Application of a poor water bacillus strain cmc7 in preparation of organic fertilizer

Abstract

The application provides an application of a paucity bacillus aquatilis (Bacillus aquatilis) strain cmc7 in preparation of organic fertilizer. Bacillus inaquosorum The paucity bacillus aquatilis strain cmc7 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No. 34133. The application also provides a method for preparing organic fertilizer by using the paucity bacillus aquatilis strain cmc7, and the paucity bacillus aquatilis strain cmc7 is used for solid-state co-fermentation of straws and poultry manure. The paucity bacillus aquatilis strain cmc7 can significantly increase the soluble sugar content in the fermentation product, and the addition of straws reduces the relative content of heavy metals in the fermentation product. The organic fertilizer has the functions of promoting plant growth, improving the physicochemical properties of soil, and adjusting the microbial community of soil, and can realize high-value utilization of agricultural wastes.

Description

Technical Field

[0001] This invention belongs to the field of bio-fertilizer fermentation technology, specifically involving the application of a Bacillus subtilis strain CMC7 in the preparation of organic fertilizer. Background Technology

[0002] Resource utilization of agricultural waste is an important direction for current green agricultural development and the circular economy. my country generates a large amount of crop straw and livestock manure annually. Traditional treatment methods, such as direct return to the field, composting, or simple fermentation, suffer from low utilization rates and high environmental pollution risks. Developing methods to convert these wastes into high-value-added products such as organic fertilizers and liquid fertilizers has significant economic and environmental benefits. Currently, there are reports on technologies using straw and / or livestock manure for microbial fermentation to prepare organic fertilizers or soil conditioners. However, existing technologies mostly focus on the degree of decomposition, organic matter content, or gas emission reduction, with less emphasis on optimizing processes using soluble sugars as the target product. A mature fermentation method capable of achieving high soluble sugar content is still lacking.

[0003] Furthermore, most commercially available or reported fermentation agents are complex strains with unclear fermentation targets. Functional strains that can simultaneously promote the accumulation of soluble sugars in straw and livestock manure mixtures are rarely reported. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a *Bacillus subtilis* (… Bacillus inaquosorum The application of strain CMC7 in the fermentation of organic fertilizer can increase the soluble sugar content in the fermentation products of straw and livestock manure.

[0005] This invention provides an application of Bacillus aquaticus strain CMC7 in increasing the soluble sugar content in the co-fermentation products of straw and livestock manure; the Bacillus aquaticus strain CMC7 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34133.

[0006] Preferably, the soluble sugar includes at least one of the following: mannose, rhamnose, galacturonic acid, glucose, galactose, xylose, and arabinose.

[0007] This invention provides an organic fertilizer obtained by solid-state co-fermentation of straw and livestock manure using *Bacillus aqueductus* strain CMC7; the volume ratio of the bacterial solution of *Bacillus aqueductus* strain CMC7 to the total dry weight of straw and livestock manure is 1 mL:(1~2) g; the OD of the bacterial solution of *Bacillus aqueductus* strain CMC7... 600 The value is 0.3 to 0.9.

[0008] Preferably, the mass ratio of straw to poultry and livestock manure is (4~8):(2~6).

[0009] This invention provides a method for preparing the organic fertilizer, wherein the *Bacillus silenstilae* strain CMC7 is co-fermented with straw and livestock manure in a solid state at 25°C to 35°C for 2 to 5 days; the water content of the solid-state co-fermentation system is 65% to 75%.

[0010] Preferably, after the solid-state co-fermentation is completed, the product of solid-state co-fermentation is further subjected to water extraction, solid-liquid separation, and collection of fermentation extract to obtain water-soluble organic fertilizer.

[0011] This invention provides an application of the organic fertilizer in at least one of promoting plant growth, improving soil physical and chemical properties, and regulating soil microbial communities.

[0012] Preferably, promoting plant growth includes increasing at least one of plant height, leaf length, leaf area, and above-ground dry weight.

[0013] Preferably, the improvement of soil physical and chemical properties includes increasing the soil organic matter content.

[0014] Preferably, the regulation of the soil microbial community includes increasing the relative abundance of bacteria such as *Microphytes*, *L.*, *Acidobacter*, and *Bryophytes*, while decreasing the relative abundance of fungi such as *Verticillium* and *Fusarium*.

[0015] This invention provides a *Bacillus aeruginosa* strain CMC7. Using this strain as a fermentation agent, the mixed fermentation of crop straw and livestock manure significantly increases the soluble sugar content in the fermentation products. Simultaneously, fermentation with *Bacillus aeruginosa* strain CMC7 maintains a high level of soluble protein, and the addition of straw reduces the relative content of heavy metals per unit mass of fermentation products, effectively mitigating the environmental risks associated with using livestock manure alone and achieving the reuse of agricultural waste. Attached Figure Description

[0016] Figure 1 The effects of Bacillus subtilis strain CMC7 on different fermentation products during solid-state fermentation were investigated. (a) represents soluble protein content, (b) represents soluble sugar content, and (c) represents colony count. LJ represents chili straw fermentation group, FQ represents tomato straw fermentation group, YG represents tobacco straw fermentation group, JF represents chicken manure fermentation group, LJJF represents chili straw and chicken manure 1:1 mixed fermentation group, FQJF represents tomato straw and chicken manure 1:1 mixed fermentation group, and YGJF represents tobacco straw and chicken manure 1:1 mixed fermentation group. Figure 2The content of heavy metal ions in the fermentation extract is shown in (a) for Zn, Fe, Cu and Mn, and (b) for Cd and As. Figure 3 The comparison of available nitrogen, phosphorus, and potassium nutrients in fermentation extract is shown in the figure. (a) is available phosphorus content, (b) is total potassium content, (c) is ammonium nitrogen content, and (d) is nitrate nitrogen content. Figure 4 The single-factor optimization experiment of fermentation conditions was conducted to determine the soluble sugar content and growth factor. Among them, (a) is the content of soluble sugar and soluble protein, (b) is the content of 7 monosaccharide components, (c) is the content of various heavy metal ions, and (d) is the content of nitrogen, phosphorus and potassium nutrients. Figure 5 The results of various physicochemical indicators in the fermentation extract under optimal conditions are shown in (a) for soluble sugar and protein content, (b) for monosaccharide content, (c) for heavy metal ion content, and (d) for nitrogen, phosphorus, and potassium nutrient content. Figure 6 The results of soil physicochemical properties were obtained after applying different extracts. Among them, (a) is the soil pH test result, (b) is the soil available potassium test result, (c) is the soil available phosphorus test result, (d) is the soil organic matter test result, (e) is the soil nitrate nitrogen test result, and (f) is the soil ammonium nitrogen test result. Figure 7 The bar chart shows the relative abundance of rhizosphere soil microbial communities at the phylum level, where (a) represents the composition of bacterial communities at the phylum level and (b) represents the composition of fungal communities at the phylum level. Figure 8 The bar chart shows the relative abundance of rhizosphere soil microbial genus-level communities, where (a) represents the composition of bacterial genus-level communities and (b) represents the composition of fungal genus-level communities.

[0017] Information on the preservation of biological materials Bacillus subtilis ( Bacillus inaquosorum The strain CMC7, with accession number CGMCC No. 34133, was deposited on April 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0018] This invention provides an application of the *Bacillus aqueductus* strain CMC7 or the fermentation agent in increasing the soluble sugar content in the co-fermentation products of straw and livestock manure. The *Bacillus aqueductus* strain CMC7 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34133. In this invention, CMC7 was isolated from the humus layer of fallen leaves and branches in a forest within the Tobacco Research Institute (Laoshan District, Qingdao City, Shandong Province) and was deposited at the China General Microbiological Culture Collection Center on April 8, 2025.

[0019] In this invention, the preferred method for culturing the *Bacillus aqueductus* strain CMC7 is to inoculate *Bacillus aqueductus* strain CMC7 into a liquid culture medium and then culture with shaking to obtain a bacterial suspension of *Bacillus aqueductus* strain CMC7. The liquid culture medium can be LB liquid medium, NB liquid medium, TSB liquid medium, or DSMZ liquid medium. In this embodiment, NB liquid culture is used for strain culture. This invention does not impose any special restrictions on the formulation of the NB liquid culture medium; any NB liquid culture medium formulation well-known in the art can be used, for example, an NB liquid culture medium formulation of 11 g / L peptone, 6 g / L sodium chloride, 3.5 g / L beef extract powder, and pH 7.0-7.2. In the fermentation agent, the viable cell concentration in the bacterial suspension is preferably not less than 1 × 10⁻⁶. 8 CFU / mL, which can be 5 × 10 8 CFU / mL, 1×10 9 CFU / mL or 5×10 9 CFU / mL. The preferred method for preparing the metabolites of the *Bacillus angulans* strain CMC7 is to inoculate *Bacillus angulans* strain CMC7 or the bacterial suspension into a liquid culture medium and then perform shaking culture. After shaking culture, solid-liquid separation is performed, and the liquid phase is collected to obtain the metabolites. The *Bacillus angulans* strain CMC7 preferably comprises bacterial cells and / or spores.

[0020] In this invention, the *Bacillus aquaticus* strain CMC7 is used to ferment straw and livestock manure simultaneously. Compared with straw fermentation alone, the addition of livestock manure can significantly increase the content of soluble sugars in the fermentation products. Compared with livestock manure fermentation alone, the addition of straw reduces the amount of heavy metals per unit mass, thereby reducing the risk of environmental pollution caused by the fermentation of livestock manure alone.

[0021] In this invention, the straw is preferably one or more of the following: solanaceous crop straw, gramineous crop straw, and leguminous crop straw, and can be one or more of the following: tobacco straw, chili straw, tomato straw, corn straw, wheat straw, and soybean straw. Although the composition ratios of different crop straws vary, they all contain components such as cellulose, hemicellulose, and pectin that can be degraded and utilized by the Bacillus subtilis strain CMC7.

[0022] In this invention, the straw preferably undergoes the following pretreatment before fermentation: drying, crushing, and passing through a 10-60 mesh sieve. The drying temperature is preferably 30-90℃. The purpose of the pretreatment is to reduce the moisture content and particle size of the straw, increase the specific surface area, facilitate bacterial adhesion and enzymatic hydrolysis, and simultaneously disrupt the cellulose crystal structure, thereby improving the release efficiency of soluble sugars during subsequent fermentation. Those skilled in the art can adaptively adjust parameters such as drying temperature, drying time, and crushing particle size according to the source, type, and initial moisture content of the straw; these adjustments require no creative effort.

[0023] In this embodiment of the invention, tobacco straw, chili straw, and tomato straw are used as fermentation raw materials. The straw is crushed and passed through a 10-mesh sieve. The results of the embodiments show that, compared with the uninoculated control, when fermenting straw alone, except for a slight increase in the soluble sugar content of tobacco straw, the soluble sugar content of the other straws and the available phosphorus content of all straws are significantly reduced. However, after fermenting straw mixed with chicken manure, the soluble sugar content significantly increases, with the mixed fermentation of tobacco straw and chicken manure showing the best effect. Therefore, the *Bacillus subtilis* strain CMC7 is more suitable for the mixed fermentation of tobacco straw and poultry manure, and can also be used for the separate fermentation of tobacco straw.

[0024] In this invention, the poultry and livestock manure is preferably one or more of chicken manure, pig manure, cow manure, sheep manure, and duck manure. All of these manures are characterized by high organic matter content, rich nitrogen and phosphorus content, and the presence of a certain amount of heavy metals. Mixing the poultry and livestock manure with straw and fermenting it using the Bacillus subtilis strain CMC7 can significantly increase the soluble sugar content. Simultaneously, the dilution effect of the straw reduces the relative content of heavy metals per unit mass of the product, effectively mitigating the environmental risks associated with using poultry and livestock manure alone.

[0025] In this invention, the soluble sugar preferably includes at least one of the following: mannose, rhamnose, galacturonic acid, glucose, galactose, xylose, and arabinose. Among the soluble sugars, xylose and arabinose are mainly derived from xylan and arabinoxylan in hemicellulose; galacturonic acid is the main building block of pectin; glucose is derived from cellulose and starch; and mannose and galactomannan are derived from mannan and galactomannan. These monosaccharides can be directly absorbed by plant roots, providing carbon and energy; they can also act as signaling molecules, inducing plant defense responses and promoting the colonization of beneficial rhizosphere microorganisms. Compared with traditional straw composting or direct return of chicken manure to the field, this invention, through inoculation with the Bacillus subtilis strain CMC7, achieves the efficient conversion of hemicellulose and pectin in straw and livestock manure into soluble monosaccharides, thereby obtaining organic fertilizer rich in active monosaccharides.

[0026] This invention provides an organic fertilizer obtained by solid-state co-fermentation of straw and livestock manure using Bacillus subtilis strain CMC7; the preferred ratio of the volume of the bacterial solution of Bacillus subtilis strain CMC7 to the total dry weight of straw and livestock manure is 1 mL: (1~2) g; the OD600 value of the bacterial solution of Bacillus subtilis strain CMC7 is 0.3~0.9.

[0027] In this invention, before solid-state co-fermentation, straw and livestock manure are preferably sterilized at 120°C to thoroughly kill pathogens and native bacteria present in the raw materials. Unlike traditional open aerobic composting systems that rely on natural fermentation by native bacteria, this invention uses only a single strain of *Bacillus hydrophilus* CMC7 for inoculation. This is a controlled solid-state pure culture fermentation dominated by a single functional strain, free from external bacterial interference. The fermentation metabolism and product composition are regulated solely by strain CMC7, which can directionally increase the accumulation of soluble sugars in the system. This method does not belong to a multi-strain natural composting system.

[0028] In this invention, the *Bacillus silagenicus* strain CMC7 is inoculated into the fermentation raw materials in the form of a bacterial suspension. The preferred inoculation amount of the *Bacillus silagenicus* strain CMC7 bacterial suspension is 50% to 100% of the total dry weight of the straw and livestock manure, and can be 60%, 70%, or 80%. The OD value of the *Bacillus silagenicus* strain CMC7 bacterial suspension... 600 The value is preferably 0.3~0.9, and can be 0.4, 0.7 or 0.8, more preferably 0.6. In the embodiments of this application, an inoculum optimization experiment was conducted on the *Bacillus subtilis* strain CMC7. With an inoculum size of 1 ml, the OD of the bacterial solution was... 600 The values ​​of cmc7 were 0.3, 0.6, 0.9, 1.2, and 1.5, respectively, indicating that the OD values ​​of the bacterial culture were... 600 The optimal effect is achieved when the OD value is 0.6. 600When the OD value is below 0.6, the initial number of bacterial strains is insufficient, fermentation starts slowly, and the increase in soluble sugar is limited; when the OD value of the bacterial culture is below 0.6, the initial number of strains is insufficient, fermentation starts slowly, and the increase in soluble sugar is limited. 600 When the value is higher than 0.6, bacterial metabolism is inhibited, and the yield of soluble sugars no longer increases significantly.

[0029] In this invention, the preferred mass ratio of straw to livestock manure is (4-8):(2-6), more preferably (6-8):(4-6), and most preferably 6:4. In the embodiments of this application, experiments were conducted using straw to livestock manure mass ratios of 5:5 and 6:4. When the mass ratio was 5:5, the soluble sugar content in the fermentation product after solid-state co-fermentation increased by approximately 230% compared to the uninoculated control group, while the soluble protein content decreased significantly compared to the control group. However, when the mass ratio was increased to 6:4, the soluble sugar content in the fermentation product after solid-state co-fermentation further increased, while the soluble protein content showed no significant difference from the control group. This indicates that increasing the straw content is beneficial for increasing the soluble sugar content of the fermentation product and reducing the consumption of soluble protein.

[0030] In this invention, it is preferred to co-ferment the Bacillus subtilis strain CMC7 with straw and livestock manure in a solid state at 25°C to 35°C for 2 to 5 days; the moisture content of the solid-state co-fermentation system is preferably 65% ​​to 75%.

[0031] In this invention, the preferred solid-state co-fermentation temperature is 25℃~35℃, specifically 27℃, 30℃, and 32℃, with 28℃ being the most preferred. In an embodiment of this application, an optimization experiment was conducted on the fermentation temperature of the *Bacillus aeruginosa* strain CMC7. Fermentation was carried out for five days at temperatures of 4℃, 12℃, 20℃, 28℃, 37℃, 43℃, and 50℃. The results showed that the soluble sugar content in the fermentation product was highest at a fermentation temperature of 28℃. When the fermentation temperature was below 28℃, the bacterial activity was inhibited by the low temperature, and the soluble sugar content decreased; similarly, when the fermentation temperature was above 28℃, the soluble sugar content also decreased. In this invention, the solid-state co-fermentation time is 2~5 days, with 3 days being the most preferred. In an embodiment of this application, an optimization experiment was conducted on the fermentation days for the *Bacillus aeruginosa* strain CMC7. Fermentation was carried out for 9 days at 28℃, and the fermentation products were tested on days 1, 3, 5, 7, and 9. The results showed that the soluble sugar content first increased and then decreased with prolonged fermentation time: from day 1 to day 3, the soluble sugar content increased with prolonged fermentation time, reaching a peak on day 3. This may be because the strain grows and metabolizes rapidly in the early stages of fermentation; in the later stages of fermentation, with the consumption of nutrients and the accumulation of metabolic products, some soluble sugars are further utilized by microorganisms as carbon sources, leading to a decrease in content. Therefore, a fermentation time of 2–5 days can balance high yield and production efficiency of soluble sugars, with 3 days being the optimal choice. Those skilled in the art can make appropriate adjustments within this range according to actual fermentation conditions such as temperature, moisture content, and inoculum size, and still obtain soluble sugar yields significantly better than the uninoculated control.

[0032] In this invention, the moisture content of the solid-state co-fermentation system is preferably 65%~75%, specifically 67%, 70%, or 73%, with 71.4% being the most preferred. Within this moisture content range, the water activity and aeration of the fermentation substrate are balanced, which is beneficial to the growth and metabolism of *Bacillus subtilis* CMC7 and the accumulation of soluble sugars. In the embodiments of this application, a moisture content optimization experiment was conducted on *Bacillus subtilis* strain CMC7. The experiments were carried out with the moisture addition controlled at 100%, 150%, 200%, 250%, and 300% of the total dry weight of straw and manure, respectively. The results showed that when the moisture addition was 250%, i.e., the moisture content of the solid-state co-fermentation system was 71.4%, the soluble sugar content in the fermentation product was the highest. When the moisture content was below 250%, the diffusion rate of bacteria and enzymes was limited; when the moisture content was above 250%, the porosity decreased, inhibiting aerobic metabolism.

[0033] In this invention, after the solid-state co-fermentation is completed, the product of the solid-state co-fermentation is preferably subjected to water extraction, solid-liquid separation, and collection of the fermentation extract to obtain a water-soluble organic fertilizer. The amount of water added is preferably 10-15 times the dry weight of the straw and livestock manure. The extraction method can be shaking, vortexing, or stirring, and the extraction time is 20-40 minutes. After extraction, solid-liquid separation is performed by centrifugation or filtration, and the supernatant is collected to obtain the liquid organic water-soluble fertilizer. In the embodiments of this application, after 1.5 g of straw and livestock manure has fermented, 20 mL of distilled water is added, and the mixture is vortexed at 2400-2500 r / min for 30 minutes, then centrifuged at 5500-8000 r / min for 4-6 minutes, and the supernatant is collected. This supernatant is an organic water-soluble fertilizer rich in soluble sugars. This fertilizer can be used directly or after dilution for drip irrigation, sprinkler irrigation, or watering, and is suitable for integrated water and fertilizer management systems.

[0034] This invention provides the application of the organic fertilizer obtained by the preparation method in at least one of promoting plant growth, improving soil physicochemical properties, and regulating soil microbial communities.

[0035] In this invention, promoting plant growth preferably includes increasing at least one of the following: plant height, leaf length, leaf area, and above-ground dry weight. The plant is preferably a member of the *Nicotiana* genus, including common cultivars such as *Nicotiana sapiens*, *Nicotiana sapiens*, *Nicotiana bensemii*, and *Nicotiana sapiens*. In this embodiment, flue-cured tobacco K326 from common tobacco was used as the test subject. Each pot was treated with the organic water-soluble fertilizer diluted ten times, applied at one-week intervals, and cultured for a total of 30 days. The results showed that, compared with the untreated group, the plant height, maximum leaf length, maximum leaf area, and above-ground dry weight of the organic water-soluble fertilizer treatment increased by 13.1%, 11.9%, 12.5%, and 15.1%, respectively, indicating that the organic fertilizer fermented by the *Bacillus subtilis* strain CMC7 of this invention can significantly promote the growth of the above-ground parts of the plant.

[0036] The improvement of soil physicochemical properties preferably includes increasing soil organic matter content. This application tested the soil after the tobacco cultivation experiment, and the results showed that, compared with the blank control group, the soil in the organic water-soluble fertilizer treatment group had significantly increased available potassium, available phosphorus, organic matter, ammonium nitrogen, and nitrate nitrogen content; compared with the uninoculated treatment group, the soil organic matter content increased significantly. This indicates that the organic fertilizer fermented by the *Bacillus subtilis* strain CMC7 described in this invention can significantly increase the organic matter content in the soil.

[0037] In this invention, the regulation of the soil microbial community preferably includes increasing the relative abundance of functional bacteria such as nitrogen-fixing, phosphorus-solubilizing, and organic matter-degrading bacteria, and / or decreasing the relative abundance of potential pathogenic fungi. In the embodiments of this application, 16S rRNA and ITS amplicon sequencing analysis was performed on rhizosphere soil. The results showed that after applying the organic fertilizer of this invention, the relative abundance of bacteria with growth-promoting functions, such as *Microphytes*, *L.*, and *Acidobacter*, significantly increased, while the relative abundance of potential pathogenic fungi, such as *Verticillium* and *Fusarium*, decreased. This indicates that the fertilizer of this invention can optimize the soil microecological environment and enhance soil health.

[0038] This invention provides a *Bacillus subtilis* strain CMC7 and its application in the preparation of organic fertilizer. The *Bacillus subtilis* strain CMC7 of this invention is inoculated into a mixture of straw and livestock manure at a temperature of 25-35°C, a moisture content of 65%-75%, and an inoculation amount of 50%-100% (strain OD). 600 Solid-state co-fermentation under conditions of 0.3–0.9 g / mL for 2–5 days significantly increased the soluble sugar content in the fermentation product compared to the uninoculated control. After response surface methodology optimization, the predicted soluble sugar value reached its highest level of 1.95 mg / mL on day 3. Simultaneously, soluble protein levels remained high, avoiding the protein content decrease issue observed in the separate fermentation of chicken manure. Furthermore, due to the dilution effect of straw, the relative content of heavy metals per unit mass of product decreased. Therefore, the strain described in this application can efficiently increase the soluble sugar content in the mixed fermentation product and reduce the consumption of soluble protein. As a single strain, the fermentation conditions are mild, the process parameters are well-defined, and it is easy to apply industrially and control quality.

[0039] This invention also provides an application of the organic fertilizer in at least one of promoting plant growth, improving soil physicochemical properties, and regulating soil microbial communities. Greenhouse pot experiments showed that after applying this fertilizer, tobacco plant height increased by 24.7%, maximum leaf area increased by 45.1%, aboveground dry weight increased by 40.0%, and underground dry weight increased by 65.2%; soil organic matter was significantly increased; in the rhizosphere microbial community, the relative abundance of functional bacteria such as nitrogen-fixing, phosphorus-solubilizing, and organic matter-degrading bacteria increased, while the relative abundance of potential pathogenic fungi decreased. Therefore, the organic fertilizer of this invention combines environmental safety with significant growth-promoting effects, can be used for soil improvement and microecological regulation, and the raw material is agricultural waste, making it low-cost and achieving resource recycling.

[0040] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a *Bacillus subtilis* strain CMC7 provided by the present invention and its application in the preparation of organic fertilizer, should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1 Fermentation experiment of Bacillus subtilis strain CMC7 1. Test strains and culture media Test strain: Bacillus aqueductus strain CMC7, preservation number CGMCC No. 34133.

[0042] NB liquid culture medium: peptone 11 g / L, sodium chloride 6 g / L, beef extract powder 3.5 g / L, pH 7.0~7.2, sterilized at 121℃ for 20 min.

[0043] NA solid medium: Add 18 g / L agar powder to NB liquid medium.

[0044] Preparation of bacterial suspension: CMC7 strain was inoculated into NB liquid medium and cultured at 28°C with shaking until the logarithmic growth phase was reached. OD was then adjusted. 600 The value is 0.5.

[0045] 2. Processing of fermentation raw materials Chili stalks, tomato stalks, tobacco stalks, and chicken manure were taken separately, dried, crushed, and passed through a 10-mesh sieve. The stalks and chicken manure were mixed in a 1:1 mass ratio, with 0.75 g of stalks and 0.75 g of chicken manure accurately weighed. All fermentation materials were sterilized at 120℃ to ensure that pathogens in the materials were killed.

[0046] 3. Fermentation method In this embodiment, a total of 7 treatment groups were set up: chili stalks alone (LJ), tomato stalks alone (FQ), tobacco stalks alone (YG), chicken manure alone (JF), a 1:1 mixture of chili stalks and chicken manure (LJ+JF), a 1:1 mixture of tomato stalks and chicken manure (FQ+JF), and a 1:1 mixture of tobacco stalks and chicken manure (YG+JF). Each treatment group was divided into an inoculated group and a non-inoculated control group. The inoculated group was treated with 1 mL of OD solution per 1.5 g of dry material. 600 =0.5% of the *Bacillus subtilis* CMC7 bacterial suspension, and the control group was added with an equal amount of sterile water. Both were fermented in a semi-closed solid state at 28°C for 5 days.

[0047] 4. Extraction of fermentation products After fermentation, add 20 mL of sterile water to the centrifuge tube and vortex at 2500 r / min for 30 min. Dilute a portion of the suspension 100,000 times and spread it on a nanoplatelet, recording the colony count. Centrifuge the remaining suspension at 8000 r / min for 6 min, and use the supernatant to determine the content of soluble sugars, soluble proteins, available phosphorus, ammonium nitrogen, nitrate nitrogen, total potassium, and heavy metal ions. Dry and weigh the remaining solid.

[0048] 5. Detection of fermentation products (1) Determination of soluble protein content (Coomassie Brilliant Blue method) Take 100 μL of sample supernatant (appropriately diluted) into a 1.5 mL microcentrifuge tube, add 1 mL of protein staining reagent (100 mg Coomassie Brilliant Blue G250 dissolved in 50 mL of 95% ethanol, mixed with 100 mL of 85% phosphate, and diluted to 1 L with distilled water), mix well, and measure the absorbance within 2-60 min. Prepare a standard curve using γ-globulin standard solution.

[0049] The results of soluble protein detection are shown below. Figure 1 (a) Compared with the uninoculated control, the soluble protein content of the three types of straw (chili pepper, tomato, and tobacco) increased after inoculation fermentation; the soluble protein content of chicken manure fermentation decreased; after straw and chicken manure were mixed in a 1:1 ratio for fermentation, the soluble protein content decreased compared with the unfermented straw, but the content was still at a higher level than that of straw fermentation alone.

[0050] (2) Determination of soluble sugar content (DNS method) Pipette 0.5 mL of the sample supernatant into a glass test tube, add 0.5 mL of DNS reagent, heat in a boiling water bath for 10 min, cool in an ice-water bath, dilute with an equal volume of distilled water, and measure the absorbance at a wavelength of 520 nm. Prepare a standard curve using glucose standard solution and calculate the soluble sugar concentration.

[0051] The results of the soluble sugar content test are shown below. Figure 1 (b) Compared with the uninoculated control, the soluble sugar content of tomato and pepper straw fermented alone after inoculation was significantly reduced; the soluble sugar content of tobacco straw increased slightly after fermentation, but remained at a low level; the soluble sugar content of chicken manure fermented alone decreased; the soluble sugar content of straw and chicken manure mixed in a 1:1 ratio during fermentation was significantly increased, with the mixed fermentation of tobacco straw and chicken manure showing the best effect.

[0052] (3) Colony count After fermentation, add 20 mL of sterile water to the centrifuge tube and vortex at 2500 r / min for 30 min. Take a portion of the suspension, dilute it 100,000 times, spread it on NA plates, and record the colony count.

[0053] The colony count results are shown in Figure 1 (c) Results showed that the number of colonies was higher after fermentation of straw alone, indicating that the strain grew well on straw; the number of colonies decreased sharply after fermentation of chicken manure alone compared with the straw group, indicating that the chicken manure environment had a certain inhibitory effect on the growth of the strain; while the number of colonies in the 1:1 mixture of straw and chicken manure was between the two, and the number of colonies in the mixture of tobacco straw and chicken manure remained at a high level, which was consistent with the increasing trend of soluble sugar content.

[0054] (4) Determination of heavy metal ion content Three types of straw fermentation extracts were diluted 20 times, and pure chicken manure and straw-chicken manure mixed fermentation extracts were diluted 40 times. The contents of elements such as Cu, Zn, Fe, Mn, Cd, and As were determined by ICP.

[0055] The results of heavy metal ion detection are shown below. Figure 2 The results showed that the content of heavy metals such as Cu, Zn, Cd, and As in the fermented extract of chicken manure alone was significantly higher than that in the fermented extract of straw. After fermentation of straw and chicken manure in a 1:1 ratio, the content of each heavy metal in the unit mass product was reduced by about 50% compared with that of chicken manure fermentation alone, effectively reducing the environmental risk of heavy metals.

[0056] (5) Determination of available phosphorus content Dilute the fermentation extract 5 times with 0.5 mol / L Na2CO3 solution, take 10 mL and put it into a 50 mL volumetric flask, add 5 mL of molybdenum antimony sulfate anti-mixing colorimetric reagent, dilute to the mark, let stand for 30 min, and then measure the color at a wavelength of 880 nm. Prepare a standard curve using phosphorus standard solution.

[0057] Results of available phosphorus determination are shown below Figure 3 (a) The results showed that the content of available phosphorus in the mixed fermentation extract was higher than that of straw fermentation alone, but lower than that of chicken manure fermentation alone.

[0058] (6) Determination of total potassium content After dilution, the fermentation extract was measured using an Optima 8000 inductively coupled plasma optical emission spectrometer (ICP).

[0059] The results of the total potassium test are shown below. Figure 3 (b) The results showed that the total potassium content in the mixed fermentation extract was higher than that of straw fermentation alone, but lower than that of chicken manure fermentation alone.

[0060] (7) Determination of ammonium nitrogen and nitrate nitrogen content Take 1 mL of fermentation extract, add 4 mL of 1 mol / L KCl solution, filter through a 0.22 μm filter membrane, and measure using a flow analyzer.

[0061] The results of ammonium nitrogen and nitrate nitrogen detection are shown below. Figure 3 (c) and Figure 3 (d) The results showed that the contents of ammonium nitrogen and nitrate nitrogen in the mixed fermentation extract were higher than those of straw fermentation alone, but lower than those of chicken manure fermentation alone.

[0062] In summary, after fermenting straw alone, compared to the uninoculated control, inoculated fermentation increased the soluble protein content, but significantly decreased the soluble sugar and available phosphorus content. Only tobacco straw showed an increase in soluble sugar content after fermentation, but it remained at a low level. After fermenting chicken manure alone, compared to the uninoculated control, the soluble protein content decreased, the soluble sugar content increased, the bacterial count in the fermentation product decreased sharply compared to straw, and the heavy metal ion content was high. After fermenting straw and chicken manure together, compared to the uninoculated control, the soluble protein content decreased, but remained at a high level, while the soluble sugar content increased significantly. Among these, the mixed fermentation of tobacco straw and chicken manure showed the best effect, with the soluble sugar content increasing by 230% compared to the uninoculated control group. Compared to fermenting chicken manure alone, the addition of straw reduced the risk of heavy metal ion contamination.

[0063] Example 2 Optimization of fermentation conditions for Bacillus subtilis strain CMC7 Example 2 determined that tobacco straw and chicken manure were the optimal raw material combination. With the soluble sugar content after fermentation as the optimization target, the combination of tobacco straw and chicken manure was successively optimized by single factor and response surface methodology.

[0064] 1. Single-factor optimization (1) Determination of fermentation material ratio: Prepare solid fermentation medium with a total amount of 1.5 g of chicken manure and tobacco straw in mass ratios of 0:10, 2:8, 4:6, 6:4, 8:2, and 10:0, and add 1 ml of OD 600 A CMC7 bacterial suspension with a value of 0.5 was used as the control, with no bacterial suspension added. The water content was controlled at 200%. The suspension was cultured at 28 °C for 5 days. The soluble sugar content was extracted with 20 mL of water and determined using the same method as above. The optimal growth ratio was evaluated using (treatment value - control value) / control.

[0065] The results of the single-factor optimization of chicken manure ratio are shown below. Figure 4 (a) The results showed that the soluble sugar content in the fermentation extract gradually increased with increasing chicken manure content, but the increase factor reached a maximum of 2.29 times when the ratio of chicken manure to tobacco straw was 4:6, and then began to decrease. Based on this, the optimal raw material ratio was determined to be 4:6 for chicken manure to tobacco straw.

[0066] (2) Optimization of inoculum size: 1 ml OD was inoculated separately. 600 CMC7 bacterial suspensions with values ​​of 0.3, 0.6, 0.9, 1.2, and 1.5 were prepared, and an equal amount of water was added to the control. The optimal material ratio obtained in the previous step was used, with 200% water added. The mixtures were cultured at 28 °C for 5 days, and the soluble sugar content of the fermentation extract was determined as above.

[0067] The results of the single-factor optimization of vaccination volume are shown below. Figure 4(b) The results showed that with the increase of the inoculum size, the soluble sugar content in the fermentation extract decreased at OD0.05. 600 The growth factor reached its maximum of 2.94 times when the value was 0.6, and then began to decline. From this, we can conclude that the OD of the inoculated bacterial culture... 600 A value of 0.6 is optimal.

[0068] (3) Moisture content optimization test: The amount of water added was controlled to be 100%, 150%, 200%, 250%, and 300% of the dry weight of straw and chicken manure, and 1 ml of OD was added. 600 The optimal material ratio was used to prepare a 0.6% CMC7 bacterial suspension, with no bacterial suspension added to the control. Fermentation was carried out at 28℃ for 5 days, and the fermentation extract was collected as above to determine the soluble sugar content.

[0069] The results of single-factor optimization for water content are shown below. Figure 4 (c) The results showed that as the water content increased from 100% to 250%, the soluble sugar content in the fermentation extract gradually increased, reaching a peak at 250%, with the maximum increase also occurring at this fold. When the water content was further increased to 300%, the soluble sugar content decreased significantly. Therefore, the optimal water content was 250%.

[0070] (4) Fermentation temperature optimization: Chicken manure and tobacco straw were mixed at a dry matter ratio of 4:6, followed by inoculation with 1 ml of a suspension of the barren water-poor Bacillus strain CMC7 with an OD600 value of 0.6, and the water content of the fermentation system was controlled at 200%. The blank control group was replaced with an equal volume of sterile water to replace the bacterial suspension. Fermentation was carried out for five days at temperatures of 4 ℃, 12 ℃, 20 ℃, 28 ℃, 37 ℃, 43 ℃, and 50 ℃. The soluble sugar content of the fermentation extract was determined as above.

[0071] The results of single-factor optimization of fermentation temperature are shown below. Figure 4 (d) The results showed that temperature changes had a significant impact on microbial metabolism. The soluble sugar content in the fermentation extract gradually increased between 4 ℃ and 28 ℃, peaking at 28 ℃, where the growth factor also reached its maximum; it gradually decreased between 37 ℃ and 50 ℃. It is possible that excessively low or high temperatures could reduce the metabolic rate and enzyme activity of the strains; therefore, 28 ℃ was determined to be the optimal fermentation temperature.

[0072] (5) Optimization of fermentation days: Optimal material ratio, moisture content 200%, inoculation OD 600 A 0.6% CMC7 bacterial suspension was prepared, with an equal volume of water added to the blank. Fermentation was carried out at 28 °C for 9 days. The fermentation extract was collected on days 1, 3, 5, 7, and 9 as above to determine the soluble sugar content. Fermentation continued before the designated number of days was reached.

[0073] The results of single-factor optimization of fermentation days are shown below. Figure 4(e) The results showed that the soluble sugar content in the fermentation extract gradually increased when the fermentation time was between 1 and 3 days; the content reached its maximum on the 3rd day, and the growth rate also reached its maximum at the same time; it decreased from 5 to 9 days. Therefore, a fermentation time of 3 days is the optimal fermentation time.

[0074] 2. Response Surface Optimization Experiment With a fixed material ratio of 6:4, select OD 600 Four factors were considered: water content (A), moisture content (B), fermentation temperature (C), and fermentation days (D). Each factor had three levels. The specific experimental factors and levels are shown in Table 1. A Box-Behnken design was used to establish the experimental scheme, and the experimental design and results are shown in Table 2. Solid-state fermentation was carried out according to the design scheme for each combination, and the soluble sugar content was measured as the response value. Design-Expert 13 software was used to perform multiple regression fitting on the experimental data, establishing a quadratic polynomial model to analyze the main effects and interaction effects of each factor.

[0075] The obtained quadratic polynomial model is: Soluble sugar content = 1.94347 + 0.112757A + 0.166751B + 0.0988629C + 0.211901D - 0.0100282AB - 0.00587236AC - 0.0172059AD - 0.0318011BC - 0.0744818BD - 0.0712875CD - 0.289998A 2 -0.282369B 2 -0.257026C 2 -0.377241D 2 .

[0076] Based on the process parameters predicted by the model for maximizing soluble sugar content, the optimal conditions are: material ratio 6:4, inoculum amount OD 600 With an optimal fermentation conditions of 0.63, a temperature of 28.5℃, a moisture content of 7.14%, and a fermentation time of 2.93 days, the predicted soluble sugar content was 1.952 mg / mL. Three repeated fermentation experiments were conducted under these predicted optimal conditions, and the actual soluble sugar content was compared with the predicted value to verify the reliability of the model. The contents of soluble sugars, proteins, monosaccharides, heavy metal ions, and nitrogen, phosphorus, and potassium nutrients in the fermentation extract under optimal conditions were measured. The results are shown in [Figure number missing]. Figure 5 .

[0077] Table 1 Experimental Factors and Levels

[0078] Table 2 Box-Behnken Experimental Design and Results

[0079] Example 3 Tobacco growth experiments were conducted using fermentation products from the Bacillus subtilis strain CMC7. 1. Experimental Materials The tested tobacco variety was K326. The tested soil was ordinary farmland soil that had not been treated with chemical fertilizers or pesticides. After air drying, it was sieved to remove stones and plant residues, and mixed with 5% vermiculite to improve its looseness. Each pot contained 220g of soil.

[0080] 2. Experimental treatment A total of three processing groups were set up: CK (Water Control): Each pot was watered with an equal amount of distilled water; X (Uninoculated extract group): The tobacco straw-chicken manure mixture (1:1) without bacterial strain was treated under the same fermentation conditions, then extracted with water, centrifuged to obtain the supernatant, diluted 10 times and then used for irrigation. V (Fermentation Extract Group): The tobacco straw-chicken manure mixture (1:1) inoculated with Bacillus subtilis CMC7 was fermented under optimized conditions, then extracted with water, centrifuged to obtain the supernatant, diluted 10 times and then used for irrigation.

[0081] Each treatment group was repeated 18 times.

[0082] 3. Experimental Procedure After tobacco seedlings were bred, when the third or fourth true leaves had just emerged, tobacco plants of similar size and growth were selected and transplanted into seedling trays, one seedling per tray. After one week of cultivation, tobacco seedlings of similar size and growth were selected and transplanted into flower pots filled with soil in advance. After observing them for one day and waiting for their growth to stabilize, the collected fermentation extract was diluted tenfold, with 40 mL added to each pot. The control group was given an equal amount of water. The fermentation extract was added once every week, and watering was carried out once in the middle of this period (i.e., on the fourth day). All treatments were kept consistent and cultivated in an artificial climate chamber at 25 ℃ for a total cultivation time of 30 days.

[0083] 4. Measurement of Tobacco Growth Indicators After cultivation, the plant height, number of effective leaves, maximum leaf length, and maximum leaf width were measured, and the maximum leaf area (leaf length × leaf width) was calculated. The above-ground parts and roots were separated, dried, and weighed for both above-ground and underground dry weight. The measurement results are shown in Table 3.

[0084] Application of the fermented extract (V) of this invention significantly increased tobacco plant height, maximum leaf length, maximum leaf area, and aboveground dry weight, with effects significantly superior to the water control and the uninoculated extract. Regarding maximum leaf width and aboveground dry weight, the fermented extract was comparable to the uninoculated extract, but significantly superior to the control. Overall, the fermented extract showed the most significant promoting effect on the aboveground growth of tobacco, while its root-promoting effect was not significantly different from that of the uninoculated extract.

[0085] Table 3. Measurement of Tobacco Growth Indicators

[0086] 5. Determination of soil physicochemical properties (1) pH value: A certain amount of soil was weighed into a centrifuge tube, pure water was added, and the soil-to-liquid ratio was kept at 1:2.5. The tube was placed on a shaker at 160 r / min for 1 h. After shaking, the tube was removed and placed on a table to stand for 30 min. Using a PXBJ-287L portable ion meter, the electrode was inserted into the soil suspension. The reading was recorded after the reading stabilized. Each treatment was repeated three times. The test results are shown in the figure. Figure 6 (a)

[0087] (2) Available potassium: Referring to "Soil Agrochemical Analysis", the flame photometer method was used. Soil samples were weighed into centrifuge tubes, and 1 mol / L NH4OAc solution was added. The soil-to-liquid ratio was 1:10. The samples were placed on a shaker at 180 r / min for 30 min, centrifuged at 5000 r / min, and the supernatant was filtered through a syringe through a 0.22 μm water film into centrifuge tubes. The readings were taken on the flame photometer. Preparation of standard curve: 100 mg / L K standard solution was pipetted into 100 mL volumetric flasks at concentrations of 0, 2, 5, 10, 20, 40, 60, 80, and 100 mL. The volume was adjusted to 1 mol / L NH4OAc solution, and the readings were taken from low to high on the flame photometer.

[0088] Where V is the volume of the influent (mL), m is the soil weight (g), and c is the concentration (μg / mL) calculated from the standard curve.

[0089] Test results are shown Figure 6 (b)

[0090] (3) Available phosphorus: Weigh a soil sample, add 0.5 mol / L NaHCO3 solution, with a solid-liquid ratio of 1 g of soil to 20 mL of solution. Shake on a shaker for 30 min at a rotation speed of 150 r / min, then centrifuge at 4000 r / min for several minutes. Filter the supernatant through a filter with a pore size of 0.22 μm using a syringe, pipette 1 mL of the filtered liquid into a 5 mL centrifuge tube, add 0.5 mL of sulfuric acid-molybdenum antimony ascorbic acid chromogenic agent, mix thoroughly to release CO2, add water to reach a constant volume of 5 mL, and mix thoroughly again. After waiting for 30 min, perform colorimetry with a microplate reader at a wavelength of 880 nm. Preparation of P standard curve: Pipette 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mL of phosphorus standard solution with a concentration of 5 mg / L into centrifuge tubes respectively, add 1 mL of 0.5 mol / L NaHCO3 solution and 0.5 mL of sulfuric acid-molybdenum antimony ascorbic acid chromogenic agent, wait until no more bubbles are generated, then bring to a constant volume of 5 mL, conduct colorimetry with the microplate reader in the same way as for the test solution, and then draw the standard curve.

[0091] Wherein ρ is the concentration obtained from the standard curve, V is the constant volume after color development, ts is the aliquot multiple, and m is the weight of soil.

[0092] The detection results are shown in Figure 6 (c).

[0093] (4) Organic matter: With reference to *Soil Agrochemical Analysis*, the potassium dichromate dilution heat method is used. Weigh the soil sample into a conical flask, add 1 mol / L 1 / 6K2Cr2O7 solution with a solid-liquid ratio of 1:20, mix well, slowly add concentrated sulfuric acid along the flask wall, shake gently to mix, let stand for 30 min, add water for dilution, add 2 drops of 1,10-phenanthroline indicator, titrate with 0.5 mol / L FeSO4 standard solution until the color of the solution changes from green to dark green and then to brick red, record the titration volume. The blank (without soil sample) follows the same procedure as the sample, and the organic matter content is calculated according to the formula.

[0094] Wherein 1.724 is the conversion coefficient from soil organic carbon to soil organic matter, 1.33 is the correction coefficient, c is the concentration of ferrous sulfate standard solution, and m is the weight of soil (g).

[0095] The detection results are shown in Figure 6 (d).

[0096] (5) Ammonium nitrogen and nitrate nitrogen: Weigh a certain amount of soil sample into a 50 mL centrifuge tube, add 1 mol / L KCl solution, the solid-liquid ratio is 1:5, tighten the cap and shake on a shaker at 180 r / min for 2 h, centrifuge at 4500 r / min for 5 min, use a disposable syringe to draw the supernatant and filter it through a 0.22 μm water membrane into a 10 mL centrifuge tube, and use a fully automatic nutrient analyzer to determine it.

[0097] Where ρ is the concentration measured by the analyzer, V is the volume of the added extract, and m is the weight of the soil.

[0098] Test results are shown Figure 6 (e) and (f).

[0099] Based on soil testing results after the greenhouse experiment, the fermented extract treatment (V) showed the most significant improvement in soil physicochemical properties. Compared with the water control (CK) and the uninoculated extract treatment (X), the soil organic matter content in the V treatment was significantly increased. Compared with the water control (CK), the contents of available phosphorus, available potassium, ammonium nitrogen, and nitrate nitrogen were all significantly increased, indicating that the fertilizer of this invention can directly supplement available nutrients and promote the accumulation of soil organic matter. The soil pH value decreased slightly after treatment, but remained within the suitable range for tobacco growth, and did not adversely affect the soil acid-base balance. In contrast, although the above indicators of the uninoculated extract treatment (X) were slightly higher than those of CK, the overall improvement was not as great as that of the V treatment. In summary, the organic water-soluble fertilizer of this invention has the effect of improving soil fertility and enhancing soil nutrient supply capacity.

[0100] 3. Soil rhizosphere microbial detection Rhizosphere soil samples underwent microbial diversity sequencing at Shanghai MajorBio Biotechnology Co., Ltd. Amplicon sequencing of the V3–V4 region of the 16S rRNA gene was performed using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3', SEQ ID NO:1) and 806R (5'-GGACTACHVGGGTWTCTAAT-3', SEQ ID NO:2). Amplicon sequencing of the ITS region of ribosomal DNA was performed using primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3', SEQ ID NO:3) and ITS2R (5'-GCTGCGTTCTTCATCGATGC-3', SEQ ID NO:4). Microbial community structure analysis was conducted on the MajorBio cloud platform (https: / / www.majorbio.com / ).

[0101] The results of the microbial community structure analysis are shown below Figure 7 . Figure 7 As shown in (a), at the phylum level, the soil bacterial communities in each treatment mainly consisted of dominant phyla such as Proteobacteria, Acidobacteriota, Actinobacteriota, Chloroflexi, Bacillota, Patescibacteria, and Bacteroidota. Actinobacteria maintained a high proportion in all treatments. Compared to the control (CK), the relative abundance of Actinobacteria increased after the application of the extract (X and V). Actinobacteria can produce various extracellular enzymes, degrading complex organic matter such as cellulose, hemicellulose, and lignin; and participate in soil organic matter formation. The relative abundance of Proteobacteria in treatment V was higher than in treatments X and CK. The increase in Proteobacteria abundance is usually related to enhanced metabolic activity of the soil microbial community, possibly through participation in the cycling of small molecules generated during organic matter degradation. The relative abundance of Bacteroidetes increased in treatments X and V compared to the control (CK). They possess strong polysaccharide-degrading capabilities, decomposing plant residues, proteins, and polysaccharide organic matter, playing a crucial role in organic matter transformation and nutrient release. Conversely, the relative abundance of Acidobacteria and Chlorconiosis decreased after application of the extract. These two phyla tend to be slow-growing and are commonly found in environments with low organic matter or stable conditions; their decrease also indicates improved soil physicochemical properties and increased organic matter.

[0102] Changes in fungal community composition at the phylum level, such as Figure 7 As shown in (b), the soil fungal community was mainly composed of dominant phyla such as Ascomycota, Basidiomycota, and Mortierellomycota. Ascomycota had the highest abundance and was the absolute dominant phylum, but its relative abundance decreased with the application of the extract. Conversely, the abundance of Chytridiomycota increased after the addition of the extract, showing a distribution of V > X > CK. Chytridiomycota can decompose various organic substances and is particularly suitable for environments rich in organic substrates; its increased abundance indicates an increase in available substrate supply. Mortierellomycota showed little change in abundance across the three treatment groups, maintaining a high level. Mortierellomycota are important growth-promoting fungi, promoting organic matter decomposition and phosphorus conversion, and producing plant growth-promoting substances. Basidiomycota showed a certain increasing trend after the application of the extract, also showing a distribution of V > X > CK. Basidiomycota contains a large number of fungi with strong degradation capabilities, playing an important role, especially in the decomposition of complex organic matter such as lignin and cellulose.

[0103] Horizontal community composition of bacteria, such as Figure 8As shown in (a), the acidibacterium-related taxa norank_f_LWQ8, Burkholderia-Caballeronia-Paraburkholderia, and Bryobacter all showed an increasing trend after the addition of the extract. Among them, the abundance of Bryobacter was higher in treatments X and V compared to CK. Furthermore, in treatment V, the relative abundance of Hyphomicrobium, Rhodanobacter, and Acidibacter were all higher than in treatments CK and X. The norank_f_LWQ8 genus typically participates in soil organic matter decomposition and carbon cycling, utilizing various complex organic substrates to promote plant growth. Bryobacterium species are closely involved in soil organic matter decomposition, utilizing polysaccharides to participate in plant residue degradation and carbon cycling, improving the growth environment for plants and microorganisms. Hyphomicrobium is a methyltrophic bacterium that utilizes simple organic compounds to participate in nitrogen cycling and organic matter transformation processes. The *L.* genus *L.* participates in soil nitrogen cycling, with some members possessing denitrification capabilities, regulating nitrate nitrogen transformation, and enhancing nitrogen cycle activity. The *Acidobacter* genus can utilize various organic acids to participate in organic matter transformation and soil carbon cycling, adapting to environments rich in organic matter.

[0104] Horizontal fungal community composition such as Figure 8 As shown in (b), the dominant genera mainly include *Gamasiella*, *Fusarium*, *Solicoccozyma*, *Fungi_gen_Incertae_sedis*, and *Lobulomycetales_gen_Incertae_sedis*. Among them, the *Fusarium* genus increased in treatment X compared to CK, but decreased in treatment V. Some species in this genus are important plant pathogens (causing root rot and wilt). Its decrease may be related to the inhibition by lipopeptide antimicrobial substances produced by CMC7 in the fermentation extract. The relative abundance of *Solicoccozyma* increased after the addition of the extract. It belongs to the yeast genus, has strong environmental adaptability, absorbs and utilizes soluble organic matter, and participates in soil organic matter transformation and nutrient cycling. In addition, the relative abundance of *Trichoderma* and *Fusicolla* increased after the application of the extract. Trichoderma is a typical biocontrol fungus that antagonizes pathogens, produces various extracellular enzymes to degrade cellulose and organic matter, and promotes plant root growth. Conversely, the relative abundance of Verticillium decreased after the addition of the extract. This genus contains various pathogens causing vascular diseases. Its decrease may be related to increased competition and antagonism among beneficial bacteria.

[0105] The results of the rhizosphere microbial composition analysis show that the application of fermentation products promoted the enrichment of some functional microbial groups. The relative abundance of bacterial communities related to nitrogen fixation, phosphorus solubilization, or organic matter degradation increased after treatment with the fermentation extract. The relative abundance of some fungal communities related to organic matter decomposition or plant interactions also increased, while the abundance of potential pathogens decreased. This indicates that the fermentation extract improved the soil microbial community structure to some extent.

[0106] 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 type of barren water bacillus ( Bacillus inaquosorum Application of strain CMC7 in increasing the soluble sugar content in the co-fermentation products of straw and livestock manure; The *Bacillus silens* strain CMC7 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34133.

2. The application according to claim 1, characterized in that, The soluble sugar includes at least one of the following: mannose, rhamnose, galacturonic acid, glucose, galactose, xylose, and arabinose.

3. An organic fertilizer, characterized in that, To utilize the barren water bacillus ( Bacillus inaquosorum The strain CMC7 was obtained through solid-state co-fermentation of straw and livestock manure. The volume ratio of the bacterial solution of the *Bacillus aqueductus* strain CMC7 to the total dry weight of straw and livestock manure was 1 mL:(1~2) g; the OD of the bacterial solution of the *Bacillus aqueductus* strain CMC7 was... 600 The value is 0.3 to 0.

9.

4. The organic fertilizer according to claim 3, characterized in that, The mass ratio of straw to poultry and livestock manure is (4~8):(2~6).

5. A method for preparing the organic fertilizer according to claim 3 or 4, characterized in that, The *Bacillus subtilis* strain CMC7 was co-fermented with straw and livestock manure at 25°C to 35°C for 2 to 5 days; the water content of the solid-state co-fermentation system was 65% to 75%.

6. The preparation method according to claim 5, characterized in that, After the solid-state co-fermentation is completed, the product of solid-state co-fermentation is further subjected to water extraction, solid-liquid separation, and collection of fermentation extract to obtain water-soluble organic fertilizer.

7. The use of the organic fertilizer of claim 3 or 4 in promoting plant growth, improving soil physical and chemical properties, and regulating soil microbial community in at least one of the following ways.

8. The application according to claim 7, characterized in that, The promotion of plant growth includes increasing at least one of the following: plant height, leaf length, leaf area, and above-ground dry weight.

9. The application according to claim 7, characterized in that, The improvement of soil physical and chemical properties includes increasing the soil organic matter content.

10. The application according to claim 7, characterized in that, The regulation of the soil microbial community includes increasing the relative abundance of bacteria such as *Microphytes*, *L.*, *Acidobacter*, and *Bryophytes*, while decreasing the relative abundance of fungi such as *Verticillium* and *Fusarium*.

Images