Bacillus splendens ckon-1 and its use in lignocellulose degradation

Abstract

The present application belongs to the technical field of microbial resource development and straw biodegradation, and discloses a Paenibacillus lautus strain CKON-1 with a preservation number of CGMCC No. 29377. The strain CKON-1 has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 25, 2023, and the address of the CGMCC is No. 1, Yihuangyuan 3rd, Beichen West Road, Chaoyang District, Beijing. The preservation number of the strain is CGMCC No. 29377. The strain has high lignocellulose degradation activity and can significantly improve the decomposition rate of straw. The strain is isolated from paddy soil and can grow in a pH value range of 5-9 and a temperature range of 15-37 DEG C, and is resistant to a salt concentration of 10%. The strain is a bacterium with a wide pH value and temperature adaptation range and salt resistance.

Description

Technical Field

[0001] This invention belongs to the field of microbial resource development and straw biodecomposition technology. Background Technology

[0002] Crop straw is a major byproduct of agricultural production, with my country producing approximately 700 million tons of various types of crop straw annually (Song Dali et al., 2018). However, current straw utilization technologies are relatively underdeveloped, with most straw still disposed of through burning or piling, causing serious environmental pollution and resource waste. Properly treating and returning straw to the field will help increase soil organic matter and nutrients, improve soil structure, thereby promoting plant growth and development, and increasing crop yield and quality (Ye Wenpei et al., 2008). Lignocellulose is a major component of plant straw and is difficult to degrade naturally (Wolfenden et al., 2001). Soil contains various microorganisms that degrade lignocellulose. To effectively decompose cellulose, hemicellulose, and lignin in straw, appropriate amounts of microbial agents that can effectively decompose these components are often applied, which can accelerate the decomposition of rice straw.

[0003] The effective decomposition of straw by microbial inoculants depends on their ability to survive in the field. However, current reports on microbial inoculants lack assessments of the suitable habitat ranges of their strains, hindering their application in the field. Therefore, isolating strains with high-efficiency lignocellulase activity and strong adaptability from the field in situ, and constructing straw-decomposing microbial inoculants based on these strains, will result in better adaptability in the field, promoting the effective decomposition of straw and meeting production needs. Summary of the Invention

[0004] In view of this, the first objective of the present invention is to provide a strain of Bacillus Brilliantus (B. Brilliantus) Paenibacillus lautus ) CKON-1, its accession number is CGMCC No.29377.

[0005] Meanwhile, this invention provides the application of Bacillus Brilliantus CKON-1 in the preparation of lignocellulose degrading agents.

[0006] In addition, the present invention also provides the application of Bacillus Brilliantus CKON-1 in the preparation of straw decomposing agents.

[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 decomposition temperature 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 Bacillus Brilliantus strain CKON-1 provided by this invention has high lignocellulose degradation activity and can significantly improve the decomposition rate of straw. This strain was isolated from paddy field soil and can grow in the pH range of 5-9 and 15-37℃. It can tolerate a salt concentration of 10%, and is a bacterium with a wide pH and temperature adaptability range and salt tolerance. Attached Figure Description

[0012] Figure 1 This is a morphological diagram of the growth of Bacillus Brilliantus CKON-1 on LB medium.

[0013] Figure 2 This is a graph showing the growth of Bacillus Brilliantus CKON-1 at different temperatures.

[0014] Figure 3 This is a graph showing the activity of lignocellulose-degrading enzymes in Bacillus Brilliantus CKON-1. 。

[0015] The cellulase hydrolysis plate EI value was 4.6.

[0016] Figure 4 This is a graph showing the activity of lignocellulose-degrading enzymes in Bacillus Brilliantus CKON-1. 。

[0017] The EI value of the xylanase hydrolysis plate was 1.2.

[0018] Figure 5 This is a graph showing the activity of lignocellulose-degrading enzymes in Bacillus Brilliantus CKON-1. 。

[0019] The EI value of the peroxidase hydrolysis plate was 3.27.

[0020] Figure 6 This is a graph showing the activity of lignocellulose-degrading enzymes in Bacillus Brilliantus CKON-1. 。

[0021] The EI value of the laccase hydrolysis plate was 3.38.

[0022] Figure 7 This image shows the effect of Bacillus Brilliantus CKON-1 on promoting the decomposition of straw. 。 Detailed Implementation

[0023] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0024] The following culture media are used in the following implementing regulations: Gao's No. 1 culture medium: 20 g soluble starch, 1 g KNO3, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g NaCl, 0.01 g FeSO4·7H2O, 0.5 g NaCl, 20 g agar, water to a final volume of 1000 mL, pH 7.2-7.4.

[0025] Beef extract peptone medium: 5 g beef extract, 10 g peptone, 5 g NaCl, 20 g agar, water to a final volume of 1000 mL, pH 7.4-7.6.

[0026] PDA medium: Potato glucose agar medium. Boil 200 g of potatoes for 30 minutes, filter and collect the filtrate. Add 20 g of glucose, 15-20 g of agar, and water to a final volume of 1000 ml. Set to natural pH.

[0027] LB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, water to a final volume of 1000 mL, natural pH.

[0028] Example 1 Isolation, identification and biological characteristics of strains I. Isolation and Purification of Strains

[0029] The sample was isolated from a rice paddy in Harbin, Heilongjiang Province in June 2021. The procedure was as follows: 1 g of sample was added to a sterile 300 ml Erlenmeyer flask containing small glass beads and 99 ml of sterile physiological saline, and incubated on a shaker at 28 ℃ for 1 h. 1 ml of the suspension was then serially diluted to 10⁻⁶. -4 Take the original solution, 10 -2 Diluent and 10 -4 The diluted solution was spread onto beef extract peptone medium, and single colonies were picked on days 5, 7 and 10, respectively. The colonies were then purified and isolated on LB medium using the streak plate method and named strain CKON-1.

[0030] The purified strain was preserved at -80°C using 20% ​​glycerol.

[0031] II. Identification of Strains 1. Morphological characteristics

[0032] Morphological characteristics of strain CKON-1, isolated from rice paddies in Harbin, Heilongjiang Province, were observed after inoculation and culture using the streak plate method. The colony characteristics of strain CKON-1 on various culture media are shown in Table 1. The average culture time was 3-4 days. On LB medium, the colonies appeared white (…). Figure 1 ).

[0033] Table 1. Colony characteristics of strain CKON-1

[0034] 2. Molecular identification PCR amplification of 16S rDNA used universal primers: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'. The amplified fragment was approximately 1473 bp in size, and the annealing temperature was 55℃.

[0035] The PCR reaction program was as follows: 95 ℃ for 5 min; 95 ℃ for 30 s, 56 ℃ for 90 s, 72 ℃ for 60 s (33 cycles); 72 ℃ for 10 min; 4 ℃.

[0036] After purification and sequencing of the amplified sequence, homology comparison was performed using NCBI's Blasten program. 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 *Bacillus splenida*. Paenibacillus lautus The 16S rDNA sequence is shown in SEQ ID NO.1 of the sequence listing.

[0037] III. Biological characteristics of the strain Determination of Bacillus splendidus ( Paenibacillus lautus The growth temperature, pH, and salt tolerance range of CKON-1 are shown in Table 2.

[0038] The Bacillus spleniculae strain CKON-1 has a growth temperature range of 15-37℃ and a growth pH range of pH 5-9, and can tolerate a concentration of 10% NaCl. This indicates that Bacillus spleniculae strain CKON-1 is a bacterium with a wide applicable pH and temperature range and salt tolerance.

[0039] Table 2. Growth temperature, pH value, and salt tolerance range of Bacillus splenium strain CKON-1

[0040] Note: + indicates tolerance, - indicates intolerance IV. Preservation of bacterial strains Bacillus splenoides isolated from rice paddies in Harbin, Heilongjiang Province ( Paenibacillus lautus CKON-1 was deposited on December 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number for this strain is CGMCC No. 29377.

[0041] Example 2 Bacillus splenoides isolated from rice paddies in Harbin, Heilongjiang Province ( Paenibacillus lautus ) Lignocellulase activity test of CKON-1 The lignocellulase activity of Bacillus Brilliantus CKON-1 was determined using the plate staining / color change method: (1) Culture of Bacillus Brilliantus: Bacillus Brilliantus CKON-1 was inoculated on LB plates by streak plating and cultured at 28°C for 3 days for later use.

[0042] (2) Inoculation of various enzyme activity test plates: Take the CKON-1 colonies that have been cultured by the strain and inoculate them in the middle of the test plate, and then incubate at 28℃ for 2 days. Then measure the colony diameter and the diameter of the color change zone.

[0043] 1) Endoglucanase activity: Carboxymethyl cellulose sodium (CMC-Na) medium (g / L): CMC-Na 15 g, K₂HPO₄ 1 g, KH₂PO₄ 1 g, MgSO₄ 0.2 g, NaCl 0.1 g, FeCl₃ 0.01 g, NaNO₃ 2.5 g, CaCl₂ 0.1 g, yeast extract 5 g, agar powder 20 g, bring to a final volume of 1000 mL, pH 7.0–7.2. After inoculation and culture for 2 days, Congo red staining is performed.

[0044] Congo red staining: Cover the culture medium with 1 mg / mL Congo red solution and stain for 10-15 min. Then, discard the Congo red solution and add 1 mol / L NaCl solution. After 15 min, discard the NaCl solution and observe the clear zone.

[0045] 2) Xylanase activity: 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 5 g, agar 20 g, adjusted to 1000 mL. After inoculation and culture for 2 days, Congo red staining was performed.

[0046] 3) Laccase activity: Guaiacin medium: Add guaiacol to PDA medium to a final concentration of 0.04%. Observe for the appearance of a reddish-brown discoloration zone after inoculation and culture.

[0047] 4) Lignin peroxidase and manganese peroxidase activities: Aniline blue medium (g / L): Add 0.01% aniline blue to BM medium (10 g yeast extract, 20 g glucose, 20 g agar, adjusted to 1000 mL). Observe for the appearance of a reddish-brown discoloration zone after inoculation and culture.

[0048] (3) Definition and determination of enzyme activity index (EI): Enzyme activity index (EI) = D / d (D: diameter of hydrolysis zone / decolorization zone / color change zone; d: colony diameter).

[0049] (4) Results: After 2 days of culture, Bacillus splendens CKON-1 showed high CMC enzyme activity (EI=4.6), laccase activity (EI=3.38), peroxidase activity (EI=3.27) and certain xylanase activity (EI=1.2), indicating that Bacillus splendens CKON-1 has a high efficiency in degrading cellulose and lignin.

[0050] Example 3 Bacillus splendidus ( Paenibacillus lautus CKON-1 is a microbial inoculant for materials. (1) Preparation of liquid seeds Select colonies of Bacillus Brilliantus CKON-1 that have grown on LB plates for 5-7 days, inoculate them into LB liquid medium, and culture at 28-30℃ with shaking at 180 rpm for 2-3 days to use as seed culture.

[0051] (2) Preparation of liquid microbial agents Inoculate the liquid seed at a mass ratio of 5% into a liquid fermentation tank, ferment at 28-30℃ for 4-6 days, and the resulting bacterial fermentation broth or the filtered bacterial fermentation broth can be used as a liquid microbial agent.

[0052] Example 4 Bacillus splendidus ( Paenibacillus lautus Application of CKON-1 as a microbial agent in the liquid decomposition of rice straw (1) Application of microbial agents After rice straw was air-dried, it was cut into sections approximately 2 cm long. In the control group, no microbial agent was applied; 2 g of straw sections were added to an Erlenmeyer flask containing 50 ml of Hutcheson's inorganic salt solution. In the treatment group, the microbial agent CKON-1 was added and mixed thoroughly. The application rate of CKON-1 was 2.5 ml / flask (OD). 600 =1).

[0053] Huchinson's inorganic salt culture medium: 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.

[0054] (2) Determination of straw decomposition rate The decomposition rate of straw was determined using the loss-in-weight method. The dry weight of the straw was measured before bottling; this is the initial dry weight of the straw. After a certain period, the undecomposed straw in the Erlenmeyer flasks was removed, washed, air-dried, and weighed; this is the dry weight of the undecomposed straw. The decomposition rate was then calculated as: Decomposition rate = (Initial dry weight of straw - Dry weight of undecomposed straw) / Initial dry weight of straw × 100%.

[0055] (3) Results The *Bacillus scintillans* CKON-1 strain provided by this invention showed in field decomposition trials that strain CKON-1 can significantly increase the decomposition rate of rice straw in liquid. One month after application of the microbial agent, the straw decomposition rate was 12.5% ​​higher than the control (Table 3). Figure 7 ).

[0056] Table 3. Determination and statistics of straw decomposition rate

[0057] The Bacillus Brilliantus CKON-1 provided by this invention has high lignocellulose degradation activity and can significantly improve the decomposition rate of straw. This strain was isolated from paddy field soil and can grow in a pH range of 5-9 and a temperature range of 15-37℃. It can tolerate a salt concentration of 10%, making it a bacterium with a wide pH and temperature adaptability range and salt tolerance.

Claims

1. Bacillus splendidus ( Paenibacillus lautus ) CKON-1, its accession number is CGMCC No.29377.

2. A microbial inoculant, characterized in that, Contains the Bacillus splendens as described in claim 1 ( Paenibacillus lautus ) CKON-1.

3. The *Bacillus splendidus* as described in any one of claims 1 to 2 ( Paenibacillus lautus Application of CKON-1 in the preparation of straw decomposing bacteria.

4. The application according to claim 3, characterized in that, The straw in question is rice straw.

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