Paenibacillus mucilaginosus S24 and application thereof in compost in-situ fermentation production of bio-organic fertilizer

Abstract

The invention belongs to the field of agricultural resource and environmental biotechnology application. The invention provides paenibacillus mucilaginosus S24, and the preservation number of the paenibacillus mucilaginosus S24 is CGMCC (China General Microbiological Culture Collection Center) NO.35709. The invention further provides a preparation method of the paenibacillus mucilaginosus S24. The strain S24 is preserved in the China General Microbiological Culture Collection Center on August 22, 2025, the preservation number of the strain S24 is CGMCC NO.35709, the center is called CGMCC for short, and the address of the center is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain S24 provided by the invention has the capabilities of dissolving phosphorus, dissolving potassium, fixing nitrogen and the like; the strain has relatively high environmental adaptability, including a wide pH suitable range (pH 4-11) and very high salt tolerance (tolerant to 6% salt concentration). In addition, the strain S24 is inoculated to compost for primary fermentation to obtain an organic fertilizer, and the organic fertilizer can be proliferated in a secondary fermentation compost to produce a biological organic fertilizer with a growth promoting effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural resources and environmental biotechnology. BACKGROUND

[0002] Bio-organic fertilizer has the effects of improving soil physical and chemical properties and enhancing soil fertility, and plays an increasingly important role in agricultural production. At present, the production of bio-organic fertilizer mainly adopts the method of adding functional microbial agents and organic fertilizer. However, there are problems such as multiple production links, high production cost, and poor compatibility of microorganisms with organic fertilizer and soil. When using compost products as carriers to adsorb functional microorganisms to produce bio-organic fertilizer, the compost used needs to be completely composted. The water content is also an important factor affecting the combination of compost and functional microorganisms. When the water content of the compost is too high, the adsorption capacity is poor and mold is easy to breed, so it is necessary to reduce the water content through natural air drying (Zhang Zhihong et al., 2010) or heat treatment (Cai Yanfei et al., 2003) and then add functional microorganisms. At the same time, the microorganisms contained in the compost will also compete with the added functional microorganisms, resulting in a rapid decrease in the number of functional microorganisms (Zhang Zhihong et al., 2010). Therefore, additional high-temperature sterilization (Wu et al., 2015) or gamma ray sterilization (Tao et al., 2020) of the compost is needed to improve the survival of functional microorganisms in the compost. SUMMARY

[0003] The present application aims to solve the problems existing in the research and production of functional bio-organic fertilizer. A new technology for producing bio-organic fertilizer by inoculating growth-promoting microorganisms in the secondary fermentation stage of compost to proliferate in the secondary fermentation pile is invented. This method combines the production processes of functional microorganisms and organic fertilizer, which is simple and low in cost.

[0004] The purpose of the present application is to provide a method for producing bio-organic fertilizer by in-situ fermentation of compost, and a strain of Paenibacillus mucilaginosus S24 and its application in plant growth promotion and composting.

[0005] The present application screens and isolates a strain S24 from the secondary fermentation pile of livestock and poultry waste compost. The strain S24 is gram-positive, rod-shaped, and can form spores. The strain S24 is identified as Paenibacillus mucilaginosus. The strain S24 was deposited with the China General Microbiological Culture Collection Center on August 22, 2025, and has the accession number CGMCC NO.35709. The address of the China General Microbiological Culture Collection Center is No.1, Beichen West Road, Haidian District, Beijing.

[0006] The application further provides an application of a Paenibacillus mucilaginosus strain S24, which is mainly applied to promoting plant growth and in-situ fermentation production of bio-organic fertilizer. Test analysis shows that the strain has the abilities of phosphorus release, potassium release, nitrogen fixation and the like; and has high environmental adaptability, including a wide pH suitable range (pH 4-11) and high salt tolerance (tolerating 6% salt concentration).

[0007] The application further provides a new technology for producing bio-organic fertilizer by inoculating microorganisms with growth promoting function in a secondary fermentation stage of compost and proliferating the microorganisms in a secondary fermentation compost, and an application of the Paenibacillus mucilaginosus strain S24 in production of bio-organic fertilizer. The bio-organic fertilizer is produced by inoculating the Paenibacillus mucilaginosus strain with growth promoting function in a secondary fermentation stage of compost and proliferating the Paenibacillus mucilaginosus strain in a secondary fermentation compost. Specifically,

[0008] The Paenibacillus mucilaginosus strain S24 is inoculated in a fermentation medium (glucose 30 g / L, K2HPO4 2 g / L, MgSO4·7H2O 2 g / L, FeCl3 0.005 g / L, CaCO3 2 g / L, beef extract 5 g / L, initial pH 7.0) to perform liquid shake flask culture, and the culture condition is as follows: culture temperature 30℃, 200 rpm of a shaker until the spore rate is more than 90%, and then the culture is ended. The compost mass is weighed, the Paenibacillus mucilaginosus strain S24 liquid is taken in an amount of 1-5% (v / w) to fully mix with the compost material, so that the S24 strain number in the compost is 1×10 6 cfu / g, and water is added to adjust the water content of the compost material to about 55%. The whole fermentation process adopts an aerobic compost mode, the secondary fermentation period of the compost is set to 10 days, the compost is completely turned over once every 2 days, and finally the product is naturally dried or dried under the condition that the temperature is not more than 35℃ until the water content is less than 30%.

[0009] Beneficial effects

[0010] The Paenibacillus mucilaginosus strain S24 of the application is isolated from a secondary fermentation compost of livestock and poultry waste, can proliferate in the compost, can be effectively compatible with the soil ecology, has no ecological toxicity, is high in safety and strong in stability.

[0011] The Paenibacillus mucilaginosus strain S24 of the present application has the abilities of phosphorus and potassium release, nitrogen fixation, and the like, and has the growth promoting effect on various crops. The strain has high environmental adaptability, including wide pH growth range (pH 4-11) and high salt tolerance (tolerance to 6% salt concentration). The strain can proliferate in the secondary fermentation pile and is used for in-situ fermentation production of bio-organic fertilizer.

[0012] The present application provides a new technology for producing bio-organic fertilizer by inoculating microorganisms with growth promoting function in the secondary fermentation stage of compost, so that the microorganisms proliferate in the secondary fermentation pile of compost. The method combines the production processes of functional microorganisms and organic fertilizer, and has simple operation, low cost, and very wide application prospect in the production of bio-organic fertilizer. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The colony morphology, temperature, pH and salt range of the Paenibacillus mucilaginosus S24 of the present application;

[0014] Figure 2 Analysis of the abilities of phosphorus and potassium release, nitrogen fixation and the like of the Paenibacillus mucilaginosus S24;

[0015] Figure 3 The content of the Paenibacillus mucilaginosus S24 in the pile before and after in-situ fermentation;

[0016] Figure 4 The growth promoting effect of the bio-organic fertilizer produced by in-situ fermentation on wheat;

[0017] Figure 5 The growth promoting effect of the bio-organic fertilizer produced by in-situ fermentation on corn. DETAILED DESCRIPTION

[0018] Example 1

[0019] I. Isolation, identification and biological characteristics of the Paenibacillus mucilaginosus strain S24

[0020] The secondary fermentation sample of cow manure compost was collected, and the silicate bacteria culture medium was used to isolate bacteria.

[0021] The preparation method of the silicate bacterial culture medium is as follows: sucrose 5 g, Na2HPO4 2 g, MgSO4·7H2O 0.5 g, FeCl3 0.005 g, CaCO3 0.1 g, bauxite 0.5 g, water 1000 mL, pH 7.0-7.5, agar 15 g, high-pressure steam sterilization at 115°C for 30 min.

[0022] The colony morphology of the strain S24 is shown in Figure 1 FIG. 1, which is a rod-shaped gram-positive bacterium capable of forming spores. The strain S24 grows at a temperature of 20-50°C, has a suitable growth pH range of 4-11, and can grow under a salt concentration (mass concentration of NaCl) of 0%-6% Figure 1 . The genomic DNA of the strain S24 is extracted, and the specific DNA sequence of the genomic DNA is amplified by PCR using 16s rDNA primers, and the strain is compared using the database of the National Center for Biotechnology Information (NCBI).

[0023] The 16s rDNA forward primer 27F is 5'-AGAGTTTGATCCTGGCTCAG-3';

[0024] The 16s rDNA reverse primer 1492R is 5'-GGTTACCTTGTTACGACTT-3';

[0025] The amplified 16S rDNA sequence has a full-length of 1414 bp. The sequence is shown in SEQ ID NO. 1. The amplified 16S rDNA sequence is compared with the gene sequences of related strains on NCBI, and it is found that the homology between the strain S24 and the model strain Paenibacillus mucilaginosus VKPM B-7519 is 99.86%. Combined with its morphological characteristics, it can be determined that the strain S24 is a Paenibacillus mucilaginosus. After identification and confirmation of the species, the strain S24 is a Paenibacillus mucilaginosus. The strain S24 has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 22, 2025, and the preservation number of the strain S24 is CGMCC NO. 35709. The address of the CGMCC is No. 1, Beichen West Road, Haidian District, Beijing.

[0026] II. Evaluation of the ability of Paenibacillus mucilaginosus strain S24 to activate nutrient elements

[0027] The ability of Paenibacillus mucilaginosus strain S24 to activate nutrient elements was evaluated using different solid media (including nitrogen fixation medium, organic phosphorus medium, inorganic phosphorus medium, potassium solubilizing medium). If the microbial strain can grow on the above culture dishes or produce transparent circles, it is considered positive; if the microbial strain has no single colony growth, it is considered negative.

[0028] The medium formula is as follows,

[0029] 1) Nitrogen fixation medium (Ashby medium): mannitol 10 g, KH2PO40.2 g, NaCl 0.2 g, CaCO35 g, MgSO4·7H2O 0.2 g, CaSO4·2H2O 0.1 g, agar 15 g, distilled water 1000 mL, 115°C high pressure steam sterilization for 30 min.

[0030] 2) Inorganic phosphorus medium: glucose 10 g, ammonium sulfate 0.5 g, NaCl 0.3 g, MgSO4·7H2O 0.3 g, MnSO40.03 g, potassium sulfate 0.3 g, Fe2SO40.03 g, Ca3(PO4)25.0 g, agar 15 g, distilled water 1000 mL, 115°C high pressure steam sterilization for 30 min.

[0031] 3) Organic phosphorus medium: glucose 10 g, ammonium sulfate 0.5 g, yeast extract powder 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, MnSO40.03 g, Fe2SO40.03 g, lecithin 0.2 g, calcium carbonate 1.0 g, agar 15 g, distilled water 1000 mL, 115°C high pressure steam sterilization for 30 min.

[0032] 4) Potassium solubilizing medium (Aleksandrov medium): glucose 5 g, magnesium sulfate 0.5 g, ferric chloride 0.005 g, calcium carbonate 0.1 g, calcium phosphate 2 g, potassium feldspar 2 g, BTB 100 mg, agar 15 g, distilled water 1000 mL, 115°C high pressure steam sterilization for 30 min.

[0033] The experimental results show that the Paenibacillus mucilaginosus strain S24 can grow on the nitrogen fixation medium, indicating that it has a certain nitrogen fixation ability. Figure 2 The strain S24 can also grow on the organic phosphorus medium and the potassium dissolution medium and produce obvious hydrolysis rings, indicating that it has high phosphorus dissolution and potassium dissolution abilities. Figure 2

[0034] III. A method for producing bio-organic fertilizer by inoculating Paenibacillus mucilaginosus strain S24 for in-situ fermentation of compost

[0035] 1) The Paenibacillus mucilaginosus strain S24 is inoculated in a fermentation medium (glucose 30 g, K2HPO4 2 g, MgSO4·7H2O 2 g, FeCl3 0.005 g, CaCO3 2 g, beef extract 5 g, water 1000 mL, pH 7.0-7.5, 115°C high-pressure steam sterilization for 30 min) for liquid shake flask culture, and the culture conditions are as follows: culture temperature 30°C, 200 rpm on a shaker until the spore rate reaches more than 90%, and then the culture is ended.

[0036] 2) The mass of the compost after the first fermentation is weighed, and the Paenibacillus mucilaginosus S24 bacterial liquid is taken in an amount of 1-5% (v / w) and mixed with the compost material to make the S24 bacterial colony number in the compost reach 1×10 6 cfu / g. At the same time, water is added to adjust the moisture content of the compost material to about 55%. The entire fermentation process adopts aerobic composting, and the composting secondary fermentation period is set to 10 days, with complete turning every 2 days. Finally, the product is naturally dried or dried at a temperature not exceeding 35°C to a moisture content of less than 30%.

[0037] IV. Proliferation of Paenibacillus mucilaginosus S24 after in-situ fermentation

[0038] 1) Five-point sampling method is used to collect samples from each level of the compost before and after the second fermentation and mix them evenly. The samples are placed at 4°C for later plate counting.

[0039] 2) 10 g of the collected sample is placed in 100 ml of sterile water with a glass rod, and after standing for 20 min, it is shaken at 200 rpm for 30 min to prepare a mother liquid bacterial suspension. 100 μl of the mother liquid is taken and added to a centrifuge tube containing 900 μl of sterile water, and shaken to disperse the sample evenly, obtaining a 10 -1 ​Dilution. Then dilute with sterile water to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Dilution. Take 100 μl of the dilution and evenly spread on silicate medium plates, 3 plates for each gradient dilution, then incubate in a 35℃ incubator for 36 h, count the number of Paenibacillus mucilaginosus in the plates, and convert the number of Paenibacillus mucilaginosus colonies in the secondary fermentation heap of compost.

[0040] The preparation method of the above silicate bacterial medium is as follows: sucrose 5 g, Na2HPO4 2 g, MgSO4·7H2O 0.5 g, FeCl3 0.005 g, CaCO3 0.1 g, bauxite 0.5 g, water 1000 ml, pH 7.0-7.5, high-pressure steam sterilization at 115℃ for 30 min.

[0041] Experimental results: Paenibacillus mucilaginosus S24 can proliferate in the secondary fermentation heap of compost, and its number increases from 1×10 6 cfu / g at the initial addition to 5.3×10 7 cfu / g Figure 3 .

[0042] Five, the growth-promoting effect of in-situ fermentation produced bio-organic fertilizer on wheat and corn

[0043] 1) Evaluation of the growth-promoting effect of in-situ fermentation produced bio-organic fertilizer on wheat

[0044] Inoculate strain S24 for in-situ fermentation production of bio-organic fertilizer (S24) / un-inoculated organic fertilizer as control (CK) with vermiculite at a ratio of 1:5, mix thoroughly, and then load into flowerpots, set 5 pots for each treatment, and scatter 9 wheat seeds in each pot, and water quantitatively. After 14 days of sowing, count the germination rate, then pull out and wash the soil, and measure the root system and plant height.

[0045] Experimental results: Compared with the control group, the wheat germination rate of the in-situ fermentation produced bio-organic fertilizer group increased by 37%, the average plant height of the wheat after 2 weeks of sowing increased by 26% compared with the control group, the root system was more developed, and the root length increased by 24.6% compared with the control group. Figure 4 ). In-situ fermentation produced bio-organic fertilizer can significantly promote the germination and growth of wheat seeds.

[0046] 2) Evaluation of the growth-promoting effect of in-situ fermentation produced bio-organic fertilizer on corn

[0047] In-situ fermentation production of biological organic fertilizer (S24) / uninfected organic fertilizer as control (CK) with vermiculite according to 1:5 ratio fully mixed and then loaded into flowerpots, 5 pots were set for each treatment, 6 corns were planted in each pot, and quantitative watering was carried out. After 14 days of sowing, the germination rate was counted, then the roots and plant height were measured.

[0048] Experimental results: In-situ fermentation production of biological organic fertilizer can promote the growth of corn. Compared with the control group, the average plant height of the corn in the in-situ fermentation production of biological organic fertilizer group increased by 30.5% compared with the control group, the root system was more developed, and the root length increased by 17.1% compared with the control group. Figure 5 In-situ fermentation production of biological organic fertilizer can significantly promote the growth of corn.

Claims

1. A strain of Paenibacillus mucilaginosus S24, with accession number CGMCCNO. 35709.

2. Use of Paenibacillus jamilae S24 according to claim 1 for the preparation of a plant growth promoting microbial agent, characterized in that: The plant growth-promoting abilities include the ability to solubilize phosphorus, potassium, and / or fix nitrogen.

3. The use of Paenibacillus jamilae S24 according to claim 1 for the preparation of an acid and alkali resistant bacterial agent, characterized in that: The pH range of the acid / base is 4-11.

4. Use of Paenibacillus jamilae S24 according to claim 1 for the preparation of a halotolerant bacterial agent, characterized in that: The NaCl mass concentration of the salt is no greater than 6%.

5. The application of the gelatinous Bacillus S24 as described in claim 1 in the in-situ fermentation of compost to produce bio-organic fertilizer.

6. The use of Paenibacillus sp. S24 according to claim 5 in the production of bio-organic fertilizer by in-situ fermentation of compost, characterized in that: The bio-organic fertilizer has a growth-promoting effect on wheat.

7. The use of Paenibacillus sp. S24 according to claim 5 in the production of bio-organic fertilizer by in-situ fermentation of compost, characterized in that: The bio-organic fertilizer has a growth-promoting effect on corn.

8. The application of the gelatinous Bacillus S24 according to claim 5 in the in-situ fermentation of compost to produce bio-organic fertilizer, characterized in that: The in-situ composting fermentation involves thoroughly mixing the Bacillus spp. S24 bacterial solution with the material after the first composting fermentation at an addition rate of 1-5% (v / w), adjusting the moisture content to 55%, and then carrying out a second fermentation using an aerobic composting method.

Images