Application of graphene oxide in straw degradation by synergistic trichoderma

Abstract

The invention discloses an application of graphene oxide in degrading straws by synergistic trichoderma. The application comprises the following steps: crushing the straws, and mixing the crushed straws with soil; spraying the trichoderma suspension into the mixture of the straws and the soil; spraying the graphene oxide hydrosol into a mixture of trichoderma, straws and soil; the concentration of the trichoderma suspension is 0.5 * 10 < 5 > cfu / mL to 5 * 10 < 5 > cfu / mL; the concentration of the graphene oxide hydrosol is 5 mg / L-50 mg / L; the water content of the mixture of the trichoderma, the straw and the soil is 50-70 wt%; trichoderma is treated through graphene oxide hydrosol, graphene oxide can promote mycelial growth of the trichoderma, meanwhile, lignin peroxidase activity and cellulase activity of the trichoderma can be promoted, and the straw degradation efficiency is improved; the expressions in the aspects of straw phenotype, mass loss rate, mycelial growth, cellulase and ligninase activity in the straw degradation process are greatly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of ecological carbon sinks, and relates to the application of graphene oxide in enhancing the degradation of straw by Trichoderma Background Art

[0002] Trichoderma spp. is a kind of filamentous fungi widely distributed in soil and humus, belonging to the Deuteromycotina. Because of its rapid growth, strong metabolism and strong environmental adaptability, it is widely used in agricultural production and biotechnology fields. Trichoderma can secrete a variety of degrading enzymes (such as cellulase, xylanase and pectinase), and efficiently decompose plant residue components such as cellulose, hemicellulose and lignin, thereby accelerating the decomposition and transformation of organic matter. The organic acids and polysaccharide substances produced by the metabolism of Trichoderma contribute to the formation of soil aggregate structure, and improve soil aeration, water retention and fertility. In addition, Trichoderma is also an important biocontrol bacterium, which can inhibit soil-borne pathogens through a variety of mechanisms.

[0003] Direct straw returning to the field is a relatively economical way of resource utilization, but it still has many disadvantages, such as long degradation time of the returned straw and high incidence of crop diseases. Straw is rich in a large amount of nutrients, and lignin, as the natural physical barrier of straw, wraps and strengthens the structure of cellulose and hemicellulose. Degradation must occur prior to cellulose and hemicellulose, and the complexity of its structure has a great impact on the process of straw degradation and resource utilization. In order to promote the effective utilization of straw resources, scientists have improved the degradation rate of straw in the field by selecting and applying straw lignin-degrading strains. However, the degradation efficiency of relevant degrading bacteria in the field is affected by various environments. Especially in difficult terrain areas, the degradation rate of straw in the field is very slow. Therefore, it is particularly important to use new technologies to improve the degradation efficiency of degrading bacteria and shorten the decomposition cycle of straw after returning to the field during the process of straw returning to the field.

[0004] As a new two-dimensional nanomaterial that has received much attention, there have been many reports on the effects of graphene entering the soil environment on plant growth and development and the structure of microbial communities. However, there are few reports on the effects of graphene on the decomposition of litter and straw in the soil environment. How to accelerate straw decomposition, increase crop yields and control diseases has become an urgent problem to be solved. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for improving the efficiency of Trichoderma in degrading straw. By treating Trichoderma with graphene oxide hydrosol, the performance of straw phenotype, mass loss rate, hyphal growth, cellulase and ligninase activities in the process of straw degradation is greatly improved.

[0006] To achieve the above object, the following technical solutions are adopted:

[0007] The application of graphene oxide in enhancing the degradation of straw by Trichoderma comprises the following steps:

[0008] (1) Crush the straw and mix it with soil;

[0009] (2) spraying the Trichoderma suspension on the mixture of straw and soil;

[0010] (3) Spraying graphene oxide hydrosol into a mixture of Trichoderma, straw and soil.

[0011] According to the above scheme, the straw in step 1 is preferably corn straw.

[0012] According to the above scheme, the Trichoderma described in step 2 is from Shanxi Agricultural University, and the strain name is BJ9. After growing in PDA medium for 5 days, the mycelium is transferred to PDB medium and cultured overnight in a rotary shaker at 28°C and 200 rpm.

[0013] According to the above scheme, the concentration of the Trichoderma suspension in step 2 is 0.5×10 5 cfu / mL-5×10 5 cfu / mL.

[0014] According to the above scheme, the concentration of the graphene oxide aqueous sol in step 3 is 5 mg / L-50 mg / L.

[0015] According to the above scheme, the water content in the mixture of Trichoderma, straw and soil in step 3 is 50-70wt%.

[0016] According to the above scheme, the graphene oxide hydrosol in step 3 is sprayed multiple times within a 60-day period, with an interval of 5-10 days.

[0017] According to the above scheme, the optimal degradation temperature in step 3 is 20-25°C.

[0018] A method for improving the efficiency of Trichoderma in degrading straw comprises spraying graphene oxide hydrosol into a mixture of Trichoderma, straw and soil.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention found that graphene oxide can promote the mycelial growth of Trichoderma, and can also promote the lignin peroxidase activity and cellulase activity of Trichoderma, thereby increasing the degradation efficiency of straw.

[0021] The present invention improves the growth of Trichoderma and the ability to degrade corn stalks through graphene oxide, thereby broadening the application scope of graphene oxide.

[0022] The technical solution provided by the present invention can help accelerate the decomposition of corn straw in large fields, improve agricultural productivity, and provide a practical technical solution for turning straw into fertilizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Surface macroscopic morphology changes of corn straw before and after degradation.

[0024] Figure 2 : Scanning electron micrographs of corn straw before and after degradation.

[0025] Figure 3 : Statistical graph of mass loss rate of corn straw before and after degradation.

[0026] Figure 4 Graphene oxide promotes the growth of Trichoderma mycelium. (A is a photograph of the mycelial growth phenotype after graphene treatment; B is a statistical graph of the mycelial growth curve after graphene treatment).

[0027] Figure 5 Graphene oxide promotes the activity of Trichoderma cellulase and lignin peroxidase. (A is a statistical chart showing the effect of graphene on the activity of Trichoderma cellulase; B is a statistical chart showing the effect of graphene on the activity of Trichoderma lignin peroxidase). DETAILED DESCRIPTION

[0028] The following examples further illustrate the technical solutions of the present invention but are not intended to limit the scope of protection of the present invention.

[0029] A specific embodiment provides a graphene oxide hydrosol, which is prepared by the following method:

[0030] Flexible graphite paper and platinum foil (flexible graphite paper, specifications, Sigma brand, Cas: 7440-44-0, size 1EA, platinum foil, specifications Aladdin brand, Cas: 7440-06-4, size 625mm) were used as the cathode and anode electrodes, respectively. The initial voltage was set to 1.60V. The intercalation reaction was carried out in 98% sulfuric acid solution at room temperature for 40 minutes. The current gradually decreased as the conductivity of graphite deteriorated. During this process, the color of the flexible graphite paper changed from silver-gray to blue. Since the blue intermediate product was light and soft and easy to break, the blue intermediate product was fixed with platinum foil as the cathode electrode for oxidation exfoliation. The anode electrode was still platinum foil. The mass concentration of the electrolyte sulfuric acid solution was controlled to 50wt% and the voltage was 2.5V to obtain the product graphene oxide. Subsequently, after suction filtration, repeated rinsing with distilled water and freeze-drying at -80℃ for 12 hours, graphene oxide powder was obtained. The graphene oxide powder was then dispersed in sterile water to obtain a graphene oxide hydrosol with a concentration of 5mg / L.

[0031] The specific implementation provides a Trichoderma strain sourced from Shanxi Agricultural University, named BJ9. After growing on PDA medium for 5 days, the mycelium was transferred to PDB medium and cultured overnight in a rotary shaker at 28°C and 200 rpm for standby.

[0032] Example 1

[0033] In this example, the decomposition bag method was used to explore the effect of graphene oxide on the decomposition of corn straw.

[0034] Accurately weigh 10 g of corn straw and put it into a nylon mesh bag with a specification of 15 cm × 10 cm and a pore size of 0.15 mm. Take the 0 - 30 cm surface soil (from the Carbon Materials Institute of Shanxi Datong University) back to the laboratory, remove roots, stones and other sundries, sieve and mix well, add tap water to make the soil water content reach 50 - 60% of the field water holding capacity (the field water holding capacity of the soil was measured in advance), and cultivate the soil in the laboratory at room temperature for 7 days to keep the soil uniform. The experimental plastic culture pot has a diameter of 12.5 cm and a height of 11.5 cm. After filling the soil or the mixture of soil and earthworm manure (5:1) into the culture pot, the decomposition bag containing the litter is obliquely inserted and buried in the soil to ensure uniform contact between the decomposition bag and the soil.

[0035] After burial, each culture pot was evenly sprayed with 30 mL of Trichoderma suspension (1×105 cfu / mL) using a sprayer (a blank control group was set), and then evenly sprayed with 30 mL of the above-mentioned graphene oxide hydrogel with a concentration of 5 mg / L or tap water using a sprayer, and placed at room temperature (20 - 25°C) for decomposition culture. Spraying was carried out every 10 days to keep the soil humidity basically unchanged, and the continuous culture was terminated after 60 days.

[0036] As Figure 1 shown, at 0 day of treatment, compared with the control, the corn straw treated with Trichoderma and the co - treatment of graphene and Trichoderma basically showed yellow, and the straw had obvious fiber structures. At 60 days of treatment, the control corn straw began to rot. Compared with the control, the degree of rotting of the corn straw treated with Trichoderma was more obvious. When graphene was added, the degree of rotting of the corn straw was the strongest, and the surface was the darkest black - brown.

[0037] Example 2

[0038] This example provides the scanning electron microscopy characterization diagrams of corn straw before and after degradation.

[0039] The corn straw tissues before and after degradation in Example 1 were cut into cross-sections with a thickness of 1 mm using a slicing machine, and then fixed with 2.5% glutaraldehyde. After washing, gradient dehydration was carried out using ethanol aqueous solutions with concentration gradients of 30%, 50%, 70%, 90% and 100%. Then, the natural drying method was adopted. After sputtering with gold, the surface morphology of the corn straw was observed using a scanning electron microscope (SEM, TESCAN MAIA 3LMH). As Figure 2 shown, when the treatment was 0 days, the surface of the corn straw was smooth, regular, flat and dense, and the structure was intact (A-C). When the treatment was 60 days, the flat structure of the corn straw in the control group was disrupted, the structure became loose, and the fiber bundles were slightly damaged, but the structure of the fiber bundle arrangement was still clearly visible ( Figure 2 D), the Trichoderma-treated ( Figure 2 E) and the Trichoderma and graphene co-treated ( Figure 2 F) corn straw surfaces were relatively rough, the internal structure of the fiber bundles was damaged, especially the fiber bundles in the Trichoderma and graphene co-treatment were severely damaged, and the arrangement of the fiber bundles became disordered ( Figure 2 F). It shows that the addition of graphene oxide and Trichoderma damaged the structure of corn straw the fastest.

[0040] Example 3

[0041] This example provides a method for detecting the mass loss rate of corn straw before and after degradation.

[0042] Repeat Example 1, with graphene oxide hydrogel concentrations of 5 mg / L, 25 mg / L and 50 mg / L respectively. Then, the corn straws treated differently at 0 days, 30 days and 60 days were collected, and their masses were weighed using a balance. And two-way repeated measures analysis of variance was used for data statistics, and a line graph was drawn. ** represents P<0.01, with significant differences. The results are as Figure 3 shown, compared with the control, the Trichoderma treatment significantly promoted the mass loss rate of corn straw. When graphene was added, graphene further promoted the mass loss rate of corn straw in a concentration-dependent manner, indicating that graphene accelerated the efficiency of Trichoderma in degrading corn straw.

[0043] Example 4

[0044] This example provides the results of the mycelial growth after treating Trichoderma with graphene.

[0045] Take 37 g of potato dextrose agar medium (AOBOX 02-023) PDA and dissolve it in 1 L of distilled water. After heating and boiling until dissolved, autoclave at 121 °C for 15 minutes and set aside. Then, in a laminar flow hood, add graphene oxide hydrogel that has been autoclaved at 121 °C for 15 minutes to the medium so that the final concentrations are 5 mg / L, 25 mg / L, and 50 mg / L respectively. The control is the PDA medium without added graphene. Then pour the medium into petri dishes to solidify. Inoculate Trichoderma mycelial blocks into the media with different treatments, and then place them in an inverted position in a biochemical incubator at 28 °C for 3 days. Use a digital vernier caliper to measure the colony diameter and analyze the results. The data was analyzed for significance using the one-tailed T-test method. Graphs were plotted using the GraphPad Prism 9.5 graphing software. *** represents P < 0.001; **** represents P < 0.0001. The results are as Figure 4 shown. Compared with the control, after treating Trichoderma with 5 mg / L, 25 mg / L, and 50 mg / L of graphene, the colony growth of Trichoderma was significantly promoted.

[0046] Example 5

[0047] This example provides the results of the effect of graphene on the cellulase activity and lignin peroxidase activity of Trichoderma.

[0048] In a laminar flow hood, take Trichoderma mycelia that have grown for 3 - 4 days and place them in potato dextrose liquid medium. Culture them in a shaker at 28 °C and 200 rpm for 16 hours. Then break the culture solution with an ultrasonic cell disruptor (Newch Bio SCIENTZ-IID) for 30 minutes, filter it through a 200-mesh nylon net, and collect the enzyme solution and store it on ice. Then use a cellulase activity detection kit (Solarbio BC2545) and a lignin peroxidase activity detection kit (Solarbio BC1610) to detect the cellulase activity and lignin peroxidase activity of the enzyme solution secreted by Trichoderma. The specific operation steps refer to the instructions of the above kits. Data analysis was performed using SPSS V.26. Significance analysis was performed using one-way ANOVA test. Graphs were plotted using the GraphPad Prism 9.5 graphing software. Different letters represent significant differences. As Figure 5 shown, compared with the control, graphene oxide significantly promoted the cellulase activity ( Figure 5 A) and lignin peroxidase activity ( Figure 5 B) of Trichoderma in a concentration-dependent manner. It indicates that graphene accelerates the efficiency of Trichoderma in decomposing corn straw and improves its degradation performance by promoting the cellulase activity and lignin peroxidase activity of Trichoderma.

[0049] The embodiments described above are only some of the embodiments of the present invention, not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art through related deductions and substitutions under the condition of the concept of the present invention without creative efforts fall within the scope of protection of the present invention.

Claims

1. Application of graphene oxide in enhancing the degradation of straw by Trichoderma, characterized in that The following steps are involved: (1) Crush the straw and mix it with soil; (2) spraying the Trichoderma suspension on the mixture of straw and soil; (3) Spraying graphene oxide hydrosol into a mixture of Trichoderma, straw and soil.

2. The application of graphene oxide in enhancing the degradation of straw by Trichoderma viride according to claim 1, wherein In step 1, the straw is preferably corn straw.

3. The application of graphene oxide in enhancing the degradation of straw by Trichoderma viride as claimed in claim 1, wherein The Trichoderma described in step 2 is from Shanxi Agricultural University, and the strain name is BJ9. After growing in PDA medium for 5 days, the mycelium is transferred to PDB medium and cultured overnight in a rotary shaker at 28°C and 200rpm.

4. The application of graphene oxide in enhancing the degradation of straw by Trichoderma viride as claimed in claim 1, wherein The concentration of the Trichoderma suspension described in Step 2 is 0.5×10 5 cfu / mL - 5×10 5 cfu / mL.

5. The application of graphene oxide according to claim 1 in enhancing the degradation of straw by Trichoderma, characterized in that The concentration of the graphene oxide aqueous sol in step 3 is 5 mg / L-50 mg / L.

6. The application of graphene oxide as claimed in claim 1 in enhancing the degradation of straw by Trichoderma, characterized in that The water content of the mixture of Trichoderma, straw and soil in step 3 is 50-70wt%.

7. The application of graphene oxide as claimed in claim 1 in enhancing the degradation of straw by Trichoderma, characterized in that The graphene oxide hydrosol in step 3 is sprayed multiple times within a 60-day period, with an interval of 5-10 days.

8. The application of graphene oxide as claimed in claim 1 in enhancing the degradation of straw by Trichoderma, characterized in that The degradation temperature in step 3 is 20-25°C.

9. A method for improving the efficiency of Trichoderma in degrading straw, characterized in that This includes crushing the straw and mixing it with the soil; Then, the Trichoderma suspension is sprayed into the mixture of straw and soil; finally, the graphene oxide hydrosol is sprayed into the mixture of Trichoderma, straw and soil.

10. The method for improving the efficiency of Trichoderma in degrading straw according to claim 9, characterized in that The concentration of the Trichoderma suspension is 0.5×10 5 cfu / mL - 5×10 5 cfu / mL; the concentration of the graphene oxide hydrogel is 5 mg / L - 50 mg / L; the water content in the mixture of Trichoderma, straw and soil is 50 - 70 wt%; the graphene oxide hydrogel is sprayed in multiple times within a 60-day cycle, and the interval is 5 - 10 days.

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