Preparation method of microbial agent for improving soil alkalinity

Abstract

The invention belongs to the field of agricultural microbial technologies and soil improvement, and particularly relates to acid-producing, alkali-reducing and yield-increasing Michelia guilliermondii, a soil conditioner and application. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.39011. According to the invention, it is found and proved for the first time that the strain has extremely strong acid production and direct reduction of the pH value of saline-alkali soil, and when a culture of the strain is applied to saline-alkali soil with the pH value of 9.01, the pH value of the soil can be significantly reduced to 7.39 within 7 days. The invention further provides an efficient soil conditioner which is formed by compounding the DY1 strain with a specific carrier soybean meal and biochemical potassium fulvate, the viable count in the product is greater than or equal to 0.5 billion CFU / g, and the organic matter content is greater than or equal to 20%. The specific combination produces a remarkable synergistic effect. Field application shows that the modifier can effectively reduce the pH value of soil and can increase the yield of crops such as rice, corn and the like by 5% or above.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology and soil improvement, specifically involving a yeast that produces acid, reduces alkali and increases yield, as well as a soil conditioner and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] my country has a vast area of ​​saline-alkali land, especially in regions like the Yellow River Delta, where soil pH values ​​often exceed 9.0, severely inhibiting crop growth. Therefore, developing efficient, environmentally friendly, and sustainable saline-alkali land improvement technologies is crucial.

[0004] Currently, methods for improving saline-alkali land mainly include physical, chemical, and biological methods. Among them, microbial improvement methods have become a hot topic in research and application due to their low cost, environmental friendliness, and long-lasting effects.

[0005] Existing microbial modification technologies mainly rely on some common bacterial groups, such as Bacillus spp. ( Bacillus These microbial agents (such as Bacillus subtilis, Bacillus licheniformis, and Bacillus mucilaginosus) primarily help plants adapt to saline-alkali stress by producing plant growth hormones, enhancing plant resistance, or subtly regulating the rhizosphere microenvironment. However, the fundamental limitation of this method lies in its relatively indirect mechanism of action. Its ability to directly neutralize high pH levels—a core hazard in saline-alkali soils—is generally weak, resulting in slow alkali reduction, insignificant effects, long actual improvement cycles, and limited yield increases.

[0006] Recently, some studies have also begun to focus on the application of yeast in agriculture. For example, Chinese patent publication number CN120272335A discloses a strain of Pichia pastoris (…). Meyerozyma guilliermondii YSY119, combined with a Bacillus subtilis strain, is designed to enhance the salt and alkali tolerance of plants. While this technology represents progress, its underlying approach, based on its disclosed content, remains focused on constructing microbial combinations to enhance plant tolerance. The patent document does not emphasize or verify the yeast strain's ability to directly and efficiently lower soil pH, nor does it address combining it with specific functional carriers to achieve synergistic effects, nor does it disclose any clear crop yield-increasing effect on saline-alkali land.

[0007] In summary, there is a significant gap and need in existing technologies: a lack of specialized microbial resources and application schemes that can directly, efficiently, and rapidly address the core problem of saline-alkali land (high pH value) and directly improve crop yields through soil improvement. Specifically: (1) Existing microbial agents (especially bacteria) have insufficient direct biological acidification capacity, resulting in unsatisfactory soil alkalinity reduction effect.

[0008] (2) Existing technologies focus on improving plant “tolerance” rather than actively and thoroughly “improving” the soil environment itself.

[0009] (3) There is a lack of research on the scientific combination of microorganisms with special functions with specific organic carriers that can provide a lasting carbon source and improve soil structure to form a long-term, synergistic improvement system.

[0010] Therefore, isolating and screening novel microbial strains with unique and strong acid-producing capabilities, and developing their new applications for directly improving saline-alkali soils and increasing yields, has become a pressing technical problem in this field. This invention was completed against this backdrop. Summary of the Invention

[0011] In view of the shortcomings of existing microbial improvement technologies for saline-alkali land, such as the weak direct bio-acidification ability of the strains used, the insignificant effect on reducing soil pH, and the limited effect on increasing crop yield, as well as the limitations of existing technologies that focus on improving plant stress resistance rather than actively improving the soil environment, this invention aims to provide a new microbial strain resource with excellent acid-producing and alkali-reducing capabilities, and to explore its new applications in directly and efficiently improving saline-alkali soil and increasing crop yield.

[0012] The technical solution adopted in this invention is as follows: In a first aspect of the invention, a *Gnaphalium guildrum* DY1 is provided, the *Gnaphalium guildrum* DY1 being classified as... Meyerozyma guilliermondii, The strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 22, 2025, with accession number CGMCC NO. 39011.

[0013] In a second aspect of the invention, the use of the aforementioned *Saccharomyces cerevisiae* strain DY1 in the preparation of formulations for reducing the pH of saline-alkali soils is provided.

[0014] Preferably, the formulation is used to reduce the pH value of saline-alkali soil with an initial pH value of not less than 9.0 to below 7.5.

[0015] Preferably, the preparation is also used to increase the yield of crops grown on the saline-alkali land.

[0016] Preferably, the crop is rice or corn.

[0017] In a third aspect of the invention, a soil conditioner for saline-alkali land is provided, the active ingredient of which comprises the aforementioned yeast strain DY1.

[0018] Preferably, the soil conditioner further comprises a carrier, the carrier comprising soybean meal and biochemical potassium humate in a mass ratio of (1~3):(1~3).

[0019] Preferably, the soil conditioner contains a viable count of at least 0.5 billion CFU / g of *Saccharomyces cerevisiae* strain DY1 and an organic matter content of at least 20%.

[0020] In a fourth aspect of the invention, a method for preparing a soil conditioner for saline-alkali land is provided, comprising the steps of: mixing a culture of yeast strain DY1 obtained by fermentation with a carrier containing soybean meal and biochemical potassium humate.

[0021] Specifically, the following steps are included: (1) Fermentation culture: The yeast strain DY1 of Gynostemma pentaphyllum was inoculated into a liquid culture medium for fermentation culture to obtain a highly active cell fermentation broth or bacterial sludge. (2) Carrier preparation: Mix soybean meal and biochemical potassium humate evenly in a predetermined ratio to obtain a mixed carrier; (3) Compounding and molding: The bacterial fermentation liquid or bacterial mud obtained in step (1) is uniformly mixed with the mixed carrier obtained in step (2), and after low-temperature drying, it is made into powder or granular products.

[0022] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) Provides a novel core microbial resource: This invention isolated and preserved a novel strain of *Saccharomyces cerevisiae* DY1. The most prominent characteristic of this strain is its exceptionally strong acid-producing ability. Experiments have shown that it can rapidly reduce the pH value from 7.05 to 4.41 in liquid acid-producing medium; when its culture is added to saline-alkali soil with a pH of 9.01, the soil pH value can be significantly reduced to 7.39 within 7 days. This highly efficient direct biological acidification ability is not possessed by commonly used bacterial amendments.

[0023] (2) A fundamental shift in the technical approach has been achieved: This invention focuses on the core application of the DY1 strain to directly and rapidly reduce soil pH, transforming it from an indirect approach of improving plant tolerance to a direct approach of actively improving soil stress. This provides a more fundamental new approach for the improvement of saline-alkali land.

[0024] (3) Significant synergistic and yield-increasing effects: This invention creatively combines the highly acid-producing DY1 strain with a specific functional carrier (soybean meal and biochemical potassium humate). This combination not only leverages the core functions of the strain in acid production and alkali reduction, but also achieves long-term energy supply, structural improvement, and nutrient supplementation through the carrier, generating a synergistic effect of "1+1>2". Field trials have shown that applying this preparation can increase the yield of crops such as rice and corn by more than 5%, realizing a complete value loop from soil improvement to yield enhancement.

[0025] (4) High degree of productization and easy application: The solid microbial preparation provided by this invention has a stable content of live bacteria and is rich in organic matter. It is easy to store, transport and apply in the field (such as basal application or furrow application). The recommended dosage is 20 kg per mu, which has good practicality and promotion prospects.

[0026] In summary, this invention achieves a series of innovations from the source (new strain), the pathway (new application) to the end (new product and yield increase effect), providing a new technical solution that is efficient, reliable and has significant economic benefits for solving the pain points of saline-alkali land improvement. Attached Figure Description

[0027] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1 Photograph of *Saccharomyces gimmickii* DY1 after crystal violet staining under an optical microscope. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. 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 invention pertains.

[0030] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0031] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0032] Example 1: Isolation, purification and identification of *Saccharomyces cerevisiae* strain DY1

[0033] 1. Sample Collection and Pretreatment: Rhizosphere soil samples were collected from a typical coastal saline-alkali land (soil pH 9.2, salt content 1.2%) in Dongying City, Shandong Province, at a depth of 0-20 cm. 5 g of soil sample was added to 45 mL of sterile physiological saline and shaken thoroughly to prepare a soil suspension.

[0034] 2. Strain Isolation and Purification: The soil suspension was serially diluted and spread onto acid-producing medium (20 g / L glucose, 5 g / L peptone, 0.5 g / L yeast extract, 0.5 g / L L-cysteine, 5 g / L NaCl, 0.5 g / L K₂HPO₄, 0.01 g / L FeSO₄·7H₂O, 0.5 g / L MgSO₄·7H₂O; the solid medium was brought to a final volume with phenol red solution and the pH was adjusted to 7.0 with 0.1 mol / L NaOH; agar 20 g / L; sterilized at 113℃ for 30 min). The culture was incubated upside down at 30℃ for 5-7 days. The single colony producing the largest clear zone was selected and repeatedly streaked onto fresh acid-producing medium plates for purification until a pure culture was obtained. This strain was designated DY1.

[0035] 3. Strain identification: Morphological observation: The DY1 strain was inoculated into LB liquid medium and cultured at 30℃ and 180 r / min for 24 hours with shaking. The bacterial culture was then stained with crystal violet and observed under an optical microscope (1000×). The cells of this strain were oval or elliptical and reproduced by budding, which is typical of yeast morphology.

[0036] Molecular biological identification: Genomic DNA was extracted from strain DY1 and used as a template to amplify its ITS rDNA sequence using universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR product was purified and sequenced, and the obtained sequence was BLAST-aligned in the NCBI database. The alignment results showed that its sequence was similar to... Meyerozyma guilliermondii The similarity to the type strain of (Saccharomyces giardinis) is as high as 99.8%.

[0037] 4. Strain preservation: Based on the above morphological and molecular identification results, this strain was identified as *Saccharomyces giardi* (…). Meyerozyma guilliermondii It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 22, 2025, with accession number CGMCC No. 39011.

[0038] Example 2: Verification of the acid-producing capacity and soil pH-lowering effect of strain DY1

[0039] 1. Liquid acid production capacity test: Prepare the acid-producing liquid culture medium: glucose 20 g / L, peptone 5 g / L, yeast extract 0.5 g / L, L-cysteine ​​0.5 g / L, NaCl 5 g / L, K₂HPO₄ 0.5 g / L, FeSO₄·7H₂O 0.01 g / L, MgSO₄·7H₂O 0.5 g / L. Adjust the pH to 7.0 with 0.1 mol / L NaOH. Sterilize at 113℃ for 30 min.

[0040] The activated DY1 strain was inoculated into an Erlenmeyer flask containing 100 mL of the culture medium at an inoculation rate of 2%, with the uninoculated culture medium serving as a blank control (CK1).

[0041] The culture medium was placed in a shaker at 28℃ and 180r / min for 3 days. Samples were taken at regular intervals each day, and the pH value of the culture medium was measured using a precision pH meter.

[0042] Results: The pH value of the blank control remained stable at around 7.05 throughout the culture period. However, the pH value of the medium inoculated with DY1 began to decrease significantly after 24 hours of culture, and dropped to 4.41 by the third day, indicating that the DY1 strain has a very strong acid-producing metabolic capacity.

[0043] 2. Soil simulation experiment to reduce soil alkalinity: Take 500 g of naturally air-dried saline-alkali soil from Dongying (initial pH 9.01) and put it into a plastic cup.

[0044] Experimental group (T1): 100 mL of liquid fermentation broth of strain DY1 was added to the soil (cultured for 3 days, viable count ≥4×10⁻⁶). 8 Mix thoroughly (CFU / mL) to bring the soil moisture content to approximately 60% of field capacity.

[0045] Control group (CK2): Add an equal volume of sterile culture medium.

[0046] Both the treatment group and the control group were placed in a constant temperature incubator at 28℃ for 7 days. After the soil was mixed, 10 grams of soil was taken, 100 ml of purified water was added, and the mixture was stirred evenly. The pH value of the soil solution was then measured.

[0047] Results: The soil pH in the control group CK2 showed no significant change over 7 days (remaining between 9.00 and 9.03). However, the soil pH in the experimental group T1 significantly decreased to 7.39 on day 7. This result directly and strongly demonstrates that strain DY1 can rapidly and effectively reduce the pH of high-pH saline-alkali soils.

[0048] Example 3: High-density liquid fermentation culture of *Saccharomyces cerevisiae* strain DY1

[0049] 1. Activation of microbial strains and preparation of seed culture Primary activation: Pick a single colony of *Saccharomyces cerevisiae* DY1 (CGMCC No. 39011) from YPD slant medium (10 g / L peptone, 5 g / L yeast extract, 20 g / L glucose, 2% agar powder, natural pH) stored at 4℃, and inoculate it into a 250 mL Erlenmeyer flask containing 50 mL of YPD liquid medium (10 g / L peptone, 5 g / L yeast extract, 20 g / L glucose, natural pH).

[0050] Culture conditions: Place in a constant temperature shaker at 28℃ and 180 rpm for 36 hours to obtain activated seed culture. At this time, the OD600 value of the seed culture should reach above 2.0, and the viable count should not be less than 1×10⁻⁶. 8 CFU / mL.

[0051] 2. High-density fermentation culture Fermentation medium: An optimized low-cost, high-yield fermentation medium was used, with the following formula: molasses 50 g / L (as the main carbon source), soybean meal 15 g / L (as a nitrogen source and growth factor), potassium dihydrogen phosphate 3 g / L, magnesium sulfate 1 g / L, and a natural pH (approximately 6.5-7.0). This formula is less expensive than traditional YPD medium and is more conducive to the accumulation of yeast biomass.

[0052] Inoculation and fermentation: Under aseptic conditions, the above seed culture was transferred to a 10L fermenter containing 5L of fermentation medium at an inoculation rate of 5% (v / v).

[0053] Fermentation control parameters: Temperature: kept constant at 28±1℃.

[0054] Aeration and stirring: To ensure the dissolved oxygen required for high-density culture, the aeration rate is set to 1:1 vvm (i.e., the ratio of gas volume to fermentation liquid volume per minute is 1:1), and the stirring speed is 200-250 rpm.

[0055] pH value: No external regulation is performed during fermentation; the pH value changes naturally through the metabolism of the microorganisms themselves.

[0056] Culture time: Continuous culture for 96-120 hours (4-5 days).

[0057] 3. Fermentation process monitoring and endpoint determination Monitoring indicators: Sampling is conducted every 12 hours; monitoring results are as follows: Viable bacteria count: determined by plate count method.

[0058] Biomass (dry weight): A certain volume of fermentation broth is taken, centrifuged, washed, dried, and then weighed for calculation.

[0059] pH value: Observe the trend of pH change in the fermentation broth.

[0060] Residual sugar content: Monitored using the Fehling's reagent method or a saccharimeter to determine the carbon source consumption.

[0061] Endpoint characteristics and results: After approximately 72 hours of cultivation, the viable cell count entered a stationary phase. The fermentation endpoint was reached on day 5 (120 hours), at which point typical indicators were: Fermentation broth pH: Due to acid production by the bacteria, it drops to the range of 4.5-5.5.

[0062] viable bacteria count: ≥ 2.0 × 10⁻⁶ 9 CFU / mL (i.e., 2 billion CFU / mL).

[0063] Dry weight of bacteria: 10-15 g / L.

[0064] When the residual sugar content is consumed to below 5 g / L, it indicates that the carbon source has been basically utilized.

[0065] 4. Fermentation broth treatment Upon reaching the endpoint, proceed with processing according to the subsequent product form (liquid or powder): If preparing liquid bacterial agents, an appropriate amount of protectant (such as 2% glycerol) can be added directly and then dispensed and stored at 4°C.

[0066] If preparing a solid powder, the fermentation broth is centrifuged in a tubular centrifuge at 4°C and 8000 rpm for 10 minutes to collect the wet mycelial sludge. This wet mycelial sludge can be directly used to compound with a carrier, or after adding a freeze-drying protectant, it can be vacuum freeze-dried to produce a high-purity mycelial powder.

[0067] Example 4: Preparation of soil conditioner (powder) containing strain DY1

[0068] 1. Carrier preparation: Crush the dried soybean meal through an 80-mesh sieve and mix it evenly with powdered biochemical potassium humate at a mass ratio of 2.33:1.

[0069] 2. Mixing and Drying: The wet bacterial sludge obtained in Example 3 was mixed with the mixed carrier from Step 1 at a mass ratio of 1:9 (based on dry matter). The mixture was then dried using a conveyor-type low-temperature dryer (inlet air temperature ≤40℃) until the moisture content was below 10%.

[0070] 3. Product Specifications: The dried material is pulverized to obtain a powdered soil conditioner. Testing showed that the viable count of *Saccharomyces cerevisiae* DY1 in the finished product remained stable at 0.52 × 10⁻⁶.8 The CFU / g is above 21.3%, and the organic matter content is 21.3%, which fully meets the design requirements.

[0071] Example 5: Field Application Effect Verification – Rice Yield Increase Trial

[0072] 1. Experimental Site and Design: The experiment was conducted in a moderately saline-alkali paddy field (pH 8.8, salt content 0.4%) in Hekou District, Dongying City, Shandong Province. Two treatments were set up: T (application of the soil conditioner prepared in Example 4) and CK (application of an equal amount of a carrier mixture without the DY1 strain). Each treatment was configured with 3 replicates in a randomized block design.

[0073] 2. Application method: Apply as a base fertilizer once during land preparation before rice transplanting. The dosage is 20 kg / mu. Mix the soil conditioner with an appropriate amount of fine soil and spread it evenly on the field surface, then immediately plow it into the soil (0-15 cm soil layer).

[0074] 3. Field Management and Data Measurement: Field management measures were completely consistent across all treatments. At the rice maturity stage, the actual yield of each plot was measured, and soil samples were collected from the topsoil to determine the pH value.

[0075] 4. Results: Soil pH value: Measured at harvest time, the soil pH value of the CK treatment was 8.65, while the soil pH value of the T treatment decreased to 8.32, showing a significant improvement effect.

[0076] Yield: The average yield of rice in the CK treatment was 521.22 kg / mu. The average yield of rice in the T treatment was 549.89 kg / mu. The yield increased by 28.67 kg / mu, with an increase rate of 5.5%, and the difference was statistically significant (p<0.05).

[0077] Example 6 Field Application Effect Verification - Maize Yield Increase Trial 1. Experimental design: The experiment was conducted in a saline-alkali cornfield in the same area of ​​Dongying. The experimental design and application method were the same as in Example 5.

[0078] result: Soil pH: The soil pH at harvest time was 8.60 in the CK treatment and decreased to 8.25 in the T treatment.

[0079] Yield: The average yield of maize in the CK treatment was 507.92 kg / mu. The average yield of maize in the T treatment was 536.36 kg / mu. The yield increase was 28.44 kg / mu, with a yield increase rate of 5.6%, which was statistically significant (p<0.05).

[0080] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Yarrowia lipolytica DY1, characterized in that, The taxonomic name of the said Quillmomyces miybeii DY1 is Meyerozyma guilliermondii, The strain has been preserved in the China General Microbiological Culture Collection Center, and the preservation date is October 22, 2025, and the preservation number is CGMCC NO. 39011.

2. A soil conditioner for saline and alkaline soils, characterized by, An active ingredient of the soil conditioner comprises the Meyerozyma guilliermondii strain DY1 according to claim 1.

3. The soil conditioner for saline land according to claim 2, characterized by, The soil conditioner further comprises a carrier, which comprises soybean meal and biochemical potassium fulvate in a mass ratio of (1-3):(1-3).

4. The soil conditioner for saline land according to claim 3, characterized by, In the soil conditioner, the viable cell count of the Meyerozyma guilliermondii strain DY1 is not less than 0.5 billion CFU / g, and the organic matter content is not less than 20%.

5. A preparation method of a soil conditioner for saline-alkali soil, characterized by comprising the following steps: mixing the culture of the Meyerozyma guilliermondii strain DY1 obtained by fermentation with a carrier comprising soybean meal and biochemical potassium fulvate.

6. The method for preparing a soil conditioner for saline land according to claim 5, wherein the soil conditioner is prepared by mixing the soil conditioner of claim 1 with water. Specifically comprising the following steps: (1) fermentation culture: inoculating the Meyerozyma guilliermondii strain DY1 into a liquid culture medium for fermentation culture to obtain a high-activity bacterial body fermentation liquor or slurry; (2) carrier preparation: uniformly mixing the soybean meal and the biochemical potassium fulvate in a predetermined ratio to obtain a mixed carrier; (3) compounding and forming: uniformly mixing the bacterial body fermentation liquor or slurry obtained in step (1) and the mixed carrier obtained in step (2), and then drying at low temperature to prepare a powdery or granular product.

7. Use of the Meyerozyma guilliermondii strain DY1 or the soil conditioner according to any one of claims 2-4 in the preparation of a preparation for reducing the pH value of saline-alkali soil.

8. Use according to claim 7, wherein the compound is ###0002### The preparation is used to reduce the pH value of saline-alkali soil with an initial pH value of not less than 9.0 to below 7.

5.

9. The use according to claim 7, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The preparation is also used to improve the yield of crops planted on the saline-alkali soil; the crops are rice or corn.

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