Cold-tolerant pseudoarthrobacter CKON-4 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 cold-resistant pseudoarthrobacter psychrotolans CKON-4, and the preservation number of the cold-resistant pseudoarthrobacter psychrotolans CKON-4 is CGMCC (China General Microbiological Culture Collection Center) No.29378. The cold-resistant pseudoarthrobacter psychrotolans CKON-4 has the advantages that the preservation number The strain CKON-4 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 25, 2023, the China General Microbiological Culture Collection Center is called CGMCC for short, the address of the China General Microbiological Culture Collection Center is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number of the strain is CGMCC No.29378. 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 4-37 DEG C, can tolerate the salt concentration of 10%, and is a bacterium with wide pH application range, salt resistance and cold resistance. The strain can play an important role in lignocellulose degrading enzyme production, straw biological decomposition and compost fermentation.

Description

Technical Field

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

[0002] Crop straw is the main waste in the agricultural production process. After reasonable treatment and returning to the field, it will be beneficial to increase soil organic matter and nutrients, improve soil structure, thus promoting plant growth and development, and increasing crop yield and quality. Lignocellulose is the main component of plant straw. Due to its tight combination with lignin and cellulose and its special physical and chemical properties, lignin has become a relatively difficult-to-degrade component in straw. Some studies have shown that the degradation of lignin can effectively improve the degradation rate of cellulose, thereby promoting the effective degradation of the overall components of straw and achieving the effective degradation of straw (Pollgioni et al. 2015; Zhang Ye et al. 2020). Therefore, it is very necessary to screen strains with good lignocellulose degradation effects, especially those with strong lignin degradation ability to break the adverse effects of lignin on the entire degradation process.

[0003] At present, many microbial strains with lignocellulose degradation activity have been isolated and reported, such as white rot fungi, Aspergillus, Trichoderma, etc. in fungi, and Bacillus, Pseudomonas, etc. in bacteria. The premise for lignocellulose-degrading microorganisms to effectively degrade straw not only lies in their reasonable lignocellulose-degrading enzyme systems, but also in their strong environmental adaptability. Therefore, screening lignocellulose-degrading microorganisms with strong environmental adaptability such as cold tolerance, salt tolerance and wide pH adaptation range will have greater advantages for the effective degradation of straw and meet the production needs. Summary of the Invention

[0004] In view of this, the first object of the present invention is to provide a cold-tolerant Pseudarthrobacter psychrotolerans CKON-4, whose preservation number is CGMCC No. 29378.

[0005] Meanwhile, the present invention provides the application of cold-tolerant Pseudarthrobacter psychrotolerans CKON-4 in the preparation of lignocellulose-degrading microbial agents.

[0006] In addition, the present invention also provides the application of cold-tolerant Pseudarthrobacter psychrotolerans CKON-4 in the preparation of straw-degrading microbial 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 degradation is 5-9.

[0009] In a specific embodiment of the present invention, the temperature of the degradation is 4-37°C.

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

[0011] The psychrotolerant Pseudarthrobacter CKON-4 provided by the present invention has high lignocellulose degradation activity and can significantly improve the decomposition rate of straw; this strain is isolated from paddy soil, can grow within the range of pH 5-9 and temperature 4-37 °C, and can tolerate a salt concentration of 10%. It is a bacterium with a wide pH adaptation range, salt tolerance and cold tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a growth morphology diagram of psychrotolerant Pseudarthrobacter CKON-4 on LB medium.

[0013] Figure 2 It is a diagram of the growth of psychrotolerant Pseudarthrobacter CKON-4 at different temperatures.

[0014] Figure 3 It is a diagram for determining the enzyme activity of the lignocellulose-degrading enzyme of psychrotolerant Pseudarthrobacter CKON-4.

[0015] Among them, the EI value of the cellulase hydrolysis zone: 1.

[0016] Figure 4 It is a diagram for determining the enzyme activity of the lignocellulose-degrading enzyme of psychrotolerant Pseudarthrobacter CKON-4.

[0017] Among them, the EI value of the xylanase hydrolysis zone: 1.38.

[0018] Figure 5 It is a diagram for determining the enzyme activity of the lignocellulose-degrading enzyme of psychrotolerant Pseudarthrobacter CKON-4.

[0019] Among them, the EI value of the peroxidase hydrolysis zone: 3.23.

[0020] Figure 6 It is a diagram for determining the enzyme activity of the lignocellulose-degrading enzyme of psychrotolerant Pseudarthrobacter CKON-4.

[0021] Among them, the EI value of the laccase hydrolysis zone: 4.25.

[0022] Figure 7 It is a diagram of the effect of psychrotolerant Pseudarthrobacter CKON-4 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: Soluble starch 20 g, KNO3 1 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, FeSO4·7H2O 0.01 g, NaCl 0.5 g, agar 20 g, make up the volume to 1000 mL with water, pH 7.2 - 7.4.

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

[0027] PDA medium: Potato dextrose agar medium. Boil 200 g of potatoes for 30 minutes, filter and take the filtrate, glucose 20 g, agar 15 - 20 g, make up the volume to 1000 ml with water, natural pH.

[0028] LB medium: Tryptone 10 g, yeast extract 5 g, NaCl 10 g, make up the volume 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: Add 1 g of the sample to a 300 ml Erlenmeyer flask containing small glass beads and 99 ml of sterile normal saline, and culture 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 and spread them on the beef extract peptone medium respectively. Pick single colonies on the 5th day, 7th day and 10th day respectively, and then use the streak plate method to purify and isolate and culture on the LB medium, named strain CKON - 4.

[0033] Preserve the obtained purified strain with 20% glycerol at -80 °C.

[0034] II. Identification of the strain

[0035] 1. Morphological characteristics

[0036] Morphological characteristics of strain CKON-4 isolated from a paddy field in Harbin, Heilongjiang. After inoculating and culturing on a medium using the streak plate method, morphological observations were carried out. The colony characteristics of strain CKON-4 on various media are shown in Table 1. The average culture time was 2 - 4 days. On LB medium, the colonies were smooth and slightly white ( Figure 1 ).

[0037] Table 1. Colony characteristics of strain CKON-4

[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.

[0041] 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.

[0042] After purification and sequencing of the amplified sequence, homology comparison was carried out through 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-4 was identified as Pseudarthrobacter psychrotolerans. Among them, the 16S rDNA sequence is shown as SEQ ID NO.1 in the sequence listing.

[0043] III. Biological characteristics of the strain

[0044] The growth temperature, pH, and salt tolerance concentration range of Pseudarthrobacter psychrotolerans strain CKON-4 were determined. The specific results are shown in Table 2.

[0045] The growth temperature range of Pseudarthrobacter psychrotolerans CKON-4 was 4 - 37°C, the growth pH range was pH 5 - 9, and it could tolerate a mass concentration of 10% NaCl. This indicates that Pseudarthrobacter psychrotolerans CKON-4 is a bacterium with a wide pH application range and salt tolerance.

[0046] Table 2. Growth temperature, pH value, and salt tolerance concentration range of Pseudarthrobacter psychrotolerans CKON-4

[0047] 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 -

[0048] Note: + indicates tolerance, - indicates intolerance

[0049] IV. Preservation of Strains

[0050] The cold-tolerant Pseudarthrobacter psychrotolerans CKON-4 isolated from a paddy field in Harbin, Heilongjiang was deposited at the General Microbiology 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, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number of this strain is CGMCC No. 29378.

[0051] Example 2

[0052] Activity test of the cellulase of the cold-tolerant Pseudarthrobacter psychrotolerans CKON-4 isolated from a paddy field in Harbin, Heilongjiang

[0053] The cellulase activity of the cold-tolerant Pseudarthrobacter psychrotolerans CKON-4 was determined by the plate dyeing / discoloration method:

[0054] (1) Cultivation of the cold-tolerant Pseudarthrobacter: Inoculate Pseudarthrobacter psychrotolerans CKON-4 on an LB plate by the streaking method and incubate at 28°C for 3 days for standby.

[0055] (2) Inoculate various enzyme activity test plates: Take the well-cultured CKON-4 colonies of the strain, inoculate them in the middle of the test plate, and then incubate at 28°C for 2 days. Then measure the colony diameter and the diameter of the discoloration circle.

[0056] 1) Endoglucanase activity:

[0057] 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, make up to 1000 mL, pH 7.0 - 7.2. After inoculation and incubation for 2 days, perform Congo red staining.

[0058] 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.

[0059] 2) Xylanase activity:

[0060] 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, volume made up to 1000 mL. After inoculation and cultivation for 2 days, Congo red staining was carried out.

[0061] 3) Laccase activity:

[0062] 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.

[0063] 4) Activities of lignin peroxidase and manganese peroxidase:

[0064] 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, volume made up to 1000 ml). Observe whether a red-brown color-changing circle appears after inoculation and cultivation.

[0065] (3) Definition and determination of enzyme activity index EI:

[0066] Enzyme activity index EI value = D / d (D: diameter of hydrolysis circle / fading circle / color-changing circle; d: diameter of colony)

[0067] (4) Results: After culturing Pseudarthrobacter psychrotolerans CKON-4 for 2 days, no CMC enzyme activity was detected (EI = 1), and it had a certain xylanase activity (EI = 1.38), a very high laccase activity (EI = 4.25) and peroxidase activity (EI = 3.23) ( Figures 3 - 6 ), indicating that Pseudarthrobacter psychrotolerans CKON-4 has a high degradation ability for hemicellulose and lignin, especially for lignin.

[0068] Example 3

[0069] Microbial inoculant using Pseudarthrobacter psychrotolerans CKON-4 as the material:

[0070] (1) Preparation of liquid seeds

[0071] Pick colonies of Pseudarthrobacter psychrotolerans CKON-4 that have grown on the LB plate for 5 - 7 days, inoculate them into the LB liquid medium, and use them as the seed liquid after shaking cultivation at 28 - 30 °C and 180 rpm for 2 - 3 days.

[0072] (2) Preparation of liquid microbial inoculant

[0073] The liquid seeds are inoculated into a liquid fermenter 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.

[0074] Example 4

[0075] Application of a microbial inoculant using Psychrobacter psychrotolerans CKON-4 in the liquid decomposition of rice straw

[0076] (1) Application of the microbial inoculant

[0077] 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-4 is added and mixed evenly. The application amount of the inoculant CKON-4 is 2.5 ml / bottle (OD 600 = 1).

[0078] 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.

[0079] (2) Determination of the straw decomposition rate

[0080] 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%.

[0081] (3) Results

[0082] The straw decomposition test of Psychrobacter psychrotolerans CKON-4 provided by the present invention in liquid shows that the strain CKON-4 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 11.7% compared with the control (Table 3, Figure 7 ).

[0083] Table 3. Determination and statistics of the straw decomposition rate

[0084]

[0085] The cold-tolerant Pseudarthrobacter CKON-4 provided by the present invention has high lignocellulose degradation activity and can significantly improve the decomposition rate of straw; this strain is isolated from paddy soil, can grow within the range of pH 5-9 and temperature 4-37 °C, and can tolerate a salt concentration of 10%, and is a bacterium with a wide pH adaptation range, salt tolerance and cold tolerance.

Claims

1. Pseudarthrobacter psychrotolerans CKON-4, with the preservation number of CGMCC No. 29378.

2. Microbial inoculant, characterized in that, Containing Pseudarthrobacter psychrotolerans CKON-4 described in claim 1.

3. An enzyme protein, characterized in that, Derived from the Pseudarthrobacter psychrotolerans strain CKON-4 described in claim 1.

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

5. Use of Pseudarthrobacter psychrotolerans CKON-4 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, wherein 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, characterized in that, The temperature of the decomposition is 4 - 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%.

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