Hypertonic-resistant paenibacillus mucilaginosus HX-02 and application thereof

Abstract

The invention discloses a high permeability resistant paenibacillus mucilaginosus HX-02 and an application thereof. The preservation number of the paenibacillus mucilaginosus HX-02 disclosed by the invention is CGMCC (China General Microbiological Culture Collection Center) The fertilizer has high permeability resistance in a high-concentration fertilizer environment. The paenibacillus mucilaginosus HX-02 bacterial powder can have a synergistic effect with the fertilizer. The paenibacillus mucilaginosus HX-02 comes from soil, is harmless to human, livestock and environment, and has the advantages of high efficiency, low toxicity, low residue and the like compared with other fertilizers. The compound fertilizer is used for developing the compound fertilizer and meets the requirements of green plant protection and agricultural sustainable development.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture, and in particular relates to a high osmotic jelly-like resistant bacillus HX-02 and an application thereof. Background Art

[0002] Fertilizers have made important contributions to the development of my country's agriculture, but the extensive development model also has many problems. Agricultural production is heavily dependent on the input of production factors, and there are problems such as excessive fertilizer application and low fertilization efficiency, which have caused serious agricultural non-point source pollution and ecological damage. Therefore, reducing the use of fertilizers and pesticides and improving the quality of agricultural products have become bottlenecks that need to be solved in my country's process of reducing fertilizers and pesticides.

[0003] At present, the main methods for reducing the use of chemical fertilizers and pesticides include precision fertilization, intensive large-scale agriculture, water-fertilizer integration, adjusting the fertilization structure, applying organic fertilizers, and applying biofertilizers. Among them, the application of organic fertilizers is the main measure to reduce the use of chemical fertilizers. Although organic fertilizers contain rich organic matter and a variety of nutrients, they can improve soil structure and increase soil fertility. However, compared with chemical fertilizers, organic fertilizers have lower nutrient content and require a larger amount of application. In addition, the production and processing costs of organic fertilizers are relatively high, which affects their widespread application. Therefore, exploring efficient, low-toxic, and environmentally friendly fertilizer active ingredients is of great significance to reducing the use of chemical fertilizers and pesticides and developing green and sustainable agriculture.

[0004] Paenibacillus jelly-like Paenibacillus mucilaginosus ) belongs to Firmicutes, Bacilales, Bacillaceae, Bacillus, and is a type of spore-forming Gram-negative bacteria that is widely distributed in nature. Paenibacillus jelly is a bacterium that can decompose silicate minerals, so some scholars call it silicate bacteria. Because this strain has the ability to decompose aluminum silicate protoplasmic minerals such as potassium feldspar and mica, it converts insoluble K, P, Si in the soil into soluble elements for plant use, and can also produce a variety of biologically active substances to promote plant growth, so it has been widely used in agriculture. In addition, Paenibacillus jelly is also widely used in mining, metallurgy, pharmaceuticals and other fields. Summary of the invention

[0005] The invention aims to provide a high osmotic jelly-like resistant Paenibacillus HX-02 and its application.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a strain HX-02 isolated from soil, which is classified and named as Paenibacillus jelly-like ( Paenibacillus mucilaginosus), has been deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, with deposit number CGMCC No.30949 and deposit date June 14, 2024.

[0007] In a second aspect, the present invention provides a bacterial agent containing the jelly-like Paenibacillus.

[0008] In a third aspect, the present invention provides a method for preparing jelly-like Paenibacillus powder, wherein the jelly-like Paenibacillus is fermented and cultured, and the fermentation liquid is centrifuged, concentrated and dried to prepare the powder.

[0009] The formula of the fermentation medium used is: 15 g starch, 2.0 g (NH4)2SO4, 2.0 g KH2PO4, 1.5 g yeast extract, 0.5 g FeCl3, 1000 mL water, pH 7.5.

[0010] In a fourth aspect, the present invention provides jelly-like Paenibacillus powder prepared according to the method.

[0011] In a fifth aspect, the present invention provides the use of the bacterial powder in the preparation of compound fertilizer (microbial compound fertilizer).

[0012] In the compound fertilizer, the number of viable bacteria of jelly-like Bacillus is 3.5×10 8 ~4.5×10 8 cfu / g (preferably 4.08×10 8 cfu / g).

[0013] The fertilizer is a mixture of (NH4)2SO4, KH2PO4 and K2SO4, wherein the mass ratio of (NH4)2SO4, KH2PO4 and K2SO4 is 1:1:1.

[0014] In a sixth aspect, the present invention provides application of the compound fertilizer in plant (crop) cultivation.

[0015] Compared with the prior art, the present invention has at least the following advantages: (I) The jelly-like Paenibacillus HX-02 of the present invention has high osmotic resistance in a high-concentration fertilizer environment. Specifically, the jelly-like Paenibacillus HX-02 survives in a high-concentration fertilizer environment within a certain range, and the effect is significant. In a 30% (NH4)2SO4+KH2PO4+ K2SO4 fertilizer mixture, the effective viable count of the jelly-like Paenibacillus HX-02 within 120 days is 3.17×10 8 cfu / g, showing significant resistance to high osmotic pressure.

[0016] (ii) Jelly-like Bacillus HX-02 powder can work synergistically with fertilizers.

[0017] (III) The jelly-like Bacillus HX-02 originates from the soil and is harmless to humans, animals and the environment. Compared with other fertilizers, it has the advantages of high efficiency, low toxicity and low residue. Using it to develop compound fertilizers meets the needs of green plant protection and sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure is a colony morphology diagram of the strain Bacillus jelly-like HX-02 of the present invention.

[0019] Figure 2 The microscopic morphology of the strain Paenibacillus jelly HX-02 of the present invention is shown in FIG.

[0020] Figure 3 The figure shows the change of effective viable bacteria count of Paenibacillus gelatinosa in mixtures of different fertilizer concentrations in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0021] The present invention aims to provide a jelly-like Paenibacillus HX-02 strain with high osmotic resistance and application thereof in a high-concentration fertilizer environment.

[0022] The present invention provides a strain HX-02 isolated from soil, which is classified and named as Paenibacillus jelly-like ( Paenibacillus mucilaginosus ), has been deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, with deposit number CGMCC No.30949 and deposit date June 14, 2024.

[0023] The present invention provides bacterial powder containing the jelly-like Paenibacillus HX-02.

[0024] The invention also provides a compound fertilizer prepared from the powder of the jelly-like Paenibacillus HX-02.

[0025] The present invention also provides a compound fertilizer prepared by using the powder of the jelly-like Paenibacillus HX-02, that is, the jelly-like Paenibacillus HX-02 can be used as a compound fertilizer by using the survival of the jelly-like Paenibacillus HX-02 in a high-concentration fertilizer environment.

[0026] The active ingredient of the compound fertilizer is the jelly-like Bacillus HX-02. In the mixture of different fertilizer concentrations, the effective live bacteria count of the jelly-like Bacillus HX-02 is maintained at 3.0×10 within 120 days. 8 cfu / g or above.

[0027] The jelly-like Paenibacillus HX-02 of the present invention has high osmotic resistance in a high-concentration fertilizer environment.

[0028] The jelly-like Paenibacillus HX-02 is prepared into bacterial powder, which can act synergistically with fertilizers.

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0030] Example 1 Isolation, purification and identification of jelly-like Paenibacillus HX-02

[0031] The present invention separates and purifies strain HX-02 from soil. The morphological characteristics of strain HX-02 are as follows: Gram staining is negative, the cells are rod-shaped, the spores are central, the spores are oval, and the spore capsules are not obviously swollen. On silicate bacterial culture medium, the colony morphology after 48 hours is relatively regular and round, slightly raised, with neat edges, transparent protrusions, smooth surface, and sticky. Figure 1 , microscopic morphology Figure 2 .

[0032] The 16S rDNA was sequenced (SEQ ID NO: 1) and the bacteria were identified as Paenibacillus jelly-like ( Paenibacillus mucilaginosus ).

[0033] Example 2 Preparation of jelly-like Paenibacillus HX-02 fermentation broth

[0034] The jelly-like fermentation broth of Paenibacillus sp. HX-02 was prepared by secondary submerged fermentation.

[0035] 1. Seed tank cultivation

[0036] Culture medium: starch 5.0 g, (NH4)2SO4 0.5 g, KH2PO4 2.0 g, yeast extract 1.5 g, FeCl3 0.5 g, water 1000 mL, pH 7.5.

[0037] Culture conditions: tank pressure 0.5MPa, culture temperature 28℃, ventilation ratio 1:0.3.

[0038] 2. Fermentation tank culture

[0039] Culture medium: starch 15 g, (NH4)2SO4 2.0 g, KH2PO4 2.0 g, yeast extract 1.5 g, FeCl3 0.5 g, water 1000 mL, pH 7.5; Culture conditions: tank pressure 0.5MPa, culture temperature 30℃, ventilation ratio 1:0.5-1.0.

[0040] Example 3 Preparation of jelly-like Paenibacillus HX-02 bacterial powder

[0041] The present invention prepares bacterial powder with high bacterial content by subjecting the fermentation broth of the jelly-like Paenibacillus sp. HX-02 strain prepared in Example 1 to a post-treatment process combining centrifugal concentration and rotary flash drying.

[0042] The jelly-like Paenibacillus HX-02 used in this example was provided by the Environmental Protection and Ecology Laboratory of the Biotechnology Center of Hebei University.

[0043] 1. Effect of different feed rates on the yield of centrifugal concentrated spores and determination of centrifugal parameters

[0044] Under the condition of centrifuge speed of 7100 r / min, the feed rate was set at four parameters of 0.3, 0.5, 0.8, and 1.0 T / h, and the effect of different feed rates on the yield was studied to determine the optimal separation parameters.

[0045] At a constant speed, changing the feed rate (flow rate) has a greater impact on the spore yield. The results are shown in Table 1.

[0046] Table 1 Effect of different feed rates on spore yield

[0047] It can be seen from Table 1 that the material feed rate has a great influence on the spore yield. A large flow rate will cause the feed liquid to stay in the centrifuge for too short a time and escape from the centrifuge with the supernatant, while a small flow rate will reduce the production efficiency. Considering the processing volume and centrifugal yield comprehensively, the flow rate is determined to be 0.5 T / h.

[0048] 2. Effects of different auxiliary materials on bacterial powder biomass and shelf life

[0049] Calcium carbonate, diatomaceous earth, peat and bran were used as adsorption raw materials. After the bacterial sludge was adsorbed with the above substances, it was placed in a constant temperature drying oven and dried at 65°C. After the moisture content was lower than 5%, it was crushed to determine the biomass of the bacterial powder.

[0050] The bacterial powder was stored at room temperature for 6 months, and its biomass was measured and the survival rate was calculated.

[0051] Survival rate = (biomass after preservation / initial biomass) × 100%

[0052] The effects of different excipients on the biomass, moisture content and shelf life of bacterial powder are shown in Table 2.

[0053] Table 2 Effects of different excipients on the survival rate of bacterial powder

[0054] As can be seen from Table 2, the biomass of the bacterial powder using bran powder as the adsorbent was the highest, reaching 297.4×10 8 cfu / g, after 1 year of storage, it was 267.7×10 8 cfu / g; using peat as the adsorbent, the biomass of bacterial powder reached 149.1×10 8 cfu / g, after 1 year of storage, it was 131.2×10 8 cfu / g, but the product color is dark brown; the biomass of bacterial powder using calcium carbonate as adsorbent is 29.5×10 8 cfu / g, but the preservation rate was the highest, with the loss amount being only 8%.

[0055] 3. Effect of flash drying material feed amount and drying temperature on the moisture content of bacterial powder

[0056] In this experimental design, the temperature was set at 125℃, 130℃ and 145℃; the feed rate was set at 50 kg / h, 75 kg / h and 110 kg / h. According to L9(3) 4 Orthogonal array test.

[0057] By studying the effects of flash dryer inlet temperature, feed rate, auxiliary material type and concentration on yield, spore survival rate, drying effect and yield, the adsorbent type and drying parameters were determined. The effects of material feed rate and drying temperature on the moisture content of bacterial powder are shown in Table 3.

[0058] Table 3 Orthogonal test results of feed amount and drying temperature

[0059] Note: K: horizontal; K i , i=1,2,3, respectively representing the average values ​​of the three levels of drying temperature and feed amount; R: range, representing the difference between the maximum average value and the minimum average value of the three levels.

[0060] From Table 3, we can see that the order of influence of each factor on the moisture content of bacterial powder is: A (drying temperature) > B (feed rate); since the lower the moisture content, the better, the trend between the three levels of each factor is A2 > A3 > A1, B2 > B1 > B3. Intuitive analysis shows that the optimal drying temperature is 130 °C and the feed rate is 75 kg / h.

[0061] 4. Experimental results

[0062] The jelly-like high-content Bacillus spp. powder was developed by combining centrifugal concentration and rotary flash drying. The optimal inlet temperature was 130°C, the optimal feed rate was 75 kg / h, and the effective viable count of the powder reached 297.4×10 8 cfu / g.

[0063] Example 4 Study on the survival of Paenibacillus jelly HX-02 in different fertilizer concentration environments

[0064] 1. Preparation of jelly-like mixtures of Paenibacillus and different fertilizers

[0065] Equal amounts of (NH4)2SO4, KH2PO4 and K2SO4 were mixed evenly, and then a certain amount of peat and protective agent were added to make 500g of a mixture of different (NH4)2SO4+KH2PO4+ K2SO4 (the mass ratio of (NH4)2SO4, KH2PO4 and K2SO4 was 1:1:1) concentrations. Finally, 22.5g of jelly-like Bacillus powder was added to develop a compound microbial fertilizer. This experiment had 5 treatments, namely T1, T2, T3, T4 and T5, each repeated 3 times and stored at room temperature. The concentrations of T1, T2, T3, T4 and T5 are as follows.

[0066] T1: [(NH4)2SO4+KH2PO4+ K2SO4]=0

[0067] T2: [(NH4)2SO4+KH2PO4+ K2SO4]=8%

[0068] T3: [(NH4)2SO4+KH2PO4+ K2SO4]=15%

[0069] T4: [(NH4)2SO4+KH2PO4+ K2SO4]=25%

[0070] T5: [(NH4)2SO4+KH2PO4+ K2SO4]=30%

[0071] 2. Determination of the number of effective viable bacteria of jelly-like Bacillus

[0072] Take out the sample, mix it thoroughly, weigh 10.0g of the sample accurately with a sterile spoon, add it to 100.0mL of sterile water, let it stand for 20min, then place it on a shaker, shake it at 200r / min for 30min to make a bacterial suspension. Use a 5mL sterile pipette to add it to 45mL of sterile water, and continue to dilute it in a gradient of 1:10 to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10-6 , 10 -7 The bacterial suspension was diluted to 10 -6 and 10 -7 Dilution, use a 0.5 mL sterile pipette to take 0.1 mL of bacterial suspension, and evenly spread it on the test medium plate of jelly-like Bacillus. Each dilution is repeated 3 times, and sterile water is used as a blank control. Incubate at 32℃ for 48h, count the number of colonies on the plate, and calculate the effective bacterial content of jelly-like Bacillus based on the average value.

[0073] 3. Experimental results

[0074] The mixtures containing jelly-like Paenibacillus with different fertilizer ratios prepared by the above method were stored at room temperature, and samples were taken at 0, 22d, 56d, 78d and 120d to determine the effective bacterial content. The storage effects of jelly-like Paenibacillus in mixtures with different fertilizer concentrations are shown in Tables 4 and Figure 1 .

[0075] Table 4 Preservation effect of jelly-like Paenibacillus in mixtures of different fertilizer concentrations

[0076] From Table 4 and Figure 3 It can be seen that the number of effective live bacteria of Bacillus gelatinus in the mixture containing 8%, 15%, 25% and 30% (NH4)2SO4+KH2PO4+K2SO4 showed a downward trend with the extension of time, but the decrease was small within 120 days. From 120 days to 170 days, except for the addition of (NH4)2SO4+KH2PO4+ K2SO4, it showed a linear decline, which determined that the shelf life of the developed compound microbial fertilizer should be 120 days.

[0077] The effects of different fertilizer concentration factors and time factors on the effective viable count of Bacillus jelly were further investigated by variance analysis, and the results are shown in Table 5.

[0078] Table 5 Variance analysis of the effects of fertilizer concentration and time factors on the number of effective live bacteria

[0079] From the two-way ANOVA in Table 5, we can see that p 时间 =0.000<0.05, it can be seen that the effect of storage time on the effective viable count has reached a significant difference level; p 浓度 =0.237>0.05, indicating that the effects of different concentrations on the number of effective live bacteria did not reach significant differences, further confirming the high osmotic resistance of the screened jelly-like Bacillus strains in fertilizers.

[0080] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Jelly-like Bacillus ( Paenibacillus mucilaginosus )HX-02, accession number CGMCCNo.30949.

2. A bacterial agent containing the jelly-like bacillus according to claim 1.

3. A method for preparing jelly-like Paenibacillus powder, characterized in that: Fermenting and culturing the jelly-like Paenibacillus according to claim 1, and concentrating the fermentation liquid by centrifugation and drying to prepare bacterial powder; The formula of the fermentation medium used is: 15 g starch, 2.0 g (NH4)2SO4, 2.0 g KH2PO4, 1.5 g yeast extract, 0.5 g FeCl3, 1000 mL water, pH 7.

5.

4. The jelly-like Paenibacillus powder prepared according to the method of claim 3.

5. Use of the bacterial powder according to claim 4 in preparing compound fertilizer.

6. A compound fertilizer, characterized in that: The invention comprises fertilizer and the bacterial powder as claimed in claim 4.

7. The compound fertilizer according to claim 6, characterized in that In the compound fertilizer, the number of viable bacteria of jelly-like Bacillus is 3.5×10 8 ~4.5×10 8 cfu / g.

8. The compound fertilizer according to claim 6 or 7, characterized in that: The fertilizer is a mixture of (NH4)2SO4, KH2PO4 and K2SO4, wherein the mass ratio of (NH4)2SO4, KH2PO4 and K2SO4 is 1:1:

1.

9. Use of the compound fertilizer according to any one of claims 6 to 9 in plant cultivation.

10. Use of the compound fertilizer according to any one of claims 6 to 9 in crop planting.

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