Bacillus, microbial inoculant and application thereof

Abstract

This invention relates to the field of microbiology, and more particularly to Bacillus, microbial agents, and their applications. This invention provides a Bacillus sp. strain with accession number CGMCC No. 36427. This invention provides a high-yield cellulase strain of Bacillus sp. and a method for rapid straw decomposition and return to the field compatible with field operations. By directionally enhancing the degradation efficiency of cellulose / hemicellulose through Bacillus sp., and combining nitrogen supplementation and water management, this method shortens the decomposition cycle while reducing the risk of nitrogen deficiency and seedling burn during return to the field, thereby improving crop emergence and root growth.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more particularly to Bacillus, microbial agents and their applications. Background Technology

[0002] Straw resources are abundant, and returning it to the field can improve soil organic matter and aggregate stability. However, the dense lignocellulose structure and high carbon-to-nitrogen ratio of straw result in a long natural degradation cycle in the field, often leading to short-term nitrogen fixation by microorganisms. This can cause problems such as uneven crop emergence and restricted root growth. Furthermore, improper humidity management can easily lead to anaerobic decay, off-odors, and pathogen proliferation. To adapt to the agricultural time window, there is an urgent need to develop a simple-to-operate microbial return technology that remains stable and effective even under fluctuating field temperature and humidity conditions.

[0003] Tobacco, as a leaf crop, generates a huge annual output of waste such as tobacco stalks. However, tobacco stalks are highly lignified and have a thick waxy outer layer, making natural decomposition slow. Furthermore, continuous cropping in tobacco fields is common, and improper straw return to the field can easily exacerbate soil-borne diseases (such as black shank and bacterial wilt) and the accumulation of nicotine residues, posing potential risks to the quality and safety of subsequent tobacco leaves. Therefore, establishing a microbial rapid decomposition and return technology adapted to the tobacco field ecology is of great practical significance for solving the resource utilization of waste in tobacco-growing areas, alleviating continuous cropping obstacles, and improving soil health in tobacco fields. Summary of the Invention

[0004] In view of this, the present invention provides Bacillus, microbial inoculants, and their applications. The present invention provides a high-yield strain of Bacillus cellulase and a method for rapid straw decomposition and return to the field compatible with field operations. By directionally enhancing the degradation efficiency of cellulose / hemicellulose through Bacillus, and combining nitrogen supplementation and water management, the decomposition cycle is shortened while reducing the risk of nitrogen deficiency and seedling burn during return to the field, thereby improving crop emergence and root growth.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides Bacillus sp., with accession number CGMCC No. 36427.

[0007] The present invention also provides microbial inoculants, comprising any one of the following and acceptable adjuvants:

[0008] (a) the above-mentioned Bacillus sp.; and / or

[0009] (b) Inactivated Bacillus sp. as described above; and / or

[0010] (c) Metabolites, derivatives, fermentation broth, cultures, exosomes, lysates, or extracts of the above-mentioned Bacillus sp.

[0011] In some embodiments of the present invention, the viable count of Bacillus sp. in the above-mentioned microbial agent is not less than 10. 8 CFU / mL.

[0012] In some embodiments of the present invention, the above-mentioned microbial agents include one or more of the following: liquid agents, solid agents, wettable powders, granules, and biochar-supported slow-release agents.

[0013] In some embodiments of the present invention, the solid microbial agent further includes excipients; the excipients include one or more of talc, bentonite, biochar and humic acid carrier.

[0014] In some embodiments of the present invention, the above-mentioned microbial inoculant further includes hemicellulose and / or pectin-degrading bacteria.

[0015] The present invention also provides the use of the above-mentioned Bacillus sp. and / or the above-mentioned microbial agents in the degradation of one or more of cellulose, lignin and nicotine.

[0016] The present invention also provides the application of the above-mentioned Bacillus sp. and / or the above-mentioned microbial agents in straw return to the field.

[0017] In some embodiments of the present invention, the straw used in the above applications includes: tobacco straw and / or corn straw.

[0018] The present invention also provides the application of the above-mentioned Bacillus sp. and / or the above-mentioned microbial agents in improving continuously cropped soils.

[0019] The present invention also provides the application of the above-mentioned Bacillus sp. and / or the above-mentioned microbial agents in inhibiting soil-borne pathogens.

[0020] The present invention also provides a straw decomposing agent, comprising: the above-mentioned Bacillus sp. and / or the above-mentioned microbial inoculant and acceptable adjuvants.

[0021] The present invention also provides a method for returning straw to the field, wherein one or more of the above-mentioned Bacillus, the above-mentioned microbial agent and the above-mentioned straw decomposing agent are applied to the pretreated straw, and the degraded straw is turned into the soil to complete the return to the field.

[0022] In some embodiments of the present invention, the above method includes the following steps:

[0023] S1: Apply one or more of the above-mentioned Bacillus, the above-mentioned microbial inoculant and the above-mentioned straw decomposing agent to the pretreated straw and mix them evenly;

[0024] S2: Supplement nitrogen source and adjust the carbon-nitrogen ratio of the straw system to 25-35;

[0025] S3: Adjust the straw moisture content to 45%~65% and plow and aerate;

[0026] S4: Turn the treated straw into the soil to complete the return to the field.

[0027] In some embodiments of the present invention, the nitrogen source in the above method includes one or more of inorganic nitrogen sources, organic nitrogen sources, and slow-release nitrogen fertilizers.

[0028] In some embodiments of the present invention, in the above method, the amount of Bacillus sp. applied is not less than 10. 8 CFU / mL; or

[0029] The viable count of Bacillus sp. in the microbial agent shall not be less than 10. 8 CFU / mL; or

[0030] The viable count of Bacillus sp. in the straw decomposing agent shall not be less than 10. 8 CFU / mL.

[0031] The beneficial effects of this invention include:

[0032] Because strain Bsp1 can continuously secrete extracellular cellulase and colonize the straw surface, it can accelerate the breaking of cellulose chains and the release of soluble sugars, thereby driving indigenous microorganisms to decompose components such as hemicellulose, thus increasing the straw weight loss rate and cellulose degradation rate. At the same time, by supplementing nitrogen and managing water and aeration, it can reduce the risk of nitrogen retention and anaerobic putrefaction in the early stage of decomposition, thereby shortening the decomposition cycle and improving crop emergence and root growth. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0034] Figure 1 This is a flowchart illustrating a method for rapid decomposition and return of straw to the field.

[0035] Figure 2This diagram illustrates the clear zone of cellulase screening plate for strain Bsp1.

[0036] Biological Preservation Instructions

[0037] Biological material: Bsp1; Classification and nomenclature: Bacillus sp.; Deposited on October 30, 2025 at the China General Microbiological Culture Collection Center (CGMCC); Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; Accession number: CGMCC No. 36427. Detailed Implementation

[0038] This invention discloses Bacillus, microbial agents, and their applications.

[0039] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0040] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0041] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0042] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0043] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0044] In Examples 1 to 3, Effect Examples 1 to 3, and Comparative Examples 1 to 2 of the present invention, the raw materials and reagents used can all be purchased from the market.

[0045]

[0046] The present invention will be further illustrated below with reference to the embodiments:

[0047] Example 1: Preparation of bacterial strain and inoculum

[0048] The strain: Bacillus sp. is a Bacillus strain isolated from tobacco samples. It was identified as Bacillus sp. by 16S rRNA sequencing. This strain is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36427.

[0049] Seed culture: In LB medium, culture at 30-37℃ and 150-200 r / min with shaking for 12-18 h to obtain the late logarithmic phase seed culture; adjust the bacterial concentration to approximately 10. 8 ~10 9 CFU / mL.

[0050] (3) Formulation: Liquid or solid bacterial agent. Liquid bacterial agent is applied directly by spraying, and the bacterial cells are evenly loaded by spraying. The moisture content is adjusted to ≤10% and then dried at low temperature to obtain powder / granules. The viable count is preferably ≥10. 8 CFU / g. Suitable carriers for solid microbial agents include talc, bentonite, biochar, humic acid, or combinations thereof.

[0051] Example 2: Method for Rapid Decomposition and Returning of Straw to the Field

[0052] (1) Straw pretreatment: After harvesting, crush the straw to 3-8 cm as soon as possible; remove obvious mud and foreign matter; if the straw is too dry (moisture content <15%), spray it with water to pre-moisten it.

[0053] (2) Application of microbial agent: Apply liquid microbial agent at a rate of 2~10 L / ton (with 10 L / ton as the base). 8 ~10 9 Apply the solid microbial agent (calculated as CFU / mL) evenly to the surface of the straw and mix thoroughly; or apply the solid agent at 0.2%~1.0% (w / w, based on the wet basis of the straw) and mix. Ensure the microbial agent is in full contact with the straw, avoiding localized excessive moisture or clumping.

[0054] (3) Matching nitrogen supplementation and carbon-nitrogen ratio regulation: In order to reduce the risk of nitrogen retention in the early stage of returning to the field, readily available nitrogen sources (urea, ammonium sulfate or their combination) can be supplemented simultaneously to adjust the carbon-nitrogen ratio of the straw system to about 25-35; the recommended urea dosage is about 2-6 kg / ton of straw (which can be adjusted according to the soil nitrogen supply level).

[0055] (4) Moisture and ventilation management: When covering the field with straw, it is recommended to maintain the straw moisture content at 45%~65% (the criterion is that it can be squeezed into a ball and water can be seen between the fingers but not dripping). Irrigate when necessary; at the same time, improve ventilation by shallow plowing / rotary tillage.

[0056] (5) (Optional) Short-term pile start-up: When conditions permit, the straw after the application of fungicide can be piled into strips or small piles (0.6~1.2 m high), and after a short-term heat generation start-up of 3~7 days, it can be plowed back into the field to shorten the field start-up period.

[0057] (6) Turning and returning to the field: Turn the treated straw into the 10-20 cm tillage layer, and combine with compaction to retain moisture or cover with film / mulch to stabilize humidity; without affecting the farming season, it is recommended to do shallow turning once every 7-15 days to break up the clumps and replenish oxygen.

[0058] Example 3 Monitoring and Evaluation Indicators of the Decomposition Process

[0059] (1) Straw weight loss rate: Take the known dry weight of the straw in the mesh bag (20 mesh nylon mesh bag) and bury it in the field at the same time. Take it out after 30 days and 60 days, wash it, dry it and weigh it to calculate the weight loss rate.

[0060] (2) Cellulose / hemicellulose: determined by Van Soest fiber analysis or acid / neutral washing method; the decrease in the treatment group and the control group were compared.

[0061] (3) Available nitrogen in soil and “risk of nitrogen deficiency”: determination of soil NH4 + -N, NO3 - -N and soluble organic carbon (DOC) were recorded, along with crop emergence rate, root length, etc.

[0062] (4) Field odor and hygiene: Record the occurrence of anaerobic putrefaction odors, maggots / flies, etc., as auxiliary criteria for ventilation and water management.

[0063] Example 1: Strain screening and verification of cellulase production capacity

[0064] To support the ability of strain Bsp1 to degrade straw lignocellulose, a cellulase plate screening and evaluation procedure was provided. Sodium carboxymethyl cellulose (CMC-Na) was used as the substrate for screening, and the extracellular cellulase secretion capacity was evaluated by observing the hydrolysis zone (clear zone).

[0065] (1) Cellulase screening medium formulation: sodium carboxymethyl cellulose 20 g / L, tryptone 2 g / L, dipotassium hydrogen phosphate 2.5 g / L, potassium dihydrogen phosphate 1.5 g / L, yeast extract 0.5 g / L, agar powder 18 g / L, add deionized water to 1 L; autoclave at 121℃ for 15 min, cool to about 50~55℃ and pour into plates (about 20 mL per plate).

[0066] (2) Inoculation and culture: After the Bsp1 strain was revived, a single colony was inoculated into liquid seed culture medium and cultured at 30-37℃ with shaking for 12-18 h; after serial dilution with sterile physiological saline, it was inoculated onto screening plates and cultured at 30-37℃ for 18-24 h.

[0067] (3) Color development and clear zone determination: The hydrolysis zone can be observed directly after incubation; or the Congo red color development method can be used: cover the plate with 0.1% (w / v) Congo red solution for 15-30 min, pour off the dye solution, and decolorize with 1 mol / L NaCl for 15-30 min. The hydrolysis zone will show a clear clear zone. The ratio of the diameter of the clear zone D to the diameter of the colony d (D / d) is used as the screening index. If necessary, use vernier calipers to measure and take pictures for record.

[0068] (4) Screening results: such as Figure 1 As shown, strain Bsp1 formed a distinct hydrolysis zone on CMC-Na plates, indicating that it has a strong extracellular cellulase secretion capacity and can be used in straw decomposition and returning to the field.

[0069] Example 2: Preparation of microbial agent and trial of returning corn straw to the field.

[0070] (1) Preparation of bacterial agent: The preserved strain Bsp1 was cultured in LB medium at 30-37℃ with shaking until the stationary phase, and the bacterial concentration was adjusted to about 10. 9 CFU / mL; prepare liquid bacterial agent for later use. If preparing a solid bacterial agent, spray the bacterial solution onto a carrier (talc / bentonite / biochar), dry at low temperature until the moisture content is ≤10%, obtaining a viable count ≥10. 8 CFU / g powder.

[0071] (2) Field treatment: Corn stalks were crushed to about 5 cm and sprayed with microbial agent at a rate of 5 L / ton in the wheat field and mixed evenly. Urea was added at 4 kg / ton of stalks to adjust the C / N ratio. Water was sprayed to make the moisture content about 55%. After 7 days, the stalks were plowed into the soil to a depth of 15 cm. The control group only received water and plowed, without applying microbial agent.

[0072] (3) Sampling and testing: The weight loss rate (30 and 60 days) was measured using the mesh bag method; the cellulose / hemicellulose content was determined using fiber analysis; and the NH4 content in the soil was measured. + -N, NO3- -N and DOC; and record the emergence rate and root length. Each treatment should have ≥3 replicates.

[0073] Table 1

[0074]

[0075] The results showed that, compared with the control group without inoculant application, the application of Bsp1 inoculant significantly accelerated straw decomposition in the field and simultaneously alleviated the risk of nitrogen deficiency in the early stages of straw return to the field. Specifically, the straw weight loss rate in the treatment group increased from 24.3% to 42.1% (an increase of approximately 73%) at 30 days, indicating faster decomposition initiation and more complete early degradation; by 60 days, the weight loss rate further increased from 50.6% to 68.4% (an increase of approximately 35%), indicating that the inoculant's effect was sustainable and promoted deeper decomposition. Simultaneously, the decrease in cellulose content at 60 days increased from 18.0% to 33.5% (an increase of approximately 86%), directly demonstrating that key structural carbon sources in straw were more effectively broken down, which is the main material basis for the increased weight loss rate. At the soil level, the NO3⁻-N concentration increased from 12.4 mg / kg to 15.8 mg / kg (an increase of approximately 27%) after 60 days, indicating that while the decomposition of straw accelerated, it did not exacerbate nitrogen fixation; instead, it promoted nitrogen mineralization and the accumulation of available nitrogen, thereby reducing the risk of seedling burn / stunted growth caused by nitrogen deficiency. The emergence rate increased by 6.3%, and the taproot length increased by 17%, indicating that the environment after straw return to the field was more conducive to early growth and better root system development.

[0076] Example 3: Preparation of microbial agent and trial of returning tobacco straw to the field

[0077] (1) Preparation of bacterial agent: The preserved strain Bsp1 was cultured in LB medium at 30-37℃ with shaking until the stationary phase, and the bacterial concentration was adjusted to about 10. 9 CFU / mL; prepare liquid bacterial agent for later use. If preparing a solid bacterial agent, spray the bacterial solution onto a carrier (talc / bentonite / biochar), dry at low temperature until the moisture content is ≤10%, obtaining a viable count ≥10. 8 CFU / g powder.

[0078] (2) Field treatment: Select tobacco stalks after harvesting, crush them to about 3-5 cm, and spray the inoculant at 8 L / ton in the tobacco field, mixing it evenly; simultaneously add 5 kg / ton urea and 3 kg / ton superphosphate to adjust the C / N ratio and supplement phosphorus; spray water to make the moisture content about 60%; after 10 days, plow the tobacco stalks into the soil to a depth of 20 cm. The control group only watered and plowed, without applying inoculant. The experimental site was a tobacco field that had been continuously cropped for 3 years, with yellow soil type and the previous crop being flue-cured tobacco variety K326.

[0079] (3) Sampling and testing: The weight loss rate of tobacco stalks (30 and 60 days) was measured using the mesh bag method; the cellulose / hemicellulose / lignin content was measured using fiber analysis; the nicotine degradation rate was measured using ultraviolet spectrophotometry; soil NH4⁺-N, NO3⁻-N, and DOC were measured; and the number of black shank pathogens (Phytophthora parasitica var. nicotianae) was determined using a plate assay; and the emergence rate, root length, and disease incidence of subsequent tobacco seedlings were recorded. Each treatment was replicated at least 3 times.

[0080] Table 2

[0081]

[0082] The results showed that, compared with the control group without inoculum, the application of Bsp1 inoculum significantly accelerated the decomposition of tobacco stalks, promoted nicotine degradation, and alleviated continuous cropping obstacles in the tobacco field environment. Specifically, the weight loss rate of tobacco stalks in the treatment group increased from 18.5% to 35.6% (an increase of approximately 92%) at 30 days, indicating that the strain had good adaptability to highly lignified tobacco stalks; by 60 days, the weight loss rate increased from 42.3% to 62.8% (an increase of approximately 48%), with cellulose and lignin reductions increasing by 107% and 131%, respectively, proving that the inoculum could effectively break down the dense lignocellulose structure of tobacco stalks. In particular, the nicotine degradation rate increased from 25.3% to 58.7% (an increase of approximately 132%) at 60 days, significantly reducing the potential inhibitory effect of nicotine residues on the soil microecology. At the soil level, soil NO3 decreased at 60 days. - -N increased from 10.8 mg / kg to 14.6 mg / kg (an increase of approximately 35%), while the number of blackleg fungus decreased by 50%, suggesting that accelerated decomposition did not exacerbate nitrogen fixation and may have inhibited pathogen reproduction by altering the rhizosphere microenvironment. The emergence rate of subsequent tobacco seedlings increased by 11.6%, and the taproot length increased by 26%, indicating a significant improvement in the environment for returning tobacco straw to the field. This effectively alleviated emergence obstacles in continuously cropped tobacco fields and provided technical support for the resource utilization of waste and soil health management in tobacco-growing areas.

[0083] Comparative Example 1: Verification of cellulase degradation by different Bacillus strains

[0084] (1) The commonly used straw composting strain reported in the literature, Bacillus licheniformis (Bs-2, purchased from China General Microbiological Culture Collection Center, CGMCC 1.265), was collected as a reference for existing technology. This strain showed a certain ability to degrade cellulose in conventional composting studies, but it had not been acclimatized to a high-salt / hyperosmolar environment. Its salt tolerance (upper limit of NaCl tolerance is about 2%) and heat tolerance (optimal growth temperature is 30~37℃, growth is inhibited above 40℃) were both weaker than the strain Bacillus sp. Bsp1 of this invention.

[0085] (2) Cellulase degradation verification (clear zone method): The same method as in Example 1, the D / d value of strain Bs-2 was 2.0±0.2, which was significantly lower than that of Bacillus sp. Bsp1 (p<0.01), and the colony growth rate was slower. The colony diameter at 24 h was only 65% ​​of that of Bacillus sp. Bsp1.

[0086] Comparative Example 2: Comparative Experiment on the Effects of Different Bacillus Straw Returning to the Field

[0087] (1) Preparation of bacterial agent: The Bacillus sp. Bsp1 strain of the present invention and the Bs-2 strain in Comparative Example 1 were prepared into liquid bacterial agents using the same method as in Effect Example 2. The concentration of the bacterial solution was adjusted to 10. 9 CFU / mL.

[0088] (2) Field treatment and testing: The experiment was set up with four treatment groups: ① no bacterial control (CK); ② control strain Bacillus sp. Bs-2 (Bs-2 group); ③ strain Bacillus sp. Bsp1 of the present invention (Bsp1 group). Each group had 3 replicates, and the experimental conditions were the same as in Example 2.

[0089] Table 3 Comparison of the field straw return effects of different Bacillus strains

[0090]

[0091] The results show that, compared with the control strain Bs-2, the present invention strain Bacillus sp. Bsp1 exhibits significant advantages in straw decomposition efficiency, nutrient conversion, and crop growth promotion. The straw weight loss rate at 30 and 60 days increased by 38.0% and 17.5%, respectively, and the cellulose degradation rate increased by 36.7%, indicating that Bsp1 can significantly accelerate early straw degradation and promote deep decomposition. Soil NO3⁻-N increased by 14.5%, effectively promoting organic nitrogen mineralization and reducing the risk of nitrogen deficiency. Emergence rate and taproot length increased by 3.7% and 8.7%, respectively, improving the physical structure and nutrient availability of the returned soil, which is beneficial to early root system formation in crops. These advantages stem from Bsp1's strong cellulase production capacity, rapid start-up characteristics, and adaptability to the field environment, providing an efficient and stable microbial solution for rapid straw return to the field.

[0092] 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. Bacillus sp., characterized by, Its accession number is: CGMCC No.36427.

2. A microbial inoculant, characterized in that, Includes any of the following and acceptable adjuvants: (a) Bacillus sp. as described in claim 1; and / or (b) Inactivated Bacillus sp. as described in claim 1; and / or (c) Metabolites, derivatives, fermentation broths, cultures, exosomes, lysates, or extracts of Bacillus sp. as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The viable count of the Bacillus sp. is not less than 10. 8 CFU / mL.

4. The use of Bacillus sp. as described in claim 1 and / or the microbial agent as described in claim 2 or 3 in the degradation of one or more of cellulose, lignin and nicotine.

5. The application of Bacillus sp. as described in claim 1 and / or the microbial inoculant as described in claim 2 or 3 in straw return to the field.

6. The application of Bacillus sp. as described in claim 1 and / or the microbial inoculants as described in claim 2 or 3 in improving continuously cropped soils.

7. The application of Bacillus sp. as described in claim 1 and / or the microbial agent as described in claim 2 or 3 in the inhibition of soil-borne pathogens.

8. A straw decomposing agent, characterized in that, include: Bacillus sp. as described in claim 1 and / or the microbial agent as described in claim 2 or 3, and acceptable adjuvants.

9. A method for returning straw to the field, characterized in that, Apply one or more of the following to the pretreated straw: Bacillus as described in claim 1, microbial agent as described in claim 2 or 3, and straw decomposing agent as described in claim 8. Then, plow the degraded straw into the soil to complete the return to the field.

10. The method as described in claim 9, characterized in that, The amount of Bacillus sp. applied shall not be less than 10 8 CFU / mL; or The viable count of Bacillus sp. in the microbial agent shall not be less than 10. 8 CFU / mL; or The viable count of Bacillus sp. in the straw decomposing agent shall not be less than 10. 8 CFU / mL.

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