Low-temperature-resistant lignin degrading bacterium and application thereof

Abstract

The invention discloses a low-temperature-resistant lignin degrading bacterium and application thereof, and relates to the technical field of biology. The invention aims to solve the problem of low straw decomposition rate of existing microorganisms at low temperature. The low-temperature-resistant lignin degrading bacterium disclosed by the invention is Trichoderma luciferase G59, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is September 18, 2024, and the preservation number is CGMCC NO.41509. The low-temperature-resistant lignin degrading bacterium disclosed by the invention has the advantages that the low-temperature-resistant lignin degrading bacterium disclosed by the invention can be used for degrading lignin, and the low-temperature-resistant lignin degrading bacterium can be used for degrading lignin; the low-temperature-resistant lignin degrading bacterium disclosed by the invention is used for degrading straws. And the degradation rate of the corn straw at 10 DEG C can reach 36.78% within 15 days. The method is applied to the field of straw degradation.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a low-temperature-resistant lignin-degrading bacterium and application thereof. Background Art

[0002] my country produces about 1 billion tons of straw annually. Heilongjiang Province is located in the northernmost part of my country, and produces 90 million tons of straw annually. The cold and low temperature conditions in Northeast China make it impossible to effectively return a large amount of straw resources to the fields, causing serious environmental pollution and waste of plant nutrients. Returning straw to the fields is an effective technical means to improve soil fertility and physical and chemical properties of the soil.

[0003] However, during the autumn harvest in Heilongjiang Province, the ambient temperature is relatively low. When the straw is returned to the fields, the activity of indigenous microorganisms is weak under the relatively low temperature natural environment conditions, and the straw cannot be quickly decomposed by the microorganisms, affecting the land preparation and sowing during the Spring Festival of the following year. In addition, the slow decomposition rate of the straw returned to the fields has brought many adverse effects on agricultural production, and has become the main obstacle to agricultural production in areas where direct straw return to the fields is promoted.

[0004] Since there are few low-temperature-resistant and efficient microbial germplasm resources (fungi / actinomycetes) that are adapted to the characteristics of the cold regions in Heilongjiang Province, low temperature conditions cannot ensure rapid decomposition of straw in the soil, and the straw decomposition rate is low. Summary of the invention

[0005] The purpose of the present invention is to solve the problem that the existing microorganisms have low straw decomposition rate at low temperatures, and to provide a low-temperature resistant lignin degrading bacterium and its application.

[0006] The present invention discloses a low-temperature-resistant lignin-degrading fungus, Trichoderma shangrilaense G59, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date being September 18, 2024, and the deposit number being CGMCC NO.41509.

[0007] The invention discloses a low-temperature-resistant lignin-degrading bacterium for degrading straw.

[0008] The present invention collects soil samples from the high-cold area of ​​Heilongjiang Province, screens microorganisms with lignocellulose degradation potential by lignin culture method and aniline blue fading method, selects efficient fungi, conducts molecular biological identification, measures lignocellulose degradation enzyme activity, and conducts applied research on corn stalk degradation.

[0009] Beneficial effects of the present invention:

[0010] The straw degrading bacteria of the present invention is Tparapiluliferum G59. The enzyme activities of lignin oxidase (Lip), laccase (Lac) and manganese peroxidase (Mnp) of the bacteria under the conditions of 10°C and 150r / min are 119.15U / mL, 57.65U / mL and 15.17U / mL respectively, and the degradation rate of corn straw can reach 36.78% within 15 days. The Tparapiluliferum G59 provided by the present invention is used for rapid decomposition of straw returned to the field, is safe for humans and animals, has no pollution to the environment, and has good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Aniline blue fading plate for G59;

[0012] Figure 2 is the phylogenetic tree of G59;

[0013] Figure 3 is the ligninase activity of strain G59;

[0014] Figure 4 This is the degradation effect of strain G59 on corn straw. DETAILED DESCRIPTION

[0015] Specific implementation method 1: In this implementation method, a low-temperature-resistant lignin-degrading fungus is Trichoderma shangrilaense G59, which is deposited in the General Microbiological Center of the China Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 18, 2024, and the deposit number is CGMCCNO.41509.

[0016] Specific embodiment 2: This embodiment is different from specific embodiment 1 in that the enzyme activities of lignin oxidase, laccase and manganese peroxidase of the lignin degrading bacteria at 10°C and 150 r / min are 119.15 U / mL, 57.65 U / mL and 15.17 U / mL respectively. The rest is the same as specific embodiment 1.

[0017] Specific implementation method three: In this implementation method, a low-temperature resistant lignin-degrading bacteria is used to degrade straw

[0018] Specific implementation method 4: This implementation method is different from specific implementation method 3 in that the straw is corn straw. The other steps are the same as those of specific implementation method 3.

[0019] The following examples are used to verify the beneficial effects of the present invention:

[0020] Embodiment 1:

[0021] 1. Isolation and screening of strains:

[0022] Take 10g of soil sample collected from the high-altitude cold area of ​​Heilongjiang Province, add it to 90mL of sterile water, shake it thoroughly to make a suspension, shake it in a constant temperature oscillator at 22℃ and 150r / min for 30min, then take it out and let it stand for 10min. The suspension is prepared. Prepare 6 sterilized test tubes, numbered 1, 2, 3, 4, 5, and 6, representing 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 , add 9mL of sterile purified water respectively. Use a pipette to draw 1mL of supernatant from the prepared suspension and place it in the test tube labeled 1, and shake it manually. After shaking, let it stand for 10 minutes and then draw 1mL of solution from it into the next gradient test tube, and dilute it step by step in this way. Take 0.2mL of bacterial solution numbered 4, 5, and 6, spread it on the lignin culture medium, and repeat 3 times for each number. Invert and culture in a constant temperature incubator at 10℃. After two weeks, select larger single colonies for plate streaking. After two purifications and separations, pick single colonies to spot on the PDA culture medium to observe the growth of different strains.

[0023] Aniline blue staining test was used to screen straw-degrading bacteria. The purified strains were inoculated onto aniline blue-PDA medium by the spot inoculation method. Three spots were inoculated on each plate, and three replicates were set. The plates were cultured in a 10°C constant temperature incubator for 72 h. The colony diameter (d, cm) and the diameter of the surrounding transparent circle (D, cm) were observed and measured, and the D / d value was calculated. The fading effect of strain G59 on aniline blue plates is shown in Tables 1 and Figure 1 , indicating that strain G59 has the ability to produce lignin oxidase and can degrade lignin.

[0024] Table 1. Fading effect of strain G59 on aniline blue plates

[0025]

[0026] 2. Identification of bacterial species

[0027] (1) Morphological identification

[0028] The isolated and purified strain was streaked on PDA medium to obtain a single colony, and its colony morphology was observed. The colony of G59 was white at the beginning, expanding in all directions, with a growth zone of white hyphae around the colony, until the whole colony finally formed multiple white patches surrounded by white hyphae and dense.

[0029] (2) Molecular Biological Identification

[0030] The high-yield strain obtained by repeated screening was sent to Shanghai Shenggong Biotechnology Co., Ltd. for ITS sequence sequencing. The strain was amplified by PCR using universal primers ITS1 and ITS4 to obtain the ITS sequence of the strain (the sequence is shown in SEQ ID NO: 1). The sequencing results were compared with the existing sequences in GenBank using BLAST software to determine the species of the strain, and phylogenetic analysis was performed using MEGA5.2 and the Neighbor-Joining method to construct a phylogenetic tree ( Figure 2 ). From the phylogenetic tree of G59, it can be seen that strain G59 is a species of Trichoderma sp. The sequence coverage of strain G59 and Trichoderma shangrilaense reached 98%. Combining the results of morphological and molecular biological identification, strain G59 was identified as Trichoderma shangrilaense G59.

[0031] 3. Determination of enzyme activity of strain G59

[0032] The screened strain G59 was used to make a plug with a diameter of 8 mm according to the method for making a plug. Fifteen 150 mL conical flasks were prepared, and 50 mL of liquid enzyme-producing culture medium was added to each conical flask. The prepared plugs were then added to each conical flask. The conical flasks were placed on a shaker and cultured at 10°C and 150 r / min. Three parallel replicates were performed for each strain. After 3 days, 2 mL of the fermentation liquid was placed in a centrifuge tube. Samples were taken every 3 days thereafter. The samples were placed in a centrifuge and centrifuged at 8000 r / min for 5 minutes. The supernatant was taken and saved as the crude enzyme solution for subsequent tests.

[0033] (1) Determination of laccase (Lac) activity by ABTS method:

[0034] Using the ABTS method, add 0.5 mL of the prepared ABTS solution to a 5 mL centrifuge tube, then add 3.3 mL of the prepared acetic acid-sodium acetate buffer, place the centrifuge tube in a constant temperature water bath, set the temperature to 30°C, and add 200 μL of crude enzyme solution with a pipette after 3 minutes. The reaction system is 4 mL, the wavelength is set to 420 nm, and the inactivated (121°C, 0.1 MPa, 30 minutes) crude enzyme solution is added under the same conditions for zero calibration. After 3 minutes of reaction, the absorbance value is immediately measured at this wavelength.

[0035] Definition of Lac enzyme activity unit: Under the above reaction conditions, the amount of enzyme required to oxidize 1 μmo LABTS per minute is one enzyme activity unit (U).

[0036] (2) Determination of lignin peroxidase (LiP) using the resveratrol oxidation method:

[0037] Add 0.4 mL of prepared resveratrol solution to a 5 mL centrifuge tube, then add 2.5 mL of prepared tartaric acid-sodium tartrate buffer, place the centrifuge tube in a constant temperature water bath to preheat, set the temperature to 30°C, use a pipette to add 1 mL of crude enzyme solution, and add 100 μL of prepared H2O2 solution to start the reaction. The reaction system is 4 mL, the wavelength is set to 310 nm, and the inactivated (121°C, 0.1 MPa, 30 min) crude enzyme solution is added under the same conditions for zero calibration. After reacting for 3 minutes, immediately place it at this wavelength to measure the absorbance value.

[0038] Definition of Lip enzyme activity unit: Under the above reaction conditions, the amount of enzyme required to oxidize 1 μmol of resveratrol per minute is one enzyme activity unit (U).

[0039] (3) Determination of manganese peroxidase (MnP):

[0040] Add 0.4 mL of prepared MnSO4 solution to a 5 mL centrifuge tube, then add 2.5 mL of prepared succinic acid-sodium succinate buffer, preheat the centrifuge tube in a constant temperature water bath, set the temperature to 37°C, use a pipette to add 1 mL of crude enzyme solution, and add 100 μL of prepared H2O2 solution to start the reaction. The reaction system is 4 mL, the wavelength is set to 240 nm, and the inactivated (121°C, 0.1 MPa, 30 min) crude enzyme solution is added under the same conditions for zero calibration. After reacting for 3 minutes, immediately place it at this wavelength to measure the absorbance value.

[0041] Mnp enzyme activity unit definition: Under the above reaction conditions, 1 μmol Mn is added per minute. 2+ Converted to Mn 3+ The amount of enzyme required is one enzyme activity unit (U).

[0042] Enzyme activity calculation formula:

[0043] U represents enzyme activity; ΔD represents absorbance; ξ represents molar extinction coefficient, M*cm, where lignin peroxidase ε310nm: 3.6×10 4 L / (mol*cm), laccase ε420nm: 9.3×10 3 L / (mol*cm). Manganese peroxidase ε240nm: 6.5×10 3 L / (mol*cm); b represents the cuvette thickness, cm; Δt represents the reaction time, min; Vtotal represents the reaction system volume, mL; Venzyme represents the volume of added enzyme, mL. The results of ligninase activity of strain G59 are shown in Figure 3 As shown by Figure 3It can be seen that strain G59 has the ability to degrade lignin, and it can produce three enzymes related to lignin degradation. The enzyme activities of Lip and Lac reached their maximum values ​​on the 9th day, which were 119.15U / mL and 57.65U / mL respectively, and the enzyme activity of Mnp reached its maximum value on the 12th day, which was 15.17U / mL. During the entire test, with the increase of culture time, the Lac, Mnp and Lac enzyme activities of strain G59 showed a trend of first increasing and then decreasing.

[0044] 4. Determination of corn straw degradation rate by strain G59

[0045] In order to verify the degradation ability of low-temperature straw degrading bacteria G59, the indoor liquid fermentation culture method was used to conduct low-temperature degradation tests with corn straw as the research object. The corn straw was washed under running tap water, dried in an oven at 80℃, and cut into 3-4cm small segments. The strain G59 was inoculated in PDB liquid culture medium and cultured on a shaker at 10℃ for 3 days to prepare bacterial liquid. Experimental group: 5% bacterial liquid was added to a conical flask containing 100mL of straw fermentation culture liquid of 10g corn straw, and the culture was statically cultured at 10℃ in a constant temperature incubator. The straw fermentation liquid with an equal amount of sterile water was set as the blank control (CK), and 3 repetitions were set for each treatment. The culture time was 15 days. During this period, the straw in the conical flask was collected every 3 days, rinsed with distilled water, and placed in a drying oven to dry to constant weight. The dry weight of the straw was weighed, and the straw degradation rate at different times was measured.

[0046] Calculation formula of straw degradation rate (η):

[0047] M is the dry weight of the straw after degradation, M0 is the initial dry weight of the straw, and η is the straw degradation rate (%) after the cultivation is completed.

[0048] Test results see Figure 4 ,Depend on Figure 4 It can be seen that adding strain G59 to the straw fermentation culture medium, under low temperature conditions, compared with the control group, the treatment group with strain G59 improved the degradation effect of corn straw, and the degradation rate of corn straw with the addition of degradation bacteria G59 in the early stage of treatment showed a rapid growth trend. On the 15th day, the straw degradation efficiency of the treatment group with G59 was the highest, reaching 36.78%, which was significantly higher than the control group without adding bacterial agents.

[0049] In summary, the lignocellulose-degrading strain G59 was screened and obtained. Through laboratory domestication, it was found that it could adapt to low temperature conditions (10°C) and could degrade corn straw under these conditions with a degradation rate of 36.78%.

[0050] The main components of the above culture medium:

[0051] Corn straw fermentation medium: corn straw 10g, (NH4)2SO4 2.0g, Na2HPO4 1.5g, MgSO4 0.2g, CaCl2 0.01g, KH2PO4 1.5g, distilled water to 1000mL, sterilize at 121℃ for 30min.

[0052] Lignin culture medium: Na2HPO4 0.2g, MgSO4 0.5g, NH4NO3 1.33g, KH2PO4 1.0g, alkaline lignin 2.0g, agar 20g, distilled water to 1000mL. Sterilize at 121℃ for 30min.

[0053] Aniline blue-PDA identification medium: Add 0.1 g / L aniline blue to PDA medium.

[0054] Potato dextrose agar medium (PDA): 12g potato powder, 20g glucose, 20g agar, 1000mL distilled water, pH 7.5-8.

[0055] Potato dextrose broth (PDB): 5 g potato powder, 20 g glucose, 1000 mL distilled water, pH 7.5-8.

[0056] Liquid enzyme production culture medium: 20 g glucose, 0.5 g magnesium sulfate, 0.05 g copper sulfate, 0.05 g manganese sulfate, pH 7.5-8.0.

Claims

1. A low temperature resistant lignin degrading bacterium, characterized in that The lignin degrading bacteria is Trichoderma shangrilaense G59, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 18, 2024, and the deposit number is CGMCC NO.41509.

2. A low-temperature resistant lignin-degrading bacteria according to claim 1, characterized in that The enzyme activities of lignin oxidase, laccase and manganese peroxidase of the lignin degrading bacteria at 10° C. and 150 r / min are 119.15 U / mL, 57.65 U / mL and 15.17 U / mL respectively.

3. A low-temperature-resistant lignin-degrading bacteria as claimed in claim 1 for degrading straw.

4. The use according to claim 3, characterized in that The straw is corn straw.

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