Lignin degrading bacterium, lignin or straw degrading method and application of lignin degrading bacterium

Abstract

The invention provides a lignin degrading bacterium, a degradation method of lignin or straw and application of the lignin degrading bacterium. The lignin degrading bacterium is adjuvant aleuria georgiana and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO. 33528. The lignin degrading bacterium can be used for degrading lignin or straw. The lignin degrading bacterium is separated from intestinal tracts of barley pests taking straws as food, and has efficient lignin and straw degrading capability.

Description

Technical Field

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

[0002] Crop straws are used as rich feed resources in China and show great application potential especially in the feeding of ruminant livestock. However, the biochemical properties of straws generally limit their practical application in production. The crude protein content of straws is low, and most of the crude protein is tightly bound to the cell wall, resulting in low digestibility and degradation rate. The straw cell wall is mainly composed of cellulose, hemicellulose, and lignin. Among them, lignin has a complex, stable, and diverse amorphous three-dimensional structure. Lignin is covalently bonded to hemicellulose, embedding the cellulose molecules therein, forming a natural barrier that makes it difficult for microorganisms to contact the cellulose molecules, thereby reducing the utilization rate and nutritional value of straws. Traditional nutrition holds that there is an inverse relationship between the lignin content and the digestibility of straws. For every 1% reduction in the lignin content in the diet, the digestibility of animals can be increased by 4% - 5%. In addition, as an important source of aromatic hydrocarbon resources, the degradation products of lignin can meet the requirements of the pharmaceutical industry for aromatic hydrocarbons, which provides a new way to reduce the dependence on non-renewable resources such as coal and petroleum. Therefore, in the process of straw treatment, the degradation of lignin is the key to improving the utilization efficiency of straws.

[0003] Currently, the commonly used methods for breaking lignin include radiation, steam explosion, puffing, grinding, acid hydrolysis, alkali treatment, oxidation treatment, and organic solvent method, etc. These methods aim to break the tight structure of lignin and cellulose to improve the nutritional value and utilization rate of straws. The Chinese patent document with the publication number CN104004201A discloses a method for degrading lignin in an acidic deep eutectic solvent. The lignin raw material, deep eutectic solvent, and water are added to a reactor, and homogeneous catalytic degradation is carried out by stirring under normal pressure and at 90 - 130°C. This invention uses the acidic deep eutectic solvent as both a solvent and a catalyst to degrade lignin, and the deep eutectic solvent can be recycled and reused, with advantages such as simple process and low cost. The Chinese patent document with the publication number CN106146757A pretreats corn straw by steam explosion method, 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 treats and oxidizes and degrades lignin with sodium chlorite.

[0004] These 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, cost, and the quality and safety of livestock feed. Therefore, seeking a safe biological method for lignin degradation is an important way for straw feed utilization. It can not only reduce environmental pollution and save energy consumption, but also turn waste into treasure and realize the reuse of straw resources. In the current field of biological lignin degradation, only white rot fungi can completely degrade lignin into CO2 and H2O under normal temperature and pressure. However, there are problems with fungal lignin degradation, such as poor environmental adaptability, long pretreatment cycle, and easy spore contamination. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a lignin-degrading bacterium, a method for degrading lignin or straw, and an application of the lignin-degrading bacterium. The lignin-degrading bacterium is isolated from the intestine of Zophobas morio that feeds on straw and is Kluyvera georgiana ( Kluyvera georgiana ), which has high lignin and straw degradation capabilities.

[0006] To solve the above problems, the first aspect of the present invention provides a lignin-degrading bacterium isolated from Zophobas morio, and the lignin-degrading bacterium is Kluyvera georgiana ( Kluyvera georgiana ), which is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC NO. 33528.

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

[0008] The second aspect of the present invention provides a method for degrading lignin, including: Culturing the above-mentioned lignin-degrading bacterium isolated from Zophobas morio; mixing alkali lignin, nutrients and water to obtain a first mixed solution, and the nutrients do not contain a carbon source; adding the cultured lignin-degrading bacterium to the first mixed solution for lignin degradation.

[0009] Preferably, the culturing of the lignin-degrading bacterium specifically includes: inoculating the lignin-degrading bacterium into an LB medium and culturing it at 20-40°C and 150-250 r / min until the logarithmic growth phase, then centrifuging and separating, washing with PBS and resuspending the bacterial solution to obtain a lignin-degrading bacterium bacterial solution.

[0010] Preferably, the nutrients include K2HPO4, M g SO4·7H2O, CaCl2, FeSO4·7H2O, MnCl2, KH2PO4 and peptone; In the first mixed solution, the alkali lignin is 2.5-3.5 g / L, K2HPO4 is 0.5-1.5 g / L, Mg The amount of FeSO4·7H2O is 0.005 - 0.015 g / L, the amount of CaCl2 is 0.05 - 0.13 g / L, the amount of FeSO4·7H2O is 0.03 - 0.08 g / L, the amount of MnCl2 is 0.01 - 0.03 g / L, the amount of KH2PO4 is 0.5 - 1.5 g / L, and the amount of peptone is 1.5 - 2.5 g / L.

[0011] Preferably, when adding the lignin-degrading bacteria obtained by culturing into the first mixed solution for lignin degradation, the temperature is 20 - 40 °C, the rotation speed is 150 - 250 r / min, and the treatment time is 12 - 120 h.

[0012] The third aspect of the present invention provides a method for degrading straw, including: Culturing the above-mentioned lignin-degrading bacteria isolated from Zophobas morio; mixing straw with water to obtain a second mixed solution; adding the lignin-degrading bacteria obtained by culturing into the second mixed solution for straw degradation.

[0013] Preferably, the culturing of the lignin-degrading bacteria specifically includes: preparing an LB medium, sterilizing it, cooling it to 20 - 30 °C, inoculating the lignin-degrading bacteria into the LB medium, and culturing it at 20 - 40 °C and 80 - 200 r / min until the logarithmic growth phase.

[0014] Preferably, when adding the lignin-degrading bacteria obtained by culturing into the mixed solution for straw degradation, the temperature is 20 - 40 °C, the humidity is 70% - 80%, and the treatment time is 5 - 30 days.

[0015] The fourth aspect of the present invention provides an application of the above-mentioned lignin-degrading bacteria isolated from Zophobas morio in lignin or straw degradation.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The lignin-degrading bacteria of the present invention are isolated from the intestine of Zophobas morio that feeds on straw. The identification results of MALDI-TOF MS and 16S rRNA show that the bacteria are Kluyvera georgiana ( Kluyvera georgiana ). It has high lignin and straw degradation ability. In the medium with lignin as the sole carbon source, after the strain is treated for 120 h, the lignin degradation rate reaches 14.8%. During the treatment process, the highest laccase activity reaches 81.7 U / L, and the highest manganese peroxidase activity reaches 254.9 U / L; in K. georgiana after treating wheat straw for 30 days, the straw loss rate reaches 9.9%. The highest laccase activity of the strain reaches 154.3 U / L; the highest manganese peroxidase activity reaches 868.2 U / L. Description of the Drawings

[0017] Figure 1 It is the growth curve of the strain and the lignin degradation curve in Example 2 of the present invention; Figure 2 It is the enzyme production situation during the lignin treatment process in Example 2 of the present invention; Figure 3 It is the influence of the treatment of lignin-degrading bacteria strains in Example 3 of the present invention on the straw weight loss; Figure 4 It is the laccase production situation during the straw treatment process in Example 3 of the present invention; Figure 5 It is the manganese peroxidase production situation during the straw treatment process in Example 3 of the present invention; Figure 6 It is the electron micrograph of the change in the straw structure after the treatment of lignin-degrading bacteria strains in Example 3 of the present invention; Figure 7 It is the electron micrograph of the change in the lignin structure after the treatment of lignin-degrading bacteria strains in Example 2 of the present invention. Detailed implementation manners

[0018] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0019] In the first aspect of the embodiments of the present invention, a lignin-degrading bacterium isolated from Zophobas morio is provided. The lignin-degrading bacterium is Kluyvera georgiana ( Kluyvera georgiana ), which was deposited on February 14, 2025, at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC NO. 33528. This lignin-degrading bacterium isolated from Zophobas morio has high lignin and straw degradation capabilities. In a medium with lignin as the sole carbon source, the lignin degradation rate reaches 14.8% after 120 h of strain treatment. During the treatment process, the highest laccase activity reaches 81.7 U / L, and the highest manganese peroxidase activity reaches 254.9 U / L; after 30 days of treating wheat straw with this lignin-degrading bacterium, the straw loss rate reaches 9.9%, the highest laccase activity of the strain reaches 154.3 U / L; the highest manganese peroxidase activity reaches 868.2 U / L.

[0020] Preferably, the 16S rRNA sequence of the lignin-degrading bacterium is as shown in SEQ ID NO.1.

[0021] The sequence structure of SEQ ID NO.1 is as follows:

[0022] In a second aspect of the embodiments of the present invention, a method for degrading lignin is provided, including: Culturing the above-mentioned lignin-degrading bacteria isolated from Zophobas morio; mixing alkali lignin, nutrients and water to obtain a first mixed solution, wherein the nutrients do not contain a carbon source; adding the cultured lignin-degrading bacteria into the first mixed solution to degrade lignin.

[0023] Compared with various physical and chemical methods, biodegradation, especially the treatment of lignin with highly efficient lignin-degrading strains, has the advantages of less pollution, environmental friendliness, and low degradation cost. Fungi are currently recognized as microorganisms with generally higher lignin-degrading ability than other strains. However, fungi grow slowly, have poor environmental adaptability, and are easily contaminated by spores. Therefore, the research on lignocellulose biodegradation carried out with fungi as the main model strains has long failed to develop a feasible industrial degradation pathway. Compared with fungi, bacteria can also secrete lignin-degrading related enzymes, and have a faster growth rate, a simple structure, and stronger adaptability to complex environments. The present invention provides a bacterial strain from the intestine of Zophobas morio Kluyvera georgiana , which has obvious straw and lignin degradation ability and can be used as a feasible industrial degradation bacterial strain for lignocellulose. The method for degrading lignin uses a bacterial strain Kluyvera georgiana , which has high-efficient lignin degradation ability. In a medium with lignin as the sole carbon source, the lignin degradation rate reaches 14.8% after the strain is treated for 120 h, 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.

[0024] Preferably, the culturing of the lignin-degrading bacteria specifically includes: inoculating the lignin-degrading bacteria into an LB medium, culturing at 20-40 °C and 150-250 r / min until the logarithmic growth phase, then centrifuging and separating, washing with PBS and resuspending the bacterial solution to obtain a lignin-degrading bacteria solution.

[0025] Preferably, the nutrients include K2HPO4, M g SO4·7H2O, CaCl2, FeSO4·7H2O, MnCl2, KH2PO4 and peptone; In the first mixed solution, 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 peptone is 1.5-2.5 g / L.

[0026] Preferably, when adding the lignin-degrading bacteria obtained by cultivation into the first mixed solution for lignin degradation, the temperature is 20 - 40°C, the rotation speed is 150 - 250 r / min, and the treatment time is 12 - 120 h.

[0027] The third aspect of the present invention provides a method for degrading straw, including: Cultivating the above-mentioned lignin-degrading bacteria isolated from Zophobas morio; mixing straw with water to obtain a second mixed solution; adding the cultivated lignin-degrading bacteria into the second mixed solution for straw degradation.

[0028] This method for degrading straw uses bacterial strains Kluyvera georgiana which have high straw degradation ability. After treating wheat straw with the bacterial strains Kluyvera georgiana for 30 days, the straw loss rate reaches 9.9%, and the highest laccase activity of the strain reaches 154.3 U / L; the highest manganese peroxidase activity reaches 868.2 U / L.

[0029] Preferably, the cultivation of the lignin-degrading bacteria specifically includes: preparing an LB medium, sterilizing it, cooling it to 20 - 30°C, inoculating the lignin-degrading bacteria into the LB medium, and culturing it at 20 - 40°C and 80 - 200 r / min until the logarithmic growth phase.

[0030] Preferably, when adding the cultivated lignin-degrading bacteria into the mixed solution for straw degradation, the temperature is 20 - 40°C, the humidity is 70% - 80%, and the treatment time is 5 - 30 days.

[0031] The fourth aspect of the present invention provides an application of the above-mentioned lignin-degrading bacteria isolated from Zophobas morio in lignin or straw degradation.

[0032] Example 1 Screening and Identification of Lignin-Degrading Strains The Zophobas morio larvae fed with wheat straw were washed twice with distilled water and 75% ethanol (30 seconds each time), and then the surface of the larvae was dried with sterile filter paper. The intestinal tissues of the larvae (including the foregut, midgut, and hindgut) were dissected on a clean bench using sterile scissors and forceps, transferred to a 50 mL centrifuge tube, and 10 mL of pre-cooled PBS was added. It was gently ground and vortex-mixed. To screen for lignin-degrading bacteria, an appropriate amount of the vortex-mixed sample was cultured in a lignin-enriched medium (alkaline lignin as the sole carbon source). The culture conditions were 28°C, with 2 days of aerobic and anaerobic culture each. Single colonies were picked and inoculated into a TSB aerobic medium (HB4114) and a triple mixed anaerobic medium, and then cultured aerobically and anaerobically at 28°C for 2 days respectively.

[0033] After the preliminary identification of bacteria by MALDI-TOF-MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry), the selected colonies were collected onto a 96-well plate. The colonies in the 96-well plate were cultured aerobically and anaerobically on alkaline lignin medium at 28 °C for 2 days. The individual colonies were further cultured on aerobic TSB medium and anaerobic triple mixed medium for 2 days. PCR amplification and DNA sequencing were performed according to the reported colony 16S rRNA gene to identify the genus or species of the bacterium. 27F and 1492R were used as primers for 16S rRNA amplification; in the GenBank database of NCBI, the 16S rRNA sequencing sequence of the bacterium was identified by BLAST search. The combined results of MALDI-TOF-MS and 16S rRNA sequencing identification showed that the isolated lignin-degrading bacterium was Kluyvera georgiana, and the 16S rRNA sequence similarity was 98.16%. The 16S rRNA sequence of this lignin-degrading bacterium is shown in SEQ ID NO.1.

[0034] The sequence structure of SEQ ID NO.1 is as follows:

[0035] The lignin-degrading bacterium was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC NO. 33528.

[0036] Example 2 Lignin Degradation Experiment (1)Preparation of Lignin Medium The liquid lignin medium contains: alkali lignin 3 g / L, K2HPO4 1 g / L, M g SO4·7H2O 0.01 g / L, CaCl2 0.08 g / L, FeSO4·7H2O 0.05 g / L, MnCl2 0.02 g / L, KH2PO4 1 g / L, peptone 2 g / L.

[0037] (2)Lignin Treatment The isolated lignin-degrading bacterium strain was inoculated into LB medium and cultured at 30 °C and 180 r / min until the logarithmic growth phase. Then, it was centrifuged at 5000 r / min for 5 min, washed twice with PBS, and the bacterial suspension was resuspended. The bacterial suspension was inoculated into the lignin medium at an inoculation amount of 5%, and continuously cultured at 30 °C and 180 r / min. The growth status of the strain was reflected by the difference in absorbance at 600 nm before centrifugation of the culture solution, and the absorbance value at 280 nm was used as the measurement index of lignin concentration. The lignin degradation rate (%) at the nth hour = (A0 - A n ) / A0×100; and the activity of lignin-degrading enzymes in the culture solution was measured. The measurement method was carried out according to the kit instructions of Beijing Solarbio Science & Technology Co., Ltd., including lignin peroxidase (Lip, BC1615), laccase (Laccase, BC1635), and manganese peroxidase (Mnp, BC1625). The culture solution after the treatment was centrifuged at 12000 r / min for 10 min, and the supernatant was quickly frozen in liquid nitrogen and then dried to a constant weight in a vacuum dryer. The dried sample was sputter-coated with gold and observed by electron microscopy. As Figure 7 shown.

[0038] (3)Treatment Effect of Lignin-degrading Bacterium Strain on Lignin As Figure 1 shown, the lignin-degrading bacterium can grow in the medium with lignin as the sole carbon source, and the growth amount reaches the maximum at 24 h; meanwhile, the results show that during the culture process, the lignin content decreases significantly and stabilizes after 96 h. In addition, the detection results of the activities of lignin-degrading enzymes show that Lip was not detected during the strain treatment process, and Mnp and Laccase play a major role in lignin degradation ( Figure 2). In the medium with lignin as the sole carbon source, the lignin degradation rate reached 14.8% after 120 h of treatment with the strain. During the treatment process, the highest laccase activity reached 81.7 U / L, and the highest manganese peroxidase activity reached 254.9 U / L.

[0039] Example 3 Straw Degradation Experiment (1) Straw collection Collect straw particles between 40 and 60 mesh, and then dry them to constant weight at 75 °C as samples for subsequent experiments.

[0040] (2) Bacterial culture Prepare 100 ml of LB medium, sterilize it at 121 °C for 20 min. After cooling to room temperature, inoculate the lignin-degrading bacteria into the liquid medium and place it in a constant-temperature shaking incubator, and culture it at 30 °C and 120 r / min until the logarithmic growth phase.

[0041] (3) Biological pretreatment of straw Add 15 ml of distilled water to a conical flask containing 5 g of ground straw, and sterilize it at 121 °C for 20 minutes. After cooling to room temperature, conduct biological pretreatment with lignin-degrading bacteria (inoculate 2 mL during the logarithmic growth phase), and statically culture it at 30 °C for 5, 10, 15, 20, 25, 30 days, ensuring a humidity of 70 - 80% during this period.

[0042] (4) Enzyme extraction and determination Extract the treated straw at 200 r / min and 30 °C for 2 h, filter the suspension through filter paper, and collect the filtrate for analyzing the enzyme production during the straw treatment process (laccase, lignin peroxidase (LiP), manganese peroxidase (MnP)).

[0043] (5) Observe the structural changes of straw by microscopy after biological pretreatment (6) Effect of lignin-degrading bacteria strain treatment on straw weight loss The results are as Figure 3 shown, where "***" indicates P <0.001; compared with the control group, the straw weight decreased significantly after treatment with the lignin-degrading bacteria strain.

[0044] (7) Enzyme production during the process of lignin-degrading bacteria strain treating straw Detect the lignin-degrading related enzymes, including laccase, manganese peroxidase, lignin peroxidase. The results are as Figure 4 , 5As shown in the figure, no Lip production was detected during the treatment process of the lignin-degrading bacterial strain, and Mnp and Laccase played a major role in lignin degradation. After 30 days of treating wheat straw with the lignin-degrading bacterial strain, the straw loss rate reached 9.9%, and the highest laccase activity of the strain reached 154.3 U / L; the highest manganese peroxidase activity reached 868.2 U / L.

[0045] (8) Influence of the treatment of lignin-degrading bacterial strain on the structural change of straw As Figure 6 shown, the treatment with the isolated strain can destroy the protrusion structure on the surface of the straw.

[0046] Obviously, the above embodiments are only examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A lignin-degrading bacterium isolated from Zophobas atratus, characterized in that, The lignin-degrading bacterium is Kluyvera georgiana ( Kluyvera georgiana ), which is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC NO. 33528.

2. The lignin-degrading bacterium isolated from Zophobas morio according to claim 1, characterized in that, The 16S rRNA sequence of the lignin-degrading bacterium is as shown in SEQ ID NO.

1.

3. A method for degrading lignin, characterized in that, Including: Culturing the lignin-degrading bacterium isolated from Zophobas morio as described 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; adding the cultured lignin-degrading bacterium into the first mixed solution to degrade lignin.

4. The method for degrading lignin according to claim 3, characterized in that: The culturing of the lignin-degrading bacterium specifically includes: inoculating the lignin-degrading bacterium into an LB medium, culturing at 20-40 °C and 150-250 r / min until the logarithmic growth phase, then centrifuging and separating, washing with PBS and resuspending the bacterial solution to obtain a lignin-degrading bacterium bacterial solution.

5. The method for degrading lignin according to claim 3, characterized in that: The nutrients include K2HPO4, M g SO4·7H2O, CaCl2, FeSO4·7H2O, MnCl2, KH2PO4 and peptone; In the first mixed solution, 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 peptone is 1.5 - 2.5 g / L.

6. The method for degrading lignin according to claim 3, characterized in that: When adding the cultured lignin-degrading bacterium into the first mixed solution to degrade lignin, the temperature is 20-40 °C, the rotation speed is 150-250 r / min, and the treatment time is 12-120 h.

7. A method for degrading straw, characterized in that, Including: Culturing the lignin-degrading bacterium isolated from Zophobas morio as described in claim 1 or 2; Mixing straw and water to obtain a second mixed solution; adding the cultured lignin-degrading bacterium into the second mixed solution to degrade straw.

8. The method for degrading straw according to claim 7, characterized in that: The culturing of the lignin-degrading bacterium specifically includes: preparing an LB medium, sterilizing it, and after cooling to 20-30 °C, inoculating the lignin-degrading bacterium into the LB medium and culturing at 20-40 °C and 80-200 r / min until the logarithmic growth phase.

9. The method for degrading lignin or straw according to claim 7, characterized in that: When adding the cultured lignin-degrading bacterium into the mixed solution to degrade straw, the temperature is 20-40 °C, the humidity is 70%-80%, and the treatment time is 5-30 days.

10. Use of a lignin-degrading bacterium isolated from Zophobas morio as described in claim 1 or 2 in the degradation of lignin or straw.

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