Straw decomposing microbial agent, preparation method and application thereof

Abstract

The application relates to a straw decomposition microbial agent, a preparation method and application thereof, and belongs to the technical field of bio-agriculture and related industries. The straw decomposition microbial agent is composed of effective components and protective agents; the effective components are composed of BA-1 microbial bodies containing graphene, SD-4 microbial bodies containing graphene and LT1 microbial bodies containing graphene. The straw decomposition microbial agent has the following advantages: the straw decomposition microbial agent can improve the seasonal decomposition efficiency of straw under the condition of direct straw returning to the field, avoids the problems of slow straw decomposition, influences the growth of the next crop, forms an empty layer in the soil to influence water and nutrient utilization and the like, shortens the straw decomposition period under the condition of straw decomposition, promotes the growth of crops, reduces diseases and improves yield.

Description

Technical Field

[0001] This invention belongs to the technical field of bio-agriculture and related industries, specifically relating to a straw decomposing microbial agent, its preparation method, and its application. Background Technology

[0002] Directly returning straw to the field is an important agronomic measure to improve soil organic matter and fertility. However, because straw is rich in cellulose, hemicellulose, and lignin, its natural decomposition is slow. Applying straw-promoting microbial agents has become a key technology to accelerate its conversion into straw. Currently, straw-promoting microbial agents are mainly made from functional microorganisms such as Bacillus subtilis, Trichoderma harzianum, and Aspergillus niger. To overcome the bottleneck of limited function of single microbial species, existing technologies generally adopt a multi-species compound strategy to achieve synergistic effects. For example, patents have developed a compound microbial agent (CN202510404791.2) composed of screened thermophilic bacterium, Guangxi thermophilic actinomycetes, and Arthrobacter spp., and an organic material composting agent (CN201310228171.5) composed of Bacillus subtilis, Bacillus licheniformis, Trichoderma kangariospora, Protozoa chrysospora, and Thermophila latum. These agents significantly enhance the degradation capacity of lignin-cellulose complexes through the synergistic effect of multiple microorganisms.

[0003] Although the concept of compound microbial agents is relatively common, existing technologies still have some problems in the direct application to the field: most existing microbial strains are mainly designed for composting scenarios. Although they have high activity of decomposing enzymes, in the direct application to the field, the microbial agents face multiple abiotic factors such as soil temperature and moisture stress, resulting in unstable decomposition effects and limited decomposition efficiency of the microbial strains. Summary of the Invention

[0004] The first objective of this invention is to disclose a straw decomposing microbial agent.

[0005] The second objective of this invention is to disclose a method for preparing the above-mentioned straw decomposing microbial agent.

[0006] The third objective of this invention is to disclose the application of the above-mentioned straw decomposing microbial agent.

[0007] The objective of this invention is achieved through the following technical solution: A straw decomposing microbial agent, wherein: the straw decomposing microbial agent is composed of an active ingredient and a protective agent acceptable to the microbial agent; the active ingredient is composed of BA-1 microbial cells containing graphene, SD-4 microbial cells containing graphene, and LT1 microbial cells containing graphene; The graphene-containing BA-1 bacterial cells were prepared by the following method: Bacillus amyloliquefaciens BA-1 CGMCC No. 36117 strain was inoculated into LB medium and cultured overnight at 30°C to obtain a seed culture; graphene was added to fresh LB medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into graphene-containing LB medium for expansion culture at 37°C and 180 rpm for 24-48 hours. After collecting the bacterial cells, skim milk powder was added and freeze-dried to obtain the graphene-containing BA-1 bacterial cells. The graphene-containing SD-4 bacterial cells were prepared by the following method: Streptomycestendae SD-4 CGMCC No.36120 strain was inoculated into Gao's No. 1 medium and cultured overnight at 30°C to obtain seed culture; graphene was added to fresh Gao's No. 1 medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into graphene-containing Gao's No. 1 medium and cultured at 28°C and 150 rpm for 48-72 hours. After collecting the bacterial cells, they were freeze-dried to obtain the graphene-containing SD-4 bacterial cells. The graphene-containing LT1 cells were prepared by the following method: Trichoderma harzianum LT1 CGMCC No. 42845 strain was inoculated into PDA medium and cultured overnight at 30°C to obtain seed culture; graphene was added to a new PDA medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into a graphene-containing PDA medium and cultured at 28°C in the dark for 3 days; after the mycelium had basically covered the culture dish, it was transferred to light conditions and cultured for another 2 days to promote sporulation; after a large number of green conidia formed on the surface of the plate, sterile deionized water was added to repeatedly rinse the surface of the culture, and the spore suspension was collected to obtain the graphene-containing LT1 cells.

[0008] The straw decomposing microbial agent described in the above technical solution has the following composition: the mass ratio of graphene-containing BA-1 cells, graphene-containing SD-4 cells, and graphene-containing LT1 cells in the effective components is (1-4):(1-4):(1-4).

[0009] The straw decomposing microbial agent described in the above technical solution, wherein the ratio of graphene-containing BA-1 cells, graphene-containing SD-4 cells, and graphene-containing LT1 cells in the active ingredient is selected from one of the following ratios: (1) BA-1 cells: SD-4 cells: LT1 cells = 1:1:1; or (2) BA-1 cells: SD-4 cells: LT1 cells = 1:1:2; or (3) BA-1 cells: SD-4 cells: LT1 cells = 1:2:1; or (4) BA-1 cells: SD-4 cells: LT1 cells = 2:1:1; or (5) BA-1 cells: SD-4 cells: LT1 cells = 4:1:2; or (6) BA-1 cells: SD-4 cells: LT1 cells = 4:2:1; or (7) BA-1 cells: SD-4 cells: LT1 cells = 1:4:2; or (8) BA-1 cells: SD-4 cells: LT1 cells = 2:1:4; or (9) BA-1 cells: SD-4 cells: LT1 cells = 2:4:1; or (10) BA-1 cells: SD-4 cells: LT1 cells = 1:2:4.

[0010] The straw decomposing microbial agent described in the above technical solution, wherein: the protective agent acceptable to the microbial agent is composed of sodium alginate, sucrose and glycerol, wherein sodium alginate accounts for 2% of the total weight of the straw decomposing microbial agent, sucrose accounts for 3% of the total weight of the straw decomposing microbial agent and glycerol accounts for 2% of the total weight of the straw decomposing microbial agent.

[0011] The method for preparing the straw decomposing microbial agent described in the above technical solution includes the following steps: (1) The BA-1 bacterial cell containing graphene, the SD-4 bacterial cell containing graphene and the LT1 bacterial cell containing graphene were prepared. The graphene-containing BA-1 bacterial cells were prepared by the following method: Bacillus amyloliquefaciens BA-1 strain was inoculated into LB medium and cultured overnight at 30°C to obtain seed culture; graphene was added to fresh LB medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into LB medium containing graphene for expansion culture at 37°C and 180 rpm for 24-48 hours; after collecting the bacterial cells, skim milk powder was added and freeze-dried to obtain BA-1 bacterial cells containing graphene; The graphene-containing SD-4 bacterial cells were prepared by the following method: *Streptomyces tangdeensis* SD-4 strain was inoculated into Gao's No. 1 medium and cultured overnight at 30°C to obtain a seed culture; graphene was added to fresh Gao's No. 1 medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into graphene-containing Gao's No. 1 medium and cultured at 28°C and 150 rpm for 48-72 hours. After collecting the bacterial cells, they were freeze-dried to obtain the graphene-containing SD-4 bacterial cells. The graphene-containing LT1 cells were prepared by the following method: Trichoderma harzianum LT1 strain was inoculated into PDA medium and cultured overnight at 30°C to obtain seed culture; graphene was added to a fresh PDA medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into a graphene-containing PDA medium and cultured at 28°C in the dark for 3 days; after the mycelium had basically covered the culture dish, it was transferred to light conditions and cultured for another 2 days to promote sporulation; after a large number of green conidia formed on the surface of the plate, sterile deionized water was added to repeatedly rinse the surface of the culture, and the spore suspension was collected to obtain the graphene-containing LT1 cells; (2) Mix the graphene-containing BA-1 cells, graphene-containing SD-4 cells and graphene-containing LT1 cells obtained in step (1) according to the mass ratio in the above technical solution, and then add 2% sodium alginate, 3% sucrose and 2% glycerol of the total weight of the straw decomposing bacteria agent. After mixing, the straw decomposing bacteria agent is obtained.

[0012] The application of the straw decomposing microbial agent described in the above technical solution in the decomposition of straw.

[0013] The present invention has the following beneficial effects:

[0014] 1. The straw decomposing microbial agent of the present invention can improve the seasonal decomposition efficiency of straw under the condition of direct straw return to the field, avoid the problems caused by slow straw decomposition affecting the growth of subsequent crops and the formation of an air gap in the soil affecting the utilization of water and nutrients; it can also shorten the straw decomposition cycle under composting conditions; promote crop growth, reduce diseases, and increase yield.

[0015] 2. The decomposition effect of the three strains combined in this invention is higher than that of the single strain, indicating that there is a synergistic effect among the three strains; the combination with a higher proportion of Bacillus amyloliquefaciens can start the decomposition process relatively quickly, while appropriately increasing the proportion of Trichoderma harzianum can obtain a higher overall decomposition efficiency.

[0016] 3. In this invention, when the graphene concentration is 20 mg / L, a high number of viable bacteria and cellulase activity can be obtained.

[0017] 4. The combination of three strains in the microbial agent of this invention gives the agent high field adaptability to cope with seasonality, diurnal temperature variation, drought, flooding and other field conditions.

[0018] Strain preservation information:

[0019] 1. This invention, Bacillus amyloliquefaciens BA-1, classified and named Bacillus amyloliquefaciens, has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.36117 and deposit date of September 29, 2025.

[0020] 2. The present invention, Streptomyces tendae SD-4, classified and named Streptomycetes tangde, has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.36120 and deposit date of September 29, 2025.

[0021] 3. The present invention, Trichoderma harzianum LT1, classified and named Trichoderma harzianum, has been deposited at the China General Microbiological Culture Collection Center (CGMCC). The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO.42845, and the deposit date is July 17, 2026. Attached Figure Description

[0022] Figure 1 To investigate the temperature and humidity adaptability of various strains of Bacillus amyloliquefaciens BA-1, Streptomyces tangde SD-4, and Trichoderma harzianum LT1.

[0023] Figure 2 To analyze the antagonistic effects between pairs of Bacillus amyloliquefaciens BA-1, Streptomyces tangde SD-4, and Trichoderma harzianum LT1 strains. Detailed Implementation

[0024] To facilitate understanding of the technical solution of the present invention, the following detailed description, in conjunction with specific embodiments, further illustrates a straw decomposing microbial agent and its application.

[0025] The culture medium and formulation used in the following embodiments of the present invention are as follows:

[0026] LB medium: peptone: 10 g, yeast extract: 5 g, NaCl: 5 g, distilled water: 1000 mL, pH: 7.2.

[0027] Gao's No. 1 culture medium: soluble starch 20 g, KNO3 1 g, NaCl 0.5 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, distilled water: 1000 mL, pH: 7.2.

[0028] PDA medium: 200 g potato, 20 g sucrose, 1000 mL distilled water.

[0029] CMC-Na medium: CMC-Na: 5 g, MgSO4·7H2O: 0.5 g, yeast extract: 1.0 g, KH2PO4: 1.0 g, NH4NO3: 1.0 g, distilled water: 1000 mL, pH: 6.8-7.2.

[0030] β-Glucoside medium: salicin: 5 g, MgSO4·7H2O: 0.5 g, yeast extract: 1.0 g, KH2PO4: 1.0 g, NH4NO3: 1.0 g, distilled water: 1000 mL, pH: 6.8-7.2.

[0031] Filter paper strip culture medium: Filter paper strips (1×2 cm): 30 strips, MgSO4·7H2O: 0.5 g, yeast extract: 1.0 g, KH2PO4: 1.0 g, NH4NO3: 1.0 g, distilled water: 1000 mL, pH: 6.8-7.2.

[0032] Example 1: Isolation and purification of three strains:

[0033] The soil used in this embodiment comes from Xinxiang, Henan Province. The soil type is alluvial soil, and the planting system is winter wheat-summer corn. Conventional fertilization is used, and all straw is returned to the field.

[0034] Take 10 g of soil from a long-term straw return plot, put it into 50 mL of sterile water, shake at 30℃ and 180 rpm for 30 minutes, let it stand for 30 minutes, take 1 mL of the supernatant, and continuously dilute it 10 times with sterile water.

[0035] Absorb 10 -3 10 -4 10 -5 10 -60.5 mL of each dilution suspension was evenly spread onto the surface of LB agar, Gao's No. 1 agar, and PDA-agar agar, respectively. Three parallel plates were prepared for each dilution, and the plates were inverted in an incubator at 30°C for 24-36 h. Single colonies exhibiting significant differences in color (e.g., white, milky white, pale yellow, yellow, orange, etc.), size (diameter 1-5 mm), shape (round, irregular, radial, etc.), surface condition (smooth, dry, wrinkled, glossy, etc.), edge morphology (regular, wavy, lobed, etc.), transparency (transparent, translucent, opaque), and degree of elevation (flat, raised, convex, etc.) were streaked into pure cultures on CMC-Na agar, β-glucosinolate agar, and filter paper strip agar, respectively. Strains with good growth (colon diameter ≥ 2 mm, regular single colony morphology) were selected and stored at low temperature.

[0036] The preliminarily isolated and purified strains were inoculated into CMC-Na liquid medium and cultured continuously at 28℃ and 180 rpm for 7 days. Cellulase activity was measured using the DNS method, and strains with high cellulase activity were further screened, yielding the three strains involved in this invention. The three strains were inoculated into LB medium, Gao's No. 1 medium, and PDA-agar medium, respectively, and cultured until the logarithmic growth phase. The cells were collected by centrifugation at 6000 rpm for 5 minutes. The genomes of the three strains were extracted using a column-based bacterial / fungal genomic DNA extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., Fungal Genomic DNA Extraction Kit DP317 and Bacterial Genomic DNA Extraction Kit DP302). PCR amplification of the bacterial and fungal strains was performed using primers 27F and 1492R, and ITS1 and ITS4, respectively. The nucleotide sequences of the primers are as follows:

[0037] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0038] 1492R:5'-GGTTACCTTGTTACGACTT-3'

[0039] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'

[0040] ITS4: 5'-TCCTCCGCTTATTGATATGC-3'

[0041] After purification, the PCR products were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequencing results were compared with sequences in the NCBI database for homology analysis. The three strains belonged to *Bacillus amyloliquefaciens*, *Streptomyces tendae*, and *Trichoderma harzianum*, respectively. All isolated strains have been deposited at the China General Microbiological Culture Collection Center (CGMCC).

[0042] Example 2: Preparation of the straw decomposing microbial agent according to the present invention patent application:

[0043] I. Preparation of graphene-containing BA-1 cells, graphene-containing SD-4 cells, and graphene-containing LT1 cells:

[0044] 1. Take Bacillus amyloliquefaciens BA-1 CGMCC No.36117 strain and inoculate it into nutrient-rich LB medium. Incubate overnight at 30°C to obtain seed culture.

[0045] Graphene was added to the new LB medium at a concentration of 20 mg / L. The resulting seed culture was then inoculated into LB medium containing graphene for expansion culture. The culture was carried out at 37°C and 180 rpm for 24-48 hours. After collecting the bacterial cells, skim milk powder was added and freeze-dried to obtain BA-1 bacterial cells containing graphene.

[0046] 2. Take Streptomyces tendae SD-4 CGMCC No.36120 strain and inoculate it into nutrient-rich Gao's No.1 medium. Incubate at 30°C overnight to obtain seed culture.

[0047] Graphene was added to the new Gao's No. 1 medium at a concentration of 20 mg / L. The obtained seed culture was inoculated into the graphene-containing Gao's No. 1 medium and cultured at 28°C and 150 rpm for 48-72 hours. After collecting the cells, they were freeze-dried to obtain SD-4 cells containing graphene.

[0048] 3. Inoculate Trichoderma harzianum LT1 CGMCC No.42845 strain into nutrient-rich PDA medium and incubate overnight at 30°C to obtain seed culture.

[0049] Graphene was added to a new PDA medium at a concentration of 20 mg / L. The resulting seed culture was inoculated into a graphene-containing PDA medium and cultured at 28°C in the dark for 3 days. After the mycelium had basically covered the culture dish, it was transferred to light conditions and cultured for another 2 days to promote conidia production. After a large number of green conidia formed on the surface of the plate, the surface of the culture was repeatedly rinsed with sterile deionized water, and the spore suspension was collected to obtain LT1 cells containing graphene.

[0050] II. Preparation of Straw Decomposing Microbial Agent:

[0051] 69.75 g of BA-1 bacterial cells containing graphene, 69.75 g of SD-2 bacterial cells containing graphene, and 139.5 g of LT1 spores containing graphene were mixed and then 6 g of sodium alginate, 9 g of sucrose, and 6 g of glycerol were added to obtain 300 g of straw decomposing microbial agent.

[0052] Example 3: The effect of straw decomposing bacteria on promoting decomposition:

[0053] The weight loss method was used to determine the decomposition-promoting effect of the microbial agent: Corn stalks of similar thickness and length were selected and cut into 3-5 cm segments. 50 g of each segment was accurately weighed and placed in a 40-mesh nylon mesh bag. Five bags were removed and dried in an oven at 85℃ for 6 hours. The weight of each bag was recorded, and the average weight was used as the initial control. Referring to the agricultural industry standard NY / T 2722-2015, two adjacent 100m² plots were divided... 2 The plots were plowed to a depth of 20 cm, with a soil moisture content of approximately 7%. One plot was treated with a microbial agent: 300 g of the prepared straw-decomposing microbial agent was mixed with 15 liters of water, and five bags of straw were completely immersed in the agent before being randomly buried 20 cm deep in the soil. The remaining agent was then evenly sprayed onto the plot surface. The other plot served as a control; five bags of straw were immersed in water before being randomly buried 20 cm deep in the soil, and an equal amount of water was evenly sprayed onto the plot surface. After 30 days, the straw was removed, rinsed with clean water until the water droplets were colorless, and dried at 85℃ for 6 hours. The weight of each bag was recorded, and the weight loss rate was calculated. The results are shown in Table 1. The straw-decomposing microbial agent showed a good promoting effect on straw decomposition under direct straw return conditions in the field, increasing the straw decomposition rate by 52.27% within 30 days compared to the control.

[0054] Table 1. Effect of straw decomposing microbial agents on promoting decomposition

[0055] Example 4: Enzyme activity detection and temperature and humidity adaptability detection of various strains in straw decomposing microbial agents:

[0056] 1. Enzyme activity detection: Each cryopreserved strain was inoculated into a nutrient-rich culture medium (bacteria were inoculated into LB medium, actinomycetes into Gao's No. 1 medium, and fungi into PDA medium), and cultured overnight at 30°C to obtain seed culture. Various inoculums were inoculated at 10% (v / v) onto CMC-Na liquid medium, β-glucosidase liquid medium, and filter paper strip liquid medium, respectively, and cultured continuously at 30℃ and 180 rpm for 7 days. The activities of CMC enzyme, β-glucosidase, and filter paper enzyme were determined using the DNS method. The results are shown in Table 2 (in Table 2, enzyme activity units are per gram of dry cell weight, and DW represents "dry weight"). All three strains showed high filter paper enzyme activity, indicating that the comprehensive degradation ability of the three strains was strong. The CMC enzyme activity of *Streptomyces harzianum* and *Streptomyces tangente* was outstanding, showing good potential for initiating cellulose degradation and destroying the internal structure of cellulose. The CMC enzyme activity of *Bacillus amyloliquefaciens* was relatively low, but its β-glucosidase activity was high. When combined with strains with high CMC enzyme activity, it can avoid the accumulation of cellobiose that inhibits CMC enzyme activity.

[0057] Table 2 Degradation capacity of each strain

[0058] 2. Temperature and Humidity Adaptability Test: Seed cultures of each strain were inoculated at a 10% (v / v) inoculum onto nutrient-rich culture media (bacteria on LB medium, actinomycetes on Gao's No. 1 medium, and fungi on PDA medium). Fermentation was carried out under different temperature and humidity conditions. After fermentation, the viable cell count was determined using a hemocytometer. The test results are as follows: Figure 1 As shown.

[0059] According to Table 2 and Figure 1 The results showed that *Bacillus amyloliquefaciens* BA-1 (CGMCC 36117), *Streptomyces tangutica* SD-4 (CGMCC 36120), and *Trichoderma harzianum* LT1 (CGMCC 42845) exhibited high cellulose degradation activity and broad temperature and humidity adaptability. *Trichoderma harzianum* LT1 and *Bacillus amyloliquefaciens* BA-1 reached 6 × 10⁶ cellulose degradation activity at 5℃ and 45℃, respectively. 7 Of the above viable bacterial counts, Bacillus amyloliquefaciens BA-1 stands out in terms of adaptability to a wide range of humidity levels. Streptomyces tangde SD-4 and Trichoderma harzianum LT1 can also maintain activity under both dry and humid conditions. Combining these three will give the inoculant higher field adaptability to cope with seasonal and diurnal temperature variations, as well as various field conditions such as drought and flooding.

[0060] Example 5: Effect of strain combination:

[0061] 1. Antagonism Test: Antagonism with *Trichoderma harzianum* is tested by spotting *Trichoderma harzianum* in the center of a solid culture medium and spotting two other bacterial strains around the outer edge of the medium. The presence of inhibition zones is observed; inhibition zone width ≤ 1 mm indicates compatibility. Antagonism between two bacterial strains is tested using the filter paper disc method. One strain is used as the substrate and evenly spread on the solid culture medium. The other strain is used as the test strain and spread on a filter paper disc, which is then placed on the surface of the solid culture medium with the substrate. The presence of inhibition zones is observed; inhibition zone width ≤ 1 mm indicates compatibility, otherwise, antagonism is indicated.

[0062] Trichoderma often exhibits antagonistic effects with Bacillus and Streptomyces, making co-cultivation and synergistic effects impossible. Before compounding, we conducted pairwise co-cultivations of three strains—Bacillus amyloliquefaciens BA-1 (CGMCC NO.36117), Streptomyces tangutica SD-4 (CGMCC NO.36120), and Trichoderma harzianum LT1 (CGMCC NO.42845)—using the aforementioned antagonistic experiments, and found no antagonistic effect (e.g., ...). Figure 2 (As shown).

[0063] 2. Strains compounding:

[0064] Bacillus amyloliquefaciens seed culture was inoculated into LB medium at a rate of 10% (v / v) and cultured at 37°C and 180 rpm for 24-48 hours. After collecting the bacterial cells, skim milk powder was added and freeze-dried.

[0065] The seed culture of *Streptomyces tangde* was inoculated into Gao's No. 1 medium at a rate of 10% (v / v) and cultured at 28°C and 150 rpm for 48-72 hours. The bacterial cells were then collected and freeze-dried.

[0066] The Trichoderma harzianum spore suspension was inoculated onto PDA medium and cultured at 28°C in the dark for 3 days. After the mycelium had basically covered the culture dish, it was transferred to light conditions and cultured for another 2 days to promote conidia production. After a large number of green conidia formed on the surface of the plate, the surface of the culture was repeatedly rinsed with sterile deionized water, and the spore suspension was collected.

[0067] The three types of bacterial powder were compounded according to different mass ratios in Table 3.

[0068] Decomposition rate test: After washing and drying the corn stalks, cut them into 2 cm uniform pieces. Resuspend the mixed inoculum powder in 1×10⁻⁶ solution. 7 CFU·ml -1 Take 50 mL of bacterial suspension and inoculate it onto 25 g of corn stalks. Mix well and incubate at room temperature. Take samples every seven days, wash, dry and weigh them. The weight loss rate reflects the decomposition rate.

[0069] The decomposition rates of the three bacterial powders were compounded and the proportions were adjusted. Table 3 shows that the decomposition effect of the compounded strains was higher than that of the single strains, indicating that there is a synergistic effect among the three strains. The combination with a higher proportion of Bacillus amyloliquefaciens can start the decomposition process relatively quickly, while appropriately increasing the proportion of Trichoderma harzianum can achieve a higher overall decomposition efficiency.

[0070] Table 3. Decomposition rate of each strain

[0071] Example 6: The reinforcing effect of graphene materials:

[0072] Three strains of bacteria, *Bacillus amyloliquefaciens* BA-1 (CGMCC NO. 36117), *Streptomyces tangde* SD-4 (CGMCC NO. 36120), and *Trichoderma harzianum* LT1 (CGMCC NO. 42845), were fermented for 12 hours in LB, Gao's No. 1, and PDA media, respectively, according to the method in Example 2. Graphene was added to the culture medium, along with 2% sodium alginate, 3% sucrose, and 2% glycerol by weight of the total bacterial inoculum as protective agents to form the inoculum. The effect of graphene concentration on viable cell count and cellulase activity is shown in Table 4. A higher viable cell count and cellulase activity were obtained when the graphene concentration was 20 mg / L.

[0073] Table 4. Effects of graphene concentration on viable bacterial count and cellulase activity

[0074] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. Any modifications, alterations, and variations made by those skilled in the art without departing from the scope of the present invention using the disclosed technical content are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of the present invention are still within the scope of the present invention.

Claims

1. A straw decomposing microbial agent, characterized in that: The straw decomposing microbial agent is composed of an active ingredient and a protective agent acceptable to the microbial agent; the active ingredient is composed of BA-1 microbial cells containing graphene, SD-4 microbial cells containing graphene, and LT1 microbial cells containing graphene. The graphene-containing BA-1 bacterial cells were prepared by the following method: Bacillus amyloliquefaciens BA-1 CGMCC No. 36117 strain was inoculated into LB medium and cultured overnight at 30°C to obtain a seed culture; graphene was added to fresh LB medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into graphene-containing LB medium for expansion culture at 37°C and 180 rpm for 24-48 hours. After collecting the bacterial cells, skim milk powder was added and freeze-dried to obtain the graphene-containing BA-1 bacterial cells. The graphene-containing SD-4 bacterial cells were prepared by the following method: Streptomyces tendae SD-4 CGMCC No.36120 strain was inoculated into Gao's No. 1 medium and cultured overnight at 30°C to obtain seed culture; graphene was added to fresh Gao's No. 1 medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into graphene-containing Gao's No. 1 medium and cultured at 28°C and 150 rpm for 48-72 hours. After collecting the bacterial cells, they were freeze-dried to obtain the graphene-containing SD-4 bacterial cells. The graphene-containing LT1 cells were prepared by the following method: Trichoderma harzianum LT1CGMCC No.42845 strain was inoculated into PDA medium and cultured overnight at 30°C to obtain seed culture; graphene was added to a new PDA medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into a graphene-containing PDA medium and cultured at 28°C in the dark for 3 days; after the mycelium had basically covered the culture dish, it was transferred to light conditions and cultured for another 2 days to promote conidia production; after a large number of green conidia formed on the surface of the plate, sterile deionized water was added to repeatedly rinse the surface of the culture, and the spore suspension was collected to obtain the graphene-containing LT1 cells.

2. The straw decomposing microbial agent according to claim 1, characterized in that: The mass ratio of graphene-containing BA-1 cells, graphene-containing SD-4 cells, and graphene-containing LT1 cells in the active ingredient is (1-4):(1-4):(1-4).

3. The straw decomposing microbial agent according to claim 2, characterized in that, The ratio of graphene-containing BA-1 cells, graphene-containing SD-4 cells, and graphene-containing LT1 cells in the active ingredient is selected from one of the following ratios: (1) BA-1 cells: SD-4 cells: LT1 cells = 1:1:1; or (2) BA-1 cells: SD-4 cells: LT1 cells = 1:1:2; or (3) BA-1 cells: SD-4 cells: LT1 cells = 1:2:1; or (4) BA-1 cells: SD-4 cells: LT1 cells = 2:1:1; or (5) BA-1 cells: SD-4 cells: LT1 cells = 4:1:2; or (6) BA-1 cells: SD-4 cells: LT1 cells = 4:2:1; or (7) BA-1 cells: SD-4 cells: LT1 cells = 1:4:2; or (8) BA-1 cells: SD-4 cells: LT1 cells = 2:1:4; or (9) BA-1 cells: SD-4 cells: LT1 cells = 2:4:1; or (10) BA-1 cells: SD-4 cells: LT1 cells = 1:2:

4.

4. The straw decomposing microbial agent according to any one of claims 1-3, characterized in that: The acceptable protectant for the microbial agent consists of sodium alginate, sucrose, and glycerol, wherein sodium alginate accounts for 2% of the total weight of the straw decomposing microbial agent, sucrose accounts for 3% of the total weight of the straw decomposing microbial agent, and glycerol accounts for 2% of the total weight of the straw decomposing microbial agent.

5. The method for preparing the straw decomposing microbial agent according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: (1) The BA-1 bacterial cell containing graphene, the SD-4 bacterial cell containing graphene and the LT1 bacterial cell containing graphene were prepared. The graphene-containing BA-1 bacterial cells were prepared by the following method: Bacillus amyloliquefaciens BA-1 strain was inoculated into LB medium and cultured overnight at 30°C to obtain seed culture; graphene was added to fresh LB medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into LB medium containing graphene for expansion culture at 37°C and 180 rpm for 24-48 hours; after collecting the bacterial cells, skim milk powder was added and freeze-dried to obtain BA-1 bacterial cells containing graphene; The graphene-containing SD-4 bacterial cells were prepared by the following method: *Streptomyces tangdeensis* SD-4 strain was inoculated into Gao's No. 1 medium and cultured overnight at 30°C to obtain a seed culture; graphene was added to fresh Gao's No. 1 medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into graphene-containing Gao's No. 1 medium and cultured at 28°C and 150 rpm for 48-72 hours. After collecting the bacterial cells, they were freeze-dried to obtain the graphene-containing SD-4 bacterial cells. The graphene-containing LT1 cells were prepared by the following method: Trichoderma harzianum LT1 strain was inoculated into PDA medium and cultured overnight at 30°C to obtain seed culture; graphene was added to a fresh PDA medium at a concentration of 20 mg / L, and the obtained seed culture was inoculated into a graphene-containing PDA medium and cultured at 28°C in the dark for 3 days; after the mycelium had basically covered the culture dish, it was transferred to light conditions and cultured for another 2 days to promote sporulation; after a large number of green conidia formed on the surface of the plate, sterile deionized water was added to repeatedly rinse the surface of the culture, and the spore suspension was collected to obtain the graphene-containing LT1 cells; (2) The graphene-containing BA-1 cells, graphene-containing SD-4 cells and graphene-containing LT1 cells obtained in step (1) are mixed according to the mass ratio of claim 2 or 3, and then 2% sodium alginate, 3% sucrose and 2% glycerol of the total weight of the straw decomposing microbial agent are added. After mixing, the straw decomposing microbial agent is obtained.

6. The application of the straw decomposing microbial agent according to claims 1-4 in the decomposition of straw.

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