Composite microbial system and application thereof in degradation of corn straw

Abstract

The invention discloses a compound bacterium system and application thereof in degradation of corn straw, and relates to the technical field of microorganisms. The compound bacterium system comprises candida guilliermondii, aspergillus terreus and funalia trogii. The multifunctional efficient degradation compound bacterial system provided by the invention can efficiently degrade cellulose, hemicellulose and lignin in tobacco straws at the same time, and has no negative influence on crop growth. In the application, the high-yield strain candida guilliermondii of the biological membrane is added, so that a biological membrane compound is formed by the compound bacterial system, the straw and the straw degrading enzyme, and the synergistic degradation of lignocellulose is promoted. The compound bacterial system shows a remarkable degradation effect and application potential, the corn straw degradation rate is 38.64%, and the compound bacterial system is remarkable in contrast, and has important significance in promoting agricultural sustainable development and ecological environment protection.

Description

Technical Field

[0001] This invention relates to a composite microbial system and its application in the degradation of corn stalks, belonging to the research field of biodegradation technology of lignocellulose. Background Technology

[0002] Straw is a renewable green biological resource with broad application prospects in biomass energy, papermaking, feed, and fertilizer. The main components of straw are fiber (primarily crude fiber), hemicellulose, lignin, soluble sugars, crude protein, crude fat, ash, and trace elements. In 2023, my country's collectable straw resources reached 734 million tons, with a CAGR of 0.46% from 2011 to 2023. Straw is both agricultural waste and an important agricultural resource. However, due to the heterogeneity and crystalline structure of lignocellulose biomass, its resource utilization is difficult and its efficiency is low. Large amounts of straw-derived lignocellulose are discarded or directly burned, causing not only resource waste but also environmental pollution. Therefore, lignocellulose conversion and utilization technologies have gradually become a research hotspot in this field. These technologies mainly include physical, chemical, physicochemical, and biological methods. Among them, biological treatment is an environmentally friendly technology that uses microorganisms to catalyze the degradation of lignocellulose, facilitating its further conversion.

[0003] Straw is a complex lignocellulose material that requires the combined action of multiple enzyme systems produced by various microorganisms to degrade. The construction of a complex microbial system essentially involves establishing a highly efficient enzyme system. By assembling strains with different functions, lignin, cellulose, and hemicellulose in straw can be degraded, thus facilitating straw degradation. For example, many microorganisms (such as fungi and bacteria) can mix together, cooperate with each other, and exert the different enzymatic hydrolytic effects of each microbial group to achieve the goal of degrading lignocellulose in plant straw.

[0004] Currently available straw composting inoculants are typically composed of bacteria (mainly Bacillus) and fungi (yeast, Trichoderma, Aspergillus) screened from the soil. These are prepared by culturing different strains separately and then mixing them in a specific ratio. However, many problems still exist in practical applications: for example, different strains may compete or antagonize each other, adversely affecting their growth and development; enzymes produced by the compound strains during growth are easily released, failing to achieve a good synergistic effect and hindering efficient straw degradation, resulting in long straw degradation times. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a composite microbial agent capable of efficiently degrading lignocellulose and its application in straw return to the field. By constructing a composite microbial agent, different functional microorganisms capable of secreting cellulase, ligninase, and hemicellulase are combined to form a synergistic effect, improving the degradation efficiency of various components. Furthermore, by producing biofilm-forming strains, the composite agent promotes the adhesion of microorganisms to straw, thereby significantly improving the resource utilization efficiency of straw.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a complex microbial system that can efficiently degrade corn stalks, comprising *Meyerozyma guilliermondii*, *Aspergillus terreus*, and *Coriolopsis trogii*.

[0008] In one embodiment of the present invention, the composite ratio of Candida guinea, Aspergillus terreus and Aster tataricus is 1-2:1-2:1-2 according to the ratio of spore number to fungal number.

[0009] In one embodiment of the present invention, the Candida guinea is Candida guinea ATCC6260.

[0010] In one embodiment of the present invention, the Aspergillus terreus is Aspergillus terreus GIM 3.547.

[0011] In one embodiment of the present invention, the *Amanita muscaria* is *Amanita muscaria* CGMCC No. 40363.

[0012] In one embodiment of the present invention, the number of effective viable bacteria in the composite bacterial strain is not less than 1×10⁻⁶. 9 CFU.

[0013] A second objective of this invention is to provide the application of the aforementioned composite microbial system in the degradation of corn stalks.

[0014] In one embodiment of the present invention, the application is to spray the compound microbial system onto corn stalks.

[0015] In one embodiment of the present invention, the application involves culturing Aspergillus terreus and Avicennia marina to a spore count ≥ 1 × 10⁻⁶. 9 A spore suspension of 1 spore / mL was used to culture Candida guinea to the logarithmic growth phase. Then, Candida guinea, Aspergillus terreus, and Aster tataricus were mixed in a certain proportion to prepare a compound bacterial solution. The compound bacterial solution was then sprayed onto straw at a concentration of 5% to 15% and allowed to stand for incubation.

[0016] In one embodiment of the present invention, the temperature for static incubation is 20–35°C.

[0017] In one embodiment of the present invention, the culture is left to stand for 10 to 30 days.

[0018] The beneficial effects of this invention are:

[0019] The composite microbial strain of this invention contains strains that produce various enzymes for degrading cellulose, hemicellulose, and lignin, including cellulase, filter paper enzyme, xylanase, laccase, lignin manganese peroxidase, and lignin peroxidase. By constructing a complex of multiple enzymes, the degradation of straw can be achieved more effectively. Furthermore, this invention adds strains capable of forming biofilms, which allows microorganisms to better adhere to straw, and the biofilm can better aggregate free lignocellulose-degrading enzymes, binding the enzymes to cells and straw, thereby enhancing straw degradation. Additionally, under environmental stress conditions, biofilm formation is more conducive to the survival of the strains, enabling the microorganisms to be better utilized in different agricultural environments and degrade straw more efficiently and rapidly. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The activity of lignocellulase in a single strain;

[0022] Figure 2 For the flat plate confrontation experiment;

[0023] Figure 3 To determine the biomass of biofilms using the crystal violet staining method;

[0024] Figure 4 Enzyme activity of compound microbial agents;

[0025] Figure 5 The degradation rate of straw components by the compound microbial agent;

[0026] Figure 6 Transmission electron microscope image of fermented straw sample;

[0027] Figure 7 Fourier transform infrared spectrum of fermented straw sample. Detailed Implementation

[0028] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0029] Source of raw materials

[0030] The *Meyerozyma guilliermondii* Y6 strain used in the examples, ATCC6260, was purchased from Ningbo Taiste Biotechnology Co., Ltd.; *Aspergillus terreus* F10 strain, GIM 3.547, was purchased from Ningbo Taiste Biotechnology Co., Ltd.; and *Coriolopsistrogii* F12 strain, CGMCCNo.40363, was purchased from the China General Microbiological Culture Collection Center.

[0031] PDA medium composition (g / L): potato 200, glucose 20.

[0032] Inorganic salt liquid culture medium composition (g / L): (NH4)2SO4, KH2PO4 2.0g, urea 0.3g, MgSO4·7H2O 0.3g, CaCl2 0.3g, FeSO4·7H2O 7.5mg, MnSO4·H2O 2.5mg, ZnSO4 2mg, CoCl2 3.0mg.

[0033] Inorganic salt liquid culture medium with straw powder as the sole carbon source: 1g straw + 5g inorganic salt culture medium (solid-liquid ratio 1:5).

[0034] In this application, the term "biofilm" refers to a biological community and its encapsulated extracellular polymers and matrix network that adhere to each other or to the surface of a tissue or object.

[0035] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or are commonly used in this industry. Example 1: Lignocellulase activity of a single strain

[0036] Aspergillus terreus (F10) and F. truncatum (F12) were inoculated onto PDA plates respectively. After the mycelium covered the plate, it was scraped off with an inoculation spatula to obtain spores with a count ≥1×10⁻⁶. 9A spore suspension of 1 spore / mL was inoculated into an inorganic salt liquid medium with straw powder as the sole carbon source for growth. *Candida guinea* (Y6), after reaching an OD600 of 0.8 in PDA medium, was also inoculated into an inorganic salt liquid medium with straw powder as the sole carbon source. After eight days of culture, fermentation broth was collected to prepare crude enzyme solutions, and the activities of cellulose-degrading enzymes (cellulosinase and filter paper enzyme), hemicellulose-degrading enzymes (xylanase activity), and lignin-degrading enzymes (laccase, lignin manganese peroxidase, and lignin peroxidase) were measured. Single-strain shake-flask enzyme activities were as follows: Figure 1 As shown, F10 has a strong ability in cellulose and hemicellulose degrading enzyme activity, F12 performs better in laccase and lignin peroxidase, and Y6 only has cellulase activity.

[0037] Example 2: Antagonistic Experiment Between Strains

[0038] Each strain of *Meyerozyma guilliermondii* Y6, *Aspergillus terreus* F10, and *Coriolopsistrogii* F12 was cultured separately on PDA solid plates. After the mycelium covered the entire plate, mycelial blocks were dug out using a punch and placed on new PDA plates for a confrontation test. *Meyerozyma guilliermondii* (Y6) that was growing well on the PDA solid plate was then spotted around *Aspergillus terreus* (F10) and *Coriolopsistrogii* (F12), and incubated at 28℃ for one week. The presence of antagonistic bands between colonies was observed. The results showed no antagonistic effect among *Meyerozyma guilliermondii* Y6, *Aspergillus terreus* F10, and *Coriolopsistrogii* F12, therefore they can be compounded into a multifunctional compound mycelial agent.

[0039] Example 3: Determination of the film-forming ability of the compound bacterial agent

[0040] The spore suspensions of Aspergillus terreus F10 and Aster spp. F12 were adjusted to a spore count ≥1×10⁻⁶. 9 After adjusting the Y6 concentration to OD600 of 0.6, inoculate 2% (v / v) of the cells into PDA medium. Incubate at 30°C for 48 hours, then remove the medium and airborne cells. Wash repeatedly with PBS, fix with 10% methanol, stain with crystal violet, wash away excess dye, dissolve in 33% acetic acid, and measure the absorbance at OD540. Figure 3 It can be seen that the biofilm content of the compound microbial community increased significantly after the addition of Y6, by 31.52% compared with Y6.

[0041] Example 4: Application of compound microbial agents in the degradation of straw-derived lignocellulose

[0042] The two-strain combination (F10+F12) and the three-strain combination (F10+F12+Y6) were compounded at a spore count ratio of 1:1 and a spore count to fungal count ratio of 1:1:1, respectively. Specifically, *Aspergillus terreus* (F10) and *Amanita muscaria* (F12) were inoculated onto PDA plates. After the mycelium covered the plate, it was scraped off with an inoculation spatula to obtain a spore count ≥1×10⁻⁶. 9 A spore suspension of *Candida yunnanensis* (Y6) was prepared and grown in PDA medium until the OD600 reached 0.8. The spores were then mixed in a specific ratio and inoculated into an inorganic salt solid medium with straw powder as the sole carbon source. The medium was then incubated at 30°C. Samples were taken at 0, 4, 8, 12, 16, and 20 days to determine the activity of lignocellulose-degrading enzymes and the straw degradation rate in both groups. The content of the three elements (cellulose, cellulose, and sulfur) was also determined using the NREL standard method. Figure 4 As can be seen, although the activity of cellulose-related enzymes decreased slightly after the addition of Y6, the activity of lignin-related enzymes increased significantly, especially manganese peroxidase, which increased by 9.63 times compared with the mold combination. Xylanase, laccase, and lignin peroxidase increased by 27.35%, 27.73%, and 52.11%, respectively. Figure 5 The left figure shows the content of straw components during the degradation process. Group F consisted of F10+F12, and group FY consisted of F10+F12+Y6. The figure shows that the most significant changes were in cellulose and hemicellulose. After 20 days, the cellulose and hemicellulose content in group FY decreased by 9.20% and 3.02% respectively compared to group F, and the straw degradation rate also increased from 35.80% to 38.64%. The F10+F12+Y6 combination achieved a straw degradation rate of 23.49% after 8 days of degradation, which is close to the degradation rate of the F10+F12 combination after 12 days (25.76%). These results demonstrate the synergistic effect of adding biofilm strains on the degradation of lignocellulose.

[0043] Example 5: Effect of compound microbial agent on the surface morphology of corn stalks

[0044] The morphological and structural changes of corn stalks before and after fermentation were observed using a scanning electron microscope. For example... Figure 6 As shown, before degradation, the surface structure of rice straw is compact and orderly, with cellulose, hemicellulose, and lignin intertwined to form a dense and complete structure. After 20 days of fermentation, the surface structure of corn stalks becomes rough, with numerous grooves appearing on the surface. The fibers are cut and arranged in an irregular, bent pattern, and the internal structure becomes loose and porous, destroying the original complex structure of natural lignocellulose. This is due to the degradation of lignocellulose by the compound microbial agent.

[0045] Example 6: Effect of compound microbial agents on functional groups on the surface of corn straw

[0046] To verify the degradation effect of the compound microbial agent on corn straw components, Fourier transform infrared spectroscopy was performed on the corn straw treated with the compound microbial agent. The results are as follows: Figure 7 As shown, in the range of 3300–3500 cm -1 The region exhibits a broad absorption band corresponding to the free stretching vibrations of the OH groups in cellulose, hemicellulose, and lignin, primarily provided by sugars. The original straw sample shows a distinct -OH absorption peak, which plateaus after 20 days. (1055 cm⁻¹) -1 The absorption peak of CO stretching vibrations related to cellulose and hemicellulose is reduced under the action of compound microbial agents, which is due to the weakening of aromatic CO bond linkages.

[0047] Example 7:

[0048] The indigenous straw-degrading microbial community was screened, and the selected Candida albicans, Aspergillus terreus, and Aster tataricus were compounded in a 1:1:1 ratio according to the method in Example 4. The mixture was then inoculated into an inorganic salt solid culture medium with straw powder as the sole carbon source and cultured in a constant temperature incubator at 30°C. The results showed that after 20 days of degradation, the degradation rate reached 38.09%, indicating that the three strains obtained from the screening can also achieve a good straw degradation effect.

[0049] Comparative Example 1:

[0050] A mixture of highly efficient lignin-degrading strains *Trichoderma harzianum* F3, cellulase-producing strain *Trichoderma asperellum* F13, and *Candida juncea* Y6 in a 1:1:1 ratio was inoculated into an inorganic salt solid medium with straw powder as the sole carbon source and cultured at 30°C. The straw degradation rate after 20 days was only 30.02%. These results indicate that the synergistic effect of adding other lignin-degrading strains with the *Candida juncea* Y6 combination on lignin degradation is somewhat different.

[0051] Comparative Example 2:

[0052] A mixture of Aspergillus terreus F10 and A. salivarius F12 strains with another biofilm-producing strain, Ligilactobacillus salivarius B7, in a 1:1:1 ratio was inoculated into an inorganic salt solid medium with straw powder as the sole carbon source and cultured at 30°C. After 20 days, the straw degradation rate was only 33.52%. These results indicate that the synergistic effect of Aspergillus terreus F10 and A. salivarius F12 strains combined with other biofilm-producing strains on lignocellulose degradation varies to some extent.

[0053] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A composite microbial system capable of efficiently degrading corn stalks, characterized in that, This includes Candida guilliermondii, Aspergillus terreus, and Coriolopsistrogii.

2. The composite microbial system capable of efficiently degrading corn stalks according to claim 1, characterized in that, According to the ratio of spore number to fungal number, the compound ratio of Candida guinea, Aspergillus terreus and Amanita burmannii is 1-2:1-2:1-2.

3. The composite microbial system capable of efficiently degrading corn stalks according to claim 1 or 2, characterized in that, The Candida jiyensis mentioned is Candida jiyensis ATCC6260.

4. The composite microbial system capable of efficiently degrading corn stalks according to claim 1 or 2, characterized in that, The Aspergillus terreus is Aspergillus terreus GIM 3.

547.

5. The composite microbial system capable of efficiently degrading corn stalks according to claim 1 or 2, characterized in that, The *Amanita muscaria* species is *Amanita muscaria* CGMCC No. 40363.

6. The composite microbial system capable of efficiently degrading corn stalks according to claim 1 or 2, characterized in that, The number of viable bacteria in the compound bacterial strain is not less than 1×10⁻⁶. 9 CFU.

7. The application of the composite microbial system according to any one of claims 1 to 6 in the degradation of corn stalks.

8. The application according to claim 7, characterized in that, The application involves spraying the compound microbial system onto corn stalks.

9. The application according to claim 8, characterized in that, The application involves culturing Aspergillus terreus and F. stearothermiae until the spore count is ≥1×10⁻⁶. 9 A spore suspension of 1 spore / mL was used to culture Candida guinea to the logarithmic growth phase. Then, Candida guinea, Aspergillus terreus, and Aster tataricus were mixed in a certain proportion to prepare a compound bacterial solution. The compound bacterial solution was then sprayed onto straw at a concentration of 5% to 15% and allowed to stand for incubation.

10. The application according to claim 9, characterized in that, Static culture involves incubating at 20–35°C for 10–30 days.

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