Low-temperature resistant compound microbial agents and their application in composting fermentation on the Qinghai-Tibet Plateau

Abstract

This invention discloses a low-temperature resistant compound microbial agent and its application in high-altitude composting fermentation. This invention relates to the field of biotechnology, specifically to a low-temperature resistant compound microbial agent and its application in high-altitude composting fermentation. The active ingredients of the compound microbial agent of this invention are *Bacillus cereus* CCTCC NO: M 20221062 and *Bacillus pumilus* CCTCC NO: M 20221061. This compound microbial agent can be applied to the high-altitude composting fermentation process under low-temperature conditions, shortening the composting fermentation time, promoting plant seed germination rate, and improving the quality of compost.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a low-temperature resistant compound microbial agent and its application in composting fermentation on the Qinghai-Tibet Plateau. Background Technology

[0002] Qinghai has a plateau continental climate with an average annual temperature of 5.7℃. January sees a low of -8.2℃, while July has a high of 17.2℃. Mixed composting of mushroom residue and animal manure is an effective way to utilize agricultural and livestock waste. However, under the low temperatures of the plateau, the composting process is time-consuming and inefficient, resulting in incomplete decomposition. To ensure the proper functioning of the composting process, research has been conducted on methods to raise the temperature of compost under low-temperature conditions, such as electric heating, biogas heating, and insulation covering. However, these methods are not yet energy-efficient or economical enough for practical composting production. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to select microbial agents suitable for low-temperature composting fermentation in plateau climates.

[0004] The present invention provides a compound microbial agent for preparing compost, wherein the active ingredients of the compound microbial agent are composed of Bacillus cereus and Bacillus pumilus.

[0005] The compound microbial agent is a compound microbial agent used for compost preparation. The Bacillus cereus is Bacillus cereus QS7, with the accession number CCTCC NO: M20221062 at the China Center for Type Culture Collection; the Bacillus pumilus is Bacillus pumilus QM6, with the accession number CCTCC NO: M 20221061 at the China Center for Type Culture Collection.

[0006] In the compound bacterial agent, the ratio of colony-forming units (CFU) of Bacillus cereus and Bacillus pumilus is 1:1.

[0007] In the compound bacterial agent, the Bacillus cereus and the Bacillus pumilus can grow at a low temperature of 10℃-15℃.

[0008] The present invention also provides a Bacillus cereus, wherein the Bacillus cereus is Bacillus cereus QS7, and its accession number at the China Center for Type Culture Collection is CCTCC NO: M 20221062.

[0009] The present invention also provides Bacillus pumilus, specifically Bacillus pumilus QM6, which has the accession number CCTCC NO: M 20221061 at the China Center for Type Culture Collection.

[0010] The present invention also provides a microbial agent for preparing compost, the microbial agent containing the above-mentioned Bacillus cereus and Bacillus pumilus.

[0011] The present invention also provides a fertilizer, which is an organic fertilizer obtained by fermenting mushroom residue and / or livestock and poultry manure as raw materials using the above-mentioned compound microbial agent, the above-mentioned Bacillus cereus or the above-mentioned Bacillus pumilus.

[0012] The present invention also provides the use of the above-mentioned compound microbial agent, the above-mentioned Bacillus cereus and Bacillus pumilus, or the above-mentioned microbial agent in any of the following:

[0013] 1) Produces cellulase;

[0014] 2) Fermentation composting under low-temperature conditions;

[0015] 3) Low temperatures accelerate the composting process;

[0016] 4) Application in promoting plant seed germination or in the preparation of products that promote plant seed germination.

[0017] The application of the above-mentioned fertilizers in promoting plant seed germination also falls within the scope of protection of this invention.

[0018] The compost is made from mushroom residue and / or livestock and poultry manure. The low temperature can be -15°C to -10°C.

[0019] The plant in question may be rapeseed.

[0020] The present invention aims to screen out mixed strains that are conducive to promoting fermentation at low temperatures and have high cellulase activity, so that they can be applied to low-temperature composting fermentation, thereby reducing composting costs and improving composting efficiency.

[0021] Preservation Instructions

[0022] Bacterial strain name: Bacillus cereus

[0023] Latin name: Bacillus cereus

[0024] Strain number: QS7

[0025] Preservation Institution: China Center for Type Culture Collection

[0026] Abbreviation for depository institution: CCTCC

[0027] Address: Wuhan University, Wuhan, China

[0028] Deposit date: July 8, 2022

[0029] Collection Center Registration Number: CCTCC NO: M 20221062

[0030] Bacterial strain name: Bacillus pumilus

[0031] Latin name: Bacillus pumilus

[0032] Strain number: QM6

[0033] Preservation Institution: China Center for Type Culture Collection

[0034] Abbreviation for depository institution: CCTCC

[0035] Address: Wuhan University, Wuhan, China

[0036] Deposit date: July 8, 2022

[0037] Collection Center Registration Number: CCTCC NO: M 20221061 Attached Figure Description

[0038] Figure 1 This is for monitoring temperature changes in the compost pile during summer. Note: Treatment 1 (mushroom residue:sheep manure volume ratio = 9:1, with added mixed bacteria), Treatment 2 (mushroom residue:sheep manure volume ratio = 9:1, without added bacteria).

[0039] Figure 2 This is for monitoring temperature changes in the compost pile during winter. Note: Treatment 3 (mixed straw:coarse manure volume ratio = 1:1, with added mixed bacteria), Treatment 4 (mixed straw:coarse manure volume ratio = 1:1, without added bacteria) Detailed Implementation

[0040] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0043] The culture medium formulations used in the following experiments are as follows:

[0044] NA solid medium (g / L): 5g beef extract, 10g peptone, 5g sodium chloride, 17g agar, add water to 1000mL, pH 7.0.

[0045] The difference between NA liquid culture medium and the aforementioned NA solid culture medium is that no agar is added.

[0046] CMC-Na medium (g / L): CMC-Na 10g, NaCl 5g, KH2PO4 1g / L, MgSO4 0.2g, yeast powder 10g, agar 18g, add double-distilled water to 1L, adjust pH to 7.0, sterilize at 121℃ for 20min.

[0047] Protectant (NB medium) (g / L): 5g beef extract, 15g peptone, 5g sodium chloride, 10% dimethyl sulfoxide, add double-distilled water to 1L, adjust pH to 7.0, sterilize at 121℃ for 20min.

[0048] Example 1: Isolation, Identification and Preservation of Low-Temperature-Resistant Bacteria

[0049] 1. Strains Isolation

[0050] Weigh 5g of cottonseed hull sample from Qinghai Huitian Agricultural Planting Base in Dabaozi, Xining City, Qinghai Province, and add it to an Erlenmeyer flask containing 45mL of sterile water. Shake for 10 minutes, perform serial dilution, and incubate on NA medium at 37℃ for 24 hours. Pick single colonies.

[0051] 2. Culture and morphological characteristics of the strain

[0052] Observe QS7: Bacterial cells are rod-shaped, with square ends, and appear in short or long chains. They are Gram-positive, non-capsulated, and motile. Colonies are relatively large in diameter, with a rough, flat, and irregular surface.

[0053] QM6: The bacteria are thin rod-shaped, Gram-positive, and have round colonies.

[0054] 3. Molecular identification

[0055] After purifying strain QS7, single colonies were selected for PCR. Universal primers for bacterial 16S rRNA gene amplification were used: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification products were then sequenced to obtain the 16S rRNA gene sequence, which is sequence 1 in the sequence listing. BLAST alignment of the sequencing results, combined with morphological analysis, confirmed that the isolated strain was *Bacillus cereus*.

[0056] After purifying strain QM6, single colonies were selected for PCR. Universal primers for bacterial 16S rRNA gene amplification were used: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification products were then sequenced to obtain the 16S rRNA gene sequence, which is sequence 2 in the sequence listing. BLAST alignment of the sequencing results, combined with morphological analysis, confirmed that the isolated strain was *Bacillus pumilus*.

[0057] 4. Preservation of QS7 and QM6 strains

[0058] The Bacillus cereus QS7 provided by this invention was deposited on July 8, 2022, at the China Center for Type Culture Collection (CCTCC; address: Wuhan University, Wuhan, China; postcode: 430072), with accession number CCTCC NO: M 20221062. Bacillus cereus QS7 is abbreviated as Bacillus cereus QS7 or QS7.

[0059] The Bacillus pumilus QM6 provided by this invention was deposited on July 8, 2022, at the China Center for Type Culture Collection (CCTCC; address: Wuhan University, Wuhan, China; postcode: 430072), with accession number CCTCC NO: M 20221061. Bacillus pumilus QM6 is abbreviated as Bacillus pumilus QM6 or QM6.

[0060] Example 2: Study on the characteristics of low-temperature resistant strains

[0061] 1. Activation of the strain

[0062] The cryopreserved Bacillus pumilus QM6 was inoculated onto a sterile NA medium plate using an inoculation loop and incubated aerobically at 37°C for 24 hours. A single colony was picked and inoculated onto the NA medium plate again, and incubated aerobically at 37°C for 24 hours. This process was repeated three times until the activated strain was ready for later experiments.

[0063] The cryopreserved Bacillus cereus QS7 was inoculated onto a sterile NA medium plate using an inoculation loop and incubated aerobically at 37°C for 24 hours. A single colony was picked and inoculated onto the NA medium plate again, and incubated aerobically at 37°C for 24 hours. This process was repeated three times until the activated strain was ready for later experiments.

[0064] 2. Growth test of Bacillus pumilus QM6 and Bacillus cereus QS7 on oyster mushroom powder and mushroom residue powder.

[0065] After activating Bacillus cereus QS7 and Bacillus pumilus QM6, respectively, sterilized toothpicks were used to inoculate them onto oyster mushroom powder and mushroom residue powder culture media. The media were then aerobically incubated at 37℃ for 24 hours, and the growth of these two strains on different media was observed. The preparation method for the oyster mushroom powder culture medium is as follows:

[0066] The preparation method of oyster mushroom powder culture medium is as follows: 2% oyster mushroom powder + 2% agar. Purchase fresh oyster mushrooms from the market, place them on a tray and dry them at 65℃ for 24 hours to completely evaporate the moisture. Then, use a grinder to grind them into powder. Calculate the required mass of oyster mushroom powder based on the total volume of the culture medium, add 2% agar, add water, sterilize, and use for subsequent experiments.

[0067] The preparation method of mushroom residue powder culture medium is as follows: 2% mushroom residue powder + 2% agar. The collected raw mushroom residue is placed in a tray and dried at 65℃ for 24 hours to completely evaporate the moisture. Then, it is ground into powder using a pulverizer. The required mass of mushroom residue powder is calculated based on the total volume of the culture medium, and 2% agar is added. Water is added, and the mixture is sterilized for subsequent experiments.

[0068] The results are shown in Table 1. Based on the growth of the two Bacillus strains after inoculation, it can be concluded that Bacillus cereus QS7 grew very well on both oyster mushroom powder and mushroom residue powder, and had strong activity, indicating that it had strong adaptability to the mushroom residue and oyster mushroom environment. Bacillus pumilus QM6 did not grow on the oyster mushroom powder culture medium, indicating that there are certain substances in the oyster mushroom powder that inhibit the growth of this bacterium, but it grew well on mushroom residue powder and can be used for the fermentation of mushroom residue.

[0069] Table 1. Growth of Bacillus cereus QS7 and Bacillus pumilus QM6 on oyster mushroom powder and mushroom residue powder.

[0070] Bacillus cereus QS7 +++ +++ Bacillus pumilus QM6 - ++

[0071] Note: +++, excellent growth; ++, very good growth; +, very poor growth; -, no growth.

[0072] 3. Low-temperature growth experiment of Bacillus pumilus QM6 and Bacillus cereus QS7

[0073] The strain activation procedure was the same as step 1 above. After purifying Bacillus cereus QS7 and Bacillus pumilus QM6, the bacteria were inoculated onto NA medium using a sterilized toothpick and cultured aerobically at 10℃ and 15℃ for 24 hours, respectively. The growth of the two strains at 10℃ and 15℃ was observed. The results showed that strain QS7 grew very well at 15℃ and very well at 10℃, indicating that this strain can maintain strong activity under low-temperature conditions, which is helpful for initiating low-temperature fermentation. Strain QM6 showed weak growth at both 10℃ and 15℃.

[0074] Table 2 Growth of QM6 and QS7 under low temperature conditions

[0075] 15℃ +++ + 10℃ ++ +

[0076] Note: +++, excellent growth; ++, very good growth; +, very poor growth; -, no growth.

[0077] 4. Antibacterial test of Bacillus pumilus QM6 and Bacillus cereus QS7

[0078] 1) Antibacterial study of QM6 against QS7

[0079] After inoculating *Bacillus pumilus* QM6 into NA liquid medium and culturing it at 37°C and 180 rpm for 24 h on a shaker, purified *Bacillus cereus* QS7 was transferred to a centrifuge tube containing 10 mL of sterile water (temperature not exceeding 50°C) using a sterile cotton swab. The absorbance was measured at 600 nm using a spectrophotometer, and the OD600 nm value was adjusted to 1.0. 0.3 mL of the above bacterial culture was added to a centrifuge tube containing 10 mL of liquid NA solid medium (temperature not exceeding 50°C), and mixed thoroughly. 5 mL of this mixture was then added to the top layer of a petri dish containing 20 mL of solid NA medium (already solidified), leveled, and allowed to solidify. Holes were then punched in the medium using a hole punch. Take 1.4 mL of liquid culture medium of strain QM6 into a centrifuge tube, centrifuge, add 200 μL of supernatant to the well, and then incubate at 37℃ for 24 h to observe the antibacterial effect of Bacillus pumilus QM6 on Bacillus cereus QS7.

[0080] 2) Antibacterial study of strain QS7 against QM6

[0081] Bacillus cereus QS7 was inoculated into NA liquid medium and cultured on a shaker at 37°C and 180 rpm for 24 h. Then, purified Bacillus simulans QM6 was transferred to a centrifuge tube containing 10 mL of sterile water (temperature not exceeding 50°C) using a sterile cotton swab. The absorbance was measured at 600 nm using a spectrophotometer, and the OD value was adjusted to 1. 0.3 mL of the above bacterial culture was added to a centrifuge tube containing 10 mL of liquid NA solid medium (temperature not exceeding 50°C), and mixed thoroughly. 5 mL of this mixture was then added to the top layer of a petri dish containing 20 mL of solidified NA medium, leveled, and allowed to solidify. After solidification, holes were punched in the medium. Take 1.4 mL of liquid culture medium of strain QS7 into a centrifuge tube, centrifuge, add 200 μL of supernatant to the well, and then incubate at 37℃ for 24 h to observe the antibacterial effect of Bacillus cereus QS7 on Bacillus pumilus QM6.

[0082] As shown in Table 3, the antibacterial results indicate that there is no mutual inhibition between strains QM6 and QS7, suggesting that these two strains can be used together without threatening the growth of the other strain.

[0083] Table 3 Mutual inhibition test between QM6 and QS7 strains

[0084] Bacillus pumilus QM6 - Bacillus cereus QS7 -

[0085] Note: The inhibition diameter includes the outer diameter of the punch, where the diameter of the inhibition zone is: - indicates no inhibition; + indicates 10-15mm; ++ indicates 15-20mm; +++ indicates 20-25mm; ++++ indicates more than 25mm.

[0086] 5. Determination of cellulase production capacity of Bacillus pumilus QM6 and Bacillus cereus QS7

[0087] Bacillus pumilus QM6 and Bacillus cereus QS7 were purified and subcultured on CMC-Na medium. The purified strains were then spotted onto CMC-Na solid medium using sterilized toothpicks and incubated at 37°C for 24 h. After staining with Congo red for 30 min, the samples were washed with 1 mol / L NaCl for 5 min, and the NaCl was discarded. The diameter of the hydrolysis zone and the colony diameter were measured using calipers, and their ratio was calculated. The ratio of the hydrolysis zone diameter to the colony diameter can be used to preliminarily determine the cellulase production capacity of different strains; the larger the D / H ratio of the Congo red hydrolysis zone, the higher the cellulase production capacity of the strain.

[0088] Table 4 shows that the D / H value of Bacillus cereus QS7 is 1.33, and the D / H value of Bacillus pumilus QM6 is 7.41, indicating that Bacillus pumilus QM6 has a higher cellulase production capacity.

[0089] Table 4. Determination of cellulase production capacity of strains

[0090]

[0091] In summary, Bacillus cereus QS7 grows better under low temperature conditions, and Bacillus pumilus QM6 has a high cellulase production capacity. Both can grow on mushroom residue powder without inhibiting each other.

[0092] 6. Temperature changes in mushroom residue and sheep manure compost piles under different treatment conditions

[0093] 6.1 Preparation of Fermentation Broth

[0094] Single colonies of Bacillus cereus QS7 were picked from agar plates and inoculated into NA liquid medium (NA liquid medium formula: 5g beef extract, 10g peptone, 5g NaCl, double-distilled water to 1L, sterilized at 121℃ for 20min). The medium was incubated at 37℃ and 180rpm for 24h to obtain Bacillus cereus QS7 seed culture. The Bacillus cereus QS7 seed culture was then added to sterilized NA liquid medium at a ratio of 1%, and incubated at 37℃ and 180rpm for 24h. The fermentation broth was collected to obtain the Bacillus cereus QS7 fermentation broth. The concentration of Bacillus cereus QS7 in the fermentation broth was 1.0 × 10⁻⁶. 9 cfu / mL or higher.

[0095] Single colonies of *Bacillus pumilus* QM6 were picked from agar plates and inoculated into NA liquid medium. The culture was incubated at 37°C and 180 rpm for 24 hours to obtain the *Bacillus pumilus* QM6 seed culture. The *Bacillus pumilus* QM6 seed culture was then added to sterilized NA liquid medium at a ratio of 1%, and the culture was incubated at 37°C and 180 rpm for 24 hours. The fermentation broth was collected to obtain the *Bacillus pumilus* QM6 fermentation broth. The concentration of *Bacillus pumilus* QM6 in the fermentation broth was 1.0 × 10⁻⁶. 9 cfu / mL or higher.

[0096] 6.2 Preparation of Compound Microbial Agent for Composting Fermentation

[0097] The content of Bacillus cereus QS7 in 6.1 was 1.0 × 10⁶. 9 The fermentation broth contained 1.0 × 10⁻⁶ cfu / g of Bacillus pumilus QM6. 9 The CFU / g fermentation broth was mixed and diluted 10 times with sterile water. The content of Bacillus cereus QS7 in this composting fermentation compound microbial agent was 1.0 × 10⁻⁶.8 The cfu / g content of Bacillus pumilus QM6 fermentation broth was 1.0 × 10⁻⁶. 8 cfu / g.

[0098] 6.3 Composting

[0099] Preparation of composting fermentation raw materials: Mix mushroom residue and sheep manure at a volume ratio of 9:1 to obtain composting fermentation raw materials.

[0100] 6.3.1 Composting fermentation using compound microbial agents

[0101] Composting was carried out at Qinghai Provincial Special Fertilizer Co., Ltd. from July to September using the 6.2 composting fermentation compound microbial agent. The above-mentioned composting fermentation raw materials were mixed evenly with the 6.2 composting fermentation compound microbial agent to obtain the compost material. The content of Bacillus cereus QS7 in the compost material was 1.0 × 10⁻⁶. 6 The cfu / g content of Bacillus pumilus QM6 fermentation broth was 1.0 × 10⁻⁶. 6 cfu / g.

[0102] The material is composted using a stacking method, with each stack having a volume of 40.5 m³. 3 (7.5m long, 4m wide, 1.35m high), with the moisture content adjusted to 55%. Insertion electrodes are installed on the top and sides of the pile, extending 40cm into the pile, and connected to digital thermometers via wires.

[0103] During the experiment, the ambient temperature and the temperature of the compost pile were measured once a day at 12 noon. Mechanical turning was used on days 0, 5, 10, 15, 20, 30, 45, and 60. After meeting national standards, the compound microbial agent-fermented compost was obtained.

[0104] 6.3.2 Natural fermentation without adding microbial agents

[0105] The only difference from 6.3.1 is that the composting fermentation compound microbial agent in 6.2 is replaced with an equal mass of sterile water. All other operations are the same, and the control composted fertilizer is obtained.

[0106] 7. Changes in germination rate and germination index of rapeseed after different compost treatments

[0107] 7.1 Treatment of composted fertilizer with compound microbial agents

[0108] The experiment was repeated three times, with each repetition as follows: 10g of the compound microbial agent composted fertilizer from step 6.3.1 was placed in an Erlenmeyer flask with 90mL of water. The mixture was filtered through filter paper, and 5mL of the filtrate was transferred to a petri dish lined with sterilized filter paper. Twenty similarly sized, plump rapeseed seeds were evenly placed in each petri dish. 5mL of distilled water was used as a control instead of the 5mL filtrate. After culturing at 25℃ for 72 hours, the number of germinated seeds and root length in each petri dish were recorded. The germination rate and seed germination index of each treatment were calculated. The formula for calculating the seed germination rate was: Germination rate = Number of seeds germinated in the specified number of days / Number of tested seeds × 100%. The formula for calculating the seed germination index was: Seed germination index = (Seed germination rate of extract × Root length) / (Germination rate of control seeds × Root length) × 100%.

[0109] 7.2 Comparison with well-rotted fertilizer treatment

[0110] The only difference from 7.1 is that the compound microbial agent composted fertilizer in 6.3.1 is replaced with the control composted fertilizer in 6.3.2; all other operations are the same.

[0111] Table 5 shows that the compound microbial agent composted fertilizer has a better effect on rapeseed seed germination than the control composted fertilizer treatment without compound microbial agent.

[0112] Table 5. Effects of different composting treatments on rapeseed germination rate and germination index.

[0113] Germination rate (%) 100.00±0.00 96.67±5.77 Seed germination index (%) 106.86±6.86 83.06±5.49

[0114] 8. Effects of fermented compost in different seasons on rapeseed seed germination rate and germination index

[0115] In summer and winter, after the compost was fermented using the method in step 6, the germination rate of rapeseed seeds was measured.

[0116] Experimental Location: Key Laboratory of Ion Beam Bioengineering, Zhengzhou, Henan Province

[0117] Treatment 1: Summer fermentation compost, room temperature (10-25℃), mushroom residue:sheep manure volume ratio = 9:1, compost with added compound microbial agent was the treatment group, where the inoculum amount of compound microbial agent (Bacillus cereus and Bacillus pumilus colony-forming units CFU ratio of 1:1) was 1.0 × 10⁻⁶. 6 CFU Bacillus cereus: 1 kg compost base (mushroom residue: sheep manure volume ratio = 9:1);

[0118] Treatment 2: Summer fermentation compost, room temperature (10-25℃), mushroom residue: sheep manure volume ratio = 9:1, compost without added compound microbial agent served as control group;

[0119] Treatment 3: Winter fermentation composting, at room temperature (-15-4℃), the compost containing a mixture of straw and cow manure in a 1:1 volume ratio, with the addition of a compound microbial agent. The inoculum amount of the compound microbial agent (Bacillus cereus and Bacillus pumilus in a 1:1 CFU ratio) was 1.0 × 10⁻⁶ CFU for Bacillus cereus. 6 CFU Bacillus cereus: 1 kg compost substrate (mushroom residue: sheep manure)

[0120] Treatment 4: Winter fermentation compost, with a volume ratio of straw to cow manure of 1:1 at room temperature (-15-4℃), and compost without the addition of compound microbial agents served as the control group.

[0121] 8.1 Composting fermentation using compound microbial agents

[0122] Qinghai Provincial Special Fertilizer Co., Ltd. conducted composting fermentation using a compound microbial agent at the Qinghai Qianziyuan Agricultural Science and Technology Expo Park from June to July (December to March). Mixed straw and cow manure were thoroughly mixed with the composting fermentation compound microbial agent to obtain the compost material. The compost material contained 1.0 × 10⁻⁶ Bacillus cereus QS7. 6 The cfu / g content of Bacillus pumilus QM6 fermentation broth was 1.0 × 10⁻⁶. 6 cfu / g.

[0123] The material is composted using a stacking method, with each stack having a volume of 18m³. 3 (4m long, 3m wide, 1.5m high) The moisture content was adjusted to 55%. A plastic film was placed over the pile surface to reduce moisture and heat loss. During the experiment, the ambient temperature and pile temperature were measured once daily at noon. Mechanical turning was used when the temperature reached its peak and began to decline continuously. After meeting the national organic fertilizer agricultural industry standard NY 525-2021, a compound microbial agent composted fertilizer was obtained.

[0124] The fermented fertilizer obtained by the above methods in summer is the well-rotted fertilizer obtained in treatment 1, and the fermented fertilizer obtained by the above methods in winter is the well-rotted fertilizer obtained in treatment 3.

[0125] 8.2 Natural fermentation without adding bacterial agents

[0126] The only difference from 8.1 is that the composting fermentation compound microbial agent is replaced with an equal mass of sterilized sterile water; all other operations are the same, resulting in control composted fertilizer. The composted fertilizer obtained by fermentation in summer using the above methods is the fertilizer obtained in treatment 2, and the composted fertilizer obtained by fermentation in winter using the above methods is the fertilizer obtained in treatment 4.

[0127] The monitoring results of temperature changes in the compost pile during summer are shown in Figure 1. Throughout the fermentation process in summer, the core temperature of different piles successively experienced three different fermentation stages: heating, high temperature, and cooling. All treatments began to heat up rapidly on the second day of composting, but the compound microbial agent treatment group 1 had the fastest heating rate. The core temperature of both treatments reached above 50℃ on the third day. Starting from the 35th day, the temperature of both groups began to gradually decrease, entering the later stage of composting. The compound microbial agent treatment group 1 cooled down faster than the control group, and its temperature was slightly lower than that of the control group. The duration of high temperature above 55℃ for both treatment groups was 29 days, and the duration of high temperature above 60℃ was 11 days for treatment group 1 and 5 days for treatment group 2, respectively. The compound microbial agent treatment group 1 reached its highest temperature of 62.8℃ on the 22nd day; the control group reached its highest temperature of 61.4℃ on the 29th day of composting, while the corresponding temperature of the compound microbial agent treatment group was 62.4℃. In summary, the addition of mixed bacteria allows the compost to heat up faster and reach a higher compost temperature, while also effectively shortening the composting time and improving composting efficiency.

[0128] Monitoring of temperature changes in the compost pile during winter composting process, results are as follows: Figure 2 As shown, during the entire fermentation process in winter, the pile temperature exhibited a trend of first rising, then falling, and then rising again. In the initial stage of fermentation, the average pile temperature for both treatments was 3℃. From day 3 onwards, the pile temperature of treatment group 3 (compound microbial agent treatment group) began to be significantly higher than that of the control group. In the later stage of fermentation, the compound microbial agent treatment group experienced a faster and lower temperature drop compared to the control group. The duration of temperatures above 50℃ was 20 days for the control group and 24 days for the compound microbial agent treatment group. The temperature in the control group did not reach 60℃, while the duration of temperatures above 60℃ in the compound microbial agent treatment group was 4 days. In conclusion, the addition of the compound microbial agent accelerated the winter composting process.

[0129] 8.3 Compound microbial agent for composting fertilizer treatment

[0130] The compound microbial agent composting fertilizer treatment experiment was conducted in triplicate, with each replicate as follows: 10g of the compound microbial agent composting fertilizer from step 8.1 (composted fertilizers obtained from treatments 1 and 3) was placed in an Erlenmeyer flask with 90mL of water. The mixture was filtered through filter paper, and 5mL of the filtrate was transferred to a petri dish lined with sterilized filter paper. Twenty similarly sized, plump rapeseed seeds were evenly placed in each petri dish. 5mL of distilled water was used as a control instead of the 5mL filtrate. After culturing at 25℃ for 72 hours, the number of germinated seeds and root length in each petri dish were recorded, and the germination rate and seed germination index of each treatment were calculated. The formula for calculating the seed germination rate is: Germination rate = Number of germinated seeds in specified days / Number of tested seeds × 100%. The formula for calculating the seed germination index is: Seed germination index = (Seed germination rate of extract × Root length) / (Germination rate of control seeds × Root length) × 100%.

[0131] 8.4 Comparison with well-rotted fertilizer treatment

[0132] The only difference from 8.3 is that the compound microbial agent composted fertilizer in 8.1 (composted fertilizer obtained from treatment 1 and treatment 3) is replaced with the control composted fertilizer in 8.2 (composted fertilizer obtained from treatment 2 and treatment 4). All other operations are the same.

[0133] As shown in Table 6, the addition of compound microbial agents to compost can achieve good germination of rapeseed seeds regardless of whether the compost is fermented in summer or winter. Under the same compost raw material conditions, the addition of compound microbial agents can greatly shorten the compost fermentation time.

[0134] Table 6. Effects of different treatments on rapeseed seed germination rate and seed germination index.

[0135] Fermentation time 60 days 91 days 83 days 146 days Germination rate (%) 100.00±0.00 96.67±5.77 98.35±1.85 95.43±2.31 Seed germination index (%) 106.86±6.86 83.06±5.49 85.46±3.25 83.32±4.63

[0136] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A compound microbial agent for fermentation composting, characterized in that, The active ingredients of the compound microbial agent consist of Bacillus cereus and Bacillus pumilus; the Bacillus cereus is Bacillus cereus. Bacillus cereus QS7, its accession number at the China Center for Type Culture Collection is CCTCC NO: M 20221062; the *Bacillus pumilus* mentioned is *Bacillus pumilus*. Bacillus pumilus QM6, whose accession number at the China Center for Type Culture Collection is CCTCC NO: M20221061; in the compound bacterial agent, the ratio of colony-forming units (CFU) of Bacillus cereus and Bacillus pumilus is 1:

1.

2. The compound microbial agent according to claim 1, characterized in that: The Bacillus cereus and the Bacillus pumilus can grow at low temperatures of 10°C-15°C.

3. A fertilizer, characterized in that, The fertilizer is an organic fertilizer obtained by fermenting mushroom residue and / or livestock and poultry manure using the compound microbial agent described in claim 1 or 2.

4. The use of the compound microbial agent according to claim 1 or 2 in any of the following: 1) Produces cellulase; 2) Fermentation composting under low-temperature conditions; 3) Accelerate the composting process.

5. The application of the fertilizer according to claim 3 in promoting rapeseed germination.

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