A halophilic and thermophilic myceliopthora strain and application thereof

Abstract

The present application relates to the field of microbial technology, and particularly relates to a salt-tolerant and heat-tolerant Myceliophthora strain and application thereof, the strain is Myceliophthora thermophila MT-ST / GP, which has been preserved in CGMCC on October 30, 2024, and the preservation number is CGMCC No.41587. The strain is outstanding in salt tolerance, and when NaCl≤10 g / L, sodium bicarbonate≤5 g / L or 30% NaCl-70% sodium bicarbonate mixed salt≤5 g / L, the average viable cell count is≥20.3, and the strain can grow stably. The strain can be used for straw field return in saline-alkali land, fermentation of high-salt agricultural waste and degradation of high-salt wastewater, and can also be used for efficient fermentation and degradation of wheat straw.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a halophilic and thermophilic strain of *Hydromycinus* and its applications. Background Technology

[0002] Myceliophthora thermophila, as a typical thermophilic filamentous fungus, has important application prospects in fields such as agricultural waste resource utilization (e.g., straw degradation and return to the field), industrial biomass conversion (e.g., cellulosic ethanol production), and environmental pollutant (e.g., lignin-based pollutants) bioremediation due to its wide suitable growth temperature range, strong extracellular secretion of cellulase, hemicellulase and other degrading enzyme systems, and high degradation efficiency of complex biomass substrates. However, in practical applications, many target processing scenarios suffer from significant salt stress problems: Saline-alkali land scenario: my country has a vast area of ​​saline-alkali land. The content of salts such as sodium chloride (NaCl) and sodium bicarbonate (baking soda) in this type of soil is high. Conventional thermophilic filamentous fungi strains are prone to problems such as decreased growth rate and reduced enzyme activity when the salt concentration exceeds 5g / L, making it difficult to meet the needs of in-situ degradation and return of straw to the field in saline-alkali land. High-salt agricultural waste scenario: Some agricultural wastes (such as straw produced in seawater irrigation areas and by-products of agricultural product pickling and processing) can have a salt content of 10-20g / L. Existing strains cannot grow and reproduce normally in such substrates, resulting in low waste degradation efficiency. Complex salt environment scenario: Salt stress in the natural environment is mostly a complex stress caused by the coexistence of multiple salts such as NaCl and baking soda. Existing studies have shown that conventional thermophilic filamentous fungi strains show a sharp drop in viable count in complex salt environments above 5 g / L, and cannot adapt to complex salt stress environments. Currently, research on thermophilic cytotoxic fungi mainly focuses on enzyme system optimization and high-temperature adaptability improvement. There is relatively little research on the screening, domestication, and application of halophilic characteristics. For example, Chinese patent CN202411479455.7 discloses a method and engineered strain for improving the cellulose degradation ability of thermophilic cytotoxic fungi, but it only improves the activity of cellulose-degrading enzymes through genetic engineering and does not involve halophilic modification, so it cannot be applied in high-salt environments; moreover, it only optimizes pure cellulose substrates and is not adapted to complex agricultural wastes such as wheat straw. Chinese patent CN113151098A discloses an alkali-resistant composite microbial pretreatment agent for wheat straw pulping and its application. However, it adopts a multi-strain composite system, which requires strict control of the strain ratio and is complicated in process. The strains do not have halophilic characteristics and cannot be used for saline-alkali land or high-salt waste treatment, thus limiting the application scenarios. Chinese patent CN113832042B discloses a strain of halophilic sacchariformis yeast and its application in the treatment of high-salt and high-COD wastewater. However, the strain is a yeast with an optimal temperature of <37℃ and no high-temperature adaptability. It also lacks cellulose degradation ability and cannot treat waste containing lignocellulose such as wheat straw and high-salt straw. The strain is significantly different in function from the strain in this application. Chinese patent CN103497942B discloses a fermentation culture scheme for a thermophilic pyridobacterium ACCC No.30572. The concentration of the salt component (ammonium sulfate) in the culture medium is limited to 1-10 g / L. This salt concentration range can only meet the basic growth requirements of the strain and does not take into account the design for tolerance to high salt environments.

[0003] It is evident that there are currently no publicly reported thermophilic *Dermocytotrichum* strains that can stably grow in a 10 g / L NaCl, 5 g / L sodium bicarbonate, or 5 g / L NaCl-sodium bicarbonate (30%:70%) complex salt environment and maintain a high viable count (average viable count ≥20) and degradation activity. Therefore, screening and obtaining a thermophilic *Dermocytotrichum* strain that is highly halophilic, adaptable to high-salt and complex-salt environments, and maintains excellent biomass degradation capabilities is of great significance for expanding the application scope of this type of strain in saline-alkali land improvement, high-salt agricultural waste treatment, and improving the efficiency of biological treatment in high-salt environments. It is also a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a halophilic and thermophilic strain of *Dermocytotrichum* and its application. This strain is highly halophilic, can adapt to high-salt and complex-salt environments, and maintains excellent biomass degradation capabilities.

[0005] The objective of this invention is achieved through the following technical solution: This invention provides a strain of Myceliophthora thermophila MT-ST / GP, which was deposited on October 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC); address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 41587.

[0006] Furthermore, the thermophilic mitochondritis MT-ST / GP can grow stably in environments with NaCl concentration ≤1-20 g / L, sodium bicarbonate concentration ≤1-6 g / L, or sodium bicarbonate mixed salt concentration ≤1-6 g / L.

[0007] This invention also provides an application of Thermophilic MT-ST / GP in the efficient fermentation and degradation of wheat straw. When applying it, wheat straw is mixed with other raw materials in a certain proportion, and the cellulose and hemicellulose in the wheat straw are metabolized and degraded by the strain. After fermentation, the degradation rate of wheat straw is ≥60-70%.

[0008] Furthermore, the raw materials include wheat straw, buckwheat hulls, corn flour, soybean meal, potatoes, and brown sugar, with the weight ratio of each raw material being wheat straw: buckwheat hulls: corn flour: soybean meal: potatoes: brown sugar = 200-220: 280-320: 28-32: 28-32: 1400-1600: 23-27, and the overall carbon-nitrogen ratio of the raw materials is adjusted to 25-30: 1.

[0009] Furthermore, the wheat straw needs to be pre-treated before fermentation: the wheat straw is cut into pieces 4-11cm in length, the buckwheat hulls are sieved to a particle size of 1-3mm, and the potatoes are peeled and cut into 0.8-2.2cm pieces. 3 For small pieces, corn flour is ground to a fineness of 70-90 mesh, and soybean meal is ground to a particle size of 2-4 mm.

[0010] Furthermore, the fermentation process parameters are as follows: the initial moisture content of the material is controlled at 63-72%, sterilized at 115-121℃ and 0.09-0.11MPa for 25-35 minutes, and after cooling to 33-42℃, the logarithmic phase bacterial solution of the strain is inoculated, with an inoculation amount of 1-3% of the total weight of the material.

[0011] This invention also provides an application of *Thermophilic mitochondritis MT-ST / GP* in the biological treatment of high-salt environments, including saline-alkali land, high-salt agricultural waste, or high-salt wastewater, in which the strain can maintain degradation activity during the treatment process.

[0012] Furthermore, the application includes mixing straw crushed to 1.5-5.5cm with the thermophilic mycorrhizal MT-ST / GP at a mass ratio of 9-11:1, spreading it on the surface layer of saline-alkali land at a depth of 4-11cm, using 1400-1600kg of straw and 140-160kg of mycorrhizal agent per hectare, maintaining a soil moisture content of 18-27%, and cultivating for 28-32 days.

[0013] Furthermore, the application includes mixing high-salt straw with high-salt wastewater with a COD value of 7000-9000 mg / L at a solid-liquid ratio of 1:4-6, adding 0.4-0.6% glucose by mass, adjusting the pH to 6.0-7.0, sterilizing at 115-121℃ for 25-35 min, inoculating with microbial agent at 4-6% of the fermentation system volume, and fermenting at a constant temperature of 48-52℃ with stirring at 90-110 r / min for 40-56 h.

[0014] Furthermore, the application includes adding 0.8-1.2% by mass of corn cob powder to high-salt wastewater, adjusting the pH to 6.5-7.5, inoculating with bacterial agent at 4-6% of the wastewater volume, and stirring at 48-52℃ and 140-160 r / min for 68-76 hours to degrade the wastewater, achieving a COD removal rate of ≥40-48%.

[0015] The beneficial effects of this invention are as follows: The thermophilic *Trichoderma* strain MT-ST / GP of this invention overcomes the halophilic limitation of conventional strains. It maintains an average viable count of 20.3-22.0 when NaCl ≤ 10 g / L, 21.3-25.0 when sodium bicarbonate ≤ 5 g / L, and 20.3-26.0 when a mixed salt of 30% NaCl + 70% sodium bicarbonate ≤ 5 g / L. It can grow stably and maintain high activity, solving the problem that the growth of existing strains is inhibited at salt concentrations above 5 g / L, and filling the technological gap in the application of thermophilic *Trichoderma* in medium-to-high salinity environments.

[0016] The strains of this invention can be directly used for returning straw to saline-alkali land (soil salinity 5-8 g / L), fermenting high-salt agricultural waste (rice straw with 10-12 g / L salt content + pickling wastewater), and degrading high-salt wastewater (10 g / L salt content), covering multiple fields of agriculture and environmental protection. For example, in the treatment of saline-alkali land, the straw cellulose degradation rate reached 65.2%, which is 103% higher than the control group. It can also reduce the soil salinity to 5.8 g / L and increase organic matter by 1.2%, achieving the dual effects of soil improvement and resource utilization.

[0017] In the fermentation of high-salt agricultural waste, the strain of this invention can achieve a COD removal rate of 42.5% and simultaneously produce 1.8 g / L of reducing sugar; in the treatment of high-salt wastewater, the COD removal rate is 44%, the degradation rate of cellulosic pollutants is 58%, and the viable count of the strain after treatment still reaches 8.5 × 10⁻⁶. 6 With a concentration of CFU / mL or higher, it not only solves the pollution problem but also transforms waste into usable materials, resulting in significant economic and environmental benefits.

[0018] According to SEM results, the surface of the wheat straw before fermentation was smooth and flat with a dense cross-sectional structure. On the 5th day of fermentation, 1-5 μm micropores appeared on the surface of the wheat straw, and 5-10 μm gaps were formed between the fiber bundles in the cross-section. On the 10th day of fermentation, dense pores of 5-20 μm formed on the surface, and the fiber bundles in the cross-section were completely broken and loose and porous. This microscopic morphological change directly proves that thermophilic *Pterygopyrosis* can efficiently decompose wheat straw cellulose and hemicellulose, with a final material weight reduction of ≥64%, which is far superior to traditional low-temperature microorganisms and solves the core problem of dense and difficult-to-degrade wheat straw fiber structure. At the same time, the progressive degradation characteristics (from surface corrosion to internal breakage) observed by SEM in this invention are highly consistent with the temperature and pH value changes during fermentation, with no degradation stagnation or sudden drop in enzyme activity. On the one hand, thermophilic... The fungus *Phytotrichum can maintain a high-temperature environment through its own metabolism, eliminating the need for additional temperature control. Furthermore, the optimized raw material ratio avoids material clumping caused by high-moisture auxiliary materials (potatoes). No undegraded areas were observed by SEM, ensuring uniform fermentation. Therefore, this invention effectively solves the pain points of temperature / pH malfunction and poor aeration in traditional processes. In addition, the introduction of buckwheat hulls shows that the material maintains a loose structure throughout SEM, solving the problems of high viscosity and poor aeration in traditional formulations. The precise control of the carbon-to-nitrogen ratio (30:1) provides a stable metabolic environment for the fungus. Moreover, no chemical reagents are added during the degradation process, and no harmful residues were found during SEM observation. The fermentation products, due to their loose and porous structure, can be directly used as feed or organic fertilizer raw materials, avoiding pollution caused by straw burning and achieving efficient resource utilization of agricultural waste. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a comparison diagram of the surface degradation of straw at different magnifications and fermentation times in this invention. Figure 2 This is a comparison diagram of the degradation of straw cross sections at different magnifications and fermentation times in this invention. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] The present invention will be described in detail below through specific embodiments.

[0027] Example 1: Screening and Identification of Strains The halophilic and thermophilic Myceliophthorathermophila strain protected by this invention is classified and named Myceliophthorathermophila, with strain number MT-ST / GP. This strain was deposited on October 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 41587. The collection center confirmed the viability of the strain on the day of deposit, and the preservation period is 30 years from the date of deposit (extendable for 5 years upon expiration), which meets the requirements for biological material preservation in the patent procedure.

[0028] 1.1 Filtering Sources This strain was isolated from the surface decomposed straw in saline-alkali land (soil salt content 5-8 g / L) in northern my country. This habitat is under long-term combined salt stress of sodium chloride (NaCl) and sodium bicarbonate (NaHCO3 sodium bicarbonate), which provides natural enrichment conditions for screening halophilic strains.

[0029] 1.2 Screening Method 1.2.1 Enrichment Culture Take 5g of decomposed straw sample from the surface of saline-alkali land and add it to a 250mL Erlenmeyer flask containing 100mL of enrichment medium. The enrichment medium is PDA liquid medium containing 5g / L NaCl (formula: 200g / L potato, 20g / L glucose, 20g / L agar, diluted with deionized water, natural pH, sterilized at 121℃ for 15min). Place it in a shaker at 50℃ and 180r / min for 48h to enrich and culture, and obtain the enriched bacterial solution.

[0030] 1.2.2 Gradient Screening and Purification Take 1 mL of the above enriched bacterial solution and serially dilute it to 10⁻¹⁰ with sterile physiological saline. -6 The culture was spread on PDA solid screening plates with different salt concentrations (salt stress gradients: NaCl 5g / L, 10g / L, 20g / L; sodium bicarbonate 5g / L, 10g / L, 20g / L; 30% NaCl + 70% sodium bicarbonate mixed salt 5g / L, 10g / L, 20g / L), with three replicates for each gradient. The plates were incubated at 50℃ for 72 h. Single colonies with good growth and typical morphology (white fluffy colonies with neat edges and pale yellow back) were selected from the plates and transferred to fresh PDA solid plates for purification and culture three times to obtain a pure strain, named MT-ST / GP.

[0031] 1.3 Strain Identification 1.3.1 Morphological identification The strain was inoculated onto PDA solid plates and cultured at 50℃ for 48 hours. Colony characteristics were observed: the colony diameter reached 3-5 cm, initially consisting of white, fluffy aerial hyphae, which later darkened to light brown. The conidia were elliptical, measuring 3-5 μm × 2-3 μm, consistent with the typical morphological characteristics of Myceliophthora thermophila.

[0032] 1.3.2 Molecular biological identification Genomic DNA was extracted from the strain and amplified by PCR. The amplified product was sequenced and submitted to the GenBank database for comparison. The results showed that its ITS sequence had 99.8% homology with the thermophilic pyriformis model strain. Combined with morphological characteristics, the strain was confirmed to be thermophilic pyriformis.

[0033] Example 2: Halophilicity test of the strain 1.1 Preparation of Experimental Materials 1.1.1 Culture medium preparation Seed culture medium: PDA liquid culture medium (potato 200g / L, glucose 20g / L, deionized water to volume, natural pH, sterilized at 121℃ for 15min); Salt stress solid medium: Based on Bengal red solid medium (5 g / L peptone, 20 g / L glucose, 0.03 g / L Bengal red, 20 g / L agar, and diluted with deionized water), different salts (NaCl, sodium bicarbonate, and a mixture of 30% NaCl and 70% sodium bicarbonate) were added according to Table 1. The amount of salts added per 70 mL of medium is shown in Table 1 below. Table 1 Salt addition amounts in solid culture medium under salt stress (70 mL)

[0034] 1.1.2 Instruments and Equipment Constant temperature shaker (temperature range 30-60℃, rotation speed 0-200r / min), ultra-clean workbench, high pressure steam sterilizer, constant temperature incubator (50℃), colony counter.

[0035] 1.2 Experimental Procedure 1.2.1 Seed liquid preparation Single colonies of strain MT-ST / GP were picked and inoculated into 100 mL of PDA liquid medium. The culture was then incubated at 50°C and 180 rpm for 48 hours to obtain a seed culture (with approximately 10⁻⁶ viable cells). 8 (CFU / mL).

[0036] 1.2.2 Salt stress plate culture Take 16 Erlenmeyer flasks and prepare 16 groups of salt stress solid culture media (3 replicate plates for each group, plus 3 blank control plates). After sterilizing at 121℃ for 15 min, pour the plates in a laminar flow hood and let them cool and solidify. The seed culture was serially diluted to 10 with sterile physiological saline. -6 Take 0.1 mL of the diluted solution and spread it evenly on each salt stress plate. Repeat the process 3 times for each plate. After incubation at 50℃ for 72 hours, the viable cell count (CFU / mL) of each plate was counted using a colony counter, and the average value was calculated.

[0037] 1.3 Test Results and Analysis 1.3.1 Results of viable cell count The viable bacterial counts on each salt stress plate are shown in Table 2 below: Table 2 Viable cell counts of strain MT-ST / GP under different salt stress conditions

[0038] 1.3.2 Results Analysis NaCl tolerance: When the NaCl concentration is ≤10g / L, the average viable count of the strain remains at 20.3-22.0, and it can grow normally; when the NaCl concentration rises to 20g / L, the viable count drops to 6.3, and growth is significantly inhibited. Baking soda tolerance: When the baking soda concentration is ≤5g / L, the average viable count of the strain reaches 21.3-25.0, and the growth status is good; when the baking soda concentration is ≥10g / L, the viable count is 0, and growth is impossible; Compound salt tolerance: When the concentration of the mixed salt of 30% NaCl + 70% sodium bicarbonate is ≤5g / L, the average viable count of the strain remains at 20.3-26.0, which can adapt to compound salt stress; when the concentration of the mixed salt is ≥10g / L, it cannot grow.

[0039] Example 3: Fermentation Capacity Test of Strains The main purpose of this embodiment is to solve the problems of "high material viscosity, poor aeration, and low degradation efficiency" in traditional wheat straw fermentation. By optimizing the raw material ratio and fermentation process, the efficient degradation ability and fermentation stability of thermophilic pyriformis on wheat straw are verified.

[0040] (a) Experimental materials Main ingredients: Shredded wheat straw (5-10cm in length, fiber content ≥70%), buckwheat hulls (1-3mm in size); Additional ingredients: corn flour (80 mesh), soybean meal (crushed to 2-4 mm particle size), fresh potatoes (peeled and cut into 1-2 cm pieces) 3 Small pieces of diced sugar and brown sugar (food grade); Culture medium: PDA medium (potato 200g / L, glucose 20g / L, agar 15g / L, natural pH); Equipment: Fermentation frame (57cm×38cm×15cm), shaker (temperature controllable 40-50℃), autoclave (121℃, 0.1MPa), pH meter (accuracy 0.01), forced-air drying oven (temperature controllable 105℃), scanning electron microscope (model SU8010, accelerating voltage 0-30keV).

[0041] (II) Raw material proportioning design To address the issues of "high material viscosity and poor aeration after sterilization" observed in previous experiments, the optimized material composition per tray in this embodiment is shown in Table 1 below (the carbon-to-nitrogen ratio is adjusted to 30:1, taking into account both the metabolic needs of the microbial strain and the physical properties of the material): Table 1 Material Proportions

[0042] (III) Detailed Experimental Procedure 1. Activation and scale-up culture of microorganisms Preparation of PDA culture medium: Weigh potatoes, glucose and agar according to the formula, add water to make up to 1000 mL, boil to dissolve and then dispense into 250 mL Erlenmeyer flasks (100 mL per flask), and sterilize at 121℃ and 0.1 MPa for 20 min. Inoculation: After the culture medium has cooled to below 45°C, inoculate with thermophilic *Hydromycinus MT-ST / GP* strain in a sterile operating table (inoculation amount is 1 cm of slant culture). 2 / bottle); Expanded culture: Place the inoculated Erlenmeyer flasks in a 45℃ constant temperature shaker and culture at 150 r / min for 24 h to obtain the logarithmic phase bacterial culture (cell concentration ≥10). 7 (CFU / mL).

[0043] 2. Solid material preparation and sterilization Material pretreatment: Weigh wheat straw, buckwheat hulls, corn flour, and soybean meal; cut potatoes into cubes; mix all solid materials evenly according to the formula. Moisture control: Dissolve 25g of brown sugar in 500mL of deionized water, and spray the sugar solution evenly into the mixture while stirring to ensure that the moisture content of the material is initially controlled at 65%-70% (it can be formed into a ball by hand and does not fall apart when released). Bagging and sterilization: Pack the mixture into high-temperature resistant polyethylene bags (approximately 800-1000g per bag), seal them, and sterilize them at 121℃ and 0.1MPa for 30 minutes. After sterilization, allow them to cool naturally to 35-40℃ (avoid high temperatures that could kill the bacteria).

[0044] 3. Inoculation and Fermentation In an aseptic environment, the cooled material was taken out and packaged in the manner of "2 bags mixed and packed into 1 fermentation box" (because each bag was not weighed during sterilization, the actual weight of each tray was different, but the ratio was consistent). 20 mL of logarithmic phase bacterial solution (inoculation amount 2%) was added to each tray, and after stirring evenly, the surface of the material was smoothed. Fermentation environment control: Place 4 fermentation frames in the upper part of the fermentation chamber (good ventilation, temperature fluctuation ≤ ±2℃), and place temperature sensors only in fermentation frames No. 1 and No. 3 (to record the material temperature in real time). The initial temperature of the fermentation chamber is set to 40℃, and no additional temperature control is required thereafter (the high temperature environment is maintained by the heat generated by the metabolism of the microorganisms).

[0045] 4. Sampling and Testing Sampling time and method: Initial sample: Before inoculation, take about 100g of the mixture (take a photo to record the appearance), and divide it into 2 portions (1 portion for pH measurement, 1 portion for moisture measurement). Fermentation samples: Take samples at 10:40 / 17:10, 8:00 / 18:00, 9:40 / 17:00, 8:20 / 17:30, and 8:00. Take 25-50g of material from each of the four fermentation boxes each time (take photos to record the appearance changes). Final sample: Remove all fermented material and record the material thickness and total weight of the four fermentation frames.

[0046] Test items and methods: Temperature detection: The temperature at the center of the material is recorded by temperature sensors in fermentation boxes No. 1 and No. 3, accurate to 0.1℃; Moisture content test: Take 25g of fresh sample and dry it in a 105℃ forced-air drying oven to constant weight. Calculate the moisture content according to the formula "Moisture content (%) = (fresh weight - dry weight) / fresh weight × 100%". Perform three parallel tests and take the average value. pH test: Take 10g of fresh sample, add 90mL of deionized water, shake for 30min and let stand for 10min, measure the pH of the supernatant with a pH meter, and take the average value of 3 parallel measurements. SEM detection: Wheat straw samples were taken before fermentation, on day 5 of fermentation, and on day 10 of fermentation. They were cut into 0.5cm×0.5cm pieces, fixed with 2.5% glutaraldehyde, dehydrated in a gradient, and sputtered with gold. The surface and cross-sectional morphology were observed at magnifications of 25×, 180×, 500×, 2000×, and 5000× under the conditions of accelerating voltage of 5keV and working current of 100pA.

[0047] (iv) Experimental Results and Analysis 1. Temperature change results (reflecting the metabolic activity of the microbial strain) The temperature changes of the materials in fermentation containers 1 and 3 during fermentation are as follows: On the day of inoculation: the temperature was 38.3-38.4℃ at 10:40 and rose to 46.0-46.2℃ at 17:10 (due to rapid proliferation and heat production of the bacterial strain). Day 2 of fermentation: The temperature reaches its peak of 46.8-49.2℃ (the optimal growth temperature range for thermophilic pyridostigma, when metabolism is most active). Fermentation days 3-4: Temperature slowly drops to 37.8-42.7℃ (the easily degradable components in the material decrease, and the metabolic rate slows down); Day 5 of fermentation: The temperature drops to 34.6-34.7℃ (degradation enters the later stage, and the activity of the strain tends to stabilize).

[0048] Conclusion: Thermophilic pyriformis can maintain a high temperature environment of 40-49℃ during fermentation through its own metabolism, adapting to the high temperature conditions required for the degradation of wheat straw. Moreover, the temperature change trend conforms to the strain growth pattern of "proliferation-metabolism-stability", and the fermentation stability is good.

[0049] 2. Results of pH and moisture content changes (reflecting the degree of material degradation) The results of the pH and moisture content changes are shown in Table 2: Table 2 Results of pH and Moisture Content Changes

[0050] As shown in Table 2, the pH value gradually increased from around 5.7 to 7.9. This was due to the decomposition of cellulose and hemicellulose in the wheat straw by the microorganisms, which produced alkaline metabolites (such as ammonia nitrogen and organic acid salts), demonstrating continuous microbial metabolism and effective degradation of organic matter. The moisture content decreased from 65%-70% to 57.52%-64.12%. This was partly due to the heat generated during fermentation causing water evaporation, and partly due to the microorganisms utilizing some water for metabolism. The decrease in moisture content was in line with the expectations for efficient fermentation (avoiding excessive moisture leading to anaerobic conditions or excessive moisture inhibiting microbial activity).

[0051] 3. Results of changes in material weight and volume (reflecting dry matter degradation) The changes in the thickness and total weight of the material in the fermentation container are shown in Table 3 (the first day of fermentation is the initial value, and the eighth day of fermentation is the final value): Table 3. Changes in the thickness and total weight of materials in the fermentation container.

[0052] As shown in the table above, the average weight of the material decreased by more than 64% after fermentation, and the volume shrank (a 1cm gap appeared around the frame). The main reasons are: ① the evaporation of moisture in the potatoes and free water in the material; ② the degradation of cellulose and hemicellulose in wheat straw by thermophilic mycorrhizal fungi into small molecules (such as glucose and xylose). Some of these small molecules were utilized or volatilized by the fungi, proving that the dry matter degradation effect was significant.

[0053] 4. SEM observation results (visual verification of degradation effect) Combination Figure 1 (Surface) and Figure 2 The morphological changes of wheat straw at different fermentation stages are shown in the SEM images of the cross-section as follows: Before fermentation: Surface (25×-5000×): The surface of the wheat straw is smooth and flat, without obvious damage, and the fiber texture is clear; Cross-section (25×-5000×): The internal structure is dense, the fiber bundles are neatly arranged, and there are no pores or cracks.

[0054] Day 5 of fermentation: Surface (500×-2000×): Obvious corrosion marks appear, surface roughness increases, and micropores (diameter 1-5μm) appear in some areas. Cross-section (500×-2000×): Gaps (5-10μm wide) appear between fiber bundles, and some fibers begin to break.

[0055] Day 10 of fermentation: Surface (2000×-5000×): Densely distributed pores (5-20μm in diameter), exposed fiber structure and fragmentation; Cross-section (2000×-5000×): The fiber bundles are completely broken, forming a loose and porous structure inside. Under 5000× magnification, traces of dissolution of the fiber cell walls can be observed.

[0056] It is evident that thermophilic cytotoxic filamentosa can effectively decompose the cellulose and hemicellulose of wheat straw, causing its surface and internal structure to gradually break down and become loose. This is corroborated by the changes in weight / moisture content, demonstrating a clear and significant degradation effect.

[0057] In summary, this embodiment successfully solved the technical pain points of traditional wheat straw fermentation by optimizing the raw material ratio (introducing buckwheat hulls to reduce viscosity and adjusting the carbon-nitrogen ratio to 30:1) and the fermentation process (activation of the inoculum at 45℃ and placement in the upper part of the fermentation chamber with ventilation), and verified the advantages of thermophilic Mycorrhizal (CGMCC No. 41587).

[0058] Example 4: Comparison of the strain of this application with existing technology strains This embodiment compares the straw degradation and soil improvement effects of the present application's Thermophilic Trichophyton MT-ST / GP (CGMCC No. 41587) with those of representative strains in the prior art under high-salt complex environments, clarifying the technical advantages of the present application's Thermophilic Trichophyton MT-ST / GP in terms of halophilicity, biomass degradation efficiency, and practical application effects.

[0059] I. Experimental Materials (a) Test strains Experimental group: The strain of *Thermophilus thermophilus* MT-ST / GP (CGMCC No. 41587) was used to prepare logarithmic-phase bacterial suspension (viable count 10⁻⁶) according to the method in Example 1. 8 (CFU / mL).

[0060] Control group 1: The "thermophilic cytotoxic strain with improved cellulose degradation ability" (hereinafter referred to as "conventional engineered strain") disclosed in Chinese patent CN202411479455.7 was cultured and prepared into a bacterial suspension (live count 10^6). 8 (CFU / mL).

[0061] Control Group 2: The "alkali-resistant compound microbial pretreatment agent" (hereinafter referred to as "compound agent") disclosed in Chinese Patent CN113151098A was used to prepare a mixed bacterial solution (live count 10) according to its disclosed ratio. 8 (CFU / mL).

[0062] Control group 3: "Halophilic yeast" (hereinafter referred to as "halophilic yeast") disclosed in Chinese patent CN113832042B, was cultured and prepared into a bacterial suspension (live count 10^6). 8 (CFU / mL).

[0063] Control group 4: Wild-type common thermophilic filamentous fungus strain that has not undergone halophilic modification (hereinafter referred to as "common wild-type strain"), isolated from conventional farmland straw, and cultured to prepare bacterial suspension (live count 10^6). 8 (CFU / mL).

[0064] (ii) Target of processing Wheat straw: cut into pieces to a length of 5-8cm, with a fiber content of 72%, meeting the requirements of the original patent application scenario.

[0065] Simulated saline-alkali soil: taken from unimproved saline-alkali land in the north, with a basic salt content of 8.5g / L (NaCl 6g / L + baking soda 2.5g / L, compound salt ratio 3:7), pH 8.3, and organic matter content of 0.8%.

[0066] (III) Experimental Equipment The constant temperature incubator, colony counter, crude fiber analyzer, soil salinity analyzer, organic matter analyzer, pH meter, etc., are consistent with the original patent embodiment.

[0067] II. Experimental Design (I) Experimental Grouping Five groups were set up, with three replicates per group. Each group treated 10 kg of soil and 1.5 kg of wheat straw (wheat straw to soil mass ratio 1:6.7). 30 mL of the corresponding bacterial solution was inoculated (inoculation amount was 0.3% of the total material weight). The specific groupings are as follows: Experimental group: simulated saline-alkali soil + wheat straw + strain MT-ST / GP (as applied) Control group 1: Simulated saline-alkali soil + wheat straw + conventional engineered bacterial strains Control group 2: Simulated saline-alkali soil + wheat straw + compound microbial agent Control group 3: Simulated saline-alkali soil + wheat straw + halophilic yeast Control group 4: Simulated saline-alkali soil + wheat straw + common wild bacterial strains Control group: Simulated saline-alkali soil + wheat straw + sterile saline solution (no bacterial strain inoculation) (II) Experimental conditions The original patented saline-alkali land treatment process was optimized and set up as follows: maintain soil moisture content of 22%-25%, natural temperature environment (average daily temperature of 25-32℃), and a cultivation cycle of 30 days, during which the soil is turned over regularly to ensure uniform aeration.

[0068] III. Experimental Procedure Soil and straw pretreatment: The simulated saline-alkali soil was sieved through a 2mm sieve to remove impurities, mixed evenly with chopped straw, and divided into 5L culture pots, with 3 pots per group.

[0069] Bacterial inoculation: Inoculate the corresponding bacterial solution according to the group, stir well, and inoculate the blank group with an equal volume of sterile physiological saline.

[0070] Cultivation and management: Monitor soil moisture content daily and maintain it at 22%-25% by spraying water for 30 days.

[0071] Sampling and testing: After the incubation period, three soil samples (5-10 cm deep) were randomly selected from each group, and the following indicators were tested respectively: (1) Viable count of strains: The dilution plating method was used to count the number of viable cells (CFU / g dry soil) using PDA medium containing 5g / L NaCl + 2g / L sodium bicarbonate. (2) Degradation rate of wheat straw: The crude fiber content of wheat straw was detected by the crude fiber determination method, and the degradation rate was calculated (degradation rate = (initial crude fiber content - residual crude fiber content) / initial crude fiber content × 100%). (3) Soil salinity: The total salt content of the soil (g / L) was determined by gravimetric method. (4) Soil organic matter content: determined by potassium dichromate oxidation-external heating method (%). (5) Soil pH value: determined by potentiometric method (soil-water ratio 1:5).

[0072] IV. Test Results and Analysis (I) Test Result Statistics Table 4. Comparative experimental results of the strains in this application and those in existing technologies (mean ± standard deviation)

[0073] (II) Results Analysis Salt preference comparison: In the experimental group, the viable count of the strain applied for in this application remained at 8.6 × 10⁶ cells / L in saline-alkali soil with a combined salt concentration of 8.5 g / L. 6 The CFU / g dry soil concentration was significantly higher than that of control group 1 (conventional engineered strain, 1.3 × 10⁻⁶). 5 CFU / g dry soil), control group 2 (compound microbial agent, 2.1×10⁻⁶) 5 CFU / g dry soil) and control group 4 (common wild strain, 0.8×10 5 (CFU / g dry soil), proving that the strain applied for has overcome the limitation of existing thermophilic filamentous strains being inhibited in growth at salt concentrations above 5 g / L; Although control group 3 (halophilic yeast) had a higher viable count (3.5 × 10⁻⁶), 6(CFU / g dry soil), but lacks cellulose degradation ability and cannot achieve straw degradation function, which is significantly different from the function of the strain in this application.

[0074] Comparison of wheat straw degradation efficiency: The degradation rate of wheat straw in the experimental group reached 65.8%, which was 130.9% higher than that of control group 1 (28.5%), 105.0% higher than that of control group 2 (32.1%), and 191.1% higher than that of control group 4 (22.6%). This demonstrates that the strain of this application still maintains excellent cellulose and hemicellulose degradation activity under high salt environment. The degradation rate of wheat straw in control group 3 (halophilic yeast) was only 10.3%, which was not significantly different from the blank group (8.5%), further proving that it had no biomass degradation capacity and could not meet the demand for straw return to the field in saline-alkali land. Although control group 2 (compound bacterial agent) is an alkali-resistant compound system, it lacks halophilic characteristics and requires strict control of the bacterial strain ratio, resulting in a degradation efficiency far lower than that of the strain in this application. Furthermore, the process is cumbersome and the actual application cost is higher.

[0075] Comparison of soil improvement effects: Soil salinity: After treatment, the soil salinity in the experimental group decreased to 5.6 g / L, a 34.1% decrease from the initial value (8.5 g / L), which was far better than that in the control groups (7.1% decrease in control group 1, 11.8% decrease in control group 2, 15.3% decrease in control group 3, and 4.7% decrease in control group 4). This proves that the strain of this application can reduce soil salt concentration through metabolic activity while degrading wheat straw, thereby improving saline-alkali land. Soil organic matter: The soil organic matter content in the experimental group increased to 1.9%, which is 137.5% higher than the initial value (0.8%). This is higher than that of each control group (control group 1 increased by 37.5%, control group 2 increased by 50.0%, control group 3 increased by 12.5%, and control group 4 increased by 25.0%), achieving the dual effect of straw resource utilization and soil fertility improvement. Soil pH: The soil pH in the experimental group dropped to 7.8, which is closer to the suitable range for crop growth (6.5-7.5), while the pH in each control group remained above 8.1, indicating limited improvement effect.

[0076] In summary, based on this application, compared to representative strains in the prior art: The strain MT-ST / GP of this application exhibits significantly better halophilicity in high-salt complex environments than conventional thermophilic filamentous engineered strains, compound inoculants, and common wild strains, thus solving the technical pain point of existing strains being inhibited in medium-high salt environments. The straw degradation efficiency of the strain applied for here far exceeds that of existing strains, and it does not require complex strain ratio control, making the process simple and the application cost lower. The strain described in this application can effectively reduce soil salinity and increase soil organic matter while achieving biomass degradation in high-salt environments, resulting in significant soil improvement effects. This comprehensive function is not found in strains such as halophilic yeast. The strain described in this application combines halophilicity, high-temperature adaptability (verified in the original patent), and high biomass degradation capacity, filling the gap in the field of biological treatment of high-salt environments and possessing significant technical advantages and practical application value.

[0077] Application Example 1: Straw Returning Treatment in Saline-Alkali Land 1.1 Processing Object Corn stalks (crushed to 2-5cm) in saline-alkali land in northern China (soil salt content 8g / L, main components are NaCl and baking soda, in a ratio of about 3:7).

[0078] 1.2 Processing Steps Preparation of bacterial agent: The MT-ST / GP strain was inoculated into cellulase liquid medium (sodium carboxymethyl cellulose 10 g / L, peptone 5 g / L, yeast extract 3 g / L, NaCl 5 g / L, sodium bicarbonate 2 g / L, pH 7.0) and cultured at 50℃ and 180 rpm for 72 h on a shaker to obtain the bacterial agent (viable count ≥ 10^6). 8 (CFU / mL) Straw treatment: Mix crushed corn straw with microbial agent at a mass ratio of 10:1, and spread evenly on the surface layer of saline-alkali land (soil depth 5-10cm). The amount used per hectare is 1500kg of straw and 150kg of microbial agent. Field management: Maintain soil moisture content at 20%-25% and cultivate at natural temperature for 30 days.

[0079] 1.3 Processing Results Sampling and testing after 30 days showed that the straw cellulose degradation rate reached 65.2%, the soil salinity decreased to 5.8 g / L, and the soil organic matter content increased by 1.2%. Compared with the control group without inoculation (cellulose degradation rate of 32.1%, and no significant change in salinity), the treatment effect was significantly improved.

[0080] Application Example 2: Fermentation of High-Salt Agricultural Waste 1.1 Processing Object Rice straw (salt content 12g / L, mainly NaCl) produced in the seawater irrigation area is mixed with a small amount of pickling wastewater (COD value 8000mg / L).

[0081] 1.2 Processing Steps Fermentation medium preparation: Crush rice straw and mix it with pickling wastewater at a solid-liquid ratio of 1:5, add 0.5% glucose (mass fraction), adjust the pH to 6.5, and sterilize at 121℃ for 30 min; Inoculation and fermentation: Inoculate with MT-ST / GP bacterial strain (10⁶ viable cells) at 5% of the fermentation system volume. 8 (CFU / mL), fermented at 50℃ for 48 hours with stirring speed of 100r / min; Product detection: After fermentation, the reducing sugar content and COD removal rate in the fermentation broth were measured.

[0082] 1.3 Processing Results After fermentation, the reducing sugar content of the fermentation broth reached 1.8 g / L (produced from the degradation of carbohydrates in straw), the COD removal rate reached 42.5%, and the viable bacterial count of the fermentation system remained at 1.2 × 10⁻⁶. 7 CFU / mL, free from bacterial contamination, enabling resource utilization and pollutant degradation of high-salt waste.

[0083] Application Example 3: Biomass Degradation of High-Salinity Wastewater 1.1 Processing Object High-salt wastewater after pretreatment of dyeing and printing wastewater (salt content 10g / L, mainly NaCl, containing a small amount of cellulose pollutants, COD value 5000mg / L).

[0084] 1.2 Processing Steps Wastewater treatment system construction: Take 1000mL of high-salt wastewater and place it in a fermenter. Add 1% (mass fraction) of corn cob powder (as a carbon source supplement) and adjust the pH to 7.0. Inoculation and degradation: Inoculate with 50 mL of MT-ST / GP bacterial agent (10⁸ viable cells). 8 (CFU / mL), degraded by stirring at 50℃ and 150r / min for 72h; Indicator testing: Regular sampling is conducted to determine the COD value, cellulose degradation rate, and viable bacterial count of the wastewater.

[0085] 1.3 Processing Results After 72 hours, the COD value of the wastewater decreased to 2800 mg / L, with a removal rate of 44%; the degradation rate of cellulosic pollutants reached 58%; and the viable bacterial count in the system was 8.5 × 10⁻⁶. 6 The CFU / mL level indicates that the strain can grow stably and play a degrading role in high-salt wastewater.

[0086] In summary, in practical applications, the inoculum size (3%-10%), culture temperature (45-55℃), pH (6.0-7.5), and treatment time (24-72h) can be adjusted according to the specific salt type (e.g., NaCl-dominated, sodium bicarbonate-dominated, or mixed salt), salt concentration, and the treatment target (straw, wastewater, waste) in the high-salt environment. All of these adjustments can achieve halophilic growth of the bacterial strains and efficient biological treatment.

[0087] The core advantage of the MT-ST / GP strain of this invention is that, compared with conventional thermophilic pyriformis strains, it can still maintain a high viable count (≥20) and biomass degradation activity in high-salt environments with NaCl≤10g / L, sodium bicarbonate≤5g / L or mixed salt≤5g / L, which significantly expands the application scope of thermophilic pyriformis strains in the field of biological treatment of high-salt environments and has good prospects for industrial application.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A strain of *Myceliophthora thermophila* MT-ST / GP, characterized in that, The thermophilic mitochondrial MT-ST / GP was deposited on October 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC); address of the depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; accession number: CGMCCNo. 41587.

2. The thermophilic pyridobacterium MT-ST / GP according to claim 1, characterized in that, The thermophilic mitochondritis MT-ST / GP can grow stably in environments with NaCl concentration ≤1-20 g / L, sodium bicarbonate concentration ≤1-6 g / L, or sodium bicarbonate mixed salt concentration ≤1-6 g / L.

3. The application of *Thermophilic mitochondritis MT-ST / GP* according to claim 1 in the efficient fermentation and degradation of wheat straw, characterized in that, When applying, wheat straw is mixed with other raw materials in a certain proportion, and the cellulose and hemicellulose in the wheat straw are metabolized and degraded by the strain. After fermentation, the degradation rate of wheat straw is ≥60-70%.

4. The application according to claim 3, characterized in that, The raw materials include wheat straw, buckwheat hulls, corn flour, soybean meal, potatoes, and brown sugar. The weight ratio of each raw material is wheat straw: buckwheat hulls: corn flour: soybean meal: potatoes: brown sugar = 200-220: 280-320: 28-32: 28-32: 1400-1600: 23-27, and the overall carbon-nitrogen ratio of the raw materials is adjusted to 25-30:

1.

5. The application according to claim 3, characterized in that, Before fermentation, wheat straw needs to be pre-treated: cut the wheat straw into pieces 4-11cm in length, sift the buckwheat hulls to a particle size of 1-3mm, and peel and cut the potatoes into 0.8-2.2cm pieces. 3 For small pieces, corn flour is ground to a fineness of 70-90 mesh, and soybean meal is ground to a particle size of 2-4 mm.

6. The application according to claim 3, characterized in that, The fermentation process parameters are as follows: the initial moisture content of the material is controlled at 63-72%, sterilization is carried out at 115-121℃ and 0.09-0.11MPa for 25-35 minutes, and after cooling to 33-42℃, the logarithmic phase bacterial solution of the strain is inoculated, with the inoculation amount being 1-3% of the total weight of the material.

7. The application of *Thermophilic MT-ST / GP* according to claim 1 in the biological treatment of high-salt environments, characterized in that, The high-salt environment includes saline-alkali land, high-salt agricultural waste, or high-salt wastewater. During the treatment process, the bacterial strain can maintain its degradation activity.

8. The application according to claim 7, characterized in that, The application involves mixing straw crushed to 1.5-5.5cm with the thermophilic mycorrhizal MT-ST / GP at a mass ratio of 9-11:1, spreading it on the surface layer of saline-alkali land at a depth of 4-11cm, using 1400-1600kg of straw and 140-160kg of mycorrhizal agent per hectare, maintaining a soil moisture content of 18-27%, and cultivating for 28-32 days.

9. The application according to claim 7, characterized in that, The application involves mixing high-salt straw with high-salt wastewater with a COD value of 7000-9000 mg / L at a solid-liquid ratio of 1:4-6, adding 0.4-0.6% glucose by mass, adjusting the pH to 6.0-7.0, sterilizing at 115-121℃ for 25-35 min, inoculating with microbial agent at 4-6% of the fermentation system volume, and fermenting at a constant temperature of 48-52℃ with stirring at 90-110 r / min for 40-56 h.

10. The application according to claim 7, characterized in that, The application involves adding 0.8-1.2% corn cob powder to high-salt wastewater, adjusting the pH to 6.5-7.5, inoculating with bacterial agent at 4-6% of the wastewater volume, and stirring at 48-52℃ and 140-160r / min for 68-76 hours to degrade the wastewater, achieving a COD removal rate of ≥40-48%.

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