Lignin-degrading bacteria, lignin or straw degradation method and application of lignin-degrading bacteria

Abstract

The application provides a lignin-degrading bacterium, a lignin or straw degradation method and application of the lignin-degrading bacterium. Kluyvera georgiana ​ The lignin-degrading bacterium is Georgiabacter sp. (Georgiabacter sp.), which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC NO. 33528. The lignin-degrading bacterium is separated from the intestine of a barleyworm which feeds on straw and has high lignin and straw degradation capacity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of lignin degradation, and particularly relates to a lignin-degrading bacterium, a lignin or straw degradation method, and application of the lignin-degrading bacterium. BACKGROUND

[0002] Crop straw is used as a rich feed resource in China, and particularly shows great application potential in the feeding of ruminant livestock. However, the biochemical characteristics of straw generally limit its practical application in production. The crude protein content of straw is low, and most of the crude protein is tightly combined with the cell wall, resulting in low digestibility and degradation rate. The cell wall of straw is mainly composed of cellulose, hemicellulose and lignin. Among them, lignin has a complex, stable and diverse amorphous three-dimensional structure. Lignin is combined with hemicellulose through covalent bonds, embedding cellulose molecules therein, forming a natural barrier, so that microorganisms are difficult to contact with the cellulose molecules, thereby reducing the utilization rate and nutritional value of straw. Traditional nutrition believes that the lignin content is inversely proportional to the digestibility of straw. For every 1% reduction in lignin content in the diet, the digestibility of the animal can be increased by 4% to 5%. In addition, as an important source of aromatic resources, the degradation products of lignin can meet the demand of the pharmaceutical industry for aromatic substances, which provides a new way to reduce the dependence on non-renewable resources such as coal and oil. Therefore, in the process of straw treatment, the degradation of lignin is the key to improving the utilization efficiency of straw.

[0003] The commonly used methods for breaking lignin at present include radiation, steam explosion, puffing, grinding, acidolysis, alkali treatment, oxidation treatment and organic solvent method. These methods aim to break the close structure of lignin and cellulose, so as to improve the nutritional value and utilization rate of straw. The Chinese patent document with the publication number CN104004201A discloses a method for degrading lignin in an acidic eutectic solvent. The lignin raw material, the eutectic solvent and water are added to a reactor, and the homogeneous catalytic degradation is carried out under the conditions of normal pressure and 90-130 DEG C. The application utilizes the acidic eutectic solvent as a solvent and a catalyst to degrade lignin. The eutectic solvent can be recycled and reused, and has the advantages of simple process and low cost. The Chinese patent document with the publication number CN106146757A pretreats corn straw by steam explosion, uses high-temperature and high-pressure steam to instantaneously release pressure to break the wrapping effect of lignin and hemicellulose on cellulose, separates and removes most of the hemicellulose and pectin, and uses sodium chlorite to oxidize and degrade lignin.

[0004] Traditional physical and chemical methods can remove some lignin from plant fiber raw materials, but they bring a series of problems such as high energy consumption, secondary pollution, high cost, and safety concerns regarding livestock feed. Therefore, seeking safe biological methods for lignin degradation is an important way to utilize straw as feed. This can not only reduce environmental pollution and save energy, but also turn waste into treasure and realize the reuse of straw resources. Currently, in the field of biological lignin degradation, only white-rot fungi can completely degrade lignin into CO2 and H2O under normal temperature and pressure. However, fungal degradation of lignin has problems such as poor environmental adaptability, long pretreatment cycle, and susceptibility to spore contamination. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a lignin-degrading bacterium, a method for degrading lignin or straw, and the application of the lignin-degrading bacterium, which is isolated from the intestine of a mealworm that feeds on straw, and is *Georgia clerophylla* (*Georgia clerophylla*). Kluyvera georgiana It has efficient lignin and straw degradation capabilities.

[0006] To address the aforementioned problems, a first aspect of the present invention provides a lignin-degrading bacterium isolated from mealworms, wherein the lignin-degrading bacterium is *Georgia clerophylla* (*Georgia clerophylla*). Kluyvera georgiana (), deposited at the China General Microbiological Culture Collection Center, accession number CGMCC NO. 33528.

[0007] Preferably, the 16S rRNA sequence of the lignin-degrading bacteria is shown in SEQ ID NO.1.

[0008] A second aspect of the present invention provides a method for degrading lignin, comprising:

[0009] The lignin-degrading bacteria isolated from mealworms were cultured; alkali lignin, nutrients and water were mixed to obtain a first mixture, wherein the nutrients did not contain a carbon source; the cultured lignin-degrading bacteria were added to the first mixture to degrade lignin.

[0010] Preferably, the cultivation of lignin-degrading bacteria specifically includes: inoculating the lignin-degrading bacteria into LB medium, culturing at 20-40℃ and 150-250 r / min to the logarithmic growth phase, then centrifuging, washing with PBS, and resuspending the bacterial solution to obtain the lignin-degrading bacterial solution.

[0011] Preferably, the nutrients include K2HPO4 and M. g SO4·7H2O, CaCl2, FeSO4·7H2O, MnCl2, KH2PO4 and peptone;

[0012] The alkali lignin is 2.5-3.5 g / L, K2HPO4 is 0.5-1.5 g / L, M g SO4·7H2O is 0.005-0.015 g / L, CaCl2 is 0.05-0.13 g / L, FeSO4·7H2O is 0.03-0.08 g / L, MnCl2 is 0.01-0.03 g / L, KH2PO4 is 0.5-1.5 g / L, and the protein peptone is 1.5-2.5 g / L.

[0013] Preferably, when the lignin-degrading bacteria obtained by culture are added to the first mixed solution for lignin degradation, the temperature is 20-40℃, the rotation speed is 150-250r / min, and the treatment time is 12-120h.

[0014] The third aspect of the present application provides a degradation method of straw, comprising:

[0015] The lignin-degrading bacteria separated from the lesser grain borer are cultured, the straw is mixed with water to obtain a second mixed solution, and the lignin-degrading bacteria obtained by culture are added to the second mixed solution for degradation of the straw.

[0016] Preferably, the culture of the lignin-degrading bacteria specifically comprises: preparing LB culture medium, sterilizing, cooling to 20-30℃, inoculating the lignin-degrading bacteria into the LB culture medium, and culturing at 20-40℃ and 80-200r / min to the logarithmic growth phase.

[0017] Preferably, when the lignin-degrading bacteria obtained by culture are added to the mixed solution for degradation of the straw, the temperature is 20-40℃, the humidity is 70%-80%, and the treatment time is 5-30 days.

[0018] The fourth aspect of the present application provides application of the lignin-degrading bacteria separated from the lesser grain borer in lignin or straw degradation.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The lignin-degrading bacteria are separated from the intestine of the lesser grain borer which feeds on straw, and the MALDI-TOF MS and 16S rRNA identification results show that the bacteria are Kluyvera georgiana. Kluyvera georgiana It has high lignin and straw degradation capacity. In the culture medium with lignin as the only carbon source, the lignin degradation rate reaches 14.8% after 120h of treatment of the strain, the highest laccase activity reaches 81.7 U / L during the treatment process, and the highest manganese peroxidase activity reaches 254.9 U / L. K. georgianaAfter 30 days of wheat straw treatment, the straw loss rate reached 9.9%, the strain laccase activity reached 154.3 U / L at most; and the manganese peroxidase activity reached 868.2 U / L at most. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the strain growth curve and lignin degradation curve in the embodiment 2 of the present application;

[0022] Figure 2 is the enzyme production in the process of lignin treatment in the embodiment 2 of the present application;

[0023] Figure 3 is the influence of the lignin degrading strain treatment on the straw weight loss in the embodiment 3 of the present application;

[0024] Figure 4 is the laccase production in the process of straw treatment in the embodiment 3 of the present application;

[0025] Figure 5 is the manganese peroxidase production in the process of straw treatment in the embodiment 3 of the present application;

[0026] Figure 6 is the change of straw structure after the lignin degrading strain treatment in the embodiment 3 of the present application.

[0027] Figure 7 is the change of lignin structure after the lignin degrading strain treatment in the embodiment 2 of the present application. DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0029] The first aspect of the embodiment of the present application provides a lignin degrading bacteria separated from the barley worm, and the lignin degrading bacteria is Kluyvera georgiana. Kluyvera georgiana), which was deposited with the China General Microbiological Culture Collection Center on February 14, 2025, and has the accession number CGMCC NO. 33528. The lignin-degrading bacteria isolated from the barley worm has high lignin and straw degradation capacity. In the culture medium with lignin as the sole carbon source, the lignin degradation rate of the strain reached 14.8% after 120 h of treatment, and the highest laccase activity reached 81.7 U / L during the treatment process, and the highest manganese peroxidase activity reached 254.9 U / L. After 30 days of treatment of the wheat straw by the lignin-degrading bacteria, the straw loss rate reached 9.9%, the highest laccase activity of the strain reached 154.3 U / L, and the highest manganese peroxidase activity reached 868.2 U / L.

[0030] Preferably, the 16S rRNA sequence of the lignin-degrading bacteria is shown in SEQ ID NO. 1.

[0031] The sequence structure of SEQ ID NO. 1 is as follows:

[0032]

[0033] The second aspect of the embodiment of the present application provides a lignin degradation method, comprising:

[0034] The lignin-degrading bacteria separated from the larvae of the Zeuzera pyrina are cultured; alkali lignin, nutrients and water are mixed to obtain a first mixed solution, the nutrients do not contain a carbon source; the lignin-degrading bacteria obtained by the culture are added to the first mixed solution to degrade the lignin.

[0035] Compared with various physical and chemical methods, biological degradation, especially the treatment of lignin by using a lignin-degrading bacterial strain, has the advantages of less pollution, environmental friendliness and low degradation cost. Fungi are currently recognized as microorganisms with higher lignin degradation capacity than other bacterial species, but fungi grow slowly, have poor environmental adaptability and are easily contaminated by spores, so the research on the biological degradation of lignocellulose by using fungi as the main model strain has not developed a feasible industrial degradation route for a long time. Compared with fungi, bacteria can also secrete lignin-degrading enzymes, and grow faster, have a simple structure and have stronger adaptability to complex environments. The present application provides a bacterial strain from the intestine of the Zeuzera pyrina Kluyvera georgiana , which has obvious straw and lignin degradation capacity and can be used as a lignocellulose feasible industrial degradation bacterial strain. The lignin degradation method uses the bacterial strain Kluyvera georgiana , which has high lignin degradation capacity, and the lignin degradation rate of the strain reaches 14.8% after 120h of treatment in a culture medium with lignin as the only carbon source, and the highest laccase activity reaches 81.7 U / L and the highest manganese peroxidase activity reaches 254.9 U / L during the treatment process.

[0036] Preferably, the culturing of the lignin-degrading bacteria specifically comprises: inoculating the lignin-degrading bacteria into an LB culture medium, culturing at 20-40℃ and 150-250r / min to the logarithmic growth phase, then centrifuging and washing with PBS to resuspend the bacterial solution to obtain a lignin-degrading bacterial solution.

[0037] Preferably, the nutrients comprise K2HPO4, M g SO4·7H2O, CaCl2, FeSO4·7H2O, MnCl2, KH2PO4 and proteose peptone;

[0038] In the first mixed solution, the alkali lignin is 2.5-3.5g / L, the K2HPO4 is 0.5-1.5g / L, the M gSO4·7H2O is 0.005-0.015g / L, CaCl2 is 0.05-0.13g / L, FeSO4·7H2O is 0.03-0.08g / L, MnCl2 is 0.01-0.03g / L, KH2PO4 is 0.5-1.5g / L, and peptone is 1.5-2.5g / L.

[0039] Preferably, when the lignin-degrading bacteria obtained by culture are added to the first mixed solution for lignin degradation, the temperature is 20-40 DEG C, the rotation speed is 150-250r / min, and the treatment time is 12-120h.

[0040] The third aspect of the application provides a straw degradation method, comprising:

[0041] The lignin-degrading bacteria separated from the Tenebrio molitor are cultured, the straw is mixed with water to obtain a second mixed solution, and the lignin-degrading bacteria obtained by culture are added to the second mixed solution for straw degradation.

[0042] The straw degradation method adopts the bacterial strain Kluyvera georgiana which has high straw degradation capacity, and the bacterial strain Kluyvera georgiana After treating the wheat straw for 30 days, the straw loss rate reaches 9.9%, the strain laccase activity reaches a maximum of 154.3 U / L, and the manganese peroxidase activity reaches a maximum of 868.2 U / L.

[0043] Preferably, the culture of the lignin-degrading bacteria specifically comprises: preparing LB culture medium and sterilizing, cooling to 20-30 DEG C, inoculating the lignin-degrading bacteria into the LB culture medium, and culturing at 20-40 DEG C and 80-200r / min to the logarithmic growth phase.

[0044] Preferably, when the lignin-degrading bacteria obtained by culture are added to the mixed solution for straw degradation, the temperature is 20-40 DEG C, the humidity is 70%-80%, and the treatment time is 5-30 days.

[0045] The fourth aspect of the application provides application of the lignin-degrading bacteria separated from the Tenebrio molitor in lignin or straw degradation.

[0046] Example 1: Screening and identification of lignin-degrading bacterial strains

[0047] The larvae of the mealworms fed with wheat straw were washed with distilled water and 75% ethanol (30 seconds each time) in turn, and then the surface of the larvae was wiped dry with sterile filter paper. The intestinal tissues (including the foregut, midgut and hindgut) of the larvae were dissected on a clean bench using sterile scissors and tweezers, and then transferred to a 50 mL centrifuge tube, 10 mL of pre-cooled PBS was added, and the mixture was ground and vortexed. In order to screen lignin-degrading bacteria, an appropriate amount of vortexed sample was cultured in a lignin-rich medium (alkali lignin as the sole carbon source). The culture conditions were 28°C, aerobic and anaerobic for 2 days each. Single colonies were inoculated into TSB aerobic medium (HB4114) and triple mixed anaerobic medium, and then cultured at 28°C for 2 days under aerobic and anaerobic conditions, respectively.

[0048] After preliminary identification of the bacteria by MALDI-TOF-MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry), the selected colonies were collected on a 96-well plate. The colonies in the 96-well plate were cultured on an alkali lignin medium at 28°C for 2 days under aerobic and anaerobic conditions. Single colonies were further cultured in aerobic TSB medium and anaerobic triple mixed medium for 2 days. According to the reported 16S rRNA gene of the colonies, PCR amplification and DNA sequencing were performed to identify the genus or species of the bacteria. 16S rRNA amplification used 27F and 1492R as primers; the 16S rRNA sequencing sequence of the bacteria was identified by BLAST search in the GenBank database in NCBI. The results of MALDI-TOF-MS and 16S rRNA sequencing identification showed that the isolated lignin-degrading bacteria were Kluyvera georgiana, and the 16s rRNA sequence similarity was 98.16%. The 16S rRNA sequence of the lignin-degrading bacteria is shown as SEQ ID NO. 1.

[0049] The sequence structure of SEQ ID NO. 1 is as follows:

[0050]

[0051] The lignin-degrading bacteria are preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO. 33528.

[0052] Example 2 lignin degradation experiment

[0053] (1) Preparation of lignin culture medium

[0054] The liquid lignin culture medium contains: alkali lignin 3 g / L, K2HPO41 g / L, MgSO4·7H2O 0.01 g / L, CaCl20.08 g / L, FeSO4·7H2O 0.05 g / L, MnCl20.02 g / L, KH2PO41 g / L, and protein peptone 2 g / L. g SO4·7H2O 0.01g / L, CaCl20.08g / L, FeSO4·7H2O 0.05g / L, MnCl20.02 g / L, KH2PO41g / L, and protein peptone 2 g / L.

[0055] (2) Lignin treatment

[0056] The isolated lignin-degrading bacterial strain was inoculated in the LB culture medium, and after 30°C, 180 r / min culture to the logarithmic growth phase, 5000 r / min centrifugation for 5 min, and after washing twice with PBS, the bacterial suspension was resuspended, and the bacterial suspension was inoculated in the lignin culture medium at a 5% inoculation amount, and continuously cultured at 30°C, 180 r / min. The difference in absorbance at 600 nm before centrifugation of the culture liquid was used to reflect the growth condition of the strain, and the absorbance value at 280 nm was used as the determination index of lignin concentration, and the lignin degradation rate (%) at the nth hour was (A0-A n ) / A0×100; and the activity of lignin-degrading enzyme in the culture liquid was determined, and the determination method was detected according to the kit instructions of Beijing Solabio Biotechnology Co., Ltd., including lignin peroxidase (Lip, BC1615), laccase (Laccase, BC1635), and manganese peroxidase (Mnp, BC1625). The culture liquid after treatment was centrifuged at 12000 r / min for 10 min, and the supernatant was quickly frozen in liquid nitrogen and placed in a vacuum drying instrument to dry to constant weight. The dried sample was sprayed with gold and observed by scanning electron microscopy. As shown in Figure 7 .

[0057] (3) Treatment effect of lignin-degrading bacterial strain on lignin

[0058] As shown in Figure 1As shown, the lignin-degrading bacteria can grow in the medium with lignin as the only carbon source, and the growth reaches the maximum at 24 h. Meanwhile, the results show that the lignin content is significantly reduced during the culture process, and tends to be stable after 96 h. In addition, the lignin-degrading enzyme activity detection results show that no Lip is detected during the treatment of the strain, and Mnp and Laccase play a major role in lignin degradation Figure 2 In the medium with lignin as the only carbon source, the lignin degradation rate reaches 14.8% after the strain is treated for 120 h, the laccase activity reaches a maximum of 81.7 U / L during the treatment process, and the manganese peroxidase activity reaches a maximum of 254.9 U / L.

[0059] Example 3 Straw degradation experiment

[0060] (1) Straw collection

[0061] Straw particles between 40 and 60 meshes were collected, and then dried to constant weight at 75°C as samples for subsequent experiments.

[0062] (2) Bacterial culture

[0063] Prepare 100 ml of LB medium, sterilize at 121°C for 20 min, and after cooling to room temperature, inoculate the lignin-degrading bacteria into the liquid medium, and place it in a constant temperature shaking incubator, cultivate at 30°C, 120 r / min to the logarithmic growth phase.

[0064] (3) Biological pretreatment of straw

[0065] Add 15 ml of distilled water to the conical flask containing 5 g of crushed straw, sterilize at 121°C for 20 minutes. After cooling to room temperature, use lignin-degrading bacteria (inoculate 2 mL at the logarithmic growth phase) for biological pretreatment, and cultivate at 30°C for 5, 10, 15, 20, 25, and 30 days, with a humidity of 70-80% during the period.

[0066] (4) Enzyme extraction and determination

[0067] Extract the treated straw at 200 r / min, 30°C for 2 h, filter the suspension through filter paper, and collect the filtrate for analysis of enzyme production during straw treatment (laccase, lignin peroxidase (LiP), and manganese peroxidase (MnP)).

[0068] (5) Observation of structural changes in straw after biological pretreatment by electron microscopy

[0069] (6) Effect of lignin-degrading bacterial strain treatment on straw weight loss

[0070] The results are as follows Figure 3As shown, "***" indicates P <0.001; Compared with the control group, the straw weight was significantly reduced after treatment with lignin-degrading bacteria strains.

[0071] (7) Enzyme production of lignin-degrading bacteria strains during straw treatment

[0072] Enzymes related to lignin degradation were detected, including laccase, manganese peroxidase (Mnp), and lignin peroxidase (Lip). The results are as follows: Figure 4 , 5 As shown, no Lip was detected during the treatment with the lignin-degrading bacteria strain. Mnp and Laccase played the main roles in lignin degradation. After 30 days of treatment with lignin-degrading bacteria on wheat straw, the straw loss rate reached 9.9%, and the strain's highest laccase activity reached 154.3 U / L; the highest manganese peroxidase activity reached 868.2 U / L.

[0073] (8) Effects of treatment with lignin-degrading bacteria strains on changes in straw structure

[0074] like Figure 6 As shown, treatment with isolated strains can disrupt the protruding structures on the surface of straw.

[0075] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A lignin-degrading fungus isolated from the mealworm, characterized in that, The lignin-degrading bacteria is Kluyvera georgiana (ATCC 33071) Kluyvera georgiana , which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC NO. 33528.

2. The lignin-degrading bacteria isolated from the lesser grain borer, as claimed in claim 1, characterized by, The 16S rRNA sequence of the lignin-degrading bacteria is shown as SEQ ID NO.

1.

3. A method of degrading lignin, characterized by, The method comprises the following steps: culturing the lignin-degrading bacteria isolated from the mealworm as claimed in claim 1 or 2; mixing alkali lignin, nutrients and water to obtain a first mixed solution, wherein the nutrients do not contain a carbon source; and adding the lignin-degrading bacteria obtained by the culturing into the first mixed solution to degrade lignin.

4. The lignin degradation method according to claim 3, wherein: The culturing of the lignin-degrading bacteria specifically comprises the following steps: inoculating the lignin-degrading bacteria into an LB culture medium, culturing at 20-40 DEG C and 150-250 r / min until the logarithmic growth phase, centrifugally separating, washing with PBS and resuspending the bacterial liquid to obtain a lignin-degrading bacteria liquid.

5. The lignin degradation method according to claim 3, wherein: The nutrients include K2HPO4, M g SO4-7H2O, CaCl2, FeSO4-7H2O, MnCl2, KH2PO4, and proteose peptone; The alkali lignin in the first mixed solution is 2.5-3.5 g / L, K2HPO4 is 0.5-1.5 g / L, M g SO4·7H2O is 0.005-0.015 g / L, CaCl2 is 0.05-0.13 g / L, FeSO4·7H2O is 0.03-0.08 g / L, MnCl2 is 0.01-0.03 g / L, KH2PO4 is 0.5-1.5 g / L, and peptone is 1.5-2.5 g / L.

6. The lignin degradation method according to claim 3, wherein: When the lignin-degrading bacteria obtained by the culturing is added into the first mixed solution to degrade lignin, the temperature is 20-40 DEG C, the rotation speed is 150-250 r / min and the treatment time is 12-120 h.

7. A method of degrading straw, characterized by, The method comprises the following steps: culturing the lignin-degrading bacteria isolated from the mealworm as claimed in claim 1 or 2; mixing straw and water to obtain a second mixed solution; and adding the lignin-degrading bacteria obtained by the culturing into the second mixed solution to degrade the straw.

8. The straw degradation method according to claim 7, wherein: The culturing of the lignin-degrading bacteria specifically comprises the following steps: preparing an LB culture medium, sterilizing, cooling to 20-30 DEG C, inoculating the lignin-degrading bacteria into the LB culture medium, and culturing at 20-40 DEG C and 80-200 r / min until the logarithmic growth phase.

9. The lignin or straw degradation method according to claim 7, wherein: When the lignin-degrading bacteria obtained by the culturing is added into the mixed solution to degrade the straw, the temperature is 20-40 DEG C, the humidity is 70%-80% and the treatment time is 5-30 days.

10. Application of the lignin-degrading bacteria isolated from the mealworm as claimed in claim 1 or 2 to lignin or straw degradation.

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