Bacillus siamensis strain fsb30, bacterial agent comprising it and its use in continuous high temperature composting

Abstract

The present application relates to the technical field of microbial fermentation, and particularly relates to a bacillus smithii FSB30, a microbial agent containing the bacillus smithii FSB30 and application of the bacillus smithii FSB30 and the microbial agent in continuous high-temperature composting. The bacteria were 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, Xibahe West Road, Beijing City, Chaoyang District. The strain not only has the ability of high-temperature fermentation and can adapt to 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, significantly shorten the fermentation period of continuous high-temperature composting from about 10 days at present to 5 days, and significantly improve the efficiency of compost fermentation. The organic fertilizer obtained by compost fermentation after inoculation of the strain has a growth-promoting effect on crops, and improves the root weight and stem leaf weight of crops.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, in particular to a bacillus siamensis FSB30, a microbial agent containing the same and application of the same in continuous high-temperature composting. BACKGROUND

[0002] In the agricultural production of China, a large amount of crop straw and stems and leaves are produced every year. As feed production raw materials for livestock and poultry, crop straw and stems and leaves indirectly promote the development of livestock and poultry breeding industry. However, if crop straw and stems and leaves and livestock and poultry manure generated by livestock and poultry breeding are not properly treated, it will cause great environmental pollution and lead to resource waste. At present, the composting method is often used to harmlessly treat these crop straw, stems and leaves and livestock and poultry manure and convert them into renewable resources, while solving the environmental pollution problem. Composting is divided into general composting and high-temperature composting. The fermentation temperature of general composting is relatively low, and the fermentation temperature of high-temperature composting is relatively high in the early stage, and the compression measures are generally used in the later stage. Continuous high-temperature composting was first proposed by Schulze, who described it as continuously maintaining high substrate temperature through external heating, thereby improving biological conversion efficiency and inhibiting the proliferation of pathogenic microorganisms. However, general composting needs at least 90 days to reach maturity, and it also needs more than 20 days to completely mature in the presence of microbial agents. Continuous high-temperature composting can greatly shorten the maturation time. The aerobic composting bioreactor can realize rapid heating and maintain high temperature, thereby shortening the fermentation time to about 10 days, and the fermentation efficiency of this fermentation method is high and the reaction conditions are easy to control.

[0003] Livestock and poultry manure and organic waste contain a variety of microorganisms, which can promote the composting of raw materials during composting. However, the fermentation effect is poor, the fermentation time is long, and the overall fermentation efficiency is low. External microbial agents have better fermentation effect and higher fermentation efficiency than direct composting fermentation using microorganisms contained therein. External microbial agents have a positive effect on the total nutrient concentration of composting. Suitable microbial agents can accelerate the degradation of lignin and cellulose in livestock and poultry manure and organic waste, and shorten the composting time. Although there are many microbial agents suitable for the fermentation and degradation of livestock and poultry manure and organic waste at present, there are few microbial agents that can adapt to the rapid continuous high-temperature composting process. The reason may be that the microorganisms in the existing microbial agents are not compatible with the characteristics of continuous high-temperature composting raw materials or the microorganisms are not suitable for the composting conditions of continuous high-temperature composting. SUMMARY

[0004] In order to solve the above technical problems, the present application provides a Bacillus smithii FSB30, a microbial inoculum comprising the same and an application of the Bacillus smithii FSB30 in continuous high-temperature composting.

[0005] In order to achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0006] The first aspect of the present application provides a Bacillus smithii FSB30, the taxonomic name of which is Bacillus smithii (Bacillus smithii) Bacillus spizizenii The strain was deposited in the China General Microbiological Culture Collection Center on August 1, 2024, the deposit number is CGMCC No. 31407, and the deposit address is No. 3, Beichen West Road, Haidian District, Beijing.

[0007] The colony of the Bacillus smithii FSB30 provided by the present application is dirty white, the surface is rough and opaque, and usually presents a relatively regular circular shape 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 FSB30 is shown in SEQ ID NO: 1.

[0008] The second aspect of the present application provides an application of the above-mentioned Bacillus smithii FSB30 in continuous high-temperature composting for producing organic fertilizer.

[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℃, and can be 60℃.

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

[0011] The third aspect of the present application provides a microbial inoculum, and the active ingredient of the microbial inoculum is the above-mentioned Bacillus smithii FSB30.

[0012] In combination with the third aspect, in the microbial inoculum, the viable bacterial count of the Bacillus smithii FSB30 is at least 10 8 CFU / mL.

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

[0014] The fourth aspect of the present application provides an application of the above-mentioned microbial inoculum in continuous high-temperature composting for producing organic fertilizer.

[0015] In a fifth aspect, the present application provides an organic fertilizer prepared by continuously high-temperature composting the Bacillus Strain FSB30 or microbial inoculum.

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

[0017] Preferably, the cellulose or lignin containing material includes one or more of a combination of livestock and poultry manure, crop straw or forestry waste, or other material that can be reused by continuously high-temperature composting.

[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 a sixth aspect, the present application provides a method for preparing an organic fertilizer, which inoculates the composting raw material with the Bacillus Strain FSB30 or microbial inoculum, continuously ferments the inoculated composting raw material at 60 to 70°C for at least 5 days, ventilates regularly during the fermentation period, maintains the moisture content of the compost at 50% to 60%, cools to room temperature after the fermentation is completed, and the resulting compost is the organic fertilizer.

[0020] Preferably, the time for continuous fermentation is 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 FSB30 provided by the present application 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 in the composting raw material that is difficult to degrade by ordinary microbial inoculums, rapidly decomposing organic matter in the compost, significantly shortening the fermentation period of continuous high-temperature composting from the current 10 days or so to 5 days, and even obtaining a seed germination index (GI index) of at least 0.9 and a carbon-nitrogen ratio (C / N) close to 16 on the third day of fermentation (indicating that the compost has been basically decomposed), a GI index of 1.14 on average and a maximum of 1.59 on the fifth day of fermentation (indicating complete decomposition), which shows that the strain FSB30 provided by the present application can significantly improve the efficiency of continuous high-temperature composting. Moreover, the organic fertilizer obtained by composting after inoculating the strain has a significant growth-promoting effect on crops, significantly increasing the root weight and stem leaf weight of crops, making the root system of crops more developed, and ensuring the vigorous growth of crops. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1This is a complete genome loop of Bacillus stearothermiae FSB30;

[0024] Figure 2 Phylogenetic tree of Bacillus stearothermiae FSB30;

[0025] Figure 3 Photographs of Bacillus stearothermiae FSB30 colonies growing on culture medium;

[0026] Figure 4 This is a photograph of Bacillus spp. FSB30 after growth on sodium carboxymethyl cellulose medium stained with Congo red (0.1% v / v).

[0027] Figure 5 A photograph of Bacillus stearothermiae FSB30 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 stenosum FSB30 and those not inoculated with FSB30.

[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 stenosum FSB30 inoculated and uninoculated FSB30.

[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 stenosum FSB30 and those not inoculated with FSB30.

[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] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application.

[0036] Livestock manure and organic waste are usually treated by composting to realize the reuse of resources. Traditional aerobic composting needs at least 3 months to complete composting, while continuous high-temperature composting (such as tank composting) can complete composting in about 10 days. Continuous high-temperature composting can make the composting raw materials quickly compost under high-temperature conditions through external heating, but no external microorganism or microbial agent for continuous high-temperature composting has been found so far, and thus the high-temperature fermentation still needs a long fermentation time to make the compost completely composted. In view of this, the present application provides a Bacillus smithii FSB30 which can be applied to continuous high-temperature composting. The strain not only tolerates high temperature and can adapt to the fermentation conditions of continuous high-temperature composting, 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 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 mixed with the livestock manure at a mass ratio of 1:5. The initial water content of the composting raw materials is adjusted to about 50%-60%, and the C / N is about 25-35:1.

[0039] The nutrient agar medium used has the following formula: 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 a pH value of 7.3.

[0040] Example 1

[0041] The present application provides a method for isolating and identifying Bacillus smithii FSB30

[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, and shaken on a vortex mixer for 60 seconds. Continuous gradient dilution is performed to 10 -6Dilute 100 μL of the solution and spread it onto nutrient agar solid medium to isolate bacteria. Incubate the culture plate at 60 °C to isolate heat-resistant microorganisms. Transfer the colonies of microorganisms that can grow at 60 °C to a new nutrient agar medium, remove morphologically redundant colonies, and screen to obtain 47 heat-resistant strains (named FSB1~FSB47).

[0043] (2) DNA was extracted and sequenced from the 47 heat-resistant strains obtained by screening. Based on the sequencing results, known duplicate strains were removed. Among the remaining strains obtained by screening, FSB30 did not have 100% homology with the identified species and was similar to Bacillus stearothermiae. Bacillus spizizenii (GCF_000227465.1) Genes with homology greater than 80% account for only 36.1% 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 FSB30 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 FSB30 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. 31407, 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 FSB30.

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

[0049] Example 3

[0050] The present application provides a ligninase activity production experiment of Bacillus smithii FSB30

[0051] The overnight cultured strain FSB30 bacterial solution was diluted 10 -6 times, 100 μL was taken and coated on a nutrient agar medium containing aniline blue (0.1 g / L) (Solarbio, Beijing, China), and cultured at 45°C in the dark for 18 hours. The medium was stained with aniline blue, but as the colonies grew, a transparent circle (as shown in Figure 5 , also known as a transparent ring) appeared around them, indicating that the strain FSB30 could secrete ligninase during growth.

[0052] Example 4

[0053] The present application provides a fermentation effect test experiment of Bacillus smithii FSB30

[0054] In the laboratory, the fermentation process of the fermentation tank was simulated using an oven: FSB30 was added to the prepared compost material at an inoculation amount of 1 wt% (mass fraction) (3 parallel compost materials), each was mixed uniformly and placed in an oven, sealed to prevent water evaporation, while the same compost material was taken without adding FSB30, the inoculated and un-inoculated compost materials were placed in the oven under the same conditions, and continuous thermophilic fermentation experiments were carried out under the same conditions, the fermentation process was as follows: the temperature of the oven was raised to 60°C (not more than 70°C) within one hour, then maintained at 60°C for 5 days, the temperature of the compost was recorded using a temperature sensor, and the compost was ventilated every 12 hours during the period, and the moisture content of the compost was maintained at 50%-60%. Then the oven was turned off and allowed to naturally cool to room temperature. At 3 days and 5 days of continuous thermophilic fermentation, samples were taken from the upper, middle and lower parts of the 3 parallel composts, 3 samples were taken at each depth, and the 3-day and 5-day continuous thermophilic fermentation samples (9 samples each) were mixed uniformly to form a composite sample, and the composite sample was sent to a third-party testing agency for testing of ingredients (moisture content was determined by vacuum oven method; total carbon and total nitrogen content were determined by 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 extract, which was used to measure pH value, electrical conductivity and seed germination index (GI): pH and EC values were measured using Five Easy Plus TM pH / EC meter (Mettler-Toledo, Shanghai, China), cucumber seeds were cultured in compost water extract at 25°C in the dark for 48 hours, root length and germination rate were measured, and GI value was calculated, deionized water was used as a control; the organic matter content in the compost was determined by potassium dichromate volumetric method; the total soluble phosphorus content was determined by UV-1900i spectrophotometer (Shimadzu Corporation, Shanghai, China); the total potassium content was determined by TAS-990 atomic absorption spectrometry (Beijing Pu Xi General Instrument Co., Ltd., Beijing, China)). The average value and standard deviation of 3 parallel compost materials were calculated for all test results, the average value of each test result is shown in Table 1, and the average carbon-nitrogen ratio and average organic matter content bar charts are shown in Figure 7 and Figure 8

[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 <Organic Fertilizer Practical Technical Regulations> standard, and the standard 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 shown in Table 1, compared with the compost inoculated with strain FSB30, the organic matter content, total nutrient content and GI index of the compost inoculated with strain FSB30 are higher than the national standard; the pH value of the compost fermented for 5 days after inoculation of strain FSB30 meets the national standard, 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 shown in Table 1, compared with the compost inoculated with strain FSB30, the organic matter content, total nutrient content and GI index of the compost inoculated with strain FSB30 are higher than the national standard; the pH value of the compost fermented for 5 days after inoculation of strain FSB30 meets the national standard, 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 shown in Table 1, compared with the compost inoculated with strain FSB30, the organic matter content, total nutrient content and GI index of the compost inoculated with strain FSB30 are higher than the national standard; the pH value of the compost fermented for 5 days after inoculation of strain FSB30 meets the national standard, 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 shown in Table 1, compared with the compost inoculated with strain FSB30, the organic matter content, total nutrient content and GI index of the compost inoculated with strain FSB30 are higher than the national standard; the pH value of the compost fermented for 5 days after inoculation of strain FSB30 meets the national standard, 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 shown in Table 1, compared with the compost inoculated with strain FSB30, the organic matter content, total nutrient content and GI index of the compost inoculated with strain FSB30 are higher than the national standard; the pH value of the compost fermented for 5 days after inoculation of strain FSB30 meets the national standard, 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 strain FSB30, which is a strain of Bacillus smithii, and the strain FSB30 has the advantages of high temperature and high efficiency, and can be used for composting.

[0062] The composts obtained by continuously thermophilic fermentation of the compost material inoculated with FSB30 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, and 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 determined, 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 FSB30 and fermented for 5 days, the root system of the wheat is more developed, the root weight, stem and leaf weight are significantly increased, and the plant height is obviously increased compared with the control group, which indicates that the compost inoculated with strain FSB30 and fermented for 5 days has obvious growth promoting effect on the wheat.

[0064] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A strain of Bacillus stearothermiae FSB30, 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. 31407, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of Bacillus stearothermia esculenta FSB30 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 FSB30 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 30 as described in claim 1.

5. The microbial agent as described in claim 4, characterized in that, The viable count of the *Bacillus stearothermiae* FSB30 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 stearothermie FSB30 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 FSB30 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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