A fungal inoculant and its use in algal mat

Abstract

The application discloses a kind of fungal inoculant and its application in algae crust, belong to the field of microorganism.The technical problem solved by the application is how to construct algae crust.The application of fungal inoculant disclosed in the application in algae crust comprises the following steps: after dark endophytic fungi and microalgae are inoculated to the surface layer of soil to be repaired, culture is carried out, the construction of algae crust is realized;the dark endophytic fungi are the strain with the preservation number CGMCC No.17463 in China General Microbiological Culture Collection Center;the microalgae are composed of Microcoleus sp., Scenedesmus sp.and Chlorella sp.The application successfully realizes the rapid formation of algae crust, improves soil carbon accumulation, solves the key technical problems such as long crust period and poor adaptability, and is beneficial to the purpose of ecological restoration of degraded soil in arid and semiarid mining areas.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a fungal agent and its application in algal crusts. Background Technology

[0002] Arid and semi-arid regions are characterized by fragile ecosystems, sparse vegetation, scarce rainfall, and intense evaporation, making their soil structure highly susceptible to disturbance and damage. Particularly in areas with concentrated coal mining, surface subsidence, wind erosion, and water erosion are exacerbated, leading to soil degradation, nutrient loss, and increased exposed surface areas, posing a serious threat to the stability of regional ecosystems. Therefore, exploring bioremediation technologies capable of rapidly restoring surface ecological functions under extreme conditions has become an important research direction in the field of ecological environment.

[0003] Biological soil crusts (BSCs) are microbial-mineral complexes formed by microorganisms such as cyanobacteria, green algae, fungi, lichens, and mosses, along with their secreted extracellular polymeric substances (EPS). They are an important component of surface ecosystems in arid and semi-arid regions. Numerous studies have shown that biosoil crusts, as pioneer communities for ecological restoration, play a crucial role in the stabilization and ecological reconstruction of damaged surface systems. Algal crusts, representing an early stage of biosoil crust development, are typically formed by filamentous cyanobacteria and green algae. Their extracellular polymeric substances exhibit significant binding and consolidation effects, enabling rapid cementation and structural stabilization of surface particles. Algal crusts play a foundational role in the formation and succession of crust communities, serving as the initial type in the biosoil crust succession process and having a decisive impact on the subsequent formation of bryophyte crusts and the succession of higher plants.

[0004] However, the formation cycle of natural algal crusts is relatively long and significantly affected by environmental stresses. Under extreme conditions such as high temperature and low moisture, algal growth is limited and EPS secretion decreases, resulting in a loose crust structure and poor stability, which restricts its application in engineered ecological restoration. Therefore, in recent years, the academic community has begun to explore the artificial construction of biological soil crusts to accelerate the surface restoration process. Among these, the "artificial algal crust" technology, which uses algae as the core building block, is considered a promising new approach to ecological restoration. Through artificial cultivation and environmental control, algal attachment, EPS enrichment, and particle consolidation can be achieved in a short period.

[0005] Therefore, developing a rapid construction method that can form a stable algal crust structure in a short period of time can not only provide new technical means for ecological restoration in arid areas and coal mining subsidence areas, but also has important scientific significance and practical value for understanding the microbial-driven process of surface structure reconstruction and carbon cycle. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to construct algal crusts.

[0007] To solve the above-mentioned technical problems, the present invention first provides a method for constructing algal crust using fungal inoculants, the method comprising: inoculating the surface layer of the soil to be repaired with fungal inoculants and then culturing it to achieve the construction of algal crust;

[0008] The fungal agent is composed of dark-colored endophytic fungi and microalgae;

[0009] The dark-colored endophytic fungus is a strain with accession number CGMCC No. 17463 from the China General Microbiological Culture Collection Center.

[0010] The microalgae are composed of Microcoleus sp.、 Scenedesmus sp. and Chlorella sp. composition.

[0011] Preferably, in the fungal inoculant, the ratio of the dark-colored endophytic fungus to the microalgae is 2.6 × 10⁻⁶. 6 CFU: 103.74 μg, or 3.9 × 10 6 CFU: 103.74 μg, or 5.2 × 10⁻⁶ μg 6 CFU: 103.74 μg or 6.5 × 10⁻⁶ 6 CFU: 103.74 μg. The microalgae are... Microcoleus sp.、 Scenedesmus sp. and Chlorella The total chlorophyll a of sp.

[0012] In one embodiment of the present invention, the ratio of the dark-colored endophytic fungus to the microalgae in the fungal agent is 2.6 × 10⁻⁶. 6 CFU: 103.74μg.

[0013] In one embodiment of the present invention, the ratio of the dark-colored endophytic fungus to the microalgae in the fungal agent is 3.9 × 10⁻⁶. 6 CFU: 103.74μg.

[0014] In one embodiment of the present invention, the ratio of the dark-colored endophytic fungus to the microalgae in the fungal agent is 5.2 × 10⁻⁶. 6 CFU: 103.74μg.

[0015] In one embodiment of the present invention, the ratio of the dark-colored endophytic fungus to the microalgae in the fungal agent is 6.5 × 10⁻⁶. 6 CFU: 103.74μg.

[0016] Preferably, in the microalgae Microcoleus sp.、 Scenedesmus sp. and Chlorella The ratio of sp. satisfies the requirement that the mass content of chlorophyll a is 1:1:1.

[0017] Preferably, the inoculation amount of the fungal agent on the surface layer of the soil to be remediated is: (2.6-6.5) × 10⁻⁶ dark-colored endophytic fungi. 6 CFU / 103.74 cm 2 The microalgae 1 μg·cm -2 Furthermore, the inoculation amount of the fungal agent on the surface layer of the soil to be remediated is: 3.9 × 10⁻⁶ of the dark-colored endophytic fungi. 6 CFU / 103.74 cm 2 The microalgae 1 μg·cm -2 Among them, the microalgae are... Microcoleus sp.、 Scenedesmus sp. and Chlorella The total chlorophyll a of sp.

[0018] Preferably, the dark-colored endophytic fungi in the fungal agent can be inoculated to a depth of 1 cm below the surface of the soil to be repaired, and the microalgae are inoculated on the surface of the soil to be repaired.

[0019] Preferably, the culture is carried out at 10-30 °C. More preferably, the culture is carried out at 25 °C.

[0020] Preferably, the light intensity for cultivation is 150–250 μmol·m⁻¹. -2 ·s -1 The air humidity was 60%, and the light-dark cycle was 14 h / 10 h. Furthermore, the light intensity for cultivation was 200 μmol·m⁻¹. -2 ·s -1 .

[0021] Preferably, the culture time is greater than or equal to 20 days. More specifically, the culture time is 28 days.

[0022] Preferably, the soil moisture content can be controlled during the cultivation process. Further, during the cultivation process, the soil moisture content is greater than or equal to 50%. Further, during the cultivation process, the soil moisture content is 65%.

[0023] The application of the method of constructing algal crusts using fungal agents to improve soil organic carbon accumulation is also within the scope of protection of this invention.

[0024] The method of constructing algal crusts using fungal agents described herein, in the rapid consolidation and / or ecological restoration of degraded soil (such as degraded land in arid mining areas), is also within the scope of protection of this invention.

[0025] The method of this invention can successfully construct algal crusts, effectively overcoming the problems of long formation cycle and poor adaptability of traditional natural algal crusts, and significantly improving the crust formation rate and ecological restoration efficiency. Specifically, when the inoculum amount of dark endophytic fungi is 3 mL (the ratio of dark endophytic fungi to algal solution is 3.9 × 10⁻⁶), the algal crust formation rate is significantly improved. 6 The optimal growth status of the algal crust was achieved when the concentration of CFU (103.74 μg) was reached: the algal crust coverage exceeded 84%, which was about 41% higher than the uninoculated control; the chlorophyll a content reached 10.2 μg·mg. -1 The soil organic carbon content increased by approximately 82%. This indicates that appropriate inoculation with dark-colored endophytic fungi (DSE) significantly promotes algal growth, enhances photosynthesis and extracellular polymer secretion, and promotes soil particle bonding and carbon fixation. Furthermore, compared to traditional algal crust construction methods without fungal inoculation, this invention shortens the algal crust formation cycle and increases soil organic carbon content to 11.7 g·kg⁻¹. - ¹, representing a 31% improvement over the control. This invention, through combined inoculation and optimized inoculation ratio, achieves rapid formation of algal crusts, increases soil carbon accumulation, and solves key technical problems such as long crust formation cycles and poor adaptability, thus contributing to the ecological restoration of degraded soils in arid and semi-arid mining areas. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 for Microcoleus sp.、 Scenedesmus sp. and Chlorella The form of sp. A, B, and C are respectively: Microcoleus sp.、 Scenedesmus sp. and Chlorella The sp. bar value is 50 μm.

[0028] Figure 2 The effects of different ratios of dark-colored endophytic fungi on the morphology of artificial algal crusts.

[0029] Figure 3This invention relates to the effect of different ratios of dark-colored endophytic fungi on the coverage of artificial algal crusts. Data with the same letter showed no significant difference (p > 0.05), while data with different letters showed significant differences (p < 0.05).

[0030] Figure 4 The effect of different ratios of dark-colored endophytic fungi on the chlorophyll a content of artificial algal crust over time.

[0031] Figure 5 The effect of different ratios of dark-colored endophytic fungi on soil organic carbon content over time.

[0032] Figure 6 Correlation analysis of algal crust coverage and soil organic carbon content. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0035] In the quantitative experiments in the following examples, at least three replicate experiments were set up. The experimental results are expressed as mean ± standard deviation, and the significance of the data was analyzed by one-way ANOVA test.

[0036] In the following examples, algal crust culture was carried out in an artificial climate chamber (RXZ intelligent artificial climate chamber).

[0037] Example 1: Rapid Construction of Artificial Algal Crust in Coal Mining Subsidence Areas

[0038] 1. Material Description

[0039] The algal crusts were collected by the inventor in the field in an area that is located in the transition zone from the Loess Plateau gully region to the Mu Us Desert.

[0040] The tested strain was a dark endophytic fungus (DSE), specifically Alternaria alternata with accession number CGMCC No. 17463 from the China General Microbiological Culture Collection Center. Alternariasp.)001, hereinafter referred to as the dark endophytic fungus CGMCC No.17463. The dark endophytic fungus CGMCC No.17463 is *Alternaria* (sp.) from Chinese Patent 201910505635.X (Authorization Announcement No. CN 110205248 B). Alternaria sp .)001.

[0041] 2. Design of cultivation scheme for algal crust

[0042] (1) Collection and preparation of algal strains

[0043] Using a small shovel, the algal crust along with a 1 cm thick layer of soil was scooped up and placed in a disposable petri dish. The dish was then transported back indoors to air dry naturally. Visible plant debris, soil clods, pebbles, etc., were manually removed. The original algal crust layer was then crushed for 40 seconds using a plant shredder and passed through a 0.25 mm soil sieve.

[0044] (2) Large-scale culture of algal strains and determination of biomass

[0045] Weigh 10 g of sieved soil and transfer it to 150 mL of BG-11 liquid culture medium. Shake at room temperature (25℃) and 160 r / min for 8 hours to allow the BG-11 culture medium to fully extract microalgae from the soil. Filter the filtrate using rapid filter paper. The obtained crust-forming algae species are... Microcoleus sp.、 Scenedesmus sp. and Chlorella sp., such as Figure 1 As shown.

[0046] Each obtained algal strain was placed in an Erlenmeyer flask containing BG-11 liquid medium and placed in an artificial climate chamber at 25°C and 150 μmol·m⁻²·m⁻²·℃. -2 ·s -1 After culturing until a distinct green color appears in the flask, it is transferred to a photobioreactor and cultured at 25°C and 200 μmol·m⁻². -2 ·s -1 The culture was then transferred to an incubator for further cultivation and expansion. The resulting mixed algal solution containing three algal species had a chlorophyll a content of 1.85 μg / mL. Microcoleus sp.、 Scenedesmus sp. and Chlorella The ratio of sp. satisfies the requirement that the mass content of chlorophyll a is 1:1:1.

[0047] (3) Algal crust culture

[0048] Plastic containers (13.3 cm long, 7.8 cm wide, and 4.8 cm high) were used as culture vessels. Each container contained 300 g of sand as the culture medium (this substrate can be any soil suitable for algal crust growth). A dark-colored endophytic fungal inoculant was mixed with the soil at a certain ratio and inoculated to a depth of 1 cm below the soil substrate. Then, an algal solution with a total chlorophyll a content of 1 μg per square centimeter was used as the inoculation standard. After inoculation, the mixture was placed in an incubator for cultivation. The relative humidity in the artificial climate chamber was set at 60%, the photoperiod was 14 h light / 10 h dark, the temperature was 25 ℃, and the soil moisture content was 18.76% (65% of the saturated moisture content of the culture medium).

[0049] The preparation method of the dark endophytic fungal inoculum is as follows: The dark endophytic fungus strain CGMCC No.17463 was inoculated into MMN liquid medium and cultured with shaking at 28 ℃ and 170 r / min for 15 days to obtain the dark endophytic fungal inoculum. The viable cell content of this inoculum was 1.3 × 10⁻⁶. 6 CFU / mL.

[0050] (4) Experimental design

[0051] Using an algal solution containing 1 μg chlorophyll a per square centimeter as the inoculation standard, the mixed algal solution was inoculated. The dark endophytic fungal agent was mixed with the soil at a certain ratio, and each treatment was replicated three times, as shown in Table 1.

[0052] Table 1 Experimental Design Table

[0053]

[0054] In treatment A, the ratio of inoculated dark-colored endophytic fungi to algal solution was 1.3 × 10⁻⁶. 6 CFU: 103.74 μg;

[0055] In treatment B, the ratio of inoculated dark-colored endophytic fungi to algal solution was 2.6 × 10⁻⁶. 6 CFU: 103.74 μg;

[0056] In treatment C, the ratio of inoculated dark-colored endophytic fungi to algal solution was 3.9 × 10⁻⁶. 6 CFU: 103.74 μg;

[0057] In treatment D, the ratio of inoculated dark-colored endophytic fungi to algal solution was 5.2 × 10⁻⁶. 6 CFU: 103.74 μg;

[0058] In treatment E, the ratio of inoculated dark-colored endophytic fungi to algal solution was 6.5 × 10⁻⁶. 6 CFU: 103.74 μg;

[0059] In treatment F, the ratio of inoculated dark-colored endophytic fungi to algal solution was 7.8 × 10⁻⁶. 6 CFU: 103.74 μg;

[0060] The inoculum volume of the algae solution was measured in units of chlorophyll a.

[0061] 3. Preparation of algal crusts

[0062] After inoculating the algal solution and bacterial agent according to step 2 (4), the mixture was kept at a temperature of 25 ℃ and a light intensity of 200 μmol·m⁻¹. -2 ·s -1 Under conditions of 60% air humidity and a light / dark cycle of 14 h / 10 h, the substrate was continuously cultured for 28 days, with water sprayed every 7 days to maintain the substrate moisture content at approximately 65% ​​of the saturated moisture content.

[0063] (1) Determination of algal crust coverage

[0064] The morphological changes of each treatment after 28 days of culture are as follows: Figure 2 As shown, different ratios of dark-colored endophytic fungi significantly affect the formation of algal crusts, with obvious differences in algal density, color intensity, and surface uniformity. With increasing inoculum size of dark-colored endophytic fungi, the algal crust coverage first increases and then decreases.

[0065] Statistical results of algal crust coverage are as follows: Figure 3 As shown in the figure, after 28 days of culture, the highest coverage was achieved with an inoculum size of 3 mL (treatment C), with an average value of 84%; followed by treatment B with an inoculum size of 2 mL. Coverage decreased after the inoculum size exceeded 4 mL, indicating that excessive fungal inoculum size may inhibit microalgal growth or disrupt the stability of the crust structure.

[0066] (2) Determination of chlorophyll a content in algal crust

[0067] Algal crust samples were collected at different cultivation times, and the chlorophyll a content under different ratios of dark-colored endophytic fungi was analyzed over time. The results are as follows: Figure 4 As shown in the figure. The results indicated that the chlorophyll a content gradually increased with the extension of culture time, reaching a peak under treatment C. After 28 days of culture, the chlorophyll a contents of treatments CK, A, B, C, D, E, and F were 5.6±1.0, 8.2±1.1, 9.2±1.6, 10.2±1.0, 8.7±1.4, 7.3±1.2, and 6.6±1.1 μg·mg, respectively. -1The chlorophyll a contents of treatments A, B, C, D, E, and F were 1.46, 1.64, 1.83, 1.56, 1.30, and 1.18 times that of treatment CK, respectively. Significance analysis showed that the chlorophyll a content of treatment C was significantly higher than that of treatments CK and F (P < 0.05), indicating that appropriate inoculation with dark-colored endophytic fungi can significantly promote photosynthesis and biomass accumulation in algae.

[0068] Comprehensive comparison shows that an inoculum volume of 3 mL results in the most vigorous algal crust growth, with high coverage, deep color, and uniform algal distribution, representing the optimal conditions for rapid formation of artificial algal crusts. Therefore, considering both the cost of fungal inoculants and the algal growth effect, an inoculum volume of 3 mL for dark-colored endophytic fungi is the recommended application condition for promoting rapid formation of artificial algal crusts.

[0069] (3) The effect of different algal crust coverage on soil organic carbon accumulation

[0070] The organic carbon content of the soil layer corresponding to each treatment was collected at different cultivation times to analyze the relationship between algal crust coverage and soil organic carbon accumulation.

[0071] The results showed that different ratios of dark endophytic fungi (DSE) inoculum significantly affected soil organic carbon content, such as... Figure 5 As shown in the figure, the organic carbon content in all treatment groups showed a continuous upward trend with the extension of culture time, but the increase varied significantly among different inoculum sizes. At 7 days, the differences between groups were small, indicating that the effect of DSE on organic carbon accumulation was not significant in the short term. At 28 days, the organic carbon content in all inoculum groups except group A was significantly higher than that at 7, 14, and 21 days (p < 0.05), indicating that the growth of algal crusts promoted organic matter transformation and carbon fixation. By day 28, the 3 mL inoculum group (group C) reached the highest organic carbon content (11.7%). The organic carbon contents of groups CK, A, B, D, E, and F were 8.9±0.2, 9.4±1.0, 10.4±0.5, 9.9±0.6, 9.6±0.8, and 9.4±0.2 g / kg, respectively. Group C had a significantly higher organic carbon content than the other treatment groups (p<0.05), being 1.31, 1.24, 1.12, 1.18, 1.21, and 1.24 times higher than groups CK, A, B, D, E, and F, respectively, showing the best improvement effect. Groups A, B, D, E, and F had organic carbon contents 1.06, 1.17, 1.11, 1.08, and 1.06 times higher than group CK, respectively. Lower inoculum amounts (groups A and B) promoted organic carbon accumulation, but the effect was relatively limited; while excessively high inoculum amounts (group DF) resulted in slightly lower organic carbon contents than group C, possibly related to mycelial competition and rapid consumption of substrate resources.

[0072] The correlation between algal crust cover and soil organic carbon content after 28 days of cultivation was analyzed. Figure 6It can be seen that there is a significant positive correlation between soil organic carbon content and algal crust coverage. As algal crust grows and coverage increases, soil organic carbon content rises significantly, indicating that the formation and expansion of algal crust can effectively promote the accumulation of organic matter.

[0073] Therefore, the artificial algal crust formed by the synergistic effect of fungi and microalgae can not only significantly increase the surface crust coverage in a short period of time, but also promote surface carbon accumulation and ecological function restoration. The optimal inoculum size of dark-colored endophytic fungi is 3 mL, which can obtain an artificial algal crust that combines rapid crust formation and high carbon fixation capacity.

[0074] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for constructing algal crusts using fungal agents, characterized in that: The method includes: inoculating the surface layer of the soil to be repaired with a fungal agent and then culturing it to achieve the construction of algal crust; The fungal agent is composed of dark-colored endophytic fungi and microalgae; The dark-colored endophytic fungus is a strain with accession number CGMCC No. 17463 from the China General Microbiological Culture Collection Center. The microalgae are composed of Microcoleus sp.、 Scenedesmus sp. and Chlorella sp. composition.

2. The method according to claim 1, characterized in that: In the fungal inoculant, the ratio of the dark-colored endophytic fungus to the microalgae is 2.6 × 10⁻⁶. 6 CFU: 103.74 μg, or 3.9 × 10⁻⁶ 6 CFU: 103.74 μg, or 5.2 × 10⁻⁶ μg 6 CFU: 103.74 μg or 6.5 × 10 6 CFU: 103.74 μg, wherein the microalgae are calculated as chlorophyll a.

3. The method according to claim 2, characterized in that: The microalgae Microcoleus sp.、 Scenedesmus sp. and Chlorella The ratio of sp. satisfies the requirement that the mass content of chlorophyll a is 1:1:

1.

4. The method according to claim 1 or 3, characterized in that: The inoculation amount of the fungal agent on the surface layer of the soil to be remediated is: (2.6-6.5) × 10⁻⁶ dark-colored endophytic fungi. 6 CFU / 103.74 cm 2 The microalgae 1 μg·cm -2 The microalgae are calculated as chlorophyll a.

5. The method according to claim 1 or 3, characterized in that: The culture was carried out at 10-30 °C.

6. The method according to claim 1 or 3, characterized in that: The light intensity for cultivation was 150–250 μmol·m⁻¹. -2 ·s -1 The air humidity is 60%, and the light-dark cycle is 14 h / 10 h.

7. The method according to claim 1 or 3, characterized in that: The culture time is greater than or equal to 20 days.

8. The method according to claim 1 or 3, characterized in that: The culture period is 28 days.

9. The application of the method according to any one of claims 1-7 in increasing soil organic carbon accumulation, or in the consolidation and / or ecological restoration of degraded soils.

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