A tobacco stalk degrading bacterium WP-2022

Abstract

The present application relates to a tobacco stalk efficient degradation bacteria WP-2022, belongs to the field of microbial technology, the preservation number is CCTCC NO:M 20241187 preserved in China typical culture preservation center.The present application discloses a tobacco stalk efficient degradation bacteria WP-2022, which has good tobacco stalk degradation capacity, can efficiently degrade cellulose and lignin in tobacco stalk at ambient temperature 22-28 DEG C, and the tobacco stalk does not need to be degraded in a specific compost environment, especially high-temperature compost, which provides an effective solution for the direct field return of tobacco stalk, helps to realize the resource utilization of stalk, and reduces environmental pollution.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a tobacco stalk efficient degradation strain WP-2022. BACKGROUND

[0002] The lignin, cellulose and hemicellulose in tobacco stalk are intertwined and mixed, and are more difficult to degrade than other ordinary crop stalks such as wheat, corn and rice. From the analysis of the components of tobacco stalk, the molecular structure of lignin is complex and irregular, and contains various types of biologically stable complex bonds, so it is not easy for microorganisms and their extracellular enzymes to combine with it. In addition, lignin does not contain easily hydrolyzed repeating units and is resistant to enzymatic hydrolysis, making it one of the most difficult microorganisms to degrade aromatic compounds. Similarly, cellulose is also a difficult-to-degrade component, and the decomposition of cellulose is further limited due to the protection of lignin. In addition, hemicellulose plays a connecting role between lignin and cellulose, forming a firm network structure, further increasing the difficulty of degradation.

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

[0004] Currently, 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, there are some limitations in existing microbial degradation technologies, for example, a small part of fungi (such as yeast) has the ability to degrade lignin or cellulose, but its degradation ability is weak. Actinomycetes can decompose lignin to some extent, but they rarely use cellulose and have a long reproduction time. Bacteria also have the ability to degrade lignin or cellulose, and compared with fungi and actinomycetes, bacteria have the advantages of more species, fast growth, and wide range of growth temperature and pH value, which have great potential for practical application. SUMMARY

[0005] The purpose of the present application is to provide a tobacco stalk efficient degradation strain WP-2022, which can efficiently degrade cellulose and lignin in tobacco stalk at the same time and improve the degradation rate of tobacco stalk.

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

[0007] Further, the 16S gene sequence of the tobacco stalk efficient degradation strain WP-2022 is shown in SEQ ID NO. 1.

[0008] Further, the separation and purification method of the tobacco stalk efficient degradation bacteria WP-2022 comprises the following steps: the collected tobacco soil sample is cultured in an enrichment medium for 1 hour; the enrichment medium culture supernatant is taken out and placed for 5 minutes, and then is subjected to oscillation culture at 28 DEG C and 180 r / min for 3 days; the cultured bacteria liquid is diluted by 10-3, 10-4 and 10-5 times according to the aseptic operation requirement; 0.1 milliliter of the diluted bacteria liquid is taken and coated on the sterile colony generation bacterial culture medium, the fungal culture medium and the actinomycete culture medium respectively, and is subjected to inverted culture at 28 DEG C for 2-3 days; after the plate grows the bacterial colony, the representative bacterial colony is selected according to the bacterial colony morphology and color, and the single bacterial colony is picked out with a sterile inoculation loop; the bacteria and the fungi are subjected to streak culture on the corresponding solid culture medium according to the aseptic operation requirement until the single bacterial colony is obtained; the single bacterial colony of the actinomycete is picked into the LB culture medium for liquid culture, and then a small amount of the bacteria liquid is taken on the actinomycete culture medium with the inoculation loop for streak culture until the single bacterial colony is obtained.

[0009] Further, the screening method of the degradation ability of the separated and purified single bacterial colony comprises the following steps: the purified single bacterial colony is inoculated in the CMC-Na culture medium, and the cultured single bacterial colony is dyed by using the Congo red dyeing method; the diameter D (cm) of the transparent circle of the bacterial colony and the diameter d (cm) of the bacterial colony are measured by using the cross intersection method, the cellulose degradation ability of the single bacterial colony is evaluated, and the bacterial strain with a larger Hc value is screened out; the screened single bacterial colony is inoculated in the PDA-aniline blue culture medium and the PDA-guaiacol culture medium, whether the color change on the culture medium is observed, so that whether the single bacterial colony has the ability to secrete lignin degradation enzyme is judged.

[0010] Further, the DNS colorimetric method is used for the carboxymethyl cellulase activity determination of the screened single bacterial colony with the lignin degradation ability, and the lignin peroxidase activity detection kit is used for the lignin enzyme activity determination of the single bacterial colony. The carboxymethyl cellulase activity of the bacterial strain reaches at least 228.10 U / mL within 16 hours, the lignin enzyme is produced after the bacterial strain is cultured for 16 h, and the enzyme activity is 10.36 U / mL. The tobacco stalk efficient degradation bacteria WP-2022 strain has the ability to efficiently degrade the cellulose and the lignin in the tobacco stalk.

[0011] The application further provides a tobacco stalk degradation bacterial agent, characterized in that the tobacco stalk degradation bacterial agent comprises the tobacco stalk efficient degradation bacteria WP-2022.

[0012] This invention also provides the use of the aforementioned tobacco straw degrading microbial agent in the degradation of tobacco straw, characterized in that the degrading bacteria can simultaneously degrade lignin and cellulose in tobacco straw. In the structure of tobacco straw, lignin is dispersed among cellulose fibers, while hemicellulose acts as a connecting component, binding lignin and cellulose together to form a robust network structure, namely a cellulose-hemicellulose-lignin complex. The tight binding of lignin and cellulose in this structure means that their degradation processes are interdependent.

[0013] The biodegrading agent of this invention effectively disrupts the cellulose-hemicellulose-lignin network structure in tobacco straw by simultaneously acting on both cellulose and lignin, the two main components. Through this method, the agent of this invention achieves highly efficient decomposition of tobacco straw, reducing environmental pollution.

[0014] Furthermore, the effective temperature range for the tobacco straw degrading microbial agent is 22-28℃. This eliminates the need for composting tobacco straw in a specific environment, particularly high-temperature composting. The agent accelerates straw degradation in the field at a natural temperature of 22-28℃, providing an effective solution for direct return of tobacco straw to the field and facilitating its resource utilization. Attached Figure Description

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

[0016] Figure 2 The degradation rate of lignocellulose components in tobacco stalks after 7 days of treatment with WP-2022 strain. Detailed Implementation

[0017] The preferred embodiments of the present invention will be described in detail below to provide a clearer understanding of the purpose, features, and advantages of the invention. It should be understood that the following embodiments are not intended to limit the scope of the invention, but are merely illustrative of the essential spirit of the technical solution of the invention.

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

[0019] Throughout this specification, references to "an 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. Therefore, the appearance of "in an embodiment" or "an embodiment" in various places throughout the specification does not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any manner in one or more embodiments.

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

[0021] The following detailed description of the WP-2022 strain of tobacco stalk degrading bacteria of the present invention, with reference to specific embodiments, is provided. The technical solution of the present invention includes, but is 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 WP-2022, a highly efficient tobacco stalk degrading bacterium. Soil samples from tobacco plantations were collected in Anshun City, Guizhou Province. The isolation and purification steps are as follows:

[0024] 10.00g of tobacco soil sample was placed in enrichment medium and cultured for 1 hour. After removing the enrichment medium and letting it stand for 5 minutes, 2mL of the supernatant was taken with a pipette and placed in a new sterilized and cooled enrichment medium. The sample was then cultured at 28℃ and 180r / min for 3 days with shaking.

[0025] Following aseptic techniques, the cultured bacterial suspensions were diluted 10⁻³, 10⁻⁴, and 10⁻⁵ times, respectively. 0.1 mL of each solution was then spread onto bacterial, fungal, and actinomycete culture media where no colonies had formed, for the isolation of bacteria, fungi, and actinomycetes, respectively. Three replicates were set up for each gradient. Bacteria, fungi, and actinomycetes were incubated upside down at 28°C for 2–3 days.

[0026] After the colonies have grown on the plates, select representative colonies based on their morphology and color, and pick out a single colony with a sterile inoculation loop. Bacteria and fungi are streaked on the corresponding solid culture medium according to aseptic operation requirements until a single colony is obtained after purification. For actinomycetes, single colonies are picked into LB medium for liquid culture, and then a small amount of bacterial solution is taken with an inoculation loop and streaked on the actinomycete medium until a single colony is obtained.

[0027] 2. Screening of the ability of the highly efficient tobacco stalk degrading bacterium WP-2022 to degrade cellulose and lignin

[0028] 2.1 Screening of the cellulose degradation ability of the highly efficient tobacco stalk degrading bacterium WP-2022: The isolated and purified strains were screened for their cellulose degradation ability. The Congo red staining method was used to screen the cellulose degradation ability; the specific screening method is as follows:

[0029] The purified strains were inoculated into CMC-Na medium in triplicate and incubated at 30°C for 2 days. The staining time was determined based on the size of the colonies.

[0030] The differential culture medium plates were stained with 0.1% Congo red solution for 15 min, and then destained with 1 mol / L sodium chloride solution for 15 min. Excess solution on the plates was removed with a pipette, and the diameter of the clear zone D (cm) and the diameter of the colony d (cm) were measured using the cross-cross method. Based on the Hc value (Hc = D / d), the ability of the strain to decompose cellulose was determined, and strains with larger Hc values ​​were screened.

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

[0032] The strains selected for cellulose degradation were inoculated onto PDA-aniline blue and PDA-guaiacol agar media using a spot inoculation method and cultured for 24 hours. The presence of a fading zone on the PDA-aniline blue medium indicated the ability to secrete MnP and LiP; and the appearance of a color development zone on the PDA-guaiacol agar medium indicated the ability to secrete Lac. This determined their ability to decompose lignin.

[0033] 2.3 The enzyme activity of the screened strains was determined using the following methods:

[0034] 1) The carboxymethyl cellulase activity of the strain was determined by the DNS colorimetric method.

[0035] 2) Ligninase activity was determined using a lignin peroxidase activity assay kit;

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

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

[0038] 3. Molecular biological identification of strains

[0039] Molecular biological identification was performed using PCR. After DNA extraction, PCR amplification and agarose gel electrophoresis were performed using universal primers for bacterial 16S rDNA. The PCR amplification products were sequenced, and the sequence was compared with the 16S rDNA sequence in GeneBank for homology. The 16S rDNA 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, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M20241187, and deposited on June 11, 2024.

[0040] 4. Tobacco Straw Degradation Test

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

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

[0043] like Figure 1 As shown, after spraying the bacterial solution onto tobacco straw for 7 days, compared with the control group (CK) which was only sprayed with sterile water, the degradation products of the tobacco straw after bacterial treatment showed a flat infrared absorption peak near 1030 cm⁻¹, indicating that the CO bonds in lignin and cellulose were destroyed and small organic molecules were released. The infrared absorption peak near 1230 cm⁻¹ decreased, indicating that the O=CC stretching vibration in the lignin-hemicellulose complex was reduced, suggesting that the complex structure of cellulose, hemicellulose, and lignin in the tobacco straw was destroyed and that cellulose, hemicellulose, and lignin separated. The vibrations of CC and CO in lignin near 1320 cm⁻¹ and CH in cellulose near 1370 cm⁻¹ decreased, indicating that some cellulose and lignin in the tobacco straw were degraded. The stretching vibrations of C=C and C=N in the aromatic skeleton near 1600 cm⁻¹ decreased, indicating that the aromatic skeleton of lignin in the tobacco straw was destroyed. The vibrations of hydrogen bonds in cellulose and carbon-hydrogen bonds in methyl and methylene groups near 2920 cm⁻¹ decreased, indicating that the hydrogen bonds in cellulose in the tobacco straw were destroyed. In conclusion, this demonstrates that strain WP-2022 can effectively degrade cellulose and lignin in tobacco straw.

[0044] 4.3 Results and Analysis

[0045] like Figure 2 As shown, after 7 days of cultivation, this 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 the untreated treatment (CK).

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

[0047] SEQ ID NO.1:

[0048] GTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGG

[0049] TGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATT

[0050] ACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACT

[0051] GAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTG

[0052] TTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGA

[0053] TTTGACGTCATCCCCACCTTTCCTCCGGTTTGTCACCGGCAGTCACCTTAG

[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] Any aspects of this invention not described in detail are well-known to those skilled in the art.

[0077] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A highly efficient tobacco stalk degrading bacterium, WP-2022, which is Bacillus subtilis, is deposited at the National Center for Type Culture Collection with accession number CCTCCM20241187.

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