Bacillus siamensis strain fsb24, bacterial agent comprising same and use thereof in continuous high-temperature composting

Abstract

The present application relates to the technical field of microbial fermentation, in particular to a bacillus siamensis FSB24, a microbial agent containing the same and application of the same in continuous high-temperature composting. The bacteria were preserved in the China General Microbiological Culture Collection Center on August 1, 2024, with a preservation number of CGMCC No. 31409 and a preservation address of No. 3, Xibahe West Road, Beijing Chaoyang District. The strain not only has the ability of high-temperature fermentation and can adapt to the specific conditions of continuous high-temperature fermentation, but also can secrete ligninase and cellulase, realize faster and more complete degradation of cellulose and lignin which are difficult to degrade in compost raw materials, rapidly decompose organic matter, shorten the fermentation period of continuous high-temperature composting from about 10 days at present to at least 3 days, and significantly improve the efficiency of compost fermentation. The organic fertilizer obtained by composting after inoculating the strain has a growth-promoting effect on crops, and improves the root weight and stem leaf weight of crops.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a strain of Bacillus stearothermia esculentus FSB24, an inoculum containing it, and its application in continuous high-temperature composting. Background Technology

[0002] my country is a major agricultural country, producing a huge amount of crop straw every year. At the same time, my country's livestock and poultry farming industry has also entered a period of rapid development. Improper handling of the large amounts of crop straw and livestock manure can cause significant environmental pollution and resource waste. Therefore, composting is commonly used to treat these organic wastes such as crop straw and fallen leaves, as well as livestock manure, in a harmless manner, transforming them into renewable resources and addressing the resulting environmental pollution problems. Composting is divided into two types: general composting and high-temperature composting. General composting has a lower fermentation temperature, while high-temperature composting has a higher initial fermentation temperature and generally uses compaction in the later stages. Continuous high-temperature composting was first proposed by Schulze, who described it as maintaining a high substrate temperature through external heating, thereby improving bioconversion efficiency and inhibiting the proliferation of pathogenic microorganisms. However, general composting requires 90-270 days to reach maturity, and even with the addition of microbial agents, it still takes 20-30 days to mature. Continuous high-temperature composting can greatly shorten the composting time. Using an aerobic composting bioreactor can achieve rapid heating and maintain a high temperature, thereby greatly shortening the fermentation time (fermentation can be completed in about 10 days). It has high fermentation efficiency, controllable reaction conditions, and can centrally collect and treat waste gas.

[0003] While livestock and poultry manure and organic waste naturally contain various microorganisms that can mature them during composting, adding external inoculants generally yields better results and requires less fermentation time than direct composting. Microbial inoculants have a positive impact on the total nutrient concentration of compost; suitable inoculants can accelerate the degradation of lignin and cellulose in livestock and poultry manure and organic waste, and shorten the compost maturation time. However, although numerous fermentation inoculants suitable for livestock and poultry manure have been developed, very few can adapt to the rapidly heating process of continuous high-temperature composting. This may be because the microorganisms in the fermentation inoculant are incompatible with the characteristics of the raw materials for continuous high-temperature composting, or the microorganisms are not adapted to the operating conditions of continuous high-temperature composting. Summary of the Invention

[0004] To address the above technical problems, this invention provides a strain of Bacillus stearothermiae FSB24, an inoculum containing it, and its application in continuous high-temperature composting. This Bacillus stearothermiae can not only adapt to the high-temperature environment of continuous high-temperature composting, but also secrete cellulase and ligninase to promote the decomposition of composting materials and improve composting efficiency.

[0005] To achieve the above-mentioned purpose of the application, the following technical solutions are adopted:

[0006] The first aspect of the present application provides a Bacillus smithii FSB24, the taxonomic name of which is Bacillus smithii (Bacillus smithii) Bacillus spizizenii The strain was deposited at the China General Microbiological Culture Collection Center on August 1, 2024, with the accession number CGMCC No. 31409, and the address of the deposit is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0007] The colony of the Bacillus smithii FSB24 provided by the present application is dirty white, rough and opaque on the surface, and usually presents a relatively regular circular or irregular branched shape. When growing in a liquid culture medium, it forms wrinkles, is an aerobic bacterium, and the suitable pH value for growth is 5.0~9.0. The 16S rDNA sequence of the strain FSB24 is shown in SEQ ID NO: 1.

[0008] In the second aspect, the present application provides an application of the above-mentioned Bacillus smithii FSB24 in the production of organic fertilizer by continuous high-temperature composting.

[0009] In combination with the second aspect, the temperature of the continuous high-temperature composting process is not more than 70℃, preferably 45~70℃, further preferably 45~60℃, which can be 60℃.

[0010] In combination with the second aspect, the continuous high-temperature composting can be carried out by tank composting.

[0011] In the third aspect, the present application provides a microbial inoculant, and the active ingredient of the microbial inoculant is the above-mentioned Bacillus smithii FSB24.

[0012] In combination with the third aspect, in the microbial inoculant, the viable bacterial count of the Bacillus smithii FSB24 is at least 10 8 CFU / mL.

[0013] Preferably, the microbial inoculant is a liquid inoculant, and further comprises a carbon source, a nitrogen source and trace elements, and the pH value is 5.0~9.0, preferably 6.0~8.0, more preferably 7.0~7.5.

[0014] In the fourth aspect, the present application provides an application of the above-mentioned microbial inoculant in the production of organic fertilizer by continuous high-temperature composting.

[0015] In the fifth aspect, the present application provides an organic fertilizer, which is prepared by using the above-mentioned Bacillus smithii FSB24 or microbial inoculant and by continuous high-temperature composting fermentation. The organic fertilizer has a growth-promoting effect on crops, can significantly increase the root weight, stem and leaf weight and plant height of crop plants, and thus indirectly increases the yield of crops.

[0016] In combination with the fifth aspect, the raw material for composting includes a cellulose or lignin containing raw material.

[0017] Preferably, the cellulose or lignin containing raw material includes one or more of a combination of livestock and poultry manure, crop straw or forestry waste, or other raw materials capable of realizing resource reutilization through continuous thermophilic composting fermentation.

[0018] Preferably, the initial moisture content of the composting raw material is about 50% to 60%, and the C / N ratio is about 25 to 35:1.

[0019] In combination with the sixth aspect, the present application provides a method for preparing an organic fertilizer, which inoculates the composting raw material with the above-mentioned Bacillus smithii FSB24 or microbial inoculum, continuously ferments the inoculated composting raw material at 60 to 70°C for at least 3 days, ventilates at regular intervals during the fermentation period, maintains the moisture content of the composting at 50% to 60%, cools to room temperature after the fermentation is completed, and the obtained compost is the organic fertilizer.

[0020] The time for continuous fermentation is preferably 3 to 5 days.

[0021] In combination with the sixth aspect, the inoculation amount of the composting raw material is about 1wt%, preferably 1wt%.

[0022] The strain FSB24 provided by the present application not only has the ability to resist high-temperature fermentation and can adapt to the specific conditions of continuous high-temperature fermentation, but also can secrete ligninase and cellulase, thereby realizing faster and more complete degradation of cellulose and lignin in the composting raw material which is difficult to be degraded by ordinary microbial inoculums, rapidly decomposing the organic matter in the compost, and significantly shortening the fermentation period of continuous high-temperature composting from the current 10 days or so to 3 to 5 days, or even obtaining a seed germination index (GI index) of at least 1.0 and a carbon-nitrogen ratio (C / N) close to 16 on the third day of fermentation (indicating that the compost is completely decomposed after 3 days of fermentation), with an average GI index of 1.42 and a maximum of 1.8 on the third day of fermentation, thereby significantly improving the efficiency of composting fermentation. Moreover, the organic fertilizer obtained by composting fermentation after inoculation of the strain has a significant growth-promoting effect on crops, significantly increases the root weight and stem leaf weight of crops, makes the root system of crops more developed, and ensures the vigorous growth of crops. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a whole genome circle diagram of Bacillus smithii FSB24;

[0024] Figure 2 is a phylogenetic tree of Bacillus smithii FSB24;

[0025] Figure 3A photograph of Bacillus stearothermiae FSB24 colonies growing on a culture medium;

[0026] Figure 4 This is a photograph of Bacillus spp. FSB24 after growth on sodium carboxymethyl cellulose medium stained with Congo red (0.1% v / v).

[0027] Figure 5 A photograph of Bacillus stearothermiae FSB24 after growth on nutrient agar medium containing aniline blue (0.1 g / L);

[0028] Figure 6 A bar chart showing the seed germination index of compost water extracts after 3 and 5 days of continuous high-temperature fermentation of compost raw materials inoculated with Bacillus stearothermia 24 and uninoculated with FSB24.

[0029] Figure 7 Bar charts showing the carbon-to-nitrogen ratio of compost piles after 3 and 5 days of continuous high-temperature fermentation with Bacillus stearothermia var. stevia FSB24 inoculated and uninoculated FSB24 inoculated compost materials;

[0030] Figure 8 Bar charts showing the organic matter content of compost piles after 3 and 5 days of continuous high-temperature fermentation of compost raw materials inoculated with Bacillus stearothermia 24 and those not inoculated with FSB24.

[0031] Figure 9 (a) is a potted photo of wheat grown for one month after applying compost at different times or without fertilizer, and (b) is a whole plant photo of wheat grown for one month after applying compost at different times or without fertilizer.

[0032] Figure 10 A bar chart showing the average root weight of wheat one month after composting was applied at different times;

[0033] Figure 11 A bar chart showing the average stem-leaf weight of wheat one month after composting was applied at different times;

[0034] Figure 12 A bar chart showing the plant height of wheat one month after composting was applied at different times. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] In the composting treatment of livestock and poultry manure and organic waste, the traditional aerobic composting (i.e. general composting) needs 90-270 days to complete the composting, while the continuous high-temperature composting (such as tank composting) only needs about 10 days to complete the composting. The continuous high-temperature composting is completed by artificial heating, but the temperature environment in the fermentation equipment used in the continuous high-temperature composting cannot continue to accelerate the composting by inoculating microorganisms with degradation ability, so that the composting time cannot be further shortened and the composting maturity cannot be further improved. In view of this, the present application provides a Bacillus smithii FSB24 which can be applied to continuous high-temperature composting. The strain not only has high temperature resistance, but also has the ability to degrade cellulose and lignin, which can greatly shorten the time of continuous composting and improve the composting maturity.

[0037] The composting material used in the following examples has the following composition:

[0038] Livestock and poultry manure (fresh cow manure, chicken manure) and wheat straw are used as composting raw materials. The wheat straw is cut into small pieces of 3-5 cm, and then mixed with the livestock and poultry manure at a mass ratio of 1:5. The initial moisture content of the composting raw materials is adjusted to about 50%-60%, and the C / N ratio is about 25-35:1.

[0039] The formula of the nutrient agar medium used is: 10 g / L of proteose peptone, 3 g / L of beef extract powder, 5 g / L of sodium chloride, 15 g / L of agar, and the pH value is 7.3.

[0040] Example 1

[0041] The present application provides a method for isolating and identifying Bacillus smithii FSB24

[0042] (1) The composting material is evenly divided into 3 parts for parallel continuous high-temperature composting experiments. When the continuous high-temperature composting reaches the high-temperature and maturation stages, 1 g of sample is taken from the top, middle and bottom of each of the 3 parallel piles, respectively. About 3 g of sample obtained from each pile is thoroughly mixed and then placed in a test tube containing 10 mL of sterile distilled water. The test tube is placed on a vortex mixer and shaken for 60 seconds. The sample is serially diluted to 10 -6 times. 100 μL of the diluted sample is spread on a culture plate containing nutrient agar to isolate bacteria. The culture plate is incubated at 60°C to isolate heat-resistant microorganisms. The microorganism colonies that can grow at 60°C are transferred to new nutrient agar medium, and the redundant colonies in terms of morphology are removed. A total of 47 heat-resistant bacterial strains (named FSB1-FSB47, respectively) are screened.

[0043] (2) DNA extraction and sequencing are performed on the 47 heat-resistant bacterial strains obtained by screening. According to the sequencing results, known duplicate strains are removed. Among the remaining strains obtained by screening, FSB24 has no 100% homology with the identified species, and has 99.9% homology with Bacillus smithii. Bacillus spizizenii(GCF_000227465.1) Genes with homology greater than 80% account for only 37.9% of the total number of genes, and the whole genome circle is as follows: Figure 1 As shown, the phylogenetic tree is as follows: Figure 2 As shown in the phylogenetic tree (the whole genome sequences of other strains were downloaded from NCBI), it was identified as Bacillus stearothermiae. Furthermore, strain FSB24 underwent further molecular identification based on its 16S sequence, and its 16S rDNA sequence is shown in SEQ ID NO.1.

[0044] The *Bacillus stearothermiae* strain FSB24 obtained by screening in this invention has colonies that are off-white, rough, and opaque, typically exhibiting a relatively regular circular or irregular branched shape. When grown in liquid culture medium, it forms wrinkles (such as...). Figure 3 As shown in the image, it is an aerobic bacterium.

[0045] The taxonomic name of this strain is Bacillus stearothermiae (Bacillus stearothermiae). Bacillus spizizenii It was deposited on August 1, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31409, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] Example 2

[0047] This invention provides an experiment on the cellulase production activity of Bacillus stearothermiae FSB24.

[0048] Dilute the overnight culture of strain FSB24 by 10 -6 Take 100 μL of the solution and spread it onto sodium carboxymethyl cellulose solid medium. Incubate in the dark at 45°C for 18 hours. After colonies grow, stain with Congo red (Macklin, Shanghai, China) at a volume ratio of 0.1%. The medium is stained with Congo red, but a clear zone appears around the colonies (e.g., ...). Figure 4 As shown in the diagram (also known as a transparent ring), this indicates that strain FSB24 can secrete cellulase during its growth.

[0049] Example 3

[0050] This invention provides an experiment on the lignin-producing enzyme activity of Bacillus stearothermiae FSB24.

[0051] Dilute the overnight culture of strain FSB24 by 10 -6 Take 100 μL and spread it on nutrient agar medium containing aniline blue (0.1 g / L) (Solarbio, Beijing, China), and incubate in the dark at 45°C for 18 hours. The medium is stained with aniline blue, but as the colonies grow, a clear zone appears around them (e.g., ...). Figure 5As shown in the diagram (also known as a transparent ring), this indicates that strain FSB24 can secrete ligninase during its growth.

[0052] Example 4

[0053] This invention provides an experiment to test the fermentation effect of Bacillus stearothermia 24 (FSB24).

[0054] In the laboratory, an oven was used to simulate the fermentation process in a fermentation tank: FSB24 was added to the prepared compost material at an inoculum rate of 1 wt% (mass percentage) (3 parallel compost materials) at room temperature. After thorough mixing, the materials were placed in the oven and sealed to prevent moisture evaporation. Simultaneously, another batch of the same compost material was prepared without FSB24. Both the inoculated and uninoculated compost materials were placed in the oven, and a continuous high-temperature fermentation experiment was conducted under the same conditions. The fermentation process was as follows: the oven temperature was raised to 60°C (not exceeding 70°C) within one hour, increasing by approximately 3°C every 5 minutes, and then maintained at 60°C for 5 days. The compost temperature was recorded using a temperature sensor. During this period, ventilation and oxygen exchange were performed every 12 hours, and the moisture content of the compost was maintained at 50%–60%. Afterward, the oven was closed, allowing it to cool naturally to room temperature. Samples were taken from the top, middle, and bottom of three parallel compost piles after 3 and 5 days of continuous high-temperature fermentation, with three samples taken from each depth. Nine samples from each of the nine samples collected after 3 and 5 days of continuous high-temperature fermentation were uniformly mixed to form composite samples. These composite samples were then sent to a third-party testing institution for component analysis (moisture content was determined using a vacuum oven method; total carbon and total nitrogen content were determined using an elemental analyzer (Flash SMART, Thermo Fisher Scientific, MA, USA); compost samples were mixed with deionized water at a 1:10 mass ratio and shaken for 1 hour to obtain compost water extracts, which were used to measure pH, conductivity, and seed germination index (GI) using Five Easy Plus™. pH and EC values ​​were determined using a pH / EC meter (Mettler-Toledo, Shanghai, China). Cucumber seeds were cultured in the compost water extract at 25°C in the dark for 48 hours, and root length and germination rate were measured. GI values ​​were calculated, with deionized water as a control. Organic matter content in the compost was determined using the potassium dichromate titration method. Total soluble phosphorus content was determined using a UV-1900i spectrophotometer (Shimadzu Corporation, Shanghai, China). Total potassium content was determined using a TAS-990 atomic absorption spectrometer (Beijing Puxi General Instrument Co., Ltd., Beijing, China). The mean and standard deviation of three parallel compost samples were calculated for all test results. The mean values ​​of each test result are shown in Table 1. The bar charts for the average carbon-to-nitrogen ratio and average organic matter content are shown in Table 2. Figure 7 and Figure 8 As shown.

[0055] Table 1

[0056]

[0057] The compost maturity evaluation is evaluated according to the standard “Organic Fertilizer (NY 525-2021)”, which is an organic fertilizer evaluation standard conforming to the standard of “Organic Fertilizer Practical Technical Regulations”, which requires that the organic matter in the organic fertilizer is greater than or equal to 30%, the total nutrient (N+P2O5+K2O) is greater than or equal to 4.0%, the pH is 5.5-8.5, the GI index is greater than or equal to 70% of the nutrient composition, and does not contain heavy metals, harmful microorganisms and their metabolites and other harmful substances.

[0058] As can be seen from Table 1, compared with the compost inoculated with strain FSB24, the organic matter content, total nutrient content and GI index of the compost inoculated with FSB24 are higher than the national standard; the pH value of the compost inoculated with FSB24 is within the standard range stipulated by the state, while the pH value of the compost without inoculation is higher than the national standard, which does not meet the standard requirements of organic fertilizer.

[0059] As can be seen from Table 1, compared with the compost inoculated with strain FSB24, the organic matter content, total nutrient content and GI index of the compost inoculated with FSB24 are higher than the national standard; the pH value of the compost inoculated with FSB24 is within the standard range stipulated by the state, while the pH value of the compost without inoculation is higher than the national standard, which does not meet the standard requirements of organic fertilizer. Figure 6 As can be seen from Table 1, compared with the compost inoculated with strain FSB24, the organic matter content, total nutrient content and GI index of the compost inoculated with FSB24 are higher than the national standard; the pH value of the compost inoculated with FSB24 is within the standard range stipulated by the state, while the pH value of the compost without inoculation is higher than the national standard, which does not meet the standard requirements of organic fertilizer. Figure 7 As can be seen from Table 1, compared with the compost inoculated with strain FSB24, the organic matter content, total nutrient content and GI index of the compost inoculated with FSB24 are higher than the national standard; the pH value of the compost inoculated with FSB24 is within the standard range stipulated by the state, while the pH value of the compost without inoculation is higher than the national standard, which does not meet the standard requirements of organic fertilizer. Figure 8 As can be seen from Table 1, compared with the compost inoculated with strain FSB24, the organic matter content, total nutrient content and GI index of the compost inoculated with FSB24 are higher than the national standard; the pH value of the compost inoculated with FSB24 is within the standard range stipulated by the state, while the pH value of the compost without inoculation is higher than the national standard, which does not meet the standard requirements of organic fertilizer.

[0060] Example 5

[0061] The present application provides a method for preparing a compost inoculated with Bacillus stratus FSB24

[0062] The composts obtained by continuously thermophilic fermentation of the compost material inoculated with FSB24 and the compost material not inoculated for 3 and 5 days respectively according to the fermentation method of Example 4 were applied in the potting soil of the just planted wheat at the rate of 100 pounds per mu of field crops, while the wheat potted without applying any fertilizer was used as the control group. The whole plant photos of the wheat grown for 1 month are shown in Figure 9 In addition, the root weight, stem and leaf weight and plant height of the wheat were measured, and the column charts are shown in Figure 10~12

[0063] As can be seen from Figure 10~12 , after applying the compost inoculated with FSB24, the root system of the wheat is more developed, the root weight and stem and leaf weight are significantly increased, and the plant height is also obviously increased. It is thus illustrated that, whether fermented for 3 days or 5 days, the compost inoculated with FSB24 has a better growth promoting effect on the wheat than the compost not inoculated.

[0064] The above merely describes the preferred embodiments of the present application, but should not be used to limit the present application, and any modification, equivalent replacement or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.​

Claims

1. A strain of Bacillus stearothermiae FSB24, characterized in that, The taxonomic name is Bacillus stearothermiae ( Bacillus spizizenii It was deposited on August 1, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31409, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of Bacillus stearothermiae FSB24 as described in claim 1 in the continuous high-temperature composting production of organic fertilizer, characterized in that, The continuous high-temperature composting time is at least 3 days.

3. The application of Bacillus stearothermia esculenta FSB24 as described in claim 2 in the continuous high-temperature composting production of organic fertilizer, characterized in that, The temperature during the continuous high-temperature composting process does not exceed 70°C.

4. A microbial inoculant, characterized in that, The active ingredient is Bacillus stearothermia 24 as described in claim 1.

5. The microbial agent as described in claim 4, characterized in that, The viable count of the *Bacillus stearothermiae* FSB24 is at least 10. 8 CFU / mL.

6. The application of the microbial agent according to claim 4 in the continuous high-temperature composting production of organic fertilizer.

7. An organic fertilizer, characterized in that, It is prepared by continuous high-temperature composting fermentation using Bacillus stearothermia 24 as described in claim 1 or the microbial agent as described in claim 4.

8. The organic fertilizer as described in claim 7, characterized in that, The raw materials used for composting include those containing cellulose or lignin.

9. A method for preparing an organic fertilizer, characterized in that, The composting raw materials are inoculated with Bacillus stearothermiae FSB24 as described in claim 1 or the microbial agent as described in claim 4. The inoculated composting raw materials are fermented continuously at 60~70℃ for at least 3 days. During the fermentation, ventilation is carried out regularly to maintain the compost moisture content at 50%~60%. After the fermentation is completed, the compost is cooled to room temperature. The resulting decomposed material is the organic fertilizer.

10. The method for preparing organic fertilizer as described in claim 9, characterized in that, The inoculation amount was 1 wt%.

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