A composite microbial agent, a preparation method thereof and application thereof in degrading wheat straw

Abstract

The application discloses a composite microbial agent, which comprises a composite microbial strain consisting of strains B5, D3 and WT5. The composite microbial agent further comprises a nutrient substrate. The application also discloses a preparation method thereof and application in degrading wheat straw. The composite microbial agent can degrade wheat straw in a drought and little rain field environment, and the degradation rate of the composite microbial agent to the wheat straw reaches 70.1% in 60 days, which is increased by 164.5% compared with a control group without adding the microbial agent. The composite microbial agent is simple to prepare and environmentally friendly to use, and has important significance for realizing resource utilization of wheat straw in a drought area.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a compound microbial agent, its preparation method, and its application in the degradation of wheat straw. Background Technology

[0002] Straw is a biomass resource rich in cellulose, hemicellulose, lignin, and various nutrients such as nitrogen, phosphorus, potassium, calcium, and magnesium, and has always been a valuable resource in agricultural production. As a core by-product of agriculture, straw has a wide range of uses, and its rational utilization brings many benefits: In the agricultural sector, it is a high-quality organic fertilizer that, when returned to the field, can improve soil structure, increase soil organic matter, and enhance soil fertility, laying the foundation for crop growth. It can also be processed into livestock feed to meet the nutritional needs of livestock and reduce breeding costs. In the energy sector, straw can be converted into biomass energy for power generation and heating, replacing some fossil fuels and reducing energy consumption and environmental pollution. From a comprehensive perspective, the rational utilization of straw can prevent air pollution caused by burning, reduce smog, and protect the ecology. Economically, it can drive the development of the straw processing industry, opening up income channels for farmers and contributing to rural economic growth. In terms of resources, it achieves recycling, improves resource utilization, promotes green and sustainable agricultural development, and facilitates the coordinated progress of rural ecological revitalization and high-quality economic development.

[0003] In recent years, my country's straw resource surplus has continued to increase, and the resources are abundant, mainly consisting of rice straw, wheat straw, corn straw, and soybean straw. However, the current utilization rate of straw is very low, and the large-scale unused straw directly leads to the waste of land resources and pollution of the planting environment. How to maximize the utilization of straw resources has become a key issue of concern.

[0004] North China, a major grain-producing region in my country, has a massive output of straw resources. In terms of straw return to the field, North China ranks first among all regions in the country in terms of direct straw return rate. However, at the same time, the large-scale straw returned to the field in North China faces the challenge of difficult degradation. On the one hand, the region is prone to drought and low rainfall in spring and autumn, which inhibits the activity of degrading microorganisms, slows down the degradation rate, and prolongs the degradation cycle. On the other hand, North China has a high planting density of crops, mainly wheat and corn, whose straw has a high fiber content and high degree of lignification, making it difficult for microorganisms to quickly degrade the complex organic components. Although microbial agents for degrading straw are currently available on the market, these products have significant shortcomings in meeting the needs of North China. Firstly, the microbial types in these agents are limited, with most products containing only one or two types of degrading bacteria. Straw degradation requires the synergistic action of multiple microbial groups, including cellulose-degrading, hemicellulose-degrading, and lignin-degrading bacteria. A single species cannot efficiently degrade the complex fiber and lignin components in wheat and corn straw, resulting in consistently low straw degradation efficiency. Secondly, they are not specifically adapted to the arid climate of North China. The peak activity of existing agents is concentrated in humid, warm environments. In the dry climate of North China, the microorganisms in these agents will experience cell dehydration due to water shortage, leading to a significant decrease in metabolic capacity, and some microorganisms may even become inactive. Simultaneously, drought inhibits the synthesis and secretion of microbial degradation enzymes. Even if a small number of microorganisms survive, they are unlikely to effectively degrade straw, rendering current agents almost ineffective and further exacerbating the technical challenges of straw degradation.

[0005] Therefore, in order to overcome the above-mentioned technical difficulties, it is necessary to find composite microorganisms that can promote the degradation of wheat straw in the arid and rain-scarce environment of North China, explore suitable nutrient substrates for the survival of such microorganisms, and then develop a composite microbial agent of "nutrient substrate-composite microbial system" that can efficiently degrade wheat straw under arid and rain-scarce conditions. This has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a compound microbial agent, its preparation method, and its application in degrading wheat straw, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of this invention provides a compound microbial agent, the compound microbial agent comprising a compound microbial system, the compound microbial system being composed of strain B5, strain D3 and strain WT5, wherein strain B5 is classified and named... Streptomyces ardesiacus B5, Latin name Streptomyces ardesiacusThe strain is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on August 22, 2025, with accession number CCTCC NO: M20251879; the strain D3 is classified as... Bacillus velezensis D3, Latin name Bacillus velezensis The strain WT5 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on August 22, 2025, with accession number CCTCC NO: M 20251880. Penicillium citrinum WT5, Latin name Penicillium citrinum It is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on August 22, 2025, with accession number CCTCC NO: M 20251881.

[0009] Furthermore, the compound microbial agent also includes a nutrient substrate, which includes a carbon source and a nitrogen source, wherein the carbon source includes glucose and the nitrogen source includes urea.

[0010] Furthermore, the nutrient matrix comprises 10-15 g / L glucose, 3-7 g / L urea, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L dipotassium hydrogen phosphate, 0.8-1.2 g / L sodium chloride, 0.3-0.7 g / L magnesium sulfate heptahydrate, and a pH of 6-7.

[0011] Furthermore, the compound microbial agent is prepared by the following steps:

[0012] (1) Preparation of B5 spore solution: Strain B5 was inoculated into a solid culture medium and cultured at 35-37℃ to produce spores. The spores were washed with sterile physiological saline to obtain the washing solution. The washing solution was filtered through multiple layers of gauze, and the filtrate was collected. The filtrate was centrifuged, the supernatant was discarded, and the spores were collected. The spore concentration was adjusted to 1×10⁻⁶ using sterile physiological saline. 7 Spore solutions with a concentration of CFU / mL or higher are classified as B5 spore solutions.

[0013] Preparation of D3 bacterial suspension: Inoculate strain D3 into liquid culture medium and incubate at 35–37°C and 160–200 rpm for 1–2 days to obtain the bacterial suspension. Centrifuge the suspension, discard the supernatant, collect the bacterial cells, and adjust the bacterial cell count to 1 × 10⁻⁶ using sterile water. 7 Bacterial suspensions with a concentration of cfu / mL or higher are classified as D3 bacterial suspensions.

[0014] Preparation of WT5 spore solution: WT5 strain was inoculated onto a solid culture medium and cultured at 26–30°C to produce spores. The spores were washed with sterile physiological saline to obtain a washing solution. The washing solution was filtered through multiple layers of gauze, and the filtrate was collected. The filtrate was centrifuged, the supernatant was discarded, and the spores were collected. The spore concentration was adjusted to 1 × 10⁻⁶ using sterile physiological saline. 7 Spore solutions with a concentration of cfu / mL or higher are considered WT5 spore solutions.

[0015] (2) Preparation of nutrient substrate: Based on 1 L of nutrient substrate, add 10-15 g of glucose, 3-7 g of urea, 0.8-1.2 g of potassium dihydrogen phosphate, 0.8-1.2 g of dipotassium hydrogen phosphate, 0.8-1.2 g of sodium chloride, and 0.3-0.7 g of magnesium sulfate heptahydrate to water, make up to 1 L with water, and adjust the pH to 6-7;

[0016] (3) Preparation of compound microbial agent: Add equal volumes of B5 spore liquid, D3 bacterial suspension and WT5 spore liquid to the nutrient substrate, mix evenly, the added volume of B5 spore liquid is 3~5% of the volume of nutrient substrate, the added volume of D3 bacterial suspension is 3~5% of the volume of nutrient substrate, and the added volume of WT5 spore liquid is 3~5% of the volume of nutrient substrate; culture at 28~30℃ and 160~200 rpm for 4~5 days, collect the fermentation broth, concentrate and dry it to obtain the powdered microbial agent product, which is the compound microbial agent.

[0017] Furthermore, the solid culture medium in step (1) includes LB solid culture medium; the liquid culture medium in step (2) includes LB liquid culture medium; the solid culture medium in step (3) includes PDA culture medium, the concentration is carried out by high-speed centrifugation, and the drying is carried out by spray drying.

[0018] A second aspect of the present invention provides a method for preparing a compound microbial agent, comprising the following steps:

[0019] (1) Preparation of B5 spore solution: Strain B5 was inoculated into a solid culture medium and cultured at 35-37℃ to produce spores. The spores were washed with sterile physiological saline to obtain the washing solution. The washing solution was filtered through multiple layers of gauze, and the filtrate was collected. The filtrate was centrifuged, the supernatant was discarded, and the spores were collected. The spore concentration was adjusted to 1×10⁻⁶ using sterile physiological saline. 7 Spore suspensions with a concentration of CFU / mL or higher are classified as B5 spore suspensions; wherein, the strain B5 is classified and named... Streptomyces ardesiacus B5, deposited at the China Center for Type Culture Collection, date of deposit: August 22, 2025, accession number: CCTCC NO: M 20251879;

[0020] Preparation of D3 bacterial suspension: Inoculate strain D3 into liquid culture medium and incubate at 35-37℃ and 160-200 rpm for 1-2 days to obtain the bacterial suspension. Centrifuge the suspension, discard the supernatant, collect the bacterial cells, and adjust the bacterial cell count to 1×10⁻⁶ using sterile water. 7 A bacterial suspension with a concentration of cfu / mL or higher is designated as a D3 bacterial suspension; wherein, the strain D3 is classified as... Bacillus velezensis D3, deposited at the China Center for Type Culture Collection, date of deposit: August 22, 2025, accession number: CCTCC NO:M 20251880;

[0021] Preparation of WT5 spore solution: WT5 strain was inoculated onto a solid culture medium and cultured at 26–30°C to produce spores. The spores were washed with sterile physiological saline to obtain a washing solution. The washing solution was filtered through multiple layers of gauze, and the filtrate was collected. The filtrate was centrifuged, the supernatant was discarded, and the spores were collected. The spore concentration was adjusted to 1 × 10⁻⁶ using sterile physiological saline. 7 A spore solution with a concentration of cfu / mL or higher is considered a WT5 spore solution; the strain WT5 is classified and named as follows: Penicillium citrinum WT5, deposited at the China Center for Type Culture Collection, date of deposit: August 22, 2025, accession number: CCTCC NO: M 20251881;

[0022] (2) Preparation of nutrient substrate: Based on 1L of nutrient substrate, add 10-15 g of glucose, 3-7 g of urea, 0.8-1.2 g of potassium dihydrogen phosphate, 0.8-1.2 g of dipotassium hydrogen phosphate, 0.8-1.2 g of sodium chloride, and 0.3-0.7 g of magnesium sulfate heptahydrate to water, make up to 1L with water, and adjust the pH to 6-7;

[0023] (3) Preparation of compound microbial agent: Add equal volumes of B5 spore liquid, D3 bacterial suspension and WT5 spore liquid to the nutrient substrate, mix evenly, the added volume of B5 spore liquid is 3~5% of the volume of nutrient substrate, the added volume of D3 bacterial suspension is 3~5% of the volume of nutrient substrate, and the added volume of WT5 spore liquid is 3~5% of the volume of nutrient substrate; culture at 28~30℃ and 160~200 rpm for 4~5 days, collect the fermentation broth, spray dry at low temperature, and obtain the powdered microbial agent product, which is the compound microbial agent.

[0024] Further, the solid culture medium in step (1) includes LB solid culture medium; the liquid culture medium in step (2) includes LB liquid culture medium; the solid culture medium in step (3) includes PDA culture medium, the concentration is carried out by high-speed centrifugation, and the drying is carried out by spray drying.

[0025] A third aspect of the present invention provides the application of the above-mentioned compound microbial agent in the degradation of straw, wherein the straw includes wheat straw.

[0026] Furthermore, the application includes the degradation of wheat straw in arid, low-rainfall field environments.

[0027] Furthermore, the drought and low rainfall are defined as no effective precipitation for more than two consecutive months, i.e., monthly precipitation <10 mm.

[0028] The beneficial effects of this invention are:

[0029] This invention screened three strains of bacteria, namely... Streptomyces ardesiacus B5 Bacillus velezensis D3 and Penicillium citrinum WT5 was compounded into a complex microbial strain for solid-state fermentation of wheat straw. On day 30 of solid-state fermentation, the degradation rate of wheat straw in the WT5 strain reached 41.1% compared to the control (CK) treatment without the strain. This demonstrates that WT5 has the ability to efficiently degrade wheat straw under solid-state fermentation conditions, which can meet the needs of promoting wheat straw degradation under arid and low-rainfall conditions.

[0030] This invention prepares a composite microbial agent by combining a composite microbial strain and a nutrient substrate. The nutrient substrate provides nutrients for the growth of microorganisms, meeting their needs for growth, metabolism, and product synthesis, preventing them from becoming inactive due to "starvation," and ensuring their activity and reproduction. Experiments have verified that the composite microbial strain B5+D3+WT5, with glucose as the carbon source and urea as the nitrogen source in the nutrient substrate, achieves a degradation rate of 54.8% on wheat straw in 30 days.

[0031] The compound microbial agent of this invention degrades wheat straw in arid and low-rainfall field environments, achieving a degradation rate of 70.1% after 60 days, which is 164.5% higher than the control group without added microbial agent. This confirms that the compound microbial agent of this invention can effectively degrade wheat straw under arid and low-rainfall conditions, overcoming the shortcomings of traditional microbial agents that are prone to microbial activity loss in arid environments, solving the problem of wheat straw degradation in arid areas, and realizing the efficient resource utilization of wheat straw.

[0032] The compound microbial agent of this invention is simple to prepare and environmentally friendly to use, and is of great significance for realizing the resource utilization of wheat straw in arid areas. Attached Figure Description

[0033] Figure 1 The transparent degradation zone of cellulose-degrading bacterium W15 (diameter ratio, i.e., degradation zone diameter / colony diameter = 2.90).

[0034] Figure 2 The transparent degradation zone of cellulose-degrading bacteria B5 (diameter ratio, i.e., degradation zone diameter / colony diameter = 3.75).

[0035] Figure 3 The transparent degradation zone of cellulose-degrading bacteria C1 (diameter ratio i.e., degradation zone diameter / colony diameter = 2.50).

[0036] Figure 4 The transparent degradation zone of cellulose-degrading bacteria C3 (diameter ratio, i.e., degradation zone diameter / colon diameter = 2.86).

[0037] Figure 5 The growth of straw wax-degrading bacteria in waxy liquid culture media of different concentrations.

[0038] Figure 6 Wheat straw that has undergone 30 days of solid-state fermentation was used as the control (CK) treatment.

[0039] Figure 7 The wheat straw after 30 days of solid-state fermentation was treated with T1.

[0040] Figure 8 Wheat straw after 30 days of solid-state fermentation under T2 treatment.

[0041] Figure 9 Wheat straw after 30 days of solid-state fermentation under T3 treatment.

[0042] Figure 10 Wheat straw after 30 days of solid-state fermentation under T4 treatment.

[0043] Figure 11 The degradation rate of wheat straw under different composite microbial strains in solid-state fermentation relative to the control (CK) treatment is shown.

[0044] Figure 12 Scanning electron microscope image of wheat straw after 30 days of solid-state fermentation for CK treatment.

[0045] Figure 13 Scanning electron microscope image of wheat straw after 30 days of solid-state fermentation in T1 treatment.

[0046] Figure 14 Scanning electron microscope image of wheat straw after 30 days of solid-state fermentation under T2 treatment.

[0047] Figure 15 Scanning electron microscope image of wheat straw after 30 days of solid-state fermentation under T3 treatment.

[0048] Figure 16 Scanning electron microscope image of wheat straw after 30 days of solid-state fermentation under T4 treatment.

[0049] Figure 17 The utilization of different carbon sources by strain D3.

[0050] Figure 18For the utilization of different nitrogen sources and compound carbon-nitrogen sources by strain D3.

[0051] Figure 19 For the utilization of different carbon sources by strain WT5 and strain B5.

[0052] Figure 20 For the utilization of different nitrogen sources and compound carbon-nitrogen sources by strain WT5 and strain B5.

[0053] Figure 21 For the degradation of wheat straw by complex microbial flora IV under different nutrient substrates. N1: Glucose, ammonium sulfate; N2: Glucose, urea; N3: Starch, ammonium sulfate; N4: Starch, urea; N5: Compound carbon-nitrogen source.

[0054] Figure 22 For the degradation effect of field wheat straw.

[0055] Figure 23 For the plate photo of strain B5.

[0056] Figure 24 For the plate photo of strain D3.

[0057] Figure 25 For the plate photo of strain WT5.

[0058] Figure 26 For the phylogenetic tree of strain B5.

[0059] Figure 27 For the phylogenetic tree of strain D3.

[0060] Figure 28 For the phylogenetic tree of strain WT5.

[0061] Different letters in the figure indicate significant differences between different treatments ( p <0.05); ** indicates 0.001 < P ≤ 0.01, with extremely significant differences; **** indicates P ≤ 0.0001, with highly significant differences.

[0062] Biological material preservation information

[0063] B5, classified as Streptomyces ardesiacus B5, with the Latin name Streptomyces ardesiacus , preserved in the China Center for Type Culture Collection, with the preservation address being Wuhan University, Wuhan, China, and the preservation date being August 22, 2025, and the preservation number being CCTCC NO: M 20251879.

[0064] D3, classified as Bacillus velezensis D3, with the Latin name Bacillus velezensisIt is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on August 22, 2025, with accession number CCTCC NO: M 20251880.

[0065] WT5, categorized as Penicillium citrinum WT5, Latin name Penicillium citrinum It is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on August 22, 2025, with accession number CCTCC NO: M 20251881. Detailed Implementation

[0066] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the scope of protection is not limited thereto. The specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0067] The culture media and chemical reagents involved in the following examples:

[0068] Sodium carboxymethyl cellulose (CMC-Na) medium: CMC-Na 15 g, ammonium nitrate 1 g, peptone 10 g, magnesium sulfate heptahydrate 0.5 g, potassium dihydrogen phosphate 1 g, water to a final volume of 1 L, agar 14 g; natural pH.

[0069] Xylan agar medium: 10 g xylan, 0.5 g magnesium sulfate heptahydrate, 2 g ammonium sulfate, 1 g potassium dihydrogen phosphate, 0.5 g sodium chloride, water to a final volume of 1 L, 15 g agar; natural pH.

[0070] Waxy medium: paraffin oil 5 g, ammonium nitrate 2 g, dipotassium hydrogen phosphate 1.5 g, potassium dihydrogen phosphate 3 g, magnesium sulfate heptahydrate 0.1 g, calcium chloride 0.01 g, disodium ethylenediaminetetraacetate dihydrate 0.01 g, water to a final volume of 1 L, agar 16 g; pH = 7.2~7.4 (adjust pH with 1 mol / L sodium hydroxide solution and 1 mol / L hydrogen chloride solution). Remove agar from the waxy liquid medium.

[0071] LB solid medium: 10 g peptone, 5 g sodium chloride, 10 g yeast extract, water to 1 L, 20 g agar; natural pH. LB liquid medium (without agar).

[0072] PDA medium: Difco TM Potato Dextrose Broth medium powder 24 g, water to a final volume of 1 L, agar 15 g; natural pH. PDB medium to remove agar.

[0073] Fermentation enzyme production medium: CMC-Na 10 g, peptone 10 g, sodium chloride 10 g, yeast extract 5 g, water to a final volume of 1 L; natural pH.

[0074] Xylan basal medium: 10 g xylan, 5 g peptone, 3 g yeast extract, 1 g potassium dihydrogen phosphate, 1 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.1 g calcium chloride, and water to a final volume of 1 L; pH = 6.5~7.0 (adjust pH with 1 mol / L sodium hydroxide solution and 1 mol / L hydrogen chloride solution).

[0075] Basic culture medium: carbon source (Table 3) 12 g / L, nitrogen source (Table 3) 5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, sodium chloride 1 g / L, magnesium sulfate heptahydrate 0.5 g / L; bacterial pH = 6.5~7.5 (adjusted with 1 mol / L sodium hydroxide solution and 1 mol / L hydrogen chloride solution), fungal pH = 5.5~6.5 (adjusted with 1 mol / L sodium hydroxide solution and 1 mol / L hydrogen chloride solution). Alternatively, a compound carbon and nitrogen source of 20 g / L, bacterial pH = 6.5~7.5 (adjusted with 1 mol / L sodium hydroxide solution and 1 mol / L hydrogen chloride solution), fungal pH = 5.5~6.5 (adjusted with 1 mol / L sodium hydroxide solution and 1 mol / L hydrogen chloride solution).

[0076] Compound carbon and nitrogen source: It is Wofang Yishengyuan organic water-soluble fertilizer purchased from Jiangsu Xielian Biotechnology Co., Ltd. It is concentrated from the fermentation broth of starch raw materials and is rich in various nutrients such as amino acids, peptides, sugars, and organic acids. The organic matter content is 220 g / L, the total N, P, and K content is 60 g / L, of which amino acids are 5 g / L, total sugars are about 90 g / L, organic acids are 30 g / L, and peptides are about 37 g / L.

[0077] 0.1% Congo Red Solution: 1 g of Congo Red, diluted with water to 1 L.

[0078] 1 mol / L sodium chloride solution: 58.5 g sodium chloride, diluted with water to 1 L.

[0079] Unless otherwise specified, the petri dishes (plates) involved in the following examples are 90 mm in diameter.

[0080] Example 1: Isolation, screening, and degradation capacity determination of functional strains

[0081] The desired functional bacteria were screened using selective culture media with different functions (sodium carboxymethyl cellulose medium, xylan agar medium, and waxy medium). Different combinations of the screened high-efficiency functional bacteria were then used to construct the most effective straw-degrading composite microbial system. The composite microbial system included cellulose-degrading bacteria, hemicellulose-degrading bacteria, and straw wax-degrading bacteria. All strains in the composite microbial system were screened from soil in Rizhao City, Shandong Province, an area characterized by drought and low rainfall (where there are more than two consecutive months of no effective precipitation, i.e., monthly rainfall <10 mm), after straw was returned to the field.

[0082] Step 1: Isolation of functional strains

[0083] The collected soil sample was mixed with sterile water at a mass ratio of 1:9 and shaken at 28℃ and 180 rpm for 30 min to prepare a soil suspension.

[0084] Step 2: Screening and purifying functional strains

[0085] Take 1 mL of the soil suspension from step one and spread it evenly on sodium carboxymethyl cellulose medium, xylan agar medium, and wax medium using the serial dilution plating method. Incubate at 28°C for 24–72 h. Pick single colonies and inoculate them again onto the corresponding medium, purify them at 28°C, and preserve them using the glycerol method.

[0086] Twenty-nine strains of cellulose-degrading bacteria, 16 strains of hemicellulose-degrading bacteria, and 24 strains of straw wax-degrading bacteria were obtained.

[0087] Step 3: Degradation capacity determination of each functional strain

[0088] (1) Determination of the degradation ability of cellulose-degrading bacteria

[0089] The cellulose-degrading bacteria obtained in step two were streaked onto solid culture media (bacteria on LB solid medium, fungi on PDA medium) and placed in an incubator (bacteria at 37°C, fungi at 28°C). After the culture medium was fully colonized, the bacteria were inoculated into liquid culture media (bacteria on LB liquid medium, fungi on PDB medium) and cultured at 28°C and 180 rpm until the bacterial suspension became turbid. Then, 4 μL of the bacterial suspension was spotted onto sodium carboxymethyl cellulose medium and cultured at 28°C until colonies appeared. 10 mL of 0.1% Congo red solution was then added to submerge the colonies for 20 min, and the 0.1% Congo red solution was discarded. 10 mL of 1 mol / L sodium chloride solution was then added to submerge the colonies for 20 min, and the 1 mol / L sodium chloride solution was discarded. The formation of a transparent degradation zone around the test strain was observed on the plate.

[0090] The cellulose-degrading bacteria obtained in step two were streaked onto solid culture media (bacteria on LB solid medium, fungi on PDA medium) and placed in an incubator (bacteria at 37°C, fungi at 28°C). After the culture medium was confluent, the bacteria were treated as follows: the bacteria were inoculated into 30 mL of LB liquid medium and cultured at 37°C and 180 rpm for 1 day to obtain the initial bacterial culture. A portion of the initial bacterial culture was serially diluted with sterile water (e.g., 10⁻⁶). -5 10 -6 10 -7 Take 100 μL of each culture and spread it onto LB solid medium. After incubating at 37°C for 2 days, perform plate counting. Calculate the initial bacterial concentration using the formula: "Initial bacterial concentration (cfu / mL) = Colony count ÷ Spread volume (mL) × Dilution factor". Centrifuge the remaining initial bacterial solution at 8000 rpm for 5 min, discard the supernatant, collect the bacterial cells, and adjust the bacterial concentration to 1 × 10⁻⁶ using sterile water. 7 The bacterial suspension was prepared at CFU / mL. Fungal treatment was as follows: spores on the culture medium were washed with sterile physiological saline to obtain the washing solution. The washing solution was filtered through three layers of gauze, and the filtrate was collected to obtain the initial spore solution. A portion of the initial spore solution was serially diluted with sterile physiological saline (e.g., 10⁻⁶ CFU / mL). -5 10 -6 10 -7 Take 100 μL of each of the following (dilution factor) and spread them onto PDA medium. After incubating at 28°C for 3 days, perform plate counting. Calculate the initial spore concentration using the formula: "Initial spore concentration (cfu / mL) = Colony count ÷ Spread volume (mL) × Dilution factor". Centrifuge the remaining initial spore solution at 8000 rpm for 5 min, discard the supernatant, collect the spores, and adjust the spore concentration to 1 × 10⁻⁶ using sterile physiological saline. 7 The spore suspension was prepared using cfu / mL. Strain B5 is a Streptomyces fungus belonging to the Actinobacteria phylum. While it is a bacterium, it forms granular spheres in liquid culture and produces sporulations. Therefore, the spore suspension was prepared as a fungus. The specific steps are as follows: Strain B5 was inoculated onto LB solid medium and incubated at 37°C. After the medium was fully colonized, the spores on the medium were washed with sterile physiological saline to obtain the washing solution. The washing solution was filtered through three layers of gauze, and the filtrate was collected to obtain the initial spore suspension. A portion of the initial spore suspension was serially diluted with sterile physiological saline (e.g., 10⁻⁶). -5 10 -6 10 -7Take 100 μL of each of the following solutions and spread them onto LB solid medium. After incubating at 37°C for 3 days, perform plate counting. Calculate the initial spore concentration using the formula: "Initial spore concentration (cfu / mL) = Colony count ÷ Spread volume (mL) × Dilution factor". Centrifuge the remaining initial spore solution at 8000 rpm for 5 min, discard the supernatant, collect the spores, and adjust the spore concentration to 1 × 10⁻⁶ using sterile physiological saline. 7 CFU / mL spore suspension. Take 500 μL of 1×10⁻⁶ CFU / mL spore suspension. 7 CFU / mL bacterial suspension / 1×10 7 CFU / mL spores were inoculated into 50 mL of fermentation enzyme-producing medium and cultured at 28℃ and 180 rpm for 3 days. The filter paper enzyme activity (FPA enzyme activity) and carboxymethyl cellulase activity (CMC enzyme activity) of each strain were determined by the 3,5-dinitrosalicylic acid method (DNS method).

[0091] The size of the transparent degradation zone, filter paper enzyme activity, and carboxymethyl cellulase activity of 29 cellulose-degrading bacteria were comprehensively evaluated, and four strains with high degradation ability in each test were screened out. The results are shown in Table 1.

[0092] (2) Determination of the degradation ability of hemicellulose-degrading bacteria

[0093] The hemicellulose-degrading bacteria obtained in step two were streaked onto solid culture media (bacteria on LB solid medium, fungi on PDA medium) and placed in an incubator (bacteria at 37°C, fungi at 28°C). After the culture medium was confluent, the bacteria were treated as follows: the bacteria were inoculated into 30 mL of LB liquid medium and cultured at 37°C and 180 rpm for 1 day to obtain the initial bacterial culture. A portion of the initial bacterial culture was serially diluted with sterile water (e.g., 10⁻⁶). -5 10 -6 10 -7 Take 100 μL of each culture and spread it onto LB solid medium. After incubating at 37°C for 2 days, perform plate counting. Calculate the initial bacterial concentration using the formula: "Initial bacterial concentration (cfu / mL) = Colony count ÷ Spread volume (mL) × Dilution factor". Centrifuge the remaining initial bacterial solution at 8000 rpm for 5 min, discard the supernatant, collect the bacterial cells, and adjust the bacterial concentration to 1 × 10⁻⁶ using sterile water. 7 The bacterial suspension was prepared at CFU / mL. Fungal treatment was as follows: spores on the culture medium were washed with sterile physiological saline to obtain the washing solution. The washing solution was filtered through three layers of gauze, and the filtrate was collected to obtain the initial spore solution. A portion of the initial spore solution was serially diluted with sterile physiological saline (e.g., 10⁻⁶ CFU / mL). -5 10 -6 10 -7Take 100 μL of each of the following (dilution factor) and spread them onto PDA medium. After incubating at 28°C for 3 days, perform plate counting. Calculate the initial spore concentration using the formula: "Initial spore concentration (cfu / mL) = Colony count ÷ Spread volume (mL) × Dilution factor". Centrifuge the remaining initial spore solution at 8000 rpm for 5 min, discard the supernatant, collect the spores, and adjust the spore concentration to 1 × 10⁻⁶ using sterile physiological saline. 7 CFU / mL spore suspension. Take 500 μL of 1×10⁻⁶ CFU / mL spore suspension. 7 CFU / mL bacterial suspension / 1×10 7 CFU / mL spore suspension was inoculated into 50 mL of xylan basal medium and cultured at 28℃ and 180 rpm for 3 days. The xylanase activity of each strain was determined by the DNS method.

[0094] The xylanase activity of 16 hemicellulose-degrading bacteria was comprehensively evaluated, and 4 strains with high xylanase activity were screened out. The results are shown in Table 1.

[0095] (3) Determination of the degradation capacity of straw wax-degrading bacteria

[0096] The straw wax-degrading bacteria obtained in step two were streaked onto solid culture media (bacteria on LB solid medium, fungi on PDA medium) and placed in an incubator (bacteria at 37°C, fungi at 28°C). After the culture medium was fully colonized, the bacteria were treated as follows: the bacteria were inoculated into 30 mL of LB liquid medium and cultured at 37°C and 180 rpm for 1 day to obtain the initial bacterial culture. A portion of the initial bacterial culture was serially diluted with sterile water (e.g., 10⁻⁶). -5 10 -6 10 -7 Take 100 μL of each culture and spread it onto LB solid medium. After incubating at 37°C for 2 days, perform plate counting. Calculate the initial bacterial concentration using the formula: "Initial bacterial concentration (cfu / mL) = Colony count ÷ Spread volume (mL) × Dilution factor". Centrifuge the remaining initial bacterial solution at 8000 rpm for 5 min, discard the supernatant, collect the bacterial cells, and adjust the bacterial concentration to 1 × 10⁻⁶ using sterile water. 7 The bacterial suspension was prepared at CFU / mL. Fungal treatment was as follows: spores on the culture medium were washed with sterile physiological saline to obtain the washing solution. The washing solution was filtered through three layers of gauze, and the filtrate was collected to obtain the initial spore solution. A portion of the initial spore solution was serially diluted with sterile physiological saline (e.g., 10⁻⁶ CFU / mL). -5 10 -6 10 -7Take 100 μL of each of the following (dilution factor) and spread them onto PDA medium. After incubating at 28°C for 3 days, perform plate counting. Calculate the initial spore concentration using the formula: "Initial spore concentration (cfu / mL) = Colony count ÷ Spread volume (mL) × Dilution factor". Centrifuge the remaining initial spore solution at 8000 rpm for 5 min, discard the supernatant, collect the spores, and adjust the spore concentration to 1 × 10⁻⁶ using sterile physiological saline. 7 CFU / mL spore suspension. Take 300 μL of 1×10⁻⁶ CFU / mL spore suspension. 7 CFU / mL bacterial suspension / 1×10 7 CFU / mL spore suspension was inoculated into 30 mL of waxy liquid culture medium of different concentrations (5 concentrations of waxy liquid culture medium: 3‰, 6‰, 9‰, 12‰, and 15‰, with different concentrations obtained by changing the paraffin oil content in the waxy liquid culture medium; for example, 3‰ waxy liquid culture medium means that the paraffin oil content is 3 g per 1 L of liquid culture medium). The culture was incubated at 28℃ and 180 rpm for 3 days to obtain bacterial suspension. The obtained bacterial suspension was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the precipitate was collected as bacterial cells. The bacterial cells were dried at 50℃ to constant weight and then weighed. The dry weight of bacterial cells of each strain in different concentrations of waxy liquid culture medium was compared.

[0097] The growth of 24 straw wax-degrading bacteria in different concentrations of wax liquid culture medium was comprehensively evaluated, and 3 strains with better growth at higher concentrations of wax liquid culture medium were screened out. The results are shown in Table 1.

[0098] Table 1. Screened cellulose-degrading bacteria, hemicellulose-degrading bacteria, and straw wax-degrading bacteria

[0099]

[0100] In Table 1, C1, C3, B5, B4, S4, and D3 are bacteria, while W15, WT1, WT5, E3, and F9 are fungi.

[0101] Clear degradation zones of 4 cellulose-degrading bacteria, as shown in the figure Figures 1-4 As shown in Table 2; the enzyme activities of 4 cellulose-degrading bacteria and 4 hemicellulose-degrading bacteria are shown in Table 2; the growth of 3 straw wax-degrading bacteria in different concentrations of waxy liquid culture medium is shown in Table 2. Figure 5 As shown; in summary, these strains have the ability to degrade straw.

[0102] Table 2 Enzyme Activities of Cellulose-Degrading and Hemicellulose-Degrading Bacteria

[0103]

[0104] Example 2 Construction and Optimization of Composite Microbial Systems

[0105] Step 1: Constructing a composite microbial system

[0106] The selected bacterial strains were streaked onto solid culture media (bacteria on LB solid medium, fungi on PDA medium) and placed in an incubator (bacteria at 37°C, fungi at 28°C). After the culture medium was confluent, the bacteria were treated as follows: the bacteria were inoculated into 30 mL of LB liquid medium and cultured at 37°C and 180 rpm for 1 day to obtain the initial bacterial culture. A portion of the initial bacterial culture was serially diluted with sterile water (e.g., 10⁻⁶). -5 10 -6 10 -7 Take 100 μL of each culture and spread it onto LB solid medium. After incubating at 37°C for 2 days, perform plate counting. Calculate the initial bacterial concentration using the formula: "Initial bacterial concentration (cfu / mL) = Colony count ÷ Spread volume (mL) × Dilution factor". Centrifuge the remaining initial bacterial solution at 8000 rpm for 5 min, discard the supernatant, collect the bacterial cells, and adjust the bacterial concentration to 1 × 10⁻⁶ using sterile water. 7 The bacterial suspension was prepared at CFU / mL. Fungal treatment was as follows: spores on the culture medium were washed with sterile physiological saline to obtain the washing solution. The washing solution was filtered through three layers of gauze, and the filtrate was collected as the initial spore solution. A portion of the initial spore solution was serially diluted with sterile physiological saline (e.g., 10⁻⁶ CFU / mL). -5 10 -6 10 -7 Take 100 μL of each of the following (dilution factor) and spread them onto PDA medium. After incubating at 28°C for 3 days, perform plate counting. Calculate the initial spore concentration using the formula: "Initial spore concentration (cfu / mL) = Colony count ÷ Spread volume (mL) × Dilution factor". Centrifuge the remaining initial spore solution at 8000 rpm for 5 min, discard the supernatant, collect the spores, and adjust the spore concentration to 1 × 10⁻⁶ using sterile physiological saline. 7 The spore suspension was prepared using cfu / mL. Strain B5 is a Streptomyces fungus belonging to the Actinobacteria phylum. While it is a bacterium, it forms granular spheres in liquid culture and produces sporulations. Therefore, the spore suspension was prepared as a fungus. The specific steps are as follows: Strain B5 was inoculated onto LB solid medium and incubated at 37°C. After the medium was fully colonized, the spores on the medium were washed with sterile physiological saline to obtain the washing solution. The washing solution was filtered through three layers of gauze, and the filtrate was collected to obtain the initial spore suspension. A portion of the initial spore suspension was serially diluted with sterile physiological saline (e.g., 10⁻⁶). -5 10 -6 10 -7Take 100 μL of each of the following solutions and spread them onto LB solid medium. After incubating at 37°C for 3 days, perform plate counting. Calculate the initial spore concentration using the formula: "Initial spore concentration (cfu / mL) = Colony count ÷ Spread volume (mL) × Dilution factor". Centrifuge the remaining initial spore solution at 8000 rpm for 5 min, discard the supernatant, collect the spores, and adjust the spore concentration to 1 × 10⁻⁶ using sterile physiological saline. 7 CFU / mL spore suspension. For each strain, take 4 μL of 1×10⁻⁶ CFU / mL spore suspension. 7 CFU / mL bacterial suspension / 1×10 7 CFU / mL spore suspension was inoculated onto solid culture medium plates (for indirect inoculation of bacteria and fungi, PDA medium and LB solid medium were mixed at a 1:1 volume ratio (PDA medium and LB solid medium were mixed before solidification), LB solid medium was used for indirect inoculation of bacteria, and PDA medium was used for indirect inoculation of fungi) for pairwise antagonism tests. Based on the principles of enzyme complementarity and non-antagonistic interaction between strains in the composite microbial system, four different composite microbial systems were obtained: Composite Microbial System I, Composite Microbial System II, Composite Microbial System III, and Composite Microbial System IV. Composite Microbial System I (W15+D3+WT5) was composed of strain W15 (… Schizophyllum commune ), strain D3 ( Bacillus velezensis ) and strain WT5 ( Penicillium citrinum Composed of; the composite microbial system II (C1+WT1+F9) consists of strain C1 ( Bacillus siamensis ), strain WT1 ( Penicillium cerradense ) and strain F9 ( Aspergillus versicolor Composed of; Complex microbial system III (C3+B4+E3) consists of strain C3 ( Bacillus tequilensis ), strain B4 ( Brevibacillus brevis ) and strain E3 ( Penicillium oxalicum Composed of; the complex microbial system IV (B5+D3+WT5) consists of strain B5 ( Streptomyces ardesiacus ), strain D3 ( Bacillus velezensis ) and strain WT5 ( Penicillium citrinum Composed of ) . Each composite microbial strain is prepared from 1×10 ppm of the corresponding strain. 7 CFU / mL bacterial suspension / 1×10 7 Prepare spore solutions of cfu / mL in equal volume ratio.

[0107] Step 2: Evaluate the ability of different composite microbial strains to degrade wheat straw.

[0108] Treatment setup: wheat straw treatment CK without microbial strain, wheat straw treatment T1 with compound microbial strain I, wheat straw treatment T2 with compound microbial strain II, wheat straw treatment T3 with compound microbial strain III, and wheat straw treatment T4 with compound microbial strain IV. Each treatment was repeated 9 times.

[0109] Wheat straw was processed into 3-4 cm pieces and dried in an oven at 50℃ until constant weight. 3 g of the dried wheat straw was weighed and adjusted to 60% moisture content using a 1:1 mixture of LB liquid medium and PDB medium (i.e., virtually no free-flowing aqueous phase, where microbial growth, metabolism, and product synthesis all occur on the surface or in the gaps of the moist solid substrate to ensure solid-state fermentation). The mixture was then placed in a 250 mL Erlenmeyer flask and sterilized at 121℃ for 30 min. After cooling to room temperature, 7.5 mL of a composite microbial culture (i.e., 1×10⁻⁶ of each of the three types of microbial cultures) was inoculated according to the treatment settings. 7 CFU / mL bacterial suspension / 1×10 7 Add 2.5 mL of cfu / mL spore solution to each sample, and add an equal volume of sterile water to the control (CK). Place the samples in a natural environment for solid-state fermentation (sealed with a breathable membrane). Take samples on days 10, 20, and 30 of solid-state fermentation (three replicates each time), dry them at 50℃ to constant weight, and weigh them again to determine the degradation rate of wheat straw. Perform scanning electron microscopy analysis on the wheat straw sample on day 30 of solid-state fermentation.

[0110] The formula for calculating the degradation rate of wheat straw is:

[0111] Wheat straw degradation rate (%) = [(Initial wheat straw dry weight - Degraded wheat straw dry weight) / Initial wheat straw dry weight] × 100%

[0112] The degradation of wheat straw in different treatments on day 30 of solid-state fermentation is compared as follows: Figures 6-10 As shown: Wheat straw treated with CK without the addition of microbial strains was generally intact and showed almost no degradation; wheat straw treated with compound microbial strains showed varying degrees of degradation, with the T4 treatment showing the most significant degradation, becoming shorter, broken, loose, and with significantly reduced toughness and hardness, making it easy to break and crumble.

[0113] The degradation rate of wheat straw in each treatment with the added compound microbial strain was compared to the control (CK) treatment without the strain at three stages (days 10, 20, and 30 of solid-state fermentation). The degradation rates were calculated as follows: (i.e., wheat straw degradation rate in the compound microbial strain treatment - wheat straw degradation rate in the CK treatment without the strain). Figure 11As shown, the wheat straw degradation rate of each treatment with the added compound microbial strain increased relative to the control (CK) treatment without the strain at days 10, 20, and 30 of solid-state fermentation. Among them, the T4 treatment showed significantly better wheat straw degradation rate than the CK treatment without the strain at days 20 and 30 of solid-state fermentation; at day 30 of solid-state fermentation, the wheat straw degradation rate of the T4 treatment reached 41.1% relative to the CK treatment without the strain.

[0114] Scanning electron microscopy results of wheat straw from each treatment on day 30 of solid-state fermentation are as follows: Figures 12-16 As shown, the wheat straw treated with CK without the addition of microbial strains showed no obvious breakage or loosening, and the overall structure had strong mechanical integrity. The wheat straw treated with various composite microbial strains had its original continuous fiber bundle structure destroyed, the surface smoothness reduced, the structure loosened, and obvious breakage and gaps appeared. In particular, the wheat straw treated with T4 was covered with cracks and holes, the fibers were obviously exposed, and the surface was covered with a thin layer of flocculent or granular degradation products.

[0115] The above results fully demonstrate that, under solid-state fermentation conditions, the composite microbial strain IV (B5+D3+WT5) has the ability to efficiently degrade wheat straw, which can meet the needs of promoting wheat straw degradation under arid and low-rainfall conditions.

[0116] Example 3: Investigation of Microbial Nutrient Substrates

[0117] Step 1: Study the carbon and nitrogen source utilization of the three strains.

[0118] Keeping the contents of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, and magnesium sulfate heptahydrate in the basal medium constant, the unique carbon source and unique nitrogen source in the formula were changed sequentially according to Table 3 by controlling variables (when considering the unique nitrogen source variable, the unique carbon source was fixed as glucose; when considering the unique carbon source variable, the unique nitrogen source was fixed as ammonium sulfate), thereby obtaining basal medium containing different carbon and nitrogen sources; at the same time, a basal medium containing a complex carbon and nitrogen source was prepared (the complex carbon and nitrogen source content was 20 g / L, and the carbon source, nitrogen source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, and magnesium sulfate heptahydrate in the basal medium were replaced).

[0119] The three strains (strain B5, strain D3, and strain WT5) in the activated composite microbial strain IV were prepared into 1×10⁻⁶ strains according to the method in Example 2. 7 CFU / mL bacterial suspension or 1×10 7 CFU / mL spore suspension. Add 1×10⁻⁶ CFU / mL to a 96-well plate containing different carbon and nitrogen sources or a basal medium containing a complex carbon and nitrogen source. 7 CFU / mL bacterial suspension (1×10⁻⁶) 7A bacterial suspension of CFU / mL was incubated with basal medium at a ratio of 2 μL to 200 μL at 37°C and 180 rpm for 24 h. The OD600 value of the bacterial suspension was then measured. 1 × 10⁻⁶ CFU / mL was added to an Erlenmeyer flask containing different carbon and nitrogen sources or a basal medium containing a complex carbon and nitrogen source. 7 CFU / mL spore solution (1×10) 7 The spore suspension (cfu / mL: basal medium = 0.3 mL: 30 mL) was cultured at 28℃ and 180 rpm for 48 h. The resulting bacterial suspension was then centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were dried at 50℃ to constant weight before weighing. Strain B5 is a Streptomyces of the Actinobacteria phylum, belonging to the bacteria category. However, because it presents as granular spheres in liquid culture and produces sporulations, the dry weight method was used for determination. This method was used to compare the growth of each strain in basal media containing different carbon and nitrogen sources or in basal media containing a complex carbon and nitrogen source, and to identify the carbon and nitrogen source utilization of each strain.

[0120] Table 3 Carbon and Nitrogen Sources

[0121]

[0122] The carbon and nitrogen source utilization of strains B5, D3, and WT5 are as follows: Figures 17-20 As shown, considering factors such as the stability of the strain in carbon and nitrogen sources, the ease of obtaining carbon and nitrogen sources, and cost, glucose, starch, ammonium sulfate, urea, and a compound carbon and nitrogen source were selected for subsequent experiments.

[0123] Step 2: Optimization of the combination of carbon and nitrogen sources

[0124] The processing settings are shown in Table 4:

[0125] Table 4. Types of carbon and nitrogen sources for each treatment

[0126]

[0127] Specific operations:

[0128] The three strains (strain B5, strain D3, and strain WT5) in the activated composite microbial strain IV were prepared into 1×10⁻⁶ strains according to the method in Example 2. 7 CFU / mL bacterial suspension or 1×10 7 CFU / mL spore solution.

[0129] Wheat straw was processed into 3-4 cm pieces and dried in an oven at 50℃ until constant weight. 3 g of dried wheat straw was placed in a 250 mL Erlenmeyer flask. Basic culture media containing different carbon and nitrogen sources and a composite carbon and nitrogen source were prepared according to Table 4. 80 mL of each medium was added to the Erlenmeyer flask and sterilized at 121℃ for 30 min. An equal volume of water was added to the control (CK). After cooling, 1×10⁻⁶ cells were inoculated at a 5 v / v% inoculum rate. 7 CFU / mL bacterial suspension / 1×10 7 CFU / mL spore suspension (i.e., 4 mL of 1×10⁻⁶ CFU / mL basal medium added to 80 mL of basal medium containing different carbon and nitrogen sources or basal medium containing a complex carbon and nitrogen source). 7 B5 spore solution (cfu / mL), 4 mL 1×10 7 CFU / mL D3 bacterial suspension and 4 mL 1×10 7 The WT5 spore solution (i.e., an equal volume ratio of B5, D3, and WT5 bacterial suspension / spore solution) was degraded at 28℃ and 180 rpm for 30 days. Samples were then taken, dried at 50℃ to constant weight, and weighed again to determine the degradation rate of wheat straw.

[0130] The formula for calculating the degradation rate of wheat straw is:

[0131] Wheat straw degradation rate (%) = [(initial dry weight of wheat straw - dry weight of degraded wheat straw) / initial dry weight of wheat straw] × 100%.

[0132] The degradation of wheat straw by the compound microbial strain IV under different nutrient substrates (containing different carbon and nitrogen sources) is as follows: Figure 21 As shown, after 30 days of combined microbial degradation, the degradation rate of wheat straw was N2>N3>N5>N1>N4>CK, with the degradation rate of wheat straw in the N2 treatment reaching 54.8%. This indicates that the basic culture medium prepared with glucose as the carbon source and urea as the nitrogen source is the optimal choice for the combined microbial strain IV as the nutrient substrate.

[0133] Example 4: Evaluation of the application of compound microbial agents (compound microbial agents constructed from nutrient substrate and compound microbial strains) in wheat straw in the field.

[0134] Preparation of compound microbial agents: Activated strains B5, D3, and WT5 were prepared into 1×10⁻⁶ solutions respectively according to the method in Example 2. 7 B5 spore solution at CFU / mL, 1×10 7 D3 bacterial suspension at cfu / mL, 1×10 7 WT5 spore suspension at cfu / mL. In a basal medium (pH 6.5) prepared with glucose as the carbon source and urea as the nitrogen source, inoculate at a ratio of 5% (1×10⁻⁶).7 CFU / mL spore solution / 1×10 7 (CFU / mL bacterial suspension: basal medium volume ratio = 1:20) Inoculate with 1×10 7 B5 spore solution with CFU / mL, 1×10 7 CFU / mL of D3 bacterial suspension and 1×10 7 CFU / mL WT5 spore suspension (i.e., based on 100mL of basal medium, 5mL of 1×10⁻⁶ spores are inoculated into 100mL of basal medium). 7 CFU / mL B5 spore solution, 5 mL 1×10 7 CFU / mL D3 bacterial suspension and 5 mL 1×10 7 The WT5 spore solution (CFU / mL) and B5, D3, and WT5 bacterial suspensions / spore solutions were mixed in equal volumes and thoroughly shaken. The mixture was then incubated at 28°C and 180 rpm for 5 days, and the fermentation broth was collected. The fermentation broth was then concentrated by high-speed centrifugation at 10,000 rpm at room temperature to reduce the solid content to 30 wt%. The concentrate was then dried using a spray drying process, with the inlet air temperature set at 180°C and the outlet air temperature at 80°C. The feeding rate and atomization rate were adjusted according to the equipment power and the above inlet and outlet air temperature parameters. After drying, the powdered bacterial agent product, namely the compound bacterial agent, was obtained.

[0135] The experiment was conducted in November 2024 in a dry and rain-scarce field environment in Yufengling Village, Beikuo Town, Lanshan District, Rizhao City, Shandong Province (the area experienced more than two consecutive months without effective rainfall, i.e., monthly rainfall <10 mm). Wheat straw from the current season was chopped to 5-10 cm, and 50 g (dry weight) of wheat straw was placed in a 20-mesh nylon bag. Experimental group (T): The above-mentioned compound microbial agent was diluted 100 times with water (1 g compound microbial agent: 100 mL water) to obtain the diluted compound microbial agent. The nylon bag containing wheat straw was immersed in the diluted compound microbial agent for 30 min (the purpose is to provide an initial moist substrate for the microorganisms in the compound microbial agent, improving their colonization rate and subsequent degradation efficiency), and then placed in a pit about 10 cm deep. The diluted compound microbial agent was then sprayed onto the surface of the wheat straw (20 mL of diluted compound microbial agent per 50 g (dry weight) of wheat straw). Control group (CK): The diluted compound microbial agent was replaced with water, and the same treatment was performed. Afterwards, the soil was covered; three bags of wheat straw were taken out every 20 days for each treatment, for a total of three times. After taking them out, the wheat straw was washed to remove soil impurities, dried at 50℃ to constant weight, and then weighed to determine the degradation rate of wheat straw.

[0136] The formula for calculating the degradation rate of wheat straw is as follows:

[0137] Wheat straw degradation rate (%) = [(initial dry weight of wheat straw - dry weight of degraded wheat straw) / initial dry weight of wheat straw] × 100%.

[0138] Wheat straw degradation rate such as Figure 22 As shown, wheat straw in both treatments showed significant degradation over time. On day 60 of degradation, the wheat straw degradation rate in the control group without the added compound microbial agent was 26.5%, while the degradation rate in the experimental group with the added compound microbial agent was 70.1%, an increase of 164.5% compared to the control group. This indicates that the compound microbial agent can effectively degrade wheat straw under arid and low-rainfall conditions.

[0139] Example 5 Identification of Functional Strains

[0140] The identification of strains B5, D3, and WT5 is as follows:

[0141] I. Morphological Identification

[0142] Strain B5 was inoculated onto LB solid medium and incubated at 37°C for 2 days. Figure 23 As shown (before spore formation), the colonies are milky white, with round colonies of different sizes. The colonies are relatively dense and tightly bound to the culture medium, making them difficult to pick up.

[0143] Strain D3 was inoculated onto LB solid medium and incubated at 37°C for 2 days. Figure 24 As shown, the colonies are pale yellow, vary in size, have a rough surface, irregular edges, are raised, and grow relatively quickly.

[0144] Strain WT5 was inoculated onto PDA medium and incubated at 28°C for 3 days. Figure 25 As shown, the colonies are light green with a yellowish-brown back, irregular in shape, wrinkled on the surface, and raised in some areas.

[0145] II. Molecular biological identification

[0146] DNA was extracted from purified strains B5, D3, and WT5, and the concentration of the extracted DNA was detected using a NanoDrop spectrophotometer. Then, the target genes of each strain were amplified by PCR. For bacteria, the 16S rRNA gene was amplified using universal primers 27F (as shown in SEQ ID NO.1) / 1492R (as shown in SEQ ID NO.2), and for fungi, the ITS region was amplified using universal primers ITS1 (as shown in SEQ ID NO.3) / ITS4 (as shown in SEQ ID NO.4). The amplified products were verified by agarose gel electrophoresis, purified, and sequenced. The resulting sequences (the 16S rRNA gene sequence of B5 is shown in SEQ ID NO.5, the 16S rRNA gene sequence of D3 is shown in SEQ ID NO.6, and the ITS gene sequence of WT5 is shown in SEQ ID NO.7) were compared using BLAST in the NCBI database. Known sequences with similarity ≥97% (bacteria) or ≥98% (fungi) were selected, and a phylogenetic tree was constructed based on the alignment results.

[0147] like Figure 26 As shown, B5 and Streptomyces ardesiacus NRRL B-1773 showed the highest homology, reaching 99.65%. For example... Figure 27 As shown, D3 and Bacillus velezensis CBMB205 showed the highest homology, reaching 99.79%. For example... Figure 28 As shown, WT5 and Penicillium citrinum AKP2-KU showed the highest homology, reaching 99.99%.

[0148] Based on colony morphology and phylogenetic tree analysis, strain B5 was identified as... Streptomyces ardesiacus It has been deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20251879; strain D3 was identified as... Bacillus velezensis It has been deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M20251880; WT5 identification is... Penicillium citrinum It has been deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20251881.

[0149] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound microbial agent, characterized in that, The compound microbial agent comprises a compound microbial system, which is composed of strain B5, strain D3, and strain WT5. ​​Strain B5 is classified and named... Streptomyces ardesiacus B5, deposited at the China Center for Type Culture Collection (CCTCC) on August 22, 2025, with accession number CCTCC NO: M 20251879; strain D3 is classified as... Bacillus velezensis D3, deposited at the China Center for Type Culture Collection (CCTCC) on August 22, 2025, with accession number CCTCC NO: M 20251880; the strain WT5 is classified and named... Penicillium citrinum WT5 is deposited at the China Center for Type Culture Collection on August 22, 2025, with accession number CCTCC NO: M 20251881.

2. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent also includes a nutrient substrate, which comprises 10-15 g / L glucose, 3-7 g / L urea, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L dipotassium hydrogen phosphate, 0.8-1.2 g / L sodium chloride, 0.3-0.7 g / L magnesium sulfate heptahydrate, and a pH of 6-7.

3. The compound microbial agent according to claim 2, characterized in that, The compound microbial agent is prepared by the following steps: (1) Preparation of B5 spore solution: Strain B5 was inoculated into PDA solid medium and cultured at 35-37℃ to produce spores. The spores were washed with sterile physiological saline to obtain the washing solution. The washing solution was filtered through multiple layers of gauze, and the filtrate was collected. The filtrate was centrifuged, the supernatant was discarded, and the spores were collected. The spore concentration was adjusted to 1×10⁻⁶ using sterile physiological saline. 7 Spore solutions with a concentration of CFU / mL or higher are classified as B5 spore solutions. Preparation of D3 bacterial suspension: Inoculate strain D3 into LB liquid medium and incubate at 35–37°C and 160–200 rpm for 1–2 days to obtain the bacterial suspension. Centrifuge the suspension, discard the supernatant, collect the bacterial cells, and adjust the bacterial cell count to 1 × 10⁻⁶ using sterile water. 7 Bacterial suspensions with a concentration of cfu / mL or higher are classified as D3 bacterial suspensions. Preparation of WT5 spore solution: WT5 strain was inoculated onto PDA solid medium and cultured at 26–30°C to produce spores. The spores were washed with sterile physiological saline to obtain a washing solution. The washing solution was filtered through multiple layers of gauze, and the filtrate was collected. The filtrate was centrifuged, the supernatant was discarded, and the spores were collected. The spore concentration was adjusted to 1 × 10⁻⁶ using sterile physiological saline. 7 Spore solutions with a concentration of cfu / mL or higher are considered WT5 spore solutions. (2) Preparation of nutrient substrate: Based on 1L of nutrient substrate, add 10-15 g of glucose, 3-7 g of urea, 0.8-1.2 g of potassium dihydrogen phosphate, 0.8-1.2 g of dipotassium hydrogen phosphate, 0.8-1.2 g of sodium chloride, and 0.3-0.7 g of magnesium sulfate heptahydrate to water, and make up to 1L with water, and adjust the pH to 6-7; (3) Preparation of compound microbial agent: Add equal volumes of B5 spore liquid, D3 bacterial suspension and WT5 spore liquid to the nutrient substrate, mix evenly, the added volume of B5 spore liquid is 3~5% of the volume of nutrient substrate, the added volume of D3 bacterial suspension is 3~5% of the volume of nutrient substrate, and the added volume of WT5 spore liquid is 3~5% of the volume of nutrient substrate; culture at 28~30℃ and 160~200 rpm for 4~5 days, collect the fermentation broth, concentrate and dry it to obtain the powdered microbial agent product, which is the compound microbial agent.

4. The compound microbial agent according to claim 3, characterized in that, The concentration in step (3) is achieved by high-speed centrifugal concentration, and the drying is achieved by spray drying.

5. A method for preparing a compound microbial agent, characterized in that, Includes the following steps: (1) Preparation of B5 spore solution: Strain B5 was inoculated into PDA solid medium and cultured at 35-37℃ to produce spores. The spores were washed with sterile physiological saline to obtain the washing solution. The washing solution was filtered through multiple layers of gauze, and the filtrate was collected. The filtrate was centrifuged, the supernatant was discarded, and the spores were collected. The spore concentration was adjusted to 1×10⁻⁶ using sterile physiological saline. 7 Spore suspensions with a concentration of CFU / mL or higher are classified as B5 spore suspensions; wherein, the strain B5 is classified and named... Streptomyces ardesiacus B5, deposited at the China Center for Type Culture Collection, date of deposit: August 22, 2025, accession number: CCTCC NO: M 20251879; Preparation of D3 bacterial suspension: Strain D3 was inoculated into LB liquid medium and cultured at 35–37°C and 160–200 rpm for 1–2 days to obtain the bacterial suspension. The suspension was centrifuged, the supernatant was discarded, and the bacterial cells were collected. The bacterial cell count was adjusted to 1 × 10⁻⁶ using sterile water. 7 A bacterial suspension with a concentration of cfu / mL or higher is designated as a D3 bacterial suspension; wherein, the strain D3 is classified as... Bacillus velezensis D3, deposited at the China Center for Type Culture Collection, date of deposit: August 22, 2025, accession number: CCTCC NO:M 20251880; Preparation of WT5 spore solution: WT5 strain was inoculated onto PDA solid medium and cultured at 26–30°C to produce spores. The spores were washed with sterile physiological saline to obtain a washing solution. The washing solution was filtered through multiple layers of gauze, and the filtrate was collected. The filtrate was centrifuged, the supernatant was discarded, and the spores were collected. The spore concentration was adjusted to 1 × 10⁻⁶ using sterile physiological saline. 7 A spore solution with a concentration of cfu / mL or higher is considered a WT5 spore solution; the strain WT5 is classified and named as follows: Penicillium citrinum WT5, deposited at the China Center for Type Culture Collection, date of deposit: August 22, 2025, accession number: CCTCC NO: M 20251881; (2) Preparation of nutrient substrate: Based on 1L of nutrient substrate, add 10-15 g of glucose, 3-7 g of urea, 0.8-1.2 g of potassium dihydrogen phosphate, 0.8-1.2 g of dipotassium hydrogen phosphate, 0.8-1.2 g of sodium chloride, and 0.3-0.7 g of magnesium sulfate heptahydrate to water, and make up to 1L with water, and adjust the pH to 6-7; (3) Preparation of compound microbial agent: Add equal volumes of B5 spore liquid, D3 bacterial suspension and WT5 spore liquid to the nutrient substrate, mix evenly, the added volume of B5 spore liquid is 3~5% of the volume of nutrient substrate, the added volume of D3 bacterial suspension is 3~5% of the volume of nutrient substrate, and the added volume of WT5 spore liquid is 3~5% of the volume of nutrient substrate; culture at 28~30℃ and 160~200 rpm for 4~5 days, collect the fermentation broth, concentrate and dry it to obtain the powdered microbial agent product, which is the compound microbial agent.

6. The method for preparing a compound microbial agent according to claim 5, characterized in that, The concentration in step (3) is achieved by high-speed centrifugal concentration, and the drying is achieved by spray drying.

7. The application of the compound microbial agent according to any one of claims 1 to 4 in the degradation of wheat straw.

8. The application according to claim 7, characterized in that, The application includes the degradation of wheat straw in arid, low-rainfall field environments.

9. The application according to claim 8, characterized in that, The drought and low rainfall are defined as no effective precipitation for more than two consecutive months, i.e., monthly precipitation <10 mm.

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