Microbial agent for aerobic composting and application thereof

Abstract

The invention relates to a microbial agent for aerobic composting. The microbial agent comprises bacillus subtilis J2, pseudoarthrobacter psychrotolans J4 and cellulosimicrobium cellulans J6, and the microbial agent is prepared from the following raw materials: bacillus subtilis J2, pseudoarthrobacter psychrotolans J4 and cellulosimicrobium cellulans J6. The microbial agent for aerobic composting is prepared from the following raw materials: bacillus subtilis J2, pseudoarthrobacter psychrotolans J4 and cellulosimicrobium cellulans J6, and the microbial agent for aerobic composting is prepared from the following raw materials: bacillus subtilis J2, pseudoarthrobacter psychrotolans J6. The invention also relates to a preparation method and application of the microbial agent. The microbial agent can quickly increase the composting temperature, effectively kill pathogenic bacteria, worm eggs and weed seeds, shorten the composting time and improve the yield and quality of organic fertilizer.

Description

Technical Field

[0001] The invention relates to a microbial agent for aerobic composting and application thereof. Background Art

[0002] Composting is a key measure for harmlessly disposing agricultural waste, utilizing it as a resource, and fertilizing it, promoting the development of circular agriculture. Traditional composting, which relies solely on the activity of microorganisms, suffers from long composting times, significant nutrient loss, and poor quality. Inoculating exogenous microbial agents during aerobic composting can effectively address these issues.

[0003] Microbial agents primarily include single-bacterial agents and composite agents. Single-bacterial agents are limited in effectiveness due to their incomplete lignocellulase enzyme system and reduced activity in high-temperature environments. Composite agents, however, are based on the mutually beneficial symbiotic effects between strains and are highly adaptable to high-temperature composting environments. They can rapidly raise compost temperatures, effectively kill pathogens, insect eggs, and weed seeds, shorten compost maturity, increase organic fertilizer yield and quality, and significantly enhance composting effectiveness. Summary of the Invention

[0004] The purpose of the present invention is to provide a microbial agent which can quickly increase the compost temperature and shorten the compost maturity time and the application thereof.

[0005] The present invention adopts the following technical solutions: A microbial agent for aerobic composting comprises Bacillus subtilis J2, Pseudomonas aeruginosa J4 and Microbacterium cellulosum J6.

[0006] Furthermore, the Bacillus subtilis ( Bacillus subtilis ) J2 was deposited in the China Center for Type Culture Collection on January 13, 2025, in Wuhan, Hubei Province, China, with the accession number CCTCC NO: M2025105.

[0007] Furthermore, the pseudoarthrobacter ( Pseudarthrobacter psychrotolerans ) J4 was deposited in the China Center for Type Culture Collection on January 13, 2025, in Wuhan, Hubei Province, China, with the deposit number CCTCC NO: M2025106.

[0008] Furthermore, the fiber microbacteria ( Cellulosimicrobium cellulans ) J6 was deposited in the China Center for Type Culture Collection on January 13, 2025, in Wuhan, Hubei Province, China, with the deposit number CCTCCNO: M2025107.

[0009] Furthermore, the total number of effective viable bacteria of the microbial agent is ≥4×10 8 CFU·mL -1 .

[0010] Furthermore, the microbial agent is prepared by the following method: (1) Bacillus subtilis J2, Pseudomonas aeruginosa J4, and Microbacterium fibrosum J6 were inoculated into LB solid medium and activated at 30°C for 2-3 days; (2) The activated strains were inoculated into LB liquid culture medium, placed in a shaker at 30°C and cultured at 180 rpm until the logarithmic growth phase; (3) The fermentation broth obtained in step (2) was centrifuged at 4°C, 6000 rpm for 10 min, the supernatant was removed, the cells were collected and diluted with sterile water to OD 600 = 1, to obtain bacterial suspensions of each strain; (4) The suspension of Bacillus subtilis J2: the suspension of Pseudomonas aeruginosa J4: the suspension of Microbacterium fibrosum J6 were mixed evenly in a volume ratio of 2-6: 6-10: 8-14 to obtain a microbial agent.

[0011] Preferably, in step (4), the volume ratio of the Bacillus subtilis J2 suspension: the pseudoarthrobacter J4 suspension: the cellulosic microbacterium J6 suspension is 4:7:13.

[0012] An application of the above-mentioned microbial agent in aerobic composting.

[0013] An application of the above-mentioned microbial agent in aerobic composting of cow dung.

[0014] The beneficial effects of the present invention are that the composite bacterial agent of the present invention can quickly decompose organic matter in compost raw materials, convert it into small molecular nutrients that are easier for plants to absorb and utilize, reduce the toxicity of compost to plants, and significantly improve the compost maturity speed and quality. It is an eco-friendly bacterial agent with broad application prospects and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Antagonism assay for strains.

[0016] Figure 2 This is the effect of strain J2 dosage on cellulase activity.

[0017] Figure 3 This is the effect of strain J6 dosage on cellulase activity.

[0018] Figure 4 This is the effect of strain J4 dosage on cellulase activity.

[0019] Figure 5 The effect of microbial agents on the temperature of aerobic composting of cow dung.

[0020] Figure 6The effect of microbial agents on the germination index during aerobic composting of cow dung. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further illustrated below through specific embodiments and drawings.

[0022] Example 1 Isolation and purification of strains The thermophilic compost samples used for strain isolation and screening were obtained from the composting laboratory of Hebei University of Science and Technology. After surface disinfection, 5 g of freshly collected thermophilic compost was weighed, added to 50 ml of sterile water, and shaken at 180 rpm for 30 minutes, followed by 1 hour of rest. One mL of the supernatant was diluted and spread onto LB solid medium. The sample was incubated in a 35°C incubator for 2–3 days. Single colonies were selected based on their morphological characteristics and purified using the three-line method. Purification was performed repeatedly until a single, pure colony was obtained.

[0023] LB solid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 18 g of agar powder, dilute to 1000 mL, adjust the pH to 7.0, and sterilize by autoclaving at 121°C.

[0024] Example 2 Screening of strains (1) Cellulose degradation test Pick pure bacterial colonies and activate them in LB liquid medium. Centrifuge the activated bacterial solution to obtain bacterial cells and dilute them with sterile water to OD 600 = 1, aspirate 10 μl and spot-inoculate into sodium carboxymethylcellulose Congo red medium. Inoculate four replicates per plate and incubate at 30°C for 48 hours. Observe the growth of the colonies. After incubation, stain with Congo red reagent and observe the appearance of a clear zone around the colonies on the medium. Measure the diameter of the clear zone (D) and the colony diameter (d) to calculate the HC value (HC = D / d).

[0025] LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, dilute to 1000 mL, adjust the pH to 7.0, and sterilize by autoclaving at 121°C.

[0026] Sodium carboxymethylcellulose Congo red medium: NaNO3 2.0 g, K2HPO4 1.0 g, KCl 0.5 g, MgSO4·7H20 0.5 g, FeSO4 0.01 g, CMC-Na 10.0 g, agar 20.0 g, pH 9.5, distilled water 1000 mL, sterilized at 121°C for 20 min.

[0027] (2) Protein degradation experiment Pick pure bacterial colonies and activate them in LB liquid medium. Centrifuge the activated bacterial solution to obtain bacterial cells and dilute them with sterile water to OD 600 = 1. Pipette 10 μl of the solution onto casein agar medium, inoculate four replicates onto each plate, and incubate at 30°C for 48 hours. Observe the growth of the colonies. After incubation, observe the presence of a clear zone around the colonies on the medium. Measure the diameter of the clear zone (D) and the colony diameter (d) to calculate the HC value.

[0028] Casein agar medium: casein 10.0 g, beef extract powder 3.0 g, disodium hydrogen phosphate Na2HPO4 2.0 g, NaCl 5.0 g, bromothymol blue 0.05 g, agar 15.0 g, pH 7.4±0.2, sterilization at 121°C for 20 min.

[0029] (3) Starch degradation experiment Pick pure bacterial colonies and activate them in LB liquid medium. Centrifuge the activated bacterial solution to obtain bacterial cells and dilute them with sterile water to OD 600 = 1, aspirate 10 μL and spot-inoculate onto starch culture medium. Inoculate four replicates onto each plate and incubate at 30°C for 48 hours. Observe the growth of the colonies. After incubation, add iodine solution and observe the appearance of a clear zone around the colonies on the culture medium. Measure the diameter of the clear zone (D) and the colony diameter (d) to calculate the HC value.

[0030] Starch culture medium: soluble starch 10.0 g, K2HPO4 1.0 g, MgSO4 1.0 g, NaCl 1.0 g, (NH4)2SO4 2.0 g, CaCO3 2.0 g, FeSO4 0.001 g, MnCl2 0.001 g, ZnSO4 0.001 g, pH 7.2±0.2, sterilization at 121°C for 20 min.

[0031] The results showed that the strains numbered J2, J4 and J6 had the ability to degrade cellulose, protein and starch. The test results are shown in Table 1.

[0032] Table 1 HC values of degradation experiments .

[0033] Example 3 Identification and preservation of strains The J2 strain was identified as Bacillus subtilis ( Bacillus subtilis The pure culture of J2 strain was deposited on January 13, 2025, at the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCC NO: M2025105.

[0034] The J4 strain was identified as Pseudomonas aeruginosa by 16SrRNA gene sequencing (SEQ ID No. 2) and NT comparison. Pseudarthrobacter psychrotolerans The pure culture of J4 strain was deposited on January 13, 2025, at the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the accession number: CCTCC NO: M2025106.

[0035] The J6 strain was identified as a cellulomicrobacterium by 16SrRNA gene sequencing (SEQ ID No. 3) and NT comparison. Cellulosimicrobium cellulans The pure culture of strain J6 was deposited on January 13, 2025, at the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the accession number: CCTCCNO: M2025107.

[0036] Example 4 Antagonism Assay Antagonism experiments were conducted with strains J2, J4, and J6. Prepare LB solid medium and sterilize it. Once cooled to 50°C, use a sterile pipette to add 0.1 mL of bacterial solution (per 20 mL of medium) to the unformed medium, mix thoroughly, and form a plate. Allow to stand horizontally until solidified. Next, inoculate another bacterial strain onto the solid medium and culture in an incubator. Finally, after 48 hours of incubation, observe for normal growth and the presence of isolation zones. If normal growth is absent and isolation zones are present, this indicates antagonism between the strains. Figure 1 The results showed that there was no antagonistic effect between the J2, J4 and J6 strains.

[0037] Example 5: Proportion of composite bacterial agent Since the crude cellulose content in livestock and poultry manure is high (30%~40%) and difficult to degrade, the cellulase activity was used as an indicator to determine the optimal ratio of the composite microbial agent through single-factor experiments.

[0038] Cellulase activity determination method: DNS method was used to determine the cellulase activity using the reducing sugar content.

[0039] Activation and culture of the strain: After partially thawing the strain frozen at -80℃ in an ice bath, use a sterile inoculation loop to pick up the unmelted ice crystals on the surface of the freezing solution, draw a line on the surface of the LB solid culture medium, and culture it upside down at 35℃ for 24 hours to obtain the activated strain. The activated strain was inoculated into LB liquid culture medium, placed in a 30℃ shaker, and cultured at 180rpm until the logarithmic growth phase to obtain the logarithmic phase strain seed liquid. The obtained bacterial solution was centrifuged at 4℃, 6000rpm for 10 minutes, the supernatant was removed, the bacteria were collected separately, and diluted with sterile water to OD600 = 1, and bacterial suspensions of three strains of bacteria were obtained respectively. The bacterial suspensions were compounded according to the proportions to obtain the composite bacterial agent solution for the test.

[0040] Determination of the dosage of J2: First, the optimized concentration of J2 in the single factor experiment is set to 0.5mL, 0.75mL, 1.00mL, 1.25mL, and 1.50mL. The experimental results are as follows: Figure 2 As the dosage of J2 increased, the cellulase activity of the mixed culture increased. The highest activity was achieved when the dosage of J2 reached 1 mL. Therefore, the optimal dosage of J2 was 1 mL. Single-factor experiments with J2 preliminarily determined the volume ratio of J2:J4:J6 in the mixed culture to be 1:2:2.

[0041] Determination of the dosage of J6: The optimized concentrations of J6 in the single factor experiment 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 shown in the figure. Figure 3 As the dosage of J6 increased, the cellulase activity of the mixed culture increased. The highest activity was achieved when the dosage of J6 reached 3.25 mL. Therefore, the optimal dosage of J6 was 3.25 mL. Single-factor experiments with J6 further determined the volume ratio of J2:J4:J6 in the mixed culture to be 1:2:3.25.

[0042] Determination of the dosage of J4: The optimized concentrations of J4 in the single factor experiment were set to 1.5mL, 1.75mL, 2.00mL, 2.25mL, and 2.50mL. The experimental results are as follows: Figure 4 As the dosage of J4 increased, the cellulase activity of the mixed culture increased. The highest activity was achieved when the dosage of J4 reached 1.75 mL. Therefore, the optimal dosage of J4 was 1.75 mL. Single-factor experiments with J6 further determined the volume ratio of J2:J4:J6 in the mixed culture to be 1:1.75:3.25.

[0043] Finally, the optimal addition ratio of J2, J4, and J6 was determined to be 4:7:13, at which point the cellulase activity of the mixed agent reached a maximum of 45.75 U·mL -1 .

[0044] Example 6 Preparation of composite bacterial agent (1) J2 ( Bacillus subtilis )、J4( Pseudarthrobacter psychrotolerans ) and J6 ( Cellulosimicrobium cellulans ) The three bacterial strains were inoculated into LB solid medium and activated at 30℃ for 2-3 days.

[0045] (2) The three activated bacterial strains were inoculated into LB liquid culture medium, placed in a shaker at 30°C and cultured at 180 rpm until the logarithmic growth phase.

[0046] (3) The fermentation broth obtained in step (2) was centrifuged at 4°C, 6000 rpm for 10 min, the supernatant was removed, the cells were collected and diluted with sterile water to OD 600 =1, and bacterial suspensions of three bacterial strains were obtained respectively.

[0047] (4) The bacterial suspension was mixed evenly in the ratio of J2:J4:J6=4:7:13, and the total number of effective viable bacteria in the bacterial suspension was ≥4×10 8 CFU·mL -1 , and obtain a composite bacterial agent.

[0048] Example 7 Effect of composite bacterial agent on aerobic composting of cow dung The microbial composite agent (marked as B), strains J2, J4 and J6 prepared in Example 6 of the present invention were inoculated into a pile with cow dung as the substrate and wheat straw as the bedding for aerobic composting. The composite agent B and the bacterial suspensions of J2, J4 and J6 (same as in step (3) of Example 6) were added at a ratio of 0.5% of the wet weight of the compost material, respectively. A control group without the addition of the agent (marked as A) was also set up.

[0049] (a) Impact on compost temperature changes According to the Technical Specification for Harmless Treatment of Livestock and Poultry Manure (GB / T 36195-2018), changes in the pile temperature can reflect changes in microbial activity in the pile and also reflect the progress of aerobic fermentation. Figure 5 As shown, the temperature trends across the five composting trials were similar. Group B, which added a composite microbial inoculant, entered a high-temperature phase exceeding 50°C on day two, while the other control groups reached 50°C or higher after four days. Group B maintained this high-temperature phase for nine days, while the other groups maintained it for five to seven days. During this high-temperature phase, Group B had the highest temperature and Group A had the lowest, with temperatures in Group B approximately 2 to 6°C higher than those in Group A. This indicates that adding microbial inoculants to the composting process can effectively accelerate the entry into the high-temperature phase, thereby increasing the high-temperature temperature and accelerating the composting process.

[0050] (b) Effect on germination index during composting The germination index GI value is a biological indicator reflecting the toxicity of compost to plant seeds and the maturity of the compost. After 18 days of composting, the GI of Group A and Group B reached more than 130%, exceeding the 70% requirement of the "Organic Fertilizer Standard" (GB NY 525-2021).

[0051] The germination index (GI value) is a biological indicator that reflects the toxicity of compost to plant seeds and the maturity of compost. The germination index changes in this composting experiment are as follows: Figure 6 As shown. As the composting process progressed, the germination index (GI) of several piles generally showed an upward trend. In terms of the overall trend of change, the GI value of the B treatment group was always higher than that of the other treatment groups. On the 17th day, the B group reached more than 130%, and on the 18th day, the germination index of the three groups J2, J4, and J6 reached more than 130%. The compost samples have reached the safe maturity standard (GI ≥ 80%). For the control group A, the inoculant of the present invention reached the maturity period faster and decomposed more organic matter. This shows that the addition of the composite microbial inoculant has a positive effect on the germination index and maturity of the compost, can effectively reduce the toxicity of the compost to plants, significantly accelerate the maturity process of the compost, and thus improve the quality of the compost.

[0052] The above is a detailed implementation plan of the present invention and the verification of the application effect of the microbial composite agent in the aerobic composting process of cow dung. However, the scope of protection of the present invention is not limited to these clearly presented contents. Whether it is an alternative operation made based on the implementation plan of the present invention or a modification of the scheme based on the ideas of the present invention, as long as its core technical concept is derived from the present invention, it is included in the scope of protection of the present invention.

Claims

1. A microbial agent for aerobic composting, characterized in that: These include Bacillus subtilis J2, Pseudarthrobacter psychrotolerans J4, and Cellulosimicrobium cellulans J6.

2. The microbial agent according to claim 1, characterized in that The deposit number of the Bacillus subtilis J2 is CCTCC NO: M2025105; the deposit number of the pseudoarthrobacter J4 is CCTCC NO: M2025106; and the deposit number of the cellulomicrobium J6 is CCTCC NO: M2025107.

3. The microbial agent according to claim 2, characterized in that The total number of effective viable bacteria of the microbial agent is ≥4×10 8 CFU·mL -1 .

4. The microbial agent according to claim 3, characterized in that It is prepared by the following method: (1) Bacillus subtilis J2, Pseudomonas aeruginosa J4, and Microbacterium fibrosum J6 were inoculated into LB solid medium and activated at 30°C for 2-3 days; (2) The activated strains were inoculated into LB liquid culture medium, placed in a shaker at 30°C and cultured at 180 rpm until the logarithmic growth phase; (3) The fermentation broth obtained in step (2) was centrifuged at 4°C, 6000 rpm for 10 min, the supernatant was removed, the cells were collected and diluted with sterile water to OD 600 = 1, to obtain bacterial suspensions of each strain; (4) The suspension of Bacillus subtilis: the suspension of Pseudomonas aeruginosa J4: the suspension of Microbacterium fibrosum J6 were mixed evenly in a volume ratio of 2-6: 6-10: 8-14 to obtain a microbial agent.

5. The microbial agent according to claim 4, characterized in that In step (4), the volume ratio of the Bacillus subtilis J2 suspension: the pseudoarthrobacter J4 suspension: the cellulosic microbacterium J6 suspension is 4:7:

13.

6. Use of the microbial agent according to any one of claims 1 to 5 in aerobic composting.

7. Use of the microbial agent according to any one of claims 1 to 5 in aerobic composting of cow dung.

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