accelerates over time.

[0077] Example 8

[0078] Degradation experiments with different substrates The grass used in the different substrate degradation experiments came from the lawn of Guangxi University, while the rice straw and corn stalks came from the Guangxi Academy of Agricultural Sciences.

[0079] Natural lignocellulose is more complex than the simple substrates used in selective culture media; it is a mixture of cellulose, hemicellulose, and lignin, typically existing in varying proportions and physical arrangements. Therefore, to accurately assess the stability of the compound microbial agent in degrading natural lignocellulose materials, this experiment selected green grass, rice straw, and corn stalks as substrates and conducted a systematic determination of their degradation efficiency. (See attached...) Figure 9 As shown, under the same experimental conditions as in Example 7, the compound microbial agent was cultured on these three different substrates for 168 h. The degradation rates of different lignocellulose sources were all above 40%, with the highest degradation rate reaching 51.4% when green grass was used as the substrate. The degradation rates of rice straw and corn straw were 43.6% and 46.4%, respectively. This demonstrates that the compound microbial agent can effectively degrade natural lignocellulose materials from different sources.

[0080] Example 9

[0081] Scanning electron microscopy (SEM) observation of compound microbial agents before and after degradation of sugarcane bagasse lignocellulose. SEM was used to observe the changes in surface morphology of lignocellulose at different degradation stages more realistically. The composite microorganisms were cultured in lignocellulose degradation liquid medium. The untreated control group and the composite microorganism-treated experimental group were respectively placed in 2 mL centrifuge tubes, centrifuged at 12000 rpm for 5 min with 20 × PBS buffer, the supernatant was discarded, and the washing was repeated 3 times. 2.50% glutaraldehyde was added, and the samples were fixed at 4℃ for 4 h, followed by repeated washing with 20 × PBS buffer 3 times. Next, the samples were dehydrated with different concentration gradients of ethanol (30%, 50%, 70%, 85%, 90%, 100%), then replaced with tert-butanol. The treated samples were frozen at -20℃ for 12 h, and then freeze-dried for 36 h. The samples were fixed to the sample stage using conductive tape and then placed in the sample chamber. The samples were then sputter-coated with gold in a vacuum to reduce the influence of charge, and observed and analyzed using a scanning electron microscope.

[0082] SEM was used to further observe the morphological changes of lignocellulose in sugarcane bagasse before and after degradation by the compound microbial agent. (See attached image.) Figure 10 As shown, sugarcane bagasse that has not undergone microbial fermentation treatment serves as the control group. The sugarcane bagasse in the control group has a smooth and flat surface, a highly dense structure, and its internal morphology is almost invisible (e.g., Figure 10 (as shown in (a)), the lignocellulose structure remains relatively intact at this stage. Lateral observation reveals tightly packed and orderly arranged fiber bundles, tightly bound together (as shown in (a)). Figure 10(b) shows the compactness and uniformity of the untreated bagasse structure. After fermentation with compound microbial agents for 48 hours, the structure of the bagasse was significantly disrupted, the surface became uneven, and numerous pores of varying sizes appeared (e.g., ...). Figure 10 (c) This may be because the compound microbial agent acts on the surface of sugarcane bagasse, causing the surface fiber fragments to be peeled off, thus gradually revealing the originally wrapped fiber bundles (as shown in the image). Figure 10 (d) As shown. After 72 hours of enzymatic hydrolysis, the surface structure was rough and irregular, exhibiting grooves of varying degrees, completely exposing the fiber bundle structure inside the sugarcane bagasse (as shown in the image). Figure 10 (as shown in (ef)). This obvious structural change is likely due to the large amount of enzymes secreted by the compound microbial agent acting on the lignocellulose network, leading to its further decomposition. After fermentation continued for 168 hours, the bagasse was further degraded, with a highly porous surface and striped grooves. The internal structure was severely damaged, and the originally bundled vascular bundles became completely exposed, with the fibers separating from each other (as shown in (ef)). Figure 10 (as shown in (gh)). The results indicate that the compound microbial agent can efficiently destroy the lignocellulose structure in sugarcane bagasse in a short time.

Claims

1. A strain of Staphylococcus warwick, characterized in that, The strain was classified and named as: Staphylococcus warwick ( Staphylococcus warneri The strain name is LH-403, the accession number is CGMCC No.36214, the depositary institution is China General Microbiological Culture Collection Center, and the deposit date is October 9, 2025.

2. The application of Staphylococcus warwick as described in claim 1 in the preparation of a compound microbial agent that promotes the degradation of lignocellulose.

3. The use of Staphylococcus warwick as described in claim 1 in the preparation of cellulase and ligninase-producing biological agents; the cellulase comprises: One or more of the following: exoglucosidase Exg, endoglucosidase Eng, β-glucosidase β-glu, and filter paper enzyme FPA; ligninases include one or more of the following: laccase Lac, lignin peroxidase LiP, and manganese peroxidase MnP.

4. A compound microbial agent, characterized in that: The compound microbial agent is composed of Staphylococcus warwick. (Staphylococcus warneri) LH-403, Trichoderma longifolia ( Trichoderma longibrachiatum Bacillus thuringiensis (Bacillus thuringiensis LMU-81 preparation; The Staphylococcus wartii (Staphylococcus warneri) LH-403 was deposited at the China General Microbiological Culture Collection Center on October 9, 2025, with accession number CGMCC No. 36214; The long-branched Trichoderma ( Trichoderma longibrachiatum It is deposited at the China Industrial Microbial Culture Collection Center, accession number CICC 41185; The Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36215.

5. The compound microbial agent as described in claim 4, characterized in that: The preparation method of the compound microbial agent is as follows: Staphylococcus warwick... (Staphylococcus warneri) LH-403, Trichoderma longifolia ( Trichoderma longibrachiatum Bacillus thuringiensis (Bacillus thuringiensis) LMU-81 was inoculated into liquid culture medium and cultured at 28 ℃, with the colony count adjusted to 1×10⁻⁶. 8 CFU / mL; mix equal volumes of each strain to obtain the compound bacterial agent.

6. The application of the compound microbial agent as described in claim 4 in the degradation of sugarcane sugar production waste.

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