A strain of Bacillus licheniformis FSB35, a bacterial agent containing the same, and its application in continuous high-temperature composting

Abstract

The present invention relates to the field of microbial fermentation technology, and in particular to a strain of Bacillus licheniformis FSB35, a bacterial agent containing the same, and its application in continuous high-temperature composting. The bacterium was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on August 1, 2024, with a deposit number of CGMCC No.31408, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. 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, and achieve faster and more complete degradation of cellulose and lignin that are difficult to degrade in compost raw materials, quickly decompose organic matter, and shorten the fermentation period of continuous high-temperature composting from the current 10 days to 5 days, significantly improving the efficiency of compost fermentation. The organic fertilizer obtained by composting and fermentation after inoculation of the strain has a growth-promoting effect on crops, and increases the plant height and stem and leaf weight of crops.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a strain of Bacillus licheniformis FSB35, a bacterial agent containing the same, and application of the same in continuous high-temperature composting. Background Art

[0002] Every year, crop cultivation produces a large amount of straw, and livestock farming also produces a large amount of livestock manure. If these crop straw and livestock manure are not handled properly, they will cause great environmental pollution and also lead to a waste of resources. Composting can make crop straw and livestock manure a resource for recycling. Composting is divided into two types: general composting and high-temperature composting. General composting has a low fermentation temperature and a slow fermentation rate, requiring at least 100 days to reach full maturity. Even with the addition of bacterial agents, it takes about 25 days to reach full maturity. High-temperature composting has a higher fermentation temperature in the early stage, and compaction measures are generally used in the later stage, which makes the composting more efficient. Continuous high-temperature composting is a composting method that continuously maintains a high substrate temperature through external heating, thereby improving bioconversion efficiency and inhibiting the proliferation of pathogenic microorganisms. It often uses aerobic composting bioreactors for high-temperature fermentation, which allows the compost raw materials to heat up quickly and maintain a high temperature, shortening the fermentation time (fermentation can be completed in about 10 days). It has the advantages of high fermentation efficiency and controllable reaction conditions.

[0003] To further improve fermentation efficiency, shorten fermentation time, and enrich the compost with nutrients, microbial fermentation agents can be introduced into the compost feedstock. Microbial inoculants have a positive impact on the total nutrient concentration of compost. Suitable microbial inoculants can accelerate the degradation of lignin and cellulose in livestock and poultry manure and organic waste, shortening the time it takes for the compost to mature. However, due to incompatibility between the microorganisms in the fermentation agents and the characteristics of the feedstock or their inability to adapt to the operating conditions of continuous high-temperature composting, there are currently few microorganisms that can adapt to continuous high-temperature composting and have a significant compost-promoting effect. Summary of the Invention

[0004] In response to the above technical problems, the present invention provides a strain of Bacillus licheniformis FSB35, a bacterial agent containing the same, and its use in continuous high-temperature composting. The Bacillus licheniformis can not only adapt to the high-temperature environment of continuous high-temperature composting, but also secrete cellulase and ligninase, significantly accelerating the decomposition of compost raw materials and improving composting efficiency.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a strain of Bacillus licheniformis FSB35, the taxonomic name of which is Bacillus licheniformis. The strain was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on August 1, 2024, with the deposit number CGMCC No. 31408, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The Bacillus licheniformis FSB35 provided by the present invention has an off-white colony with a smooth, uneven surface, shallowly porous or radially shaped, and burr-like edges. When grown in liquid culture, it forms white granules. It is an aerobic bacterium with an optimal growth pH of 5.0 to 9.0. The 16S rDNA sequence of strain FSB35 is shown in SEQ ID NO: 1.

[0008] In a second aspect, the present invention provides a use of the above-mentioned Bacillus licheniformis FSB35 in producing organic fertilizer by continuous high-temperature composting.

[0009] In combination with the second aspect, the temperature of the continuous high-temperature composting process does not exceed 70°C, preferably 45-70°C, more preferably 45-60°C, and can be 60°C.

[0010] In combination with the second aspect, the continuous high-temperature composting can be fermented in a tank composting manner.

[0011] In a third aspect, the present invention provides a microbial agent, the active ingredient of which is the above-mentioned Bacillus licheniformis FSB35.

[0012] In combination with the third aspect, in the microbial agent, the viable count of the Bacillus licheniformis FSB35 is at least 10 8 CFU / mL.

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

[0014] In a fourth aspect, the present invention provides a use of the above-mentioned microbial agent in the production of organic fertilizer by continuous high-temperature composting.

[0015] In a fifth aspect, the present invention provides an organic fertilizer produced by continuous high-temperature composting and fermentation using the aforementioned Bacillus licheniformis FSB35 or a microbial agent. This organic fertilizer promotes the growth of crops, significantly increasing plant height and stem and leaf weight, thereby indirectly increasing crop yields.

[0016] In conjunction with the fifth aspect, the raw materials used for composting include raw materials containing cellulose or lignin.

[0017] Preferably, the raw materials containing cellulose or lignin include one or more of livestock and poultry manure, crop straw or forestry waste, or other raw materials that can be recycled through continuous high-temperature composting and fermentation.

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

[0019] In a sixth aspect, the present invention provides a method for preparing an organic fertilizer, wherein the compost raw materials are inoculated with the above-mentioned Bacillus licheniformis FSB35 or microbial agent, and the inoculated compost raw materials are continuously fermented at 60-70°C for 3-5 days. During the fermentation period, ventilation is carried out regularly to maintain the moisture content of the compost at 50%-60%. After the fermentation is completed, the compost is cooled to room temperature, and the obtained decomposed material is the organic fertilizer.

[0020] In combination with the sixth aspect, the inoculation amount of the compost raw material is about 1 wt%, preferably 1 wt%.

[0021] The strain FSB35 provided by the present invention not only has the ability to withstand high temperature fermentation and can adapt to the specific conditions of continuous high temperature fermentation, but also can secrete ligninase and cellulase, thereby achieving faster and more complete degradation of cellulose and lignin that are difficult to degrade by ordinary microbial agents in the compost raw materials, so that the organic matter in the compost is quickly decomposed, and the fermentation cycle of continuous high temperature composting is greatly shortened from the current 10 days to 5 days. On the 5th day of fermentation, a seed germination index (GI index) greater than 100% and a carbon-nitrogen ratio close to 16 (indicating that the fermentation can be completely decomposed in 5 days) can be obtained, which significantly improves the fermentation efficiency of the compost. Moreover, the organic fertilizer obtained by compost fermentation after inoculation of the strain has a significant growth-promoting effect on crops, and the plant height and stem and leaf weight of the crops are significantly improved, and the root system is also more developed. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the circle map of the whole genome of Bacillus licheniformis FSB35;

[0023] Figure 2 is the phylogenetic tree of Bacillus licheniformis FSB35;

[0024] Figure 3 This is a photo of the colony of Bacillus licheniformis FSB35 growing on the culture medium;

[0025] Figure 4 This is a photograph of Bacillus licheniformis FSB35 grown on sodium carboxymethylcellulose medium stained with Congo red (0.1% by volume);

[0026] Figure 5This is a photograph of Bacillus licheniformis FSB35 grown on a nutrient agar medium containing aniline blue (0.1 g / L);

[0027] Figure 6 The bar graphs show the seed germination index of the compost water extracts corresponding to the compost materials inoculated with Bacillus licheniformis FSB35 and those not inoculated with FSB35 after continuous high temperature fermentation for 3 days and 5 days;

[0028] Figure 7 The bar graphs show the carbon-nitrogen ratios of the composts inoculated with Bacillus licheniformis FSB35 and those not inoculated with FSB35 after continuous high-temperature fermentation for 3 and 5 days.

[0029] Figure 8 The bar graphs show the organic matter content of the compost after continuous high-temperature fermentation for 3 days and 5 days with and without Bacillus licheniformis FSB35 inoculation.

[0030] Figure 9 , (a) is a photo of a potted wheat plant with compost applied at different periods or without fertilizer applied at one month's growth; (b) is a photo of the whole wheat plant with compost applied at different periods or without fertilizer applied at one month's growth;

[0031] Figure 10 This is a bar graph of the average stem and leaf weight of wheat after applying compost at different periods of growth for one month;

[0032] Figure 11 This is a bar graph of wheat plant height after applying compost at different periods of time for one month. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0034] Traditional aerobic composting requires at least 100 days to reach full maturity, while continuous high-temperature composting (such as tank composting) can reach full maturity in only about 10 days, significantly improving fermentation efficiency. However, the temperature environment during continuous high-temperature composting often makes it impossible to further accelerate the maturity by inoculating microorganisms with degradation capabilities, which in turn results in an inability to further shorten the composting time and improve the maturity. In view of this, the present invention provides a strain of Bacillus licheniformis FSB35 that can be used in continuous high-temperature composting. This strain is not only heat-resistant but also has the ability to degrade cellulose and lignin, significantly shortening the continuous composting time and achieving full maturity in just 5 days.

[0035] The composition of the compost material used in the following examples is:

[0036] Livestock and poultry manure (fresh cow manure, chicken manure) and wheat straw are used as compost raw materials. The wheat straw is cut into small segments of 3 to 5 cm and then fully mixed with livestock and poultry manure at a mass ratio of 1:5. The initial moisture content of the compost raw materials is adjusted to about 50% to 60%, and the C / N ratio is about 25 to 35:1.

[0037] The formula of the nutrient agar medium used is: peptone 10 g / L, beef extract powder 3 g / L, sodium chloride 5 g / L, agar 15 g / L, and pH 7.3.

[0038] Example 1

[0039] The present invention provides a method for isolating and identifying Bacillus licheniformis FSB35.

[0040] (1) The compost material was divided into three equal parts for parallel continuous high-temperature composting experiments. When the continuous high-temperature composting reached the high temperature and mature stage, 1 g of sample was taken from the top, middle and bottom of the three parallel piles respectively. After about 3 g of sample from each compost was fully mixed, it was placed in a test tube containing 10 mL of sterile distilled water, placed on a vortex mixer and shaken for 60 seconds, and continuously diluted to 10 -6 The dilution was diluted 200 μL and 100 μL was spread on a nutrient agar plate to isolate the bacteria. The plate was incubated at 60°C to isolate heat-resistant microorganisms. Microbial colonies that could grow at 60°C were transferred to a new nutrient agar medium, and morphologically redundant colonies were removed. A total of 47 heat-resistant strains (named FSB1 to FSB47) were screened and obtained.

[0041] (2) DNA was extracted and sequenced from the 47 thermotolerant strains screened. Known duplicate strains were removed based on the sequencing results. Among the remaining strains screened, FSB35 was not 100% homologous to the identified species. The genes with a homology greater than 80% to Bacillus licheniformis (GCF_000011645.1) accounted for only 40.16% of the total number of genes. The whole genome was circled as follows: Figure 1 The phylogenetic tree is shown in Figure 2 As shown in the figure (the complete genome sequences of other strains in the phylogenetic tree were downloaded from NCBI), it was determined to be Bacillus licheniformis. In addition, strain FSB35 was further molecularly identified based on the 16S sequence, and its 16S rDNA sequence is shown in SEQ ID NO.1.

[0042] The strain FSB35 of Bacillus licheniformis screened by the present invention has a grayish white colony with a smooth and uneven surface, a shallow hole or radial shape, and a "burr-like" edge. When grown in a liquid culture medium, it forms a wrinkled mold (such as Figure 3 is an aerobic bacterium.

[0043] The taxonomic name of this strain is Bacillus licheniformis. It was deposited in the General Microbiology Center of China Culture Collection Administration on August 1, 2024. The deposit number is CGMCC No. 31408. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0044] Example 2

[0045] The present invention provides an experiment on the cellulase activity of Bacillus licheniformis FSB35.

[0046] The overnight culture of strain FSB35 was diluted 10 -6 100 μL was spread on a sodium carboxymethyl cellulose solid medium and cultured in the dark at 45°C for 18 hours. After the colonies grew, they were stained with Congo red (Macklin, Shanghai, China) at a volume ratio of 0.1%. The medium was stained with Congo red, but a transparent zone appeared around the colonies (such as Figure 4 The results show that the strain FSB35 can secrete cellulase during its growth.

[0047] Example 3

[0048] The present invention provides an experiment on the activity of ligninase produced by Bacillus licheniformis FSB35.

[0049] The overnight culture of strain FSB35 was diluted 10 -6 100 μL was spread on a nutrient agar medium containing aniline blue (0.1 g / L) (Solarbio, Beijing, China) and incubated in the dark at 45°C for 18 hours. The medium was stained with aniline blue, but as the colonies grew, a transparent zone appeared around them (e.g. Figure 5 As shown in the figure, it can also be called transparent ring), indicating that strain FSB35 can secrete ligninase during its growth.

[0050] Example 4

[0051] The present invention provides a fermentation effect test experiment of Bacillus licheniformis FSB35

[0052] In a laboratory oven, the fermentation process in a fermenter was simulated: FSB35 was added to the prepared compost at room temperature at a rate of 1 wt% (by mass) (three replicates). The mixture was then placed in an oven and sealed to prevent evaporation. Simultaneously, the same compost, but without FSB35, was taken. Both the inoculated and uninoculated composts were placed in the oven and subjected to continuous high-temperature fermentation under the same conditions. The oven temperature was raised to 60°C (maximum 70°C) within one hour, increasing by 3°C every five minutes. The temperature was then maintained at 60°C for five days. A temperature sensor was used to record the compost temperature. Ventilation was performed every 12 hours during this period, and the moisture content of the compost was maintained between 50% and 60%. The oven was then closed and allowed to cool naturally to room temperature. On days 3 and 5 of continuous high-temperature fermentation, samples were taken from the upper, middle, and lower parts of three parallel compost piles, with three samples taken at each depth. The subsamples (nine each) collected on days 3 and 5 of continuous high-temperature fermentation were evenly mixed to form composite samples. The composite samples were sent to a third-party testing agency for composition testing (water content was determined using a vacuum oven method; total carbon and total nitrogen contents were determined using an elemental analyzer (Flash SMART, Thermo Fisher Scientific, MA, USA); the compost samples were mixed with deionized water at a mass ratio of 1:10 and shaken for 1 hour to obtain compost water extracts for measurement of pH, conductivity, and seed germination index (GI) using Five Easy Plus™). pH and EC values ​​were measured using a pH / EC meter (Mettler Toledo, Shanghai, China). Cucumber seeds were incubated in a compost water extract at 25°C in the dark for 48 h. Root length and germination rate were measured, and GI values ​​were calculated, with deionized water used as a control. The organic matter content in the compost was determined using the potassium dichromate volumetric 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 the three replicate compost materials were calculated for all test results. The mean values ​​for each test result are shown in Table 1. The bar graphs of the average carbon-nitrogen ratio and average organic matter content are shown in Table 1, respectively. Figure 7 and Figure 8 shown.

[0053] Table 1

[0054]

[0055] The compost maturity evaluation is carried out in accordance with the standard "Organic Fertilizer" (NY 525-2021). "Organic Fertilizer" is an organic fertilizer evaluation standard that complies with the "Practical Technical Regulations for Organic Fertilizers" issued by the Ministry of Agriculture and Rural Affairs of China. This standard requires that organic fertilizers contain organic matter ≥30%, total nutrients (N+P2O5+K2O) ≥4.0%, pH 5.5-8.5, GI index ≥70%, and do not contain harmful substances such as heavy metals, harmful microorganisms and their metabolites.

[0056] As shown in Table 1, compared with the compost not inoculated with strain FSB35, the organic matter content, total nutrient content and GI index of the compost inoculated with FSB35 were higher than the national standards; the pH value of the compost inoculated with FSB35 was within the national standard range, while the pH value of the compost not inoculated was higher than the national standard and did not meet the standard requirements for organic fertilizer.

[0057] Depend on Figure 6 It can be seen that the average GI index of the compost inoculated with strain FSB35 after 3 days of continuous high-temperature fermentation is 0.65, and the average GI index after 5 days of continuous high-temperature fermentation is 1.29, indicating that the compost inoculated with strain FSB35 has reached the level of complete decomposition after 5 days of continuous high-temperature fermentation (it is judged to be completely decomposed when the GI index is greater than 70%); and the GI index of the water extract of the compost of the uninoculated compost after 5 days of fermentation under the same conditions is also higher than 70%, but its degree of maturity is still lower than the degree of maturity of the compost inoculated with strain FSB35 after 5 days of continuous high-temperature fermentation, indicating that the strain FSB35 provided by the present invention has excellent high-temperature decomposition-promoting ability, and can make the compost quickly decompose in a short time and have a higher degree of decomposition. Figure 7 It can be seen that the carbon-nitrogen ratio of the compost inoculated with strain FSB35 is close to 16 after 5 days of continuous high-temperature fermentation (the closer the carbon-nitrogen ratio is to 16 (it is generally believed that when the carbon-nitrogen ratio of compost is close to 16, that is, the carbon-nitrogen ratio of microorganisms, the compost is considered mature), the higher the degree of maturity. When the carbon-nitrogen ratio drops to about 16, the compost can be considered basically mature). This shows that the licheniformis FSB35 provided by the present invention only needs 5 days of continuous high-temperature fermentation to meet the maturity requirements, and the maturity reaches more than 100%, which significantly accelerates the compost maturity process. Figure 8 It can be seen that the organic matter content of the compost inoculated with strain FSB35 at 3 days and 5 days of continuous high-temperature fermentation was lower than that of the uninoculated compost, indicating that strain FSB35 has a good degradation effect on organic matter in the compost.

[0058] Example 5

[0059] The present invention studies the effect of compost inoculated with Bacillus licheniformis FSB35 on wheat growth

[0060] The compost inoculated with FSB35 and the compost not inoculated were fermented at high temperature for 3 and 5 days respectively according to the fermentation method of Example 4. The compost was applied to the soil of the newly planted wheat pot at a rate of 100 catties per mu of field crops. At the same time, the wheat pot without any fertilizer was used as the control group. Figure 9 In addition, the stem and leaf weight and plant height of wheat were measured, and the bar graphs are as follows: Figures 10-11 shown.

[0061] Depend on Figures 10-11 As can be seen, after applying the compost inoculated with FSB35 and fermented for 5 days, the stem and leaf weight and plant height of wheat increased significantly. This shows that after 5 days of fermentation, the compost inoculated with FSB35 has a better growth-promoting effect on wheat than the uninoculated compost.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A strain of Bacillus licheniformis FSB35, characterized in that: The taxonomic name is Bacillus licheniformis. It was deposited in the General Microbiology Center of China Culture Collection Administration on August 1, 2024. The deposit number is CGMCC No. 31408. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. Use of the Bacillus licheniformis FSB35 according to claim 1 in producing organic fertilizer by continuous high-temperature composting.

3. The use of Bacillus licheniformis FSB35 in producing organic fertilizer by continuous high-temperature composting according to claim 2, characterized in that: The temperature of the continuous high-temperature composting process is 60-70°C.

4. A microbial agent, characterized in that: The active ingredient is the Bacillus licheniformis FSB35 described in claim 1.

5. The microbial agent according to claim 4, wherein The viable count of the Bacillus licheniformis FSB35 is at least 10 8 CFU / mL.

6. Use of the microbial agent according to claim 4 in producing organic fertilizer by continuous high-temperature composting.

7. An organic fertilizer, characterized in that The product is prepared by continuous high-temperature composting and fermentation using the Bacillus licheniformis FSB35 described in claim 1 or the microbial agent described in claim 4.

8. The organic fertilizer according to claim 7, wherein Raw materials used for composting include materials containing cellulose or lignin.

9. A method for preparing an organic fertilizer, characterized in that: The compost raw materials are inoculated with the Bacillus licheniformis FSB35 described in claim 1 or the microbial agent described in claim 4, and the inoculated compost raw materials are continuously fermented at 60-70° C. for at least 5 days. During the fermentation, ventilation is performed regularly to maintain the moisture content of the compost at 50%-60%. After the fermentation is completed, the compost is cooled to room temperature, and the obtained decomposed material is the organic fertilizer.

10. The method for preparing an organic fertilizer according to claim 9, wherein The inoculum amount was 1 wt%.

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