Bacillus subtilis and application thereof
By using Bacillus subtilis strain DW-14, the problem of insufficient cellulase and protease secretion capacity in existing technologies has been solved, achieving efficient fermentation of livestock and poultry manure and preparation of organic fertilizer, thereby improving soil quality and crop production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEBEI FUHUA ORGANIC FERTILIZER CO LTD
- Filing Date
- 2026-04-16
- Publication Date
- 2026-07-03
AI Technical Summary
Existing Bacillus subtilis strains have insufficient cellulase and protease secretion capabilities, resulting in long fermentation cycles of livestock and poultry manure, insufficient organic fertilizer decomposition, and low effective viable bacteria count, which fails to effectively improve soil and increase crop yields.
Using Bacillus subtilis strain DW-14, its cellulase and protease activities were enhanced through specific culture media and fermentation conditions. This strain was then applied to the fermentation of sheep manure to prepare organic fertilizer. Combined with appropriate fermentation and aging steps, a highly efficient microbial organic fertilizer was produced.
It improves the cellulose degradation rate and protein decomposition efficiency of organic fertilizer, shortens the fermentation cycle, increases the number of effective live bacteria and organic matter content of organic fertilizer, improves soil structure, and increases crop yield and quality.
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a Bacillus subtilis strain and its applications. Background Technology
[0002] The rapid development of livestock and poultry farming has generated a large amount of livestock and poultry manure waste. Sheep manure, as an important type, is produced in large quantities and widely distributed. If it is not effectively treated and is indiscriminately piled up or discharged, it will not only waste resources such as nitrogen and phosphorus, but also cause serious pollution to the surrounding soil, water bodies, and atmosphere due to the large number of pathogens, parasite eggs, and malodorous substances contained in the manure, thus damaging the ecological environment. Turning livestock and poultry manure into organic fertilizer through microbial fermentation is an important way to realize the resource utilization of agricultural waste and turn waste into treasure. It can not only solve the pollution problem of livestock and poultry manure, but also replenish soil organic matter and improve soil structure, meeting the development needs of green agriculture and ecological agriculture.
[0003] Traditional manure composting relies on natural microorganisms, resulting in long fermentation cycles, slow temperature rise, and strong odors. Utilizing microbial inoculants is an effective way to solve these problems. By adding exogenous functional microorganisms, the decomposition of organic matter can be accelerated, the composting cycle shortened, and the quality of composting improved. For example, cellulase secreted by Bacillus subtilis can efficiently degrade large cellulose molecules in crop straw and livestock manure, while proteases can break down proteins into smaller peptides and amino acids. This not only accelerates the composting process of livestock manure but also enhances the nutritional availability of organic fertilizer.
[0004] Currently, existing Bacillus subtilis strains exhibit low enzyme activity, particularly insufficient secretion of cellulase and protease. This results in low degradation efficiency of organic matter in livestock and poultry manure, long fermentation cycles, and insufficient composting of the organic fertilizer. When applied to fermented livestock and poultry manure, the resulting organic fertilizer has low effective viable bacteria count and insufficient organic matter content. Furthermore, its colonization ability after being applied to the soil is weak, failing to achieve the expected soil improvement and crop yield increase effects.
[0005] Therefore, it is necessary to develop a Bacillus subtilis strain and its applications to address the aforementioned shortcomings. Summary of the Invention
[0006] The purpose of this invention is to provide a Bacillus subtilis strain and its application. This strain has a high cellulase and protease secretion capacity, which can improve fermentation efficiency and organic fertilizer quality. The resulting organic fertilizer can improve soil, inhibit crop diseases, and increase yield and quality when applied to crop planting.
[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A type of Bacillus subtilis ( Bacillus subtilisThe sample was classified and named Bacillus subtilis DW-14, with accession number CGMCC No. 37076, accession date December 15, 2025, and deposited by the China General Microbiological Culture Collection Center.
[0008] It also provides the application of Bacillus subtilis DW-14 in the preparation of microbial organic fertilizer.
[0009] Furthermore, using sheep manure as a fermentation raw material, Bacillus subtilis DW-14 was applied as a fermentation bacteria in the fermentation of sheep manure.
[0010] The steps for using Bacillus subtilis DW-14 to ferment sheep manure and prepare organic fertilizer are as follows: (1) Bacillus subtilis DW-14 was inoculated into a culture medium and cultured to prepare a bacterial agent; (2) Add the microbial agent from step (1) to the sheep manure and crush it; (3) Aerate and ferment the crushed material from step (2) for 18 to 22 days; (4) After fermentation, the product is aged once and then crushed again. After crushing, it is aged a second time. (5) The sheep manure that has been aged twice in step (4) is put into a mixer and stirred evenly, then screened by a screening machine, and finally packaged.
[0011] Furthermore, the content of each component of the culture medium in step (1) is 7.5 g / L soluble starch, 10 g / L soybean meal, and 15 g / L disodium hydrogen phosphate, and the pH of the culture medium is 7.5; Furthermore, the inoculum amount of Bacillus subtilis DW-14 in step (1) is 3% to 5%, and fermentation is carried out at a speed of 150 to 170 r / min for 40 to 60 h.
[0012] Furthermore, in step (4), the first aging time is 6 to 8 days, and the second aging time is 8 to 12 days.
[0013] Compared with the prior art, the beneficial technical effects of the present invention are as follows: The Bacillus subtilis DW-14 of this invention exhibits excellent enzyme activity, with both cellulase and protease activities at high levels. The cellulose degradation rate reaches 62.32%, which can efficiently degrade macromolecular organic matter in livestock and poultry manure such as sheep manure, accelerating the fermentation and maturation process. It is easy to achieve large-scale, low-cost industrial cultivation and is suitable for industrial application.
[0014] The microbial organic fertilizer prepared by this invention has a high number of effective live bacteria, rich organic matter content, 100% parasite egg mortality rate, low heavy metal content, and excellent and stable fertilizer quality.
[0015] After the microbial organic fertilizer of the present invention is applied to the soil, Bacillus subtilis DW-14 can effectively colonize the soil. On the one hand, it can significantly increase the soil organic matter content, improve soil compaction and salinization, and enhance the soil's water and fertilizer retention capacity and fertilizer utilization rate. On the other hand, it can inhibit the absorption of nitrate nitrogen, heavy metals and pesticides by crops, thereby purifying and restoring the soil and improving the soil ecological environment. Detailed Implementation
[0016] The core of this invention is to provide a Bacillus subtilis strain and its application. This strain has high cellulase and protease secretion capabilities, which can improve fermentation efficiency and organic fertilizer quality. The resulting organic fertilizer can be applied to crop cultivation to improve soil, inhibit crop diseases, and increase yield and quality.
[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Example
[0018] Fermentation medium preparation: Add each component sequentially to deionized water according to the ratio of 7.5 g / L soluble starch, 10 g / L soybean meal, and 15 g / L disodium hydrogen phosphate. Stir continuously for 30 min under 30℃ water bath conditions to ensure complete dissolution of each component. Then adjust the initial pH of the medium to 7.5 using 1 mol / L hydrochloric acid and 1 mol / L sodium hydroxide solution. Pour the prepared medium into Erlenmeyer flasks, filling each flask to 1 / 3 of its volume. Sterilize using an autoclave at 121℃ and 0.1 MPa for 20 min. After sterilization, remove and cool to below 30℃ for later use. Strain activation: Take the frozen bacterial suspension of Bacillus subtilis DW-14, inoculate it onto LB solid medium plates using the streak method, and incubate it in a constant temperature incubator at 35℃ for 24h. Pick single colonies with uniform morphology and good growth from the plate, inoculate them into LB liquid medium, and incubate them in a shaker at 35℃ and 160r / min for 12h until the OD600 value of the bacterial suspension is 0.8-1.0 to obtain the seed culture for later use. Fermentation culture of the strain: The seed liquid prepared above was inoculated into the sterilized fermentation medium at an inoculation rate of 4% under aseptic conditions. The Erlenmeyer flask was placed in a constant temperature shaker at 35°C and shaken at a speed of 160 r / min for fermentation culture. The fermentation cycle was 48 h. After fermentation, the fermentation broth was centrifuged at 4℃ and 8000 r / min for 10 min. The supernatant was used as the crude enzyme solution. The activities of cellulase, neutral protease, and alkaline protease in the crude enzyme solution, as well as the degradation rate of cellulose by the strain, were detected using current national standard methods. The results showed that under these conditions, the cellulase activity of Bacillus subtilis DW-14 fermentation broth was 266.48 U / mL, the neutral protease activity was 228.41 U / mL, the alkaline protease activity was 170.76 U / mL, and the cellulose degradation rate was 62.32%. Example
[0019] Preparation of sheep manure microbial organic fertilizer using Bacillus subtilis DW-14: The process includes the following steps: After the sheep manure raw material arrives, Bacillus subtilis DW-14 fermentation liquid prepared in Example 1 is added to it at a rate of 1‰ of the sheep manure mass; after thorough mixing, it is pulverized using a pulverizer. The crushed material is put into a fermentation tank for aeration and fermentation. The material temperature naturally rises from 35℃ to 75℃, and then drops to below 30℃. The fermentation time is 20 days. After the fermented material is removed from the tank, it is placed in an aging area for aging for 7 days. The material after the first aging is crushed again, and then aged a second time for 10 days. After secondary aging, the material is mixed evenly by a mixer, then large particles of impurities are removed by a screening machine, and finally packaged to obtain the finished microbial organic fertilizer.
[0020] According to the current national standards for agricultural organic fertilizers, the prepared microbial organic fertilizer product underwent comprehensive testing. The test results were: effective viable bacteria count 9.1 × 10⁻⁶. 8 CFU / g, organic matter 44.70%, total potassium 2.55%, total phosphorus 1.40%, total nitrogen 1.91%, total nutrients 5.90%, pH 8.2, moisture 21.1%, parasite egg mortality rate 100%, fecal coliform count <3 / g, total mercury 0.10mg / kg, total arsenic 1.8mg / kg, total lead 18.4mg / kg, total chromium 22.3mg / kg, total cadmium 1.3mg / kg. Example
[0021] An apple orchard with flat terrain and uniform soil fertility was selected. The basic soil fertility was 12.3 g / kg organic matter, 85 mg / kg available nitrogen, 22 mg / kg available phosphorus, and 98 mg / kg available potassium. The fertilizer used in the experiment was Bacillus subtilis DW-14 sheep manure microbial organic fertilizer prepared in Example 2. The conventional fertilizers used were commercially available urea, diammonium phosphate, and compound fertilizer.
[0022] The experiment employed a randomized block design, with three experimental groups and one control group. Each treatment covered an area of approximately 150 m² and contained 15 apple trees. Field management practices, including irrigation, pruning, and pest and disease control, remained consistent across treatments, with the only difference being the fertilization method. Specific treatment protocols are as follows: Experiment 1: At the end of August, during the late stage of fruit enlargement, 15 kg of the microbial organic fertilizer prepared in Example 2 was applied to each plant using the trench application method. During the remaining growth stages, 0.45 kg of urea, 0.63 kg of diammonium phosphate, and 1.07 kg of compound fertilizer were applied according to the conventional fertilization plan. Experiment 2: In late August, during the fruit enlargement stage, 20 kg of the microbial organic fertilizer prepared in Example 2 was applied to each plant using the trench application method. During the remaining growth stages, the conventional fertilization plan was followed. Experimental Group 3: At the end of August, during the late stage of fruit enlargement, 25 kg of the microbial organic fertilizer prepared in Example 2 was applied to each plant using the trench application method. During the remaining growth stages, the conventional fertilization plan was followed. Control group: No microbial organic fertilizer was applied throughout the growth cycle. Instead, 0.45 kg of urea, 0.63 kg of diammonium phosphate, and 1.07 kg of compound fertilizer were applied according to the conventional fertilization plan.
[0023] After the apples ripened in mid-to-late October, they were harvested uniformly. Relevant indicators for each treatment were investigated and statistically analyzed, as follows: Disease incidence rate: The number of apple trees affected by canker in each treatment was counted, the incidence rate was calculated as follows: incidence rate = number of affected trees / total number of trees × 100%, and the disease reduction rate was also calculated. Soil fertility index: After harvesting, soil samples from the 0-20cm topsoil layer were collected at each treatment test site using a five-point sampling method to determine the soil organic matter content. The determination method was carried out in accordance with NY / T 1121.6-2006. Yield indicators: The fruits of apple trees in each treatment were harvested and weighed separately, and the yield per tree and yield per acre were calculated to determine the yield increase rate. Fruit quality indicators: 50 fruits from each treatment were randomly selected, and the transverse diameter of the fruits was measured using vernier calipers. The percentage of superior fruits (the proportion of fruits with a diameter of 80 mm or more) was calculated. Economic benefits: Statistics on fertilizer input and yield returns for each treatment were compiled to calculate the cost savings and efficiency gains. Cost savings and efficiency gains = experimental group gains - control group gains - additional fertilizer input for the experimental group.
[0024] Experimental results: Soil fertility: The soil organic matter content in the control group was 12.1 g / kg, in group 1 it was 15.8 g / kg, in group 2 it was 16.9 g / kg, and in group 3 it was 17.22 g / kg. The soil organic matter content in the experimental groups increased by an average of 4.54 g / kg compared with the control group, and the soil looseness was significantly improved, and the water and fertilizer retention capacity was improved. Yield indicators: The apple yield of the control group was 3352.5 kg / mu, while that of Experimental Group 1 was 3583.61 kg / mu, an increase of 231.11 kg / mu, or 6.89%; Experimental Group 2 was 3605.25 kg / mu, an increase of 252.75 kg / mu, or 7.54%; and Experimental Group 3 was 3623.96 kg / mu, an increase of 271.46 kg / mu, or 8.1%. The apple yields of the experimental groups were significantly higher than those of the control group, with an average increase rate of 7.51%. Fruit quality: The rate of superior apples in the control group was 68.2%, in experimental group 1 it was 75.5%, in experimental group 2 it was 78.3%, and in experimental group 3 it was 80.7%. The rate of superior apples in the experimental groups was significantly higher than that in the control group, and the marketability of the fruit was improved. Economic benefits: According to the calculation, the average cost saving and efficiency improvement of the experimental groups was 159.4 yuan / mu, among which experimental group 2 had the best cost saving and efficiency improvement effect, reaching 186.3 yuan / mu, with significant economic benefits.
[0025] The results show that Bacillus subtilis DW-14 provided by this invention, when applied to sheep manure fermentation to prepare microbial organic fertilizer and then applied to the soil, can effectively colonize the soil. On the one hand, it can significantly increase the soil organic matter content, improve soil compaction and salinization, and enhance the soil's water and fertilizer retention capacity and fertilizer utilization rate. On the other hand, it can inhibit the absorption of nitrate nitrogen, heavy metals, and pesticides by crops, thereby purifying and restoring the soil and improving the soil ecological environment.
[0026] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.
[0027] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A Bacillus subtilis, characterized in that: The Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ) DW-14, the preservation number is CGMCC No. 37076, the preservation date is December 15, 2025, and the preservation unit is China General Microbiological Culture Collection Center.
2. The application of Bacillus subtilis DW-14 as described in claim 1 in the preparation of microbial organic fertilizer.
3. Use according to claim 2, characterized in that: Sheep manure was used as the fermentation raw material, and Bacillus subtilis DW-14 was used as the fermentation bacteria in the fermentation of sheep manure.
4. Use according to claim 3, characterized in that: The steps for using Bacillus subtilis DW-14 to ferment sheep manure and prepare organic fertilizer are as follows: (1) Bacillus subtilis DW-14 was inoculated into a culture medium and cultured to prepare a bacterial agent; (2) Add the microbial agent from step (1) to the sheep manure and crush it; (3) Aerate and ferment the crushed material from step (2) for 18 to 22 days; (4) After fermentation, the product is aged once and then crushed again. After crushing, it is aged a second time. (5) The sheep manure that has been aged twice in step (4) is put into a mixer and stirred evenly, then screened by a screening machine, and finally packaged.
5. Use according to claim 4, characterized in that: The components of the culture medium in step (1) are 7.5 g / L soluble starch, 10 g / L soybean meal, and 15 g / L disodium hydrogen phosphate, and the pH of the culture medium is 7.
5.
6. Use according to claim 4, characterized in that: The inoculum amount of Bacillus subtilis DW-14 in step (1) is 3% to 5%, and fermentation is carried out at a speed of 150 to 170 r / min for 40 to 60 h.
7. Use according to claim 4, characterized in that: In step (4), the first aging time is 6 to 8 days, and the second aging time is 8 to 12 days.