Paenibacillus splendidus CKON-1 and application thereof in lignocellulose degradation

Abstract

The invention belongs to the technical field of microbial resource development and straw biological decomposition. The invention discloses a paenibacillus splendidus strain CKON-1, and the preservation number of the paenibacillus splendidus strain CKON-1 is CGMCC (China General Microbiological Culture Collection Center) No. 29377. The strain CKON-1 has been preserved in the China General Microbiological Culture Collection Center on December 25, 2023, the center is called CGMCC for short, the address of the center is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number of the strain is CGMCC No.29377. The strain disclosed by the invention has very high lignocellulose degradation activity, and the decomposition rate of straws can be remarkably improved; the strain is separated from rice field soil, can grow in the pH value range of 5-9 and the temperature range of 15-37 DEG C, can tolerate the salt concentration of 10%, and is a bacterium with wide pH value and temperature application range and salt tolerance.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial resource development and straw biological decomposition. Background Art

[0002] Crop straw is the main by-product in the process of agricultural production. In China, about 700 million tons of various crop straws are produced annually (Song Dali et al., 2018). At present, the comprehensive utilization technology of straw is relatively backward, and most of the straws are still disposed of by burning and piling up, causing serious environmental pollution and waste of resources. After reasonable treatment and returning to the field, it will be beneficial to increase soil organic matter and nutrients, improve soil structure, thereby promoting plant growth and development, and improving crop yield and quality (Ye Wenpei et al., 2008). Lignocellulose is the main component of plant straws and is difficult to degrade in the natural state (Wolfenden et al., 2001). There are various microorganisms in the soil that can degrade lignocellulose. In order to effectively decompose cellulose, hemicellulose and lignin in straws, an appropriate amount of microbial inoculants that can effectively decompose the above components is often applied, which can accelerate the decomposition of rice straws.

[0003] The prerequisite for the effective decomposition of straws by microbial inoculants is that these inoculants can survive in the field. However, the currently reported microbial inoculants lack the evaluation of the suitable growth range of the strains therein, which is not conducive to the application in the field. Therefore, isolating strains with high lignocellulase activity and strong adaptability from in-situ fields, and constructing straw-decomposing microbial inoculants therefrom will have better adaptability in the field, promote the effective decomposition of straws in the field, and meet the production requirements. Summary of the Invention

[0004] In view of this, the first object of the present invention is to provide a Paenibacillus lautus CKON-1 with a preservation number of CGMCC No. 29377.

[0005] Meanwhile, the present invention provides the application of Paenibacillus lautus CKON-1 in the preparation of lignocellulose-degrading inoculants.

[0006] In addition, the present invention also provides the application of Paenibacillus lautus CKON-1 in the preparation of straw-decomposing inoculants.

[0007] In a specific embodiment of the present invention, the straw is rice straw.

[0008] In a specific embodiment of the present invention, the pH value of the decomposition is 5-9.

[0009] In a specific embodiment of the present invention, the temperature of the decomposition is 15-37 °C.

[0010] In a specific embodiment of the present invention, the mass concentration of the decomposed NaCl is not higher than 10%.

[0011] The Paenibacillus lautus strain CKON-1 provided by the present invention has high lignocellulose degradation activity and can significantly improve the decomposition rate of straw. This strain was isolated from paddy soil, can grow within the range of pH 5-9 and 15-37 °C, and can tolerate a salt concentration of 10%. It is a bacterium with a wide pH and temperature adaptation range and salt tolerance. Description of the Drawings

[0012] Figure 1 It is a growth morphology diagram of Paenibacillus lautus CKON-1 on LB medium.

[0013] Figure 2 It is a diagram of the growth of Paenibacillus lautus CKON-1 at different temperatures.

[0014] Figure 3 It is a diagram for measuring the enzyme activity of the lignocellulose-degrading enzyme of Paenibacillus lautus CKON-1.

[0015] Among them, the EI value of the cellulase hydrolysis zone plate: 4.6.

[0016] Figure 4 It is a diagram for measuring the enzyme activity of the lignocellulose-degrading enzyme of Paenibacillus lautus CKON-1.

[0017] Among them, the EI value of the xylanase hydrolysis zone plate: 1.2.

[0018] Figure 5 It is a diagram for measuring the enzyme activity of the lignocellulose-degrading enzyme of Paenibacillus lautus CKON-1.

[0019] Among them, the EI value of the peroxidase hydrolysis zone plate: 3.27.

[0020] Figure 6 It is a diagram for measuring the enzyme activity of the lignocellulose-degrading enzyme of Paenibacillus lautus CKON-1.

[0021] Among them, the EI value of the laccase hydrolysis zone plate: 3.38.

[0022] Figure 7 It is a diagram of the effect of Paenibacillus lautus CKON-1 on promoting the decomposition of straw. Specific Embodiments

[0023] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified.

[0024] In the following implementation regulations, the following culture media are used:

[0025] Gause's No. 1 medium: 20 g of soluble starch, 1 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, 0.5 g of NaCl, 20 g of agar, made up to 1000 mL with water, pH 7.2 - 7.4.

[0026] Beef extract peptone medium: 5 g of beef extract, 10 g of peptone, 5 g of NaCl, 20 g of agar, made up to 1000 mL with water, pH 7.4 - 7.6.

[0027] PDA medium: Potato dextrose agar medium, 200 g of potato, boiled for 30 minutes and then filtered to obtain the filtrate, 20 g of glucose, 15 - 20 g of agar, made up to 1000 ml with water, natural pH.

[0028] LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, made up to 1000 mL with water, natural pH.

[0029] Example 1

[0030] Isolation, identification and biological characteristics of the strain

[0031] I. Isolation and purification of the strain

[0032] Isolated from a paddy field in Harbin, Heilongjiang in June 2021. The process is as follows: 1 g of the sample was added to a 300 ml Erlenmeyer flask containing sterilized small glass beads and 99 ml of sterile normal saline, and cultured on a shaker at 28°C for 1 h. Take 1 ml of the suspension and dilute it to 10 -4 , take the original solution, 10 -2 dilution and 10 -4 dilution were respectively spread on the beef extract peptone medium, and single colonies were picked on the 5th day, 7th day and 10th day respectively, and then purified and isolated by the streak plate method on the LB medium, named strain CKON - 1.

[0033] The obtained purified strain was stored at -80°C using 20% glycerol.

[0034] II. Identification of the strain

[0035] 1. Morphological characteristics

[0036] Morphological characteristics of strain CKON-1 isolated from a paddy field in Harbin, Heilongjiang. After inoculating and culturing on a medium using the streak plate method, morphological observations were made. The colony characteristics of strain CKON-1 on various media are shown in Table 1. The average culture time was 3 - 4 days. On LB medium, the colonies were white( Figure 1 ).

[0037] Table 1. Colony characteristics of strain CKON-1

[0038]

[0039] 2. Molecular identification

[0040] For PCR amplification of 16S rDNA, universal primers were used: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3. The amplified fragment size was approximately 1473 bp, and the annealing temperature was 55°C. The PCR reaction program was: 95°C for 5 min; 95°C for 30 s, 56°C for 90 s, 72°C for 60 s (33 cycles); 72°C for 10 min; 4°C.

[0041] After purification and sequencing of the amplified sequence, homology comparison was performed using the Blastn program of NCBI. Based on the sequence alignment results of the 16S rDNA fragment and combined with the morphological characteristics of the strain, strain CKON-1 was identified as Paenibacillus lautus. Among them, the 16S rDNA sequence is shown as SEQ ID NO.1 in the sequence listing.

[0042] III. Biological characteristics of the strain

[0043] The growth temperature, pH, and salt tolerance concentration range of Paenibacillus lautus CKON-1 were determined. The specific results are shown in Table 2.

[0044] The growth temperature range of Paenibacillus lautus strain CKON-1 was 15 - 37°C, the growth pH range was pH 5 - 9, and it could tolerate a mass concentration of 10% NaCl. This indicates that Paenibacillus lautus strain CKON-1 is a bacterium with a wide range of applicable pH values and temperatures and salt tolerance.

[0045] Table 2. Growth temperature, pH value, and salt tolerance concentration range of Paenibacillus lautus strain CKON-1

[0046] Growth temperature Tolerance Growth pH Tolerance Tolerant salt concentration Tolerance 4℃ - pH 4 - 1% NaCl + 15℃ +++ pH 5 + 2% NaCl + 25℃ +++ pH 6 + 5% NaCl + 37℃ +++ pH 7 + 7% NaCl + 45℃ - pH 8 + 10% NaCl + 55℃ - pH 9 + 12% NaCl - - pH 10 - 15% NaCl -

[0047] Note: + indicates tolerance, - indicates intolerance

[0048] IV. Preservation of Strains

[0049] Paenibacillus lautus CKON-1, isolated from a paddy field in Harbin, Heilongjiang, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 25, 2023. The center is abbreviated as CGMCC, and its address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number of this strain is CGMCC No. 29377.

[0050] Example 2

[0051] Activity Test of Cellulase from Paenibacillus lautus CKON-1 Isolated from a Paddy Field in Harbin, Heilongjiang

[0052] The cellulase activity of Paenibacillus lautus CKON-1 was determined by the plate dyeing / discoloration method:

[0053] (1) Cultivation of Paenibacillus lautus: Paenibacillus lautus CKON-1 was inoculated on an LB plate by the streaking method and cultured at 28°C for 3 days for standby.

[0054] (2) Inoculation of various enzyme activity test plates: Take the well-cultured CKON-1 colonies of the strain and inoculate them in the middle of the test plate, and then culture at 28°C for 2 days. Then measure the colony diameter and the diameter of the discoloration circle.

[0055] 1) Endoglucanase activity:

[0056] Carboxymethyl cellulose sodium CMC-Na medium (g / L): CMC-Na 15g, K2HPO4 1g, KH2PO4 1g, MgSO4 0.2g, NaCl 0.1g, FeCl3 0.01g, NaNO3 2.5g, CaCl2 0.1g, yeast powder 5g, agar powder 20g, volume made up to 1000 mL, pH 7.0 - 7.2. After inoculation and culture for 2 days, Congo red staining was carried out.

[0057] Congo red staining: On the medium with colonies grown, cover it with a 1 mg / mL Congo red solution. After staining for 10 - 15 min, pour off the Congo red solution, add a 1 mol / L NaCl solution, pour off the NaCl solution after 15 min, and then observe the transparent circle.

[0058] 2) Xylanase activity:

[0059] Xylan medium (g / L): xylan 5 g, K2HPO4 1.31 g, KCl 0.5 g, MgSO4·7H2O 0.5 g, NaNO3 3 g, FeSO4·7H2O 0.01 g, tryptone 10 g, yeast extract powder 5 g, agar 20 g, made up to 1000 mL. After inoculation and cultivation for 2 days, Congo red staining was carried out.

[0060] 3) Laccase activity:

[0061] Guaiacol medium: Add guaiacol with a final concentration of 0.04% to the PDA medium. Observe whether a red-brown color-changing circle appears after inoculation and cultivation.

[0062] 4) Activities of lignin peroxidase and manganese peroxidase:

[0063] Aniline blue medium (g / L): Add aniline blue with a final concentration of 0.01% to the BM medium (yeast extract 10 g, glucose 20 g, agar 20 g, made up to 1000 mL). Observe whether a red-brown color-changing circle appears after inoculation and cultivation.

[0064] (3) Definition and determination of enzyme activity index EI:

[0065] Enzyme activity index EI value = D / d (D: diameter of hydrolysis circle / fading circle / color-changing circle; d: diameter of colony)

[0066] (4) Results: After 2 days of cultivation of Paenibacillus lautus CKON-1, it had high CMC enzyme activity (EI = 4.6), laccase enzyme activity (EI = 3.38), peroxidase enzyme activity (EI = 3.27) and certain xylanase activity (EI = 1.2), indicating that Paenibacillus lautus CKON-1 has high degradation ability for cellulose and lignin.

[0067] Example 3

[0068] Microbial inoculant using Paenibacillus lautus CKON-1 as the material

[0069] (1) Preparation of liquid seeds

[0070] Pick the colonies of Paenibacillus lautus CKON-1 that have grown on the LB plate for 5 - 7 days, inoculate them into the LB liquid medium, and culture them at 28 - 30 °C with shaking at 180 rpm for 2 - 3 days for use as the seed liquid.

[0071] (2) Preparation of liquid microbial inoculant

[0072] The liquid seeds are inoculated into the liquid fermentation tank at a mass ratio of 5%, and fermented at 28 - 30 °C for 4 - 6 days. The obtained bacterial fermentation broth or the fermentation broth of the filtered bacteria can be used as a liquid microbial inoculant.

[0073] Example 4

[0074] Application of a microbial inoculant using Paenibacillus lautus CKON-1 in the liquid decomposition of rice straw

[0075] (1) Application of the microbial inoculant

[0076] After the rice straw is air-dried, it is cut into straw segments about 2 cm long. In the control group, no inoculant is applied. 2 g of straw segments are added to a triangular flask containing 50 ml of Hutchinson's inorganic salt solution. In the treatment group, the microbial inoculant CKON-1 is added and mixed evenly. The application amount of the inoculant CKON-1 is 2.5 ml / bottle (OD 600 = 1).

[0077] Hutchinson's inorganic salt culture solution: KH2PO4 1.0 g, NaCl 0.1 g, MgSO4·7H2O 0.3 g, NaNO3 2.5 g, CaCl2 0.1 g, FeCl3 0.01 g, distilled water 1000 mL.

[0078] (2) Determination of the straw decomposition rate

[0079] The decomposition rate of the straw is determined by the weight loss method. The dry weight of the straw is measured before bottling, which is the initial straw dry weight. After a certain period of time, the un-decomposed straw in the triangular flask is picked out, washed, air-dried naturally and weighed, which is the dry weight of the un-decomposed straw. Then the decomposition rate is calculated. Decomposition rate = (Initial straw dry weight - Dry weight of un-decomposed straw) / Initial straw dry weight × 100%.

[0080] (3) Results

[0081] The Paenibacillus lautus CKON-1 provided by the present invention shows in the field decomposition promotion test that the strain CKON-1 can significantly improve the decomposition rate of rice straw in liquid. The straw decomposition rate after applying the inoculant for 1 month is increased by 12.5% compared with the control (Table 3, Figure 4 ).

[0082] Table 3. Determination and statistics of straw decomposition rate

[0083]

[0084]

[0085] The Paenibacillus lautus CKON-1 provided by the present invention has high lignocellulose degradation activity and can significantly improve the decomposition rate of straw. This strain was isolated from paddy field soil, can grow within the range of pH 5-9 and temperature 15-37°C, and can tolerate a salt concentration of 10%. It is a bacterium with a wide pH and temperature adaptation range and salt tolerance.

Claims

1. Paenibacillus lautus CKON-1, with the preservation number of CGMCC No. 29377.

2. Microbial inoculant, characterized in that, Containing Paenibacillus lautus CKON-1 described in claim 1.

3. An enzyme protein, characterized in that, Derived from Paenibacillus lautus CKON-1 described in claim 1.

4. Use of Paenibacillus lautus CKON-1 described in any one of claims 1 to 3 in the preparation of a lignocellulose-degrading microbial agent.

5. Use of Paenibacillus lautus CKON-1 described in any one of claims 1 to 3 in the preparation of a straw-decomposing microbial agent.

6. The application according to claim 5, characterized in that, The straw is rice straw.

7. The application according to claim 5, characterized in that, The pH value of the decomposition is 5 - 9.

8. The application according to claim 5, wherein The temperature of the decomposition is 15 - 37 °C.

9. The application according to claim 5, characterized in that, The mass concentration of NaCl in the decomposition is not higher than 10%.

Images