Microbial inoculant for aerobic composting and use thereof

Abstract

The present application relates to a kind of microbial inoculants for aerobic composting, comprising Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ) J2, Pseudonocardia (Pseudonocardia) Pseudarthrobacter psychrotolerans ) J4 and Fibrobacter (Fibrobacter) Cellulosimicrobium cellulans ) J6.The present application also relates to the preparation method and application of the above microbial inoculants.The microbial inoculants can quickly increase the composting temperature, effectively kill pathogenic bacteria, insect eggs and weed seeds, shorten the composting time, and improve the yield and quality of organic fertilizer.

Description

Technical Field

[0001] This invention relates to a microbial inoculant for aerobic composting and its application. Background Technology

[0002] Composting is a key measure to achieve the harmless, resource-based, and fertilizer-based utilization of agricultural waste, and to promote the development of circular agriculture. Traditional composting relies solely on the action of microorganisms, which suffers from problems such as long composting time, significant nutrient loss, and poor quality. Inoculating the aerobic composting process with exogenous microbial agents can effectively solve these problems.

[0003] Microbial inoculants mainly include single-strain inoculants and compound inoculants. Single-strain inoculants have limited effectiveness due to incomplete lignocellulase enzyme systems and reduced activity in high-temperature environments. Compound inoculants, on the other hand, are based on the mutualistic symbiotic effect between strains, have a strong ability to adapt to high-temperature composting environments, can rapidly increase composting temperature, effectively kill pathogens, insect eggs, and weed seeds, shorten composting time, increase organic fertilizer yield and quality, and significantly improve composting efficiency. Summary of the Invention

[0004] The purpose of this invention is to provide a microbial agent that can rapidly increase composting temperature and shorten composting time, and its application.

[0005] The present invention adopts the following technical solution:

[0006] A microbial inoculant for aerobic composting includes Bacillus subtilis J2, Pseudomonas aeruginosa J4, and fibrous microbacteria J6.

[0007] Furthermore, the Bacillus subtilis ( Bacillus subtilis J2 was deposited at the China Center for Type Culture Collection on January 13, 2025, located in Wuhan, Hubei Province, China, with accession number CCTCC NO:M2025105.

[0008] Furthermore, the aforementioned *Pseudomonas* ( Pseudarthrobacter psychrotolerans J4 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan, Hubei Province, China, on January 13, 2025, with accession number CCTCC NO:M2025106.

[0009] Furthermore, the fibrous microbes ( Cellulosimicrobium cellulans J6 was deposited at the China Center for Type Culture Collection on January 13, 2025, located in Wuhan, Hubei Province, China, with accession number CCTCCNO:M2025107.

[0010] Furthermore, the total number of viable bacteria in the microbial agent is ≥4×10⁻⁶. 8 CFU·mL -1 .

[0011] Furthermore, the microbial inoculant is prepared by the following method:

[0012] (1) Bacillus subtilis J2, Pseudomonas aeruginosa J4 and Microfibrillariae J6 were inoculated into LB solid medium and activated at 30°C for 2-3 days;

[0013] (2) The activated strains were inoculated into LB liquid medium and placed in a shaker at 30°C and shaken at 180 rpm until the logarithmic growth phase.

[0014] (3) Centrifuge the fermentation broth obtained in step (2) at 4°C and 6000 rpm for 10 min, remove the supernatant, collect the cells separately, and dilute with sterile water to OD. 600 =1, to obtain bacterial suspensions of each strain;

[0015] (4) Mix the Bacillus subtilis J2 suspension : Pseudomonas aeruginosa J4 suspension : Fibroblasts J6 suspension in a volume ratio of 2~6 : 6~10 : 8~14 to obtain the microbial agent.

[0016] Preferably, in step (4), the volume ratio of Bacillus subtilis J2 suspension: Pseudomonas aeruginosa J4 suspension: Fibromicrobacterium J6 suspension is 4:7:13.

[0017] The application of the above-mentioned microbial inoculant in aerobic composting.

[0018] Application of the above-mentioned microbial agent in aerobic composting of cow manure.

[0019] The beneficial effects of this invention are as follows: the compound microbial agent of this invention can rapidly decompose the organic matter in compost raw materials, transforming it into small molecule nutrients that are more easily absorbed and utilized by plants, reducing the toxicity of compost to plants, and significantly improving the composting speed and quality. It is an eco-friendly microbial agent with broad application prospects and market value. Attached Figure Description

[0020] Figure 1 This was to determine the antagonistic effect of the strain.

[0021] Figure 2 The effect of the dosage of strain J2 on cellulase activity.

[0022] Figure 3 The effect of strain J6 dosage on cellulase activity.

[0023] Figure 4 The effect of strain J4 dosage on cellulase activity.

[0024] Figure 5The effect of microbial inoculants on the temperature of aerobic composting of cow manure.

[0025] Figure 6 The effect of microbial inoculants on the germination index during aerobic composting of cow manure. Detailed Implementation

[0026] The technical solution of the present invention will be further described below through specific embodiments and accompanying drawings.

[0027] Example 1: Isolation and purification of strains

[0028] The high-temperature compost samples used for isolating and screening strains were obtained from compost at the Hebei University of Science and Technology's composting experimental field. After surface sterilization, 5g of the freshly collected high-temperature compost samples were weighed, added to 50ml of sterile water, and placed on a shaker at 180rpm for 30min, followed by standing for 1h. 1mL of the supernatant was diluted and spread onto LB agar. The samples were incubated at 35℃ for 2-3 days. Single colonies were selected for isolation based on morphological characteristics, and strains were purified using the triplet method. This process was repeated several times until pure single colonies were obtained.

[0029] LB solid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 18g agar powder, bring to a final volume of 1000mL, adjust pH to 7.0, and autoclave at 121℃.

[0030] Example 2 Screening of strains

[0031] (1) Cellulose degradation test

[0032] Pure bacterial colonies were picked and activated in LB liquid medium. The activated bacterial solution was then centrifuged to obtain bacterial cells, which were diluted with sterile water to an OD value of 1. 600 =1, take 10 μL and spot it onto sodium carboxymethyl cellulose Congo red medium. Inoculate 4 replicates on each plate and incubate at 30°C for 48 h. Observe the growth of colonies. After incubation, stain with Congo red reagent and observe whether a clear zone appears around the colonies on the medium. Measure the diameter of the clear zone (D) and the diameter of the colony (d), and calculate the HC value (HC=D / d).

[0033] LB liquid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, bring to a final volume of 1000mL, adjust pH to 7.0, and autoclave at 121℃.

[0034] Carboxymethyl cellulose sodium Congo red medium: NaNO3 2.0g, K2HPO4 1.0g, KCl 0.5g, MgSO4·7H2O 0.5g, FeSO4 0.01g, CMC-Na 10.0g, agar 20.0g, pH 9.5, distilled water 1000mL, sterilized at 121℃ for 20min.

[0035] (2) Protein degradation experiment

[0036] Pure bacterial colonies were picked and activated in LB liquid medium. The activated bacterial solution was then centrifuged to obtain bacterial cells, which were diluted with sterile water to an OD value of 1. 600 =1, take 10 μL and spot it onto casein agar medium, inoculate 4 replicates on each plate, incubate at 30°C for 48 h, and observe the growth of colonies. After incubation, observe whether a clear zone appears around the colonies on the medium, measure the diameter of the clear zone (D) and the diameter of the colony (d), and calculate the HC value.

[0037] Casein agar medium: 10.0g casein, 3.0g beef extract powder, 2.0g disodium hydrogen phosphate Na2HPO4, 5.0g NaCl, 0.05g bromothymol blue, 15.0g agar, pH 7.4±0.2, sterilized at 121℃ for 20min.

[0038] (3) Starch degradation experiment

[0039] Pure bacterial colonies were picked and activated in LB liquid medium. The activated bacterial solution was then centrifuged to obtain bacterial cells, which were diluted with sterile water to an OD value of 1. 600 =1, take 10 μL and spot it into starch medium, inoculate 4 replicates on each plate, incubate at 30℃ for 48 h, and observe the growth of colonies. After incubation, add iodine solution and observe whether a clear zone appears around the colonies on the medium. Measure the diameter of the clear zone (D) and the diameter of the colony (d), and calculate the HC value.

[0040] Starch culture medium: 10.0g soluble starch, 1.0g K2HPO4, 1.0g MgSO4, 1.0g NaCl, 2.0g (NH4)2SO4, 2.0g CaCO3, 0.001g FeSO4, 0.001g MnCl2, 0.001g ZnSO4, pH 7.2±0.2, sterilized at 121℃ for 20min.

[0041] The results showed that strains numbered J2, J4, and J6 had the ability to degrade cellulose, protein, and starch. The results are shown in Table 1.

[0042] Table 1 HC values ​​from degradation experiments

[0043] .

[0044] Example 3 Identification and Preservation of Strains

[0045] Strain J2 was identified as Bacillus subtilis by 16S rRNA gene sequencing (SEQ ID No. 1) and NT comparison. Bacillus subtilis The pure culture of strain J2 was deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on January 13, 2025, with accession number CCTCC NO: M2025105.

[0046] Strain J4 was identified as *Pseudorobacter* by 16S rRNA gene sequencing (SEQ ID No. 2) and NT comparison. Pseudarthrobacter psychrotolerans The pure culture of strain J4 was deposited at the China Center for Type Culture Collection on January 13, 2025, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M2025106.

[0047] Strain J6 was identified as a fibrobacterium (Fibriophyte) by 16S rRNA gene sequencing (SEQ ID No. 3) and NT comparison. Cellulosimicrobium cellulans The pure culture of strain J6 was deposited at the China Center for Type Culture Collection on January 13, 2025, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCCNO:M2025107.

[0048] Example 4 Antagonistic Effect Determination

[0049] Antagonistic experiments were conducted on strains J2, J4, and J6. LB solid medium was prepared and sterilized. After cooling to 50°C, 0.1 mL of bacterial suspension (per 20 mL of medium) was added to the unformed medium using a sterile pipette and mixed thoroughly to form plates. These plates were then allowed to solidify horizontally. Next, another bacterial strain was inoculated onto this solid medium and incubated. Finally, after 48 hours of incubation, normal growth and the formation of an isolation zone were observed. If normal growth was not observed and an isolation zone was formed, it indicated antagonistic activity between the strains. Figure 1 The results showed that no antagonistic effect was observed among strains J2, J4, and J6.

[0050] Example 5: Formulation of Compound Microbial Agent

[0051] Because livestock and poultry manure contains high levels of crude cellulose (30%~40%) and is difficult to degrade, cellulase activity was used as an indicator to determine the optimal ratio of compound microbial agents through single-factor experiments.

[0052] Cellulase activity assay: The DNS method was used to determine cellulase activity based on the reducing sugar content.

[0053] Activation and culture of the strain: After partial thawing of the strain stored at -80℃ in an ice bath, the unmelted ice crystals on the surface of the cryopreservation solution were collected using a sterile inoculation loop and streaked onto the surface of LB solid medium. The culture was then inverted at 35℃ for 24 h to obtain the activated strain. The activated strain was inoculated into LB liquid medium and cultured at 30℃ with shaking at 180 rpm until the logarithmic growth phase, obtaining the seed culture of the logarithmic growth phase strain. The obtained bacterial culture was centrifuged at 4℃, 6000 rpm for 10 min, the supernatant was removed, and the bacterial cells were collected and diluted with sterile water to OD0.05. 600 =1, and bacterial suspensions of 3 strains were obtained. The bacterial suspensions were then compounded according to the specified ratio to obtain the experimental compound bacterial agent solution.

[0054] The dosage of J2 was determined as follows: The optimal concentrations for the single-factor experiment of J2 were initially set to 0.5 mL, 0.75 mL, 1.00 mL, 1.25 mL, and 1.50 mL. The experimental results are as follows: Figure 2 As the dosage of J2 increased, the cellulase activity of the mixed bacterial solution also increased, reaching its highest level when the dosage of J2 reached 1 mL. Therefore, the optimal dosage of J2 is 1 mL. Through single-factor experiments with J2, the volume ratio of J2:J4:J6 in the mixed bacterial solution was preliminarily determined to be 1:2:2.

[0055] The dosage of J6 was determined as follows: The optimal concentrations for the single-factor experiment of J6 were set to 2 mL, 2.5 mL, 2.75 mL, 3.00 mL, 3.25 mL, and 3.50 mL. The experimental results are as follows: Figure 3 As the dosage of J6 increased, the cellulase activity of the mixed bacterial solution also increased, reaching its highest level when the dosage of J6 reached 3.25 mL. Therefore, the optimal dosage of J6 is 3.25 mL. Through single-factor experiments with J6, the volume ratio of J2:J4:J6 in the mixed bacterial solution was further determined to be 1:2:3.25.

[0056] The dosage of J4 was determined as follows: The optimal concentrations for the single-factor experiment of J4 were set to 1.5 mL, 1.75 mL, 2.00 mL, 2.25 mL, and 2.50 mL. The experimental results are as follows: Figure 4 As the dosage of J4 increased, the cellulase activity of the mixed bacterial solution also increased, reaching its highest level when the dosage of J4 reached 1.75 mL. Therefore, the optimal dosage of J4 is 1.75 mL. Through single-factor experiments with J6, the volume ratio of J2:J4:J6 in the mixed bacterial solution was further determined to be 1:1.75:3.25.

[0057] The optimal dosage ratio of J2, J4, and J6 was ultimately determined to be 4:7:13, at which point the cellulase activity of the mixed inoculant reached a maximum of 45.75 U·mL. -1 .

[0058] Example 6 Preparation of Compound Microbial Agent

[0059] (1) J2 ( Bacillus subtilis J4 Pseudarthrobacter psychrotolerans ) and J6 ( Cellulosimicrobium cellulans The three strains were inoculated into LB solid medium and activated at 30°C for 2-3 days.

[0060] (2) The three activated strains were inoculated into LB liquid medium and placed in a shaker at 30°C and shaken at 180 rpm until the logarithmic growth phase.

[0061] (3) Centrifuge the fermentation broth obtained in step (2) at 4°C and 6000 rpm for 10 min, remove the supernatant, collect the cells separately, and dilute with sterile water to OD. 600 =1, and bacterial suspensions of 3 strains were obtained respectively.

[0062] (4) Mix the bacterial suspension evenly in a ratio of J2∶J4∶J6=4∶7∶13 to obtain a total effective viable bacterial count of ≥4×10⁻⁶. 8 CFU·mL -1 A compound microbial agent was obtained.

[0063] Example 7: Effects of compound microbial agents on aerobic composting of cow manure

[0064] The microbial compound inoculant (labeled as B), strains J2, J4, and J6 prepared in Example 6 of this invention were inoculated into a pile with cow dung as substrate and wheat straw as bedding material for aerobic composting. The inoculant B and the bacterial suspensions of J2, J4, and J6 (same as step (3) in Example 6) were added at a ratio of 0.5% of the wet weight of the compost material. Another control group (labeled as A) was set up without adding inoculant.

[0065] (a) Effect on composting temperature changes

[0066] According to the "Technical Specification for Harmless Treatment of Livestock and Poultry Manure" (GB / T 36195-2018), changes in the temperature of the manure pile can reflect changes in the activity of microorganisms within the pile, and also indicate the progress of aerobic fermentation. For example... Figure 5As shown, the temperature change trends in the five composting experiments were similar. The group with the added compound microbial inoculant (B) entered a high-temperature stage above 50°C from day 2; while the other control groups reached above 50°C after 4 days. The high-temperature period in group B lasted for 9 days, while the high-temperature period in the other groups lasted 5-7 days. During the high-temperature period, group B had the highest temperature, and group A had the lowest, with group B being approximately 2-6°C higher than group A. This indicates that adding microbial inoculants during composting can effectively promote the compost pile to enter the high-temperature stage more quickly, is more conducive to increasing the high-temperature stage during composting, and accelerates the composting process.

[0067] (b) Effect on germination index during composting

[0068] Germination index (GI) is a biological indicator that reflects the toxicity of compost to plant seeds and the degree of compost maturity. After 18 days of composting, the GI of both Group A and Group B reached over 130%, exceeding the 70% requirement of the "Organic Fertilizer Standard" (GB NY 525-2021).

[0069] Germination index (GI value) is a biological indicator reflecting the toxicity of compost to plant seeds and the degree of compost maturity. The changes in germination index in this composting experiment are shown below. Figure 6 As shown, the germination index (GI) of the various compost piles generally increased as the composting process progressed. Overall, the GI value of treatment group B was consistently higher than that of the other treatment groups. On day 17, group B reached over 130%, and on day 18, the germination index of groups J2, J4, and J6 also reached over 130%, indicating that the compost samples had reached the safe maturity standard (GI ≥ 80%). Compared to control group A, the inoculant of this invention reached maturity faster, resulting in greater decomposition of organic matter. This indicates that the addition of the compound microbial inoculant has a positive impact on the germination index and maturity of compost, effectively reducing the toxicity of compost to plants, significantly accelerating the composting process, and thus improving compost quality.

[0070] The above describes the detailed embodiments of the present invention and the verification of the application effect of the microbial compound agent in the aerobic composting process of cow manure. However, the scope of protection of the present invention is not limited to these explicitly presented contents. Any alternative operations made based on the embodiments of the present invention, or modifications made according to the ideas of the present invention, as long as their core technical concept originates from the present invention, are included within the scope of protection of the present invention.

Claims

1. A microbial inoculant for aerobic composting, characterized by, It comprises: Bacillus subtilis (Bacillus subtilis) J1 with the preservation number CCTCC NO: M2025105 Bacillus subtilis Pseudonocardia sp. J2 with the preservation number CCTCC NO: M2025106 Pseudarthrobacter psychrotolerans Microbacterium sp. J4 with the preservation number CCTCC NO: M2025107 Cellulosimicrobium cellulans Microbacterium sp. J6 with the preservation number CCTCC NO: M2025108 2. The microbial inoculant of claim 1, wherein, The total number of effective viable bacteria of the microbial agent is ≥4×10 8 CFU·mL -1 .

3. The microbial inoculant of claim 2, wherein, It is prepared by the following method: (1) Bacillus subtilis J2 and Pseudomonas pseudoarthritis Pseudarthrobacter psychrotolerans J4 and fibrobacterium J6 were inoculated into LB solid medium and activated at 30°C for 2-3 days. (2) The activated strains are inoculated into LB liquid medium respectively, and placed in a 30℃ shaking table, and cultured at 180rpm until the logarithmic growth phase; (3) The fermentation broth obtained in step (2) was centrifuged at 4°C, 6000 rpm for 10 min, and the supernatant was removed to collect the bacterial cells and dilute with sterile water to OD 600 = 1 to obtain a bacterial suspension of each strain; (4) The volume ratio of Bacillus subtilis bacterial suspension: Pseudoclavibacter sp. is 2-6:6-10:8-14, and the mixture is uniformly mixed to obtain the microbial inoculum. Pseudarthrobacter psychrotolerans The volume ratio of J4 bacterial suspension: fiber microbacterium J6 bacterial suspension is 2-6:6-10:8-14, and the mixture is uniformly mixed to obtain the microbial inoculum.

4. The microbial inoculant of claim 3, characterized by, In step (4), the Bacillus subtilis J2 suspension: Pseudorabies Pseudarthrobacter psychrotolerans The volume ratio of J4 bacterial suspension to J6 fibrous microbacterial suspension is 4:7:

13.

5. The microbial inoculant according to any one of claims 1-4 for use in aerobic composting.

6. The microbial inoculant according to any one of claims 1-4 for use in aerobic composting of cow dung.

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