Functional strain for degrading straws, composite flora and application of functional strain and composite flora

Abstract

The invention provides a functional strain for degrading straws, a composite flora and application of the functional strain and the composite flora, and belongs to the technical field of microorganisms. According to the invention, three cellulose degradation strains with excellent phenotypes are screened from soil of a test field subjected to long-term straw returning from Shenyang Ecological Experimental Station Station (41 degrees 31 'N, 123 degrees 24' E) of Chinese Academy of Sciences in black soil areas in Northeast China, and are finally identified as Bacillus cereus CN8, Bacillus cereus CN10 and Bacillus thuringiensis CN16. Experiments show that the combined application of the functional strains disclosed by the invention can efficiently degrade the straws under a medium-low temperature (15 DEG C) liquid culture condition, and has great significance in improving the utilization rate of the straws and improving the soil environment.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a functional bacterial strain for degrading straw, a composite bacterial community and applications thereof. Background Art

[0002] Crop straw is an important biomass resource. Directly returning straw to the field can make straw resources fertilizer, increase soil organic matter content, and improve soil fertility. However, straw is a complex structure formed by the interweaving of cellulose, hemicellulose, and lignin. Therefore, the complex structure makes it very difficult to degrade. Under natural conditions, the decomposition rate of straw is slow. Returning a large amount of straw to the field will affect the rooting and survival of crops and cause inconvenience in production management. Therefore, the development of straw rapid decomposition technology is an urgent issue to be solved in production practice.

[0003] Straw-degrading microorganisms are an important driving force in the straw degradation process. However, most commercially available straw-degrading microbial agents are adapted to medium-high temperatures (15-50°C). Low temperatures significantly reduce the activity of microorganisms and inhibit their metabolic activities, including enzyme activity and the speed of biochemical reactions, thereby reducing the straw degradation rate. Therefore, the development of efficient straw-degrading bacteria suitable for medium-low temperature conditions is of great significance for improving straw utilization. Summary of the invention

[0004] The purpose of the present invention is to provide a functional strain for degrading corn straw, a composite bacterial community and applications thereof. The functional strain of the present invention is an efficient corn straw degrading bacterium under medium and low temperature (15°C) conditions, which is of great significance for improving the utilization rate of corn straw.

[0005] The present invention provides a functional strain for degrading straw, which is Bacillus cereus ( Bacillus cereus ) CN8; the preservation number of the Bacillus cereus CN8 is CGMCC No.30118.

[0006] The present invention also provides a composite bacterial community for degrading straw, including Bacillus cereus ( Bacillus cereus )CN8, Bacillus cereus ( Bacillus cereus ) CN10 and Bacillus thuringiensis ( Bacillus thuringiensis ) CN16; the preservation number of the Bacillus cereus CN8 is CGMCC No.30118; the preservation number of the Bacillus cereus CN10 is CGMCC No.30119; the preservation number of the Bacillus thuringiensis CN16 is CGMCC No.30120.

[0007] Preferably, the ratio of the effective viable counts of Bacillus cereus CN8, Bacillus cereus CN10 and Bacillus thuringiensis CN16 is 1:1:1.

[0008] The present invention also provides a bacterial agent for degrading straw, comprising the functional strain or the composite bacterial community described in the above scheme.

[0009] Preferably, the OD of the functional strain or composite bacterial community in the bacterial agent is 600nm It is 0.5~0.7.

[0010] The present invention also provides the use of the functional strain, the composite bacterial community or the bacterial agent described in the above scheme in degrading straw and / or degrading cellulose.

[0011] Preferably, the degradation environment temperature is 15-28°C.

[0012] The present invention also provides a method for degrading straw, comprising the following steps: after returning the straw to the field, applying the functional strain, the composite bacterial community or the bacterial agent described in the above scheme to the soil.

[0013] Preferably, before the applying, the method further comprises adjusting the carbon-nitrogen ratio of the soil to 25:1.

[0014] Preferably, the ambient temperature during the application is 15-28°C.

[0015] The present invention provides a functional strain for degrading straw, which is Bacillus cereus ( Bacillus cereus )CN8; the deposit number of the Bacillus cereus CN8 is CGMCC No.30118. The functional strain of the present invention is a cellulose-degrading strain with excellent phenotype, which can survive at a relatively low temperature (4°C) and can efficiently degrade straw under relatively low temperature conditions, which is of great significance for improving straw utilization and improving the soil environment. The present invention screened three cellulose-degrading strains with excellent phenotypes from the soil of a long-term straw return test site at the Shenyang Ecological Experimental Station of the Chinese Academy of Sciences (41°31′N, 123°24′E) in the black soil region of Northeast China, and finally identified them as Bacillus cereus ( Bacillus cereus )CN8, Bacillus cereus ( Bacillus cereus ) CN10 and Bacillus thuringiensis ( Bacillus thuringiensis )CN16. Experiments show that the combined application of the functional strains of the present invention can efficiently degrade straw under medium-low temperature (15°C) liquid culture conditions, which is of great significance for improving straw utilization and soil environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0017] Figure 1 This is a diagram showing the results of the Congo red identification culture medium rescreening in Example 1 of the present invention; Figure 2 is a standard curve of glucose absorbance in Example 1 of the present invention; Figure 3 This is a graph showing the results of measuring the CMC enzyme activity of the cellulose degrading strain in Example 1 of the present invention; Figure 4 This is a graph showing the straw degradation rate of the cellulose-degrading strain under medium-low temperature liquid culture conditions in Example 1 of the present invention; Figure 5 This is the phylogenetic tree of the medium-low temperature straw degrading bacteria CN8, CN10, and CN16 in Example 1 of the present invention; Figure 6 This is a diagram showing the microscopic morphological observation results of the low-temperature straw-degrading bacteria CN8, CN10 and CN16 in Example 1 of the present invention; Figure 7 This is a diagram showing the results of the antagonistic cultivation of the medium- and low-temperature straw-degrading strain in Example 2 of the present invention; Figure 8 The results of straw degradation rate of different combinations in Example 2 of the present invention are shown in FIG. 1 , wherein a) is in liquid culture conditions, and b) is a buried bag experiment; Fig. 9 This is a graph showing the straw degradation rate of composite bacterial community D at different carbon-nitrogen ratios under liquid culture conditions in Example 3 of the present invention; Fig.10 This is a graph showing the straw degradation rate results of composite bacterial community D at different carbon-nitrogen ratios under soil bag burial conditions in Example 3 of the present invention; Fig.11 This is the result diagram of straw degradation rate when composite bacterial community D is applied; Fig.12 These are diagrams showing the straw degradation effect of composite flora D in an application example. The left picture shows the straw after 30 days of application of composite flora D, and the right picture shows the straw after 30 days of application of the control group (distilled water).

[0018] Biological Deposit Description Bacillus cereus ( Bacillus cereus) CN8 is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC), with the deposit number CGMCC No.30118, the deposit date is March 25, 2024, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the status is alive; Bacillus cereus ( Bacillus cereus ) CN10 is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC), with the deposit number CGMCC No.30119, the deposit date is March 25, 2024, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the status is alive; Bacillus thuringiensis ( Bacillus thuringiensis ) CN16 is deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), with the deposit number CGMCC No.30120, the deposit date is March 25, 2024, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the status is alive. DETAILED DESCRIPTION

[0019] The present invention provides a functional strain for degrading straw, which is Bacillus cereus ( Bacillus cereus ) CN8; the preservation number of the Bacillus cereus CN8 is CGMCC No.30118.

[0020] The present invention also provides a composite bacterial community for degrading straw, including Bacillus cereus ( Bacillus cereus )CN8, Bacillus cereus ( Bacillus cereus ) CN10 and Bacillus thuringiensis ( Bacillus thuringiensis ) CN16; the preservation number of the Bacillus cereus CN8 is CGMCC No.30118; the preservation number of the Bacillus cereus CN10 is CGMCC No.30119; the preservation number of the Bacillus thuringiensis CN16 is CGMCC No.30120.

[0021] The functional strains of the present invention are isolated from the soil of a long-term straw return test site at the Shenyang Ecological Experimental Station of the Chinese Academy of Sciences in the Northeast Black Soil Region (41°31′N, 123°24′E). The colonies of Bacillus cereus CN8 are opaque, milky white, round, with an uneven surface and no bulges; the colonies of Bacillus cereus CN10 are transparent, white, oval or round, with an uneven surface and no bulges; and the colonies of Bacillus thuringiensis CN16 are opaque, milky white, round, with an uneven surface and no bulges.

[0022] The functional strain of the present invention is a cellulose-degrading strain with excellent phenotype, which can survive at a relatively low temperature (4°C) and can efficiently degrade straw under relatively low temperature conditions, and is of great significance for improving the utilization rate of straw and improving the soil environment.

[0023] In the composite flora of the present invention, Bacillus cereus CN8, Bacillus cereus CN10 and Bacillus thuringiensis CN16 can synergistically degrade straw, have strong straw degradation ability, can degrade straw by 26.33%, and the degradation ability is better than that of single strains CN8, CN10, CN16, and also significantly better than the degradation effect of strains in pairs (CN8+CN10, CN8+CN16, CN10+CN16). The composite flora of the present invention can maintain high activity at low temperatures, has no adverse effects on the environment, and has high universality. This result enriches the beneficial microbial resources of cold land, such as the black soil in Northeast China, improves the utilization rate of straw resources in medium and low temperature habitats, and has good application prospects.

[0024] In the specific implementation process of the present invention, the ratio of the effective live bacteria count of Bacillus cereus CN8, Bacillus cereus CN10 and Bacillus thuringiensis CN16 is 1:1:1. Under this ratio, Bacillus cereus CN8, Bacillus cereus CN10 and Bacillus thuringiensis CN16 can synergistically play the role of degrading straw, and have the advantage of high degradation rate.

[0025] The present invention also provides a bacterial agent for degrading straw, comprising the functional strain or the composite bacterial community described in the above scheme.

[0026] In the specific implementation process of the present invention, the OD of the functional strain or composite bacterial community in the bacterial agent is 600nm 0.5~0.7, further to 0.6, OD 600nm ≈0.6, the functional strain is in a vigorous growth logarithmic phase and has the best growth state; the effective viable count of the functional strain or composite bacterial community in the bacterial agent is 10 8 CFU / mL, further 8*10 8 CFU / mL.

[0027] In the specific implementation process of the present invention, the bacterial agent includes a bacterial suspension; when the bacterial agent includes a composite bacterial community, the preparation method of the bacterial solution includes: liquid culturing Bacillus cereus CN8, Bacillus cereus CN10 and Bacillus thuringiensis CN16 respectively to obtain seed solutions of each single bacteria respectively; the seed solutions of the single bacteria are mixed to obtain; the mixing includes mixing in equal proportions of effective live bacteria; the mixing of the seed solutions of the single bacteria includes: centrifuging the seed solutions of each single bacteria respectively, collecting the precipitate and resuspending the precipitate to obtain bacterial suspensions of each single bacteria respectively; mixing the bacterial suspensions of each single bacteria to obtain the bacterial agent; the culture medium used for the liquid culture includes LB liquid culture medium.

[0028] The bacterial agent of the present invention can maintain activity at low temperatures and stably degrade straw.

[0029] The present invention also provides the use of the functional strain, the composite bacterial community or the bacterial agent described in the above scheme in degrading straw and / or degrading cellulose.

[0030] In the specific implementation process of the present invention, the degradation environment temperature is 15~28°C.

[0031] The present invention also provides a method for degrading straw, comprising the following steps: after returning the straw to the field, applying the functional strain, the composite bacterial community or the bacterial agent described in the above scheme to the soil.

[0032] In the specific implementation process of the present invention, before the application, the carbon-nitrogen ratio of the soil is adjusted to 25:1; the composite bacterial community has a high-efficiency degradation ability at a carbon-nitrogen ratio of 25:1. In one embodiment of the present invention, when applying in the field, the carbon-nitrogen ratio is adjusted by applying urea.

[0033] In the specific implementation process of the present invention, the application is applied to the soil surface; the application includes spot application or spraying; the ambient temperature during the application is 15-28°C; the application time is ≥20 days, and further ≥30 days; the present invention has no special restrictions on the application amount, and the conventional application amount in this field can be used; before the application, the straw is returned to the field; the straw includes corn straw; the depth of the straw buried in the soil is ≥15cm, which is conducive to the decomposition of the straw and the subsequent growth of the root system of the crop; the soil applied includes black soil. In one embodiment of the present invention, the black soil is the black soil in Shenyang, China.

[0034] To further illustrate the present invention, a functional bacterial strain for degrading straw, a composite bacterial community and applications thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] In an embodiment of the present invention, the culture medium involved is specifically as follows: Carboxymethyl cellulose (CMC) medium (1 L), with water as solvent, had the following formula: 2.5 g dipotassium hydrogen phosphate, 2.5 g disodium hydrogen phosphate, 20 g sodium carboxymethyl cellulose, 2 g peptone and 0.5 g yeast extract powder, pH 7.2 ± 0.2.

[0036] Congo red identification medium (1L), with water as solvent, has the following formula: 2g ammonium sulfate, 0.5g magnesium sulfate heptahydrate, 1g potassium hydrogen phosphate, 0.5g sodium chloride, 2g sodium carboxymethyl cellulose and 20g agar.

[0037] LB medium (1 L), with water as solvent, had the following formula: 10 g of tryptone and 5 g of yeast extract, and 10 g of sodium chloride.

[0038] Straw culture medium (1 L), with water as solvent, had the following formula: 5 g corn straw, 10 g tryptone, 5 g yeast extract and 10 g sodium chloride.

[0039] Example 1 Isolation of Potentially Functional Strains from Black Soil Region of Northeast China Using Plate Condition Screening Specific steps: 1. Sample collection: Soil samples were collected from the long-term straw return test site of the Shenyang Ecological Experimental Station of the Chinese Academy of Sciences (41°31′N, 123°24′E) (no-tillage + 100% corn straw coverage, plot area of ​​8 m*15 m, soil total carbon content of 11.05 g / kg, soil total nitrogen content of 9.42 g / kg). The long-term straw return and acclimatization process is more targeted for the screening of target strains.

[0040] 2. Initial screening of strains: Add sterile saline (10 g soil, 100 mL 0.85% saline) to the collected soil samples, place them on a shaker (150 rpm, 1 h), let them stand for 1 h, aspirate the supernatant and prepare 10 -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 100 µL of soil suspension from each of the six dilution gradients was taken and spread on a CMC solid culture medium plate, inverted, and cultured at 28°C for 72 h. The strain grown in this step can be preliminarily identified as a cellulose-degrading strain.

[0041] 3. Streak separation: The cellulose-degrading strain obtained in step (3) was selected and colonies of different morphologies were streaked and purified on LB medium plates for three generations to obtain a purified single strain. A total of 10 cellulose-degrading strains were purified in this step. The obtained single strain was inoculated into LB liquid medium, cultured to the logarithmic growth phase, and then stored in a -80°C refrigerator in a cryopreservation tube using glycerol as a protective agent.

[0042] 4. Strain rescreening: Confirm the cellulose degradation ability of a single strain using Congo red culture medium. Pick a single colony and inoculate it on a Congo red identification culture medium plate, invert it, and culture it at 28°C for 72 hours. Measure the diameter of the transparent circle and the colony. The diameter of the transparent circle is used to characterize the cellulose degradation ability of the strain ( Figure 1 , Table 1).

[0043] Table 1 Size of transparent zone in Congo red identification medium rescreening

[0044] Note: D / d represents the ability of the strain to decompose cellulose.

[0045] 5. Determination of CMC enzyme activity of degradation strains: Select a single colony from the plate, inoculate it into LB liquid medium, and shake and culture until OD 600nm ≈0.6 (150 rpm, 15°C), centrifuge the bacteria (4000 rpm, 10 min), and the supernatant is the crude enzyme solution. The CMC enzyme activity was determined by the 3,5-dinitrosalicylic acid (DNS) colorimetric method, using 1% sodium carboxymethyl cellulose (CMC-Na) as the substrate for enzyme activity determination. The reaction principle of this method is that cellulase can hydrolyze cellulose to produce reducing sugars such as glucose, and the resulting reducing sugar reacts with DNS to produce orange 3-amino-5-nitrosalicylic acid. The color depth is related to the amount of reducing sugar, so the amount of reducing sugar can be used to indicate the activity of cellulase. Add 1.5 mL of citric acid buffer containing 1% CMC-Na to the test tube, and add 0.5 mL of crude enzyme solution, react at 50°C for 30 minutes, add DNS reagent to terminate the reaction, put the test tube in a boiling water bath for 5 minutes, take it out and cool it to room temperature after the reaction, add distilled water to make up to 25 mL, and measure its OD value at 540 nm. Use the glucose absorbance standard curve ( Figure 2 ) (Drawing of glucose absorbance standard curve: Take 6 dry and clean stoppered test tubes, add 1 mg / mL glucose solution and distilled water in sequence according to Table 2, shake well to prepare glucose solutions of different concentrations. Take 3 mL of glucose solution in a test tube, add 1.5 mL of DNS colorimetric agent, boil in water bath for 10 min, immediately terminate the reaction with cold water, use test tube No. 0 as blank control to adjust to zero, and measure the OD value of the solution at a wavelength of 540 nm.) Calculate the CMC enzyme activity of a single strain ( Figure 3 ).

[0046] Table 2 Preparation of glucose standard solution

[0047] 6. Temperature gradient culture experiment: In order to explore the minimum growth temperature of the degradation strains, four temperature gradients (4℃, 10℃, 15℃, 20℃) were set up. The dominant strains after liquid activation were divided into zones and streaked on carboxymethyl cellulose solid culture medium plates and cultured at a constant temperature (5 d-25 d). The growth of the strains at different temperatures was observed. The results showed (Table 3) that the cellulose-degrading strains could maintain a good growth state at 15℃, and the four bacterial strains CN8, CN9, CN10, and CN16 had the best growth state.

[0048] Table 3 Growth of strains at different temperatures

[0049] Note: “++++++”: the strain can multiply rapidly and in large quantities, “++++”: the strain can grow rapidly, “+++”: the strain is in good growth condition, “++”: the strain grows slowly, “+”: the strain grows extremely slowly, “-”: the strain stops growing.

[0050] 7. Determination of straw degradation rate of single strain under different temperature gradient conditions: The bacterial solution of the single strain was cultured to OD 600nm ≈0.6, centrifuged (8000 rpm, 10 min), washed three times with sterile water, and then resuspended the cells in sterile water to prepare the OD 600nm =1.2 bacterial suspension. Add 1.2 mL of bacterial suspension to 20 mL of straw culture medium and culture at constant temperature for 30 days (150 rpm, 10℃, 15℃, 20℃). After the culture, wash the straw with distilled water, dry it at 80℃ to constant weight, weigh it, and calculate the weight loss rate. The experiment was repeated three times. The results showed that ( Figure 4 ), cellulose-degrading strains have the ability to degrade straw, among which CN8, CN10, and CN16 are the three strains with the highest degradation rates under medium and low temperature (15°C), with degradation rates of 25.67%, 24.67%, and 24.33%, respectively. Therefore, CN8, CN10, and CN16 are considered to be three medium and low temperature straw-degrading strains with excellent phenotypes. As the culture temperature increases, the degradation rate still increases, but in order to be more suitable for application in the medium and low temperature areas in the north, we choose the lowest temperature with the highest degradation rate, i.e. 15°C, for further research.

[0051] 8. Classification and identification of strains with straw degradation ability: First, identification was performed using 16SrDNA, i.e., PCR amplification was performed using the universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGYTACCTTGTTACGACTT-3', SEQ ID NO.2) of prokaryotic 16SrDNA (specific PCR method can be found in the patent "A method for rapid extraction of a small amount of total soil DNA" (patent application number CN 200510120584.7, published on July 4, 2007) and sequencing, and then compared with the international NCBI GenBank (www.ncbi.nlm.nih.gov) nucleotide database. The comparison results are shown in Table 4. The nucleotide homology is 99%. Sequences with higher homology were selected and a phylogenetic tree was constructed using MEGA 11.0 ( Figure 5 ); second, morphological identification using an optical microscope ( Figure 6 ) and growth characteristics identification.

[0052] Table 4 Homology comparison results of medium-low temperature straw-degrading bacteria CN8, CN10 and CN16

[0053] The morphological characteristics are as follows (Table 5): CN8 colonies are opaque, milky white, round, with a rough surface and no protrusions; CN10 colonies are transparent, white, oval or round, with a rough surface and no protrusions; CN16 colonies are opaque, milky white, round, with a rough surface and no protrusions. For physiological and biochemical analysis, refer to Table 1.

[0054] Table 5 Basic characteristics of strains

[0055] “+”: positive reaction, “-”: negative reaction.

[0056] Ten cellulose-degrading strains were initially screened from the soil of a long-term straw return experiment site at the Shenyang Ecological Experimental Station of the Chinese Academy of Sciences (41°31′N, 123°24′E) in the black soil region of Northeast China by using the plate screening method. Three strains with excellent phenotypes were further screened and finally identified as Bacillus cereus ( Bacillus cereus )CN8, Bacillus cereus ( Bacillus cereus )CN10, Bacillus thuringiensis ( Bacillus thuringiensis ) CN16 three strains. Experiments show that the above three bacterial strains can efficiently degrade straw under low-temperature liquid culture conditions, which is of great significance to improving the utilization rate of straw and the soil environment in the black soil area of ​​Northeast China.

[0057] The strains are preserved as follows: Bacillus cereus ( Bacillus cereus )CN8, Bacillus cereus ( Bacillus cereus ) CN10 and Bacillus thuringiensis ( Bacillus thuringiensis ) CN16 was cultured on LB liquid or solid medium at 28°C and could be stored at 4°C for a short period of time after culture.

[0058] If long-term storage is required, glycerol freezing tubes or freeze-drying tubes (for specific methods, please refer to: Zhao Bin, He Shaojiang, Microbiology Experiment, First Edition, Science Press. Beijing, 2002: P202-205) are more appropriate for preserving strains.

[0059] Example 2 Study on the degradation of straw by the new composite bacterial community D under liquid culture and bagging conditions under medium and low temperature conditions The above three strains of bacteria were subjected to antagonism experiments, and composite bacterial communities were prepared in different combinations to measure the corresponding straw degradation capabilities. The experiments showed that the above three strains of bacteria can degrade straw under individual and composite culture conditions, and can degrade straw under low temperature conditions, which is of great significance for improving the utilization rate of straw resources and increasing the content of organic matter in the black soil region of Northeast China.

[0060] The following are the experimental steps for the new composite bacterial community D in degrading straw under low temperature conditions: 1. The three strains obtained in Example 1 were co-cultured (28°C) by cross-streaking on a plate to observe whether the strains were antagonistic. The experimental results showed that there was no antagonism between the three strains CN8, CN10, and CN16, and there was no antagonism between the three strains CN8, CN10, and CN16. The coexistence of strains is a prerequisite for constructing a composite bacterial community ( Figure 7 ).

[0061] 2. Select three single colonies of the strains from the plate, inoculate them into LB liquid medium, and shake and culture until OD 600nm ≈0.6 (150 rpm, 15°C), centrifuged (8000 rpm, 10 min), washed with sterile water three times, and then resuspended in sterile water to prepare bacterial suspension. The effective viable count was 8*10 8 CFU / mL. Composite bacterial communities were constructed in the following combinations with equal ratios of effective viable counts: composite bacterial community A: CN8+CN10 (1:1), composite bacterial community B: CN8+CN16 (1:1), composite bacterial community C: CN10+CN16 (1:1), composite bacterial community D: CN8+CN10+CN16 (1:1:1).

[0062] 3. Determination of straw degradation rate under liquid culture conditions: 1.2 mL of the bacterial suspension of the composite flora was inoculated into 20 mL of straw culture medium and cultured at a constant temperature (15°C, 150 rpm) for 30 days. After the culture, the straw was washed with distilled water, dried at 80°C to constant weight, weighed, and the weight loss rate was calculated. The experiment was repeated three times, and the same volume of sterile distilled water as the treatment group was used as the blank control (CK) ( Figure 8 ).

[0063] 4. Determination of straw degradation rate under soil bag burial conditions (bag burial experiment): In order to simulate the natural environment, the black soil and corn straw used in the experiment were not sterilized. In a 50 mL glass test tube containing an equal mass of non-sterile black soil, 0.5 g corn straw was wrapped in a nylon mesh bag and buried at 15 cm, 1.2 mL of bacterial suspension was added, and the 15°C constant temperature was kept for 30 days. After the culturing, the straw was washed with distilled water, dried at 80°C to constant weight, weighed, and the weight loss rate was calculated. The experiment was repeated six times ( Figure 8 ).

[0064] From the experimental results ( Figure 8 ) It can be seen that after the three medium- and low-temperature straw-degrading strains were combined in pairs, the straw degradation rates of CN8+CN10, CN8+CN16, and CN10+CN16 under liquid culture conditions and soil buried bag conditions were 20.5%~24.03%, 15%~19.33%, and 11.33%~17.22%, respectively, while the composite bacterial community D (CN8+CN10+CN16) of the three bacteria had the highest degradation rate, which could degrade straw by 26.27%~26.33% after 30 days of cultivation under 15°C liquid culture conditions and soil buried bag conditions. From the above analysis, it can be seen that the three strains have a synergistic effect, see Figure 8 b) in the above.

[0065] Example 3 Exploring the optimal carbon-nitrogen ratio for efficient straw degradation using the new composite bacterial community D under medium and low temperature conditions In the process of returning straw to the field, the carbon-nitrogen ratio is crucial to the efficiency of microbial straw degradation. In order to continuously improve the ability of the bacterial system to effectively degrade cellulose in a medium-low temperature environment, this example uses the selected low-temperature resistant cellulose-degrading composite bacterial community D as the research object, and sets up four carbon-nitrogen ratio gradients (15:1, 20:1, 25:1, 30:1) of corn straw degradation experiments to determine the carbon-nitrogen ratio with the best degradation effect of the bacterial system.

[0066] The following are the experimental steps for exploring the optimal carbon-nitrogen ratio of the new composite bacterial community D for efficient straw degradation under low temperature conditions: 1. The total carbon content of straw is determined according to the standard T / NAIA 070-2021 Determination of the total carbon content of plants; the total nitrogen content of straw is determined according to the standard NY / TT 2017-2011 Determination of nitrogen, phosphorus and potassium in plants; the ratio of total carbon to total nitrogen in straw is the carbon-nitrogen ratio. According to the carbon-nitrogen ratio of straw, (NH4)2SO4 is used as a supplementary nitrogen source, and four ratios of 15:1, 20:1, 25:1, and 30:1 are set for degradation experiments under liquid culture conditions. The calculation shows how much mass of (NH4)2SO4 needs to be added to adjust the carbon-nitrogen ratio of the entire culture system to 15:1, 20:1, 25:1, and 30:1, respectively, and a blank control is set (sterile distilled water of the same volume as the treatment group is applied, CK).

[0067] 2. Corn straw degradation experiment under liquid culture and soil bag conditions: Except for different carbon-nitrogen ratio gradients, other factors remain unchanged. For the above-mentioned composite bacterial community D, refer to the experimental steps in Example 2 to explore the straw degradation rate under liquid culture and soil bag conditions at different carbon-nitrogen ratio gradients.

[0068] The results show that ( Fig. 9 , Fig.10 ), under the condition of C / N=25:1, the degradation effect of composite flora D reached the best. After 25 days of constant temperature liquid shaking culture at 15℃, the degradation efficiency reached 26.33%. The results of the black soil buried bag degradation experiment further verified the efficient degradation ability of composite flora D under a carbon-nitrogen ratio of 25:1. After 56 days of constant temperature static culture at 15℃, the weight loss rate of corn straw reached 49.22%.

[0069] Example 4 Application of composite bacterial community D to efficiently degrade straw under medium and low temperature conditions In order to further verify the actual application effect of composite bacterial community D, this example conducted a field straw bag burying experiment in Jiangning District, Nanjing City, Jiangsu Province from September 25, 2024 to October 28, 2024.

[0070] The following are the application steps of the new composite bacterial community D for efficient straw degradation under medium and low temperature conditions: 1. September is the harvest period for summer-sown corn, and the average temperature in Nanjing from the end of September to the end of October is about 15-23°C. The stubble and straw after corn harvest also return to the field at this time. Therefore, this is a suitable time for applying the bacterial community of the present invention. Therefore, we conducted a field straw bag burying experiment in Jiangning District, Nanjing City, Jiangsu Province from September 25, 2024 to October 28, 2024.

[0071] 2. Select single colonies of CN8, CN10 and CN16 from the plates, inoculate them into LB liquid medium, and shake and culture until OD 600nm≈0.6 (150 rpm, 15°C), centrifuged (8000 rpm, 10 min), washed with sterile water three times, and then resuspended in sterile water to prepare a bacterial suspension (OD 600nm =1.2). Take 20 mL of bacterial suspension of the three strains respectively and construct composite bacterial community D in equal proportions: CN8+CN10+CN16 (1:1:1).

[0072] 3. Accurately weigh 8 g of corn stalks in a nylon mesh bag, bury the nylon mesh bag in the wild soil at a depth of 15 cm without weeds, and apply 60 mL of complex bacterial community D on the soil by spot application. No complex bacterial community D was added to the control treatment. Each treatment was repeated 9 times. The mesh bag was taken out at 10 d, 20 d, and 30 d after the bag was buried, and 3 replicates were taken each time. Bring it back to the laboratory, rinse it with distilled water, dry it (55℃, 24 h), weigh it, and calculate the weight loss rate of the stalks. Apply the complex bacterial community D once on the 15th day of the bag-burying experiment, and the application amount is the same as the first application amount.

[0073] The results showed that when the composite bacterial community D was applied for 10 days, the straw degradation rate was low, about 5%; after 20 days of application of the composite bacterial community D, the straw degradation rate began to increase, reaching 17%; after 30 days of application of the composite bacterial community D, the straw degradation rate reached 21.37%, which was 13.37% higher than the 8% of the control treatment ( Fig.11 ). And it can be clearly observed that compared with the blank control (administered with the same volume of sterile distilled water as the treatment group, CK) ( Fig.12 Compared with the right figure in the figure, in the treatment with composite bacterial flora D, the straw was obviously fragmented ( Fig.12 The left picture in the figure shows that there are more debris, indicating that the application of composite bacterial community D promotes the degradation of corn straw in the field. It can be seen that during the one-month field bag burial experiment, the application of composite bacterial community D increased the degradation rate of corn straw compared with the in-situ soil, which is conducive to the return of corn straw to the field.

[0074] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A functional bacterial strain for degrading straw, characterized in that: Bacillus cereus ( Bacillus cereus ) CN8; the preservation number of the Bacillus cereus CN8 is CGMCC No.30118.

2. A composite bacterial community for degrading straw, characterized in that: Including Bacillus cereus ( Bacillus cereus )CN8, Bacillus cereus ( Bacillus cereus ) CN10 and Bacillus thuringiensis ( Bacillus thuringiensis )CN16; The deposit number of the Bacillus cereus CN8 is CGMCC No.30118; The deposit number of the Bacillus cereus CN10 is CGMCC No.30119; The deposit number of the Bacillus thuringiensis CN16 is CGMCC No.30120.

3. The composite bacterial flora according to claim 2, characterized in that The ratio of the effective live bacteria counts of Bacillus cereus CN8, Bacillus cereus CN10 and Bacillus thuringiensis CN16 is 1:1:

1.

4. A bacterial agent for degrading straw, characterized in that: It includes the functional strain described in claim 1 or the composite bacterial community described in claim 2 or 3.

5. The bacterial agent according to claim 4, characterized in that The OD of the functional strain or composite bacterial community in the bacterial agent 600 nm It is 0.5~0.

7.

6. Use of the functional strain according to claim 1, the composite bacterial community according to claim 2 or 3, or the microbial agent according to claim 4 or 5 in degrading straw and / or cellulose.

7. The use according to claim 6, characterized in that: The degradation environment temperature is 15-28°C.

8. A method for degrading straw, characterized in that: The following steps are involved: After returning the straw to the field, the functional strain according to claim 1, the composite bacterial community according to claim 2 or 3, or the microbial agent according to claim 4 or 5 is applied to the soil.

9. The method according to claim 8, characterized in that Before the application, the carbon-nitrogen ratio of the soil is adjusted to 25:

1.

10. The method according to claim 8, characterized in that The ambient temperature during the application is 15-28°C.

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