Pseudomonas killieri Z3 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 strain of Pseudomonas kielensis Z3, and the preservation number of the Pseudomonas kielensis Z3 is CGMCC (China General Microbiological Culture Collection Center) No.29379. The invention further discloses a preparation method of the Pseudomonas kielensis Z3. The strain Z3 is 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.29379. 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 15-45 DEG C, can tolerate the salt concentration of 5%, and is a bacterium with wide pH value and temperature application range and salt tolerance. 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 biodegradation. 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 treated by burning and stacking, resulting in 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, thus 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 their 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 strain of Pseudomonas kielensis Z3 with a preservation number of CGMCC No. 29379.

[0005] Meanwhile, the present invention provides the application of Pseudomonas kielensis Z3 in the preparation of lignocellulose-degrading inoculants.

[0006] In addition, the present invention also provides the application of Pseudomonas kielensis Z3 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 - 45 °C.

[0010] In a specific embodiment of the present invention, the mass concentration of NaCl in the decomposition is not higher than 5%.

[0011] The Pseudomonas kiliani Z3 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 - 45 °C, and can tolerate a salt concentration of 5%. 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 Pseudomonas kiliani strain Z3 on LB medium.

[0013] Figure 2 It is a diagram showing the growth of Pseudomonas kiliani strain Z3 at different temperatures

[0014] Figure 3 It is a diagram for measuring the enzyme activity of lignocellulose - degrading enzymes of Pseudomonas kiliani strain Z3.

[0015] Among them, the EI value of the cellulase hydrolysis zone plate: 4.43.

[0016] Figure 4 It is a diagram for measuring the enzyme activity of lignocellulose - degrading enzymes of Pseudomonas kiliani strain Z3.

[0017] Among them, the EI value of the xylanase hydrolysis zone plate: 3.91.

[0018] Figure 5 It is an effect diagram of promoting the decomposition of straw by Pseudomonas kiliani strain Z3. Detailed Embodiments

[0019] 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.

[0020] In the following embodiments, the following media are used:

[0021] 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, made up to 1000 mL with water, pH 7.2 - 7.4.

[0022] Nutrient agar medium: beef extract 5 g, peptone 10 g, NaCl 5 g, agar 20 g, made up to 1000 mL with water, pH 7.4 - 7.6.

[0023] PDA medium: Potato Dextrose Agar medium. Weigh 200 g of potatoes, boil for 30 minutes, filter to obtain the filtrate, add 20 g of dextrose, 15 - 20 g of agar, make up the volume to 1000 ml with water, and adjust the natural pH.

[0024] LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, make up the volume to 1000 mL with water, and adjust the natural pH.

[0025] Example 1

[0026] Isolation, identification and biological characteristics of the strain

[0027] I. Isolation and purification of the strain

[0028] 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 the culture on the LB medium, named strain Z3.

[0029] Preserve the obtained purified strain with 20% glycerol at -80 °C.

[0030] II. Identification of the strain

[0031] 1. Morphological characteristics

[0032] The morphological characteristics of strain Z3 isolated from a paddy field in Harbin, Heilongjiang. After inoculating and culturing on the medium using the streak plate method, morphological observation was carried out. The colony characteristics of strain Z3 on various media are shown in Table 1. The average culture time is 3 - 4 days. On the LB medium, the colonies are smooth and milky white( Figure 1 ).

[0033] Table 1. Colony characteristics of strain Z3

[0034]

[0035] 2. Molecular identification

[0036] The universal primers used for PCR amplification of 16S rDNA are: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3. The size of the amplified fragment is about 1473 bp, and the annealing temperature is 55 °C.

[0037] The PCR reaction program was as follows: 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.

[0038] After the amplified sequence was purified and sequenced, homology comparison was performed through the Blastn program of NCBI. According to the sequence alignment results of the 16S rDNA fragment and combined with the morphological characteristics of the strain, strain Z3 was identified as Pseudomonas kielensis. Among them, the 16S rDNA sequence is shown as SEQ ID NO.1 in the sequence listing.

[0039] III. Biological characteristics of the strain

[0040] The growth temperature, pH value, and salt tolerance concentration range of Pseudomonas kielensis Z3 were determined, and the specific results are shown in Table 2.

[0041] The growth temperature range of Pseudomonas kielensis Z3 was 15 - 45°C, the growth pH value range was pH 5 - 9, and it could tolerate 5% NaCl by mass concentration. This indicates that Pseudomonas kielensis Z3 is a bacterium with a wide range of applicable pH values and temperatures and salt tolerance.

[0042] Table 2. Growth temperature, pH value, and salt tolerance concentration range of Pseudomonas kielensis Z3

[0043] 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 -

[0044] Remarks: + indicates tolerance, - indicates intolerance

[0045] IV. Preservation of the strain

[0046] Pseudomonas kielensis Z3 isolated from a paddy field in Harbin, Heilongjiang was deposited at the China General Microbiological Culture Collection Center 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. 29379.

[0047] Example 2

[0048] Activity test of the cellulase from Pseudomonas kielensis Z3 isolated from a paddy field in Harbin, Heilongjiang

[0049] The cellulase activity of Pseudomonas kielensis Z3 was determined by the plate dyeing / discoloration method:

[0050] (1) Culture of Pseudomonas kiliani: Inoculate Pseudomonas kiliani Z3 on an LB plate by the streaking method and incubate it at 28 °C for 3 days for later use.

[0051] (2) Inoculation of various enzyme activity test plates: Take the well-cultured Z3 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 color-changing circle.

[0052] 1) Endoglucanase activity:

[0053] 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 powder 5 g, agar powder 20 g, make up to 1000 mL, pH 7.0 - 7.2. After inoculation and incubation for 2 days, perform Congo red staining.

[0054] Congo red staining: On the medium with grown colonies, cover with 1 mg / mL Congo red solution. After staining for 10 - 15 min, pour off the Congo red solution, add 1 mol / L NaCl solution, pour off the NaCl solution after 15 min, and then observe the clear zone.

[0055] 2) Xylanase activity:

[0056] 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, make up to 1000 mL. After inoculation and incubation for 2 days, perform Congo red staining.

[0057] 3) Laccase activity:

[0058] 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 incubation.

[0059] 4) Lignin peroxidase and manganese peroxidase activities:

[0060] 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, make up to 1000 mL). Observe whether a red-brown color-changing circle appears after inoculation and incubation.

[0061] (3) Definition and determination of the enzyme activity index EI:

[0062] Enzyme activity index EI value = D / d (D: diameter of hydrolysis zone / fading zone / color-changing zone; d: diameter of colony)

[0063] (4) Results: After Pseudomonas kielensis Z3 was cultured for 2 days, it had relatively high CMC enzyme activity (EI = 4.43) and xylanase activity (EI = 3.91) (as Figure 2 shown), and no laccase enzyme activity (EI = 1) and peroxidase enzyme activity (EI = 1) were detected, indicating that Pseudomonas kielensis Z3 has a high degradation ability for cellulose and hemicellulose.

[0064] Example 3

[0065] Microbial inoculant using Pseudomonas kielensis Z3 as the material

[0066] (1) Preparation of liquid seeds

[0067] Pick the colonies of Pseudomonas kielensis Z3 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, then use them as the seed liquid.

[0068] (2) Preparation of liquid microbial inoculant

[0069] Inoculate the liquid seeds into the liquid fermentation tank at a mass ratio of 5%, ferment at 28 - 30 °C for 4 - 6 days, and the obtained bacterial fermentation broth or the fermentation broth of the filtered bacteria can be used as the liquid microbial inoculant.

[0070] Example 4

[0071] Application of microbial inoculant using Pseudomonas kielensis Z3 as the material in the liquid decomposition of rice straw

[0072] (1) Application of microbial inoculant

[0073] After the rice straw is air-dried, cut it into straw segments about 2 cm long. In the control group, no inoculant is applied. Add 2 g of straw segments into a triangular flask containing 50 ml of Hutchinson's inorganic salt solution. In the treatment group, add the microbial inoculant Z3 and mix well. The application amount of the inoculant Z3 is 2.5 ml / bottle (OD 600 = 1).

[0074] 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.

[0075] (2) Determination of straw decomposition rate

[0076] The weight loss method was used to determine the decomposition rate of straw. The dry weight of the straw was measured before being put into the bottle, which was the initial dry weight of the straw. After a certain period of time, the undecomposed straw in the Erlenmeyer flask was picked out, washed, air-dried naturally and weighed, which was the dry weight of the unrotted straw. Then the decomposition rate was calculated as follows: Decomposition rate = (Initial dry weight of straw - Dry weight of unrotted straw) / Initial dry weight of straw × 100%.

[0077] (3) Results

[0078] The experiment on promoting the decomposition of straw by Pseudomonas kilensis Z3 provided by the present invention showed that strain Z3 could significantly improve the decomposition rate of rice straw in liquid. The decomposition rate of straw after 1 month of applying the bacterial agent was increased by 11.2% compared with the control (Table 3, Figure 4 ).

[0079] Table 3. Determination and statistics of straw decomposition rate

[0080]

[0081] Pseudomonas kilensis Z3 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 temperature 15 - 45°C, and can tolerate a salt concentration of 5%. It is a bacterium with a wide range of pH and temperature adaptation and salt tolerance.

Claims

1. Pseudomonas kielensis Z3, with the preservation number of CGMCC No. 29379.

2. Microbial inoculant, characterized in that, Containing Pseudomonas kielensis Z3 described in claim 1.

3. Enzyme protein, characterized in that, From Pseudomonas fluorescens Z3 described in claim 1.

4. Use of Pseudomonas kielensis Z3 according to any one of claims 1 to 3 in the preparation of a lignocellulose-degrading microbial agent.

5. Use of Pseudomonas kielensis Z3 according to 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 - 45 °C.

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

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