A humification preparation containing bacillus licheniformis, a preparation method and application thereof

Abstract

The application discloses a humification preparation containing bacillus licheniformis and a preparation method and application thereof, and belongs to the technical field of microorganisms. The humification preparation comprises the bacillus licheniformis and thermus arciformis. The bacillus licheniformis is preserved in the China General Microbiological Culture Collection Center on June 27, 2025, and the address of the preservation center is No. 3, Xili Beichen, Chaoyang District, Beijing, and the preservation number is CGMCC No. 35039. The bacillus licheniformis is separated from a coastal saline-alkali soil, has high-temperature resistance and salt tolerance, and can secrete protease and lipase. After the bacillus licheniformis and the thermus arciformis are mixed, the synergistic effect is obvious, duck-derived red mud can be effectively converted into humic acid, and resourceful high-value utilization of the duck-derived red mud is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a decomposed preparation containing Bacillus licheniformis, a preparation method and an application thereof. Background Art

[0002] Duck red mud refers to a mixture of feathers, visceral debris, blood, and washing wastewater produced during duck slaughtering. This reddish-brown mud with a distinct fishy odor forms after solid-liquid separation and oxidation stabilization pretreatment. Its primary components are keratin, hemoglobin, amino acids, lipids, minerals, and nitrogen, phosphorus, and potassium nutrients. It may also contain harmful substances such as pathogenic microorganisms and viruses. Duck red mud has a high moisture content, poor dehydration properties, fine particles, low density, high organic matter content, and high pollutant levels. Improper handling or direct discharge without treatment can seriously harm the ecological environment and human health.

[0003] Currently, the main methods for treating and disposing of duck-sourced red mud are open-air dumping, landfilling, or biological treatment using methods such as black soldier flies and earthworms. Due to its colloidal structure and moisture content exceeding 95%, duck-sourced red mud is difficult to dehydrate and treat, and is prone to secondary pollution. The organic matter content of duck-sourced red mud is typically 60% to 80%, primarily consisting of organic debris and bacterial cells, making it difficult to effectively degrade and recycle it. Summary of the Invention

[0004] The present invention aims to solve the problems existing in the prior art and optimizes the separation to obtain a strain of Bacillus licheniformis, which has high temperature resistance and salt resistance and can work together with Thermus toxoplasma to effectively degrade protein and fat in duck-derived red mud.

[0005] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0006] A decomposed preparation containing Bacillus licheniformis, which comprises Bacillus licheniformis fermentation liquid and Thermus toxoplasma fermentation liquid, with the volume ratio of the two being 2:1.

[0007] Furthermore, the Bacillus licheniformis strain was deposited at the China General Microorganisms Center, China Culture Collection Administration, on June 27, 2025, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 35039. The Thermus arciformis strain, CGMCC No. 1.6992, was purchased from the China General Microorganisms Center, China Culture Collection Administration, with the original deposit date being November 1, 2007. Thermus arciformis is available for purchase through the collection's strain catalog, without the need for additional deposits.

[0008] The Bacillus licheniformis of the present invention is separated from coastal saline-alkali land.

[0009] Isolation method: Take 10g soil sample, place it in sterilized LB medium, shake culture, take the diluted proliferation liquid, and inoculate the sample on 1% solid LB medium by spreading method for culture. After the culture is completed, select single colonies and transfer to 2% Na + concentration in the culture medium and gradually increase the Na + Concentration, transferred to 5% Na + The concentration was then diluted and spread, and the strain with the best growth potential was separated and preserved to obtain the target strain Bacillus licheniformis.

[0010] Morphological characteristics of the strain: The colonies cultured in LB medium at 60℃ for 16 hours are off-white, mostly single, with a few arranged in pairs, and have circular protrusions on the colonies, slightly translucent around, smooth surface, and sticky when picked (see Figure 1 ). The Gram staining results of the colonies are as follows Figure 2 As shown, it can be observed that the strain is rod-shaped and the bacteria are purple in Gram stain, indicating that the strain is a Gram-positive bacterium.

[0011] A method for preparing a decomposed preparation containing Bacillus licheniformis comprises the following steps:

[0012] After thawing, Bacillus licheniformis and Thermus toxoplasma were inoculated into LB medium for activation culture. After activation culture, the bacterial cakes of each strain were selected and added into LB liquid medium at an inoculum volume of 1.5% for fermentation culture. When the effective viable bacteria count in the liquid medium reached 5×10 8 CFU / mL, the fermentation was stopped to obtain two fermentation bacterial solutions, and then the two fermentation bacterial solutions were mixed in a volume ratio of 2:1 to obtain a decomposed preparation.

[0013] The present invention also provides the use of the decomposition preparation containing Bacillus licheniformis in fermenting and decomposing protein and fat-containing organic waste. The decomposition preparation containing Bacillus licheniformis is added at 1-3% of the total weight of the organic waste. The main ingredient of the protein and fat-containing organic waste is duck-derived red mud, and the auxiliary ingredients are corn hair, peanut shells, and corn cobs.

[0014] Furthermore, the mass ratio of duck-derived red mud, corn hair, peanut shells, and corn cobs in the protein- and fat-containing organic waste is 11:3:3:1.

[0015] Furthermore, the duck-source red mud is a reddish-brown mud formed by processing a mixture of feathers, visceral residues, blood and cleaning wastewater generated during the duck slaughtering process in a slaughterhouse, with a moisture content of 70-75%; the corn hair is not crushed, in a natural state, and has a moisture content of 10-12%; the peanut shell is not crushed, in a natural form, and has a moisture content of 12-15%; the corn cob is 8-12 cm long and has a moisture content of 25-30%.

[0016] Furthermore, the specific application steps include:

[0017] Mix duck-derived red mud, corn hair, peanut shells and corn cobs in proportion to form a mixed material; use inorganic nutrient solution to adjust the moisture content of the mixed material to 60-70%; inoculate a composting preparation containing Bacillus licheniformis; turn the pile every 2-3 days until composting is completed, and turn it over to cool it down when the temperature exceeds 73°C during the composting process.

[0018] Furthermore, the inorganic nutrient solution contains 0.1% potassium dihydrogen phosphate, 0.1% urea and 1-3% soda ash.

[0019] Beneficial effects

[0020] The present invention screened out a strain of Bacillus licheniformis, which has high temperature resistance and salt resistance, and can secrete protease and lipase. The secreted lipase directly degrades duck fat to produce glycerol and fatty acids, provides precursors for humic acid synthesis, and effectively degrades duck-derived red mud.

[0021] The Bacillus licheniformis and Thermus toxoplasma are used in combination to achieve synergistic effects and significantly decompose duck-derived red mud. During the decomposition process, the high temperature of about 70°C is maintained for 55-60 days, effectively removing pathogenic microorganisms in the duck-derived red mud. At the same time, organic matter such as the duck-derived red mud is converted into humic acid and nutrients that are easily absorbed and utilized by plants, thereby realizing high-value resource utilization of the duck-derived red mud. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a morphological diagram of Bacillus licheniformis;

[0023] Figure 2 This is the Gram staining image of Bacillus licheniformis;

[0024] Figure 3 This is a diagram of the high temperature resistance experiment of Bacillus licheniformis;

[0025] Figure 4 This is a diagram showing the temperature change of the pile during the process of composting organic wastes such as duck-derived red mud using the composting preparation of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0027] Example 1

[0028] A decomposed preparation containing Bacillus licheniformis, which comprises Bacillus licheniformis fermentation liquid and Thermus toxoplasma fermentation liquid, with the volume ratio of the two being 2:1.

[0029] The Bacillus licheniformis was deposited at the General Microbiology Center of the China Culture Collection Administration on June 27, 2025, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC No. 35039; the strain number of the Thermus toxoplasma was CGMCC No. 1.6992, purchased from the General Microbiology Center of the China Culture Collection Administration.

[0030] In this example, Bacillus licheniformis was isolated from coastal saline-alkali land.

[0031] Isolation method: Take 10g soil sample, place it in sterilized LB medium, shake culture, take the diluted proliferation liquid, and inoculate the sample on 1% solid LB medium by spreading method for culture. After the culture is completed, select single colonies and transfer to 2% Na + concentration in the culture medium and gradually increase the Na + Concentration, transferred to 5% Na + The concentration was then diluted and spread, and the strain with the best growth potential was separated and preserved to obtain the target strain Bacillus licheniformis.

[0032] Morphological characteristics of the strain: The colonies cultured in LB medium at 60℃ for 16 hours are off-white, mostly single, with a few arranged in pairs, and have circular protrusions on the colonies, slightly translucent around, with a smooth surface, and are sticky or jelly when picked (see Figure 1 ). The Gram staining results of the colonies are as follows Figure 2 As shown, it can be observed that the strain is rod-shaped and the bacteria are purple in Gram stain, indicating that the strain is a Gram-positive bacterium.

[0033] High temperature resistance identification: Inoculate Bacillus licheniformis into LB medium, culture at 37℃ for 6h, divide into 10mL test tubes, shake on a water bath shaker at 30℃, 40℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃ for half an hour, take out and cool, dilute 10mL with sterile water, and 6 times, take 200 μl and spread on LB plate, culture at 37℃ for 24h, and count the growing colonies (see Figure 3 ). It can be seen that the strain can withstand high temperatures of 70℃.

[0034] A method for preparing a decomposed preparation containing Bacillus licheniformis comprises the following steps:

[0035] After thawing, Bacillus licheniformis and Thermus toxoplasma were inoculated into LB medium for activation culture. After activation culture, the bacterial cakes of each strain were selected and added into LB liquid medium at an inoculum volume of 1.5% for fermentation culture. When the effective viable bacteria count in the liquid medium reached 5×10 8 CFU / mL, the fermentation was stopped to obtain two fermentation bacterial solutions, and then the two fermentation bacterial solutions were mixed in a volume ratio of 2:1 to obtain a decomposed preparation.

[0036] Comparative Example 1

[0037] Compared with Example 1, this comparative example is the same as Example 1, except that the volume ratio of Bacillus licheniformis fermentation broth to Thermus toxoplasma fermentation broth is 1:1, and the other raw materials and steps are the same as Example 1.

[0038] Comparative Example 2

[0039] Compared with Example 1, this comparative example is the same as Example 1, except that the volume ratio of the fermentation bacteria liquid of Bacillus licheniformis and Thermus toxoplasma is 1:2, and the other raw materials and steps are the same as Example 1.

[0040] Comparative Example 3

[0041] Compared with Example 1, this comparative example uses only Bacillus licheniformis bacterial solution, and the rest of the raw materials and steps are the same as those of Example 1.

[0042] Comparative Example 4

[0043] Compared with Example 1, this comparative example uses only the fermentation broth of Thermus toxoplasma, and the other raw materials and steps are the same as those of Example 1.

[0044] Performance Testing

[0045] Functional identification of strains:

[0046] Protease production identification: Use an inoculating needle to inoculate the isolated and purified Bacillus licheniformis strains and Thermus toxoplasma onto protein plates, observe the clear zone after culturing for 24 hours, and measure the ratio of clear zone to colony diameter (D / d). The two strains are then inoculated into LB liquid medium and cultured at 37°C and 200 r / min for 24 hours to obtain seed liquid. The seed liquid is added to the fermentation medium at a 5% inoculum volume and cultured at 37°C and 200 r / min for 24 hours. The fermentation broth is collected and centrifuged, and the supernatant is collected. The protease activity of the two strains is determined using the enzyme activity determination method specified in GB / T 23527-2009 "Protease Preparations". Protein plate medium: 10 g casein, 1 g yeast extract, 20 g agar powder, 1000 mL distilled water, adjust the pH to 7.3, and sterilize at 116°C for 30 minutes.

[0047] Lipase production identification: The isolated and purified Bacillus licheniformis and Thermus toxoplasma were inoculated onto fat plates using an inoculating needle and cultured for 24 h. The colony clear zone diameter to colony diameter ratio (D / d) in the culture medium was measured. The two strains were then inoculated into LB liquid medium and cultured at 37°C and 200 r / min for 24 h to obtain seed liquid. The seed liquid was added to the fermentation medium at a 5% inoculum volume and cultured at 37°C and 200 r / min for 24 h. The fermentation broth was collected and centrifuged, and the supernatant was collected. The lipase activity of the two strains was determined by the acid-base titration method specified in GB / T 23535-2009 "Determination of Enzyme Activity of Lipase Preparations". Fat plate culture medium: 5.0% soy peptone, 15.0% tryptone, 5.0% sodium chloride, 3.0% yeast extract, 20.0% agar powder, and 10.0% neutral tributyrin. The pH was adjusted to 7.5 and the culture was sterilized at 121°C for 15 min.

[0048] Table 1 Enzymatic hydrolysis ability of each strain

[0049]

[0050] Application of the decomposed preparation containing Bacillus licheniformis of the present invention

[0051] The application of the decomposed preparation prepared in Example 1 and Comparative Examples 1-4 of the present invention is as follows: duck-source red mud, corn hair, peanut shells and corn cobs are mixed in proportion to form a mixed material, wherein the main ingredient is duck-source red mud, and the auxiliary ingredients are corn hair, peanut shells and corn cobs. The masses of duck-source red mud, corn hair, peanut shells and corn cobs are 550kg, 150kg, 150kg and 50kg respectively. The duck-source red mud is a reddish-brown mud formed by the treatment of feathers, visceral residues, blood and cleaning wastewater generated during the slaughtering of ducks in a slaughterhouse, with a moisture content of 70-75%. The corn hair is not crushed and in its natural state, with a moisture content of 10-12%. The peanut shells are Unbroken, natural shape, moisture content 12-15%, corn cob length 8-12cm, moisture content 25-30%; the mixed material is piled up statically for 5 days; the moisture content of the mixed material is adjusted to 60% using an inorganic nutrient solution containing 0.1% potassium dihydrogen phosphate, 0.1% urea and 1% soda ash; then inoculated with a decomposition preparation containing Bacillus licheniformis, the decomposition preparation containing Bacillus licheniformis is added at 1% of the total weight of the mixed material; the pile is piled until the decomposition is completed, the pile is turned over every 2-3 days during the pile process, and the temperature is turned over and cooled when it exceeds 73°C; after the pile is completed, the corn cobs are screened and recovered, and the finished product is naturally aged for 7-10 days. The temperature change of the pile during the pile treatment of the decomposition preparation prepared in Example 1 is shown in FIG. Figure 4 .

[0052] The mixed materials after composting with different composting preparations were tested, and the specific test results are shown in Table 2.

[0053] Table 2 Decomposed material index detection

[0054]

[0055] As shown in Table 2, the composting preparation prepared using Example 1 of the present invention can effectively remove pathogenic microorganisms from red mud through continuous high-temperature composting, convert organic matter such as duck-derived red mud into humic acid that is easily absorbed and utilized by plants, and significantly increase the organic matter content of the waste. Furthermore, the composting material prepared in Example 1 of the present invention has low electrical conductivity, that is, it contains a low concentration of soluble salts.

[0056] It should be noted that the above embodiments are only some of the preferred embodiments of the present invention, and not all of them. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

Claims

1. A decomposed preparation containing Bacillus licheniformis, characterized in that: The decomposed preparation comprises a fermentation liquid of Bacillus licheniformis and a fermentation liquid of Thermus toxoplasma, with a volume ratio of 2:1; The Bacillus licheniformis was deposited in the General Microbiology Center of the China Culture Collection Administration on June 27, 2025, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC No. 35039; the strain number of the Thermus toxoplasma was CGMCC No. 1.6992, which was purchased from the General Microbiology Center of the China Culture Collection Administration.

2. A method for preparing a decomposed preparation containing Bacillus licheniformis according to claim 1, characterized in that: The method comprises the following steps: After thawing, Bacillus licheniformis and Thermus toxoplasma were inoculated into LB medium for activation culture. After activation culture, the bacterial cakes of each strain were selected and added into LB liquid medium at an inoculum volume of 1.5% for fermentation culture. When the effective viable bacteria count in the liquid medium reached 5×10 8 CFU / mL, the fermentation was stopped to obtain two fermentation bacterial solutions, and then the two fermentation bacterial solutions were mixed in a volume ratio of 2:1 to obtain a decomposed preparation.

3. A use of the decomposition preparation containing Bacillus licheniformis according to claim 1 in fermenting and decomposing organic waste containing protein and fat, characterized in that: The decomposed preparation containing Bacillus licheniformis is added at 1-3% of the total weight of the organic waste. The main material of the organic waste containing protein and fat is duck-derived red mud, and the auxiliary materials are corn hair, peanut shells and corn cobs.

4. The use of the decomposing preparation containing Bacillus licheniformis according to claim 3 in fermenting and decomposing organic waste containing protein and fat, characterized in that: The mass ratio of duck-derived red mud, corn hair, peanut shells and corn cobs in the protein- and fat-containing organic waste is 11:3:3:

1.

5. The use of the decomposing preparation containing Bacillus licheniformis according to claim 3 in fermenting and decomposing organic waste containing protein and fat, characterized in that: The duck-source red mud is a reddish-brown mud formed by treating a mixture of feathers, visceral residues, blood, and cleaning wastewater generated during the duck slaughtering process at a slaughterhouse, with a moisture content of 70-75%. The corn hair is uncrushed, in a natural state, and has a moisture content of 10-12%; the peanut shell is uncrushed, in a natural state, and has a moisture content of 12-15%; the corn cob is 8-12 cm long and has a moisture content of 25-30%.

6. The use of the decomposing preparation containing Bacillus licheniformis according to claim 3 in fermenting and decomposing organic waste containing protein and fat, characterized in that: The specific application steps include: Duck-derived red mud, corn hair, peanut shells and corn cobs are mixed in proportion to form a mixture; the moisture content of the mixture is adjusted to 60-70% using an inorganic nutrient solution; a composting preparation containing Bacillus licheniformis is inoculated; the pile is turned over every 2-3 days until composting is complete, and when the temperature exceeds 73°C during the composting process, the mixture is turned over to cool it down.

7. The use of the decomposing preparation containing Bacillus licheniformis according to claim 6 in fermenting and decomposing organic waste containing protein and fat, characterized in that: The inorganic nutrient solution contains 0.1% potassium dihydrogen phosphate, 0.1% urea and 1-3% soda ash.

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