A strain of streptomyces carbonophilus and a complex microbial agent, a preparation method and applications thereof

Abstract

This paper describes a strain of *Streptomyces thermophilus* and its compound inoculum, its preparation method, and its application. It belongs to the field of microbial technology. The strain is classified and named as *Streptomyces thermophilus*. Streptomyces thermocarboxydus The strain, named G-4-7, with accession number CGMCC No. 36417, was used in combination with *Bacillus brevis* (…). Brevibacillus agri G-5-2, thermophilic Chaetomium ( Chaetomium thermophilum A highly efficient lignin-degrading compound microbial agent can be prepared. The compound microbial agent can efficiently degrade sugarcane bagasse lignin in a high-temperature environment. The enzyme systems of each strain complement each other, and the degradation efficiency is synergistically improved. This can promote the resource utilization of lignin-rich sugarcane bagasse and provide a technical basis for the green treatment of agricultural waste.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically a thermophilic carbon monoxide-loving Streptomyces and its compound inoculant, preparation method and application. Background Technology

[0002] Economic development and population growth have driven the expansion of agricultural production, resulting in a large amount of agricultural waste. Sugarcane is the world's most widely cultivated sugar crop and also its most important energy and feed crop, possessing high ecological and economic value. Following Brazil and India, China is the world's third-largest sugarcane producer. Guangxi is my country's largest sugarcane-growing region, averaging 50 million tons of sugarcane produced in recent years, with each ton of sugarcane producing 280 kilograms of bagasse after processing. Globally, approximately 181 million tons of bagasse are produced annually, its main component being lignin cellulose. Most of this bagasse is discarded or incinerated. If these agricultural wastes are not properly disposed of, they will cause resource waste and environmental pollution.

[0003] Due to the recalcitrant nature of lignocellulose, especially its lignin component, aerobic composting of solid waste rich in lignocellulose often exhibits low degradation efficiency. To promote lignocellulose degradation and shorten the composting process, the addition of exogenous microbial agents is commonly used. The high-temperature period of composting is a crucial stage for the biological decomposition of organic matter and ensuring the harmlessness of the compost; however, microorganisms are often affected by high temperatures during this stage, resulting in a general decrease in population size and activity, and a short residence time in the high-temperature period, thus limiting the rapid decomposition of organic matter.

[0004] thermophilic carbon monoxide streptomyces ( Streptomyces thermocarboxydus It belongs to the phylum Actinobacteria, family Streptomycetes. It is a thermophilic bacterium, with an optimal growth temperature typically between 50-65℃. Its internal enzymes and metabolic systems possess natural thermal stability. This makes it an ideal candidate for developing biomass conversion technologies and realizing the resource utilization of waste, providing new solutions for a sustainable circular economy and biomanufacturing. Existing literature includes, for example: 1. Chinese Patent, A novel thermophilic carbon monoxide-loving Streptomyces strain and its application; Publication No.: CN103484396A; the novel strain CF1 can rapidly increase the temperature of the fermentation pile during the cold season in northern regions and maintain the pile at a high temperature above 50℃ for 15 days, shortening the fermentation cycle; 2. Chinese Patent, A thermophilic carbon monoxide-loving low-temperature subspecies of Streptomyces Dstr3-3 and its application; Publication No.: CN104988095A; the strain Dstr3-3 produces lignin peroxidase and manganese peroxidase, but not laccase. 3. Chinese Patent, A thermophilic garden waste decomposing bacterium ST3 and its application; Publication No.: CN105567607A; The thermophilic carbon monoxide-loving Streptomyces ( Streptomyces thermocarboxydusST3 is a cellulose-degrading microbial strain screened from garden waste compost. It can produce a large amount of cellulase within a temperature range of 40-70℃, with an enzyme activity as high as 30.05 U / mL. 4. Chinese Patent, A method for producing cellulase using *Streptomyces comatus* and a liquid fermentation medium; Publication No.: CN106191012A; *Streptomyces comatus* produces cellulase at a concentration of 0.80 IU / mL. Although there are some existing records of *Streptomyces comatus* secreting lignocellulase, its specific enzyme activity for degrading lignin has not been analyzed and studied in detail.

[0005] On the other hand, although utilizing complex microbial systems to synergistically degrade lignocellulose is considered a more effective strategy than using single strains, existing technologies have significant shortcomings. First, many microbial agents are not specifically optimized for the component characteristics of sugarcane bagasse, exhibiting strong generality but poor specificity. Second, conventional complex microbial agents lack heat resistance and cannot maintain their effectiveness during the high-temperature fermentation period. Furthermore, existing artificial complex methods often involve simple combinations of strains, failing to fully consider the metabolic complementarity and synergistic stability of strains on sugarcane bagasse, a specific substrate, under high-temperature conditions. Therefore, isolating and screening natural strains with heat resistance from specific environments, and constructing a complex microbial community capable of specifically and efficiently synergistically degrading lignocellulose in sugarcane bagasse, is of urgent practical significance and enormous application value for solving current technical challenges in the resource utilization of sugarcane bagasse and improving the high-value utilization level of agricultural waste.

[0006] Sugarcane bagasse contains a high lignin content of 20-32%, of which phenolic lignin (guaiacol and syringyl) accounts for 70-85%. Phenolic lignin contains a large number of free hydroxyl groups, which are the core targets of degrading enzymes such as manganese peroxidase (MnP) and lignin peroxidase (LiP). Therefore, screening and constructing composite microbial agents capable of efficiently degrading lignin cellulose under high-temperature conditions has become a key challenge for improving the degradation efficiency of sugarcane bagasse. Summary of the Invention

[0007] This invention addresses the problem of low lignin degradation efficiency in sugarcane bagasse by providing a thermophilic carbon monoxide-loving *Streptomyces* strain, its compound inoculum, preparation method, and application. This thermophilic carbon monoxide-loving *Streptomyces* strain is combined with other lignin-degrading strains to obtain a more efficient compound inoculum. The enzyme systems of the various strains in the compound inoculum are complementary, resulting in a synergistic improvement in degradation efficiency and producing a more comprehensive range of lignin-degrading enzymes with higher enzyme activity. The technical solution of this invention is as follows:

[0008] A strain of *Streptomyces thermophilus*, classified and named as: *Streptomyces thermophilus* (… Streptomyces thermocarboxydusThe strain name is G-4-7, the accession number is CGMCC No.36417, the depositary institution is the China General Microbiological Culture Collection Center, and the deposit date is October 30, 2025.

[0009] The application of the thermophilic carbon monoxide-loving Streptomyces in the preparation of a compound microbial agent that promotes lignin degradation.

[0010] The application of the thermophilic carbon monoxide-loving Streptomyces in the preparation of lignin-producing biological agents, wherein the lignin-producing enzymes include one or more of laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP).

[0011] A compound microbial agent, the compound microbial agent being composed of thermophilic carbon monoxide-loving Streptomyces ( Streptomyces thermocarboxydus G-4-7, Soil-borne Bacillus ( Brevibacillus agri G-5-2 and thermophilic Chaetomium ( Chaetomium thermophilum )preparation;

[0012] The thermophilic carbon monoxide streptomyces ( Streptomyces thermocarboxydus G-4-7 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 30, 2025, with accession number CGMCC No. 36417.

[0013] The soil short-spore bacteria ( Brevibacillus agri G-5-2 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 30, 2025, with accession number CGMCC No. 36418.

[0014] The thermophilic Chaetomium ( Chaetomium thermophilum The sample is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC 3.17990.

[0015] The preparation method of compound microbial agent is as follows: Thermophilic streptomyces (… Streptomyces thermocarboxydus G-4-7, Soil-borne Bacillus ( Brevibacillus agri G-5-2 and thermophilic Chaetomium ( Chaetomium thermophilum Inoculate separately into liquid culture medium and incubate at 50°C, adjusting the colony count to 1×10⁻⁶. 8 CFU / mL; mix equal volumes of each strain to obtain the compound bacterial agent.

[0016] 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.

[0017] The beneficial effects of this invention are:

[0018] 1. Compared with existing strains, especially existing thermophilic streptomyces strains, strain G-4-7 has a stronger secretion capacity of LiP enzyme and MnP enzyme for phenolic structural lignin in sugarcane bagasse, and has particular potential for application in the rapid degradation of sugarcane bagasse lignin during high-temperature composting.

[0019] 2. The compound microbial agent prepared by this invention is prepared from three strains. There is no antagonism between the strains and their enzyme systems are complementary, which synergistically improves the degradation efficiency. They can secrete a large amount of ligninase, especially lignin peroxidase LiP and manganese peroxidase MnP, which are particularly suitable for degrading natural lignin in sugarcane bagasse with phenolic structure. The compound microbial agent achieves a relative degradation rate of 55.6% for sugarcane bagasse lignocellulose. Attached Figure Description

[0020] Figure 1 The diagrams show the aniline blue-LB bleaching test results for strains G-4-7 and G-5-2; Figure 1 A is a diagram of the aniline blue-LB fading test for strain G-4-7; Figure 1 B is a diagram of the aniline blue-LB fading test for strain G-5-2;

[0021] Figure 2 These are images showing the colony morphology of strain G-4-7 from both sides. Figure 2 (a) shows the frontal colony morphology. Figure 2 (b) shows the colony morphology on the reverse side;

[0022] Figure 3 These are images showing the colony morphology of strain G-5-2 from both sides. Figure 3 (a) shows the frontal colony morphology. Figure 3 (b) shows the colony morphology on the reverse side;

[0023] Figure 4 Electrophoresis images of PCR products from strains G-4-7 and G-5-2;

[0024] Figure 5 Phylogenetic tree diagrams of strains G-4-7 and G-5-2; Figure 5 A is the phylogenetic tree diagram of strain G-4-7; Figure 5 B is the phylogenetic tree diagram of strain G-5-2;

[0025] Figure 6 Bar chart showing the ligninase activity of strain G-4-7, strain G-5-2, thermophilic Chaetomium, and compound inoculum;

[0026] Figure 7 Bar chart showing the degradation rate of sugarcane bagasse lignocellulose by strain G-4-7, strain G-5-2, thermophilic Chaetomium, and compound microbial agent.

[0027] As shown in the uploaded microbial preservation certificate and microbial survival certificate, the strain preservation information is as follows:

[0028] thermophilic carbon monoxide streptomyces ( Streptomyces thermocarboxydus G-4-7 was deposited on October 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 36417; and is referred to as strain G-4-7 in the following examples.

[0029] Soil brevicorbacterium ( Brevibacillus agri G-5-2 was deposited on October 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 36418; and is referred to as strain G-5-2 in the following examples.

[0030] thermophilic chamomile ( Chaetomium thermophilum The specimen, with accession number CGMCC 3.17990, was purchased by the applicant from the China General Microbiological Culture Collection Center. In the following examples, it is abbreviated as *Chaetoceros thermophilus*. Detailed Implementation

[0031] Preparation of culture media and solutions used in the examples

[0032] 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.

[0033] LB solid medium (g·L) -1 ): LB liquid medium with 20.00 g agar.

[0034] 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.

[0035] PDA medium (g·L) -1 ): PDB medium with 20.00 g agar.

[0036] Initial screening liquid culture medium (g·L) -1): Alkali lignin 5.00 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.

[0037] Initial screening of solid culture medium (g·L) -1 ): Initial screening liquid culture medium with 20.00 g agar.

[0038] Aniline blue-LB medium (g·L) -1 ): LB medium with 0.10 g aniline blue.

[0039] 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.

[0040] Reagent solution:

[0041] 50% glycerol: 50g glycerol, 50g H2O, sterilize by moist heat at 121℃ for 20min.

[0042] 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.

[0043] 10 mM resveratrol: Weigh 1.682 g of resveratrol, dissolve it in distilled water, and bring the volume to 1 L.

[0044] 10 mM H2O2: Measure 1.02 mL of 30% H2O2 and add distilled water to bring the volume to 1 L.

[0045] 0.05 M succinate buffer (pH 4.50): Mix 0.05 M succinate solution and 0.05 M sodium succinate solution until the pH reaches 4.50. (0.05 M succinate solution: Weigh 0.59 g of succinate, dissolve in distilled water, and bring to a final volume of 100 mL; 0.05 M sodium succinate solution: Weigh 1.35 g of sodium succinate, dissolve in distilled water, and bring to a final volume of 100 mL).

[0046] 15 mM MnSO4: Weigh 2.535 g MnSO4, dissolve in distilled water, and bring the volume to 1 L.

[0047] 0.60 mM ABTS: Weigh 0.033 g ABTS, dissolve in distilled water, and bring the volume to 100 mL.

[0048] 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.

[0049] Example 1

[0050] The screening process for strains G-4-7 and G-5-2:

[0051] 1. Preparation of compost samples

[0052] Sugarcane leaves and sugarcane filter mud (wet weight) were thoroughly mixed at a weight ratio of approximately 1:1.5, adjusting the initial carbon-to-nitrogen ratio to around 25, with an initial moisture content of approximately 55% (wet weight). A composting experiment was conducted. The experimental materials were placed in a 60L plastic container with a breathable bottom, the container was wrapped with insulating film, and several holes were drilled in the bottom. The container was placed in a well-ventilated room to ensure insulation and uniform oxygen supply during the composting process. Temperature was measured daily. Once the compost reached its high-temperature phase (≥50℃), samples were taken using a five-point sampling method, mixed thoroughly, and temporarily stored in a laboratory freezer at -20℃ for subsequent microbial culture screening.

[0053] 2. Enrichment and Separation Purification

[0054] (1) Enrichment

[0055] Weigh 10g of high-temperature compost sample and add it to a 250mL Erlenmeyer flask containing 90mL of 0.9% sterile physiological saline. Place the flask in a shaker at 50℃ and 180rpm and let it stand. Then, take 5mL of the bacterial suspension and inoculate it into 100mL of primary screening liquid culture medium. Incubate the flask in a shaker at 50℃ and 130rpm for 1-5 days.

[0056] (2) Separation and purification

[0057] Under aseptic conditions, following a concentration gradient dilution method, 1 mL of the enriched bacterial suspension was added to a test tube containing 9 mL of sterile water and mixed thoroughly to obtain a concentration gradient of 10. -1 The bacterial culture was diluted sequentially using this method to obtain a concentration gradient of 10. -1 -10 -6 The bacterial culture dilution. Take 100 µL of each of the 10... -4 10 -5 and 10 -6The diluted bacterial culture solution was inoculated onto the surface of the initial screening solid culture medium and spread evenly. It was then placed in an inverted incubator at 50°C and observed periodically. After colony growth, single colonies were picked based on their morphological characteristics for streak isolation. This process was repeated several times until a pure strain was obtained.

[0058] (3) Preservation of microbial strains

[0059] The purified bacterial strains were inoculated into LB liquid medium and cultured in a shaker at 150 rpm and 50°C until the logarithmic growth phase. Under aseptic conditions, the bacterial suspension was mixed with an equal volume of sterile 50% glycerol in cryovials and stored at -20°C. The fungi were inoculated into PDA slant agar and stored at 4°C using the slant cryopreservation method.

[0060] Example 2

[0061] Secondary screening of strains G-4-7 and G-5-2

[0062] The purified strains were inoculated onto aniline blue-LB medium, with three replicates for each strain. The medium was then incubated at 50°C for 1–5 days. During this period, the changes in the bleaching zone on the medium were observed. The formation of a bleaching zone indicates that the strain has the ability to produce peroxidase. By measuring the diameter of the bleaching zone, the ability of the strain to produce MnP and LiP under high-temperature conditions can be preliminarily assessed.

[0063] As attached Figure 1 As shown in A, strain G-4-7: bleaching zone diameter (D / mm): 20.44 ± 1.75; colony diameter (d / mm): 5.77 ± 0.33; D / d: 3.54 ± 0.13.

[0064] As attached Figure 1 As shown in B, strain G-5-2: bleaching zone diameter (D / mm): 24.73 ± 1.20 mm, colony diameter (d / mm): 6.82 ± 0.24, D / d: 3.62 ± 0.06.

[0065] Example 3

[0066] Molecular biological identification of strains G-4-7 and G-5-2

[0067] Morphological and molecular identification of strain G-4-7: Appendix Figure 2As shown: Single colonies of strain G-4-7 on LB solid medium are small, round, and raised, with a dry, opaque, milky-white surface, exhibiting a dense, velvety texture. The bacterial cells are tightly bound to the medium and are not easily picked up. These colony characteristics are consistent with the general features of the *Streptomyces* genus. After Gram staining, strain G-4-7 appears purple and has slender, branched hyphae, classifying it as a Gram-positive bacterium.

[0068] After strain G-4-7 was cultured 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 4 As shown, specific bands of the expected size are displayed.

[0069] As attached Figure 5 As shown in Figure A: Through phylogenetic tree analysis, strain G-4-7 is related to *Streptomyces thermophilus* (…). Streptomyces thermocarboxydus The strain G-4-7 is most closely related to *Streptomyces thermophilus* and has been preliminarily identified as *Streptomyces*. Strain G-4-7 belongs to the phylum Actinomycetota, class Actinobacteridae, order Actinomycetales, family Streptomycetaceae, and genus *Streptomyces*. (Streptomyces) .

[0070] Morphological and molecular identification of strain G-5-2: as shown in the attached document. Figure 3 As shown: Single colonies formed by strain G-5-2 on LB solid medium are round, raised, milky white, with a moist and smooth surface, and are easily picked up. The colony characteristics are consistent with the general features of the genus Bacillus. After Gram staining, strain G-5-2 appears purple and short rod-shaped under a microscope, indicating it is a Gram-positive bacterium.

[0071] After strain G-5-2 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 4 As shown, specific bands of the expected size are displayed.

[0072] As attached Figure 5 As shown in B: Through phylogenetic tree analysis, strain G-5-2 is related to *Bacillus brevis*. (Brevibacillus agri) The strain G-5-2 is most closely related to the bacteria and has been preliminarily identified as *Bacillus spp.* Strain G-5-2 belongs to the phylum Firmicutes. (Bacillota), class Bacillia, order Brevibacillales, family Brevibacillaceae, genus Bacillota ( Brevibacillus ).

[0073] Example 4

[0074] Antagonism experiment between strains

[0075] The culture medium was prepared as follows: PDA medium (g·L) -1 Weigh out 200.00 g of peeled potatoes, 20.00 g of glucose, and 20.00 g 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 1000 mL and sterilize at 121°C for 20 minutes.

[0076] A plate antagonism experiment was conducted on *Chaetoceros thermophilus*, strain G-4-7, and strain G-5-2. The antagonism between strains G-4-7 and G-5-2 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.

[0077] To verify the antagonism between strain G-4-7 and *Chaetoceros thermophilus*, and between strain G-5-2 and *Chaetoceros thermophilus*, strains G-4-7 and G-5-2 can be mixed with solid culture medium and poured into plates. Then, *Chaetoceros thermophilus* can be plugged using a sterile punch and inoculated into the bacterial plates. 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.

[0078] 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.

[0079]

[0080] Note: "-" indicates no antagonistic effect, and "+" indicates antagonistic effect.

[0081] Example 5

[0082] The preparation method of compound microbial agents includes the following steps:

[0083] (1) Strains G-4-7 and G-5-2 were cultured in LB liquid medium for 2 days at 50℃ and 180 rpm. The bacterial count in the liquid medium was determined by viable plate counting method. The bacterial count was adjusted with sterile water to reach 1×10⁻⁶. 8 CFU / mL;

[0084] (2) The activated thermophilic Chaetomium was inoculated onto a PDA solid plate, and the fungal spores on the surface of the PDA plate were collected. The plate was then placed in PDB liquid medium and cultured at 50°C and 180 rpm for 7 days. The spore concentration was measured using a hemocytometer and adjusted to 1×10⁻⁶ with sterile water. 8 CFU / mL concentration;

[0085] (3) Mix strain G-4-7, strain G-5-2 and thermophilic Chaetomium in equal proportions to obtain a compound bacterial agent.

[0086] Example 6

[0087] Enzyme activity assay

[0088] (1) Preparation of crude enzyme solution

[0089] The thermophilic Chaetomium, strain G-4-7, strain G-5-2, and the compound bacterial agent were inoculated into lignin enzyme-producing medium and cultured at 50℃ and 180 rpm for 5 days. 5 mL of fermentation broth was centrifuged at 4℃ and 8000 rpm for 10 min, and the supernatant was the crude enzyme solution.

[0090] (2) Enzyme activity assay

[0091] LiP activity assay: 1.50 mL of 0.10 M tartaric acid buffer, 1 mL of 10 mM resveratrol, and 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 oxidize resveratrol to produce 1 μmol of veratraldehyde per minute.

[0092] MnP activity assay: MnP can convert Mn 2+ Oxidized to Mn 3+ Add 2 mL of 0.05 M succinate buffer, 0.50 mL of 15 mM MnSO4, and 0.4 mL of crude enzyme solution sequentially, and finally add 0.10 mL of 10 mM H2O2 to start the reaction at 30 °C. Detect the absorbance change of the reaction solution at 240 nm wavelength within the first 3 minutes. One unit of enzyme activity is defined as the oxidation of 1 μmol Mn per minute. 2+ The required amount of enzyme.

[0093] Lac activity assay: 0.50 mL of 0.60 mM ABTS, 2 mL of 0.05 M citrate buffer, and finally 1 mL of crude enzyme solution were added sequentially. The reaction was started at 25°C, and the absorbance change of the reaction solution at 420 nm wavelength was detected in the first 3 minutes. One unit of enzyme activity is defined as the amount of enzyme required to catalyze 1 μmol of ABTS per minute.

[0094] The formulas for calculating the activities of LiP, MnP, and Lac enzymes are as follows: 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). The results are attached. Figure 6 As shown.

[0095] The activity of laccase (Lac) in the compound microbial agent was determined to be 9.42 U / mL; the activity of lignin peroxidase (LiP) was 22.70 U / mL; and the activity of manganese peroxidase (MnP) was 19.50 U / mL.

[0096] The activity of laccase (Lac) in strain G-4-7 was determined to be 3.48 U / mL; the activity of lignin peroxidase (LiP) was 19.54 U / mL; and the activity of manganese peroxidase (MnP) was 17.25 U / mL.

[0097] The activity of laccase (Lac) in strain G-5-2 was determined to be 0.33 U / mL; the activity of lignin peroxidase (LiP) was 15.77 U / mL; and the activity of manganese peroxidase (MnP) was 8.85 U / mL.

[0098] The activity of laccase (Lac) in *Chaetomium thermophilum* was determined to be 8.85 U / mL; the activity of lignin peroxidase (LiP) was 3.52 U / mL; and the activity of manganese peroxidase (MnP) was 2.44 U / mL.

[0099] Example 7

[0100] Determination of Lignocellulose Degradation Rate in Sugarcane Bagasse

[0101] 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.

[0102] To demonstrate and verify the practical application effect of the compound microbial agent in degrading lignocellulose, thermophilic Chaetomium, strain G-4-7, strain G-5-2, and the compound microbial agent were inoculated at a 5% inoculum into a liquid culture medium with sugarcane bagasse as the sole carbon source for sugarcane bagasse liquid fermentation experiments. Each experiment was repeated in triplicate, with a control medium without inoculum. After culturing at 50 ℃ in a constant temperature shaker for 7 days, the culture was centrifuged, and the supernatant was discarded. The residue was repeatedly washed with a mixture of dilute nitric acid and dilute hydrochloric acid, followed by multiple washes with distilled water to elute the bacterial cells. Finally, the precipitate was dried to constant weight. The relative degradation rate of lignocellulose was calculated using the weight loss method: Lignocellulose degradation rate (%) = [(weight of sugarcane bagasse - weight of residual sugarcane bagasse) / weight of sugarcane bagasse] × 100%. The experimental results are attached. Figure 7 As shown.

[0103] The results showed that the sugarcane bagasse degradation rate of strain G-4-7 was 47.7%; that of strain G-5-2 was 39.2%; and that of *Chaetoceros thermophilus* was 33.40%. Compared with single-strain agents, the compound-strain agent significantly improved the sugarcane bagasse degradation rate, reaching 55.6%.

Claims

1. A compound microbial agent, characterized in that, The compound microbial agent is composed of thermophilic streptomyces (Streptomyces thermophilus). Streptomyces thermocarboxydus G-4-7, Soil-borne Bacillus ( Brevibacillus agri G-5-2 and thermophilic Chaetomium ( Chaetomium thermophilum )preparation; The thermophilic carbon monoxide streptomyces ( Streptomyces thermocarboxydus G-4-7 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 30, 2025, with accession number CGMCC No. 36417. The soil short-spore bacteria ( Brevibacillus agri G-5-2 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 30, 2025, with accession number CGMCC No. 36418. The thermophilic Chaetomium ( Chaetomium thermophilum The sample is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC 3.17990.

2. The compound microbial agent according to claim 1, characterized in that, The preparation method of compound microbial agent is as follows: Thermophilic streptomyces (… Streptomyces thermocarboxydus G-4-7, Soil-borne Bacillus ( Brevibacillus agri G-5-2 and thermophilic Chaetomium ( Chaetomium thermophilum Inoculate separately into liquid culture medium and incubate at 50°C, adjusting the colony count to 1×10⁻⁶. 8 CFU / mL; mix equal volumes of each strain to obtain the compound bacterial agent.

3. The application of the compound microbial agent as described in claim 1 in the degradation of sugarcane sugar production waste.

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