Straw in-situ decomposition quality-improving microbial agent
By using a compound inoculant of Stenotrophomonas maltophilia and Neurospora crassa, straw is decomposed into organic matter under low temperature conditions, solving the problem of low decomposition efficiency in the in-situ straw return technology and achieving efficient straw decomposition and soil organic matter accumulation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF MICROBIOLOGY CHINESE ACAD OF SCI
- Filing Date
- 2024-11-21
- Publication Date
- 2026-05-22
AI Technical Summary
In the low-temperature conditions of Northeast China, the in-situ straw return technology has low decomposition efficiency and long decomposition cycle. Furthermore, existing microbial agents have failed to effectively promote the conversion of recalcitrant carbon into organic matter, affecting soil quality and crop growth.
A compound microbial agent consisting of Stenotrophomonas maltophilia JGDQ29-2 and Neurospora crassa N7 was used to directionally convert the organic carbon produced by straw decomposition into organic matter under low-temperature conditions, thereby increasing the straw decomposition rate and soil organic matter content.
It significantly improves straw decomposition rate and soil organic matter content under low temperature conditions, promotes the benign cycle of soil ecosystem, shortens the decomposition cycle, and improves arable land quality.
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Figure CN122071673A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of straw microbial decomposition technology. Background Technology
[0002] During the decomposition of straw, lignocellulose, the main component of straw, is converted into CO2 or CH4 by microorganisms and emitted into the atmosphere. Only a small portion of organic carbon is converted into recalcitrant substances and remains in the soil, forming soil organic matter.
[0003] Straw return to the field mainly includes different forms such as in-situ return and off-field composting followed by return. Off-field composting involves piling straw in one place, where microorganisms ferment it under high temperatures to decompose it into organic fertilizer, which is then applied back to the field. However, off-field composting has high transportation costs, and the composting process can lead to fertilizer and water loss, significant leakage, and water pollution. In-situ straw return reduces water and wind erosion, prevents soil compaction, promotes a healthy cycle in the soil ecosystem, and is convenient and low-cost, making it the most promising method for widespread application.
[0004] Northeast China, a crucial grain-producing region, suffers from low temperatures and prolonged periods of land freezing, which limit microbial growth and enzyme activity. This results in slow, lengthy, and inefficient decomposition of straw after it is returned to the field. Given the large volume of straw returned in this region, developing highly efficient, low-temperature decomposition agents that break down lignocellulose and promote organic matter formation is crucial for implementing in-situ straw return technology, preventing soil degradation, and significantly improving arable land quality. Common in-situ straw return techniques suffer from low decomposition efficiency under low-temperature conditions, impacting spring planting and crop growth. Furthermore, typical straw decomposition agents focus on rapid straw decomposition and increasing the decomposition rate, converting lignocellulose into CO2 or CH4 emissions without considering the generation and accumulation of recalcitrant carbon, thus failing to demonstrate the direct effect of microbial agents on improving soil organic matter. Summary of the Invention
[0005] In view of the above problems, the present invention provides a microbial inoculant for in-situ decomposition and quality improvement of straw, comprising Stenotrophomonas maltophilia JGDQ29-2 and Neurospora crassa N7; the preservation number of Stenotrophomonas maltophilia JGDQ29-2 is CGMCC No. 27979; the preservation number of Neurospora crassa N7 is 40738.
[0006] Furthermore, the viable count of the Stenotrophomonas maltophilia JGDQ29-2 is not less than 1 × 10⁻⁶. 9 cfu / mL.
[0007] Furthermore, the number of spores of Neurospora crassa N7 is not less than 1 × 10⁻⁶. 7 / mL.
[0008] The present invention also provides the application of the aforementioned straw in-situ decomposition and quality-improving microbial agent in the in-situ decomposition of straw and / or the enhancement of soil organic matter content.
[0009] Furthermore, the straw is corn straw or rice straw.
[0010] Furthermore, the temperature for in-situ decomposition of the straw is 4 to 25°C.
[0011] The microbial agent provided by this invention can capture and directionally convert the organic carbon produced after straw decomposition into recalcitrant carbon such as organic matter in the low-temperature environment of black soil, promote the efficient decomposition and quality improvement of straw in situ, and increase the straw decomposition rate and the organic matter content in the soil under low-temperature conditions. Attached Figure Description
[0012] Figure 1 This is a diagram of the antagonistic experiment of microbial components in the inoculant. Detailed Implementation
[0013] The technical solution of the present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the raw materials used in the present invention are all conventional commercially available products; unless otherwise specified, the methods used in the present invention are all conventional methods in the art.
[0014] Stenotrophomonas maltophilia JGDQ29-2, with accession number CGMCC No. 27979, was deposited on July 24, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0015] Neurospora crassa N7, with accession number CGMCC No. 40738, was deposited on July 24, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0016] Example 1
[0017] Antagonistic effects between microorganisms in the inoculant
[0018] The plate confrontation culture method was used to test whether there is an antagonistic effect between Neurospora crassa N7 and Stenotrophomonas maltophilia JGDQ29-2.
[0019] First, activate Neurospora crassa N7 on a PDA plate and incubate it at room temperature for one week.
[0020] After the fungus culture is complete, activate the Stenotrophomonas maltophilia JGDQ29-2 monoclonal strain one day in advance using liquid LB medium.
[0021] Take more than 10 μL of activated Stenotrophomonas maltophilia JGDQ29-2 bacterial suspension and spot it on both sides of a WA plate. Inoculate the center of the plate with Neurospora crassa N7 fungal hyphae. At the same time, set up a plate without bacterial suspension as a control. Incubate at room temperature and observe whether the diameter of fungal colonies is affected by bacterial growth compared with the control.
[0022] If the diameter of fungal colonies remains unchanged regardless of the presence or absence of bacteria, it indicates that there is no antagonistic effect between Neurosporacrassa N7 and Stenotrophomonas maltophilia JGDQ29-2; otherwise, it indicates that there is an antagonistic effect between the two.
[0023] WA medium (Waksman's agar): peptone 15 g / L, glucose 10 g / L, beef extract 3 g / L, NaCl 5 g / L, agar 15 g / L.
[0024] like Figure 1 As shown, the results of the confrontation culture on WA plates demonstrate that, compared with the control group which was inoculated with a single fungal hyphae of the test fungus in the middle of the plate, the hyphae of Neurospora crassa N7 spread to the bacterial colony and did not show any antagonistic reaction with the bacterial Stenotrophomonas maltophilia JGDQ29-2, which can be used for the development of compound bacterial agents.
[0025] Example 2
[0026] Low-temperature decomposition ability of microbial agents in straw-inorganic salt culture medium
[0027] Neurospora crassa N7 strain was activated using Vogel's agar plates and cultured at room temperature for 7 days. Spores were collected by rinsing the plates with sterile water to prepare a spore suspension. After dilution, the spore concentration was counted using a hemocytometer, and the final concentration was recorded as 1×10⁻⁶. 5 Inoculate straw-based inorganic salt medium with a concentration of 1 spore / mL. In addition, Stenotrophomonas maltophilia JGDQ29-2 was inoculated into 10 mL of LB liquid medium and cultured overnight at 37°C. 1% of the overnight culture was then inoculated into straw-based inorganic salt medium.
[0028] Inoculate with the above-mentioned Neurospora crassa N7 strain (containing no less than 1×10⁻⁶ spores). 7 ( / mL) and Stenotrophomonas maltophilia JGDQ29-2 strain (containing a viable count of not less than 1×10⁻⁶). 9 Microbial inoculum was prepared by culture medium containing cfu / mL; and cultured at 25℃ and 15℃ with shaking at 150 rpm for 21 days. After 21 days of culture, 2 mL of the culture medium was taken into a sterile centrifuge tube and centrifuged at 12000 rpm for 5 min. The supernatant was the crude enzyme solution, and the crude enzyme solution was used to determine the activity of laccase.
[0029] The method for preparing the straw inorganic salt culture medium is as follows: first, cut the corn straw into straw segments about 2cm long, then cut each straw segment into four pieces, weigh 2g of the cut straw pieces, add 10mL of 5×M9 solution and 38mL of distilled water into a 150mL shake flask, and autoclave at 115℃ for 20min. After cooling, add the following filtered and sterilized solution: 500 μL NaNO3 (2.5 g / 10 mL), 500 μL CaCl2 (0.11 g / 10 mL), 100 μL MgSO4·7H2O (2.465 g / 10 mL), 50 μL FeCl3·6H2O (0.162 g / 10 mL), 50 μL CuSO4·5H2O (2.496 g / 10 mL), 50 μL ZnSO4·7H2O (0.288 g / 10 mL), and 50 μL MnSO4·H2O (0.34 g / 10 mL).
[0030] 5×M9 solution: 64g Na2HPO4·7H2O, 15g KH2PO4, 2.5g NaCl, 5g NH4Cl, dissolve in water and bring to a final volume of 1000mL, then adjust the pH to 7.2.
[0031] Laccase activity assay:
[0032] Take 25 μL of crude enzyme solution and add it to 160 μL of 0.2 mol / L sodium acetate buffer (pH = 4.5). Start the reaction with 15 μL of 20 mM ABTS. Measure the absorbance at 420 nm and record the change in absorbance over 3 minutes.
[0033] Enzyme activity unit definition: The amount of enzyme required to oxidize 1 μmol / L of substrate ABTS per minute per milliliter of crude enzyme solution is defined as one enzyme activity unit.
[0034] There was no significant difference in laccase activity and decomposition rate between 25℃ and 15℃, indicating that the utilization efficiency of straw by the applied microbial agent was not affected by low temperature. Under the low temperature condition of 15℃, the straw decomposition rate of the microbial agent after 21 days of shake flask culture can reach more than 40%.
[0035] Table 1. Determination of laccase activity in crude fermentation solution of inoculant using straw as the sole carbon source.
[0036] 25℃ 15℃ Laccase activity (U / mL) 0.05±0.01 0.0385±0.015
[0037] Example 3
[0038] Soil pot experiment on in-situ decomposition of straw
[0039] Spores of Neurospora crassa N7 were inoculated into a 250 mL shake flask containing 50 mL of Vogel's liquid medium and incubated on a shaker at 25 °C and 150 rpm for 48 h. At least 15 mL of the bacterial culture was then inoculated into 500 mL of Vogel's liquid medium and incubated on a shaker at 25 °C and 150 rpm for 48 h.
[0040] Stenotrophomonas maltophilia JGDQ29-2 was inoculated into 10 mL of LB liquid medium and cultured overnight at 37°C. The seed culture was then transferred to 500 mL of LB liquid medium at a 1% inoculation rate and cultured on a shaker at 37°C and 200 rpm for 12 h.
[0041] The bacterial agent contains 1×10⁻⁶ N7 of Neurospora crassa. 7 1 spore / cell, corresponding to an inoculum size of 1×10⁶ for Stenotrophomonas maltophilia JGDQ29-2. 8 The two strains of bacterial sludge were mixed evenly according to different treatment groups, and distilled water was added to make up to 50 mL to obtain the bacterial solution to be applied.
[0042] Garden soil was sieved through a 4-mesh sieve to remove stones and other debris. 10g of straw was weighed, mixed thoroughly with 50mL of prepared bacterial solution, and then combined with 1.8kg of garden soil. The mixture was then placed in a box measuring 11.8cm x 12.6cm x 15cm. The box was placed in an indoor environment (4-8℃) or at room temperature, and watered regularly to maintain soil moisture at no less than 40%. The control group (CK) used 50mL of sterile water to mix with the straw. After 30 days of incubation at room temperature, the soil in the box was sieved through a 4-mesh sieve to remove undecomposed straw. The removed straw was rinsed with 80mL of distilled water, dried in a 60℃ oven, and the straw decomposition rate was measured. A 10g sample of soil from the box was taken using a five-point sampling method, sieved through a 100-mesh sieve, and analyzed for organic matter content using a potassium dichromate oxidation-spectrophotometric method.
[0043] The method for determining the straw decomposition rate is as follows: After washing the straw using the above treatment method, drying it at 60℃, and weighing the remaining straw, the decomposition rate is calculated using the following formula:
[0044]
[0045] Where W0 is the original dry weight of straw, and W1 is the dry weight of straw after decomposition.
[0046] The method for determining the organic matter content is as follows: After air-drying the soil sample, pass it through a 0.25 mm sieve. Weigh a certain amount of soil sample into a test tube, add 10 mL of 0.4 mol / L potassium dichromate-sulfuric acid solution and shake well. Place a glass funnel at the mouth of the test tube, insert it into an iron wire furnace, and place it in oil at 170-180℃. Let the solution in the test tube boil for 5 minutes. After cooling, transfer the liquid and soil sample in the test tube to an Erlenmeyer flask. Also transfer the washing solution used to clean the test tube and funnel to the Erlenmeyer flask, bringing the final volume to 50-60 mL. Add 3 drops of o-phenanthroline indicator and titrate the remaining potassium dichromate with ferrous sulfate solution. Take about 0.2 g of ignited pumice powder or soil as a blank test. The formula for calculating soil organic matter is as follows:
[0047]
[0048] In the formula, SOM is the mass fraction of soil organic matter (g / kg); c is the concentration of ferrous sulfate standard solution (mol / L); V0 is the volume of ferrous sulfate standard solution consumed in the blank test (mL); V is the volume of ferrous sulfate standard solution consumed in the sample test (mL); 0.003 is the millimolecular mass of 1 / 4 carbon atom (g); 1.10 is the oxidation correction coefficient; m is the mass of the dried sample (g); 1.724 is the conversion factor of organic matter to organic carbon; and 1000 is the coefficient for converting the result to content per kilogram.
[0049] Test results in a soil pot experiment under low-temperature conditions (4–8℃) showed that the in-situ decomposition rate of straw in the control group without inoculant application was 10.33±1.96%, while the decomposition rate in the experimental group with inoculant application reached 41.65±3.02%, an increase of approximately four times. After 30 days of in-situ decomposition of straw, the total organic matter (SOM) content in the control group was 1.86±0.16 g / kg, while that in the experimental group was 3.00±0.40 g / kg, representing a 61.1% increase in soil organic matter content. Soil pot experiments verified that the straw decomposition carbon-sinking inoculant of this invention can effectively improve straw decomposition efficiency and soil organic matter content under low-temperature conditions (4–8℃), promoting the accumulation of organic matter in the soil.
[0050] Table 2 Evaluation of the effect of microbial agents on in-situ decomposition and quality improvement of straw under low temperature (4-8℃) conditions in soil.
[0051] control group experimental group Decomposition rate (%) 10.33±1.96 41.65±3.02 Organic matter content (g / Kg) 1.86±0.16 3.00±0.40
[0052] Test results in a soil pot experiment under laboratory temperature conditions showed that the in-situ decomposition rate of straw in the control group without inoculant application was 58.53±6.37%, while the decomposition rate in the experimental group with inoculant application reached 87.30±4.51%, an increase of 49.2%. After 30 days of in-situ decomposition, the total organic matter content of the control group was 2.28±0.34 g / kg, while that of the experimental group was 3.26±0.90 g / kg, an increase of 43.2%. Soil pot experiments verified that the straw decomposition and carbon-collecting inoculant of this invention can effectively improve straw decomposition efficiency and soil organic matter content, promoting the accumulation of organic matter in the soil.
[0053] Table 3 Evaluation of the effect of microbial agents on in-situ decomposition and quality improvement of straw in soil under room temperature conditions.
[0054] control group experimental group Decomposition rate (%) 58.53±6.37 87.30±4.51 Organic matter content (g / Kg) 2.28±0.34 3.26±0.90
[0055] Example 4
[0056] In-situ decomposition of corn straw by microbial agents
[0057] To accurately determine the in-situ decomposition performance of corn stalks after returning them to the field, this invention employs a nylon mesh bag method to measure the in-situ decomposition of corn stalks. Experimental and control groups were placed in cornfields during the spring planting season. A 40×60cm mesh bag was used. 40g of dry corn stalks were mixed with 200mL of experimental microbial agent and soaked for 30 minutes. This mixture was then stirred with a certain amount of soil sample, placed in the mesh bag, and positioned in the cornfield. The experimental microbial agent contained *Neurospora crassa* strain N7 (containing at least 1×10⁻⁶ spores). 7 ( / mL) and Stenotrophomonas maltophilia JGDQ29-2 strain (containing a viable count of not less than 1×10⁻⁶). 9A composite microbial culture medium (cfu / mL) was prepared. The control group used 200 mL of sterile water mixed with straw. The experimental results were consistent with those described in Example 3.
[0058] In cornfields in Northeast China, after two months of experiments in May and June, the decomposition rate of total corn stalks in nylon mesh bags was measured. The decomposition rate of the control group without microbial agent application was 62.4±6.7%, while the decomposition rate of the experimental group with microbial agent application was 72.3±5.9%, an increase of 15.9%. Soil samples were taken from the nylon mesh bags using the five-point sampling method. The organic matter content of the control group was 6.9±0.9%. After the straw was decomposed in situ with the microbial agent, organic matter accumulated, reaching 8.1±0.5%, an increase of 17.7%.
[0059] Example 5
[0060] The in-situ decomposition of rice straw in paddy fields using the invented microbial agent
[0061] To accurately determine the in-situ decomposition performance of rice straw, this invention employs a PVC pipe method. A perforated PVC pipe with a diameter of 12-16 cm, a length of 23-25 cm, and a thickness of 3-5 mm is selected. 34 g of rice straw is weighed and mixed thoroughly with 170 mL of microbial agent, then mixed with a certain amount of soil sample. The mixture is placed inside the PVC pipe, covered with a mesh bag, and positioned in the field. The experimental microbial agent contains *Neurospora crassa* strain N7 (containing at least 1 × 10⁻⁶ spores). 7 ( / mL) and Stenotrophomonas maltophilia JGDQ29-2 strain (containing a viable count of not less than 1×10⁻⁶). 9 The experimental group used a compound microbial culture medium (cfu / mL). The control group used 170 mL of sterile water mixed with straw. Both groups were introduced into rice paddy fields during the spring plowing season. Nine parallel groups were designed for each group. Sampling was carried out regularly to determine the straw decomposition rate and soil organic matter content, and to evaluate the in-situ decomposition effect of the microbial agent on rice straw and the soil improvement effect.
[0062] In rice paddies in Northeast China, after two months of experiments in May and June, the decomposition rate of total rice straw in PVC pipes was measured. The decomposition rate of the control group without microbial agents was 47.9±5.0%, while the decomposition rate of the experimental group with microbial agents was 62.5±4.2%, an increase of 30.6%. Soil samples were taken from the PVC pipes using the five-point sampling method. The organic matter content of the control group was 8.5±1.6%. After the straw was decomposed in situ with microbial agents, organic matter accumulated, reaching 9.1±0.2%, an increase of 7.3%.
[0063] In summary, the invented microbial agent promoted straw decomposition and increased soil organic matter during field experiments of in-situ return of corn and rice straw, demonstrating soil improvement effects. Furthermore, comparative analysis of microbial diversity showed that the microorganisms in the agent exhibited good colonization ability in both corn and rice fields, further indicating that the increased decomposition efficiency and organic matter content in the experimental groups are directly related to the functional microorganisms in the agent.
Claims
1. A microbial inoculant for in-situ decomposition and quality improvement of straw, characterized by: Including Stenotrophomonas maltophilia JGDQ29-2 and Neurospora crassa N7; The accession number of Stenotrophomonas maltophilia JGDQ29-2 is CGMCC No. 27979; The accession number for Neurospora crassa N7 is 40738.
2. The straw in-situ decomposition and quality-improving microbial agent according to claim 1, characterized in that, The viable count of the Stenotrophomonas maltophilia JGDQ29-2 strain is not less than 1 × 10⁻⁶. 9 cfu / mL.
3. The straw in-situ decomposition and quality-improving microbial agent according to claim 1, characterized in that, The number of spores of Neurospora crassa N7 is not less than 1 × 10⁻⁶. 7 / mL.
4. The application of the straw in-situ decomposition and quality improvement microbial agent according to any one of claims 1 to 3 in the in-situ decomposition of straw and / or the improvement of soil organic matter content.
5. The application of the straw in-situ decomposition and quality-improving microbial agent according to any one of claims 1 to 3 in the in-situ decomposition of straw and / or soil improvement, characterized in that, The straw is either corn straw or rice straw.
6. The application of the straw in-situ decomposition and quality-improving microbial agent according to any one of claims 1 to 3 in the in-situ decomposition of straw and / or soil improvement, characterized in that, The temperature for in-situ decomposition of the straw is 4 to 25°C.