A strain for degrading lignin and its application

Abstract

The present invention relates to a corynebacterium, which is Pseudomonas sp. Strain MZK-2-7. The Pseudomonas MZK-2-7 can grow rapidly and stably using an inexpensive culture medium, has a short fermentation period, and a high lignin degradation efficiency. This bacterium can be used for the biological degradation of straw, improving the utilization rate of straw, and has high economic value and ecological value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a strain for degrading lignin and its application. Background Art

[0002] China is a large agricultural country and one of the countries with the richest straw resources. The scientific development and utilization of crop straw resources can not only alleviate the shortage of rural feed, fertilizer, fuel and industrial raw materials, but also is an urgent requirement for protecting the rural ecological environment and promoting the sustainable and coordinated development of agriculture.

[0003] The main components of straw are cellulose, hemicellulose and lignin. Although lignin accounts for a low proportion, its protective effect on straw is very strong. To improve the degradation rate of straw, it is necessary to first remove the protective effect of lignin on cellulose and hemicellulose.

[0004] Currently, the commonly used lignin treatment methods mainly include physical methods, chemical methods, biological methods or a combination of several methods. Although these methods have increased the utilization rate of biomass resources to a certain extent, there are still many drawbacks in the operation process. Physical methods include ultrasonic method, microwave radiation, γ-ray radiation. It mainly generates short-term local high temperature, high pressure and rapid temperature change inside lignin to destroy the structural stability of lignin and cause chemical reactions. However, the effect of this method is unstable and the noise is unfavorable to the environment, making it difficult to be amplified and applied industrially. Chemical methods use some strong acids and alkalis to break some chemical bonds in lignin to achieve the degradation of lignin. However, this method is not friendly to the environment. In addition, there are problems such as high cost and difficulty in recycling reagents after use. The biological method is relatively mild compared with the above two methods and the treatment effect is also relatively good. However, it also has problems such as long treatment time and low decomposition efficiency.

[0005] Therefore, it is of great significance to develop an effective method for isolating lignin-degrading bacteria and screen lignin-degrading bacteria with high degradation efficiency and short fermentation period. Summary of the Invention

[0006] Problems to be Solved by the Invention

[0007] The biological method has strict requirements for the culture conditions of strains and the degradation process is relatively slow. Therefore, the current research on biological treatment methods mainly focuses on finding a strain with high degradation ability.

[0008] Solutions for Solving the Problems

[0009] The present invention provides a corynebacterium, which is Pseudomonas sp. MZK-2-7. The preservation number of the corynebacterium is CCTCC NO: M 20221892. It is preserved in the China Center for Type Culture Collection, with the preservation date being December 7, 2022, and the preservation address being Wuhan University, Wuhan, China.

[0010] Preservation description:

[0011] Preservation number: CCTCC NO: M 20221892;

[0012] Preservation unit code: CCTCC - China Center for Type Culture Collection;

[0013] Preservation date: December 7, 2022;

[0014] Preservation address: Wuhan University, Wuhan, China;

[0015] Taxonomic name: Pseudomonas sp.;

[0016] Preservation status: Alive.

[0017] Preferably, the 16S rDNA gene sequence of the corynebacterium is as shown in SEQ ID NO.1.

[0018] The present invention provides a method for isolating and screening the above-mentioned corynebacterium, which is characterized by comprising the following steps:

[0019] (1) Sampling and collection;

[0020] (2) Strain isolation;

[0021] (3) Strain screening;

[0022] (4) Strain identification;

[0023] (5) Strain cultivation.

[0024] Preferably, the formula of the lignin medium in step (2) is: per liter of the medium, it contains 1 - 4 g of alkaline lignin, 0.2 - 2 g of KH 2 PO 4 、0.2 - 2 g of K 2 HPO 4 、0.5 - 5 g of NaCl, 0.1 - 1 g of MgCl 2 ·6H 2 O, 0.2 - 2 g of ammonium chloride, 0.04 - 0.4 g of CaCl 2 、1 mL of trace elements, 15 g of agar, 100 - 500 μL of vitamin B 12, the trace element formula is boric acid 300 - 2100 mg / L, cobalt chloride hexahydrate 190 - 1400 mg / L, manganese chloride tetrahydrate 50 - 350 mg / L, zinc chloride 40 - 280 mg / L, nickel chloride hexahydrate 20 - 140 mg / L, sodium molybdate dihydrate 15 - 110 mg / L, copper chloride dihydrate 2 - 14 mg / L, ferrous sulfate heptahydrate 1 - 8 g / L, and the vitamin B 12 The concentration of the solution is 100 mg / L.

[0025] Preferably, in step (3), the screening is carried out using a PDA - aniline blue plate for color development.

[0026] Preferably, step (5) includes the following steps: inoculating the corynebacterium into a medium and performing fermentation culture at 30 - 60 °C.

[0027] The present invention provides a bacterial agent, comprising the above - mentioned corynebacterium and / or the fermentation broth of the corynebacterium.

[0028] The present invention provides the application of the above - mentioned corynebacterium in lignin degradation.

[0029] The present invention provides the application of the above - mentioned bacterial agent in lignin degradation.

[0030] The present invention provides a method for degrading lignin using the above - mentioned corynebacterium, comprising the following steps: inoculating the corynebacterium into a medium containing lignin and performing fermentation culture.

[0031] The present invention provides a method for degrading lignin using the above - mentioned bacterial agent, comprising the following steps: inoculating the bacterial agent into a medium containing lignin and performing fermentation culture.

[0032] Effects of the invention

[0033] The present invention expands the strain library of lignin - degrading bacteria, which is beneficial to further improving the in - depth application of bacteria in the field of lignin degradation. Pseudomonas sp. MZK - 2 - 7 can grow rapidly and stably using a cheap medium, has a short fermentation period, and a high lignin degradation efficiency. This bacterium can be used for the biodegradation of straw to improve the straw utilization rate, and has high economic value and ecological value. Brief description of the drawings

[0034] Figure 1 It is the colony morphology diagram of the strain Pseudomonas sp. MZK - 2 - 7 of the present invention.

[0035] Figure 2 It is the aniline blue plate color development diagram of the strain Pseudomonas sp. MZK - 2 - 7 of the present invention.

[0036] Figure 3 It is the 16S identification electrophoresis diagram of the strain Pseudomonas sp. MZK - 2 - 7 of the present invention.

[0037] Figure 4 This is the phylogenetic tree of the strain Pseudomonas MZK-2-7 of the present invention.

[0038] Figure 5 This is the lignin degradation efficiency of the strain Pseudomonas MZK-2-7 of the present invention.

[0039] Figure 6 This is the growth curve of the strain Pseudomonas MZK-2-7 of the present invention. Detailed implementation manners

[0040] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following provides a detailed description by listing specific examples. Among them, the drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0041] The present invention provides a corynebacterium, which is Pseudomonas sp. MZK-2-7. The preservation number of the corynebacterium is CCTCC NO: M 20221892, and it is preserved in the China Center for Type Culture Collection on December 7, 2022, with the preservation address being Wuhan University, Wuhan, China.

[0042] In some embodiments, the 16S rDNA gene sequence of the corynebacterium is as shown in SEQ ID NO.1.

[0043] SEQ ID NO.1

[0044] CCGTGGTACCGTCCTCCTTGCGGTTAGACTAGCTACTTCTGGAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTTTGTACCGACCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCCGAGGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGCATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTATCGCGTTAGCTGCGCCACTAAGATCTCAAGGATCCCAACGGCTAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATCAGTCCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCACCCTCTACCGTACTCTAGCTCAGTA

[0045] GTTTTGGATGCAATTCCCAGGTTGAGCCCGGGGCTTTCACATCCAACTTGCTGAACCACCTAC

[0046] GCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTTCGTATTACCGCGGCTGCTGGC

[0047] ACGAAGTTAGCCGGTGCTTATTCTGTTGGTAACGTCAAAACAGCAAGGTATTAACTTACTGCCC

[0048] TTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCGGCATGGCTGGAT

[0049] CAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCT

[0050] CAGTTCCAGTGTGACTGATCATCCTCTCAGACCAGTTACGGATCGTCGCCTTGGTGGGCCATTA

[0051] CCCCACCAACTAGCTAATCCGACCTAGGCTCATCTGATAGCGTGAGGTCCGAAGATCCCCCACT

[0052] TTCTCCCGTAGGACGTATGCGGTATTAGCGTTCCTTTCGAAACGTTATCCCCCACTACCAGGCA

[0053] GATTCCTAGGCATTACTCACCCGTCCGCCGCTGAATCATGGAGCAAGCTCCACTCATCCGCTCG

[0054] ACTTGCATGTGTAG

[0055] The present invention provides a method for isolating and screening the above-mentioned Corynebacterium, comprising the following steps:

[0056] (1) Sampling and collection;

[0057] (2) Strain isolation;

[0058] (3) Strain screening;

[0059] (4) Strain identification;

[0060] (5) Strain cultivation.

[0061] In some embodiments, the above separation and screening method further includes strain preservation.

[0062] In some embodiments, the formulation of the lignin medium in step (2) is as follows: per liter of the medium, it contains 1 - 4 g of alkaline lignin, 0.2 - 2 g of KH 2 PO 4 、0.2 - 2 g of K 2 HPO 4 、0.5 - 5 g of NaCl, 0.1 - 1 g of MgCl 2 ·6H 2 O, 0.2 - 2 g of ammonium chloride, 0.04 - 0.4 g of CaCl 2 、1 mL of trace elements, 15 g of agar, 100 - 500 μL of vitamin B 12 , the formulation of the trace elements is boric acid 300 - 2100 mg / L, cobalt chloride hexahydrate 190 - 1400 mg / L, manganese chloride tetrahydrate 50 - 350 mg / L, zinc chloride 40 - 280 mg / L, nickel chloride hexahydrate 20 - 140 mg / L, sodium molybdate dihydrate 15 - 110 mg / L, copper chloride dihydrate 2 - 14 mg / L, ferrous sulfate heptahydrate 1 - 8 g / L, and the concentration of the vitamin B 12 solution is 100 mg / L.

[0063] In some embodiments, the formulation of the lignin medium in step (2) is as follows: per liter of the medium, it contains 2 g of alkaline lignin, 0.45 g of KH 2 PO 4 、0.45 g of K 2 HPO 4 、0.9 g of NaCl, 0.15 g of MgCl 2 ·6H 2 O, 0.4 g of ammonium chloride, 0.07 g of CaCl 2 、1 mL of trace elements, 15 g of agar, 100 μL of vitamin B 12 ; the formulation of the trace elements is boric acid 300 mg / L, cobalt chloride hexahydrate 190 mg / L, manganese chloride tetrahydrate 50 mg / L, zinc chloride 42 mg / L, nickel chloride hexahydrate 24 mg / L, sodium molybdate dihydrate 18 mg / L, copper chloride dihydrate 2 mg / L, ferrous sulfate heptahydrate 1.1 g / L, and the concentration of the vitamin B 12 solution is 100 mg / L.

[0064] In some embodiments, in step (3), the screening is performed using a PDA - aniline blue plate for color development.

[0065] In some embodiments, step (5) includes the following steps: inoculating Corynebacterium into the medium and performing fermentation culture at 30 - 60 °C.

[0066] In some embodiments, the method for isolating and screening the above-mentioned Corynebacterium includes the following steps:

[0067] (1) Sampling

[0068] Take fresh rumen fluid from the rumen of live cattle;

[0069] (2) Strain isolation and screening

[0070] Use a lignin screening plate to screen for microorganisms that can grow using alkaline lignin as the sole carbon source, and then use an aniline blue plate to further screen for dominant strains that can produce a clear zone;

[0071] (3) Strain identification

[0072] Use 27F / 1492R as primers for strain identification, then perform agarose gel electrophoresis, and perform sanger sequencing on the PCR products with normal electrophoresis results. Conduct BLAST analysis on the sequencing results and generate a phylogenetic tree;

[0073] (4) Strain preservation

[0074] Shake the bacteria in a lignin screening liquid medium and preserve the bacteria with 10-30% glycerol.

[0075] The present invention provides a bacterial agent, comprising the above-mentioned Corynebacterium and / or the fermentation broth of the Corynebacterium.

[0076] The present invention provides the application of the above-mentioned Corynebacterium in lignin degradation.

[0077] The present invention provides the application of the above-mentioned bacterial agent in lignin degradation.

[0078] The present invention provides a method for degrading lignin using the above-mentioned Corynebacterium, comprising the following steps: inoculating the Corynebacterium into a medium containing lignin and performing fermentation culture.

[0079] The present invention provides a method for degrading lignin using the above-mentioned bacterial agent, characterized by comprising the following steps: inoculating the bacterial agent into a medium containing lignin and performing fermentation culture.

[0080] Example 1

[0081] (1) Sampling

[0082] Take fresh rumen fluid from the cattle on Huangshan Farm. The sampling process should be strictly carried out under aseptic conditions to avoid contamination. The obtained sample is filled into a sterile glass bottle, placed in an ice box for preservation, and brought back to the laboratory for processing within 6 hours.

[0083] (2) Strain isolation

[0084] Dilute the fresh rumen fluid with 1× salt solution to 10 8 times, take 200 μL and evenly coat it on the lignin screening plate, and then place the plate in a biochemical incubator at 39 °C for incubation. After 24 h, monoclonal colonies grow on the plate. Pick monoclonal colonies with different morphologies and streak them on the lignin screening plate. Repeat this step 5-6 times until only monoclonal colonies with one morphology grow on the plate and the subculture is stable.

[0085] The formula of 1× salt solution is: 0.45 g KH 2 PO 4 per liter, 0.45 g K 2 HPO 4 per liter, 0.9 g NaCl, 0.15 g MgCl 2 .6H 2 O per liter, 0.4 g ammonium chloride, 0.07 g CaCl 2 per liter, pH 6.5. Use 20 mL glass test tubes, dispense 5 mL into each tube, sterilize at 121 °C for 30 min and set aside for use.

[0086] The formula of the lignin screening plate is: 2 g alkaline lignin per liter, 0.45 g KH 2 PO 4 per liter, 0.45 g K 2 HPO 4 per liter, 0.9 g NaCl, 0.15 g MgCl 2 .6H 2 O per liter, 0.4 g ammonium chloride, 0.07 g CaCl 2 per liter, 1 mL of trace elements, 15 g of agar, sterilize at 121 °C for 30 min, when cooled to about 60 °C, add 100 μL of vitamin B12, pour the plate, about 20 mL for each plate, and set aside for use.

[0087] The formula of trace elements is: boric acid 300 mg / L, cobalt chloride hexahydrate 190 mg / L, manganese chloride tetrahydrate 50 mg / L, zinc chloride 42 mg / L, nickel chloride hexahydrate 24 mg / L, sodium molybdate dihydrate 18 mg / L, copper chloride dihydrate 2 mg / L, ferrous sulfate heptahydrate 1.1 g / L.

[0088] The formula of B12 solution is: 100 mg B12 per liter. Filter and sterilize with 0.22 μm and add before use.

[0089] (3) Strain screening: Screening by color development on PDA-aniline blue plate

[0090] Lignin-degrading bacteria with laccase activity can form a transparent halo on PDA-aniline blue plates. The lignin-degrading bacteria with the identified strain are inoculated into the lignin screening liquid medium and cultured at 39 °C and 150 rpm for 48 h - 72 h. The lignin-degrading bacteria are inoculated on the PDA-aniline blue plates, and three parallel experiments are carried out for each lignin-degrading bacteria. The inoculation amount is 0.5 μL, and it is placed in an incubator at 28 °C for cultivation. Observe the above plates after overnight cultivation, measure the diameter of the transparent halo and the diameter of the colony, and calculate the ratio of the two to represent the degradation activity of the strain.

[0091] The formula of the PDA-aniline blue plate is: 200 g of peeled potatoes, after boiling in water for 30 min, the filtered liquid is added with 10 g of glucose, 2.5 g of yeast powder, 5 g of peptone, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, 15 g of agar, and made up to 1 L. Then 0.3 g / L of aniline blue is added to it.

[0092] (4) Strain identification

[0093] Pick the above monoclonal and dissolve it in 10 μL of sterile water. Take 2 μL of the bacterial solution as a template and perform PCR with 27F / 1492R as primers respectively. The remaining 8 μL of the bacterial solution is added to 2 mL of the lignin screening liquid medium for cultivation. The PCR products are subjected to agarose gel electrophoresis, and the samples with a single band and the correct size (27F / 1492R: about 1500 bp) in the electrophoresis results are sent for testing. The samples with normal sequencing are subjected to sequence alignment.

[0094] Primer information:

[0095] Primer Name Sequence (5' to 3') Function 27F AGAGTTTGATCCTGGCTCAG Full-length 16s amplification 1492R TACGGCTACCTTGTTACGACTT Full-length 16s amplification

[0096] PCR reaction system

[0097]

[0098]

[0099] PCR reaction program

[0100]

[0101] Electrophoresis information: 1% agarose gel, 120 V voltage, electrophoresis time 30 min.

[0102] Use NCBI to perform BLAST analysis on the returned sequence information, select the Fast Minimum Evolution method to construct a phylogenetic tree, and the relevant parameter settings are: maximum sequence difference = 0.75, and the sequence label is the classification name.

[0103] (5) Strain preservation

[0104] The selected strains were cultured in 50 mL of lignin liquid medium by shaking for subsequent determination of lignin degradation efficiency. When the bacteria grew to the logarithmic phase, three aliquots of 750 μL of the bacterial solution were taken and added to three tubes containing 250 μL of 50% glycerol, and then stored in a -80 °C refrigerator.

[0105] Example 2

[0106] (1) Determination of alkaline lignin degradation efficiency

[0107] Lignin has a maximum absorption peak at 320 nm. By measuring the absorbance value at this wavelength, the lignin content can be calculated.

[0108] The glycerol bacteria of the lignin-degrading bacteria of the identified strains were cultured by shaking in lignin and LB liquid media. After culturing at 39 °C and 150 rpm for 48 h, the protein concentration was measured. Pseudomonas sp. MZK-2-7 was inoculated into the alkaline lignin liquid medium at the final protein concentrations of 10 μg / mL and 50 μg / mL, respectively, and the alkaline lignin medium without inoculated bacterial solution was used as a blank control. The experimental group and the control group were cultured at 39 °C and 150 rpm. Every 24 h, 1 mL of the culture was taken, centrifuged at 12,000 g for 5 min, the supernatant was used to measure the lignin absorbance, and the precipitate was used for protein concentration measurement. The spectrophotometer was preheated for more than 30 min, the wavelength was adjusted to 320 nm, and distilled water was used to zero the instrument. The absorbance value just after inoculation was measured as the initial value of the lignin content and denoted as A0. The changes in the absorbance values of the cultures were continuously measured, and the data were denoted as A1, A2, A3, and A4, respectively. ΔA1 = A1 - A0, ΔA2 = A2 - A0, and so on. The lignin degradation efficiency per day can be calculated.

[0109] Subtracting the degradation efficiency of the control group from that of the experimental group gives the net degradation efficiency of MZK-2-7 for alkaline lignin. Using this value as the ordinate and the culture time as the abscissa, the lignin degradation efficiency graph of MZK-2-7 can be obtained ( Figure 5 ).

[0110] (2) Determination of bacterial protein by Bradford method

[0111] Take out the Bradford staining solution and equilibrate it to room temperature. Take out the BSA samples and thaw them at room temperature. Prepare a series of BSA protein standards with concentrations of 0 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, and 400 μg / mL respectively. Add 1 mL of 1 M NaOH to the cell pellet of Pseudomonas sp. MZK-2-7 and heat it in a metal bath at 100 °C for 15 min, then centrifuge at 12,000 g for 10 min. Take 25 μL each of the standard solution and the sample to be tested and add them to 1.5 mL centrifuge tubes. Add 1 mL of Bradford working solution to each tube and mix quickly. Take 900 μL of the liquid in the sample tube and measure the absorbance value at A595 using a spectrophotometer. Plot a standard curve with the OD values measured for the standard group as the ordinate and the corresponding protein concentrations as the abscissa. Substitute the OD value measured for the sample into the standard curve to calculate the protein concentration of the sample.

[0112] The growth of the strain was represented by the protein concentration, and a growth curve of MZK-2-7 was obtained by plotting the number of days of culture as the abscissa and the protein concentration as the ordinate ( Figure 6 ).

[0113] Example 3

[0114] 1. Determination of straw degradation efficiency

[0115] (1) Activation of the strain

[0116] Inoculate a loopful of the slant culture of Pseudomonas sp. MZK-2-7 onto the lignin screening solid medium and culture it at 39 °C for 24 - 48 h to obtain the activated strain;

[0117] (2) Preparation of the bacterial suspension

[0118] Inoculate the activated strain into 4 mL of the lignin screening liquid medium and culture it on a shaker at 39 °C and 200 rpm for 12 - 16 h to obtain the overnight culture; Take 2 mL of the overnight culture and inoculate it into 100 mL of the lignin screening liquid medium and culture it on a shaker at 39 °C and 200 rpm for 1 - 2 days to obtain the bacterial suspension;

[0119] (3) Treatment of straw

[0120] Dry the straw at 65 °C, crush it; Sieve it through a 20-mesh sieve and dry it again at 65 °C to obtain the straw powder;

[0121] (4) Preparation of the straw degradation medium:

[0122] The formula of the straw degradation medium is as follows: Each liter of the medium contains 100 g of straw powder (20 mesh), 1.35 g of KH 2 PO 4 、1.35 g of K2 HPO 4 、2.70 g NaCl, 0.45 g MgCl 2 ·6H 2 O, 1.20 g ammonium chloride, 0.21 g CaCl 2 、3 mL trace elements; the formula of the trace elements is boric acid 300 mg / L, cobalt chloride hexahydrate 190 mg / L, manganese chloride tetrahydrate 50 mg / L, zinc chloride 42 mg / L, nickel chloride hexahydrate 24 mg / L, sodium molybdate dihydrate 18 mg / L, copper chloride dihydrate 2 mg / L, ferrous sulfate heptahydrate 1.1 g / L.

[0123] Prepare 6 500-ml conical flasks, and prepare 150 ml of fermentation medium in each conical flask according to the above formula. Sterilize at 121 °C for 90 minutes. After cooling to room temperature, add 45 μL of sterile vitamin B 12 ,the vitamin B 12 solution has a concentration of 100 mg / L.

[0124] (5) Inoculation and cultivation:

[0125] Take the bacterial suspension in step (2) and add it to 3 of the above conical flasks containing the straw degradation medium according to an inoculation ratio of 5%. The remaining 3 flasks are used as controls. The above 6 conical flasks are cultured at a temperature of 39 °C and a rotation speed of 150 rpm. Terminate the reaction at a predetermined time point, recover the degraded straw and dry it to a constant weight at 65 °C. Use the weight loss method to detect the degradation products. After one week of degradation, the weight loss rate of the straw reaches 27.47%.

[0126] 2. Determination of lignin, cellulose, and hemicellulose contents in straw

[0127] Experimental operation steps, methods, and result evaluation:

[0128] (1) Specimen preparation (performed according to the provisions of NY / T 3492)

[0129] Cut short: Cut the crop straw short simply to facilitate subsequent drying and grinding.

[0130] Drying: Place a clean weighing pan in an electric blast drying oven at (45 ± 3) °C. After drying for 3 hours, take it out and cool it to room temperature in a room temperature environment (20 - 30 °C, relative humidity less than 50%, it can be cooled in a desiccator). Place the cut straw in the dried weighing pan and put it in an electric blast drying oven at (45 ± 3) °C. After drying for 36 hours, take it out and cool it to room temperature. Then put it back in an electric blast drying oven at (45 ± 3) °C. After drying for 1 hour, take it out and cool it to room temperature, and weigh the mass of the weighing device (accurate to 0.1 g). Repeat this operation until the mass difference of consecutive weighings does not exceed 1% of the sample mass. For easy drying, the thickness of the sample laid should not exceed 10 mm.

[0131] Crushing and sieving: The dried sample is crushed using a cutting-type crushing device and sieved using an experimental sieve with a size of 850 μm. If there is oversize material, repeat the crushing and sieving until all passes through the 850 μm test sieve. Combine all the undersize materials and mix them thoroughly. Avoid overheating the sample during the crushing process, and the crushing device should operate at the lowest possible speed to reduce dust loss.

[0132] Quartering: Quarter the crushed and sieved sample using the coning and quartering method, and repeat the quartering process until the sample amount required for analysis is obtained (not less than 50 g).

[0133] Storage and identification: The sample should be stored in a sealed container and should be labeled with information such as the type of sample with a unique identifier.

[0134] (2) Extraction

[0135] Water extraction: Weigh approximately 3 g of the sample (accurate to 0.1 mg) into a pre-weighed filter paper tube. Place the filter paper tube into the extraction thimble of a Soxhlet extractor, connect it to a receiving flask that has been dried to a constant weight. Add 190 mL of water from the upper end of the condenser of the extractor, heat it on an electric heating mantle, and make the water continuously reflux for extraction (4 - 5 times / h), generally for 6 - 8 h. After the extraction is completed, turn off the heating mantle and cool the Soxhlet extractor to room temperature.

[0136] Ethanol extraction; Connect the extraction thimble after water extraction to a receiving flask that has been dried to a constant weight. Add 190 mL of ethanol from the upper end of the condenser of the extractor, heat it on an electric heating mantle, and make the ethanol continuously reflux for extraction (6 - 10 times / h), generally for 16 - 24 h. After the extraction is completed, turn off the heating mantle and cool the Soxhlet extractor to room temperature. Dry the biomass sample after two-step extraction (i.e., the sample without extractives) in a drying oven at 45 ± 3 °C to a constant weight, and weigh the mass of the sample accurate to 0.1 mg.

[0137] (3) Two-step acid hydrolysis

[0138] Constant weight of crucible: Place the glass sand core crucible (G4) in a muffle furnace and calcine it to constant weight at 575 ± 25 °C. After taking the crucible out of the muffle furnace, put it in a desiccator to cool, and weigh it accurately to 0.1 mg.

[0139] Hydrolysis with concentrated acid: Weigh 300.0 mg (accurate to 0.1 mg) of the extract-free sample into a pressure-resistant tube, and conduct more than 2 parallel experiments for each sample. Immediately add 3.00 mL of 72% sulfuric acid solution to the pressure-resistant tube and mix well immediately. Place the pressure-resistant tube in a water bath at 30 ± 3 °C and stir it once every 5 - 10 min. After keeping the temperature constant for 60 min, take it out, add 84.00 mL of water to the pressure-resistant tube, and tighten the lid to mix well.

[0140] Sugar recovery rate: Prepare sugar standard solutions for calculating the recovery rate, including D(+)-glucose, D(+)-xylose, D(+)-galactose, L(+)-arabinose, and L(+)-mannose. The concentration of the sugar recovery standard solution should be close to the sugar concentration of the test sample. Weigh the mass of each sugar accurately to 0.1 mg, add 10.0 mL of water, then add 348 μL of 72% sulfuric acid solution, tighten the lid to mix well. Filter it with a microporous filter, dispense it into sample bottles, and store it frozen. Thaw and shake it well before use.

[0141] Hydrolysis with dilute acid: Place the pressure-resistant test tubes containing the sample and the sugar recovery standard solution in an autoclave and hydrolyze them at 121 °C for 1 hour. Wait for the hydrolysis product to cool to room temperature, filter it through a glass sand core crucible (G4), collect about 50 mL of the filtrate with a conical flask, and transfer it to a stoppered container for storage at 0 - 4 °C. Determine the acid-soluble lignin within 6 hours.

[0142] (4) Determination

[0143] Acid-soluble lignin: Use the above-mentioned filtered dilute acid hydrolysis solution to measure the absorbance value of the liquid sample at 320 nm with a UV-visible spectrophotometer. Use water as the blank for the UV-visible spectrophotometer and dilute it with water until the absorbance is 0.7 - 1.0. Record the dilution factor and record the absorbance value accurately to 0.001.

[0144] Acid-insoluble lignin: Rinse the acid-insoluble residue remaining in a 100 mL stoppered pressure-resistant test tube with more than 50 mL of hot water, so that all the residue remains in the glass filter crucible (G4), and dry it with a vacuum filter. Dry the glass filter crucible (G4) and the acid-insoluble residue at 105 ± 3 °C to a constant weight, record the mass of the glass filter crucible and the acid-insoluble residue, accurate to 0.1 mg. Place the glass filter crucible (G4) and the acid-insoluble residue in a muffle furnace and calcine at (575 ± 25) °C for at least 3 hours until all the organic matter is ashed. Control the heating rate at 10 °C / min to prevent the sample from burning and mechanical loss of the sample caused by strong air currents. After ashing, cool to 105 °C, take out and place in a desiccator to cool. Weigh the crucible and the ash accurately to 0.1 mg.

[0145] Carbohydrates: Chromatographic conditions

[0146] Item Condition High Performance Liquid Chromatograph Shimadzu High Performance Liquid Chromatograph: Equipped with a differential refractive index detector Chromatographic Column Rezex ROA-Organic Acid H+(8%) Chromatographic Column 300×7.8mm Mobile Phase 5mM Sulfuric Acid Flow Rate 0.6mL / min Column Temperature 50℃ Injection Volume 10μL Detector Differential Refractive Index Detector, Detector Temperature 50°C

[0147] Drawing of the standard curve: Prepare a mixed standard solution of D-cellobiose, D(+)-glucose, and D(+)-xylose according to the concentration ranges recommended in the following table, and use the five-point calibration method. Respectively pipette 10 μL of the standard solution into a high-performance liquid chromatograph, and measure the response value (peak area) of the standard solution under the above chromatographic conditions. Draw a standard curve with the concentration as the abscissa and the peak area as the ordinate.

[0148] The recommended mass concentration ranges of the sugar standard solutions are as follows:

[0149] Component Mass Concentration Range, mg / mL D-Cellobiose 0.1～4.0 D(+) Glucose 0.1～4.0 D(+) Xylose 0.1～4.0

[0150] Determination of the sample solution: Take 10 mL of the above-filtered dilute acid hydrolysis solution into a 50 mL centrifuge tube, slowly add 1 g of calcium carbonate to neutralize the hydrolysis solution to pH 5 - 6. Centrifuge the mixture at 5000 g for 10 min, filter the supernatant with a 0.22 μm microporous filter, and then perform high-performance liquid chromatography analysis. Calculate the content of various sugars. If the content of cellobiose in the sample is higher than 3 mg / mL, it indicates that the hydrolysis is incomplete. If there is a peak before cellobiose, it indicates that the sugars in the sample have undergone excessive hydrolysis.

[0151] (5) Result calculation

[0152] ① Content of acid-soluble lignin

[0153] The content of acid-soluble lignin ASL in the sample, expressed as a mass percentage (%), is calculated according to the following formula.

[0154]

[0155] In the formula: ASL---Content of acid-soluble lignin in the sample, unit is mass percentage (%);

[0156] -- Average UV-visible absorbance value of the filtrate at 320 nm;

[0157] V --- Volume of the filtrate, with a value of 87 mL;

[0158] N --- Dilution factor of the filtrate;

[0159] L --- Thickness of the cuvette, in centimeters (cm);

[0160] ε --- Absorptivity of acid-soluble lignin at 320 nm, taking 30 L / (g·cm) for corn stover and 25 L / (g·cm) for other raw materials;

[0161] w 0 --- Mass of the sample before extraction, in grams (g);

[0162] w 1 ---- Mass of the sample after extraction, in grams (g);

[0163] w ef ---- Mass of the sample without extractives taken, in grams (g).

[0164] ② Acid-insoluble lignin content

[0165] The acid-insoluble lignin content AIL in the sample, expressed as a mass percentage (%), is calculated by the following formula

[0166]

[0167] In the formula: AIL --- Acid-insoluble lignin content in the sample, in mass percentage (%);

[0168] m 1 --- Mass of the glass sand core crucible, in g;

[0169] m 2 ---- Mass of the glass sand core crucible and acid-insoluble residue, in g;

[0170] m 3 --- Mass of the glass sand core crucible and ash, in g.

[0171] ③ Lignin content

[0172] The total lignin content Lig in the sample, expressed as a mass percentage (%), is calculated by the following formula

[0173] Lig = ASL + AIL

[0174] In the formula: Lig --- Total lignin content in the sample, in mass percentage (%);

[0175] ASL---Acid-soluble lignin content in the sample, in mass percentage (%).

[0176] AIL---Acid-insoluble lignin content in the sample, in mass percentage (%).

[0177] ④Cellulose content and hemicellulose content

[0178] Calculate the sugar recovery rate according to the following formula:

[0179]

[0180] where C i1 is the mass concentration measured by HPLC of the i-th sugar recovery solution prepared, and C i0 is the mass concentration of the i-th sugar solution prepared.

[0181] Determine the mass Z i of glucose and xylose, expressed in mass fraction (%).

[0182]

[0183] where C i represents the mass concentration of the i-th monosaccharide measured by HPLC, in mg / mL.

[0184] F: is the dehydration correction factor, 0.88 for xylose and arabinose, and 0.9 for glucose, galactose and mannose.

[0185] V: the volume after dilute acid digestion, 87 mL.

[0186] W 1 : the mass of the sample after extraction, in g.

[0187] R i : the sugar recovery rate calculated above.

[0188] W ef : the mass of the sample without extractives weighed.

[0189] W 0 : the mass of the sample before extraction, in g.

[0190] Content of cellulose: Cel = Z glucose

[0191] Z glucose: the content of glucose in the sample, in mass percentage (%).

[0192] Content of hemicellulose: Hem = Z xylose

[0193] Z xylose: the content of xylose in the sample, in mass percentage (%)

[0194] The degradation efficiency is shown in Table 1. The strain Pseudomonas MZK-2-7 of the present invention can efficiently degrade lignin in straw in a short time, while the degradation rates of cellulose and hemicellulose are low, with high efficiency and specificity.

[0195] Table 1 Straw degradation efficiency of the strain Pseudomonas MZK-2-7 of the present invention

[0196] Sample Name Control Group Experimental Group Lignin Content (g) 2.2646 1.9479 Cellulose Content (g) 3.8443 3.7735 Hemicellulose Content (g) 1.6882 1.6304 Lignin Degradation Efficiency (%) / 13.98 Cellulose Degradation Efficiency (%) / 1.84 Hemicellulose Degradation Efficiency (%) / 3.42

[0197] It should be understood that the above embodiments are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various modifications and changes can be made based on the above embodiments. Similarly, the technical features of the above embodiments can be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. A Corynebacterium characterized in that The corynebacterium is Pseudomonas (< Pseudomonas sp. ) MZK-2-7, and the preservation number of the corynebacterium is CCTCC NO: M 20221892. It is preserved in the China Center for Type Culture Collection, with the preservation date being December 7, 2022, and the preservation address being Wuhan University, Wuhan, China.

2. The Corynebacterium according to claim 1 characterized in that the 16S rDNA gene sequence of the Corynebacterium is as shown in SEQ ID NO.

1.

3. A bacterial agent characterized in that it comprises the Corynebacterium according to any one of claims 1-2 and / or the fermentation broth of the Corynebacterium.

4. Use of the Corynebacterium according to any one of claims 1-2 in lignin degradation.

5. Use of the bacterial agent according to claim 3 in lignin degradation.

6. A method for degrading lignin using the Corynebacterium according to any one of claims 1-2 characterized in that it comprises the following steps inoculating the Corynebacterium into a medium containing lignin and performing fermentation culture.

7. A method for degrading lignin using the bacterial agent according to claim 3 characterized in that it comprises the following steps inoculating the bacterial agent into a medium containing lignin and performing fermentation culture.

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