A composite bacterial agent for decomposing soybean straw and its application

Abstract

The invention belongs to the field of microbial technology, and specifically relates to a soybean straw decomposition composite bacterial agent and its application. The soybean straw decomposition composite bacterial agent is composed of Sonora Desert Bacillus Bacillus sonoran DF‑1, Bacillus licheniformis Bacillus lichen-like DF‑8 and Bacillus subtilis Bacillus subtle DF‑22. The three Bacillus strains in the composite microbial agent have good synergistic degradation functions. Applying it to soybean straw composting can quickly increase the composting temperature, prolong the high temperature period, effectively accelerate the composting maturity process, and achieve efficient degradation of soybean straw, which has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a soybean straw decomposing composite bacterial agent and application thereof. Background Art

[0002] In recent years, with the significant increase in soybean planting area and yield, soybean straw and other by-products have been produced in large quantities and are showing an increasing trend. If soybean straw and other by-products are not utilized, it will not only cause a waste of resources, but also may cause environmental pollution. Therefore, the resource utilization of soybean straw needs to be solved urgently.

[0003] The main components of soybean straw are lignocellulose composed of macromolecules such as cellulose, hemicellulose and lignin, which account for about 60% of its total solid content. The dense and ordered complex structure of lignocellulose makes soybean straw difficult to degrade, which seriously reduces its utilization efficiency. Microorganisms are the key drivers of straw degradation. If microorganisms with degradation functions can be introduced, it will help accelerate the degradation of straw. However, the complex composition and structure of straw determine that the microorganisms involved in the decomposition are of various types and functions, and there are interactions between multiple species. At present, the existing straw decomposing agents often have a single species. Although they can improve the degradation efficiency of straw, their effects are unstable due to their single function and limited adaptability, which limits the promotion and application of straw decomposing agents.

[0004] Therefore, it is urgent to develop a composite microbial agent containing multiple microbial strains to give full play to the synergy and adaptability of microorganisms to improve the degradation efficiency of soybean straw and promote the resource utilization of soybean straw. Summary of the invention

[0005] The purpose of the present invention is to provide a soybean straw decomposing composite bacterial agent and application thereof, so as to overcome the shortcomings of the prior art. The composite bacterial agent of the present invention is based on the synergistic effect of microorganisms. Through mutual promotion and functional synergy between different microbial species, the problems of the prior art such as single bacterial agent type and unstable effect in the straw degradation process are avoided, and the efficient degradation of soybean straw is achieved, which has good practical application value.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In a first aspect, the present invention provides a soybean straw decomposition composite bacterial agent, wherein the soybean straw decomposition composite bacterial agent is composed of Sonora Desert Bacillus Bacillus sonorensis DF-1, Bacillus licheniformis Bacillus licheniformis DF-8 and Bacillus subtilis Bacillus subtilisDF-22; the Sonora Desert Bacillus DF-1, Bacillus licheniformis DF-8 and Bacillus subtilis DF-22 have all been sent to the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit date of June 5, 2024, and a deposit address of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0008] Among them, the Sonora Desert Bacillus DF-1 has a preservation number of CGMCC No.30858; the licheniformis DF-8 has a preservation number of CGMCC No.30859; and the subtilis DF-22 has a preservation number of CGMCC No.30860.

[0009] In some other embodiments, the volume ratio of the Sonora Desert Bacillus DF-1, Bacillus licheniformis DF-8 and Bacillus subtilis DF-22 is 1:(1-2):(1-2).

[0010] In some other embodiments, the mixing volume ratio of the Sonora Desert Bacillus DF-1, Bacillus licheniformis DF-8 and Bacillus subtilis DF-22 is 1:1:1.

[0011] In some other embodiments, the total viable count of Sonora Desert Bacillus DF-1, Bacillus licheniformis DF-8 and Bacillus subtilis DF-22 is higher than 1×10 9 CFU / mL.

[0012] In a second aspect, the present invention provides use of the soybean straw decomposing composite bacterial agent described in the first aspect in straw decomposition.

[0013] In some other embodiments, the applied temperature is 30-50°C.

[0014] In some other embodiments, the straw is soybean straw.

[0015] In a third aspect, the present invention provides the use of the soybean straw decomposing composite bacterial agent described in the first aspect in promoting the straw composting process.

[0016] In some other embodiments, the promoting the soybean straw composting process is promoting the composting process or reducing the phytotoxicity of the compost product.

[0017] In some other embodiments, the applied temperature is 30-50° C., and the straw is soybean straw.

[0018] Beneficial effects of the present invention:

[0019] (1) The present invention utilizes the mutual promotion and functional synergy between the three Bacillus species, namely, Sonora Desert Bacillus DF-1, Bacillus licheniformis DF-8 and Bacillus subtilis DF-22, to provide a new composite bacterial agent resource for the decomposition of soybean straw.

[0020] (2) The present invention provides an effective path and data support for the screening and compounding of Bacillus, and has important guiding significance for the scientific combination of Bacillus composite inoculant products.

[0021] (3) The composite bacterial agent provided by the present invention can significantly accelerate the composting process of soybean straw and accelerate the maturity of the compost, showing good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 The colony phenotypes of the 22 soybean straw decomposing strains screened in Example 1 of the present invention on the LB medium plate;

[0024] Figure 2 The confrontation growth conditions of the five soybean straw decomposing strains selected in Example 2 of the present invention on the LB medium plate;

[0025] Figure 3 The soybean straw degradation rate after compounding the five soybean straw decomposing strains selected in Example 2 of the present invention;

[0026] Figure 4 The phylogenetic tree is constructed after 16SrDNA sequencing and identification of three soybean straw decomposing strains in the combination of strains with strong degradation functions in Example 2 of the present invention;

[0027] Figure 5 are the filter paper enzyme activity, carboxymethyl cellulase activity and soybean straw degradation rate at different times of the single bacterial agent and the composite bacterial agent in Example 3 of the present invention, wherein Figure 5 (a) is the filter paper enzyme activity, Figure 5 (b) is the activity of carboxymethyl cellulase, Figure 5 (c) in the figure is the degradation rate at different times;

[0028] Figure 6 The effect of the composite bacterial agent in Example 4 of the present invention on the changes in temperature, pH and EC value during soybean straw composting and the effect on the seed germination rate of the soybean straw compost product; wherein, Figure 6 (a) shows the effect of the composite bacterial agent on the temperature during soybean straw composting. Figure 6 (b) shows the effect of the composite bacterial agent on pH during soybean straw composting. Figure 6 (c) shows the effect of compound microbial agent on EC value during soybean straw composting. Figure 6 (d) is the effect of the composite microbial agent on the seed germination rate of soybean straw compost product;

[0029] Figure 7 This is a physical picture of the decomposition effect of the blank control and the compound bacterial agent added in Example 3 of the present invention. DETAILED DESCRIPTION

[0030] As introduced in the background technology, the straw decomposing microbial agents in the prior art often have a single strain, and although they can also accelerate the decomposition, they have a single function and limited adaptability, resulting in unstable effects of the microbial agents, which limits the promotion and application of the straw decomposing microbial agents. In order to solve the above technical problems, the present invention proposes a soybean straw decomposing composite microbial agent and its application.

[0031] Bacillus can form spores in harsh environments, has a wide range of adaptability to acidity, alkali and temperature differences, and has strong survival ability in harsh environments. By screening Bacillus that have the functions of efficiently degrading cellulose, oil, starch, protein, etc. and can survive under medium and high temperature conditions in the natural environment, the compound strain has a straw degradation effect that exceeds that of a single strain. The composite strain is used for soybean straw composting, which not only has efficient degradation, but also has a continuous decomposition effect on soybean straw, achieving the maximum decomposition of soybean straw.

[0032] The present invention provides a soybean straw decomposition composite bacterial agent, which is composed of Sonora Desert Bacillus Bacillus sonorensis DF-1, Bacillus licheniformis Bacillus licheniformis DF-8, Bacillus subtilis Bacillus subtilis DF-22 bacterial liquid; the Sonora Desert Bacillus DF-1, Bacillus licheniformis DF-8 and Bacillus subtilis DF-22 have all been sent to the General Microbiology Center of China Microorganism Culture Collection Committee, with a preservation date of June 5, 2024, and a preservation address of Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; among them, the Sonora Desert Bacillus DF-1 preservation number is CGMCC No.30858; the Bacillus licheniformis DF-8 preservation number is CGMCC No.30859; the Bacillus subtilis DF-22 preservation number is CGMCC No.30860.

[0033] The soybean straw decomposing composite bacterial agent of the present invention is selected and composed according to the following principles:

[0034] (1) The strains were all derived from decomposed soybean straw. Strains that can grow normally under medium and high temperature conditions were selected for molecular identification to complete the initial screening.

[0035] (2) The strains are subjected to degradation culture medium experiments to select strains that can grow normally in a variety of degradation culture media. A confrontation experiment is also conducted to select strains with fusion phenotypes to complete rescreening.

[0036] (3) The strains were used to perform filter paper strip degradation experiments, and the top five strains with the highest degradation rates were selected for compounding to complete the straw degradation experiment. The degradation ability of the composite bacterial community was significantly higher than that of a single bacteria and showed a synergistic effect of multiple bacteria. The combination with the highest degradation rate was selected as the combination of composite bacterial agents.

[0037] The preparation method of the composite bacterial agent of the present invention:

[0038] The activated strains DF-1, DF-8 and DF-22 were inoculated into LB liquid medium and cultured at 37°C and 180 r / min for 18-24 h. The OD values ​​of the three strains were adjusted. 600 If the values ​​are the same, adjust the OD 600 The three bacterial solutions were mixed in equal amounts and the OD 600 Adjust the value to 3 (greater than 2×10 8 CFU / mL) is the soybean straw decomposing composite bacterial agent.

[0039] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0040] Example 1

[0041] Screening of bacterial strains for decomposing soybean straw

[0042] 1.1 Screening and isolation of soybean straw decomposition strains

[0043] Soybean straw naturally decomposed in the field was used as the bacterial source. It was placed in a triangular flask containing sterile water and shaken at 180 r / min at room temperature for 30 min to make a suspension. The suspension was allowed to settle for 30 min, and 10 mL of the upper suspension was taken and gradiently diluted with sterile water and spread on LB medium. The temperature was used as the screening condition. The culture conditions were 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, pH 7.0, and cultured at 45°C for 8 h. Single colonies were picked and streaked for purification twice, and a total of 160 strains were screened.

[0044] 1.2 Rescreening of degradation function of soybean straw decomposing strains

[0045] The strains with extensive degradation ability were screened by degrading proteins, fats, cellulose and starch. The culture conditions were 37°C. The culture medium formula for screening protein-degrading strains was as follows: 10 g casein, 3 g beef extract powder, 5 g NaCl, 2 g Na2HPO4, 15 g agar, 0.05 g bromothymol blue, pH 7.0, and 1000 mL distilled water. The culture medium formula for screening fat-degrading strains was as follows: 10 g peptone, 5 g beef extract, 5 g NaCl, 10 g sesame oil, 1 mL 1.6% neutral red aqueous solution, 15 g agar, pH 7.0, and 1000 mL distilled water (heat the oil agar and water first, and add the neutral red aqueous solution after adjusting the pH). The culture medium formula for screening cellulose-degrading strains is: 10 g peptone, 3 g yeast extract, 5 g sodium chloride, 10 g CMC-Na, 1 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 0.2 g Congo red, 15 g agar, pH 7.0, 1000 mL distilled water. The culture medium formula for screening starch-degrading strains is: 10 g peptone, 5 g beef extract, 5 g NaCl, 2 g soluble starch, 15 g agar, pH 7.0, 1000 mL distilled water. A total of 22 strains with protein, fat, cellulose, and starch degradation functions were screened out. The selected strains were cultured in LB medium at 37°C for 24 h and the colonies were observed. Figure 1 .

[0046] Whatman No. 1 filter paper was used to further verify the degradation ability of each strain. The activated strain was cultured to OD 600 =1, added to the filter paper strip culture medium at a 2% inoculation rate, and cultured on a shaker at 37°C and 150 r / min for 7 days. The degradation rate of the filter paper strips was counted, and different amounts of commercial cellulase were used as controls. The filter paper strip culture medium formula is: Whatman No. 1 filter paper 1×5 cm, 3 strips are added to the inorganic salt culture medium, and sterilized at 121°C for 20 min. The inorganic salt culture medium formula used is: ammonium sulfate 4 g, potassium dihydrogen phosphate 1 g, magnesium sulfate 0.3 g, sodium chloride 0.1 g, calcium chloride 0.1 g, ferric chloride 0.01 g, and purified water 1 L. The strains with the top 5 filter paper strip degradation rates were screened out. The degradation rates of filter paper strips were DF-8 (16.08%) > DF-22 (13.33%) > DF-10 (12.42%) > DF-1 (11.58%) > DF-5 (9.50%). The degradation rates of the first four strains were between 20u (11.50%) and 50u (18.00%) of commercial cellulase.

[0047] Example 2

[0048] Preparation of composite microbial agent for decomposing soybean straw

[0049] 2.1 Antagonism between soybean straw decomposing strains

[0050] The strain confrontation experiment was used to verify whether there was antagonism between the strains. By cross-colony colonization, the screened strains were crossed horizontally and vertically on the LB medium plate, and cultured at 37°C for 18 h to obtain a colony cross-culture plate. Whether there was a blank area at the intersection of the two strains was observed to determine whether there was antagonism between the bacteria. The results are shown in Figure 2 The five strains obtained through repeated screening were able to grow together without any mutual antagonism.

[0051] 2.2 Synergy between soybean straw decomposition strains

[0052] The straw degradation effect of the strain combination was used to investigate whether there was a synergistic effect between the re-screened strains. The strain combination scheme is shown in Table 1. Using soybean straw as the substrate, the OD of the fermentation liquid of a single strain was 600 After the value is adjusted to 1 (greater than 1×10 8 CFU / mL) were mixed in equal volume ratios and added to the straw culture medium at an inoculum of 5%, and sterile water was used instead of the bacterial agent as a control. The formula of the straw culture medium is: 5 g soybean straw, 100 mL inorganic salt culture solution, and sterilized at 121℃ for 20 min. The formula of the inorganic salt culture solution used is: 4 g ammonium sulfate, 1 g dipotassium hydrogen phosphate, 0.3 g magnesium sulfate, 0.1 g sodium chloride, 0.1 g calcium chloride, 0.01 g ferric chloride, 1 L pure water, pH 7.2. The degradation conditions are 37℃, 150 r / min in a shaking table for 7 d. After the end, the remaining straw was rinsed with distilled water in a 100-mesh filter to remove the bacteria and soluble substances attached to the surface of the straw, dried at 105℃ to constant weight, and the dry weight of the straw residue was weighed to calculate the straw degradation rate.

[0053] Table 1 Strain compounding scheme

[0054]

[0055] The straw degradation rates of single strains and compound strains were calculated by the formula of straw relative degradation rate RD = (m0-m1) / m0×100% (m0 is the dry weight of the remaining straw in the control group, m1 is the dry weight of the remaining straw in the treatment group). The straw degradation rates of strain diversity are shown in the figure. Figure 3 As shown in the data, the combination of strains DF-1, DF-8 and DF-22 had the highest straw degradation rate of 27.20%, which was higher than the straw degradation rates of single strains, double strains and multiple strains, showing a significant synergistic degradation effect with a streamlined combination.

[0056] The strains DF-1, DF-8 and DF-22 were phenotypically identified, genetically identified, sequenced and phylogenetically identified using 16S rDNA sequencing, and the phylogenetic tree was constructed. Figure 4 , strains DF-1, DF-8, and DF-22 were taxonomically identified as Bacillus sonoranus, Bacillus licheniformis, and Bacillus subtilis.

[0057] Gene sequence

[0058] The gene sequence of Sonora Desert Bacillus CGMCC No.30858DF-1 identified by 16S rRNA method is:

[0059] tcgagcgaaccgacgggagcttgctcccttaggttagcggcggacgggtgagtaacacgtgggtaacctg

[0060] cctgtaagactgggataactccgggaaaccggggctaataccggatgcttgattgaaccgcatggttcaa

[0061] ttataaaaggtggcttttagctaccacttacagatggacccgcggcgcattagctagttggtgaggtaac

[0062] ggcgtcaccaaggcgacgatggcggtagcgcggacctgagaagggtgatcggccacactgggactgagac

[0063] acggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaac

[0064] gccggcgtgagtgatgaaggttttcggatcgtaaaacttctgttgttagggaagaacaagtaccgttcga

[0065] acagggcggtgccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaata

[0066] cgtaggtggcaagcgttgtccggaattattgggcgtaaagcgcgcgcaggcggtttcttaagtctgatgt

[0067] gaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtgga

[0068] attccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtct

[0069] gtaactgacgctgaggcgcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaa

[0070] acgatgagtgctaagtgttagagggtttccgccctttagtgctgcagcaaacgcattaagcactccgcct

[0071] ggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtgg

[0072] tttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacacccctagagatagggctt

[0073] ccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtggagatgttgggttaa

[0074] gtccccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctataggtggactgc

[0075] cggtgacacaaccggaggaaggtggggatgaacgtcaaatcatcatgccccttatgacctgggctacaca

[0076] cgtgctacaatgggcagaacaaagggcagcgaagccgcgaggctaagccaatcccacaaatctgctctca

[0077] gttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgc

[0078] ggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcgg

[0079] tgaggtaacc (SEQ ID NO:1).

[0080] The gene sequence of Bacillus licheniformis CGMCC No. 30859 DF-8 identified by 16S rRNA method is:

[0081] ctggctccaaaaggttacctcaccgacttcgggtgttacaaactctcgtggtgtgacgggcggtgtgtac

[0082] aaggcccgggaacgtattcaccgcggcatgctgatccgcgattactagcgattccagcttcacgcagtcg

[0083] agttgcagactgcgatccgaactgagaacagatttgtgggattggcttaaagcctcgcggcttcgctgcc

[0084] ctttgttctgcccattgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatcccc

[0085] accttcctccggtttgtcaccggcagtcaccttagagtgcccaactgaatgctggcaactaagatcaagg

[0086] gttgcgctcgttgcgggacttaacccaacattctcacgacacgagctgacgacaaccatgcaccacctgt

[0087] cactctgcccccgaaggggaagccctatctctagggttgtcagaggatgtcaagacctggtaaggttctt

[0088] cgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggcccccgtcaattcctttgagtttcag

[0089] tcttgcgaccgtactccccaggcggagtgcttaatgcgtttgctgcagcactaaagggcggaaaccctct

[0090] aacacttagcactcatcgtttacggcgtggactaccagggtatctaatcctgttcgctccccacgctttc

[0091] gcgcctcagcgtcagttacagaccagagagtcgccttcgccactggtgttcctccacatctctacgcatt

[0092] tcaccgctacacgtggaattccactctcctcttctgcactcaagttccccagtttccaatgaccctcccc

[0093] ggttgagccgggggctttcacatcagacttaagaaaccgcctgcgcgcgctttacgcccaataattccgg

[0094] acaacgcttgccacctacgtattaccgcggctgctggcacgtagttagccgtggctttctggttaggtac

[0095] cgtcaaggtaccgccctattcgaacggtacttgttcttccctaacaacagagttttacgatccgaaaacc

[0096] ttcatcactcacgcgggcgttgctccggtcagactttcgtccatttgcggaagattccctactgctgcct

[0097] cccgtaggagtctgggccgtgtctcagtcccagtgtggccgatcaccctctcaggtcggctacgcatcgt

[0098] cgccttggtgagccgttacctcaccaactagctaatgcgccgcgggtccatctgcaagtggtagctaaaa

[0099] gccaccttttatgattgaaccatgcggttcaatcaagcatccggtattagccccggtttcccggagttat

[0100] cccagtcttacaggcaggttacccacgtgttactcacccgtccgccgctgacctaagggagcaagctccc

[0101] gtcggtccgctcga (SEQ ID NO:2).

[0102] The gene sequence of Bacillus subtilis CGMCC No.30860 DF-22 identified by 16S rRNA method is:

[0103] cctagactgcaagtcgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaaca

[0104] cgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtttgaa

[0105] ccgcatggttcaaacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagt

[0106] tggtgaggtaacggctcaccaaggcaacgatgcgtaaccgacctgagagggtgatcggccacactgggac

[0107] tgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacgr

[0108] agcaacsccgcgtgagtgatgaaggtyttcggatcgtaaagctctgttgwtagsgaagaacaagtaccgt

[0109] tcgaatagggcggtaccttgacggtacctarccagaaagccacggctaactacgtgccagcagccgcggt

[0110] aatacgtaggtggcaagcgttgtcyggaattattgggcgtaaagggctcgcaggcggtttcttaagtctg

[0111] atgtgaaagcccccggctcaaccggggagrgtcattggaaactggggaacttgagtgcagaagaggagag

[0112] tggaattccacgtgtagcggtgaaatgygtagagatgtggaggaacaccagtggcgaaggcgactctctg

[0113] gtctgtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgcc

[0114] gtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactcc

[0115] gcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcat

[0116] gtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagatagg

[0117] acgtccccttcgggsgcaragtgacasgtgstgcatggttgtcgtcagctcgtgtcgtggagatgttggg

[0118] ttaagtccccgcaacragcgcmacmcgtwgatcttagattgccagcattcmsttgggcactctaaggtga

[0119] ctgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctaca

[0120] cacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttct

[0121] cagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgcc

[0122] gcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtc

[0123] ggtgaggtaacctcttaggagccagcccaccg (SEQ ID NO: 3).

[0124] 2.3 Preparation of single bacterial inoculant

[0125] Take the frozen DF-1, DF-8 and DF-22 bacterial liquids and streak them on LB solid culture medium respectively, place them in a 37℃ incubator for overnight culture to obtain single colonies, pick single colonies and put them in a triangle flask containing 100 mL LB liquid culture medium, culture them at 37℃ 180r / min overnight for activation, take 2 mL and inoculate them in a 500 mL triangle flask containing 200 mL LB liquid culture medium, culture them at 37℃ 180r / min for 24 h, and use a spectrophotometer to measure the OD of the three bacterial liquids respectively 600 The value was adjusted to 3 (greater than 2 × 10 8 CFU / mL), that is, a single bacterial agent.

[0126] 2.4 Preparation of composite bacterial agent

[0127] The fermentation broths of the single strains were mixed at a volume ratio of 1:1:1 and the OD 600 Adjust the value to 3 (greater than 2×10 8 CFU / mL) is a composite bacterial agent.

[0128] Example 3

[0129] Verification of the synergistic degradation function of composite bacterial agents

[0130] The preparation of the single bacterial agent and the composite bacterial agent refers to Example 2, and the synergy of the composite bacterial agent is verified by the degradation enzyme activity and straw degradation rate of the single bacterial agent and the composite bacterial agent.

[0131] 3.1 Degradation enzyme activity of composite bacterial agent

[0132] The method for determining the activity of filter paper degrading enzyme is as follows: take 10 mL of bacterial solution into a 15 mL centrifuge tube, centrifuge at 4°C and 10,000 rpm for 10 min, take 2 portions of 0.5 mL of supernatant, add them to 2 20 mL stoppered test tubes respectively, and then add 1.5 mL of citric acid buffer respectively. Add 2.0 mL of DNS reagent to the first test tube and preheat it in a 50°C water bath for 5 min. Add 1 Whatman No. 1 filter paper with a specification of 5×1 cm to each of the two test tubes, shake well and put it in a 50°C water bath, and react at a constant temperature for 60 min. After taking it out, add 2 mL of DNS reagent to the second test tube, boil the No. 1 and No. 2 test tubes in a boiling water bath for 5 min, take them out, cool them to room temperature, shake well and add water to the 15 ml mark, use the first test tube as a control, and measure the absorbance at 540 nm.

[0133] The results of filter paper degradation enzyme activity are shown in Figure 5 In (a), the filter paper degrading enzyme activities of DF-1, DF-8 and DF-22 single bacterial agents were 2.43 U / mL, 1.19 U / mL and 0.15 U / mL, respectively, while the filter paper degrading enzyme activity of the composite bacterial agent was 4.26 U / mL, which was significantly higher than that of the single bacterial agent, indicating that the composite bacterial agent has a synergistic effect in the filter paper degradation function.

[0134] The method for determining the activity of CMC-Na enzyme was the same as that for the filter paper degradation enzyme, except that the degradation object was replaced by 1.5 mL 1% CMC-Na solution instead of 5×1 cm Whatman No. 1 filter paper. Figure 5 In (b), the CMCase of DF-1, DF-8 and DF-22 single bacterial agents were 15.73 U / mL, 6.26 U / mL and 1.97 U / mL respectively, while the CMCase of the composite agent was 23.27 U / mL, which was significantly higher than that of the single bacterial agent. The composite agent had a significant synergistic effect in the degradation enzyme activity, that is, it achieved the effect of 1+1+1>3.

[0135] 3.2 Degradation rate of straw by composite microbial agent

[0136] Soybean straw was used as the substrate, and DF-1, DF-8, DF-22 single bacteria agents and composite bacteria agents were inoculated into the straw culture medium at an inoculation rate of 5%. The formula of the straw culture medium was: 5 g soybean straw, 100 mL inorganic salt culture solution, and sterilized at 121℃ for 20 min. The formula of the inorganic salt culture solution used was: 4 g ammonium sulfate, 1 g dipotassium hydrogen phosphate, 0.3 g magnesium sulfate, 0.1 g sodium chloride, 0.1 g calcium chloride, 0.01 g ferric chloride, 1 L pure water, pH 7.2. The degradation conditions were shaker culture at 37℃ and 150 r / min for 7 days. After 1 d, 3 d, 5 d and 7 d of degradation, the remaining straw was rinsed with distilled water in a 100-mesh filter to remove the bacteria and soluble substances attached to the surface of the straw, and dried at 105℃ to constant weight. The dry weight of the straw residue was weighed and the straw degradation rate was calculated. The calculation formula is RD = (m0-m1) / m0×100% (m0 is the remaining dry weight of straw in the control group, and m1 is the remaining dry weight of straw in the treatment group).

[0137] The results are as follows Figure 5 As shown in (c), the straw degradation rate increases with time. On the 7th day, the straw degradation rates of DF-1, DF-8 and DF-22 single bacteria agents were 13.57%, 14.31% and 9.77%, respectively, and the straw degradation rate of the composite agent reached 27.20%. The straw degradation rates of the composite agent were 2.01 times, 1.90 times and 2.78 times that of DF-1, DF-8 and DF-22 single bacteria agents, respectively. This shows that the composite agent has significant synergy in straw degradation function.

[0138] Example 4

[0139] Soybean straw decomposition compound microbial agent promotes compost maturity

[0140] The preparation of the composite bacterial agent refers to Example 2. A foam insulation box with an internal specification of 33×22×23 cm was used as a composting container. Cow dung, distiller's grains, and soybean straw were crushed and mixed in a ratio of 2:1:2. The total amount was 1 kg and the moisture content was adjusted to 55%-65%. 1×10 9 cfu / kg of compound bacteria were inoculated, and sterile water was used instead of bacteria as a control. The mixture was stirred evenly, and piled in a constant temperature box at 37℃. The internal temperature of the pile was measured daily, the pile was turned over regularly, and samples were taken to measure the pH and EC values. The changes in temperature, pH and EC values ​​reflected the promoting effect of the compound bacteria on the composting process. The composting was carried out for 35 days in total. At the end of the composting, the effect of the compost product on seed germination was measured. The seed germination index was used to reflect the reducing effect of the compound bacteria on the toxicity of compost plants.

[0141] 4.1 The effect of compound microbial agents on the composting process

[0142] The results of compost temperature changes are shown in Figure 6 In (a), the high temperature period began on the second day, and the highest temperature of the group treated with the compound bacteria reached 68°C, 13°C higher than the highest temperature of the control group. The temperature of the group treated with the compound bacteria rose faster than that of the control group, and the high temperature lasted longer, indicating that the compound bacteria can accelerate the composting reaction process and shorten the composting cycle. The results of compost pH changes are shown in Figure 6 In (b), the pH change trend of the treatment group with compound bacteria is similar to that of the control group, indicating that the compound bacteria has little effect on the pH of the compost. Figure 6 In (c), the EC values ​​of the treatment group connected with the composite bacterial agent and the control group during the fermentation process showed a trend of first increasing and then decreasing. During the stabilization period and cooling period of the composting, the EC of the treatment group connected with the composite bacterial agent decreased more rapidly, indicating that the decomposition process was accelerated.

[0143] 4.2 Effect of compound microbial agents on reducing phytotoxicity of compost products

[0144] Seed germination rate and seed germination index are important indicators of the maturity of the reactor compost. According to the national standard NY525-2021, when the GI is greater than 80%, it can be considered that the compost has no phytotoxicity or is mature. Figure 6 As shown in (d), the seed germination index of the compost product treated with the composite bacterial agent was 99.05%, reaching the maturity standard and 10.4% higher than that of the control group, indicating that the composite bacterial agent can significantly reduce the phytotoxicity of the compost and promote the maturity of the compost.

[0145] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. A soybean straw decomposition composite bacterial agent, characterized in that: The soybean straw decomposition composite bacterial agent is made from Sonora Desert Bacillus Bacillus sonorensis DF-1, Bacillus licheniformis Bacillus licheniformis DF-8 and Bacillus subtilis Bacillus subtilis DF-22 composition: The Sonora Desert Bacillus DF-1, Bacillus licheniformis DF-8 and Bacillus subtilis DF-22 have all been sent to the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit date of June 5, 2024, and a deposit address of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Among them, the Sonora Desert Bacillus DF-1 has a preservation number of CGMCC No. 30858; the licheniformis DF-8 has a preservation number of CGMCC No. 30859; the subtilis DF-22 has a preservation number of CGMCC No. 30860; The mixing volume ratio of the Sonora Desert Bacillus DF-1, Bacillus licheniformis DF-8 and Bacillus subtilis DF-22 is 1:(1-2):(1-2); The total number of viable bacteria of Sonora Desert Bacillus DF-1, Bacillus licheniformis DF-8 and Bacillus subtilis DF-22 is higher than 1×10 9 CFU / mL.

2. The soybean straw decomposing composite bacterial agent according to claim 1, characterized in that: The mixing volume ratio of the Sonora Desert Bacillus DF-1, the Bacillus licheniformis DF-8 and the Bacillus subtilis DF-22 is 1:1:

1.

3. Use of the soybean straw decomposing composite bacterial agent according to claim 1 or 2 in straw decomposition.

4. The use according to claim 3, characterized in that The applied temperature is 30-50°C.

5. The use according to claim 3, characterized in that The straw is soybean straw.

6. Use of the soybean straw decomposing composite bacterial agent according to claim 1 or 2 in promoting the straw composting process.

7. The use according to claim 6, characterized in that The method of promoting the straw composting process is to reduce the phytotoxicity of the compost product.

8. The use according to claim 6, characterized in that The applied temperature is 30-50° C., and the straw is soybean straw.

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