Hypha three-dimensional carbon net composite carbon sequestration fungicide as well as preparation method and application thereof

Abstract

The invention discloses a hypha three-dimensional carbon net composite carbon sequestration fungicide and a preparation method and application thereof. The preparation method comprises the following steps: firstly, placing aspergillus niger hyphae in a carbonization furnace for carbonization to obtain a hypha three-dimensional carbon net; and then putting the mycelium three-dimensional carbon net into a bacillus megatherium liquid, oscillating at constant temperature, filtering, and drying to obtain the mycelium three-dimensional carbon net composite carbon sequestration fungicide. When the compound is applied to improvement of soil fertility, the soil fertility can be comprehensively improved through multiple ways of regulating and controlling a soil carbon library, optimizing an acid-base environment, improving a physical structure, improving nutrient reserves and the like, and a basis is provided for application of the compound as a novel soil conditioner to agricultural production and promotion of soil health and sustainable agricultural development.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, and more specifically to a three-dimensional carbon network composite carbon-fixing agent of mycelium, its preparation method and application. Background Technology

[0002] Biochar is a carbon-rich material produced by the pyrolysis of biomass under anaerobic or micro-anaerobic conditions. It possesses core characteristics such as high stable carbon content, slow decomposition rate, and porous structure, which can promote the polymerization of organic compounds. Therefore, it has become an excellent soil conditioner with both carbon sequestration and soil physical structure improvement value. Due to its alkaline properties, large specific surface area, well-developed pore structure, and strong adsorption capacity, biochar has wide applications in soil improvement. Studies have confirmed that it can significantly increase soil porosity, reduce soil bulk density, alleviate soil acidification, and enhance soil water retention capacity. Simultaneously, by improving nutrient retention capacity and driving nutrient cycling, it provides support for environmental remediation and sustainable agricultural practices.

[0003] Soil microorganisms are the core driving force for maintaining soil fertility, playing an irreplaceable role in key biochemical processes such as nutrient transformation, element cycling, and energy flow, directly determining the stability of ecosystem functions. Biochar can optimize the microbial living environment through its own characteristics: its huge surface area and complex pore network can provide an ideal colonization microenvironment for microorganisms, protecting them from environmental stress and predation by natural enemies. Scanning electron microscopy imaging has confirmed that bacterial-mycorrhizal colonization clusters can form in the pores of biochar, effectively reducing interspecies competition among microorganisms. In addition, the stable carbon pool characteristics of biochar can support long-term carbon sequestration, and its easily decomposable carbon sources can directly stimulate microbial activity; at the same time, its surface oxygen-containing functional groups can fix heavy metals, reducing the toxicity of heavy metals to plants and the inhibition of microbial activity. However, the biochar preparation process may produce harmful compounds such as benzene, phenols, and polycyclic aromatic hydrocarbons. If the content exceeds the standard, it will significantly inhibit soil biological activity and reduce soil productivity. Therefore, screening suitable biochar precursors and application rates to achieve a balance between "enhancing soil fertility and reducing ecological risks" is a key research direction.

[0004] Fungal hyphae, as a novel biomass precursor, possess significant advantages: short growth cycle, abundant sources, and low cost. Furthermore, they can form a unique three-dimensional network structure. During pyrolysis, the resulting dehydration and water loss allow the carbonized products to form a more porous system, resulting in a higher specific surface area compared to traditional biomass-based biochar. Chemically, fungal hyphae contain anthraquinones, aromatic compounds, and sterols, among other compounds. These substances provide abundant functional groups that enhance the complexation between biochar and soil particles, further optimizing its soil amendment potential. Compared to traditional biochar, mycelium-based biochar also exhibits easier control and greater adaptability.

[0005] Currently, mycelium-based biochar has been extensively studied in the field of water pollutant adsorption, but research on the preparation of biochar using fungal mycelia as carbon precursors and its application in improving soil fertility has not yet been reported. Given the significant advantages of mycelium-based biochar in terms of structure, chemical properties, and cost, systematically exploring its potential and mechanisms for improving soil fertility is of great theoretical and practical significance for developing new and efficient soil conditioners and promoting sustainable agricultural development. Summary of the Invention

[0006] In view of this, the present invention provides a three-dimensional carbon network composite carbon-fixing agent of mycelium and its preparation method, providing a basis for its application as a novel soil conditioner in agricultural production and promoting soil health and sustainable agricultural development.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: First, this invention provides a method for preparing a three-dimensional carbon network composite carbon-fixing agent of mycelia, comprising the following steps: (1) Carbonize the mycelium of Aspergillus niger in a carbonization furnace to obtain a three-dimensional carbon network of mycelium, and grind it for later use; (2) The three-dimensional carbon network of mycelium prepared in step (1) is added to the Bacillus megaterium solution, shaken at a constant temperature for 12 hours, filtered and dried to obtain the three-dimensional carbon network composite carbon fixation agent of mycelium.

[0008] Preferably, the carbonization in step (1) uses programmed temperature increase, as follows: First, raise the furnace temperature to 200°C, then evacuate the carbonization furnace and purge it with nitrogen to eliminate oxygen; then raise the furnace temperature to 500°C and maintain it until there is no smoke, then stop heating and allow it to cool naturally to room temperature.

[0009] Preferably, the grinding process in step (1) is followed by sieving through a 2mm sieve.

[0010] Preferably, the OD in the Bacillus megaterium culture in step (2) 600 ≥1.0 (indicating that the bacterial activity and quantity meet the adsorption requirements), the ratio of the three-dimensional carbon network of hyphae to the bacterial solution is 0.02 g / mL.

[0011] Preferably, in step (2), the constant temperature oscillation temperature is 30°C and the oscillation frequency is 150 rpm. Continuous oscillation ensures that the biochar and bacterial solution are in full contact, so that Bacillus megaterium can achieve efficient adsorption with the help of the porous structure of biochar to form a stable carbon-bacterial composite system.

[0012] Preferably, vacuum freeze drying is used in step (2) to avoid high temperature damage to the activity of Bacillus and the porous structure of biochar.

[0013] The present invention also provides a three-dimensional carbon network composite carbon-fixing agent of mycelium prepared by the method described above.

[0014] This invention also provides a three-dimensional carbon network composite carbon-fixing agent of mycelium prepared by the method described above, or the application of the three-dimensional carbon network composite carbon-fixing agent of mycelium in soil fertility improvement.

[0015] Furthermore, the mycelial three-dimensional carbon network composite carbon-fixing agent was applied to the soil to be improved at a rate of 21.2 g / kg by soil weight.

[0016] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a three-dimensional carbon network composite carbon-fixing agent of mycelium, its preparation method and application, which has the following beneficial effects: The technical solution of this invention uses fungal mycelial biochar as a carrier and loads a biological agent as the final product. When applied to soil, it can significantly improve soil fertility, increase soil organic carbon content, regulate soil pH, reduce soil bulk density, and increase total nitrogen and phosphorus reserves. Furthermore, the biochar, as a slow-release nutrient reservoir, can further support microbial growth and enhance community diversity, indirectly promoting soil nutrient cycling. In summary, the fungal mycelial-based biochar prepared by the technical solution of this invention can comprehensively improve soil fertility through multiple pathways, including regulating the soil carbon pool, optimizing the pH environment, improving physical structure, and increasing nutrient reserves. This provides a basis for its application as a novel soil conditioner in agricultural production, promoting soil health and sustainable agricultural development. Attached Figure Description

[0017] 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 Scanning electron microscope image of the three-dimensional carbon mesh composite bacterial agent prepared in Example 1; Figure 2 The effects of different amounts of biochar on pH (a), soil organic carbon (b), total soil nitrogen (c), total phosphorus (d), available nitrogen (e), available phosphorus (f), and soil bulk density (g); Figure 3 The overall growth of potted scallions (a) and the growth of a single plant (b). Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 Preparation of a three-dimensional carbon network composite carbon-fixing inoculant based on mycelium: The culture and pretreatment procedures for Aspergillus niger are as follows: The strain is inoculated into an Erlenmeyer flask containing 100 mL of potato-glucose-liquid (PDL) medium, wherein the PDL medium formulation contains 200 g / L of PDL. -1 Potato extract, 20g L -1 D-glucose; then the flasks were incubated at 28°C for 7 days. After the incubation period, the mycelium in the system was separated using a sieve; to remove dust and water-soluble contaminants from the surface of the mycelium, it was washed sequentially with ultrapure water and 10wt.% hydrochloric acid solution; finally, the washed mycelium was freeze-dried under vacuum and stored under suitable conditions for later use.

[0021] Preparation of three-dimensional network structured biochar: Three-dimensional network structure biochar (hereinafter referred to as "prepared biochar") was prepared by freeze-dried Aspergillus niger mycelium in a tubular carbonization furnace. The temperature of the carbonization furnace was controlled by a step heating method. First, the furnace temperature was raised to 200℃. Then, the furnace was evacuated and filled with nitrogen three times to eliminate oxygen. Then, the furnace temperature was raised to 500℃ and held at 500℃ for 1 hour until smokeless state was reached. Then, the heating program was terminated and the furnace was allowed to cool naturally. The prepared biochar was then ground and sieved through a 2mm sieve. The three-dimensional network structure biochar composite microbial agent uses biochar prepared from Aspergillus niger as a carrier and Bacillus megaterium as a functional strain. The specific preparation process is as follows: First, a quantitative amount of Bacillus megaterium is inoculated into 100 mL of LB liquid medium. The inoculated medium is then placed in a 30℃ constant temperature shaker and cultured at a shaking rate of 150 rpm for 12 hours. During this period, the absorbance (OD) of the bacterial solution is monitored using a UV spectrophotometer. 600 ), awaiting OD 600After reaching a concentration of ≥1.0, 2g of three-dimensional network biochar prepared from Aspergillus niger was added to the bacterial culture. The culture was then maintained at 30℃ and 150rpm for 12 hours with continuous shaking to ensure full contact between the biochar and the bacterial culture. This allowed Bacillus megaterium to achieve efficient adsorption through the porous structure of the biochar, forming a stable biochar-bacterial composite system. After the composite culture was completed, a sieve filtration system was used to separate the three-dimensional network biochar that had adsorbed Bacillus megaterium. Finally, the composite bacterial agent was dried in a vacuum freeze dryer. Once completely dry, it was transferred to a sterile sealed container and stored at 4℃ in a dry, light-protected environment to ensure the long-term stability and functional activity of the composite bacterial agent.

[0022] Experimental Example Material: Test Soil: The test soil was collected from the experimental field of Jilin Agricultural University, located in the semi-humid region of Northeast China (43°48′43.57′′N, 125°23′38.50′′E). This region has a temperate semi-humid climate with an average annual temperature of 4.6℃ and an average annual precipitation of 600-700 mm. According to the Chinese soil classification system, the soil is classified as black soil under the suborder of semi-humid temperate semi-leached soils, equivalent to black soil in the USDA soil classification. In September 2024, soil samples were randomly collected from the 0-20 cm soil layer and combined into composite samples. After sampling, visible organic residues were manually removed. The moist soil from the field was naturally air-dried and then sieved through a 2 mm sieve for subsequent pot experiments. The basic soil properties are as follows: SOC content 17.36 g kg. -1 TN content: 1.81 g / kg -1 AN content: 132.21 mg / kg -1 AP content: 0.45 mg / kg -1 The pH value is 6.30.

[0023] Commercial biochar: purchased from Hubei Jinri Ecological Energy Co., Ltd.; Bacillus megaterium is commercially available.

[0024] First, the basic characteristics of the three-dimensional network structure biochar prepared in Example 1 and commercial biochar were compared, and the results are shown in Table 1: Table 1 Basic Properties of Biochar

[0025] Experimental Design: The pot experiment was conducted in a greenhouse. The tested scallion seeds were Qingcongwang No. 8, produced by Shanghai Huihe Seed Industry Co., Ltd. Soil samples were divided into three treatments: CK treatment (no biochar added), A1 treatment (21.2g of commercial biochar per kilogram of soil), and B1 treatment (21.2g of the mycelial three-dimensional carbon network composite carbon-fixing inoculant prepared in Example 1 per kilogram of soil). Each treatment was repeated three times. Each sample was mixed with the corresponding group's soil sample according to the weight ratio and then added to the planting bags used in the pot experiment.

[0026] Select scallion seeds of similar size and saturation from the seed collection. Disinfect the seeds with a 0.5% sodium hypochlorite solution for 20 minutes. After rinsing the seeds with distilled water, place them in petri dishes and soak them in distilled water for 24 hours in a 30℃ artificial climate incubator. Select seeds with uniform white sprouting, and select 30 seeds per group, evenly placing them in petri dishes lined with absorbent cotton. Add 2 mL of distilled water to each group and place them in a 25℃ artificial climate incubator. Replace the absorbent cotton and add 2 mL of distilled water every 48 hours. The artificial climate incubator has a 12-hour light / dark cycle, a relative humidity of 70%, and a light intensity of 3000 lx.

[0027] Five healthy scallion seedlings of similar growth were transplanted per bag, ensuring even distribution within the bags. The seedlings were allowed to recover for 3 days. Equal amounts of distilled water were added to treatment groups A1 and B1, as well as the control group, every 7 days. The pot experiment lasted 60 days, maintaining a soil moisture content of 60%. Soil samples were collected after the experiment. After air-drying, the soil samples were passed through a 2 mm sieve for soil property analysis.

[0028] Soil analysis: Soil organic matter (SOC) concentration was determined using the potassium dichromate oxidation-external heating method; total nitrogen (TN) was determined using the Kjeldahl distillation method; total phosphorus (TP) was measured by fusion with NaOH followed by molybdenum-antimony colorimetric method; available nitrogen (AN) was determined by alkaline diffusion method; available phosphorus (AP) was extracted with NaHCO3 followed by molybdenum-antimony colorimetric method; soil pH was determined using a pH meter in a 1:2.5 (w / v) soil / water suspension; and soil bulk density (BD) was determined using the cut-ring method.

[0029] Statistical analysis: All data were preprocessed using Microsoft Excel 2010 and analyzed using IBM SPSS Statistics 25 (IBM Corporation, Armonk, NY, USA). Two-way ANOVA was used to analyze significant differences in carbon content under different biochar addition rates. The Student-Newman-Keuls (SNK) test was used to assess the significance of differences between treatments at a significance level of [missing value].P <0.05, different letters represent significant differences.

[0030] The results are as follows: 1) Scanning electron microscopy test results of mycelial three-dimensional carbon network composite carbon fixation agent The microstructure of the three-dimensional carbon network composite carbon-fixing agent prepared in the examples was observed using scanning electron microscopy (SEM) to verify the composite effect of Bacillus megaterium on biochar. The results are as follows: Figure 1 As shown. From Figure 1 It can be observed that the biochar prepared from Aspergillus niger exhibits a loose, porous three-dimensional network structure with numerous irregular pores and grooves on the surface. The pore size is uniformly distributed, and this porous structure provides ample physical sites for microbial colonization, making it an ideal microbial carrier. When combined with Bacillus megaterium, a large number of short rod-shaped microbial individuals are clearly observed adhering to the pores and surface areas of the biochar. Their morphological characteristics (approximately 0.5–1.0 μm wide and 1.5–2.0 μm long) highly match the typical morphology of Bacillus megaterium. Some of these short rod-shaped microorganisms not only adhere tightly to the outer surface of the biochar but also embed themselves within the pores. Furthermore, a slight mucus-like substance is visible around the microorganisms, further enhancing the binding stability between the microorganisms and the biochar carrier.

[0031] Furthermore, high-magnification SEM images showed that the attached bacterial cells were morphologically intact and structurally clear, without obvious shriveling or cracking, and no large-scale aggregation of free bacterial cells was observed. This indicates that *Bacillus megaterium* was not simply suspended in the system, but rather stably colonized within the porous structure of biochar through physical adsorption and biological processes. In summary, SEM microscopic morphology analysis directly confirms that *Bacillus megaterium* has been successfully incorporated into the surface and pores of the biochar carrier, and the microstructure of the three-dimensional carbon network composite bacterial agent has been successfully constructed.

[0032] 2) The effect of biochar addition on soil carbon content Figure 2 The results showed that, compared with the blank control (CK), the addition of commercial biochar (A1) and the mycelial three-dimensional carbon network composite carbon-fixing agent prepared in Example 1 (B1) both significantly increased the soil organic carbon content, and the improvement effect of treatment B1 was significantly better than that of treatment A1. This is because biochar has natural anti-degradation properties, which can produce a "negative initiation effect" on the decomposition of native soil organic carbon. At the same time, it can protect the carbon in soil aggregates through physical action, and the biochar prepared in this experiment showed better efficiency in the above processes.

[0033] Regarding soil pH regulation, both treatments A1 and B1 significantly increased soil pH, with treatment B1 showing the most pronounced effect. Numerous studies have shown that biochar is generally alkaline and has a particularly significant effect on increasing the pH of acidic soils, a finding consistent with this experiment. The increase in soil pH mainly stems from two factors: firstly, the ash and carbonates formed during biochar pyrolysis play a role; secondly, the COO2 contained in biochar, derived from the original biomass, contributes to the pH increase. - O - Functional groups and cationic components such as metal oxides can effectively neutralize hydrogen ions in the soil. At the same time, the release of alkaline cations can make it easier for exchangeable Al³⁺ and H⁺ on the soil surface to detach from negatively charged soil particles, further promoting an increase in pH. B1 performs better under the synergy of these effects.

[0034] Regarding soil nitrogen and phosphorus nutrients (total nitrogen, total phosphorus, available nitrogen, and available phosphorus), compared with the control (CK), the B1 treatment significantly increased the content of these nutrients; while the nutrient content in the A1 treatment was higher than that in the CK, it was significantly lower than that in the B1 treatment. The increase in soil nitrogen and phosphorus content in this study may be driven by multiple factors: firstly, biochar (especially B1) can directly release nutrients; secondly, its porous surface enhances the adsorption and retention capacity of nutrients; and thirdly, the application of biochar increases phosphatase activity, promoting the mineralization of organic phosphorus. Previous studies have confirmed that adding biochar to soil can increase the content of soil organic carbon and the levels of nitrogen forms such as ammonium and nitrates. This indicates that biochar can act as a "slow-release nutrient reservoir," providing support for microbial growth and enhancing microbial community diversity, with B1 playing a more significant role in nutrient supply and regulation.

[0035] Furthermore, compared to the control (CK), both treatments A1 and B1 significantly reduced soil bulk density, with B1 showing a superior reduction effect. Numerous studies have shown that biochar can reduce soil bulk density, which is closely related to its ability to enhance soil porosity and nutrient retention. These structural changes provide physical barriers, making it easier for soil microorganisms to access nutrients and form symbiotic relationships within the biochar pores, thereby optimizing the microbial microenvironment. B1, however, exhibits a stronger effect in improving soil physical structure.

[0036] 3) Determination of agronomic indicators and phenotypic observation of potted scallions After a 60-day potted scallion experiment, the plant height, fresh weight, stem diameter, and root length of each group of potted scallions were systematically measured, and phenotypic observations were conducted simultaneously. At the end of the experiment, the most balanced and representative whole-pot sample from each group was photographed. Figure 3 a) Showing the overall growth of potted scallions; at the same time, 3 typical scallion plants were selected from each group for photography ( Figure 3(b, showing the growth of individual plants). Image analysis results show that the growth of scallions in treatment groups A1 and B1 is significantly better than that in the control group (CK group). Among them, the scallions in treatment group B1 have the most vigorous growth, which directly reflects the promoting effect of compound microbial agent on scallion growth, and the effect of treatment B1 is particularly outstanding.

[0037] 4) To further clarify the effect of compound microbial agents on the growth of scallions, the plant height, fresh weight, stem diameter and root length of each group of scallions were quantitatively measured, and the results are shown in Table 2.

[0038] Table 2 Agronomic Indicators of Potted Scallions Plant height (cm) Fresh weight (g) Stem diameter (cm) Root length (cm) CK 41.9±0.14 7.9±0.10 0.44±0.01 2.11±0.15 A1 61.2±0.25 18.1±0.22 0.77±0.03 4.82±0.17 B1 65.7±0.19 18.3±0.28 0.82±0.02 3.92±0.11 The data in Table 2 shows that: Plant height: The plant heights of the A1 and B1 treatment groups (61.2±0.25cm and 65.7±0.19cm, respectively) were significantly higher than those of the CK group (41.9±0.14cm), and the plant height of the B1 treatment group was significantly better than that of the A1 treatment group.

[0039] Fresh weight: The fresh weights of the A1 and B1 treatment groups (18.1±0.22g and 18.3±0.28g, respectively) were significantly higher than those of the CK group (7.9±0.10g), while the fresh weight of the B1 treatment group was slightly higher than that of the A1 treatment group.

[0040] Stem diameter: The stem diameters of the A1 and B1 treatment groups (0.77±0.03cm and 0.82±0.02cm, respectively) were significantly better than those of the CK group (0.44±0.01cm). Among them, the stem diameter of the B1 treatment group was the most outstanding, followed by the A1 treatment group.

[0041] Root length: The root lengths of the A1 and B1 treatment groups (4.82±0.17cm and 3.92±0.11cm, respectively) were significantly longer than those of the CK group (2.11±0.15cm).

[0042] The embodiments and experimental examples in this specification are described in a progressive manner, with each experimental example highlighting the differences between the embodiments and comparative examples. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a three-dimensional carbon network composite carbon-fixing inoculant, characterized in that, Includes the following steps: (1) Carbonize the mycelium of Aspergillus niger in a carbonization furnace to obtain a three-dimensional carbon network of mycelium, and grind it for later use; (2) The three-dimensional carbon network of mycelium prepared in step (1) is added to the Bacillus megaterium solution, shaken at a constant temperature for 12 hours, filtered and dried to obtain the three-dimensional carbon network composite carbon fixation agent of mycelium.

2. The method for preparing a three-dimensional carbon network composite carbon-fixing agent of mycelium according to claim 1, characterized in that, In step (1), carbonization is performed using a programmed temperature increase, as detailed below: First, raise the furnace temperature to 200℃, evacuate the carbonization furnace and then purge it with nitrogen; then raise the furnace temperature to 500℃ and maintain it until there is no smoke, then stop heating and let it cool naturally to room temperature.

3. The method for preparing a three-dimensional carbon network composite carbon-fixing agent of mycelium according to claim 1, characterized in that, After grinding in step (1), the material is passed through a 2mm sieve.

4. The method for preparing a three-dimensional carbon network composite carbon-fixing agent of mycelium according to claim 1, characterized in that, Step (2) OD in Bacillus megaterium culture 600 ≥1.0, the ratio of mycelial three-dimensional carbon network to bacterial solution is 0.02 g / mL.

5. The method for preparing a three-dimensional carbon network composite carbon-fixing agent of mycelium according to claim 1, characterized in that, In step (2), the constant temperature oscillation temperature is 30℃ and the oscillation frequency is 150 rpm.

6. The method for preparing a three-dimensional carbon network composite carbon-fixing agent of mycelium according to claim 1, characterized in that, In step (2), vacuum freeze drying is used for drying.

7. A three-dimensional carbon network composite carbon-fixing agent of mycelium prepared by the method according to any one of claims 1-6.

8. The application of a mycelial three-dimensional carbon network composite carbon-fixing agent prepared by the method of any one of claims 1-6 or the mycelial three-dimensional carbon network composite carbon-fixing agent of claim 7 in improving soil fertility.

9. The application according to claim 8, characterized in that, The mycelial three-dimensional carbon network composite carbon-fixing agent was applied to the soil to be improved at a rate of 21.2 g / kg by soil weight.

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