Tobacco stem efficient degradation complex microbial inoculant as well as preparation method and application thereof
By constructing the complex bacteria agent J-2024-1, combined with the synergistic effects of multiple functional microorganisms, the problem of difficulty in effectively degrading tobacco straw in the existing technology has been solved, efficient degradation and safety for crop growth have been achieved, and the resource utilization efficiency of straw has been significantly improved.
Patent Information
- Application Number
- CN202411576767.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-06
- Publication Date
- 2025-03-11
AI Technical Summary
Existing microbial agents are difficult to effectively degrade protein, cellulose and lignin in tobacco straw, and are unstable under extreme environmental conditions, which may have toxic effects on crops and affect growth.
Complex bacterial agent J-2024-1 was constructed, including Bacillus sausage ZY2, Acinetobacteria indica LXX-2022-12 and Bacillus YP-2023-01. These strains can secrete cellulase, ligninase and protease, improve degradation efficiency through synergy, and ensure the stability and safety of bacterial agents through screening and antagonism experiments.
The complex bacteria agent J-2024-1 can significantly improve the degradation rate of tobacco straw, reaching 40.30%, and has no negative impact on crop growth, demonstrating its application potential and practical feasibility in the agricultural field.
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Figure CN119592443A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to a highly efficient tobacco stalk degradation composite bacterial agent and a preparation method and application thereof. Background Art
[0002] A large amount of straw is produced in current agricultural production, and as a renewable resource, its utilization rate is still low. Therefore, effectively degrading straw can not only reduce agricultural waste and environmental pollution, but also transform it into a valuable resource.
[0003] The chemical structures and physical properties of different components in straw vary, resulting in different levels of difficulty in their degradation. Cellulose and hemicellulose are relatively easy to decompose, while lignin has a strong resistance to degradation due to its complex structure. In addition, the protein in straw will be hydrolyzed into small molecules such as amino acids and peptide chains during microbial degradation. These substances provide a continuous nitrogen source and support the rapid reproduction of microorganisms, thereby accelerating the degradation of other components (such as cellulose and lignin). Although the degradation processes of cellulose, lignin and protein are interdependent, the degradation of each component requires different types of microorganisms and their specific enzymes, so it is difficult for a single strain of bacteria to play a comprehensive role in the complex straw degradation environment. Therefore, it is intended to solve this problem by constructing a composite bacterial agent.
[0004] However, the symbiotic relationship between microorganisms is very complex, and there are phenomena such as nutrient competition, space competition and inhibition of secondary metabolites. This mutual inhibition weakens the overall activity of the bacterial community, resulting in a decrease in the degradation efficiency of the composite bacterial agent. In addition, although some composite bacterial agents perform well under indoor conditions, they are unstable in natural environments or extreme conditions (such as high temperature, high humidity or pH changes). Whether they can survive and function effectively for a long time after application is a key issue, and some microorganisms may even rapidly reduce their activity after application, resulting in a decrease in their degradation ability.
[0005] Although the application of microbial technology in agriculture has achieved remarkable results, research on specific organic matter (such as lignin, cellulose and protein) is still insufficient. The existing microbial agents do not fully consider the degradation characteristics of various organic matter and the synergistic effects of microorganisms when designing, resulting in many agents being unable to effectively improve the degradation efficiency of straw. Composite agents must not only efficiently degrade straw, but also ensure safety for crops. In practical applications, if certain strains or their metabolites in the agents are toxic to crops, it may lead to inhibition of crop growth or the occurrence of diseases.
[0006] Therefore, providing a composite bacterial agent that can simultaneously and efficiently degrade protein, cellulose and lignin in tobacco straw without negatively affecting crop growth is an urgent problem to be solved by the present invention. Summary of the invention
[0007] The present invention provides a highly efficient tobacco stalk degradation composite bacterial agent and its preparation method and application, aiming to solve the problems existing in the above-mentioned background technology. By constructing a composite bacterial agent, different functional microorganisms that can secrete cellulase, ligninase and protease are combined together to form a synergistic effect, improve the degradation efficiency of various components, accelerate the release of carbon and nitrogen sources, promote the proliferation and activity of microorganisms, and comprehensively decompose the complex structure in the straw, thereby significantly improving the resource utilization efficiency of the straw.
[0008] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:
[0009] In a first aspect, the present invention discloses a composite bacterial agent for efficiently degrading tobacco stalks. The composite bacterial agent is named composite bacterial agent J-2024-1, which includes Bacillus zanthoxyli ZY2, Acinetobacter indonesiensis LXX-2022-12 and Bacillus YP-2023-01.
[0010] In a preferred embodiment of the present invention, the preservation number of the Bacillus zanthoxylum ZY2 is CCTCC NO: M2021897, the preservation number of the Acinetobacter indica LXX-2022-12 is CCTCC NO: M 202319831, and the preservation number of the Bacillus YP-2023-01 is CCTCC NO: M 20241188.
[0011] In a preferred embodiment of the present invention, the composite bacterial agent is fermented by adding seed liquid of Bacillus zanthoxylum ZY2, Acinetobacter indica LXX-2022-12 and Bacillus YP-2023-01 at a live bacteria count ratio of 1:1:1.
[0012] In a preferred embodiment of the present invention, the effective viable bacteria count in the composite bacterial agent is not less than 1×1010 CFU.
[0013] In a second aspect, the present invention discloses a method for preparing a composite bacterial agent for efficient tobacco stem degradation as described in the first aspect, comprising the following steps:
[0014] 1) After enrichment and culture of tobacco-growing soil samples, the supernatant was collected and shaken, and the diluted bacterial solution was spread on a sterile culture medium for culture, and representative colonies were selected for streaking and purification until a single colony was obtained;
[0015] 2) The isolated and purified strains were inoculated on skim milk powder medium, with 3 replicates for each strain, cultured at 37°C, and the size of the hydrolysis transparent zone was observed to screen out the strain with the strongest protein degradation ability;
[0016] 3) Inoculate a single strain on CMC-Na medium, with 3 replicates per strain, culture at 30°C, and then detect using Congo red clearing zone staining to select the cellulose-degrading strain with the largest clearing zone;
[0017] 4) The strains were inoculated on PDA-aniline blue and PDA-guaiacol culture media, with 3 replicates for each strain, and cultured at 30°C to observe whether fading or coloring circles were produced, and to select the strain with the strongest lignin degradation ability;
[0018] 5) An antagonistic experiment is performed on the three strains of bacteria screened. If there is no antagonistic effect, the seed liquid of the three strains of bacteria is added at a ratio of 1:1:1 of live bacteria count for fermentation, that is, compounded into the composite bacterial agent J-2024-1.
[0019] In a third aspect, the present invention discloses the use of the highly efficient tobacco stalk degradation composite bacterial agent as described in the first aspect in degrading tobacco stalks.
[0020] In a preferred embodiment of the present invention, the composite bacterial agent is sprayed on the cigarette rod.
[0021] Preferably, the composite bacterial agent is sprayed on the tobacco stem at an inoculation rate of 6%.
[0022] In a preferred embodiment of the present invention, the composite bacterial agent degrades protein, cellulose and lignin in straw.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] The multifunctional and efficient degradation composite bacterial agent J-2024-1 provided by the present invention can simultaneously and efficiently degrade protein, cellulose and lignin in tobacco straws, and has no negative impact on crop growth. In actual field applications, J-2024-1 showed significant degradation effects and application potential, with a tobacco straw degradation rate of 40.30%, significantly higher than the control, which is of great significance for promoting sustainable agricultural development and ecological environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0026] Figure 1 This is a comparison chart of the degradation rate of the composite bacterial agent J-2024-1 and single bacteria;
[0027] Figure 2 This is the decomposition rate of tobacco stalks degraded by the composite bacterial agent J-2024-1;
[0028] Figure 3 This is the Fourier transform infrared spectrum of tobacco straw treated with the composite bacterial agent J-2024-1.
[0029] Figure 4 Antagonism results between strains of composite bacterial agent J-2024-1. DETAILED DESCRIPTION
[0030] The following examples are provided to further illustrate the present invention, but not to limit the present invention. Specific experimental conditions and methods are not specified in the following examples, and the technical means adopted are conventional means well known to those skilled in the art.
[0031] Example 1 Preparation of composite bacterial agent
[0032] 1. Isolation and purification of degradation strains
[0033] Tobacco soil samples were collected from Anshun City, Guizhou Province. 10.00 g of fresh soil samples were weighed and cultured in enrichment medium for 1 hour. After standing for 5 minutes, 2 mL of supernatant was placed in a new sterilized and cooled enrichment medium and cultured at 28°C and 180 r / min for 3 days. According to the requirements of aseptic operation, the bacterial solution was diluted to 10 -3 , 10 -4 , 10 -5 0.1 mL was spread on sterile culture medium and inverted at 28°C for 2-3 days. Three parallel experiments were set for each gradient. Representative colonies were selected according to colony morphology and color and purified by streaking until a single colony was obtained.
[0034] 2. Strain Function Screening Method
[0035] 2.1 Screening of protein degradation ability
[0036] The isolated and purified strains were inoculated on skimmed milk powder culture medium, with 3 replicates for each strain, and cultured at 37°C for 24 hours. The size of the hydrolysis transparent zone was observed to screen the strain with the strongest protein degradation ability.
[0037] 2.2 Screening of cellulose degradation ability
[0038] The strains were inoculated on CMC-Na medium with 3 replicates for each strain. After culturing at 30°C for 24 hours, the strains were detected by Congo red transparent zone staining to screen the cellulose-degrading strains with the largest transparent zone.
[0039] 2.3 Lignin degradation ability screening
[0040] The strains were inoculated on PDA-aniline blue and PDA-guaiacol medium, with 3 replicates per strain, and cultured at 30°C for 24 hours to observe whether a fading circle (ability to secrete MnP and LiP) or a color circle (ability to secrete Lac) was produced, and the strain with the strongest lignin degradation ability was selected.
[0041] 3. Molecular Biological Identification of the Strains
[0042] The 16S rDNA was amplified by PCR, and the different functional strains screened were identified by molecular biology. After the strain DNA was extracted, PCR amplification and agarose gel electrophoresis were performed using 16S rDNA universal primers, and the PCR products were sequenced and compared with the sequences in the GeneBank database for homology to determine the species of the strain.
[0043] The 16S rRNA identification showed that they were Bacillus zanthoxyli ZY2, Acinetobacter indonesiensis LXX-2022-12 and Bacillus YP-2023-01. Antagonistic experiments were performed on the three strains, and the results showed that there was no antagonistic effect between the three strains (such as Figure 4 ). The seed liquid of the above three strains of bacteria was added in a ratio of 1:1:1 in terms of live bacteria count for fermentation, that is, compounded into the composite bacterial agent J-2024-2.
[0044] Test Example 1 Bacterial Antagonism Experiment
[0045] The different functional single strains isolated and purified were subjected to a cross-streak inoculation experiment on sterile LB solid culture medium: first, the strain to be tested was inoculated along a straight line at one end of the plate; then, the strain to be tested was inoculated in the same way at the other end, so that the streaked areas of the two bacteria were close to each other but not overlapping. The inoculated plates were cultured at 28°C until the strains formed obvious colonies or bacterial ribbons, and then the growth inhibition zone at the junction of the two bacteria was observed. If there was an inhibition zone, it indicated the presence of antagonism. Each group of experiments was repeated 3 times to ensure reliable results.
[0046] The results showed that there was no antagonism between Bacillus zanthoxyli ZY2, a highly efficient protein-degrading strain, Acinetobacter indonesiensis LXX-2022-12, a highly efficient cellulose-degrading strain, and Bacillus YP-2023-01, a highly efficient lignin-degrading strain. Figure 4 ), so it can be compounded into the multifunctional composite bacterial agent J-2024-1.
[0047] Test Example 2 Comparative experiment on the ability of composite bacterial agent and single bacteria
[0048] Select the strains without antagonistic effects to form a composite bacteria, and mix them with the single bacteria at a 6% inoculation rate (strain content ≥ 1×10 10 CFU) was sprayed on 500g tobacco stems, mixed with an appropriate amount of soil and poured into pots. The straw was taken out at 0d, 7d, 15d, 21d and 35d, rinsed with distilled water, and then the attached bacteria and CaCO3 precipitation were removed with dilute acid solution. Finally, it was rinsed with distilled water for 3 times and dried at 80℃ to constant weight. The straw degradation rate was calculated by weight loss method: Straw degradation rate % = (W0-Wt) / W0×100%
[0049] Wherein, W0 is the initial dry weight of straw, and Wt is the dry weight of straw after cultivation for t time.
[0050] The degradation rates of composite bacterial agent J-2024-1 and single bacterial strains are as follows Figure 1 As shown. The ZY2 strain is a protein-degrading bacterium, so it has a poor degradation effect on straw. In contrast, the degradation rates of the lignin-degrading bacteria YP-2023-01 and the cellulose-degrading bacteria LXX-2022-12 were higher than those of the CK (control group). Although there was no significant difference in the degradation rates between the two strains, the degradation rate of the cellulose-degrading bacteria LXX-2022-12 was slightly higher, showing its potential in degrading straw. From the perspective of the degradation process, the degradation effect of the composite bacterial agent J-2024-1 was better than that of a single strain at each time point, indicating that the composite bacterial agent has a more stable and effective degradation ability, which may be related to the synergistic effect of its diverse functional strains, which can more comprehensively decompose various organic substances in the straw.
[0051] Test Example 3: Performance verification of composite microbial agents and study of field application effects
[0052] The prepared composite bacteria were inoculated at a rate of 6% (strain content ≥ 1×10 10CFU) were inoculated into liquid culture medium with tobacco stem as the sole carbon source. Each composite bacteria was repeated 3 times. Samples were taken at 7, 14 and 28 days after inoculation for Fourier transform infrared spectroscopy analysis to verify the degradation effect of the composite bacteria on components such as lignin and cellulose.
[0053] In the field experiment, tobacco straw was selected as the material, and the composite bacterial agent J-2024-1 treatment group and the single bacterial treatment group were set as the control, with 3 replicates in each group. 5% bacterial solution or sterile water was added to each replicate, and the effective viable bacterial count of the bacterial solution was ≥1×10 -8 The degradation effect in the field was evaluated by calculating the straw decomposition rate. To further verify the effect of the composite microbial agent on the growth of flue-cured tobacco, the treated straw was returned to the field, and flue-cured tobacco was planted. The growth of flue-cured tobacco was recorded according to the Survey and Measurement Method of Tobacco Agronomic Traits (YC / T 142—2010).
[0054] The results are as follows Figure 2 As shown: After 105 days of treatment, the composite agent showed excellent straw degradation efficiency compared with the control. The degradation rate of the composite agent continued to be significantly higher than that of the control group (CK), indicating that it maintained efficient degradation performance throughout the treatment cycle. On the 105th day, the degradation rate of the composite agent reached the highest value, with the degradation rate of the composite agent treatment group being 40.30%, while that of the control group was 26.75%. During the same period, the degradation rate of the J-2024-1 agent was better than the degradation rates of the composite strains F and G reported in the literature, which were 32.96% and 36.24%, respectively (Wang Ziyuan, 2024). These results further indicate that the composite agent J-2024-1 has a persistent and efficient degradation ability and shows a wide range of application potential.
[0055] Experimental Example 4 Degradation effect of composite bacterial agent J-2024-1 on tobacco straw components
[0056] In order to verify the degradation effect of the composite bacterial agent J-2024-1 on tobacco straw components, the tobacco straw treated with the composite bacterial agent J-2024-1 was scanned by Fourier transform infrared spectroscopy. Figure 3 As shown, it was found that 1050cm -1 and 1109cm -1 The absorption peaks at 616cm are related to Si-O and CO stretching vibrations, respectively. On the 7th day, these absorption peaks increased, indicating that the silicified layer was destroyed, cellulose and hemicellulose were exposed and began to degrade. On the 14th and 28th days, the absorption peaks weakened as the small molecular sugars were further degraded or mineralized. -1 The absorption peak at 1423cm -1 、1652cm -1 、1740cm -1The changes in the absorption peaks of the same bands showed that the degradation of lignin, cellulose, and hemicellulose was accompanied by the generation and decomposition of aliphatic compounds and carbonyl-containing intermediates. The changes in the absorption peaks of 3100-3254cm-1 reflected the generation and degradation of alcohols and phenols. The results showed that the composite bacterial agent J-2024-1 could effectively degrade the main components in tobacco straw.
[0057] Experimental Example 5 Effect of Composite Bacterial Agent J-2024-1 on Tobacco Growth
[0058] High-quality composite microbial agents not only need to have efficient straw degradation performance, but also need to ensure that there is no negative impact on crop growth. To verify this effect, the present invention returns the straw treated with the composite microbial agent and the straw of the control group to the field, and plants flue-cured tobacco. Through the investigation of the agronomic traits of flue-cured tobacco clusters, including indicators such as the number of leaves, maximum leaf length, maximum leaf width, plant height and stem girth, the results are shown in Table 1. There is no significant difference between the composite microbial agent treatment group and the control group in these growth indicators. This shows that the composite microbial agent has no adverse effects on the growth of flue-cured tobacco, further confirming its application potential and practical feasibility in the agricultural field.
[0059] Table 1 Effects of compound bacterial agent J-2024-1 on agronomic traits of flue-cured tobacco at the cluster stage
[0060]
[0061] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A highly efficient tobacco stalk degradation composite bacterial agent, characterized in that: The composite bacterial agent is named as composite bacterial agent J-2024-1, which includes Bacillus zanthoxyli ZY2, Acinetobacter indonesiensis LXX-2022-12 and Bacillus YP-2023-01.
2. The high-efficiency tobacco stem degradation composite bacterial agent according to claim 1, characterized in that: The preservation number of the Zanthoxylum bungeanum ZY2 is CCTCC NO: M 2021897, the preservation number of the Indian Acinetobacter LXX-2022-12 is CCTCC NO: M202319831, and the preservation number of the Bacillus YP-2023-01 is CCTCC NO: M 20241188.
3. The high-efficiency tobacco stem degradation composite bacterial agent according to claim 1, characterized in that: The composite bacterial agent is fermented by adding seed liquid of Bacillus zanthoxylum ZY2, Acinetobacter indica LXX-2022-12 and Bacillus YP-2023-01 in a live bacteria count ratio of 1:1:
1.
4. The high-efficiency tobacco stem degradation composite bacterial agent according to claim 1, characterized in that: The effective live bacteria count in the composite bacterial agent is not less than 1×1010 CFU.
5. A method for preparing the highly efficient tobacco stem degradation composite bacterial agent according to any one of claims 1 to 4, characterized in that: The steps include: 1) After enrichment and culture of tobacco-growing soil samples, the supernatant was collected and shaken, and the diluted bacterial solution was spread on a sterile culture medium for culture, and representative colonies were selected for streaking and purification until a single colony was obtained; 2) The isolated and purified strains were inoculated on skim milk powder medium, with 3 replicates for each strain, cultured at 37°C, and the size of the hydrolysis transparent zone was observed to screen out the strain with the strongest protein degradation ability; 3) Inoculate a single strain on CMC-Na medium, with 3 replicates per strain, culture at 30°C, and then detect using Congo red clearing zone staining to select the cellulose-degrading strain with the largest clearing zone; 4) The strains were inoculated on PDA-aniline blue and PDA-guaiacol culture media, with 3 replicates for each strain, and cultured at 30°C to observe whether fading or coloring circles were produced, and to select the strain with the strongest lignin degradation ability; 5) An antagonistic experiment is performed on the three strains of bacteria screened. If there is no antagonistic effect, the seed liquid of the three strains of bacteria is added at a ratio of 1:1:1 of live bacteria count for fermentation, that is, compounded into the composite bacterial agent J-2024-1.
6. Use of the high-efficiency tobacco stem degradation composite bacterial agent as described in any one of claims 1 to 4 in degrading tobacco stems.
7. The use according to claim 6, characterized in that: The composite bacterial agent is sprayed on the tobacco stem.
8. The use according to claim 7, characterized in that: The composite bacterial agent is sprayed on the tobacco stem at an inoculation rate of 6%.
9. The use according to claim 6, characterized in that: The composite bacterial agent degrades protein, cellulose and lignin in straw.