Bacillus stutzeri FSB30, fungicide containing same and application of bacillus stutzeri FSB30 in continuous high-temperature composting

Abstract

The invention relates to the technical field of microbial fermentation, in particular to bacillus stutzeri FSB30, a fungicide containing the bacillus stutzeri FSB30 and application of the bacillus stutzeri FSB30 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.31407, 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 greatly shortened to 5 days from the current about 10 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 FSB30, a bacterial agent containing the same and application of the same in continuous high-temperature composting. Background Art

[0002] In my country's agricultural production, a large amount of crop straw and stems are produced every year. Crop straw and stems are used as raw materials for livestock and poultry feed production, which indirectly promotes the development of livestock and poultry farming. However, if crop straw and stems and livestock and poultry manure produced by livestock and poultry farming are not properly handled, it will cause great environmental pollution and waste resources. At present, composting is often used to harmlessly treat these organic wastes such as crop straw, stems and 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 early fermentation temperature of high-temperature composting is high. 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 biological conversion and inhibiting the proliferation of pathogenic microorganisms. However, general composting takes at least 90 days to reach maturity, and it takes more than 20 days to fully mature when bacterial agents are added. Continuous high-temperature composting can greatly shorten the time of decomposition. The aerobic composting bioreactor can achieve rapid temperature rise and maintain a high temperature state, thereby shortening the fermentation time to about 10 days. This fermentation method has high fermentation efficiency and easy to control reaction conditions.

[0003] Livestock and poultry manure and organic waste contain a variety of microorganisms, which can promote the maturity of compost raw materials during the composting process, but the fermentation effect is poor and the fermentation time required is long, which makes the overall fermentation efficiency low. External microbial agents have better fermentation effect and higher fermentation efficiency than using the microorganisms contained in the compost directly for composting fermentation. External microbial agents have a positive effect on the total nutrient concentration of compost. Suitable microbial agents can accelerate the degradation of lignin and cellulose in livestock and poultry manure and organic waste and shorten the compost maturity time. Although there are many microbial agents suitable for the fermentation and degradation of livestock and poultry manure and organic waste, there are few reports on microbial agents that can adapt to continuous high-temperature composting with rapid heating process. The reason may be that the microorganisms in the existing microbial agents are incompatible with the characteristics of continuous high-temperature composting raw materials or the microorganisms are not adapted to the composting 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 FSB30, 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 FSB30, whose taxonomic name is Bacillus spizizenii. The strain was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on August 1, 2024, with a deposit number of CGMCC No. 31407, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The colony of the Bacillus stutzeri FSB30 provided by the present invention is dirty white, with a rough and opaque surface, usually presenting a relatively regular circular or irregular branched shape, and forming wrinkles when growing in a liquid culture medium. It is an aerobic bacterium, and the pH value suitable for growth is 5.0-9.0. The 16S rDNA sequence of the strain FSB30 is shown in SEQ ID NO: 1.

[0008] In a second aspect, the present invention provides a use of the above-mentioned Bacillus stinzeri FSB30 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 FSB30.

[0012] In combination with the third aspect, in the microbial agent, the viable count of Bacillus stutzeri FSB30 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 FSB30 or microbial agent. The organic fertilizer has a growth-promoting effect on the growth of crops, can significantly increase the root weight, stem and leaf weight and plant height of crop plants, and indirectly increase the yield of crops.

[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 FSB30 or a microbial agent, fermenting the inoculated compost raw material continuously at 60-70°C for at least 5 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] Preferably, the continuous fermentation time is 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 FSB30 provided by the present invention not only has the ability of high temperature fermentation resistance, 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 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, and even on the third day of fermentation, a seed germination index (GI index) of at least 0.9 and a carbon-nitrogen ratio close to 16 (indicating that the fermentation has been basically decomposed for 3 days) can be obtained. The GI index on the fifth day of fermentation is 1.14 on average, and can reach up to 1.59, reaching complete decomposition, indicating that the strain FSB30 provided by the present invention can significantly improve the efficiency of continuous high temperature composting fermentation. Moreover, the organic fertilizer obtained by composting and 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 1This is the whole genome circle map of Bacillus stutzeri FSB30;

[0024] Figure 2 is the phylogenetic tree of Bacillus stutzeri FSB30;

[0025] Figure 3 This is a photo of the colonies of Bacillus stutzeri FSB30 growing on the culture medium;

[0026] Figure 4 This is a photograph of Bacillus stutzeri FSB30 grown on sodium carboxymethylcellulose medium stained with Congo red (0.1% volume ratio);

[0027] Figure 5 This is a photograph of Bacillus stutzeri FSB30 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 FSB30 and those not inoculated with FSB30 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 FSB30 and not inoculated with FSB30 after continuous high-temperature fermentation for 3 and 5 days;

[0030] Figure 8 The bar graphs of organic matter content in the composts inoculated with Bacillus Steinerneri FSB30 and not inoculated with FSB30 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] Livestock and poultry excrement and organic waste are mostly harmlessly treated by composting, so as to realize the reuse of resources. Traditional aerobic composting needs at least 3 months to complete decomposition, while continuous high temperature composting (such as tank composting) only needs about 10 days to complete decomposition. Continuous high temperature composting makes the compost raw material quickly decompose under high temperature conditions by means of external heating, but the external microorganisms or microbial agents that can be used for continuous high temperature composting have not yet been found, so the microorganisms contained in the compost raw material itself can only be relied on for high temperature fermentation, and still need a long fermentation time to make the compost fully decomposed. In view of this, the present invention provides a strain of Bacillus sternii FSB30 that can be applied to continuous high temperature composting, which is not only resistant to high temperatures, but also can adapt to the fermentation conditions of continuous high temperature composting, and also has the ability to degrade cellulose and lignin, which can greatly shorten the time of continuous composting and improve the compost 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 FSB30

[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 -6The dilution was diluted 100 μL and spread on nutrient agar solid medium to isolate 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 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. The known duplicate strains were removed based on the sequencing results. Among the remaining strains screened, FSB30 was not 100% homologous to the identified species, and the genes with a homology greater than 80% to Bacillus spizizenii (GCF_000227465.1) accounted for only 36.1% 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 FSB30 was further molecularly identified based on the 16S sequence, and its 16S rDNA sequence is shown in SEQ ID NO.1.

[0044] The strain FSB30 of Bacillus stinii screened by the present invention has a dirty white colony with a rough and opaque surface, and generally presents a relatively regular round 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.31407. 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 FSB30

[0048] Dilute the overnight culture of strain FSB30 by 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 FSB30 can secrete cellulase during growth.

[0049] Example 3

[0050] The present invention provides an experiment on the ligninase activity of Bacillus stutzeri FSB30.

[0051] Dilute the overnight culture of strain FSB30 by 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 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 FSB30 can secrete ligninase during growth.

[0052] Example 4

[0053] The present invention provides a fermentation effect test experiment of Bacillus stutzeri FSB30

[0054] In the laboratory, an oven was used to simulate the fermentation process of the fermentation tank: FSB30 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 adding FSB30. Both the inoculated and uninoculated compost materials were placed in the oven, and continuous high-temperature fermentation experiments were 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 performed every 12 hours during the period, and the water content of the compost was maintained 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] 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.

[0058] As shown in Table 1, compared with the compost not inoculated with strain FSB30, the organic matter content, total nutrient content and GI index of the compost inoculated with strain FSB30 were higher than the national standards; the pH value of the compost fermented for 5 days after inoculation with strain FSB30 met the national standards, while the pH value of the compost not inoculated with bacteria was higher than the national standard and did not meet the standard requirements for organic fertilizers.

[0059] Depend on Figure 6 It can be seen that the GI index of the compost inoculated with strain FSB30 after 5 days of continuous high-temperature fermentation is as low as 0.91 (i.e. 91%), and as high as close to 1.60 (the highest is 1.59, i.e. 159%), indicating that the compost inoculated with strain FSB30 has reached the degree of complete maturity after 5 days of continuous high-temperature fermentation (it is judged to be completely mature when the GI index is greater than 70%); while the average GI index of the water extract of the compost of the uninoculated compost is still less than 90% when fermented under the same conditions for 5 days, and the lowest is only 60%, indicating that the strain FSB30 provided by the present invention has excellent high-temperature decomposition-promoting ability, and can make the compost quickly mature in a short time. Figure 7 It can be seen that the carbon-nitrogen ratio of the compost inoculated with strain FSB30 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 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 FSB30 provided by the present invention only needs 5 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 FSB30 at 3 days and 5 days of continuous high-temperature fermentation was lower than that of the uninoculated compost, indicating that strain FSB30 has a good degradation effect on organic matter in the compost.

[0060] Example 5

[0061] The present invention studies the effect of compost inoculated with Bacillus stutzeri FSB30 on wheat growth

[0062] The compost material inoculated with FSB30 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.

[0063] Depend on Figures 10-12 It can be seen that after applying the compost inoculated with FSB30 and fermented for 5 days, compared with the control group, the root system of wheat was more developed, the root weight and stem and leaf weight were significantly increased, and the plant height was also significantly increased, indicating that the compost inoculated with strain FSB30 and fermented for 5 days has a significant growth-promoting effect on wheat.

[0064] 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 FSB30, 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. 31407. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Bacillus stinii FSB30 described in claim 1 in producing organic fertilizer by continuous high-temperature composting.

3. The use of Bacillus stinii FSB30 in producing organic fertilizer by continuous high temperature composting as claimed in claim 2, 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 FSB30 described in claim 1.

5. The microbial agent according to claim 4, characterized in that The viable count of Bacillus stutzeri FSB30 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 FSB30 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 material is inoculated with the Bacillus stinkelii FSB30 described in claim 1 or the microbial agent described in claim 4, and the inoculated compost raw material is continuously fermented at 60-70° C. for at least 5 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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