Microbial composition, microbial inoculant and application in under-forest earthworm breeding
By using a microbial combination of Bacillus aureus and Bacillus kochia to treat fresh cow manure, the problems of long processing time and environmental pollution caused by high-temperature composting fermentation have been solved, and stable earthworm farming and efficient resource utilization have been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG FOREST SCI RES INST
- Filing Date
- 2026-06-25
- Publication Date
- 2026-07-28
AI Technical Summary
In existing technologies, fresh cow manure cannot be directly consumed by earthworms due to its high ammonia concentration and high pH value. It requires high-temperature composting and fermentation pretreatment, which results in long processing time, significant nutrient loss, foul odor, and leachate. Furthermore, earthworms may escape or die when raised directly on unfermented cow manure.
A microbial composition of Bacillus aryabhattai and Bacillus kochii was used. The pH value was reduced by air drying, rinsing and inoculation with Bacillus aryabhattai. After static treatment, Bacillus kochii was inoculated to decompose macromolecular organic matter, forming an earthworm farming method that does not require pre-composting.
It enables rapid improvement of the palatability and environment of fresh cow manure without the need for pre-composting, increases earthworm feeding efficiency and survival rate, avoids the defects of traditional fermentation, and improves resource utilization efficiency.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, and particularly relates to a microbial composition, inoculant, and its application in forest earthworm farming. Background Technology
[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.
[0003] With the continuous development of my country's livestock industry and the expansion of livestock and poultry production scale, livestock and poultry excrement is constantly increasing, which can easily cause pollution of rural surface water and groundwater, bringing many problems to farmers' living environment. Many livestock farms, areas, and households with concentrated and large-scale development have resulted in mixed human and animal living conditions and manure everywhere, which has become a major source of non-point source pollution in rural areas.
[0004] Earthworm farming is an important way to treat organic waste and produce high-quality protein feed and organic fertilizer. Cow manure is a common substrate for earthworm farming. Current technology typically uses high-temperature composting to pre-treat fresh cow manure, allowing it to decompose and cool before use for earthworm farming. However, the composting process is time-consuming and involves nitrogen loss and the generation of foul-smelling gases, which not only reduces the nutrient value of the cow manure but also increases the environmental burden. Some studies have attempted to use unfermented cow manure directly for earthworm farming, but the high concentration of ammonia and the alkaline environment in the manure often cause earthworms to escape, grow slowly, or even die in large numbers, leading to farming failures. Summary of the Invention
[0005] In order to solve at least one of the technical problems existing in the background art, the present invention provides a microbial composition, a fungal agent, and its application in forest earthworm farming.
[0006] The present invention adopts the following technical solution: A first aspect of the present invention provides a microbial composition comprising Bacillus aureus (Bacillus argentea). Bacillus aryabhattai )8' and Bacillus kochia ( Bacillus kochii )7'; The preservation number of Bacillus argentea 8' is CGMCC No. 26116, and the preservation number of Bacillus kochiae 7' is CGMCC No. 26118.
[0007] A second aspect of the present invention provides a microbial inoculant comprising the above-described microbial composition.
[0008] A third aspect of the present invention provides a method for preparing the above-mentioned microbial inoculant, comprising the following steps: Bacillus argentea 8' and Bacillus kochia 7' were inoculated into liquid culture medium and cultured to obtain Bacillus argentea 8' and Bacillus kochia 7' bacterial suspensions, respectively.
[0009] In a fourth aspect, the present invention provides the application of the above-described microbial composition or the above-described microbial agent in the non-composting fermentation treatment of fresh cow manure for earthworm farming under forest cover.
[0010] A fifth aspect of the present invention provides a method for cultivating earthworms under fresh cow manure, wherein the fresh cow manure is treated with the microbial composition or the microbial agent described above; the treatment includes bed preparation and feeding. The bed preparation management includes selecting understory forest land, laying fresh cow manure, air-drying and rinsing it, inoculating it with Bacillus aureus 8', letting it stand for treatment, and completing the bed preparation treatment when the pH value of the cow manure drops below 8.5, followed by earthworm inoculation. The feeding management includes simultaneously inoculating fresh cow manure fed in spots during earthworm farming with Bacillus kochiae 7'.
[0011] Compared with the prior art, the beneficial effects of the present invention are: This invention achieves a synergistic improvement effect on fresh cow manure by using a combination of *Bacillus aureus* 8' and *Bacillus kochia* 7'. *Bacillus aureus* 8' has a strong acid-producing capacity, which can rapidly lower the pH value of fresh cow manure and improve the living environment for earthworms; *Bacillus kochia* 7' can decompose large molecular organic matter in cow manure, reducing ammonia production. The two strains work together to bring fresh cow manure to a state suitable for direct earthworm consumption without the need for pre-composting fermentation. This is achieved through a three-stage synergistic strategy: air-drying to remove some moisture, rinsing to remove soluble salts and ammonia nitrogen, and rapid acid production and pH reduction by *Bacillus aureus* 8'. This avoids the nutrient loss and environmental pollution problems of traditional fermentation processes and improves the resource utilization efficiency of cow manure. Detailed Implementation
[0012] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0013] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.
[0014] As described in the background section, fresh cow manure, due to its high ammonia concentration and high pH value, cannot be directly consumed by earthworms in existing technologies. It typically requires high-temperature composting and fermentation pretreatment, a process that suffers from drawbacks such as long processing time, significant nutrient loss, foul odor, and leachate production. Some studies have attempted to use unfermented cow manure directly for earthworm rearing, but without any active modification to the manure, this resulted in mass earthworm mortality and escapes, necessitating frequent replacements to maintain the rearing process and preventing stable, continuous earthworm production.
[0015] Based on this, the present invention provides a microbial composition comprising Bacillus aureus (Bacillus argentea). Bacillus aryabhattai )8' and Bacillus kochia ( Bacillus kochii )7'; The *Bacillus argentea* 8' was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 11, 2022, with accession number CGMCC No. 26116, and its 16S rDNA sequence is shown in SEQ ID NO. 1; the *Bacillus kochii* 7' was deposited at the same center on November 11, 2022, with accession number CGMCC No. 26118, and its 16S rDNA sequence is shown in SEQ ID NO. 2.
[0016] Furthermore, the *Bacillus argentea* 8' and *Bacillus kochia* 7' are placed separately.
[0017] The *Bacillus kohlii* 8' exhibits strong acid-producing ability, rapidly reducing the pH value of cow dung and producing extracellular polysaccharides, thus improving the adaptability of earthworms. Through the metabolic action of *Bacillus kohlii* 7', it decomposes large-molecule organic matter in fresh cow dung, reduces ammonia production, increases the content of small-molecule sugars, amino acids, and other attractant substances, thereby improving the feeding speed and conversion efficiency of earthworms on fresh cow dung.
[0018] Furthermore, the ratio of viable bacteria of Bacillus aureus 8' to Bacillus kochia 7' is 10:1 to 30:1, preferably 20:1.
[0019] The present invention also provides a microbial agent comprising the above-mentioned microbial composition.
[0020] Furthermore, the viable count of the *Bacillus argentea* 8' is 2 × 10⁻⁶. 9 CFU / mL ~ 3×10 9 CFU / mL; the viable count of the *Bacillus kochia* 7' was 1 × 10⁻⁶. 8 CFU / mL ~ 2×10 8 CFU / mL.
[0021] The present invention also provides a method for preparing the above-mentioned microbial inoculant, wherein Bacillus aureus 8' and Bacillus kochia 7' are respectively inoculated into liquid culture medium and cultured to obtain Bacillus aureus 8' bacterial suspension and Bacillus kochia 7' bacterial suspension, respectively.
[0022] Furthermore, the *Bacillus argentea* 8' and *Bacillus kochia* 7' are placed separately.
[0023] This invention also provides the application of the above-mentioned microbial composition and microbial agent in the non-composting fermentation treatment of fresh cow manure for earthworm farming under forest cover.
[0024] Furthermore, the processing of fresh cow manure includes one or more of the following 1)-5): 1) Lower the pH value of cow manure; 2) Reduce the ammonia concentration in cow manure; 3) Increase polysaccharide content; 4) Increase small molecule organic matter; 5) Increase the amount of food-stimulating substances.
[0025] The present invention also provides a method for cultivating earthworms under fresh cow manure, wherein the fresh cow manure is treated with the above-mentioned microbial composition or the above-mentioned microbial agent; the treatment includes bed preparation and feeding. The bed preparation management includes selecting understory forest land, laying fresh cow manure, air-drying and rinsing it, inoculating it with Bacillus aureus 8', letting it stand for treatment, and completing the bed preparation treatment when the pH value of the cow manure drops below 8.5, followed by earthworm inoculation. The feeding management includes simultaneously inoculating fresh cow manure fed in spots during earthworm farming with Bacillus kochiae 7'.
[0026] This invention eliminates the need for pre-fermentation of fresh cow manure. Through a synergistic treatment of "air drying + rinsing + Bacillus subtilis 8'", it avoids the problems of foul odor and leachate pollution in traditional fermentation processes.
[0027] This invention involves inoculating the earthworm bed with Bacillus aureus 8' during the bed preparation stage. This strain rapidly reduces the pH value of cow manure through acid production and generates extracellular polysaccharides, improving the earthworms' adaptability. During the feeding stage, Bacillus kochiae 7' is inoculated to decompose macromolecular organic matter, reduce ammonia, and increase food-attracting substances, forming a two-stage microbial synergistic system of "bed pretreatment + feeding palatability improvement," ensuring the earthworms' efficient utilization of fresh cow manure throughout the entire cycle.
[0028] This invention makes full use of the natural conditions of shade, moisture retention, and ventilation under the forest canopy, and works synergistically with the compost-free fermentation process. It is easy to operate, low in cost, and suitable for large-scale promotion and application.
[0029] Furthermore, in the aforementioned bed-laying management, the laying thickness is 10-15cm; Alternatively, air dry until the moisture content is 40%-50%; rinse 2-3 times, each time with water just enough to moisten the surface of the cow manure and allow some water to seep in, in order to remove some of the soluble salts and ammonia nitrogen from the cow manure.
[0030] Alternatively, the inoculation amount of Bacillus aureus 8' is 0.1%-0.5% of the mass of cow manure. After inoculation, the moisture content of the cow manure should be maintained at 50%-65%, and the manure should be left to stand for 3-5 days.
[0031] Furthermore, in the feeding management, the inoculation amount of Bacillus kochiae 7' is 0.05%-0.2% of the mass of the cow manure being fed.
[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0033] Example 1 1.1 Specific preparation method of microbial agent Bacillus argentea 8' and Bacillus kochia 7', preserved on slant agar plates, were activated using the three-zone streak method and then plated onto LB agar plates (10g peptone; 10g NaCl; 5g yeast extract; 15-20g agar; 1000ml water; pH 7.0-7.2, aliquoted, sterilized at 121℃ for 20min). Single colonies were picked and transferred to 10ml of the corresponding LB liquid medium and incubated at 37℃ and 180rpm for 24h. Seed cultures were then inoculated at a 1% inoculum into 50ml of the corresponding liquid medium for fermentation, and incubated at 37℃ and 180rpm for 2d to obtain Bacillus argentea 8' and Bacillus kochia 7' bacterial suspensions, respectively.
[0034] 1.2 Site Selection The chosen location is a poplar forest in Liangshan, Jining City, Shandong Province, with approximately 85% shade from the trees, flat terrain, and good drainage.
[0035] 1.3 Bed Making Management After clearing away weeds, lay fresh cow manure as the breeding bed, with a width of 1.0 m and a thickness of 12 cm. Let the fresh cow manure air dry naturally for 2 days, reducing its moisture content to about 45%. Then, rinse the cow manure with clean water three times, until the surface of the manure is moist and slightly seeping into the soil. After rinsing, let it stand for 1 day to allow excess water to drain naturally.
[0036] Bacillus aryabhattai 8' (CGMCC No. 26116) bacterial suspension (viable count ≥ 2 × 10⁻⁶) 9 A 0.15% (CFU / mL) solution of cow manure by weight was evenly sprayed onto the surface of the manure. The manure moisture content was adjusted to 60%. After 4 days of treatment, the manure was inoculated with "Daping No. 2" earthworms at a density of 3.0 kg / m². 2 After inoculation, maintain the substrate moisture at 50%-65%. For ease of statistical analysis, 140g of different materials were placed in nylon mesh bags, and 20 earthworms with approximately the same weight and distinct clitellum were placed in each bag. Survival rate, number of eggs laid, and weight gain were investigated, with each treatment repeated three times.
[0037] 1.4 Material Feeding Processing Feeding begins on the 7th day after inoculation. A point feeding method is used, with a feeding point set up every 30 cm on both sides of the breeding bed, and about 1.2 kg of fresh cow manure is placed at each point.
[0038] Immediately after each feeding, evenly sprinkle Bacillus kochii 7' (CGMCC No. 26118) bacterial solution (approximately 1×10⁻⁶ viable bacteria count) onto the cow manure. 8 The inoculation rate is 0.15% of the weight of the cow manure fed to the cow. The cow is then fed every 5-7 days, with simultaneous inoculation of the above-mentioned microbial agent.
[0039] Example 2 The difference between this embodiment and Embodiment 1 is that the inoculum size of *Bacillus aureus* 8' is 0.45% of the mass of cow dung. Example 3 The difference between this embodiment and Embodiment 1 is that the inoculation amount of Bacillus kochiae 7' is 0.15% of the mass of the cow dung fed to the animal.
[0040] Example 4 The difference between this embodiment and Embodiment 1 is that the viable count of *Bacillus aureus* 8' is 2.5 × 10⁻⁶. 9 CFU / mL; viable count of Bacillus kochiae 7' was 1.5 × 10⁻⁶. 8 CFU / mL.
[0041] Comparative Example 1 Using the same forest site and earthworm species as in Example 1, fresh cow manure without any treatment was directly laid on the bed to a thickness of 12 cm without air drying, rinsing, or inoculation with Bacillus oryzae 8'.
[0042] Feed fresh cow dung, without inoculating with Bacillus kochiae 7'.
[0043] Comparative Example 2 The difference from Example 1 is that: when making the bed, Bacillus aureus 8' is not inoculated, but only air-drying and rinsing are performed.
[0044] Comparative Example 3 The difference between this comparative example and Example 1 is that it is not inoculated with Bacillus kochiae 7'. Comparative Example 4 The difference between this comparative example and Example 1 is that no rinsing step is performed during the bed preparation process; instead, the product is directly inoculated with Bacillus aureus 8' after air drying.
[0045] Comparative Example 5 The difference between this comparative example and Example 1 is that the inoculation amount of Bacillus argentis 8' is 8% of the mass of cow dung.
[0046] Comparative Example 6 The difference between this comparative example and Example 1 is that the inoculation amount of Bacillus kochiae 7' is 0.6% of the mass of the cow dung fed to the animal.
[0047] Comparative Example 7 The difference between this comparative example and Example 1 is that Bacillus aureus 8' is replaced with Bacillus subtilis.
[0048] Comparative Example 8 The difference between this comparative example and Example 1 is that Bacillus kochiae 7' is replaced with Bacillus subtilis.
[0049] Comparative Example 9 Fermentation is carried out using a strip-cutting method, where fresh cow dung is piled into a 1.5m trapezoidal heap with holes punched downwards at the top, five holes per square meter. Aerobic fermentation is then carried out, and the heap is turned over every two days once the temperature rises above 55℃. The fermentation process is complete when the material stops heating up. The fermented cow dung is then used for earthworm farming.
[0050] Effect evaluation After 20 days of breeding, the growth of earthworms and the effect of cow manure treatment were evaluated.
[0051] Table 1. Comparison of earthworm farming effects and cow manure treatment effects in different embodiments and comparative examples.
[0052] In Example 1, the earthworms had an average body weight of 0.63g, a survival rate of 100%, and laid 105 eggs. The earthworms were vigorous, had bright body color, and did not escape or die due to excessive ammonia or salt content.
[0053] The seedbed in Comparative Example 1 had a distinct ammonia odor. After the seedlings were introduced, almost no earthworms entered the bed; a large number of earthworms escaped or died, resulting in a survival rate of only 18.3%, and their weight decreased significantly. This demonstrates the necessity and effectiveness of the present invention in improving bed preparation and palatability through physical regulation combined with microbial synergy.
[0054] In Comparative Example 2, earthworms showed poor adaptability in the initial inoculation stage, with a small number escaping. Although the palatability-improving microbial agent used in the later stages of rearing played a role, the overall earthworm growth rate and reproduction rate were lower than in Example 1. This indicates that the two microbial agents used in the bed preparation and feeding stages each have their own focus and work together; neither is dispensable. The indicators in Comparative Example 3 were also significantly lower than in Example 1. This shows that *Bacillus aureus* 8' and *Bacillus kochia* 7' each have their own focus in the bed preparation and feeding stages, and their combined use is essential for achieving the best treatment effect; neither can be omitted.
[0055] Comparative Example 4 omitted the rinsing step, Comparative Example 5 increased the inoculum size of Bacillus aureus 8' to 8%, and Comparative Example 6 increased the inoculum size of Bacillus kochiae 7' to 0.6%. Compared with Example 1, the survival rate of Comparative Example 4 decreased to 68.6%, indicating some escape, which shows that the rinsing step is crucial for removing soluble salts and ammonia nitrogen from cow manure. Although no escape was observed in Comparative Examples 5-6, all indicators were lower than in Example 1, and the inoculum was wasted, indicating that a higher inoculum size is not necessarily better. The preferred inoculum size range of the present invention is reasonable and economical.
[0056] Comparative Example 7 replaced *Bacillus argentea* 8' with *Bacillus subtilis*, and Comparative Example 8 replaced *Bacillus Kochellae* 7' with *Bacillus subtilis*. Compared to Example 1, the pH of the cow manure in Comparative Example 7 was 8.8 (higher than 8.5 in Example 1), and the survival rate decreased to 80.3%; the average body weight of Comparative Example 8 was 0.52 g (lower than 0.63 g in Example 1). This indicates that *Bacillus subtilis* cannot replace the specific strain screened in this invention.
[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A microbial composition, characterized in that, Including Bacillus argentea ( Bacillus aryabhattai )8' and Bacillus kochia ( Bacillus kochii )7'; The preservation number of Bacillus argentea 8' is CGMCC No. 26116, and the preservation number of Bacillus kochiae 7' is CGMCC No. 26118.
2. The microbial composition according to claim 1, characterized in that, The viable count ratio of *Bacillus aureus* 8' to *Bacillus kochia* 7' is 10:1 to 30:
1.
3. A microbial inoculant, characterized in that, Includes the microbial composition as described in claim 1 or 2.
4. The microbial agent as described in claim 3, characterized in that, The viable count of the *Bacillus aureus* 8' was 2 × 10⁻⁶. 9 CFU / mL ~ 3×10 9 CFU / mL; the viable count of the *Bacillus kochia* 7' was 1 × 10⁻⁶. 8 CFU / mL ~ 2×10 8 CFU / mL.
5. A method for preparing a microbial inoculant according to any one of claims 3-4, characterized in that, Includes the following steps: Bacillus argentea 8' and Bacillus kochia 7' were inoculated into liquid culture medium and cultured to obtain Bacillus argentea 8' and Bacillus kochia 7' bacterial suspensions, respectively.
6. The application of a microbial composition according to any one of claims 1-2 or a microbial agent according to any one of claims 3-4 in the non-composting fermentation treatment of fresh cow manure for earthworm farming under forest cover.
7. The application as described in claim 6, characterized in that, The processing of fresh cow manure includes one or more of the following 1)-5): 1) Lower the pH value of cow manure; 2) Reduce the ammonia concentration in cow manure; 3) Increase polysaccharide content; 4) Increase small molecule organic matter; 5) Increase the amount of food-stimulating substances.
8. A method for cultivating earthworms under forest canopy using fresh cow dung, characterized in that, Fresh cow manure is treated with the microbial composition as described in any one of claims 1-2 or the microbial agent as described in any one of claims 3-4; the treatment includes bed preparation and feeding. The bed preparation management includes selecting understory forest land, laying fresh cow manure, air-drying and rinsing it, inoculating it with Bacillus aureus 8', letting it stand for treatment, and completing the bed preparation treatment when the pH value of the cow manure drops below 8.5, followed by earthworm inoculation. The feeding management includes simultaneously inoculating fresh cow manure fed in spots during earthworm farming with Bacillus kochiae 7'.
9. The method as described in claim 8, characterized in that, In the aforementioned bed-laying management, the laying thickness is 10-15cm; Alternatively, the product can be air-dried to a moisture content of 40%-50% and then rinsed 2-3 times. Alternatively, the inoculation amount of Bacillus aureus 8' is 0.1%-0.5%, and after inoculation, the moisture content of the cow manure is kept at 50%-65%, and it is left to stand for 3-5 days.
10. The method as described in claim 8, characterized in that, In the feeding management, the inoculation amount of Bacillus kochiae 7' is 0.05%-0.2% of the mass of the cow manure being fed.