CN120966698A - Bacillus mucilaginosus S24 and application thereof in composting in-situ fermentation production of bio-organic fertilizer - Google Patents

Abstract

The invention belongs to the application field of agricultural resources and environmental biotechnology. The invention provides a bacillus mucilaginosus (Paenibacillus mucilaginosus) S24 with a preservation number of CGMCC NO.35709. The strain S24 is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms (CGMCC) at the 8 th month 22 of 2025, wherein the preservation number of the strain S24 is CGMCC No.35709, and the center address is North Star Xiyu No. 1,3 in the Chaoyang area of Beijing city. The strain S24 provided by the invention has the capabilities of phosphate dissolution, potassium dissolution, nitrogen fixation and the like, and has higher environmental adaptability, including wide pH adaptation range (pH 4-11) and very high salt tolerance (tolerance of 6% salt concentration). In addition, the strain S24 is inoculated to the organic fertilizer obtained after primary fermentation of the compost, and can be proliferated in a secondary fermentation pile body to produce the biological organic fertilizer with the growth promoting effect.

Description

Bacillus mucilaginosus S24 and application thereof in composting in-situ fermentation production of bio-organic fertilizer

Technical Field

The invention belongs to the application field of agricultural resources and environmental biotechnology.

Background

The biological organic fertilizer has the functions of improving the physical and chemical properties of soil, enhancing the fertility of soil and the like, and plays an increasingly important role in agricultural production. At present, the production of the bio-organic fertilizer is mainly carried out by adopting a mode of mixing and adding functional microbial inoculum and organic fertilizer, and the problems of multiple production links, high production cost, poor compatibility of microorganisms, organic fertilizer and soil and the like exist. When the compost product is used as a carrier to adsorb functional microorganisms to produce the bio-organic fertilizer, the used compost needs to be thoroughly decomposed, the water content is also an important factor influencing the combination of the compost and the functional microorganisms, when the water content of the compost is too high, the adsorption capacity is poor, mould is easy to grow, and the water content needs to be reduced by natural air drying (Zhang Zhigong, etc. 2010) or heat treatment (Cai Yanfei, etc. 2003) and the functional microorganisms are added. Meanwhile, the microorganisms contained in the compost themselves compete with the added functional microorganisms, so that the number of the functional microorganisms is rapidly reduced (Zhang Zhigong, etc., 2010), and thus, the compost needs to be subjected to operations such as additional high-temperature sterilization (Wu et al, 2015) or gamma-ray sterilization (Tao et al, 2020) to improve the viability of the functional microorganisms in the compost.

Disclosure of Invention

Aiming at the problems existing in the research and production of the current functional bio-organic fertilizer, the invention provides a novel technology for producing the bio-organic fertilizer by inoculating microorganisms with growth promoting function in a secondary fermentation stage of composting and proliferating the microorganisms in a secondary fermentation stack of the composting. The method combines the production process of functional microorganisms and organic fertilizers into one, and has simple operation and low cost.

The invention aims to provide a method for producing a bio-organic fertilizer through composting in-situ fermentation, a bacillus mucilaginosus (Paenibacillus mucilaginosus) S24 and application thereof in plant growth promotion and composting.

The invention screens and separates a strain S24 from the livestock and poultry waste compost secondary fermentation pile body, gram staining of the strain S24 is positive, and cells are rod-shaped, so that spores can be formed. Through identification, the strain S24 is Paenibacillus mucilaginosus (Paenibacillus mucilaginosus), the strain S24 is preserved in China general microbiological culture Collection center (CGMCC) No.35709 in 8-22-2025, the preservation number of the strain S24 is CGMCC, the center is called CGMCC, and the center address is North Star Xiyu No. 1 and 3 in the Chaoyang area of Beijing.

The invention also provides an application of the bacillus mucilaginosus (Paenibacillus mucilaginosus) strain S24, which is mainly applied to promoting plant growth and in-situ fermentation production of the bio-organic fertilizer. The test analysis shows that the strain has the capabilities of phosphate dissolution, potassium dissolution, nitrogen fixation and the like, and has higher environmental adaptability, including wide pH adaptation range (pH 4-11) and very high salt tolerance (tolerance of 6% salt concentration).

The invention also provides a novel technology for producing the bio-organic fertilizer by inoculating microorganisms with growth promoting functions in the secondary fermentation stage of the compost, and the application of the bacillus mucilaginosus (Paenibacillus mucilaginosus) strain S24 in the production of the bio-organic fertilizer. The method comprises the following steps:

The Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) strain S24 is inoculated into a fermentation medium (glucose 30 g/L,K₂HPO₄ 2g/L,MgSO₄·7H₂O 2g/L,FeCl₃ 0.005g/L,CaCO₃ 2g/L, beef extract 5g/L, initial pH 7.0) and subjected to liquid shake flask culture under the conditions that the culture temperature is 30 ℃, and the culture is finished when the spore rate reaches more than 90% by shaking table 200 and rpm. Weighing the mass of the compost, measuring and taking the Paenibacillus mucilaginosus S24 bacterial liquid according to the addition amount (v/w) of 1-5%, fully and uniformly mixing the Paenibacillus mucilaginosus S24 bacterial liquid with the compost material, so that the number of S24 bacterial colonies in the compost reaches 1X 10 ⁶ cfu/g, and simultaneously adding water to adjust the water content of the compost material to be about 55%. The whole fermentation process adopts an aerobic composting mode, the secondary fermentation period of composting is set to be 10 d, every 2 d is thoroughly turned over once, and finally, the product is naturally air-dried or air-dried under the condition of not exceeding 35 ℃ until the water content is below 30%.

Advantageous effects

The bacillus mucilaginosus (Paenibacillus mucilaginosus) strain S24 is separated from the livestock and poultry waste compost secondary fermentation pile body, can be proliferated in the pile body after being added into the compost, can be harmoniously compatible with soil ecology effectively, and has no ecological toxicity, high safety and strong stability.

The bacillus mucilaginosus (Paenibacillus mucilaginosus) strain S24 has the capabilities of phosphate dissolving, potassium dissolving, nitrogen fixing and the like, and has growth promoting effect on various crops. The strain has high environmental adaptability, including wide pH adaptive range (pH 4-11) and high salt tolerance (6% salt concentration tolerance). The strain can be proliferated in a secondary fermentation pile body and is used for producing the bio-organic fertilizer by composting in-situ fermentation.

The invention provides a novel technology for producing a biological organic fertilizer by inoculating microorganisms with growth promoting functions in a secondary fermentation stage of composting and enabling the microorganisms to proliferate in a secondary fermentation pile of the composting. The method combines the production process of the functional microorganism and the organic fertilizer into one, has simple operation and low cost, and has very wide application prospect in the production of the biological organic fertilizer.

Drawings

FIG. 1 shows colony morphology, growable temperature, pH and salinity range of Bacillus mucilaginosus (Paenibacillus mucilaginosus) S24 according to the present invention;

FIG. 2 shows the analysis of the phosphate-solubilizing, potassium-solubilizing, nitrogen-fixing and other abilities of Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) S24;

FIG. 3 shows the content of Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) S24 in the heap before and after in situ fermentation;

FIG. 4. Promoting growth of wheat with bio-organic fertilizer produced by in situ fermentation;

FIG. 5. Promoting growth of corn with bio-organic fertilizer produced by in situ fermentation.

Detailed Description

Example 1

1. Isolation, identification and biological Properties of Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) Strain S24

Collecting a cow dung compost secondary fermentation sample, and separating bacteria by adopting a silicate bacteria culture medium.

The preparation method of the silicate bacteria culture medium comprises the steps of 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 of 7.0-7.5, agar 15 g and high-pressure steam sterilization at 115 ℃ of 30 min.

Bacterial strain S24, which belongs to the gram-positive bacteria of the rod shape, can form spores as shown in FIG. 1. The strain grows at 20-50deg.C, has a suitable growth pH range of 4-11, and can grow under the condition of 0% -6% salt (NaCl mass concentration) (figure 1). Extracting genome DNA of the strain, carrying out PCR amplification on the genome DNA by using a 16s rDNA primer, and carrying out strain comparison by using a national center for biotechnology information (National Center for Biotechnology Information, abbreviated as NCBI) database.

16S rDNA forward primer 27F:5 '-AGAGTTTGATCCTGGGCTCAG-3';

16s rDNA reverse primer 1499R:5 '-GGTTACCTTGTTACGACTT-3';

The amplified 16S rDNA sequence has the full length of 1414 bp. The sequence is shown in a sequence table SEQ ID NO. 1. Comparing the amplified 16S rDNA sequence with the gene sequence of related strain at NCBI, the homology of the strain S24 with the model strain Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) VKPM B-7519 is found to be 99.86%. By combining the morphological characteristics, it can be determined that the strain S24 is a Paenibacillus mucilaginosus (Paenibacillus mucilaginosus). After identifying and confirming the species, paenibacillus mucilaginosus (Paenibacillus mucilaginosus). The strain S24 is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms (CGMCC) at the 8 th month 22 of 2025, wherein the preservation number of the strain S24 is CGMCC No.35709, and the center address is North Star Xiyu No.1, 3 in the Chaoyang area of Beijing city.

2. Evaluation of the ability of Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) Strain S24 to activate nutrient elements

Different solid media (including nitrogen fixation medium, organic phosphorus medium, inorganic phosphorus medium, potassium dissolving medium) were used to evaluate the ability of paenibacillus mucilaginosus (Paenibacillus mucilaginosus) strain S24 to activate nutrient elements. If the microbial strain is able to grow on the dish or a transparent ring is produced, the result is considered positive, and if the microbial strain does not grow as a single colony, the result is considered negative.

The formula of the culture medium is as follows,

1) Nitrogen fixation medium (Ashby medium) mannitol 10 g,KH₂PO₄ 0.2 g,NaCl 0.2 g,CaCO₃ 5 g,MgSO₄·7H₂O 0.2 g,CaSO₄·2H₂O 0.1 g, agar 15 g, distilled water 1000 ml, and steam sterilization at 115 ℃ under high pressure 30min.

2) Inorganic phosphorus culture medium, glucose 10 g, ammonium sulfate 0.5 g,NaCl 0.3 g,MgSO ₄∙7H₂O 0.3 g,MnSO₄ 0.03.03 and g, potassium sulfate 0.3 g, fe ₂SO₄ 0.03 g,Ca₃(PO₄)₂ 5.0.0 and g, agar 15 and g, distilled water 1000 mL, and high pressure steam sterilization at 115 ℃ 30 and min.

3) The organic phosphorus culture medium comprises glucose 10 g, ammonium sulfate 0.5 g, yeast extract 0.5 g,NaCl 0.3 g,KCl 0.3 g,MgSO₄∙7H₂O 0.3 g,MnSO₄ 0.03 g,Fe₂SO₄ 0.03 g, lecithin 0.2 g, calcium carbonate 1.0 g, agar 15g, distilled water 1000 mL, and steam sterilization at 115 ℃ under high pressure 30 min.

4) Potassium decomposing culture medium (Aleksandrov culture medium) comprising glucose 5g, magnesium sulfate 0.5 g, ferric chloride 0.005 g, calcium carbonate 0.1 g, calcium phosphate 2 g, potassium feldspar 2 g, BTB 100mg, agar 15g, distilled water 1000 mL, and steam sterilizing at 115deg.C under high pressure 30 min.

As a result of the experiment, paenibacillus mucilaginosus (Paenibacillus mucilaginosus) strain S24 can grow on a nitrogen fixation medium, which shows that the Paenibacillus mucilaginosus has a certain nitrogen fixation capacity (figure 2). Strain S24 can also grow on organophosphorus and potassium solubilizing media and produce distinct hydrolysis circles, indicating its higher phosphate and potassium solubilizing capacity (fig. 2).

3. Method for producing bio-organic fertilizer by composting in-situ fermentation through inoculating Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) strain S24

1) The bacillus mucilaginosus (Paenibacillus mucilaginosus) strain S24 is inoculated into a fermentation medium (glucose 30 g,K₂HPO₄ 2g,MgSO₄·7H₂O 2 g,FeCl₃ 0.005 g,CaCO₃ 2 g, beef extract 5g, water 1000 mL, pH 7.0-7.5,115 ℃ and high-pressure steam sterilization 30 min) and subjected to liquid shake flask culture under the conditions that the culture temperature is 30 ℃ and the culture is carried out by a shaking table 200 rpm until the spore rate reaches more than 90%, and the culture is ended.

2) Weighing the mass of the material after primary fermentation of the compost, measuring and taking the Paenibacillus mucilaginosus S24 bacterial liquid according to the addition amount (v/w) of 1-5%, and fully and uniformly mixing the Paenibacillus mucilaginosus S24 bacterial liquid with the compost material, so that the number of S24 bacterial colonies in the compost reaches 1X 10 ⁶ cfu/g, and simultaneously adding water to adjust the water content of the compost material to be about 55%. The whole fermentation process adopts an aerobic composting mode, the secondary fermentation period of composting is set to be 10 d, every 2d is thoroughly turned over once, and finally, the product is naturally air-dried or air-dried under the condition of not exceeding 35 ℃ until the water content is below 30%.

4. Proliferation of Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) S24 after in situ fermentation

1) Samples are collected from each layer of the pile body before and after the secondary fermentation of the compost by a five-point sampling method and are uniformly mixed, and the samples are placed at 4 ℃ to be counted by a flat plate.

2) The collected sample 10 g is placed in 100 ml sterile water with glass strains, kept stand for 20 min, and a shaking table 200 rpm is fully vibrated for 30 min to prepare mother liquor bacterial suspension. 100 μl of mother liquor was added to a centrifuge tube containing 900 μl of sterile water, and mixed by shaking to disperse the sample thoroughly to obtain 10 ^-1 dilution. Then diluted with sterile water in a gradient to obtain 10 ^-2、10^-3、10^-4、10^-5、10^-6 dilutions. And (3) uniformly coating 100 mu l of diluent on a silicate culture medium plate, coating 3 plates on each gradient diluent, then counting the number of the paenibacillus mucilaginosus in the plates after culturing in a 35 ℃ incubator for 36 h, and converting the colony number of the paenibacillus mucilaginosus in the compost secondary fermentation heap.

The preparation method of the silicate bacteria culture medium comprises sterilizing sucrose 5 g,Na₂HPO₄ 2 g,MgSO₄·7H₂O 0.5 g,FeCl₃ 0.005 g,CaCO₃ 0.1 g, bauxite 0.5g with 1000 ml water and sterilizing with high pressure steam at pH7.0-7.5,115 deg.C for 30 min.

As a result of the experiment, paenibacillus mucilaginosus (Paenibacillus mucilaginosus) S24 was able to proliferate in compost secondary fermentation piles in an amount ranging from 1X 10 ⁶ cfu/g to 5.3X 10 ⁷ cfu/g at the time of initial addition (FIG. 3).

5. Promoting growth of wheat and corn by bio-organic fertilizer produced by in-situ fermentation

1) Evaluation of growth promoting effect of bio-organic fertilizer produced by in-situ fermentation on wheat

And (3) fully and uniformly mixing the biological organic fertilizer (S24)/non-inoculated organic fertilizer produced by in-situ fermentation of the inoculated strain S24 serving as a Control (CK) and vermiculite according to a ratio of 1:5, filling the mixture into a flowerpot, repeating 5 pots for each treatment, dispersing 9 wheat seeds in each pot, and quantitatively watering. And counting the emergence rate after sowing for 14 days, then pulling out soil, cleaning the soil, and measuring the root system and the plant height.

The experimental result shows that compared with the control group, the wheat emergence rate of the bio-organic fertilizer group produced by in-situ fermentation is improved by 37%, the average plant height of wheat after sowing for 2 weeks is improved by 26% compared with the control group, the root system is more developed, and the root system length is improved by 24.6% compared with the control group. (FIG. 4). The bio-organic fertilizer produced by in-situ fermentation can obviously promote germination and growth of wheat seeds.

2) Evaluation of growth promoting effect of bio-organic fertilizer produced by in-situ fermentation on corn

And (3) fully and uniformly mixing the biological organic fertilizer (S24)/non-inoculated organic fertilizer produced by in-situ fermentation of the inoculated strain S24 serving as a Control (CK) and vermiculite according to a ratio of 1:5, filling the mixture into a flowerpot, setting 5 pots for repetition for each treatment, dispersing 6 corn seeds in each pot, and quantitatively watering. And counting the emergence rate after sowing for 14 days, then pulling out soil, cleaning the soil, and measuring the root system and the plant height.

The experimental result shows that the bio-organic fertilizer produced by in-situ fermentation can promote the growth of corn, compared with a control group, the average plant height of corn of the bio-organic fertilizer group produced by in-situ fermentation is improved by 30.5% compared with the control group, the root system is more developed, and the root system length is improved by 17.1% compared with the control group (figure 5). The bio-organic fertilizer produced by in-situ fermentation can obviously promote the growth of corn.

Claims (8)

1. A strain of Paenibacillus mucilaginosus S24, with accession number CGMCCNO. 35709. 2. The application of the gelatinous Bacillus S24 of claim 1 in the preparation of a plant growth promoter, characterized in that: the plant growth promoter includes the ability to solubilize phosphorus, potassium and/or fix nitrogen. 3. The application of the gelatinous Bacillus S24 according to claim 1 in the preparation of acid- and alkali-resistant bacterial agents, characterized in that: the pH value range is 4-11. 4. The application of the gelatinous Bacillus S24 according to claim 1 in the preparation of salt-tolerant bacterial agents, characterized in that: the NaCl mass concentration of the salt is not 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 application of Bacillus spp. S24, a gelatinous spore-forming bacterium according to claim 5, in the in-situ fermentation of compost to produce bio-organic fertilizer, characterized in that: the bio-organic fertilizer has a growth-promoting effect on wheat. 7. 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 bio-organic fertilizer has a growth-promoting effect on corn. 8. The application of Bacillus spp. S24 as described in claim 5 in the production of bio-organic fertilizer through in-situ composting fermentation, 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 in an aerobic composting manner.