Fermenting bacteria, fermentation compound agents containing them, preparation methods and applications of compound agents

Abstract

This invention belongs to the field of bio-fermentation technology, and particularly relates to fermenting bacteria, a fermentation compound agent prepared based on these fermenting bacteria, and specific preparation methods and applications of the fermentation compound agent. The fermenting bacteria are selected from *Bacillus licheniformis* CY-1 and *Alcaligenes faecalis* CY-2; the *Bacillus licheniformis* CY-1 is deposited at CGMCC with accession number CGMCC No. 21241; the *Alcaligenes faecalis* CY-2 is deposited at CGMCC with accession number CGMCC No. 21242. This invention achieves effective composting of easily perishable waste through the specific combination of *Bacillus licheniformis* CY-1 and *Alcaligenes faecalis* CY-2. During the composting process, it effectively fixes nitrogen and reduces emissions, improving the quality, economic value, and utility of the treated easily perishable waste compost product.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, and particularly relates to fermenting bacteria, fermentation compound agents prepared based on these fermenting bacteria, and specific preparation methods and applications of the fermentation compound agents. Background Technology

[0002] In recent years, with the promotion and implementation of the garbage classification system, my country has greatly improved the disposal and resource utilization rate of perishable waste. Perishable waste is rich in organic matter such as starch, cellulose, protein, and lipids, and after processing, it can be made into organic fertilizer or soil conditioner, thus achieving harmless treatment and efficient resource utilization of perishable waste. Therefore, utilizing aerobic fermentation by microorganisms to produce organic fertilizer, soil conditioner, and other resource-based products has become the mainstream method for the resource utilization of perishable waste both domestically and internationally.

[0003] Fermentation of perishable waste is one of the main methods of aerobic fermentation. However, the current problems of low-quality organic fertilizer and high energy consumption in perishable waste treatment equipment are actually caused by the low aerobic degradation efficiency of perishable waste. If the processing time is insufficient, the quality of the organic fertilizer will not meet the standards; if the processing time is increased, the energy consumption will be even greater. Therefore, the key to solving the problems of low-quality organic fertilizer and high energy consumption is to improve the degradation efficiency of kitchen waste.

[0004] On the other hand, in recent years, solar-assisted composting fermentation technology, developed based on traditional composting treatment, has been widely promoted and used in many parts of China. Compared with traditional aerobic composting treatment technology, aerobic composting fermentation technology using solar-powered sunrooms for kitchen waste increases the ambient temperature and improves the efficiency of aerobic fermentation treatment of kitchen waste. It has certain advantages, especially in the low-temperature winter season, for the rapid heating and fermentation of kitchen waste. However, due to the high oil, high salt, and high water content of easily perishable waste such as kitchen waste, most solar-powered sunroom-assisted fermentation still suffers from problems such as low treatment efficiency, substandard organic fertilizer, and odor pollution. Some composting sites can lead to new water or air pollution, causing greater environmental impact and higher treatment costs. Therefore, this invention aims to improve composting efficiency, reduce odor pollution and nitrogen loss, and improve compost quality by preparing highly efficient microbial agents and their application methods in the composting of easily perishable waste, thereby improving the volume reduction, deodorization, and maturity of easily perishable waste.

[0005] Existing technologies, such as CN108148790A, disclose a mixed bacterial strain for the degradation of livestock and poultry manure and its application method. This method uses multiple strains of bacilli to treat easily decomposable livestock and poultry manure, achieving pollution-free and odorless emissions, and the treated manure can be used as organic fertilizer for landscaping. However, it has significant drawbacks. For example, the odor-free effect is achieved by decomposing large amounts of organic matter and releasing large quantities of ammonia and hydrogen sulfide, which are then fully released to achieve odorlessness. However, this treatment significantly reduces the nitrogen content of the easily decomposable waste, resulting in a substantial decrease in its actual effective value.

[0006] Therefore, how to rationally combine fermentation bacteria to effectively treat perishable waste while preserving its nutritional value for effective recycling is an important research direction in current bio-fermentation technology. Summary of the Invention

[0007] To address the limitations of existing microbial fermentation technologies, which are currently focused on waste treatment and unable to simultaneously achieve high-value utilization of perishable waste, this invention provides fermenting bacteria, a fermentation compound agent based on these bacteria, a preparation method for the fermentation compound agent, and its application technology.

[0008] The purpose of this invention is:

[0009] I. Two fermentation bacteria with specific functions were obtained through screening;

[0010] II. Effective fermentation treatment of perishable waste is achieved through the synergistic cooperation of specific functional fermenting bacteria and their supporting application technologies.

[0011] Third, ensure that the perishable waste after fermentation is pollution-free and odorless, while retaining high nutritional value;

[0012] IV. Significantly improves the efficiency of fermentation and degradation of perishable waste.

[0013] To achieve the above objectives, the present invention adopts the following technical solution.

[0014] Fermenting bacteria,

[0015] The fermenting bacteria are selected from Bacillus licheniformis and Alcaligenes faecalis;

[0016] The Bacillus licheniformis is classified and named Bacillus licheniformis (Bacillus licheniformis). Bacillus licheniformis CY-1, depositary institution: CGMCC, depositary accession number: CGMCC No.21241, deposit date: November 26, 2020;

[0017] The alkaloid bacillus described is classified and named Alcaligenes faecalis (Alcaligenes faecalis). Alcaligenes faecalis CY-2, depositary institution: CGMCC, deposit number: CGMCC No.21242, deposit date: November 26, 2020;

[0018] Specifically, the preservation unit, CGMCC, is officially known as the China General Microbiological Culture Collection Center, and its address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0019] Fermentation compound microbial agent,

[0020] The fermentation compound microbial agent contains Bacillus licheniformis and Alcaligenes faecalis.

[0021] As a preferred option

[0022] The viable count of Bacillus licheniformis and Alcaligenes faecalis in the fermentation compound microbial agent is ≥10. 8 CFU / mL.

[0023] Preparation method of fermentation compound microbial agent,

[0024] The method includes:

[0025] Colonies of Bacillus licheniformis and Alcaligenes faecalis were picked and cultured in a culture medium at 37±1 ℃ until a viable count of ≥10⁻⁶ was obtained. 8 The fermentation compound microbial agent is obtained by mixing the bacterial broth obtained from Bacillus licheniformis culture and the bacterial broth obtained from Alcaligenes faecalis culture at a volume ratio of 1:(0.9-1.1).

[0026] Application of fermentation compound microbial agents,

[0027] The fermentation compound microbial agent is used for composting easily perishable waste.

[0028] As a preferred option

[0029] The specific method for composting easily perishable waste is as follows:

[0030] Take at least 0.15 wt% of the fermentation compound microbial agent based on the weight of the perishable waste, and add it evenly to the perishable waste for treatment for at least 18 days.

[0031] For the technical solution of this invention, the invention first provides two specific fermenting bacteria, namely Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2. The whole genome sequencing of Bacillus licheniformis CY-1 is shown in SEQ ID NO:1, and the whole genome sequencing of Alcaligenes faecalis CY-2 is shown in SEQ ID NO:2. Common Bacillus licheniformis and Alcaligenes faecalis are relatively common fermenting bacteria, and there are precedents for their use in composting easily decomposable waste. However, the existing use of Bacillus licheniformis and Alcaligenes faecalis alone or in combination for composting still suffers from low efficiency and / or high emissions of odorous gases such as ammonia and hydrogen sulfide, as well as significant nitrogen nutrient loss, resulting in a long actual composting cycle, poor compost product quality, and significant environmental pollution and carbon emissions.

[0032] The specific Bacillus licheniformis and Alcaligenes faecalis used in this invention achieve composting efficiency more than double compared to conventional Bacillus licheniformis and / or Alcaligenes faecalis, as well as natural composting, significantly reducing the time required for compost maturation. Furthermore, the combined use of the specific Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 effectively suppresses ammonia emissions during composting. While a small number of nitrogen-fixing Bacillus megaterium and genetically engineered specific Alcaligenes faecalis can produce nitrogenase activity under anaerobic conditions with nitrates, thus exhibiting some nitrogen-fixing activity, typical waste composting occurs under aerobic conditions. Since decomposition is essentially an oxidative degradation process, Alcaligenes faecalis does not typically produce the corresponding nitrogen-fixing effect in conventional composting of easily perishable waste.

[0033] Regarding the technical solution of this invention, the specific Bacillus and Alcaligenes faecalis used have a mutually promoting effect, as demonstrated in the co-propagation experiment, which showed a significant synergistic effect.

[0034] The specific Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 used in this invention were both collected from easily perishable waste material in a fermentation pile in a sunroom in Sanmen County, Taizhou City, Zhejiang Province. The culture medium used for isolating Bacillus licheniformis CY-1 was agar plates (composition: 20 g / L agar; sterilization conditions: 121 ℃ for 15 min), and the culture medium used for isolating Alcaligenes faecalis CY-2 was calcium phytate plates (composition: 30 g / L glucose, 25 g / L tryptone, 5 g / L calcium phytate, 20 g / L agar, 0.5 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 0.03 g / L anhydrous manganese sulfate, 0.5 g / L potassium chloride; sterilization conditions: 121 ℃ for 15 min). Specifically, the collected easily perishable waste material was placed in sterile sampling bags and brought back to the laboratory for pulverization. 5 g of each sample was weighed and then 45 g of the sample was added to each sample. mL of sterile water was placed in Erlenmeyer flasks, and 5 sterile glass beads were added to each flask (for each sample). The flasks were incubated at 60 °C on a shaker for 2 h. The supernatant was then aspirated and serially diluted 10-fold for further incubation. Finally, 10 mL of the supernatant was collected. -4 100 μL of the diluted solution was spread onto the agar and calcium phytate plates mentioned above, and incubated at 37 °C for 72 h. After the colonies with obvious hydrolysis zones grew on the plates, they were inoculated onto fresh, identical plates and purified and incubated at 37 ± 1 °C for 72 h. The purified Bacillus licheniformis CY-1 was isolated from the agar plate, and the purified Alcaligenes faecalis CY-2 was isolated from the calcium phytate plate. In further propagation experiments, perishable waste material after high-temperature sterilization was crushed into powder and used to prepare a culture medium (components: 20 g / L glucose, 60 g / L perishable waste material powder, 2 g / L calcium phytate, 20 g / L agar, 0.3 g / L ammonium sulfate, 0.3 g / L magnesium sulfate heptahydrate, 0.01 g / L anhydrous manganese sulfate, 0.2 g / L potassium chloride; sterilization conditions: 121 ℃ for 15 min). Single colonies of the isolated and purified Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 were inoculated into the above culture medium and incubated at 37 ℃ and 180 r / min for 24 h. The viable count was ≥10. 9The CFU / mL concentration indicates that *Bacillus licheniformis* CY-1 can decompose some components of easily perishable waste into effective nutrients required for the growth of *Alcaligenes faecalis* CY-2, thereby effectively promoting the growth of *Alcaligenes faecalis* CY-2. Simultaneously, the presence of *Alcaligenes faecalis* accelerates the growth and development efficiency of *Bacillus licheniformis* CY-1. This is because, after isolating *Bacillus licheniformis* CY-1, its propagation on calcium phytate plates was less effective than the cultivation on calcium phytate plates and agar plates during the isolation process. Therefore, it can be seen that *Bacillus licheniformis* CY-1 and *Alcaligenes faecalis* CY-2 can actually produce a synergistic effect during the composting of easily perishable waste.

[0035] The beneficial effects of this invention are:

[0036] This invention achieves effective composting of perishable waste through the specific combination of Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2. During the composting process, nitrogen can be effectively fixed, thereby improving the economic and utility value of the treated perishable waste. Attached Figure Description

[0037] Figure 1 The figure shows the experimental results of the number of composting days and the compost temperature when the fermentation compound microbial agent was used in the actual composting treatment of perishable waste.

[0038] Figure 2 A graph showing the change in fresh weight of perishable waste pile when the fermentation compound microbial agent is used in actual perishable waste composting treatment.

[0039] Figure 3 This is a graph showing the change in ammonia emissions when the fermentation compound microbial agent is used in the actual composting of perishable waste. Detailed Implementation

[0040] The present invention will be further described clearly and in detail below with reference to specific embodiments and the accompanying drawings. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0041] Unless otherwise specified, all raw materials used in the embodiments of the present invention are commercially available or obtainable by those skilled in the art; unless otherwise specified, all methods used in the embodiments of the present invention are methods mastered by those skilled in the art.

[0042] Example 1

[0043] Screening and purification of Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-1:

[0044] (1) Sampling

[0045] We collected perishable waste from a fermentation pile in a sunroom in Sanmen County, Taizhou City, and stored the samples in sterile sampling bags before bringing them back to the laboratory.

[0046] (2) Separation

[0047] After pulverizing the collected samples, weigh out 5 g of each sample and add it to an Erlenmeyer flask containing 45 mL of sterile water. Add 5 sterile glass beads to the Erlenmeyer flask and incubate on a shaker at 60 ℃ for 2 h. Dilute to 10⁻⁶. -4 A 100 μL sample of the diluted solution was spread onto an agar plate, and another 100 μL sample was spread onto a calcium phytate plate. Both were incubated at 37 °C for 72 h.

[0048] The agar plate formulation is: 20 g / L agar; the agar plates are sterilized at 121 ℃ for 15 min.

[0049] The phytate tablet formulation is as follows: 30 g / L glucose, 25 g / L tryptone, 5 g / L calcium phytate, 20 g / L agar, 0.5 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 0.03 g / L anhydrous manganese sulfate, and 0.5 g / L potassium chloride; the phytate tablets are sterilized at 121 °C for 15 min.

[0050] After colonies with obvious hydrolysis zones grow on the agar plates, the colonies on the agar plates are Bacillus licheniformis CY-1 colonies. Based on the colony morphology, colonies with different morphologies from Bacillus licheniformis CY-1 colonies on the calcium phytate plate are isolated, which are Alcaligenes faecalis CY-2 colonies, thus obtaining purified Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 strains.

[0051] The purified Bacillus licheniformis CY-1 strain and Alcaligenes faecalis CY-2 strain were respectively deposited at the China General Microbiological Culture Collection Center (CGMCC).

[0052] Bacillus licheniformis ( Bacillus licheniformis CY-1, with accession number CGMCC No. 21241, accession date: November 26, 2020;

[0053] Alcaligenes faecalis ( Alcaligenes faecalis CY-2, deposited on November 26, 2020, with accession number CGMCC No. 21242.

[0054] In addition, whole-genome sequencing was performed on the purified CY-1 strain and Alcaligenes faecalis CY-2 strain. The sequencing results are shown in the table below. In the table, SEQ ID NO: 1 represents the whole-genome sequencing result of Bacillus licheniformis CY-1, and SEQ ID NO: 2 represents the whole-genome sequencing result of Alcaligenes faecalis CY-2.

[0055] Serial Number sequence SEQ ID NO:1 CCTTCGGCGGCTGGCTCCAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAGCCTCGCGGCTTCGCTGCCCTTTGTTCTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGAAGCCCTATCTCTAGGGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTTGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTATTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCTAACAACAGAGTTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCTAAAAGCCACCTTTTATGATTGAACCATGCGGTTCAATCAAGCATCCGGTGTTAACCCCGGTTTCCCGGAGTTATCCCAGTCTTACCGGCAGGTTACCCACGTGGTACTCACCCGTCCGCCGCTGAACTAAGGGAGCAAGCTCCCGTCGGTCCGCTCGACTTGGATGTATTAAGCACGCCGGCAGCGTTCGTCCTGA SEQ ID NO:2 GTTTCTGAGATTGGCTCCCCCTCGCGGGTTGGCGACCCTCTGTCCCGACCATTGTATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCTCTTGCGTAGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCCGGTTCTCTTGCGAGCACGGCCAAATCTCTTCGGCTTTCCAGACATGTCAAGGGTAGGTAAGGTTTTTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTGCGCTACTAAGGCCTAACGGCCCCAACAGCTAGTTGACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGTCTGAGCGTCAGTATTATCCCAGGGGGCTGCCTTCGCCATCGGTATTCCTCCACATATCTACGCATTTCACTGCTACACGTGGAATTCTACCCCCCTCTGACATACTCTAGCTCGGCAGTTAAAAATGCAGTTCCAAGGTTGAGCCCTGGGATTTCACATCTTTCTTTCCGAACCGCCTACACACGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCTGCAGATACCGTCAGCAGTATCCCGTATTAGGGGATACCTTTTCTTCTCTGCCAAAAGTACTTTACAACCCGAAGGCCTTCATCATACACGCGGGATGGCTGGATCAGGGTTTCCCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGGTCGTCCTCTCAAACCAGCTACGGATCGTTGCCTTGGTGAGCCTTTACCCCACCAACTAGCTAATCCGATATCGGCCGCTCCAATAGTGAGAGGTCTTGCGATCCCCCCCTTTCCCCCGTAGGGCGTATGCGGTATTAGCCACTCTTTCGAGTAGTTATCCCCCGCTACTGGGCACGTTCCGATATATTACTCACCCGTCCGCCACTCGCCGCCAAGAGAGCAAGCTCTCTCGCGCTGCCGTTCGACTTGCATGTGTAAAGCATCCCGCTAGCGTTCAATCTGAGCCA .

[0056] Example 2

[0057] Preparation of fermentation compound microbial agent:

[0058] Single colonies of *Bacillus licheniformis* CY-1 isolated and purified in Example 1 were picked and placed on the agar plate medium described in Example 1, and single colonies of *Alcaligenes faecalis* CY-2 were picked and placed on the calcium phytate plate medium described in Example 1. Both were incubated at 37±1 °C until a viable count ≥10⁻⁶ was obtained. 8 The fermentation compound bacterial agent is obtained by mixing the bacterial solution of Bacillus licheniformis CY-1 culture and the bacterial solution of Alcaligenes faecalis CY-2 culture at a volume ratio of 1:1.

[0059] The fermentation compound inoculum prepared in this example is labeled as FCba-1.

[0060] Example 3

[0061] Preparation of fermentation compound microbial agent:

[0062] Single colonies of *Bacillus licheniformis* CY-1 and *Alcaligenes faecalis* CY-2 isolated and purified in Example 1 were picked and co-cultured in compost culture medium (composition: 20 g / L glucose, 60 g / L easily degradable waste powder, 2 g / L calcium phytate, 20 g / L agar, 0.3 g / L ammonium sulfate, 0.3 g / L magnesium sulfate heptahydrate, 0.01 g / L anhydrous manganese sulfate, 0.2 g / L potassium chloride; sterilization conditions: 121 ℃ for 15 min) at 37±1 ℃ until a viable count ≥10⁻⁶ was obtained. 8 The fermentation compound bacterial agent is obtained by preparing a bacterial solution of CFU / mL.

[0063] The fermentation compound inoculum prepared in this example is labeled as FCba-2.

[0064] Comparative Example 1

[0065] Preparation of fermentation inoculum:

[0066] A single colony of *Bacillus licheniformis* CY-1 isolated and purified in Example 1 was picked and placed on the agar plate medium described in Example 1, and incubated at 37±1 °C until a viable count of ≥10⁻⁶ was obtained. 8 The fermentation agent is obtained by dissolving the bacterial solution in CFU / mL.

[0067] The fermentation agent prepared in this example is labeled as Fa-1.

[0068] Comparative Example 2

[0069] Preparation of fermentation inoculum:

[0070] A single colony of *Alcaligenes faecalis* CY-2 isolated and purified in Example 1 was picked and placed on the calcium phytate plate medium described in Example 1, and cultured at 37±1 °C until a viable count of ≥10⁻⁶ was obtained. 8 The fermentation agent is obtained by dissolving the bacterial solution in CFU / mL.

[0071] The fermentation agent prepared in this example is labeled as Fa-2.

[0072] Comparative Example 3

[0073] Preparation of fermentation compound microbial agent:

[0074] Microbial agent labeling volume ratio FCba-31 The volume ratio of Bacillus licheniformis CY-1 bacterial suspension to Alcaligenes faecalis CY-2 bacterial suspension was 1:0.8. FCba-32 The volume ratio of Bacillus licheniformis CY-1 bacterial suspension to Alcaligenes faecalis CY-2 bacterial suspension was 1:0.9. FCba-33 The volume ratio of Bacillus licheniformis CY-1 bacterial suspension to Alcaligenes faecalis CY-2 bacterial suspension was 1:1.1. FCba-34 The volume ratio of Bacillus licheniformis CY-1 bacterial suspension to Alcaligenes faecalis CY-2 bacterial suspension was 1:1.2.

[0075] Comparative Example 4

[0076] Soil samples were collected from experimental fields of the Zhejiang Academy of Agricultural Sciences. *Bacillus licheniformis* and *Alcaligenes faecalis* were isolated and purified from the soil as described in Example 1. Using conventional morphological, nucleic acid, and optical characterization methods, the isolated strains were confirmed to be common *Bacillus licheniformis* species. Bacillus licheniformis ) and soil alkaloidobacterium ( Alcaligenes faecalis );

[0077] The above-mentioned soil Bacillus licheniformis and soil Alcaligenes faecalis were prepared into a fermentation compound microbial agent by implementing the method described in step 2.

[0078] The bacterial agent prepared in this example is labeled FCba-4.

[0079] Comparative Example 5

[0080] Purchased CICC Center Bacillus licheniformis strain ( Bacillus licheniformis, (Catalyst ID: CICC 10037) and Alcaligenes faecalis strain ( Alcaligenes faecalis, (Number: CICC 20141), prepared as a fermentation compound microbial agent according to the method described in Example 2.

[0081] The bacterial agent prepared in this example is labeled FCba-5.

[0082] Comparative Example 6

[0083] Purchased CICC Center Bacillus licheniformis strain ( Bacillus licheniformis, (Catalyst ID: CICC 10037) and Alcaligenes faecalis strain ( Alcaligenes faecalis, (Case No.: CICC 20141), prepared as a fermentation compound microbial agent according to the method described in Example 3. However, during the experiment, it was actually shown that the final fermentation compound microbial agent contained *Alcaligenes faecalis* (…). Alcaligenes faecalis, The viable count of strain CICC 20141 was significantly lower than that of Example 3, indicating that the conventional Bacillus licheniformis strain ( Bacillus licheniformis, (Catalyst ID: CICC 10037) and Alcaligenes faecalis strain ( Alcaligenes faecalis, (CICC 20141) is not suitable for direct joint propagation. The resulting inoculum had a significantly unbalanced proportion of viable bacteria, therefore no further application trials were conducted.

[0084] Application Examples

[0085] The microbial agent was evenly sprayed into the perishable waste pile at a mass ratio of 0.002:1, that is, 2 g of microbial agent was sprayed per 1000 g of perishable waste pile. The microbial agent label was used as the test group label, and the perishable waste pile without microbial agent spraying was used as the blank control (CK) for comparison.

[0086] The stack temperature testing results for Examples 2, 3, Comparative Examples 1, 2, 4, and 5 are as follows:

[0087] from Figure 1As can be seen from the figures, the compound microbial agents prepared in Examples 2 and 3 of this invention can rapidly increase the composting temperature, reflecting the dynamic changes in the temperature of the aerobic fermentation pile of perishable waste after the addition of the compound microbial agent. As can be seen from the figures, after the addition of the compound microbial agent, the temperature of the perishable waste pile rapidly increased and continued to rise after the second day, reaching a maximum of about 75 ℃. The pile temperature of 55 ℃ was maintained for 14 days before the pile temperature rapidly decreased. In contrast, the temperature of the perishable waste pile in the control group (CK) without any added microbial agent rose significantly slowly, and the maximum temperature only reached about 58 ℃. It is evident that adding the screened compound functional microbial agent to perishable waste has a significant effect on promoting the rapid temperature rise and fermentation of the aerobic pile of perishable waste. Comparing the FCba-1 and FCba-2 inoculants in Examples 2 and 3, it can be seen that the FCba-2 inoculant heats up significantly faster and reaches peak efficiency more quickly. Research shows that in Example 3, the ratio of inoculants cultivated in the composting medium is closer to the ratio of Bacillus licheniformis CY-1 to Alcaligenes faecalis CY-2 of 1:0.96, indicating that this ratio may be more beneficial for composting of perishable waste. Furthermore, cultivation in the composting medium may further enhance certain functionalities of the inoculants, allowing them to manifest in the actual composting process. In contrast, the Fa-1 and Fa-2 inoculants used in Comparative Examples 1 and 2 demonstrate that the specific Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 used in this invention do not perform well when used alone as fermentation inoculants; their fermentation capabilities are actually synergistic.

[0088] A comparison with FCba-1, FCba-4, and FCba-5 reveals that, under the same culture and usage conditions, the specific Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 of this invention are significantly different from conventional Bacillus licheniformis and Alcaligenes faecalis, as well as commercially available Bacillus licheniformis CICC10037 and Alcaligenes faecalis CICC20141 used for fermentation. They exhibit obvious specificity and have a stronger effect on the composting of perishable waste.

[0089] experimental group Germination Index experimental group Germination Index FCba-1 112 % Fa-2 89 % FCba-2 117 % FCba-4 98 % Fa-1 93 % FCba-5 102 % / / CK 70 %

[0090] As can be seen from the table above, the Bacillus licheniformis-specific test groups (FCba-1 and FCba-2) of this invention are significantly superior to other test groups, combined with Figure 2The recorded results show that the perishable waste pile treated by this invention has a higher degree of decomposition. On the other hand, a germination index of 50% is sufficient to determine that the perishable waste pile has reached the decomposition standard. Therefore, regarding rapid decomposition, the lowest time to reach a 50% germination index was recorded. Specifically, the FCba-1 and FCba-5 experimental groups reached the decomposition standard on day 7, the Fa-1 experimental group on day 11, the Fa-2 experimental group on day 12, the FCba-4 experimental group on day 9, the FCba-5 experimental group on day 9, and the CK experimental group on day 15. Therefore, it can be seen that the specific Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 of this invention can also be effectively used for rapid decomposition and fermentation.

[0091] In addition, the composting process of perishable waste produces unpleasant gases such as ammonia and hydrogen sulfide, which not only pollute the surrounding air but also lead to the loss of nitrogen (and accompanying sulfur) in the compost material. Therefore, reducing ammonia emissions and nitrogen loss in the compost material is a key technology in the aerobic fermentation process of perishable waste. This study observed the cumulative ammonia emissions of the compost pile at different times by adding compound microbial agents to the perishable waste pile.

[0092] The test comparison results of Examples 2, 3, 4, and 5 are as follows: Figure 3 As shown. From Figure 3 As can be seen, with the extension of composting time, in the first week, the ammonia emission in the FCba-1 and FCba-2 experimental groups of this invention continued to rise. In the first 8 days of composting, the ammonia emission in the compost material with added FCba-1 and FCba-2 compound microbial agents was higher than that in the control. This was mainly because the compost heated up and fermented quickly after the addition of compound microbial agents, causing a large amount of organic nitrogen to be rapidly mineralized into ammonia and volatilized into the air. As the composting time was further extended, the ammonia emission in the compost material with added compound microbial agents gradually stabilized, while the ammonia emission in the control without added microbial agents rose rapidly. This indicates that the addition of the FCba-1 and FCba-2 fermentation compound microbial agents of this invention has the effect of nitrogen retention and deodorization in the aerobic composting fermentation of easily decomposable waste.

[0093] A comparison of FCba-1, FCba-2, FCba-4, and FCba-5 shows that the specific Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 of this invention are superior to common soil Bacillus licheniformis (…). Bacillus licheniformis ) and soil alkaloidobacterium ( Alcaligenes faecalisCompared to the commonly available Bacillus licheniformis CICC10037 and Alcaligenes faecalis CICC20141 used for fermentation, it has a good nitrogen retention and deodorization effect, which can effectively reduce nitrogen loss, improve the utility and economic value of composting, and retain more nutrients. This is also close to the germination index test results.

[0094] experimental group Germination Index experimental group Germination Index FCba-1 112 % CK 70 % FCba-31 97 % FCba-33 107 % FCba-32 111 % FCba-34 91 %

[0095] As can be seen from the table above, in order to maintain the relatively superior composting fermentation effect of the fermentation compound microbial agent of the present invention, the mixing ratio of Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 should be reasonably controlled at 1:(0.9-1.1). Meanwhile, in the same ammonia volatilization test, the FCba-31 test group was significantly worse than the other test groups, indicating that the presence of Bacillus licheniformis CY-1 alone cannot produce a good nitrogen fixation effect. While the FCba-34 test group was slightly better than the FCba-5 test group, it was only close to the FCba-5 test group, indicating that the actual nitrogen fixation and deodorization effect is uniquely produced by the combined effects of the specific Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 of the present invention.

Claims

1. A fermentation compound microbial agent, characterized in that, Composed of fermenting bacteria; The fermenting bacteria are selected from Bacillus licheniformis and Alcaligenes faecalis; The Bacillus licheniformis is classified and named Bacillus licheniformis (Bacillus licheniformis). Bacillus licheniformis CY-1, depositary institution: CGMCC, depositary accession number: CGMCC No.21241, deposit date: November 26, 2020; The alkaloid bacillus described is classified and named Alcaligenes faecalis (Alcaligenes faecalis). Alcaligenes faecalis CY-2, depositary institution: CGMCC, deposit number: CGMCC No.21242, deposit date: November 26, 2020; The fermentation compound microbial agent is prepared by picking colonies of Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 into a culture medium and incubating them at 37±1 ℃ until a viable count of ≥10⁻⁶ is obtained. 8 The CFU / mL bacterial solution was obtained by mixing the bacterial solution obtained from Bacillus licheniformis CY-1 culture and the bacterial solution obtained from Alcaligenes faecalis CY-2 culture at a volume ratio of 1:0.9 to 1.

1.

2. The method for preparing the fermentation compound microbial agent according to claim 1, characterized in that, The method includes: S1. Bacillus licheniformis CY-1 and Alcaligenes faecalis CY-2 were isolated and purified in culture medium, respectively. S2. Collect colonies of *Bacillus licheniformis* CY-1 and *Alcaligenes faecalis* CY-2 into a culture medium and incubate at 37±1℃ until a viable count ≥10⁻⁶ is obtained. 8 CFU / mL bacterial suspension; S3. The bacterial solution obtained by cultivating Bacillus licheniformis CY-1 and the bacterial solution obtained by cultivating Alcaligenes faecalis CY-2 are mixed at a volume ratio of 1:0.9 to 1.1 to obtain the fermentation compound bacterial agent.

3. The method for preparing the fermentation compound microbial agent according to claim 2, characterized in that, The culture medium used for the isolation and purification of Bacillus licheniformis CY-1 in S1 was agar plates, the composition of which was 20 g / L agar. The culture medium used for the isolation and purification of Alcaligenes faecalis CY-2 in S1 was calcium phytate plates, the composition of which was 30 g / L glucose, 25 g / L tryptone, 5 g / L calcium phytate, 20 g / L agar, 0.5 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 0.03 g / L anhydrous manganese sulfate, and 0.5 g / L potassium chloride. The sterilization conditions for both the agar plates and the calcium phytate plates were 121°C and 15 min.

4. The application of the fermentation compound microbial agent according to claim 1, characterized in that, The fermentation compound microbial agent is used for composting easily perishable waste.

5. The application of the fermentation compound microbial agent according to claim 4, characterized in that, The specific method for composting easily perishable waste is as follows: Take at least 0.15 wt% of the fermentation compound microbial agent based on the weight of the perishable waste, and add it evenly to the perishable waste for treatment for at least 18 days.

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