Trichoderma harzianum strain HMS1 and its application in degradation of low-rank lignite

Abstract

The application discloses a strain of Trichoderma harzianum HMS1 and application of the strain in degradation of low-grade lignite, and belongs to the technical field of microorganisms. The strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 42173. The strain has heat resistance, the optimal growth temperature is 35-38 DEG C, and the highest growth temperature is 44 DEG C, so that the strain is suitable for high-temperature environment or reactor operation, and cooling energy consumption is significantly reduced. The strain can directly use untreated low-grade lignite as the only carbon source, and the final degradation rate reaches 49+ / -0.5% through solid culture by means of a plate method for 21 days, the final degradation rate reaches 50.4+ / -0.8% through liquid culture by means of a shaking flask method for 7 days, and the final degradation rate reaches 51.3+ / -1.2% through liquid culture by means of a shaking flask method for 14 days. The strain overcomes the problem that the activity of the existing mesophilic strain is significantly reduced or even inactivated in a high-temperature environment (greater than 35 DEG C), and is particularly suitable for lignite biotransformation treatment and high-temperature composting in a hot and humid area (such as Yunnan, the eastern part of Inner Mongolia, etc. in China) where lignite resources are rich.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Trichoderma Ethiopianum HMS1 and its application in the degradation of low-grade lignite. Background Technology

[0002] my country possesses abundant lignite resources, with proven reserves exceeding 100 billion tons, accounting for approximately 13% of total coal reserves. These resources are primarily distributed in eastern Inner Mongolia and Yunnan. However, lignite is generally of low quality, characterized by high moisture content (25%-60%), high ash content (10%-30%), and low calorific value (typically <16.7 MJ / kg), classifying it as a typical low-rank coal. These characteristics pose significant challenges to its direct utilization: high transportation costs, low combustion efficiency, high risk of spontaneous combustion, and the alkali metals in the ash can easily trigger boiler slagging and corrosion. Therefore, developing technologies for effectively processing low-grade lignite is of great strategic importance.

[0003] Microbial degradation is one approach to treating low-grade lignite. Currently reported highly efficient lignite-degrading strains mainly originate from coal mining soils and decaying wood environments, including white-rot fungi (such as *Trametes versicolor* and *Protozoa*), some bacteria (such as *Bacillus*), and a few other fungi (such as *Aspergillus*, *Penicillium*, and *Trichoderma*). High temperatures can increase the reaction rate of microbial degradation; thermotolerant strains maintain enzyme activity under high-temperature conditions, significantly improving lignite degradation efficiency and shortening reaction time. However, most highly efficient lignite-degrading fungi are mesophilic, with their optimal growth and degradation temperatures typically between 25-30℃, even those of the *Trichoderma* genus (…). Trichoderma Extensively studied strains (such as Trichoderma reesei) T. reesei Trichoderma harzianum T. harzianum These mesophilic strains are also primarily adapted to this temperature range; their activity significantly decreases or even becomes inactive at high temperatures (>35°C). Furthermore, during periods of high ambient temperature (such as summer) or in regions with high ambient temperature (tropical / subtropical regions), the microbial degradation process itself generates heat, causing the reaction system to continuously heat up. Therefore, to maintain a specific operating temperature—the optimal temperature for the microorganisms—in the reactor, maintaining mesophilic conditions (25-30°C) requires additional cooling energy compared to maintaining high-temperature conditions (>35°C), which also increases operating costs. Therefore, thermophilic strains adapted to high temperatures have a greater advantage.

[0004] In terms of raw materials, lignite, especially low-grade lignite, has a complex structure, high degree of condensation, poor hydrophilicity, and contains inhibitory substances, resulting in low bioavailability. This leads most biodegradation research to heavily rely on chemical oxidation pretreatment steps to improve the biodegradability rate of lignite, such as using strong oxidants like nitric acid (HNO3), hydrogen peroxide (H2O2), and ozone (O3) to oxidize and degrade the macromolecular structure of coal. While this is currently the most effective method to improve biodegradation efficiency, it is also the most expensive, environmentally risky, and corrosive method. Chemical pretreatment (especially nitric acid and hydrogen peroxide) brings high chemical costs, equipment corrosion, safety hazards, and environmental problems such as nitrogen-containing / acid-containing wastewater and waste residue, severely restricting the economic viability and sustainability of the technology.

[0005] Therefore, developing microbial strains that can operate efficiently at high temperatures and directly degrade low-grade lignite without chemical pretreatment is a key problem that urgently needs to be solved in this field. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide an Ethiopian Trichoderma HMS1 strain and its application in the degradation of low-grade lignite.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of Trichoderma esculenta (Ethiopian Trichoderma). Trichoderma aethiopicum HMS1, the Ethiopian Trichoderma, is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 42173.

[0008] The optimal growth temperature for *Trichoderma esculenta* of this invention is 35-38°C, with a growth temperature range of 4-44°C. It cannot grow above 44°C, but will not die. After being cultured at 50°C for 3 days, it can continue to grow when placed within the optimal growth temperature range, thus belonging to the category of heat-resistant fungi. The optimal pH is 5.5-6. At 38°C, it forms green filamentous colonies on potato dextrose agar (PDA) plates in 2 days. On lignite-based media with only one carbon source, it forms green colonies in 3 days.

[0009] In a second aspect, the present invention provides a microbial agent containing the aforementioned Trichoderma Ethiopianum HMS1, wherein the microbial agent is a powder, fermentation broth, or suspension.

[0010] A third aspect of the invention provides the use of the aforementioned Trichoderma Ethiopianum HMS1 or its culture medium or an inoculum containing it in the preparation of high-temperature degraded lignite products.

[0011] In a fourth aspect, the present invention provides a product for the high-temperature degradation of lignite, comprising the aforementioned Trichoderma Ethiopianum HMS1 or its culture medium or an inoculum containing it.

[0012] A fifth aspect of the invention provides a method for high-temperature degradation of lignite, comprising culturing the aforementioned *Trichoderma Ethiopianense* HMS1 in a culture medium containing lignite.

[0013] Furthermore, the cultivation temperature is 30-40℃.

[0014] Furthermore, the culture medium containing lignite is a lignite-only carbon source culture medium, and its composition is as follows: 15-25 parts lignite powder, 0.5-5 parts CaSO4, 0.5-5 parts NH4Cl, 0.5-5 parts KH2PO4, 10-20 parts agar powder, and 900-1100 parts distilled water.

[0015] Furthermore, the preparation method of the culture medium with lignite as the sole carbon source is as follows: Sterilize components A and B separately at 121℃ for 10-20 min, and after cooling to 50-60℃, mix the two components evenly to obtain lignite-only carbon source culture medium. Component A: Dissolve 20 parts of lignite powder in 200 parts of distilled water; Component B: Dissolve 1 part of CaSO4, 1 part of NH4Cl, 1 part of KH2PO4, and 15 parts of agar powder in 800 parts of distilled water.

[0016] Furthermore, the culture medium containing lignite is a PDA or PDB culture medium.

[0017] Under plate culture (solid) conditions, with lignite as the sole carbon source, the degradation rate reached 49±0.1% after 21 days. Under shake flask culture (liquid) conditions, with lignite as the sole carbon source, the degradation rate reached 50.4±0.8% after 7 days and 51.3±1.2% after 14 days.

[0018] Furthermore, the culture is an aerobic culture.

[0019] The beneficial effects of this invention are: 1. This invention successfully isolated and obtained a new strain of Trichoderma Ethiopia ( Trichoderma aethiopicum The strain HMS1 (CGMCC No. 42173) exhibits thermophilic characteristics: its optimal growth temperature is 35-38℃, with a maximum of 44℃. Growth ceases at 50℃, but the strain does not die and can continue to grow within its target temperature range. It is suitable for high-temperature environments or reactor operation, significantly reducing cooling energy consumption. It is particularly suitable for lignite bioconversion in regions rich in lignite resources and with hot climates (such as Yunnan and eastern Inner Mongolia in my country), and can be used in high-temperature biological treatment processes such as composting.

[0020] 2. The strain of this invention does not require any chemical or physical pretreatment. It directly uses low-grade lignite as the sole carbon source. The final degradation rate reaches 49±0.5% after 21 days of solid-state culture using the plate method, 50.4±0.8% after 7 days of liquid-state culture using the shake flask method, and 51.3±1.2% after 14 days.

[0021] 3. When the strain of the present invention is used in production, the process is simplified and environmentally friendly, eliminating expensive, dangerous and polluting chemical pretreatment steps (such as nitric acid, hydrogen peroxide and hydrogen hydration), simplifying the process flow, significantly reducing the costs of raw materials, equipment, safety protection and waste treatment, and improving the economy and sustainability of the technology. Attached Figure Description

[0022] Figure 1 Here is a morphological photograph of strain HMS1 on a PDA plate; Figure 2 A photograph of the morphology of strain HMS1 on a plate of lignite-only carbon source medium. Figure 3 A phylogenetic tree constructed based on ITS sequences; Figure 4 This is a growth curve of strain HMS1 on PDA medium; Figure 5 The graph shows the degradation rate of lignite by strain HMS1 in plate culture. Figure 6 This is a graph showing the degradation rate of lignite by strain HMS1 in shake flask culture.

[0023] Figure 7 The absorbance of lignite degradation solution after shake-flask culture of strain HMS1 was measured. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0025] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0026] The experimental materials used in the embodiments of this invention, unless otherwise specified, are all conventional experimental materials in the art and can be purchased through commercial channels. The PDA culture medium used in this invention is prepared according to the Chinese Pharmacopoeia. The only difference between PDB and PDA is that PDB does not contain agar. The lignite used in this invention is lignite mined from Xinjiang lignite mines in my country. It has been physically crushed and dried, and the ash content was measured using the method of "GB / T212-2008 Industrial Analysis Methods for Coal". The ash content of the lignite powder is 27.6±0.1%. According to the national standard "GB / T 15224.1-2018 Coal Quality Classification Part 1: Ash Content", the coal is classified as medium-ash coal, belonging to low-grade coal with low calorific value.

[0027] Example 1: Isolation and Identification of Strains HMS1 1. Sample collection: The sample was collected from the dust near our humic acid production workshop.

[0028] 2. Separation and purification Serial dilution method: Take 1g of sample and serially dilute with sterile water to 10⁻⁶. -4 To determine the concentration, obtain a diluted solution. Take 100 μL of the diluted solution and spread it onto a plate containing only lignite as the carbon source.

[0029] The formulation for a culture medium using lignite as the sole carbon source is as follows: Component A: Dissolve 20g of lignite powder in 200mL of distilled water; Component B: Dissolve 1g of CaSO4, 1g of NH4Cl, 1g of KH2PO4, and 15g of agar powder in 800mL of distilled water.

[0030] Components A and B were sterilized separately in an autoclave at 121°C for 15 minutes. After cooling to approximately 55°C, the two components were mixed thoroughly, poured into petri dishes, and the resulting culture medium plates containing lignite as the sole carbon source were obtained.

[0031] The coated plates were placed in an incubator at 35°C. Colonies growing on the lignite-only carbon source medium were observed daily. The bacteria were isolated and purified using repeated streak plating to obtain a pure strain, designated HMS1.

[0032] 3. Morphological observation: The purified HMS1 strain was inoculated into the center of a PDA plate and incubated at 35-38°C. After 2 days, typical green filamentous colonies formed (e.g., ...). Figure 1 Preliminary identification indicates it is Trichoderma.

[0033] HMS1 was inoculated onto a plate of lignite-based carbon source medium and incubated at 35-38℃ for 3 days, after which green colonies formed (e.g., Figure 2 This confirms that it can grow using lignite as the sole carbon source.

[0034] 4. Molecular biological identification: Plates containing HMS1 cells grown to the logarithmic growth phase were sent to Riboxin Biotechnology Co., Ltd. for sequencing. The ITS rDNA region was amplified using readily available universal fungal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The obtained ITS sequences were BLAST-aligned in the NCBI database, showing similarity to *Trichoderma esculenta* (Ethioplasma esculenta). Trichoderma aethiopicum The similarity reached 99.83%. A phylogenetic tree was constructed using this sequence (e.g.,...). Figure 3 Further confirmation that it is *Trichoderma esculenta* (Ethiopian *Trichoderma*) Trichoderma aethiopicum The sequence has been submitted to NCBI GenBank, accession number PV997898.

[0035] 5. Strain Preservation: The identified strain *Trichoderma Ethiopianum* HMS1 was sent to the China General Microbiological Culture Collection Center (CGMCC) for preservation. Classification and Nomenclature: *Trichoderma Ethiopianum* Trichoderma aethiopicum Deposit date: August 19, 2025; Deposit number: CGMCC No. 42173; Deposit location: China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0036] Example 2: Determination of growth characteristics of strain HMS1 1. Raw material preparation: The culture medium used in this example is potato dextrose agar (PDA).

[0037] 2. Determination method (colony diameter method): HMS1 spores were selected and a spore suspension was prepared (concentration of 10). 9 (spores / mL). Take 5 μL of spore suspension and spot it onto the center of a PDA plate (6 cm in diameter). Set up 3 parallel experiments.

[0038] 3. Thermophilic characteristics of strain HMS1 Plates inoculated with strain HMS1 were placed in incubators at 4℃, 20℃, 25℃, 28-45℃ (temperature intervals of 1℃), 46℃, and 50℃ for incubation. The growth status was reflected by measuring the colony diameter.

[0039] Experimental results show that the strain has a growth temperature range of 4-44℃. This strain has thermophilic characteristics: the optimal growth temperature is 35-38℃, the maximum is 44℃, growth stops at 50℃ but it will not die, and it can continue to grow when it is moved into the growth temperature range.

[0040] 4. Determination of growth characteristics of strain HMS1 Incubate the plates in an incubator at 35-38℃ (optimal temperature range). Measure the colony diameter (hyphae length) every 12 hours until the mycelium completely covers the plate. Plot a growth curve based on the colony diameter at different time points; the results are shown below. Figure 4 .

[0041] Figure 4 The results showed that strain HMS1 grew rapidly on PDA.

[0042] Example 3: Determination of lignite degradation performance of strain HMS1 1. Raw material preparation Culture medium base: Potato glucose agar (PDA) and potato glucose broth (PDB).

[0043] Lignite pretreatment: The lignite powder used has not undergone any chemical (nitric acid, hydrogen peroxide, etc.) or physical pretreatment.

[0044] 2. Plate culture method (solid): Add 1g of sterile lignite powder and 1mL of HMS1 spore suspension (10) to each petri dish. 9 The samples were divided into four groups according to different incubation temperatures (samples / mL) and 4 mL of PDA medium, with three replicates in each group. The samples were incubated at 25, 30, 38, and 44°C, and samples were taken on days 0, 4, 6, 10, 12, 14, 17, and 21.

[0045] Residue Measurement: The culture mixture was washed with distilled water and transferred to a mortar, where it was thoroughly ground to release the undegraded lignite powder encapsulated by the mycelium. The residue was collected by filtering with filter paper, and the mycelium was carefully removed from the residue. The residue was dried to constant weight and weighed.

[0046] The formula for calculating the degradation rate is as follows: Degradation rate (%) = [(Initial lignite powder weight - Residue weight) / Initial lignite powder weight] × 100% The results of cultivation at 38℃ are shown below. Figure 5 The degradation rate gradually increased from 0 to 6 days, and the growth rate slowed down and stabilized after 7 days. The final degradation rate reached 49±0.5% after 21 days.

[0047] Compared to strain HMS1 cultured at 38℃, the final degradation rates after 21 days were 36±0.2%, 41±0.4%, and 42±0.3% at 25℃, 30℃, and 44℃, respectively.

[0048] 3. Shake flask culture method (liquid): Add 100 mL of sterile PDB medium, 2 g of sterile lignite powder, and 0.5 g of polyvinyl alcohol (PVA, used to disperse mycelia, reduce mycelial balling rate, and improve lignite degradation effect) to a 250 mL Erlenmeyer flask. Inoculate with 1 mL of HMS1 spore suspension (10 9 The samples were cultured in triplicate (samples / mL) at 25℃, 30℃, 38℃, and 44℃ in shakers at 160 rpm. Samples were taken on days 0, 1, 2, 3, 4, 5, 7, 10, and 14.

[0049] The methods for measuring residue and calculating degradation rate are the same as the corresponding steps in step 2 of this embodiment, the plate culture method (solid).

[0050] The results of cultivation at 38℃ are shown below. Figure 6 The degradation rate increases rapidly from 0 to 4 days (rapid degradation period), from 0% to 47.0±0.7%; it enters a stable period from 4 to 14 days, with the degradation rate fluctuating slightly before stabilizing. The final degradation rate reaches 50.4±0.8% after 7 days and 51.3±1.2% after 14 days.

[0051] Compared to strain HMS1 cultured at 38℃, the final degradation rates after 14 days were 37±0.5%, 43±0.8%, and 44±0.6% at 25℃, 30℃, and 44℃, respectively.

[0052] By comparing the degradation efficiency of the two methods mentioned above in this embodiment, the degradation rate of shake flask culture (liquid) is significantly higher than that of plate culture (solid). This indicates that the liquid shake flask environment is more conducive to the contact between mycelia and lignite particles, material exchange and oxygen transfer, which ultimately makes it more conducive for the HMS1 strain to quickly exert its lignite degradation ability.

[0053] 4. Absorbance test of degradation solution The HMS1 lignite degradation solution, cultured at 38℃ for 14 days using the shake-flask method, was allowed to stand. The supernatant was then diluted with pure water at a ratio of 1 ml: 5 ml, and the absorbance curve was measured using a UV spectrophotometer. The results are shown below. Figure 7 .

[0054] Figure 7The results showed that the degradation solution had an absorption peak at 230 nm, indicating the presence of conjugated enones or conjugated dienes in the sample, which is highly consistent with the chemical composition of lignite. The absorbance curve showed a slow decrease in the long-wavelength direction (>300 nm), continuing into the near-infrared region (1000 nm) with measurable absorption, indicating the presence of highly conjugated electronic systems or large aromatic ring structures in the sample, such as α,β-unsaturated carbonyl groups (e.g., carboxyl and ketone groups in humic acid) and extended aromatic rings. These molecules have large conjugated systems and small electronic transition gaps, resulting in a wide absorption spectrum extending from the ultraviolet region to the visible and even near-infrared regions. This curve clearly reflects the rich organic matter content of the lignite degradation solution and demonstrates the good degradation effect of HMS1 on lignite.

[0055] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A strain of Trichoderma esculenta (Ethiopian Trichoderma) Trichoderma aethiopicum HMS1, characterized in that, The *Trichoderma esculenta* described is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 42173.

2. A fungal agent containing *Trichoderma esculenta* HMS1 as described in claim 1, characterized in that, The microbial agent is a powder, fermentation broth, or suspension.

3. The application of the Ethiopian Trichoderma HMS1 or its culture medium or an inoculum containing it as described in claim 1 in the preparation of high-temperature degraded lignite products.

4. A product for high-temperature degradation of lignite, characterized in that, Includes the Ethiopian Trichoderma HMS1 as described in claim 1, or its culture medium or an inoculum containing it.

5. A method for high-temperature degradation of lignite, characterized in that, This includes culturing the Ethiopian Trichoderma HMS1 of claim 1 in a culture medium containing lignite.

6. The method for high-temperature degradation of lignite according to claim 5, characterized in that, The incubation temperature is 30-44℃.

7. The method for high-temperature degradation of lignite according to claim 5, characterized in that, The culture medium containing lignite is a lignite-only carbon source culture medium, and its composition is as follows: 15-25 parts lignite powder, 0.5-5 parts CaSO4, 0.5-5 parts NH4Cl, 0.5-5 parts KH2PO4, 10-20 parts agar powder, and 900-1100 parts distilled water.

8. The method for high-temperature degradation of lignite according to claim 7, characterized in that, The preparation method of the culture medium with lignite as the sole carbon source is as follows: Sterilize components A and B separately at 121℃ for 10-20 min, and after cooling to 50-60℃, mix the two components evenly to obtain lignite-only carbon source culture medium. Component A: Dissolve 20 parts of lignite powder in 200 parts of distilled water; Component B: Dissolve 1 part of CaSO4, 1 part of NH4Cl, 1 part of KH2PO4, and 15 parts of agar powder in 800 parts of distilled water.

9. The method for high-temperature degradation of lignite according to claim 5, characterized in that, The culture medium containing lignite is a PDA or PDB culture medium.

10. The method for high-temperature degradation of lignite according to claim 5, characterized in that, The culture described is an aerobic culture.

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