Tobacco stem efficient degrading bacterium WP-2022

Abstract

The invention relates to a tobacco stem efficient degrading bacterium WP-2022, which belongs to the technical field of microorganisms, has the preservation number of CCTCC NO: M 20241187, and is preserved in the China Center for Type Culture Collection. The efficient tobacco stalk degrading bacterium WP-2022 has good tobacco stalk degrading capacity, cellulose and lignin in tobacco stalks can be efficiently degraded at the environment temperature of 22-28 DEG C, the tobacco stalks do not need to be degraded in a specific composting environment, especially high-temperature composting is mainly used, and the tobacco stalks are not prone to being degraded. An effective solution is provided for directly returning the tobacco straws to the field, resource utilization of the straws is facilitated, and meanwhile, environmental pollution is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a tobacco stem efficient degradation bacterium WP-2022. Background Art

[0002] Tobacco straw contains a mixture of lignin, cellulose and hemicellulose, making it more difficult to degrade than other common crop straws, such as wheat, corn and rice. From the analysis of tobacco straw components, the molecular structure of lignin is complex and irregular, containing various biologically stable complex bond types, so it is not easy for microorganisms and their extracellular enzymes to bind to it. In addition, lignin does not contain easily hydrolyzed and repeated units, and is resistant to enzymatic hydrolysis, making it one of the aromatic compounds that is difficult for microorganisms to degrade. Similarly, cellulose is also a difficult-to-degrade component, and the protection of cellulose by lignin further limits the decomposition of cellulose. In addition, hemicellulose acts as a connector between lignin and cellulose, forming a strong network structure, which further increases the difficulty of degradation.

[0003] Therefore, it is necessary to develop a microbial agent that can efficiently degrade cellulose and lignin at the same time, so as to solve the problem of the protective layer formed by lignin and the difficulty in degrading lignin and cellulose themselves.

[0004] At present, the existing degradation technologies mainly rely on the action of microorganisms, including fungi, bacteria and actinomycetes. These microorganisms decompose lignin and cellulose by secreting extracellular enzymes. However, the existing microbial degradation technology has some limitations. For example, a small number of microorganisms (such as yeast) in fungi have the ability to degrade lignin or cellulose, but their degradation ability is weak. Actinomycetes can decompose lignin to a certain extent, but they rarely use cellulose and have a long reproduction time. Bacteria also have the ability to degrade lignin or cellulose. Compared with fungi and actinomycetes, bacteria have the advantages of many species, fast growth, and a wide range of growth temperature and adaptable pH values, and have great practical application potential. Summary of the invention

[0005] The purpose of the present invention is to provide a tobacco stalk efficient degradation bacterium WP-2022, which can simultaneously and efficiently degrade cellulose and lignin in tobacco stalks and improve the tobacco stalk degradation rate.

[0006] The invention provides a tobacco stalk efficient degrading bacterium WP-2022, which is a tobacco stalk efficient degrading bacterium, has a preservation number of CCTCC NO: M20241187, a preservation date of June 11, 2024, and is preserved by the China Center for Type Culture Collection.

[0007] Furthermore, the 16S gene sequence of the highly efficient tobacco stalk degrading bacteria WP-2022 is shown in SEQ ID NO.1.

[0008] Furthermore, the separation and purification method of the tobacco stalk efficient degradation bacteria WP-2022 comprises the following steps: collecting tobacco-growing soil samples and culturing them in an enrichment medium for 1 hour; taking out and standing for 5 minutes, taking the supernatant of the enrichment medium culture and oscillating and culturing it at 28°C and 180 rpm for 3 days; diluting the cultured bacterial solution by 10-3, 10-4, and 10-5 times according to the requirements of aseptic operation; taking 0.1 ml of the diluted bacterial solution and applying it on a bacterial culture medium, a fungal culture medium, and an actinomycete culture medium without colony formation, respectively, and inverting and culturing it at 28°C for 2 to 3 days; after colonies grow on the plate, selecting representative colonies according to the morphology and color of the colonies, and picking out single colonies with a sterile inoculation loop; bacteria and fungi are streaked and cultured on the corresponding solid culture medium according to the requirements of aseptic operation until single colonies are purified; actinomycetes pick single colonies into LB culture medium for liquid culture, and then use an inoculation loop to dip a small amount of bacterial solution and streak culture on the actinomycete culture medium until single colonies are obtained.

[0009] Furthermore, the method for screening the degradation ability of the isolated and purified single colony comprises the following steps: inoculating the purified single colony into a CMC-Na culture medium, and staining the cultured single colony using a Congo red staining method; measuring the transparent circle diameter D (cm) and the colony diameter d (cm) of the colony using a cross method, evaluating the cellulose degradation ability of the single colony, and screening out strains with larger Hc values; inoculating the screened single colony into a PDA-aniline blue culture medium and a PDA-guaiacol culture medium, observing whether color changes occur on the culture medium, and thereby judging whether the single colony has the ability to secrete lignin degrading enzymes.

[0010] Furthermore, the single colony screened out with the ability to degrade lignin was tested for carboxymethyl cellulase activity using the DNS colorimetric method, and the ligninase activity of the single colony was tested using a lignin peroxidase activity detection kit. The carboxymethyl cellulase activity of the strain reached at least 228.10 U / mL within 16 hours, and the strain produced ligninase after 16 hours of cultivation, with an enzyme activity of 10.36 U / mL. The WP-2022 strain of tobacco stalk efficient degrading bacteria has the ability to efficiently degrade cellulose and lignin in tobacco stalks.

[0011] The present invention also provides a tobacco straw degrading bacterial agent, characterized in that the tobacco straw degrading bacterial agent comprises the tobacco straw efficient degrading bacteria WP-2022.

[0012] The present invention also provides the use of the tobacco straw degrading bacterial agent in degrading tobacco straw, characterized in that the degrading bacteria can degrade lignin and cellulose in the tobacco straw at the same time. In the structure of tobacco straw, lignin is distributed between cellulose in a dispersed state, and hemicellulose serves as a connecting component to combine lignin and cellulose together to form a solid network structure, i.e., a cellulose-hemicellulose-lignin complex. The close combination of lignin and cellulose in this structure means that their degradation processes are interdependent.

[0013] The degradation agent of the present invention can effectively destroy the cellulose-hemicellulose-lignin network structure in tobacco straw by acting on the two main components of cellulose and lignin at the same time. In this way, the agent of the present invention can achieve efficient decomposition of tobacco straw and reduce environmental pollution.

[0014] Furthermore, the tobacco straw degradation microbial agent has an action environment temperature of 22-28° C. Instead of composting tobacco straw in a specific environment, especially high-temperature composting, the tobacco straw degradation microbial agent can accelerate the degradation of straw at 22-28° C. in the natural field, which provides an effective solution for the direct return of tobacco straw to the field and helps to realize the resource utilization of straw. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the Fourier transform infrared spectra of tobacco straw before and after 7 days of degradation by WP-2022 strain;

[0016] Figure 2 This is the degradation rate of lignocellulose components in tobacco stems after 7 days of treatment with WP-2022 strain. DETAILED DESCRIPTION

[0017] The preferred embodiments of the present invention will be described in detail below so that the purpose, features and advantages of the present invention can be more clearly understood. It should be understood that the following embodiments are not intended to limit the scope of the present invention, but are only intended to illustrate the essential spirit of the technical solution of the present invention.

[0018] In the following description, certain specific details are set forth for the purpose of illustrating the various disclosed embodiments to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with the present application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0019] References throughout the specification to "one embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0020] The purpose of the present invention is to provide a tobacco stalk efficient degradation bacterium WP-2022, which can simultaneously and efficiently degrade cellulose and lignin in tobacco stalks and improve the tobacco stalk degradation rate.

[0021] The tobacco stem efficient degradation bacteria WP-2022 of the present invention is further introduced in detail below in conjunction with specific embodiments. The technical solutions of the present invention include but are not limited to the following embodiments.

[0022] 1. Isolation and purification of WP-2022, a highly efficient tobacco stalk-degrading bacterium

[0023] 1.1 Isolation and purification of tobacco stalk efficient degradation bacteria WP-2022. Tobacco-growing soil samples were collected in Anshun City, Guizhou Province. The isolation and purification steps are as follows:

[0024] 10.00 g of tobacco-growing soil sample was weighed and cultured in an enrichment medium for 1 h. The enrichment medium was taken out and allowed to stand for 5 min. 2 mL of supernatant was taken out with a pipette and placed in a new sterilized and cooled enrichment medium. The culture was shaken at 28 °C and 180 r / min for 3 d.

[0025] According to the requirements of aseptic operation, the cultured bacterial solution was diluted 10-3, 10-4, and 10-5 times, and 0.1 mL was applied to the bacterial culture medium, fungal culture medium, and actinomycete culture medium without colony formation, respectively, for the separation of bacteria, fungi, and actinomycetes. Three groups of parallels were set for each gradient. Bacteria, fungi, and actinomycetes were all inverted and cultured at 28°C for 2 to 3 days.

[0026] After colonies grow on the plate, select representative colonies according to their morphology and color, and use a sterile inoculation loop to pick out a single colony. For bacteria and fungi, streak culture on the corresponding solid culture medium according to the sterile operation requirements until a single colony is purified. For actinomycetes, pick a single colony into LB culture medium for liquid culture, and then use an inoculation loop to pick up a small amount of bacterial liquid and streak culture on the actinomycete culture medium until a single colony is obtained.

[0027] 2. Screening of the ability of WP-2022 strain to degrade cellulose and lignin

[0028] 2.1 Screening of the cellulose degradation ability of the tobacco stalk efficient degrading bacteria strain WP-2022, the isolated and purified strains were screened for their cellulose degradation ability. The cellulose degradation ability was screened using the Congo red staining method, and the specific screening method is as follows:

[0029] The purified strains were inoculated into CMC-Na medium, with 3 replicates for each strain, and cultured in a constant temperature incubator at 30°C for 2 days. The staining time was determined according to the colony size.

[0030] The identification culture medium plate was stained with 0.1% Congo red dye for 15 minutes, and then decolorized with 1 mol / L sodium chloride solution for 15 minutes. The excess solution on the plate was aspirated with a pipette, and the diameter of the transparent circle D (cm) and the diameter of the colony d (cm) were measured by the cross method. According to the Hc value (Hc=D / d), it was determined that the strain had the ability to decompose cellulose, and the strain with a larger Hc value was screened out.

[0031] 2.2 Screening of the ability of the tobacco stalk efficient degrading bacteria strain WP-2022 to degrade lignin. The isolated and purified strains were screened for their lignin degradation ability. The specific screening method is as follows:

[0032] The strains screened for cellulose degradation were inoculated on PDA-aniline blue medium and PDA-guaiacol medium by dot grafting and cultured for 24 hours to observe whether a fading circle was produced on the PDA-aniline blue medium, indicating that the strains had the ability to secrete MnP and LiP; and whether a coloring circle appeared on the PDA-guaiacol medium, indicating that the strains had the ability to secrete Lac, to determine whether the strains had the ability to decompose lignin.

[0033] 2.3 The enzyme activity of the screened strains was determined. The specific determination method is as follows:

[0034] 1) Determination of carboxymethyl cellulase activity of strains using DNS colorimetric method

[0035] 2) using a lignin peroxidase activity detection kit to measure ligninase activity;

[0036] The strain has a carboxymethyl cellulase activity of at least 228.10 U / mL within 16 hours, and produces ligninase with an enzyme activity of 10.36 U / mL after being cultured for 16 hours.

[0037] The results showed that the tobacco straw efficient degrading bacteria strain WP-2022 has the ability to efficiently degrade cellulose and lignin in tobacco straw.

[0038] 3. Molecular Biological Identification of Strains

[0039] The PCR method was used for molecular biological identification. After the strain DNA was extracted, PCR amplification and agarose gel electrophoresis were performed using bacterial 16SrDNA universal primers, and the PCR amplification product was sequenced. The measured sequence was compared with the 16S rDNA sequence in GeneBank for homology, and the 16SrRNA was identified as Bacillus subtilis and named Bacillus subtilis WP-2022. The 16S rDNA sequence of the strain is shown in SEQ ID NO.1 below, and it is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO:M20241187 and a deposit date of June 11, 2024.

[0040] 4. Tobacco straw degradation test

[0041] 4.1 Tobacco straw was selected as the test material, and two treatments were set up: treatment with the tobacco straw efficient degradation bacteria strain WP-2022 and sterile water treatment (CK). Each treatment was repeated 3 times, and 5% bacterial solution or sterile water was added to each repetition. The effective viable count of the bacterial solution was ≥1×10-8. Each treatment was cultured at a constant temperature of 28°C, and the degradation rates of lignin and cellulose in tobacco straw were measured after 7 days.

[0042] 4.2 Determination of cellulose, hemicellulose, and lignin The acid washing method was used, and Fourier transform infrared spectroscopy was used to analyze the changes in chemical groups during the degradation process to further demonstrate the degradation effect.

[0043] like Figure 1 As shown in the figure, after the bacterial solution was sprayed on tobacco straw for 7 days, compared with the case of spraying only sterile water (CK) before culture, the infrared absorption peak of the degradation products of tobacco straw after culture with bacterial agent was gentle near 1030cm-1, indicating that the CO bonds in lignin and cellulose were destroyed and small molecular organic matter was released; the infrared absorption peak near 1230cm-1 decreased, and the O=CC stretching vibration in the lignin-hemicellulose complex decreased, indicating that the composite structure of cellulose, hemicellulose and lignin in tobacco straw was destroyed, and cellulose, hemicellulose and lignin were separated; the vibrations of CC and CO in lignin near 1320cm-1 and CH in cellulose near 1370cm-1 decreased, indicating that part of cellulose and lignin in tobacco straw were degraded; the stretching vibrations of C=C and C=N in the aromatic skeleton near 1600cm-1 decreased, and the aromatic skeleton of lignin in tobacco straw was destroyed; the vibrations of hydrogen bonds in cellulose and carbon-hydrogen bonds in methyl and methylene near 2920cm-1 decreased, indicating that the hydrogen bonds of cellulose in tobacco straw were destroyed. In summary, it was demonstrated that strain WP-2022 could effectively degrade cellulose and lignin in tobacco straw.

[0044] 4.3 Results and Analysis

[0045] like Figure 2 As shown in Figure 2, after 7 days of cultivation, the strain significantly degraded lignin and cellulose in tobacco straw ( Figure 2 ), the lignin degradation rate was 8.12%, and the cellulose degradation rate was 27.01%, both significantly higher than those of the treatment without bacterial agent application (CK).

[0046] In the study of tobacco straw degradation, the highly efficient tobacco straw degrading bacteria strain WP-2022 showed excellent efficiency.

[0047] SEQ ID NO.1:

[0048] GTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGG

[0049] TGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATT

[0050] ACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACT

[0051] GAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTG

[0052] TTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGA

[0053] TTTGACGTCATCCCCCACCTTTCCTCCGGTTTGTCACCGGCAGTCACCTTAG

[0054] AGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCG

[0055] GGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCAC

[0056] CTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGG

[0057] ATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATG

[0058] CTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGC

[0059] GACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGG

[0060] GGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACC

[0061] AGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGT

[0062] TACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACG

[0063] CATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTT

[0064] CCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAG

[0065] ACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACG

[0066] CTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCT

[0067] TTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTC

[0068] TTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGG

[0069] CGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCC

[0070] TCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCT

[0071] CTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACT

[0072] AGCTAATGCGCCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTT

[0073] ATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTC

[0074] CCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCG

[0075] TCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCCTCGACTGC

[0076] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0077] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A highly efficient tobacco stem degradation bacterium WP-2022, which is a Bacillus subtilis strain, is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M20241187.

2. According to claim 1, the efficient tobacco stem degrading bacteria WP-2022, the 16S gene sequence of the efficient tobacco stem degrading bacteria WP-2022 is shown in SEQ ID NO.

1.

3. The highly efficient tobacco stem degrading bacteria WP-2022 according to claim 1 is characterized in that: The separation and purification method comprises the following steps: 1) Cultivate the collected tobacco-growing soil samples in an enrichment medium for 1 hour; 2) After being taken out and allowed to stand for 5 minutes, the supernatant of the enriched medium culture was taken and shake-cultured at 28°C and 180 rpm for 3 days; 3) diluting the cultured bacterial solution obtained in step 2); 4) Take 0.1 ml of the diluted bacterial solution and spread it on the bacterial culture medium, fungal culture medium, and actinomycete culture medium without colony formation, and invert and culture at 28°C for 2 to 3 days; 5) Use a sterile inoculating loop to pick out a single colony; 6) For bacteria and fungi, use aseptic technique to streak culture on solid culture medium until a single colony is purified. For actinomycetes, pick a single colony into LB medium for liquid culture, then use an inoculation loop to pick up a small amount of bacterial liquid and streak culture on actinomycete medium until a single colony is obtained.

4. The highly efficient tobacco stem degrading bacteria WP-2022 according to claim 3 is characterized in that: The method for screening the degradation ability of the isolated and purified single bacterial colonies comprises the following steps: 1) inoculating the purified single colony into CMC-Na medium, and staining the cultured colony using Congo red staining method, 2) Using the cross method to measure the diameter D of the transparent circle of the colony and the diameter d of the colony, calculate the ratio Hc=D / d, evaluate the cellulose degradation ability of the colony, and screen out the strain with a larger Hc value; 3) The selected strains were inoculated into PDA-aniline blue medium and PDA-guaiacol medium, and the color change on the medium was observed to determine whether the strains had the ability to decompose lignin.

5. The highly efficient tobacco stem degrading bacteria WP-2022 according to claim 4 is characterized in that: The carboxymethyl cellulase activity of the screened single bacterial colonies having the ability to decompose lignin was determined using the DNS colorimetric method.

6. The high-efficiency tobacco stem degrading bacteria WP-2022 according to claim 4 is characterized in that: The ligninase activity of the screened single bacterial colony with the ability to decompose lignin is determined using a lignin peroxidase activity detection kit.

7. A tobacco straw degradation bacterial agent, characterized in that: The tobacco straw degrading bacterial agent includes the tobacco straw efficient degrading bacteria WP-2022 according to claim 1 or 2.

8. Use of the tobacco straw degrading bacterial agent according to claim 7 in degrading tobacco straw, characterized in that: The degrading bacteria can degrade lignin and cellulose in tobacco straw simultaneously.

9. The use according to claim 8, characterized in that The tobacco straw degradation bacterial agent acts in an environment with a temperature of 22-28°C.

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