Bacillus stutzeri FSB24, fungicide containing same and application of bacillus stutzeri FSB24 in continuous high-temperature composting

Abstract

The invention relates to the technical field of microbial fermentation, in particular to bacillus stutzeri FSB24, a fungicide containing the bacillus stutzeri FSB24 and application of the bacillus stutzeri FSB24 in continuous high-temperature composting. The strain is preserved in the China General Microbiological Culture Collection Center on August 1, 2024, the preservation number is CGMCC No.31409, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain not only has high-temperature fermentation capacity and can adapt to specific conditions of continuous high-temperature fermentation, but also can secrete ligninase and cellulase, so that cellulose and lignin which are difficult to degrade in compost raw materials can be degraded more quickly and more completely, organic matters are decomposed quickly, and the yield of the compost raw materials is increased. The fermentation period of continuous high-temperature composting is shortened from about 10 days at present to at least 3 days, and the composting fermentation efficiency is remarkably improved. The organic fertilizer obtained through compost fermentation after inoculation of the strain has a growth promoting effect on crops, and the root weight and stem leaf weight of the crops are increased.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a strain of Bacillus stutzeri FSB24, a bacterial agent containing the same and application of the same in continuous high-temperature composting. Background Art

[0002] my country is a large agricultural country, and the amount of crop straw produced every year is huge. At the same time, my country's livestock and poultry farming industry has also entered a stage of vigorous development. If the large amount of straw produced by planting crops and the livestock and poultry manure produced by livestock and poultry farming are not handled properly, it will cause great environmental pollution and waste of resources. Therefore, composting is often used to harmlessly treat these organic wastes such as crop straw and fallen leaves and livestock and poultry manure and convert them into renewable resources, while solving the environmental pollution problems caused by them. Composting is divided into general composting and high-temperature composting. The fermentation temperature of general composting is low, and the fermentation temperature of high-temperature composting is high in the early stage. In the later stage, compaction measures are generally adopted. Continuous high-temperature composting was first proposed by Schulze, who described continuous high-temperature composting as maintaining a high substrate temperature continuously through external heating, thereby improving the efficiency of bioconversion and inhibiting the proliferation of pathogenic microorganisms. However, it takes 90 to 270 days for general composting to reach maturity, and even with the addition of bacterial agents, it takes 20 to 30 days to mature. Continuous high-temperature composting can greatly shorten the decomposition time. The use of aerobic composting bioreactors can achieve rapid heating and maintain a high temperature state, thereby greatly shortening the fermentation time (fermentation can be completed in about 10 days). The fermentation efficiency is high, the reaction conditions are controllable, and the waste gas can be collected and treated in a centralized manner.

[0003] Although livestock and poultry manure and organic waste contain a variety of microorganisms that can mature them during the composting process, adding external inoculants usually has better results than direct composting and requires shorter fermentation time. Microbial inoculants have a positive effect 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 and shorten the time for composting to mature. However, although there are countless fermentation agents suitable for livestock and poultry manure, there are very few microbial fermentation agents that can adapt to the rapid heating process of continuous high-temperature composting. The reason may be that the microorganisms in the fermentation agents are incompatible with the characteristics of continuous high-temperature composting raw materials, or the microorganisms are not adapted to the operating conditions of continuous high-temperature composting. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a strain of Bacillus stutzeri FSB24, a bacterial agent containing the same and the application of the same in continuous high-temperature composting. The Bacillus stutzeri can not only adapt to the high-temperature environment of continuous high-temperature composting, but also secrete cellulase and ligninase to promote the maturity of compost raw materials and improve 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 spizizenii FSB24, whose taxonomic name is Bacillus spizizenii. The strain was deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee on August 1, 2024, with a deposit number of CGMCC No. 31409, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The colony of the Bacillus stinii FSB24 provided by the present invention is dirty white, with a rough and opaque surface, usually presenting a relatively regular circular or irregular branched shape, forming wrinkles when growing in a liquid culture medium, and is an aerobic bacterium with a suitable pH value of 5.0 to 9.0 for growth. The 16S rDNA sequence of the strain FSB24 is shown in SEQ ID NO: 1.

[0008] In a second aspect, the present invention provides a use of the above-mentioned Bacillus stinzeri 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 does not exceed 70°C, preferably 45-70°C, more preferably 45-60°C, and may 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 stutzeri FSB24.

[0012] In combination with the third aspect, in the microbial agent, the viable count of Bacillus stutzeri FSB24 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 an application 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, which is prepared by continuous high-temperature composting and fermentation using the above-mentioned Bacillus stinii FSB24 or microbial agent. The organic fertilizer has a growth-promoting effect on crop growth, can significantly increase the root weight, stem and leaf weight and plant height of crop plants, and thus indirectly increase 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 material containing cellulose or lignin includes one or more combinations of livestock and poultry manure, crop straw or forestry waste, and may also be other raw materials that can be recycled by 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, comprising inoculating a compost raw material with the above-mentioned Bacillus stinkelii FSB24 or a microbial agent, fermenting the inoculated compost raw material continuously at 60-70°C for at least 3 days, ventilating the compost regularly during the fermentation period to maintain a moisture content of 50%-60%, and cooling the compost to room temperature after the fermentation is completed. The resulting decomposed material is the organic fertilizer.

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

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

[0022] The strain FSB24 provided by the present invention not only has the ability of high temperature fermentation, 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 common microbial agents in compost raw materials, so that the organic matter in the compost is rapidly decomposed, and the fermentation cycle of continuous high temperature composting is greatly shortened from the current 10 days to 3-5 days, and even on the third day of fermentation, a seed germination index (GI index) of at least 1.0 and a carbon-nitrogen ratio close to 16 (indicating that fermentation can be completely decomposed in 3 days) can be obtained, and the GI index on the third day of fermentation is 1.42 on average, up to 1.8, which significantly improves the efficiency of compost fermentation. Moreover, the organic fertilizer obtained by compost fermentation after inoculation of the strain has an obvious growth-promoting effect on crops, significantly improves the root weight and stem and leaf weight of crops, makes the root system of crops more developed, and ensures the vigorous growth of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the circle map of the whole genome of Bacillus stutzeri FSB24;

[0024] Figure 2 is the phylogenetic tree of Bacillus stutzeri FSB24;

[0025] Figure 3 This is a photo of the colonies of Bacillus stutzeri FSB24 growing on the culture medium;

[0026] Figure 4 This is a photograph of Bacillus stutzeri FSB24 grown on sodium carboxymethylcellulose medium stained with Congo red (0.1% volume ratio);

[0027] Figure 5 This is a photograph of Bacillus stutzeri FSB24 grown on a nutrient agar medium containing aniline blue (0.1 g / L);

[0028] Figure 6 The bar graphs are the seed germination index of the compost water extracts corresponding to the compost raw materials inoculated with Bacillus Steinerneri FSB24 and those not inoculated with FSB24 after continuous high temperature fermentation for 3 days and 5 days;

[0029] Figure 7 The bar graphs of carbon-nitrogen ratios of the composts inoculated with Bacillus Steinerneri FSB24 and not inoculated with FSB24 after continuous high-temperature fermentation for 3 and 5 days;

[0030] Figure 8 The bar graphs of organic matter content in the compost piles inoculated with Bacillus Steinerneri FSB24 and without FSB24 after continuous high temperature fermentation for 3 days and 5 days;

[0031] Fig. 9 , (a) is a photo of a potted wheat plant with compost applied at different periods or without fertilizer applied after one month of growth, (b) is a photo of the whole plant of wheat plant with compost applied at different periods or without fertilizer applied after one month of growth;

[0032] Fig.10 This is a bar graph of the average root weight of wheat after applying compost at different periods of growth for one month;

[0033] Fig.11 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;

[0034] Fig.12 This is a bar graph of plant height of wheat after applying compost at different periods of growth for one month. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution 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 used to limit the present invention.

[0036] In the composting of livestock and poultry manure and organic waste, traditional aerobic composting (i.e., general composting) requires 90 to 270 days to complete decomposition, while continuous high-temperature composting (such as tank composting) only takes about 10 days to complete decomposition. Continuous high-temperature composting is completed by artificial heating, but the temperature environment in the fermentation equipment used for continuous high-temperature composting makes it impossible to continue to accelerate decomposition by inoculating microorganisms with degradation ability, resulting in the composting time cannot be further shortened and the decomposition degree cannot be further improved. In view of this, the present invention provides a strain of Bacillus stutzeri FSB24 that can be applied to continuous high-temperature composting. The strain is not only resistant to high temperatures, 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 composition of the compost material used in the following examples is:

[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 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 composting raw materials is adjusted to about 50% to 60%, and the C / N is about 25 to 35:1.

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

[0040] Example 1

[0041] The present invention provides a method for separating and identifying Bacillus stutzeri FSB24

[0042] (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. After about 3 g of sample obtained 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 for 60 seconds, and continuously diluted to 10 -6 The dilution was diluted 200 μL and 100 μL of the dilution was spread on a culture plate with nutrient agar to isolate the bacteria. The culture plate was incubated at 60°C to isolate heat-resistant microorganisms. The microbial colonies that could grow at 60°C were transferred to a new nutrient agar medium, and the morphologically redundant colonies were removed. A total of 47 heat-resistant strains (named FSB1 to FSB47) were screened.

[0043] (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, FSB24 was not 100% homologous to the identified species, and genes with a homology greater than 80% to Bacillus spizizenii (GCF_000227465.1) accounted for only 37.9% of the total number of genes. Figure 1 The phylogenetic tree is shown in Figure 2 As shown in the figure (the whole genome sequences of other strains in the phylogenetic tree were downloaded from NCBI), it was determined to be Bacillus stutzeri. In addition, the strain FSB24 was further molecularly identified based on the 16S sequence, and its 16S rDNA sequence is shown in SEQ ID NO.1.

[0044] The strain FSB24 of Bacillus stutzeri screened by the present invention has a dirty white colony with a rough and opaque surface, and generally presents a relatively regular circular or irregular branched shape. When grown in a liquid culture medium, it forms a wrinkled mold (such as Figure 3 is an aerobic bacterium.

[0045] The taxonomic name of this strain is Bacillus spizizenii. It was deposited in the General Microbiology Center of China Culture Collection Administration on August 1, 2024. The deposit number is CGMCC No.31409. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] Example 2

[0047] The present invention provides an experiment on the cellulase activity of Bacillus stutzeri FSB24.

[0048] The overnight culture of strain FSB24 was diluted 10 -6 100 μL was spread on 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 circle appeared around the colonies (such as Figure 4 As shown, it can also be called transparent ring), indicating that strain FSB24 can secrete cellulase during growth.

[0049] Example 3

[0050] The present invention provides an experiment on the ligninase activity of Bacillus stutzeri FSB24.

[0051] The overnight culture of strain FSB24 was diluted 10 -6100 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 h. The medium was stained with aniline blue, but as the colonies grew, a transparent zone appeared around them (e.g. Figure 5 As shown, it can also be called transparent ring), indicating that strain FSB24 can secrete ligninase during growth.

[0052] Example 4

[0053] The present invention provides a fermentation effect test experiment of Bacillus stutzeri FSB24

[0054] In the laboratory, an oven was used to simulate the fermentation process of the fermentation tank: FSB24 was added to the prepared compost material at room temperature according to the inoculation amount of 1wt% (mass percentage) (3 parallel compost materials), and each was mixed and placed in an oven, sealed to prevent water evaporation. At the same time, the same compost material was taken but without FSB24, and both the inoculated and uninoculated compost materials were placed in the oven. A continuous high-temperature fermentation experiment was carried out under the same conditions. The fermentation process was: the oven temperature was raised to 60°C (maximum not exceeding 70°C) within one hour, and increased by 3°C every 5 minutes, and then maintained at 60°C for 5 days. The compost temperature was recorded using a temperature sensor, and ventilation and oxygenation were carried out every 12 hours during the period, and the water content of the compost was kept at 50% to 60%. The oven was then closed to allow it to naturally drop to room temperature. At 3 and 5 days of continuous high-temperature fermentation, samples were taken from the upper, middle and lower parts of 3 parallel piles, and 3 samples were taken at each depth. The sub-samples (9 samples each) collected at 3 and 5 days of continuous high-temperature fermentation were evenly mixed as composite samples, and the composite samples were sent to a third-party testing agency for component testing (water content was determined by vacuum oven method; total carbon and total nitrogen content 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 measuring pH, conductivity and seed germination index (GI): Five Easy Plus TM pH and EC values ​​were determined using a pH / EC meter (Mettler-Toledo, Shanghai, China). Cucumber seeds were cultured in a compost water extract at 25 °C in the dark for 48 h, and the root length and germination rate were measured, and the GI value was calculated. Deionized water was used as a control. The organic matter content in the compost was determined using the potassium dichromate volumetric method. The total soluble phosphorus content was determined using a UV-1900i spectrophotometer (Shimadzu Corporation, Shanghai, China). The total potassium content was determined using a TAS-990 atomic absorption spectrometer (Beijing Puxi General Instrument Co., Ltd., Beijing, China). The average and standard deviation of the three parallel compost materials were calculated for all test results. The average values ​​of each test result are shown in Table 1. The bar graphs of the average carbon-nitrogen ratio and the average organic matter content are shown in Table 2. Figure 7 and Figure 8 shown.

[0055] Table 1

[0056]

[0057]

[0058] The maturity evaluation of compost is carried out in accordance with the standard "Organic Fertilizer" (NY 525-2021). "Organic Fertilizer" is an organic fertilizer evaluation standard that meets the "Practical Technical Regulations for Organic Fertilizer" standard. This standard requires that organic fertilizer contains organic matter ≥30%, total nutrients (N+P2O5+K2O) ≥4.0%, pH 5.5-8.5, GI index ≥70%, and does not contain harmful substances such as heavy metals, harmful microorganisms and their metabolites.

[0059] As can be seen from Table 1, compared with the compost not 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 standards; the pH value of the compost inoculated with FSB24 is within the standard range prescribed by the state, while the pH value of the compost not inoculated with bacteria is higher than the national standard and does not meet the standard requirements for organic fertilizers.

[0060] Depend on Figure 6 It can be seen that the GI index of the compost inoculated with strain FSB24 after continuous high-temperature fermentation for 3 days is as low as 1.0 (i.e. 100%) and as high as 1.8 (i.e. 180%). This indicates that the compost inoculated with strain FSB24 has reached the level of complete maturity after continuous high-temperature fermentation for 3 days (it is considered to be fully mature when the GI index is greater than 70%). However, when the compost not inoculated is fermented under the same conditions for 3 days, the GI index of the compost water extract is still lower than 90% at the highest and only 40% at the lowest. This indicates that the strain FSB24 provided by the present invention has excellent high-temperature decomposition-promoting ability and can make the compost mature quickly in a short time. Figure 7 It can be seen that the carbon-nitrogen ratio of the compost inoculated with strain FSB24 is close to 16 after 3 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 the compost is close to 16, that is, the carbon-nitrogen ratio of the 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), indicating that the Bacillus stinica FSB24 provided by the present invention only needs 3 days of continuous high-temperature fermentation to reach the maturity requirement, which significantly accelerates the compost maturity process. Figure 8 It can be seen that the organic matter content of the compost inoculated with strain FSB24 at 3 days and 5 days of continuous high-temperature fermentation was lower than that of the uninoculated compost, indicating that strain FSB24 has a good degradation effect on organic matter in the compost.

[0061] Example 5

[0062] The present invention studies the effect of compost inoculated with Bacillus stutzeri FSB24 on wheat growth

[0063] The compost material inoculated with FSB24 and the compost material not inoculated were fermented at high temperature for 3 and 5 days respectively according to the fermentation method of Example 4, and the compost obtained was applied to the soil of the newly planted wheat potted plants at a rate of 100 catties per mu of field crops. At the same time, the wheat potted plants without any fertilizer were used as the control group. The whole wheat plant after one month of growth is shown in the figure below. Fig. 9 In addition, the root weight, stem and leaf weight and plant height of wheat were measured, and the bar graphs are shown in Figures 10-12 shown.

[0064] Depend on Figures 10-12 It can be seen that after applying the compost inoculated with FSB24, the root system of wheat became more developed, the root weight, stem and leaf weight increased significantly, and the plant height also increased significantly. This shows that whether it is fermented for 3 days or 5 days, the compost inoculated with FSB24 has a better growth-promoting effect on wheat than the uninoculated compost.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus stutzeri FSB24, characterized in that: The taxonomic name is Bacillus spizizenii. It was deposited in the General Microbiology Center of China Culture Collection Administration on August 1, 2024. The deposit number is CGMCC No. 31409. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Bacillus stinii FSB24 according to claim 1 in producing organic fertilizer by continuous high-temperature composting.

3. The use of Bacillus stinii FSB24 as claimed in claim 2 in producing organic fertilizer by continuous high temperature composting, characterized in that: The temperature of the continuous high temperature composting process does not exceed 70°C.

4. A microbial agent, characterized in that: The active ingredient is the Bacillus stutzeri FSB24 described in claim 1.

5. The microbial agent according to claim 4, characterized in that The viable count of Bacillus stutzeri FSB24 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 invention is prepared by using the Bacillus stinkelii FSB24 described in claim 1 or the microbial agent described in claim 4 through continuous high-temperature composting and fermentation.

8. The organic fertilizer according to claim 7, characterized in that 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 stinkelii FSB24 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 3 days, and ventilation is performed regularly during the fermentation to keep 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, characterized in that: The inoculation amount was 1 wt%.

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