White soil straw-degrading bacteria and their application in corn straw return to the field
By screening and combining four actinomycete strains, a compound microbial agent was prepared, which solved the problem of slow degradation of corn straw in alkaline soil areas, significantly improved the straw degradation rate and soil fertility, and realized the resource utilization of waste.
Patent Information
- Application Number
- CN202610100312.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2025-01-27
- Filing Date
- 2026-01-26
- Publication Date
- 2026-05-26
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microorganisms, and more particularly to straw-degrading bacteria in alkaline soil and their application in returning corn straw to the field. Background Technology
[0002] Albic soil is an important soil type for grain production. It is formed on slightly sloping hills under forest and meadow vegetation in temperate semi-humid and humid regions, on a light-colored topsoil and a clayey bottom, through processes such as limonization. It consists of a dark humus surface layer (black soil layer), a grayish-white subsurface layer (limonite layer), and a dark brown clayey sedimentary layer. Therefore, improving low-yield albic soil is of significant strategic importance for ensuring food security. Returning corn stalks to the field is a common method to improve soil fertility. However, due to the cold climate in albic soil areas, the degradation rate of directly returned stalks is slow, with an annual degradation rate of only about 40%. Therefore, research is needed on highly efficient straw-degrading bacteria for albic soil. Summary of the Invention
[0003] The purpose of this invention is to provide a highly efficient corn stalk degrading bacterium, namely the actinomycete Streptomyces sp. GS9 (accession number GDMCC No: 65857).
[0004] The purpose of this invention is to provide a highly efficient corn stalk degrading bacterium, namely the actinomycete Streptomyces canus MD31 (accession number GDMCC No: 65843).
[0005] The purpose of this invention is to provide a highly efficient corn stalk degrading bacterium, namely the actinomycete Streptomyces fagopyri MD55 (accession number GDMCC No: 65844).
[0006] The purpose of this invention is to provide a highly efficient corn stalk degrading bacterium, namely the actinomycete Streptomyces cinnabarigriseus MD63 (accession number GDMCC No: 65845).
[0007] The purpose of this invention is to provide a highly efficient compound microbial agent for degrading corn stalks, comprising a combination of Streptomyces sp. GS9 (GDMCC No.: 65857), Streptomyces canus MD31 (GDMCC No.: 65843), Streptomyces fagopyri MD55 (GDMCC No.: 65844), and Streptomyces cinnabarigriseus MD63 (GDMCC No.: 65845).
[0008] In a preferred embodiment of the present invention, the method for preparing the compound microbial agent is to prepare each actinomycete in the compound microbial agent into a strain with an effective viable count of not less than 10. 9 A bacterial suspension of 100 cells / mL is prepared by mixing the individual bacterial suspensions in equal volumes to obtain a compound bacterial agent. Preferably, the effective viable count of the compound bacterial agent is not less than 10. 9 per ml.
[0009] In a preferred embodiment of the present invention, the method for preparing the actinomycete culture is as follows: The actinomycete strain is streaked onto ISP 2 solid medium and cultured at 28-30℃ for 5-7 days. After sporulation, the spores are washed off with sterile water, and then inoculated into ISP 2 liquid medium at an inoculation rate of 3-5%. The medium is then cultured at 30-40℃ for 5-10 days under a shaker at 180-210 rpm, yielding an effective viable count of not less than 10⁻⁶. 9 The bacterial suspension with a count of CFU / mL is obtained, preferably with an effective viable count of not less than 10 CFU / mL. 9 Add the actinomycete culture at a concentration of 3-5% per mL to the fermentation medium and culture at 30-40℃ for 7-10 days at 180-210 rpm.
[0010] In a preferred embodiment of the present invention, the ISP 2 solid culture medium is composed of 0.4% glucose, 1% malt extract, 0.4% yeast extract, and 2% agar, with a pH of 7.0-7.4.
[0011] In a preferred embodiment of the present invention, the ISP 2 liquid culture medium is formulated as follows: 0.4% glucose, 1% malt extract, 0.4% yeast extract, pH 7.0-7.4.
[0012] In a preferred embodiment of the present invention, the fermentation culture medium is composed of: 0.3% yeast extract, 0.3% dipotassium hydrogen phosphate, 0.03% magnesium sulfate, 0.01% ferrous sulfate, 0.04% zinc sulfate, 0.01% calcium chloride and 0.0015% Tween 20.
[0013] Another objective of this invention is to provide the application of the highly efficient corn straw degrading bacteria or compound microbial agent described herein in the return of corn straw to the field.
[0014] In a preferred embodiment of the present invention, the specific steps of the application are as follows: corn stalks inoculated with degrading bacteria or compound bacterial agents are turned over and buried in the soil at a depth of 20-30cm 10-15 days before corn sowing.
[0015] In a preferred embodiment of the present invention, the effective viable count of the corn stalk degrading bacteria or the compound microbial agent is not less than 10. 9 per ml.
[0016] In a preferred embodiment of the present invention, the inoculation amount of the corn stalk degrading bacteria or compound microbial agent on the corn stalk is 3-5%.
[0017] In a preferred embodiment of the present invention, the corn stalks are flattened and cut into 1-5cm segments before being inoculated with the compound microbial agent, and then dried at 70-80℃.
[0018] In a preferred embodiment of the present invention, the soil is albic soil.
[0019] In a preferred embodiment of the present invention, the soil is alkaline soil, and the soil at a depth of 20-30cm is an alkaline soil alkaline layer.
[0020] In a preferred embodiment of the present invention, the organic matter content of the white clay white clay layer is less than 12 g / kg.
[0021] In a preferred embodiment of the present invention, the total nitrogen content of the white clay white clay layer is less than 1 g / kg.
[0022] In a preferred embodiment of the present invention, the total phosphorus content of the white clay white clay layer is less than 0.5 g / kg.
[0023] In a preferred embodiment of the present invention, the total potassium content of the white clay white clay layer is less than 7 g / kg.
[0024] In a preferred embodiment of the present invention, the temperature of the white clay layer is 1-25℃.
[0025] In a preferred embodiment of the present invention, the pH value of the white clay white clay layer is 5-11.
[0026] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; the remainder is weight / weight percentage.
[0027] Unless otherwise stated, the present invention is tested using the following methods:
[0028] (1) The determination of major components such as cellulose, lignin, hemicellulose, and ash was carried out in accordance with the Van der Waals washing method.
[0029] (2) Cellulose degradation rate (%) = (Cellulose amount before treatment - Cellulose amount after treatment) / Cellulose amount before treatment × 100%;
[0030] Hemicellulose degradation rate (%) = (hemicellulose amount before treatment - hemicellulose amount after treatment) / hemicellulose amount before treatment × 100%;
[0031] Lignin degradation rate (%) = (Lignin content before treatment - Lignin content after treatment) / Lignin content before treatment × 100%.
[0032] Straw degradation rate (%) = (straw weight before returning to the field - residual straw weight after returning to the field) / straw weight before returning to the field × 100%.
[0033] (3) Methods for detecting soil components
[0034] Organic matter (SOM): Methods in "Soil Agrochemical Analysis (Third Edition)" (edited by Bao Shidan, published by China Agriculture Press in 2000, pp. 34-35); Total nitrogen (TN) was determined according to the LY / T 1228-2015 standard ("Determination of Nitrogen in Forest Soils"); Total phosphorus (TP) and total potassium (TK) were determined according to the HJ 832-2017 standard ("Digestion of Total Metal Elements in Soils and Sediments by Microwave Digestion / Inductively Coupled Plasma Mass Spectrometry"); Soil moisture content: water meter.
[0035] Compared with the prior art, the present invention has the following beneficial technical effects:
[0036] 1. The four corn straw-degrading bacteria strains screened in this invention have strong degradation capabilities for cellulose, hemicellulose, and lignin. The compound microbial agent obtained by combining these four strains exhibits higher straw degradation efficiency than a single strain. The degrading bacteria or compound microbial agent can tolerate low and high temperatures (10-40℃) and complex acidic and alkaline environments (pH tolerance range 5-11), making it suitable for the complex environment of returning straw to alkaline soils and significantly improving the straw degradation rate.
[0037] 2. This invention realizes the resource utilization of waste, is easy to operate, has low cost, and is suitable for industrial production. Attached Figure Description
[0038] Figure 1 The invention relates to the degradation effect of highly efficient corn stalk degrading bacteria on corn stalks;
[0039] Figure 2 Field trial results of the high-efficiency corn stalk degradation compound microbial agent of this invention;
[0040] Figure 3 The present invention relates to the effect of a highly efficient corn stalk degradation compound microbial agent on improving soil fertility. Detailed Implementation
[0041] The present invention will be further described below with reference to the embodiments.
[0042] The ISP 2 solid culture medium consists of 0.4% glucose, 1% malt extract, 0.4% yeast extract, and 2% agar, with a pH of 7.0-7.4.
[0043] The ISP 2 liquid culture medium is composed of 0.4% glucose, 1% malt extract, and 0.4% yeast extract, with a pH of 7.0-7.4.
[0044] The fermentation medium consists of: 0.3% yeast extract, 0.3% dipotassium hydrogen phosphate, 0.03% magnesium sulfate, 0.01% ferrous sulfate, 0.04% zinc sulfate, 0.01% calcium chloride, and 0.0015% Tween 20.
[0045] Example 1 Screening of highly efficient corn straw degrading bacteria
[0046] 1. Screening of actinomycetes with straw lignin degradation capabilities
[0047] Five sampling points were selected from each of the following soil samples: albic soil layer from the 852 Farm in Baoqing County, Shuangyashan City, Heilongjiang Province; soil under straw piles from the albic soil layer; and soil under straw piles from Beiji Town, Mohe City, Heilongjiang Province. From each sampling point, five points were randomly selected, and the samples were merged and processed. Soil samples were air-dried in a cool place, ground into a fine powder in a mortar, placed in Erlenmeyer flasks, and a suitable amount of sterile glass beads were added. The flasks were then shaken at 180 rpm for 0.5 hours. The powder was then diluted and spread onto ISP2 solid medium for separation. The cultures were incubated upside down in a 28°C incubator. The strains were purified and passaged 3-5 times. Afterward, Congo red staining was used, and strains producing large transparent zones were selected for subsequent filter paper decomposition analysis. The filter paper decomposition results showed that strains GS9, MD31, MD55, and MD63 exhibited excellent performance (+++), with the filter paper losing its original shape and almost decomposing into a paste.
[0048] Molecular biological identification was performed on the four active strains, and the specific results are as follows: strain GS9 belongs to the genus Streptomyces sp., strain MD31 belongs to the genus Streptomyces canus, strain MD55 belongs to the genus Streptomyces fagopyri, and strain MD63 belongs to the genus Streptomyces cinnabarigriseus.
[0049] Strain 1 was named Streptomyces sp. GS9, with accession number GDMCC No: 65857. The depository is Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is January 24, 2025.
[0050] Strain 2 was named Streptomyces canus MD31. Its accession number is GDMCC No: 65843. The depositary institution is Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is January 22, 2025.
[0051] Strain 3 was named Streptomyces fagopyri MD55. Its accession number is GDMCC No:65844. The depositary institution is Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is January 22, 2025.
[0052] Strain 4 was named Streptomyces cinnabarigriseus MD63. Its accession number is GDMCC No:65845. The depositary institution is Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is January 22, 2025.
[0053] 2. Study on antagonistic effects among strains
[0054] To determine the antagonistic effect between strains, strains 1, 2, 3, and 4 were streaked onto ISP2 solid medium and cultured at 30°C for 5 days. The colony growth was then observed, and the results showed that the four strains did not have any antagonistic effect and could be used to construct a mixed bacterial community.
[0055] Experimental Example 1: Study on the degradation effect of the highly efficient corn stalk degrading bacteria of the present invention on corn stalks
[0056] The corn stalks were collected from the Heilongjiang Academy of Agricultural Sciences Science and Technology Park. The composition of the corn stalks is shown in Table 1. The flattened and cut corn stalks into 1cm pieces were dried at 80℃ and set aside for later use.
[0057] Table 1
[0058] Element content(%) Cellulose 39.7 hemicellulose 29.1 Lignin 8.6 Other substances 22.6
[0059] 1. Preparation of bacterial culture
[0060] Preparation of GS9 bacterial suspension: GS9 strain, stored at -80℃, was streaked onto ISP 2 solid medium and incubated at 28℃ for 5 days. After sporulation, the spores were washed off with sterile water and then inoculated into ISP 2 liquid medium at a 3% inoculation rate. The medium was then incubated at 30℃ for 10 days under a shaker at 180 rpm to obtain GS9 bacterial suspension. The viable count in the suspension was not less than 10⁻⁶. 9 per mL.
[0061] Preparation of MD31 bacterial suspension: MD31 strain, stored at -80℃, was streaked onto ISP 2 solid medium and incubated at 28℃ for 5 days. After sporulation, the spores were washed off with sterile water and then inoculated into ISP 2 liquid medium at a 3% inoculum. The medium was then incubated at 30℃ for 10 days under a shaker at 180 rpm to obtain MD31 bacterial suspension. The effective viable count in the suspension was not less than 10. 9 per mL.
[0062] Preparation of MD55 bacterial suspension: MD55 strain stored at -80℃ was streaked onto ISP 2 solid medium and incubated at 28℃ for 5 days. After sporulation, the spores were washed off with sterile water, and then inoculated into ISP 2 liquid medium at a 3% inoculum. The medium was incubated at 30℃ for 10 days under a shaker at 180 rpm to obtain MD55 bacterial suspension. The effective viable count in the suspension was not less than 10. 9 per mL.
[0063] Preparation of MD63 bacterial suspension: MD63 strain, stored at -80℃, was streaked onto ISP 2 solid medium and incubated at 28℃ for 5 days. After sporulation, the spores were washed off with sterile water, and then inoculated into ISP 2 liquid medium at a 3% inoculum. The medium was then incubated at 30℃ for 10 days under a shaker at 180 rpm to obtain MD63 bacterial suspension. The effective viable count in the suspension was not less than 10⁻⁶. 9 per mL.
[0064] Preparation of compound microbial agent: GS9 bacterial solution, MD31 bacterial solution, MD55 bacterial solution, and MD63 bacterial solution are mixed in equal volume ratios to obtain a compound microbial agent. The effective viable count in each bacterial solution is not less than 10. 9 per mL.
[0065] 3. Degradation test study
[0066] GS9, MD31, MD55, MD63 bacterial solutions, and a compound bacterial agent were added to 150 ml of fermentation medium at an inoculum rate of 3%. 3 g of pulverized 40-mesh corn stalks were added, and the pH was adjusted to 7.0. The mixture was incubated at 30℃ for 7 days at 210 rpm / min. The fermented corn stalks were then collected, dried, and the contents of cellulose, hemicellulose, and lignin in the dried product were determined. The degradation rate was calculated. (See attached table). Figure 1 .
[0067] Experiment Example 2: Field Trial of High-Efficiency Corn Straw Degrading Compound Microbial Agent
[0068] 1. Preparation of compound microbial agents for field trials
[0069] The GS9, MD31, MD55, and MD63 bacterial cultures prepared in Experiment Example 1 were added to 150 ml of fermentation medium at an inoculation rate of 3%, the pH was adjusted to 7.0, and the cultures were incubated at 30°C for 7 days at 210 rpm to obtain expanded GS9, MD31, MD55, and MD63 bacterial cultures.
[0070] The expanded culture solutions of GS9, MD31, MD55, and MD63 were mixed in equal volumes to obtain a compound microbial agent for field trials.
[0071] 2. Field trials
[0072] The field experiment was conducted in the Heilongjiang Academy of Agricultural Sciences Science and Technology Park, located in a cold-temperate continental climate zone with annual precipitation between 500 and 550 mm, a frost-free period of approximately 140 days, and an accumulated temperature ≥10℃ of approximately 2700℃. During the experiment, the soil temperature at a depth of 20 cm ranged from 1.55 to 24.22℃. The experimental field was divided into four plots, each with a soil area of 7.14 m². 2 The soil samples were collected from the Heilongjiang Academy of Agricultural Sciences Science and Technology Park, and the soil pH value was 6.66.
[0073] The corn stalks were collected from the Heilongjiang Academy of Agricultural Sciences Science and Technology Park. The composition of the corn stalks is shown in Table 1. The flattened and cut corn stalks into 1cm pieces were dried at 80℃ and set aside for later use.
[0074] Set up 4 groups:
[0075] Group A: Take 600g of soil, add 24ml of water and mix well;
[0076] Group B: Take 40g of corn stalks, add 24ml of water and mix well, then add it to 600g of soil and mix well.
[0077] Group C: Take 40g of corn stalks and 0.18g of Wobao straw composting agent (purchased from Henan Wobao Biotechnology Co., Ltd., with an effective live bacteria count of not less than 100 million / g), add 24ml of water and stir well, then add to 600g of soil and mix well.
[0078] Group D: Take 1.2 ml of the compound microbial agent for field trials prepared in step 1, dilute it with 22.8 ml of water, mix it with 40 g of straw, and then add it to 600 g of soil and mix well.
[0079] Water was added to each group to adjust the soil moisture content to 60%. A nylon mesh bag method was used; the nylon mesh bags were 34cm × 36cm in size and had an 80-mesh aperture. Samples from groups A, B, C, and D were placed in the nylon mesh bags, sealed with nylon thread, and buried in a 20cm layer of alkaline soil. Straw and soil samples were collected every 28 days for analysis.
[0080] Under the condition of direct return of corn stalks to the field, the degradation rates of corn stalks buried in albic soil for 28 days, 56 days, 84 days, 112 days, and 140 days are as follows: Figure 2 When corn stalks were directly returned to the field for one quarter, the degradation rate of stalks in group D was significantly higher than that in groups C and B.
[0081] The results of soil organic matter (SOM), total nitrogen (TN), total phosphorus (TP), and total potassium (TK) tests in each group are shown below. Figure 3 Compared with the soil control group, the addition of the compound microbial agent of the present invention significantly increased the levels of soil organic matter, total nitrogen, total phosphorus and total potassium.
[0082] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.
Claims
1. A highly efficient corn straw degrading bacterium, namely the actinomycete Streptomyces sp. GS9 (accession number GDMCC No:65857).
2. A highly efficient corn stalk degrading bacterium, namely the actinomycete Streptomyces canus MD31 (accession number GDMCC No:65843).
3. A highly efficient corn straw degrading bacterium, namely the actinomycete Streptomyces fagopyri MD55 (accession number GDMCCNo: 65844).
4. A highly efficient corn straw degrading bacterium, namely the actinomycete Streptomyces cinnabarigriseus MD63 (accession number GDMCC No: 65845).
5. A highly efficient corn stalk degradation compound microbial agent, comprising a combination of Streptomyces sp. GS9 (accession number GDMCC No: 65857), Streptomyces canus MD31 (accession number GDMCC No: 65843), Streptomyces fagopyri MD55 (accession number GDMCC No: 65844), and Streptomyces cinnabarigriseus MD63 (accession number GDMCC No: 65845).
6. The compound microbial agent as described in claim 5, wherein the preparation method of the compound microbial agent is to prepare each of the four actinomycetes in the compound microbial agent into individuals with an effective viable count of not less than 10. 9 A compound bacterial agent is prepared by mixing the various bacterial solutions at a ratio of 10 / mL with the bacterial culture of actinomycetes. Preferably, the compound bacterial agent has an effective viable count of not less than 10. 9 per ml.
7. The compound bacterial agent as described in claim 6, wherein the method for preparing the actinomycete bacterial solution is as follows: The actinomycete strain is streaked onto ISP2 solid medium and cultured at 28-30℃ for 5-7 days. After sporulation, the spores are washed off with sterile water, and then inoculated into ISP2 liquid medium at an inoculation rate of 3-5%. The solution is then cultured at 30-40℃ for 5-10 days under a shaker at 180-200 rpm to obtain the bacterial solution, wherein the effective viable count in the bacterial solution is not less than 10. 9 per mL.
8. The application of the highly efficient corn straw degrading bacteria as described in any one of claims 1-4 or the compound microbial agent as described in claim 5 in the return of corn straw to the field.
9. The application as described in claim 8, wherein the specific steps of the application are as follows: corn stalks inoculated with degrading bacteria or compound microbial agents are turned over and buried in the soil at a depth of 20-30cm 10-15 days before corn sowing.
10. The application as described in any one of claims 8-9, wherein the soil is albic soil, and the soil at a depth of 20-30cm is an albic soil albic layer.